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SURVIVAL OF BACILLUS CEREUS IN ARTIFICIALLY CONTAMINATED UHT MILK AND INFANT MILK
Kong Wan Wei
(36541)
QR Bachelor of Science with Honours
(Resource Biotechnology) - B3 2015K82
2015
82
Pusat Khidmat Maklumat Akademik UN IVERSITI MALAYSI A SARAWAK
Survival of Bacillus cereus in Artificially Contaminated UHT Milk and Infant Milk
Kong Wan Wei (36541)
This project is submitted in full fulfilment of the requirement for the Degree of Bachelor of Science with Honours (Resource Biotechnology)
Supervisor Dr Lesley Maurite BiIung Co-supervisor Dr Hashimatul Fatma Hashim
Resource Biotechnology Department of Molecular Biology
Faculty of Resource Science and Technology Universiti Malaysia Sarawak
2015
Acknowledgement
My greatest gratitude goes to my supervisor Dr Lesley Maurice Bilung for her
guidance support and ad vices through out the whole course of my fmal year project I would
like to thanks my co-supervisor Dr Hashimatul Fatma Hashim for her guidance and support
throughout my fmal year project
My appreciation also goes to the postgraduates especially Ernie Suhaiza Pui Chai
Fung and Cindy Lai Sin Chai for their guidance help and patience in assisting me to fmish my
fmal year project In addition I also appreciate the helps and patience of lab assistants
especially Mr Aziz Ajim and Miss Kamaliawati Yusop during my final year project
Not forgetting my family (dad mum Fion Wan Ru Alice and Eddie) for their
unending love and encouragement I also appreciate the help and support from my friends and
coursemates while conducting my fmal year project
UNIVERSITI MALAYSIA SARAWAK
Grade
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Final Year Project Report IV I Masters 0 PhD D
DECLARATION OF ORIGINAL WORK
This declaration is made on the ~ day of-~~2015
Students Declaration
I Kong Wan Wei 36541 of Faculty of Resource Science and Technology hereby declare that the work entitled Survival of Bacillus cereus in Artificially Contaminated UHT Milk and Infant Milk is my original work I have not copied from any other students work or from any other sources except where due reference or acknowledgement is made explicitly in the text nor has any part been written for me by another person
rune 10 J lOIS (005 ~o ~ti (~b6t) Date submitted Name of the student (Matric No)
Supervisors Declaration
I Lesley Maurice Bilung hereby certifies that the work entitled Survival of Bacillus cereus in Artificially Contaminated UHT Milk and Infant Milk was prepared by the above named student and was submitted to the FACULTY as a gtI middotpartiaUfull fulfillment for the confennent of Bachelor of Science with Honours and the aforementioned work to the best of my knowledge is the sAid students work
Lesley Mamce Bilunl (PhD) Senior Lecturer
Received for examination by
Department of Molecular BioIoc) Fa4ulty of Resource Science ud Tlwology
Universiti Malaysia Sarawak Date 1
0 r~
(Name of the supervisor)
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research was done)
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P sat Khidmat Maklumat Akademik UN VERSITI MALAYSIA ARAWAK
Table of Contents
Acknowledgement
Declaration II
Table of Contents IV
List of Abbreviations VI
List ofTables VII
List of Figures IX
Abstract XI
10 Introduction
20 Literature Review 4
21 Bacillus cereus 4
211 Morphological and biochemical characteristics 4
21 2 B cereus food borne disease 4
213 Isolation and identification of Bcereus 5
214 Production of enterotoxins 5
215 Effect of temperature and pH on growth and survival of B 6
cereus
22 UHT milk 7
23 Infant rriilk 7
24 Polymerase Chain Reaction (PCR) nd agarose gel electrophoresis 8
IV
30 Materials and Methods 10
31 The milk 10
32 Inocula preparation 10
33 Survival and growth of B cereus in UHT milk and infant milk 10
331 Effect of temperature 10
332 Effect ofpH 11
34 Determination of growth and survival of B cereus in UHT milk and 11
infant milk samples
35 Genomic Deoxyribonucleic acid (DNA) extraction 12
36 PCR 12
37 Statistical analysis 12
40 Results 14
50 Discussion 42
60 Conclusion 51
70 References 52
v
I
List of Abbreviations
ANOVA Analysis of Variance
B cereus Bacillus cereus
BHI Brain Heart Infusion
CBA-P Columbia base-blood polymyxin B agar
CFU colony-forming unit
FDA Food and Drug Administration
gyrB gyrase B
HCl Hydrogen chloride
MYP Mannitol Egg Yolk agar
microlitre
ml millilitre
NaOH Sodium hydroxide
PEMBA Polymyxin B-egg yolk-mannitol bromothymol blue agar
PCR Polymerase Chain Reaction
UHT ultra-high temperature
VP Voges-Proskauer
VI
List of Tables
Table 1 Mixture volumes ofPCR for detection of B cereus 13
Table 2 Thermal cycling profile ofPCR for detection ofB cereus 13
Table 3 B cereus colony count from day 0 to day 7 in UHT milk stored at 14
temperature range of4 middotC to 7 middotC
Table 4 B cereus colony count from day 0 to day 7 in infant milk stored at 15
temperature range of 4 middotC to 7 c
Table 5 B cereus colony count from day 0 to day 7 in UHT milk stored at 16
temperature range of 10 middotC to 13 middotC
Table 6 B cereus colony count from day 0 to day 7 in infant milk stored at 17
temperature range of 10 middotC to 13 middotC
Table 7 B cereus colony count from day 0 to day 7 in UHT milk stored at 25degC 18
Table 8 B cereus colony count from day 0 to day 7 in infant milk stored at 25 19
DC
Table 9 B cereus colony count from day 0 to day 7 in UHT milk stored at 35degC 20
Table 10 B cereus colony count from day 0 to day 7 in infant milk stored at 35 20
DC
Table 11 B cereus colony count from day 0 to day 7 in UHT milk stored at 45degC 22
Table 12 B cereus colony count from day 0 to day 7 in infant milk stored at 45 22
DC
Table 13 B cereus colony count from day 0 to day 7 in UHT milk stored at 55degC 24
VII
Table 14 B cereus colony count from day 0 to day 7 in infant milk stored at 55 24
0c
Table 15 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 29
ofpH 620 stored at refrigerated temperature
Table 16 B cereus colony count from day 0 to day 7 in infant milk with initial pH 30
of pH 620 stored at refrigerated temperature
Table 17 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 31
of pH 640 stored at refrigerated temperature
Table 18 B cereus colony count from day 0 to day 7 in infant milk with initial pH 32
of pH 640 stored at refrigerated temperature
Table 19 B cereus colony count from day 0 to day 7 in UHT milk with initia l pH 33
of pH 660 stored at refrigerated temperature
Table 20 B cereus colony count from d-ay 0 to day 7 in infant milk with initial pH 34
of pH 660 stored at refrigerated temperature
Table 21 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 35
of pH 680 stored at refrigerated temperature
Table 22 B cereus colony count from day 0 to day 7 in infant milk with initial pH 36
of pH 680 stored at refrigerated temperature
VIII
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
List of Figures
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 4 middotC to 7 c
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 10 middotC to 13 middotC
15
17
The survival curve of B cereus
infant milk stored at 25 c
The survival curve of B cereus
infant milk stored at 35 c
The survival curve of B cereus
infant milk stored at 45 c
The survival curve of B cereus
infant milk stored at 55 middotC
in artificially contaminated UHT and 19
in artificially contaminated UHT and 21
in artificially contaminated UHT and 23
in artificially contaminated UHT and 25
The survival curve of B cereus in aiiifIcially contaminated UHT milk 27
stored at 4 middotC -7 middotc 10 middotC-13 middotC 25 middotc 35 middotC 45 middotC and 55 middotC
The survival curve of B cereus in artificially contaminated infant milk 28
stored at 4 middotC -7 middotC 10 middotC-13 middotC 25 middotC 35 middotC 45 middotC and 55 c
The survival curve of B cereus in artifIcially contaminated UHT and 30
infant milk with initial pH of pH 620 stored at refrigerated temperature
(4 QC-7 QC)
IX
Figure 10 The survival curve of B cereus in artificially contaminated UHT and 32
infant milk with initial pH of pH 640 stored at refrigerated temperature
(4 middotC-7 C)
Figure 11 The survival curve of B cereus in artificially contaminated UHT and 34
infant milk with initial pH of pH 660 stored at refrigerated temperature
(4 middotC-7 C)
Figure 12 The survival curve of B cereus in artificially contaminated UHT and 36
infant milk with initial pH of pH 680 stored at refrigerated temperature
(4 middotC-7 C)
Figure 13 The survival curve of B cereus in artificially contaminated UHT milk 38
with different initial pH stored at refrigerated temperature (4 middotC-7 C)
Figure 14 The survival curve of B cereus in artificially contaminated infant milk 39
with different initial pH stored- at refrigerated temperature (4 middotC-7 middotC)
Figure 15 B cereus colonies grown on MYP Agar 40
Figure 16 Morphology of B cereus isolated from UHT milk and infant milk 40
samples
Figure 17 Amplification of the gyrB gene (475 bp) for the confirmation of B 41
cereus in UHT milk and infant milk samples
x
Survival ofBacillus cereus in Artificially Contaminated UHT Milk and Infant Milk
Kong Wan Wei
Resource Bioteclmology Department of Molecular Biology
Faculty of Resource Science and Teclmology Universiti Malaysia Sarawak
Abstract
The inappropriate storage temperature and pH enable the survival of Bacillus cereus a known food pathogen in processed milk products resulting in foodborne diseases In the present study UHT milk and infant milk purchased from local supermarkets in Kuching Sarawak were inoculated with 103 CFUml of B cereus Selected storage temperatures of 4 C-7 C 10 C-13 C 25 C 35 C 45 C and 55 C were used to investigate the effects of temperature on the survival of B cereus Initial pH of 620640 660 and 680 at refrigerated temperature (4 C-7 C) were used to study the effects of pH on the survival of B cereus Enumeration of colony counts were investigated daily for the duration of seven days Gram staining and Polymerase Chain Reaction (PCR) targeting gyrB gene were performed for the confirmation of B cereus isolates The present findings revealed that the survival of B cereus in UHT milk and infant mil k showed significant differences (pltO05) in different temperatures and pH At 25 C there were significant difference (plt005) between the survival of B cereus in UHT milk and infant milk B cereus were able to survive under all the experimental temperatures However growth was detected at all storage temperatures except 55 c Survival of B cereus in artificially contaminated UHT milk and infant milk with different initial pH stored at referigerated temperature were also recorded The results from this study can be treated as background data for the implementation of safety measure to avoid food poisoning caused by B cere liS
Key words Bacillus cereus survival milk temperature pH
Abstrak
Suhll penyimpanan dan pH yang tidak sesllai membolehkall kehidupan Bacillus cereus sejenis pathogen makanan dalam produk susu diproses dan mellyebabkan penyakit bawaan makanall Dalam kajian ini SIISII UHT dan SIISU bayi yang dibeli daripada pasar raya tempatan di Kuching Sarawak telah diinoklliasi dengan B cerells sebanyak J(i CFUlml Suhll penyimpanan 4 C-7 c 10 C-3 c 25 c 35 middotc 45 c dan 55 c telah digllnakan IIntllk mengkaji kesan slIhll terhadap kehidupan B cerells Pada suhll peti sejuk (4 C-7 C) pH 620 pH 640 pH 660 dan pH 680 telah digunakan sebagai pH permulaal IIntuk mengkaji kesan pH terhadap keflidupan B cereus Penggiraall kololli telah dijalankan setiap hari selama IIIjuh hari Pewarnaan Gram dan penggllnaan reaksi rantai polimerase (PCR) yang menyasarkan gen gyrB telah mengesahkan kewujlldan koloni B cereus Kajian ini menllnjukkan buhawa kehidupan B cereus dalam SIISU UHT dan susu bayi menunjukkan perbezaan yang ketara (plt005) di suhu penyimpanan dan pH yang berbeza Perbezaan yang ketara (plt005) berlaku anlara kehidupan B cereus dalam susu UHT dengan kehidllpan B cereus dalam SIISII bayi pada sllhu 25 c B cereus dapal menunjllkkan kehidupan dalam SIISU UHT dan SIISU bayi yang telah disimpan pada semua suhll eksperimen Walau bagaimanapun pertumbllhan dapat dikesan pada semua sllhll eksperimen keclali 55 c Selain itll kehidllpan B cerells dalam SUSII UHT dan SUSII bayi yang tercemar secara bllalan pada slIhll peli sejuk telah dicatat pada peJbagai pH Hasil daripadu kajian ini boleh dianggap sebagai dala lalar belakallg tllltllk pelaksanuan langkah keselamatan bagi mengelakkan keracunall makanall daripada B cereus
Kata kunci Bacillus cerellS keflidllpan SIISU sllltl pH
XI
10 Introduction
Population growth and increase in consciousness about health are some of the factors that
increase demand for milk and milk products in Malaysia In Malaysian market ultra-high
temperature (UHT) milk and infant milk powders come under a variety of brands and are
either locally produced or imported Milk provides calcium protein vitamin A vitamin B 12
riboflavin and zinc to the human diet Consumption of milk helps to build and maintain bone
health as well as to reduce the risk of osteoporosis Infant milk provides major nutrients to
infants and it is essential for the growth of newborns
Milk is a high moisture and high nutritious medium which is also suitable for the
growth of mircoorganisms Most of the people believed that processed milk samples are
totally safe for consumption but actually there are many ways that leads to milk
contamination Contamination happened duripg milk processing as Bacillus cereus (B cereus)
strains which were not found in raw milk samples was present in the processed milk samples
(Banyk6 and Vyletelova 2009) Microorganisms contaminate the milk and affects the milk
quality by infecting the mammary gland udder and teat surfaces milking equipment and
storage tank (Gleeson et al 2013) Moreover improper processing and the usage of raw milk
that did not meet the microbiology and physio-chemical standards lead to the defects in raw
and UHT milk (Abdul-Hadi et al 2014) Inappropriate food storage and handling of
contaminated food encourage the growth of B cereus to the levels which can cause food
poisoning (Granum 2005) In order to maintain the quality and safety of processed milk
products consumers are suggested to store these products at temperature of 4 middotC or [less (Boor
and Fromm 2006)
Food pOlsonmg which may even have lethal effect on human is due to the
inappropriate food preparation and food exposure to 30 middotC (Sandra et at 2012) From year
1998 until 2008 there were 235 cases of foodbome disease outbreaks associated with B
cereus in United States (Bennett et at 2013) In 2011 the number of B cereus foodborne
diseases in Europe showed an increase of 1222 (MesseJhausser et ai 2014) The number
reported shows that B cereus poses a threat to the public health as it is often found in
contaminated food However the number of B cereus foodbome poisoning is believed to be
under reported because this type of food poisoning is mild and reporting these cases is not an
compulsory act (EFSA 2005)
B cereus was recognized as foodbome pathogen since 1955 and it is the cause for
about 2 of all foodbome disease worldwide (Lesley et at 2013) Garcia-Arribas and Kramer
(1990) mentioned that B cereus contribute to economic loss by causing contamination
spoilage and food-poisoning B cereus are found in dairy products meats spices and cereals
(Browne and Dowds 2002) as well as in infant foods and dried milk products (Becker et aI
1994)
Most of the patients involved in B cereus outbreaks were infants or elders (Becker et
ai 1994) The immune system of infants are still developing so they are highly susceptible to
B cereus food poisoning after consuming contaminated infant milk Although infant milk
basically present in low moisture powder form the vegetative cells and spores of
microorganisms are able to survive after drying but with their metabolism reduced (I LSI
Europe 2011) This poses threat to infants because some consumers keep the reconstituted
infant formula at room temperature for a long period of time as they wrongly believed that
infant formula are sterilised and are completely free of B cereus (ILSI Europe 2011)
2
B cereus is conunonly related to dairy products because they are able to survive at
different environmental stresses such as pasteurization (Foltys and Kirchnerova 2006 Sadek
et (II 2006) Schelderman et al (2006) discussed that aerobic spore former such as B cereus
has increased tolerance towards low temperature low pH sterilization and UHT treatments
and these conditions increase the concern in food industry In year 2011 there were food
poisoning associated with UHT milk provided from 1 Malaysia Milk Progranune and B cereus
were detected in these UHT milk (Yusof 2011) In 2013 infant milk formula product from
Fonterra were tested positive for Clostridium botulinum which is known as an endospore
former (Frediansyah 2014) The botulism scare was later confirmed as false alarm and the
actual organism was Clostridium 5porogenes However these pathogens have alarmed public
on the safety ofUHT milk and infant milk Currently there are no known studies in Malaysia
examining the factors influencing the survival of B cereus in UHT milk and infant milk
Hence the objectives of this study were
I To study the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk stored at different temperature for seven days
II To examine the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk with different initial pH stored at refrigerated temperature for seven
days
3
20 Literature Review
21 Bacillus cereus
211 Morphological and biochemical characteristics
Bacillus cereus is a Gram positive motile rod-shaped aerobic or facultative anaerobe and
aerobic spore forming foodborne pathogen belonging to Bacillus genus that is normally found
in soil and vegetation (Tortora et al 2010 Food Standards Australia New Zealand 2013
Gleeson et al 2013) This bacterium is mesophile or psychrotroph This bacterium is closely
related to B thuringiensis B mycoides B weihenstephanensis B pseudomycoides and B
anthracis (Gleeson et al 2013) The ability of B cereus to produce endospores which are heat
resistant increases the survivality of B cereus at unfavourable conditions such as high
temperatures extreme pH values or envirorunents with insufficient nutrients compared to
other bacterial enteropathogens (Di Pinto et al 2013 Gleeson et al 2013) For the
biochemical characteristics B cereus are positive for motility Voges-Proskauer (VP)
reaction starch hydrolysis citrate catalase and negative for oxidase tests (Shinagawa 1990
Choma ef al 2000) B cereus is shown as slender bacilli with square end singly or in short
chains under Gram-staining (Bottone 2010)
212 B cereus food borne disease
Rice dishes and meat or poultry dishes were the common sources for B cereus outbreaks
(Bennett et al 2013) Rice and pasta contaminated with B cereus are the causes for emetic
syndrome while the causes for diarrheal illness are desserts meat dishes and dairy products
(Granum 2005) Endospores of B cereus were able to survive in improperly cooked rice and
they caused Fried Rice Syndrome (Sandra et al 2012) In Malaysia B cereus was fIrst
4
Pusat Khidmat Maklumat Ak d Ut lVERSTTJ MA AYSIA SARAWAK
suspected as the cause for food poisoning which affected 114 female students in Klang
(Rampal et al 1984) In order to confIrm that gastroenteritis is caused by B cereus the
number of B cereus iso lated from suspected food need to exceed 105 co lony- forming unit per
gram (CFUg) (Tortora et al 20[0)
213 Isolation and identification of B cereus
Mannitol egg yolk polymyxin agar (MYF) Kim-Geopfert agar Columbia base-blood
polymyxin B agar (CBA-P) and Polymyxin B-egg yolk-mannitol bromothymol blue agar
(PEMBA) are some of the selective media suitable for isoIation and identifIcation of B cereus
(van Netten and Kramer 1992) The inability to ferment mannitol causes B cereus colonies to
appear as pink on MYP and lecithinase activity enables the observation of precipitated zone
around colonies (Pacheco-Sanchez and Massaguer 2007) Food and Drug Administration
(FDA) recommended MYP agar as the stapdard media for identifIcation of B cereus
214 Production of enterotoxins
The two forms of illness associated with B cereus enterotoxins are diarrheal and emetic
syndromes Onset of watery diarrhea and abdominal pain which resemble Clostridium
pefringens intoxications occur 8 hours to 16 hours after ingestion of contaminated food are
the symptoms of diarrheal outbreak (Shinagawa 1990 Tortora et ai 2010) This diarrheal
syndrome occurs after the ingestion of vegetattve cells which later produce a protein
enterotoxin in the gastrointinal tract (Bottone 2010) This type of enterotoxin is heat-labile
(Bello et al 2014) Haemolysin BL (HBL) non-haemolytic enterotoxin and cytotoxin K are
the three types of enterotoxins produced by this bacterium (Samarzija et al 2012 Food
Standards Australia New Zealand 2013) Reis et al (2013) concluded that hblD was the
5
common HBL gene of B cereus detected in milk and dairy products leading to diarrheal form
ofB cereus foodborne disease
The symptoms of emetic outbreaks are similar to Staphylococcus aureus food
intoxication and occur 05 hour to 6 hours upon ingestion of contaminated food
(Messelhausser et al 2014) The ingestion of food contaminated with preformed cereulide
which is also known as the emetic toxin leads to emetic syndrome (Bottone 2010) This heatshy
stable toxin causes nausea and vomiting in the infected patients (Bello et al2014)
215 Effect of temperature and pH on growth and survival of B cereus
Generally B cereus grows between 4 C and 55 C but exhibit optimum growth between 30 C
and 40 C (Schraft and Griffiths 2006 Food Standards Australia New Zealand 2013) Growth
of B cereus had been detected at temperature ranging from 7 C to 45 C and significant
growth was observed at 45 c in steril ized raw milk samples (Bellow et al 2007) In the
absence of oxygen or at temperature below 10 C emetic B cereus are believed to show
incapability in growth and production of toxin (EFSA 2005) At temperature below 10 C
with the limitation of other factors the lag time and generation times of B cereus are longer
(EFSA 2005)
As mentioned by Food Standards Australia New Zealand (2013) B cereus is able to
grow at pH between pH 49 and pH 100 and the optimum pH is between pH 60 and pH 70
Bello et al (2014) concluded that B cereus was unable to grow at pH 63 to pH 68 for 24
hour in pasteurized whole cow milk and the growth was inhibited at pH 68 for a period of 48
hours Byaruhanga et af (1999) studied that mague base inoculated with B cereus for 24
hours at 3S C was able to grow when pH decreases from pH 65 to pH 50 Brain Heart
6
--~~~----~----~----------
Infusion (BHI) medium at pH 88 inhibits growth of emetic and diarrheal strains of B cereus
better than pH 50 of the same medium (Garcia-Arribas and Kramer 1990)
22 UHT milk
Milk treated with UHT pasteurization is heated to 138 middotC for 2 seconds and aseptically placed
into a sterile substantial container which can be stored without refrigeration for six months to
a year (Ledenbach and Marshall 2009) UHT treatment minimized heat denaturation and
produces long shelf life milk that has similar taste and color to pasteurized milk (Harding
1995) UHT milk is generally contaminated during technological processing because food
pathogens are destroyed by the UHT temperature (Vyletelova et al 2002) Longer storage
period causes the drop of UHT milk pH and enzymes may be the reason for changes in UHT
milk (Hassan et al 2009)
Among the 110 UHT milk samples tested by Vidal-Martins et al (2005) 118
(1 3110) samples were positive for B cereus Researchers Rezende-Lago et al (2007)
identified B cereus in four (13 3) of the UHT milk samples tested In the study performed
by Reis et al (2013) among the 260 samples of UHT milk pasteurized milk and powdered
milk tested in Brazil a total of 63 isolates of B cereus were found and 12 of them were from
UHT milk
23 Infant milk
Cows milk and soy protein are the major component of infant milk or infant formulas For the
production of infant formula from milk the milk is heat-treated concentrated and dried while
infant fonnula from other ingredients is dry-blended wetted and dried again or only dry-
blended (Becker et al 1994) The sublethally injured pathogens can recover after the process
7
of drying because this process caused only little damage to the cell (Miller et ai 2012) During
the process of drying components such as lactose protein and milk fat that are present in
milk-based infant formula have the potential to provide protection to the bacteria (Mi1er et ai
20 12) Infant formulas are given to newborns and the content of nutrition is similar to breast
milk (Becker et al 1994) Currently infant formulas in the market are available in powder
liquid concentration and ready-to-feed After rehydration and inappropriate storage lowshy
moisture foods such as infant milk powder should be consumed within a short time because
growth of microorganisms may occur (lLSI Europe 2011)
Becker et al (1994) tested 92 infant milk formula samples and 52 of the samples
were B cereus positive Another study by Rowan et al (1997) tested 100 samples of infant
milk formula from United Kingdom and 17 of the samples were B cereus positive In the
study done by Haughton et al (20 I 0) B cereus were detected in 59 out of 100 infant milk
formula samples from Ireland These studies showed that B cereus is commonly found in
most of the infant milk formula and this poses threat to infants who are highly susceptible
24 Polymerase Chain Reaction (PCR)
As ment ioned by Sandra et al (2012) PCR allows rapid detection of pathogens Schraft and
Griffiths (2006) mentioned that the detection of B cereus targetting B cereus per se and
specific sub-groups have been performed by PCR Yamada et al (1999) designed PCR primer
set for gyrB fragment of B cereus and used this primer set in the detection of B cereus in
boiled rice Confirmation of B cereus in typical colonies are performed using PCR techniques
(Rahimi el al 2013) Thousands to millions copies of specific DNA sequence are produced
from the amplification of a single or a few copies of a piece of DNA by PCR (Joshi and
8
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
Pusat Khidmat Maklumat Akademik UN IVERSITI MALAYSI A SARAWAK
Survival of Bacillus cereus in Artificially Contaminated UHT Milk and Infant Milk
Kong Wan Wei (36541)
This project is submitted in full fulfilment of the requirement for the Degree of Bachelor of Science with Honours (Resource Biotechnology)
Supervisor Dr Lesley Maurite BiIung Co-supervisor Dr Hashimatul Fatma Hashim
Resource Biotechnology Department of Molecular Biology
Faculty of Resource Science and Technology Universiti Malaysia Sarawak
2015
Acknowledgement
My greatest gratitude goes to my supervisor Dr Lesley Maurice Bilung for her
guidance support and ad vices through out the whole course of my fmal year project I would
like to thanks my co-supervisor Dr Hashimatul Fatma Hashim for her guidance and support
throughout my fmal year project
My appreciation also goes to the postgraduates especially Ernie Suhaiza Pui Chai
Fung and Cindy Lai Sin Chai for their guidance help and patience in assisting me to fmish my
fmal year project In addition I also appreciate the helps and patience of lab assistants
especially Mr Aziz Ajim and Miss Kamaliawati Yusop during my final year project
Not forgetting my family (dad mum Fion Wan Ru Alice and Eddie) for their
unending love and encouragement I also appreciate the help and support from my friends and
coursemates while conducting my fmal year project
UNIVERSITI MALAYSIA SARAWAK
Grade
Please tick (J)
Final Year Project Report IV I Masters 0 PhD D
DECLARATION OF ORIGINAL WORK
This declaration is made on the ~ day of-~~2015
Students Declaration
I Kong Wan Wei 36541 of Faculty of Resource Science and Technology hereby declare that the work entitled Survival of Bacillus cereus in Artificially Contaminated UHT Milk and Infant Milk is my original work I have not copied from any other students work or from any other sources except where due reference or acknowledgement is made explicitly in the text nor has any part been written for me by another person
rune 10 J lOIS (005 ~o ~ti (~b6t) Date submitted Name of the student (Matric No)
Supervisors Declaration
I Lesley Maurice Bilung hereby certifies that the work entitled Survival of Bacillus cereus in Artificially Contaminated UHT Milk and Infant Milk was prepared by the above named student and was submitted to the FACULTY as a gtI middotpartiaUfull fulfillment for the confennent of Bachelor of Science with Honours and the aforementioned work to the best of my knowledge is the sAid students work
Lesley Mamce Bilunl (PhD) Senior Lecturer
Received for examination by
Department of Molecular BioIoc) Fa4ulty of Resource Science ud Tlwology
Universiti Malaysia Sarawak Date 1
0 r~
(Name of the supervisor)
II
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research was done)
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III
P sat Khidmat Maklumat Akademik UN VERSITI MALAYSIA ARAWAK
Table of Contents
Acknowledgement
Declaration II
Table of Contents IV
List of Abbreviations VI
List ofTables VII
List of Figures IX
Abstract XI
10 Introduction
20 Literature Review 4
21 Bacillus cereus 4
211 Morphological and biochemical characteristics 4
21 2 B cereus food borne disease 4
213 Isolation and identification of Bcereus 5
214 Production of enterotoxins 5
215 Effect of temperature and pH on growth and survival of B 6
cereus
22 UHT milk 7
23 Infant rriilk 7
24 Polymerase Chain Reaction (PCR) nd agarose gel electrophoresis 8
IV
30 Materials and Methods 10
31 The milk 10
32 Inocula preparation 10
33 Survival and growth of B cereus in UHT milk and infant milk 10
331 Effect of temperature 10
332 Effect ofpH 11
34 Determination of growth and survival of B cereus in UHT milk and 11
infant milk samples
35 Genomic Deoxyribonucleic acid (DNA) extraction 12
36 PCR 12
37 Statistical analysis 12
40 Results 14
50 Discussion 42
60 Conclusion 51
70 References 52
v
I
List of Abbreviations
ANOVA Analysis of Variance
B cereus Bacillus cereus
BHI Brain Heart Infusion
CBA-P Columbia base-blood polymyxin B agar
CFU colony-forming unit
FDA Food and Drug Administration
gyrB gyrase B
HCl Hydrogen chloride
MYP Mannitol Egg Yolk agar
microlitre
ml millilitre
NaOH Sodium hydroxide
PEMBA Polymyxin B-egg yolk-mannitol bromothymol blue agar
PCR Polymerase Chain Reaction
UHT ultra-high temperature
VP Voges-Proskauer
VI
List of Tables
Table 1 Mixture volumes ofPCR for detection of B cereus 13
Table 2 Thermal cycling profile ofPCR for detection ofB cereus 13
Table 3 B cereus colony count from day 0 to day 7 in UHT milk stored at 14
temperature range of4 middotC to 7 middotC
Table 4 B cereus colony count from day 0 to day 7 in infant milk stored at 15
temperature range of 4 middotC to 7 c
Table 5 B cereus colony count from day 0 to day 7 in UHT milk stored at 16
temperature range of 10 middotC to 13 middotC
Table 6 B cereus colony count from day 0 to day 7 in infant milk stored at 17
temperature range of 10 middotC to 13 middotC
Table 7 B cereus colony count from day 0 to day 7 in UHT milk stored at 25degC 18
Table 8 B cereus colony count from day 0 to day 7 in infant milk stored at 25 19
DC
Table 9 B cereus colony count from day 0 to day 7 in UHT milk stored at 35degC 20
Table 10 B cereus colony count from day 0 to day 7 in infant milk stored at 35 20
DC
Table 11 B cereus colony count from day 0 to day 7 in UHT milk stored at 45degC 22
Table 12 B cereus colony count from day 0 to day 7 in infant milk stored at 45 22
DC
Table 13 B cereus colony count from day 0 to day 7 in UHT milk stored at 55degC 24
VII
Table 14 B cereus colony count from day 0 to day 7 in infant milk stored at 55 24
0c
Table 15 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 29
ofpH 620 stored at refrigerated temperature
Table 16 B cereus colony count from day 0 to day 7 in infant milk with initial pH 30
of pH 620 stored at refrigerated temperature
Table 17 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 31
of pH 640 stored at refrigerated temperature
Table 18 B cereus colony count from day 0 to day 7 in infant milk with initial pH 32
of pH 640 stored at refrigerated temperature
Table 19 B cereus colony count from day 0 to day 7 in UHT milk with initia l pH 33
of pH 660 stored at refrigerated temperature
Table 20 B cereus colony count from d-ay 0 to day 7 in infant milk with initial pH 34
of pH 660 stored at refrigerated temperature
Table 21 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 35
of pH 680 stored at refrigerated temperature
Table 22 B cereus colony count from day 0 to day 7 in infant milk with initial pH 36
of pH 680 stored at refrigerated temperature
VIII
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
List of Figures
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 4 middotC to 7 c
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 10 middotC to 13 middotC
15
17
The survival curve of B cereus
infant milk stored at 25 c
The survival curve of B cereus
infant milk stored at 35 c
The survival curve of B cereus
infant milk stored at 45 c
The survival curve of B cereus
infant milk stored at 55 middotC
in artificially contaminated UHT and 19
in artificially contaminated UHT and 21
in artificially contaminated UHT and 23
in artificially contaminated UHT and 25
The survival curve of B cereus in aiiifIcially contaminated UHT milk 27
stored at 4 middotC -7 middotc 10 middotC-13 middotC 25 middotc 35 middotC 45 middotC and 55 middotC
The survival curve of B cereus in artificially contaminated infant milk 28
stored at 4 middotC -7 middotC 10 middotC-13 middotC 25 middotC 35 middotC 45 middotC and 55 c
The survival curve of B cereus in artifIcially contaminated UHT and 30
infant milk with initial pH of pH 620 stored at refrigerated temperature
(4 QC-7 QC)
IX
Figure 10 The survival curve of B cereus in artificially contaminated UHT and 32
infant milk with initial pH of pH 640 stored at refrigerated temperature
(4 middotC-7 C)
Figure 11 The survival curve of B cereus in artificially contaminated UHT and 34
infant milk with initial pH of pH 660 stored at refrigerated temperature
(4 middotC-7 C)
Figure 12 The survival curve of B cereus in artificially contaminated UHT and 36
infant milk with initial pH of pH 680 stored at refrigerated temperature
(4 middotC-7 C)
Figure 13 The survival curve of B cereus in artificially contaminated UHT milk 38
with different initial pH stored at refrigerated temperature (4 middotC-7 C)
Figure 14 The survival curve of B cereus in artificially contaminated infant milk 39
with different initial pH stored- at refrigerated temperature (4 middotC-7 middotC)
Figure 15 B cereus colonies grown on MYP Agar 40
Figure 16 Morphology of B cereus isolated from UHT milk and infant milk 40
samples
Figure 17 Amplification of the gyrB gene (475 bp) for the confirmation of B 41
cereus in UHT milk and infant milk samples
x
Survival ofBacillus cereus in Artificially Contaminated UHT Milk and Infant Milk
Kong Wan Wei
Resource Bioteclmology Department of Molecular Biology
Faculty of Resource Science and Teclmology Universiti Malaysia Sarawak
Abstract
The inappropriate storage temperature and pH enable the survival of Bacillus cereus a known food pathogen in processed milk products resulting in foodborne diseases In the present study UHT milk and infant milk purchased from local supermarkets in Kuching Sarawak were inoculated with 103 CFUml of B cereus Selected storage temperatures of 4 C-7 C 10 C-13 C 25 C 35 C 45 C and 55 C were used to investigate the effects of temperature on the survival of B cereus Initial pH of 620640 660 and 680 at refrigerated temperature (4 C-7 C) were used to study the effects of pH on the survival of B cereus Enumeration of colony counts were investigated daily for the duration of seven days Gram staining and Polymerase Chain Reaction (PCR) targeting gyrB gene were performed for the confirmation of B cereus isolates The present findings revealed that the survival of B cereus in UHT milk and infant mil k showed significant differences (pltO05) in different temperatures and pH At 25 C there were significant difference (plt005) between the survival of B cereus in UHT milk and infant milk B cereus were able to survive under all the experimental temperatures However growth was detected at all storage temperatures except 55 c Survival of B cereus in artificially contaminated UHT milk and infant milk with different initial pH stored at referigerated temperature were also recorded The results from this study can be treated as background data for the implementation of safety measure to avoid food poisoning caused by B cere liS
Key words Bacillus cereus survival milk temperature pH
Abstrak
Suhll penyimpanan dan pH yang tidak sesllai membolehkall kehidupan Bacillus cereus sejenis pathogen makanan dalam produk susu diproses dan mellyebabkan penyakit bawaan makanall Dalam kajian ini SIISII UHT dan SIISU bayi yang dibeli daripada pasar raya tempatan di Kuching Sarawak telah diinoklliasi dengan B cerells sebanyak J(i CFUlml Suhll penyimpanan 4 C-7 c 10 C-3 c 25 c 35 middotc 45 c dan 55 c telah digllnakan IIntllk mengkaji kesan slIhll terhadap kehidupan B cerells Pada suhll peti sejuk (4 C-7 C) pH 620 pH 640 pH 660 dan pH 680 telah digunakan sebagai pH permulaal IIntuk mengkaji kesan pH terhadap keflidupan B cereus Penggiraall kololli telah dijalankan setiap hari selama IIIjuh hari Pewarnaan Gram dan penggllnaan reaksi rantai polimerase (PCR) yang menyasarkan gen gyrB telah mengesahkan kewujlldan koloni B cereus Kajian ini menllnjukkan buhawa kehidupan B cereus dalam SIISU UHT dan susu bayi menunjukkan perbezaan yang ketara (plt005) di suhu penyimpanan dan pH yang berbeza Perbezaan yang ketara (plt005) berlaku anlara kehidupan B cereus dalam susu UHT dengan kehidllpan B cereus dalam SIISII bayi pada sllhu 25 c B cereus dapal menunjllkkan kehidupan dalam SIISU UHT dan SIISU bayi yang telah disimpan pada semua suhll eksperimen Walau bagaimanapun pertumbllhan dapat dikesan pada semua sllhll eksperimen keclali 55 c Selain itll kehidllpan B cerells dalam SUSII UHT dan SUSII bayi yang tercemar secara bllalan pada slIhll peli sejuk telah dicatat pada peJbagai pH Hasil daripadu kajian ini boleh dianggap sebagai dala lalar belakallg tllltllk pelaksanuan langkah keselamatan bagi mengelakkan keracunall makanall daripada B cereus
Kata kunci Bacillus cerellS keflidllpan SIISU sllltl pH
XI
10 Introduction
Population growth and increase in consciousness about health are some of the factors that
increase demand for milk and milk products in Malaysia In Malaysian market ultra-high
temperature (UHT) milk and infant milk powders come under a variety of brands and are
either locally produced or imported Milk provides calcium protein vitamin A vitamin B 12
riboflavin and zinc to the human diet Consumption of milk helps to build and maintain bone
health as well as to reduce the risk of osteoporosis Infant milk provides major nutrients to
infants and it is essential for the growth of newborns
Milk is a high moisture and high nutritious medium which is also suitable for the
growth of mircoorganisms Most of the people believed that processed milk samples are
totally safe for consumption but actually there are many ways that leads to milk
contamination Contamination happened duripg milk processing as Bacillus cereus (B cereus)
strains which were not found in raw milk samples was present in the processed milk samples
(Banyk6 and Vyletelova 2009) Microorganisms contaminate the milk and affects the milk
quality by infecting the mammary gland udder and teat surfaces milking equipment and
storage tank (Gleeson et al 2013) Moreover improper processing and the usage of raw milk
that did not meet the microbiology and physio-chemical standards lead to the defects in raw
and UHT milk (Abdul-Hadi et al 2014) Inappropriate food storage and handling of
contaminated food encourage the growth of B cereus to the levels which can cause food
poisoning (Granum 2005) In order to maintain the quality and safety of processed milk
products consumers are suggested to store these products at temperature of 4 middotC or [less (Boor
and Fromm 2006)
Food pOlsonmg which may even have lethal effect on human is due to the
inappropriate food preparation and food exposure to 30 middotC (Sandra et at 2012) From year
1998 until 2008 there were 235 cases of foodbome disease outbreaks associated with B
cereus in United States (Bennett et at 2013) In 2011 the number of B cereus foodborne
diseases in Europe showed an increase of 1222 (MesseJhausser et ai 2014) The number
reported shows that B cereus poses a threat to the public health as it is often found in
contaminated food However the number of B cereus foodbome poisoning is believed to be
under reported because this type of food poisoning is mild and reporting these cases is not an
compulsory act (EFSA 2005)
B cereus was recognized as foodbome pathogen since 1955 and it is the cause for
about 2 of all foodbome disease worldwide (Lesley et at 2013) Garcia-Arribas and Kramer
(1990) mentioned that B cereus contribute to economic loss by causing contamination
spoilage and food-poisoning B cereus are found in dairy products meats spices and cereals
(Browne and Dowds 2002) as well as in infant foods and dried milk products (Becker et aI
1994)
Most of the patients involved in B cereus outbreaks were infants or elders (Becker et
ai 1994) The immune system of infants are still developing so they are highly susceptible to
B cereus food poisoning after consuming contaminated infant milk Although infant milk
basically present in low moisture powder form the vegetative cells and spores of
microorganisms are able to survive after drying but with their metabolism reduced (I LSI
Europe 2011) This poses threat to infants because some consumers keep the reconstituted
infant formula at room temperature for a long period of time as they wrongly believed that
infant formula are sterilised and are completely free of B cereus (ILSI Europe 2011)
2
B cereus is conunonly related to dairy products because they are able to survive at
different environmental stresses such as pasteurization (Foltys and Kirchnerova 2006 Sadek
et (II 2006) Schelderman et al (2006) discussed that aerobic spore former such as B cereus
has increased tolerance towards low temperature low pH sterilization and UHT treatments
and these conditions increase the concern in food industry In year 2011 there were food
poisoning associated with UHT milk provided from 1 Malaysia Milk Progranune and B cereus
were detected in these UHT milk (Yusof 2011) In 2013 infant milk formula product from
Fonterra were tested positive for Clostridium botulinum which is known as an endospore
former (Frediansyah 2014) The botulism scare was later confirmed as false alarm and the
actual organism was Clostridium 5porogenes However these pathogens have alarmed public
on the safety ofUHT milk and infant milk Currently there are no known studies in Malaysia
examining the factors influencing the survival of B cereus in UHT milk and infant milk
Hence the objectives of this study were
I To study the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk stored at different temperature for seven days
II To examine the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk with different initial pH stored at refrigerated temperature for seven
days
3
20 Literature Review
21 Bacillus cereus
211 Morphological and biochemical characteristics
Bacillus cereus is a Gram positive motile rod-shaped aerobic or facultative anaerobe and
aerobic spore forming foodborne pathogen belonging to Bacillus genus that is normally found
in soil and vegetation (Tortora et al 2010 Food Standards Australia New Zealand 2013
Gleeson et al 2013) This bacterium is mesophile or psychrotroph This bacterium is closely
related to B thuringiensis B mycoides B weihenstephanensis B pseudomycoides and B
anthracis (Gleeson et al 2013) The ability of B cereus to produce endospores which are heat
resistant increases the survivality of B cereus at unfavourable conditions such as high
temperatures extreme pH values or envirorunents with insufficient nutrients compared to
other bacterial enteropathogens (Di Pinto et al 2013 Gleeson et al 2013) For the
biochemical characteristics B cereus are positive for motility Voges-Proskauer (VP)
reaction starch hydrolysis citrate catalase and negative for oxidase tests (Shinagawa 1990
Choma ef al 2000) B cereus is shown as slender bacilli with square end singly or in short
chains under Gram-staining (Bottone 2010)
212 B cereus food borne disease
Rice dishes and meat or poultry dishes were the common sources for B cereus outbreaks
(Bennett et al 2013) Rice and pasta contaminated with B cereus are the causes for emetic
syndrome while the causes for diarrheal illness are desserts meat dishes and dairy products
(Granum 2005) Endospores of B cereus were able to survive in improperly cooked rice and
they caused Fried Rice Syndrome (Sandra et al 2012) In Malaysia B cereus was fIrst
4
Pusat Khidmat Maklumat Ak d Ut lVERSTTJ MA AYSIA SARAWAK
suspected as the cause for food poisoning which affected 114 female students in Klang
(Rampal et al 1984) In order to confIrm that gastroenteritis is caused by B cereus the
number of B cereus iso lated from suspected food need to exceed 105 co lony- forming unit per
gram (CFUg) (Tortora et al 20[0)
213 Isolation and identification of B cereus
Mannitol egg yolk polymyxin agar (MYF) Kim-Geopfert agar Columbia base-blood
polymyxin B agar (CBA-P) and Polymyxin B-egg yolk-mannitol bromothymol blue agar
(PEMBA) are some of the selective media suitable for isoIation and identifIcation of B cereus
(van Netten and Kramer 1992) The inability to ferment mannitol causes B cereus colonies to
appear as pink on MYP and lecithinase activity enables the observation of precipitated zone
around colonies (Pacheco-Sanchez and Massaguer 2007) Food and Drug Administration
(FDA) recommended MYP agar as the stapdard media for identifIcation of B cereus
214 Production of enterotoxins
The two forms of illness associated with B cereus enterotoxins are diarrheal and emetic
syndromes Onset of watery diarrhea and abdominal pain which resemble Clostridium
pefringens intoxications occur 8 hours to 16 hours after ingestion of contaminated food are
the symptoms of diarrheal outbreak (Shinagawa 1990 Tortora et ai 2010) This diarrheal
syndrome occurs after the ingestion of vegetattve cells which later produce a protein
enterotoxin in the gastrointinal tract (Bottone 2010) This type of enterotoxin is heat-labile
(Bello et al 2014) Haemolysin BL (HBL) non-haemolytic enterotoxin and cytotoxin K are
the three types of enterotoxins produced by this bacterium (Samarzija et al 2012 Food
Standards Australia New Zealand 2013) Reis et al (2013) concluded that hblD was the
5
common HBL gene of B cereus detected in milk and dairy products leading to diarrheal form
ofB cereus foodborne disease
The symptoms of emetic outbreaks are similar to Staphylococcus aureus food
intoxication and occur 05 hour to 6 hours upon ingestion of contaminated food
(Messelhausser et al 2014) The ingestion of food contaminated with preformed cereulide
which is also known as the emetic toxin leads to emetic syndrome (Bottone 2010) This heatshy
stable toxin causes nausea and vomiting in the infected patients (Bello et al2014)
215 Effect of temperature and pH on growth and survival of B cereus
Generally B cereus grows between 4 C and 55 C but exhibit optimum growth between 30 C
and 40 C (Schraft and Griffiths 2006 Food Standards Australia New Zealand 2013) Growth
of B cereus had been detected at temperature ranging from 7 C to 45 C and significant
growth was observed at 45 c in steril ized raw milk samples (Bellow et al 2007) In the
absence of oxygen or at temperature below 10 C emetic B cereus are believed to show
incapability in growth and production of toxin (EFSA 2005) At temperature below 10 C
with the limitation of other factors the lag time and generation times of B cereus are longer
(EFSA 2005)
As mentioned by Food Standards Australia New Zealand (2013) B cereus is able to
grow at pH between pH 49 and pH 100 and the optimum pH is between pH 60 and pH 70
Bello et al (2014) concluded that B cereus was unable to grow at pH 63 to pH 68 for 24
hour in pasteurized whole cow milk and the growth was inhibited at pH 68 for a period of 48
hours Byaruhanga et af (1999) studied that mague base inoculated with B cereus for 24
hours at 3S C was able to grow when pH decreases from pH 65 to pH 50 Brain Heart
6
--~~~----~----~----------
Infusion (BHI) medium at pH 88 inhibits growth of emetic and diarrheal strains of B cereus
better than pH 50 of the same medium (Garcia-Arribas and Kramer 1990)
22 UHT milk
Milk treated with UHT pasteurization is heated to 138 middotC for 2 seconds and aseptically placed
into a sterile substantial container which can be stored without refrigeration for six months to
a year (Ledenbach and Marshall 2009) UHT treatment minimized heat denaturation and
produces long shelf life milk that has similar taste and color to pasteurized milk (Harding
1995) UHT milk is generally contaminated during technological processing because food
pathogens are destroyed by the UHT temperature (Vyletelova et al 2002) Longer storage
period causes the drop of UHT milk pH and enzymes may be the reason for changes in UHT
milk (Hassan et al 2009)
Among the 110 UHT milk samples tested by Vidal-Martins et al (2005) 118
(1 3110) samples were positive for B cereus Researchers Rezende-Lago et al (2007)
identified B cereus in four (13 3) of the UHT milk samples tested In the study performed
by Reis et al (2013) among the 260 samples of UHT milk pasteurized milk and powdered
milk tested in Brazil a total of 63 isolates of B cereus were found and 12 of them were from
UHT milk
23 Infant milk
Cows milk and soy protein are the major component of infant milk or infant formulas For the
production of infant formula from milk the milk is heat-treated concentrated and dried while
infant fonnula from other ingredients is dry-blended wetted and dried again or only dry-
blended (Becker et al 1994) The sublethally injured pathogens can recover after the process
7
of drying because this process caused only little damage to the cell (Miller et ai 2012) During
the process of drying components such as lactose protein and milk fat that are present in
milk-based infant formula have the potential to provide protection to the bacteria (Mi1er et ai
20 12) Infant formulas are given to newborns and the content of nutrition is similar to breast
milk (Becker et al 1994) Currently infant formulas in the market are available in powder
liquid concentration and ready-to-feed After rehydration and inappropriate storage lowshy
moisture foods such as infant milk powder should be consumed within a short time because
growth of microorganisms may occur (lLSI Europe 2011)
Becker et al (1994) tested 92 infant milk formula samples and 52 of the samples
were B cereus positive Another study by Rowan et al (1997) tested 100 samples of infant
milk formula from United Kingdom and 17 of the samples were B cereus positive In the
study done by Haughton et al (20 I 0) B cereus were detected in 59 out of 100 infant milk
formula samples from Ireland These studies showed that B cereus is commonly found in
most of the infant milk formula and this poses threat to infants who are highly susceptible
24 Polymerase Chain Reaction (PCR)
As ment ioned by Sandra et al (2012) PCR allows rapid detection of pathogens Schraft and
Griffiths (2006) mentioned that the detection of B cereus targetting B cereus per se and
specific sub-groups have been performed by PCR Yamada et al (1999) designed PCR primer
set for gyrB fragment of B cereus and used this primer set in the detection of B cereus in
boiled rice Confirmation of B cereus in typical colonies are performed using PCR techniques
(Rahimi el al 2013) Thousands to millions copies of specific DNA sequence are produced
from the amplification of a single or a few copies of a piece of DNA by PCR (Joshi and
8
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
Acknowledgement
My greatest gratitude goes to my supervisor Dr Lesley Maurice Bilung for her
guidance support and ad vices through out the whole course of my fmal year project I would
like to thanks my co-supervisor Dr Hashimatul Fatma Hashim for her guidance and support
throughout my fmal year project
My appreciation also goes to the postgraduates especially Ernie Suhaiza Pui Chai
Fung and Cindy Lai Sin Chai for their guidance help and patience in assisting me to fmish my
fmal year project In addition I also appreciate the helps and patience of lab assistants
especially Mr Aziz Ajim and Miss Kamaliawati Yusop during my final year project
Not forgetting my family (dad mum Fion Wan Ru Alice and Eddie) for their
unending love and encouragement I also appreciate the help and support from my friends and
coursemates while conducting my fmal year project
UNIVERSITI MALAYSIA SARAWAK
Grade
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DECLARATION OF ORIGINAL WORK
This declaration is made on the ~ day of-~~2015
Students Declaration
I Kong Wan Wei 36541 of Faculty of Resource Science and Technology hereby declare that the work entitled Survival of Bacillus cereus in Artificially Contaminated UHT Milk and Infant Milk is my original work I have not copied from any other students work or from any other sources except where due reference or acknowledgement is made explicitly in the text nor has any part been written for me by another person
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Lesley Mamce Bilunl (PhD) Senior Lecturer
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Department of Molecular BioIoc) Fa4ulty of Resource Science ud Tlwology
Universiti Malaysia Sarawak Date 1
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P sat Khidmat Maklumat Akademik UN VERSITI MALAYSIA ARAWAK
Table of Contents
Acknowledgement
Declaration II
Table of Contents IV
List of Abbreviations VI
List ofTables VII
List of Figures IX
Abstract XI
10 Introduction
20 Literature Review 4
21 Bacillus cereus 4
211 Morphological and biochemical characteristics 4
21 2 B cereus food borne disease 4
213 Isolation and identification of Bcereus 5
214 Production of enterotoxins 5
215 Effect of temperature and pH on growth and survival of B 6
cereus
22 UHT milk 7
23 Infant rriilk 7
24 Polymerase Chain Reaction (PCR) nd agarose gel electrophoresis 8
IV
30 Materials and Methods 10
31 The milk 10
32 Inocula preparation 10
33 Survival and growth of B cereus in UHT milk and infant milk 10
331 Effect of temperature 10
332 Effect ofpH 11
34 Determination of growth and survival of B cereus in UHT milk and 11
infant milk samples
35 Genomic Deoxyribonucleic acid (DNA) extraction 12
36 PCR 12
37 Statistical analysis 12
40 Results 14
50 Discussion 42
60 Conclusion 51
70 References 52
v
I
List of Abbreviations
ANOVA Analysis of Variance
B cereus Bacillus cereus
BHI Brain Heart Infusion
CBA-P Columbia base-blood polymyxin B agar
CFU colony-forming unit
FDA Food and Drug Administration
gyrB gyrase B
HCl Hydrogen chloride
MYP Mannitol Egg Yolk agar
microlitre
ml millilitre
NaOH Sodium hydroxide
PEMBA Polymyxin B-egg yolk-mannitol bromothymol blue agar
PCR Polymerase Chain Reaction
UHT ultra-high temperature
VP Voges-Proskauer
VI
List of Tables
Table 1 Mixture volumes ofPCR for detection of B cereus 13
Table 2 Thermal cycling profile ofPCR for detection ofB cereus 13
Table 3 B cereus colony count from day 0 to day 7 in UHT milk stored at 14
temperature range of4 middotC to 7 middotC
Table 4 B cereus colony count from day 0 to day 7 in infant milk stored at 15
temperature range of 4 middotC to 7 c
Table 5 B cereus colony count from day 0 to day 7 in UHT milk stored at 16
temperature range of 10 middotC to 13 middotC
Table 6 B cereus colony count from day 0 to day 7 in infant milk stored at 17
temperature range of 10 middotC to 13 middotC
Table 7 B cereus colony count from day 0 to day 7 in UHT milk stored at 25degC 18
Table 8 B cereus colony count from day 0 to day 7 in infant milk stored at 25 19
DC
Table 9 B cereus colony count from day 0 to day 7 in UHT milk stored at 35degC 20
Table 10 B cereus colony count from day 0 to day 7 in infant milk stored at 35 20
DC
Table 11 B cereus colony count from day 0 to day 7 in UHT milk stored at 45degC 22
Table 12 B cereus colony count from day 0 to day 7 in infant milk stored at 45 22
DC
Table 13 B cereus colony count from day 0 to day 7 in UHT milk stored at 55degC 24
VII
Table 14 B cereus colony count from day 0 to day 7 in infant milk stored at 55 24
0c
Table 15 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 29
ofpH 620 stored at refrigerated temperature
Table 16 B cereus colony count from day 0 to day 7 in infant milk with initial pH 30
of pH 620 stored at refrigerated temperature
Table 17 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 31
of pH 640 stored at refrigerated temperature
Table 18 B cereus colony count from day 0 to day 7 in infant milk with initial pH 32
of pH 640 stored at refrigerated temperature
Table 19 B cereus colony count from day 0 to day 7 in UHT milk with initia l pH 33
of pH 660 stored at refrigerated temperature
Table 20 B cereus colony count from d-ay 0 to day 7 in infant milk with initial pH 34
of pH 660 stored at refrigerated temperature
Table 21 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 35
of pH 680 stored at refrigerated temperature
Table 22 B cereus colony count from day 0 to day 7 in infant milk with initial pH 36
of pH 680 stored at refrigerated temperature
VIII
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
List of Figures
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 4 middotC to 7 c
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 10 middotC to 13 middotC
15
17
The survival curve of B cereus
infant milk stored at 25 c
The survival curve of B cereus
infant milk stored at 35 c
The survival curve of B cereus
infant milk stored at 45 c
The survival curve of B cereus
infant milk stored at 55 middotC
in artificially contaminated UHT and 19
in artificially contaminated UHT and 21
in artificially contaminated UHT and 23
in artificially contaminated UHT and 25
The survival curve of B cereus in aiiifIcially contaminated UHT milk 27
stored at 4 middotC -7 middotc 10 middotC-13 middotC 25 middotc 35 middotC 45 middotC and 55 middotC
The survival curve of B cereus in artificially contaminated infant milk 28
stored at 4 middotC -7 middotC 10 middotC-13 middotC 25 middotC 35 middotC 45 middotC and 55 c
The survival curve of B cereus in artifIcially contaminated UHT and 30
infant milk with initial pH of pH 620 stored at refrigerated temperature
(4 QC-7 QC)
IX
Figure 10 The survival curve of B cereus in artificially contaminated UHT and 32
infant milk with initial pH of pH 640 stored at refrigerated temperature
(4 middotC-7 C)
Figure 11 The survival curve of B cereus in artificially contaminated UHT and 34
infant milk with initial pH of pH 660 stored at refrigerated temperature
(4 middotC-7 C)
Figure 12 The survival curve of B cereus in artificially contaminated UHT and 36
infant milk with initial pH of pH 680 stored at refrigerated temperature
(4 middotC-7 C)
Figure 13 The survival curve of B cereus in artificially contaminated UHT milk 38
with different initial pH stored at refrigerated temperature (4 middotC-7 C)
Figure 14 The survival curve of B cereus in artificially contaminated infant milk 39
with different initial pH stored- at refrigerated temperature (4 middotC-7 middotC)
Figure 15 B cereus colonies grown on MYP Agar 40
Figure 16 Morphology of B cereus isolated from UHT milk and infant milk 40
samples
Figure 17 Amplification of the gyrB gene (475 bp) for the confirmation of B 41
cereus in UHT milk and infant milk samples
x
Survival ofBacillus cereus in Artificially Contaminated UHT Milk and Infant Milk
Kong Wan Wei
Resource Bioteclmology Department of Molecular Biology
Faculty of Resource Science and Teclmology Universiti Malaysia Sarawak
Abstract
The inappropriate storage temperature and pH enable the survival of Bacillus cereus a known food pathogen in processed milk products resulting in foodborne diseases In the present study UHT milk and infant milk purchased from local supermarkets in Kuching Sarawak were inoculated with 103 CFUml of B cereus Selected storage temperatures of 4 C-7 C 10 C-13 C 25 C 35 C 45 C and 55 C were used to investigate the effects of temperature on the survival of B cereus Initial pH of 620640 660 and 680 at refrigerated temperature (4 C-7 C) were used to study the effects of pH on the survival of B cereus Enumeration of colony counts were investigated daily for the duration of seven days Gram staining and Polymerase Chain Reaction (PCR) targeting gyrB gene were performed for the confirmation of B cereus isolates The present findings revealed that the survival of B cereus in UHT milk and infant mil k showed significant differences (pltO05) in different temperatures and pH At 25 C there were significant difference (plt005) between the survival of B cereus in UHT milk and infant milk B cereus were able to survive under all the experimental temperatures However growth was detected at all storage temperatures except 55 c Survival of B cereus in artificially contaminated UHT milk and infant milk with different initial pH stored at referigerated temperature were also recorded The results from this study can be treated as background data for the implementation of safety measure to avoid food poisoning caused by B cere liS
Key words Bacillus cereus survival milk temperature pH
Abstrak
Suhll penyimpanan dan pH yang tidak sesllai membolehkall kehidupan Bacillus cereus sejenis pathogen makanan dalam produk susu diproses dan mellyebabkan penyakit bawaan makanall Dalam kajian ini SIISII UHT dan SIISU bayi yang dibeli daripada pasar raya tempatan di Kuching Sarawak telah diinoklliasi dengan B cerells sebanyak J(i CFUlml Suhll penyimpanan 4 C-7 c 10 C-3 c 25 c 35 middotc 45 c dan 55 c telah digllnakan IIntllk mengkaji kesan slIhll terhadap kehidupan B cerells Pada suhll peti sejuk (4 C-7 C) pH 620 pH 640 pH 660 dan pH 680 telah digunakan sebagai pH permulaal IIntuk mengkaji kesan pH terhadap keflidupan B cereus Penggiraall kololli telah dijalankan setiap hari selama IIIjuh hari Pewarnaan Gram dan penggllnaan reaksi rantai polimerase (PCR) yang menyasarkan gen gyrB telah mengesahkan kewujlldan koloni B cereus Kajian ini menllnjukkan buhawa kehidupan B cereus dalam SIISU UHT dan susu bayi menunjukkan perbezaan yang ketara (plt005) di suhu penyimpanan dan pH yang berbeza Perbezaan yang ketara (plt005) berlaku anlara kehidupan B cereus dalam susu UHT dengan kehidllpan B cereus dalam SIISII bayi pada sllhu 25 c B cereus dapal menunjllkkan kehidupan dalam SIISU UHT dan SIISU bayi yang telah disimpan pada semua suhll eksperimen Walau bagaimanapun pertumbllhan dapat dikesan pada semua sllhll eksperimen keclali 55 c Selain itll kehidllpan B cerells dalam SUSII UHT dan SUSII bayi yang tercemar secara bllalan pada slIhll peli sejuk telah dicatat pada peJbagai pH Hasil daripadu kajian ini boleh dianggap sebagai dala lalar belakallg tllltllk pelaksanuan langkah keselamatan bagi mengelakkan keracunall makanall daripada B cereus
Kata kunci Bacillus cerellS keflidllpan SIISU sllltl pH
XI
10 Introduction
Population growth and increase in consciousness about health are some of the factors that
increase demand for milk and milk products in Malaysia In Malaysian market ultra-high
temperature (UHT) milk and infant milk powders come under a variety of brands and are
either locally produced or imported Milk provides calcium protein vitamin A vitamin B 12
riboflavin and zinc to the human diet Consumption of milk helps to build and maintain bone
health as well as to reduce the risk of osteoporosis Infant milk provides major nutrients to
infants and it is essential for the growth of newborns
Milk is a high moisture and high nutritious medium which is also suitable for the
growth of mircoorganisms Most of the people believed that processed milk samples are
totally safe for consumption but actually there are many ways that leads to milk
contamination Contamination happened duripg milk processing as Bacillus cereus (B cereus)
strains which were not found in raw milk samples was present in the processed milk samples
(Banyk6 and Vyletelova 2009) Microorganisms contaminate the milk and affects the milk
quality by infecting the mammary gland udder and teat surfaces milking equipment and
storage tank (Gleeson et al 2013) Moreover improper processing and the usage of raw milk
that did not meet the microbiology and physio-chemical standards lead to the defects in raw
and UHT milk (Abdul-Hadi et al 2014) Inappropriate food storage and handling of
contaminated food encourage the growth of B cereus to the levels which can cause food
poisoning (Granum 2005) In order to maintain the quality and safety of processed milk
products consumers are suggested to store these products at temperature of 4 middotC or [less (Boor
and Fromm 2006)
Food pOlsonmg which may even have lethal effect on human is due to the
inappropriate food preparation and food exposure to 30 middotC (Sandra et at 2012) From year
1998 until 2008 there were 235 cases of foodbome disease outbreaks associated with B
cereus in United States (Bennett et at 2013) In 2011 the number of B cereus foodborne
diseases in Europe showed an increase of 1222 (MesseJhausser et ai 2014) The number
reported shows that B cereus poses a threat to the public health as it is often found in
contaminated food However the number of B cereus foodbome poisoning is believed to be
under reported because this type of food poisoning is mild and reporting these cases is not an
compulsory act (EFSA 2005)
B cereus was recognized as foodbome pathogen since 1955 and it is the cause for
about 2 of all foodbome disease worldwide (Lesley et at 2013) Garcia-Arribas and Kramer
(1990) mentioned that B cereus contribute to economic loss by causing contamination
spoilage and food-poisoning B cereus are found in dairy products meats spices and cereals
(Browne and Dowds 2002) as well as in infant foods and dried milk products (Becker et aI
1994)
Most of the patients involved in B cereus outbreaks were infants or elders (Becker et
ai 1994) The immune system of infants are still developing so they are highly susceptible to
B cereus food poisoning after consuming contaminated infant milk Although infant milk
basically present in low moisture powder form the vegetative cells and spores of
microorganisms are able to survive after drying but with their metabolism reduced (I LSI
Europe 2011) This poses threat to infants because some consumers keep the reconstituted
infant formula at room temperature for a long period of time as they wrongly believed that
infant formula are sterilised and are completely free of B cereus (ILSI Europe 2011)
2
B cereus is conunonly related to dairy products because they are able to survive at
different environmental stresses such as pasteurization (Foltys and Kirchnerova 2006 Sadek
et (II 2006) Schelderman et al (2006) discussed that aerobic spore former such as B cereus
has increased tolerance towards low temperature low pH sterilization and UHT treatments
and these conditions increase the concern in food industry In year 2011 there were food
poisoning associated with UHT milk provided from 1 Malaysia Milk Progranune and B cereus
were detected in these UHT milk (Yusof 2011) In 2013 infant milk formula product from
Fonterra were tested positive for Clostridium botulinum which is known as an endospore
former (Frediansyah 2014) The botulism scare was later confirmed as false alarm and the
actual organism was Clostridium 5porogenes However these pathogens have alarmed public
on the safety ofUHT milk and infant milk Currently there are no known studies in Malaysia
examining the factors influencing the survival of B cereus in UHT milk and infant milk
Hence the objectives of this study were
I To study the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk stored at different temperature for seven days
II To examine the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk with different initial pH stored at refrigerated temperature for seven
days
3
20 Literature Review
21 Bacillus cereus
211 Morphological and biochemical characteristics
Bacillus cereus is a Gram positive motile rod-shaped aerobic or facultative anaerobe and
aerobic spore forming foodborne pathogen belonging to Bacillus genus that is normally found
in soil and vegetation (Tortora et al 2010 Food Standards Australia New Zealand 2013
Gleeson et al 2013) This bacterium is mesophile or psychrotroph This bacterium is closely
related to B thuringiensis B mycoides B weihenstephanensis B pseudomycoides and B
anthracis (Gleeson et al 2013) The ability of B cereus to produce endospores which are heat
resistant increases the survivality of B cereus at unfavourable conditions such as high
temperatures extreme pH values or envirorunents with insufficient nutrients compared to
other bacterial enteropathogens (Di Pinto et al 2013 Gleeson et al 2013) For the
biochemical characteristics B cereus are positive for motility Voges-Proskauer (VP)
reaction starch hydrolysis citrate catalase and negative for oxidase tests (Shinagawa 1990
Choma ef al 2000) B cereus is shown as slender bacilli with square end singly or in short
chains under Gram-staining (Bottone 2010)
212 B cereus food borne disease
Rice dishes and meat or poultry dishes were the common sources for B cereus outbreaks
(Bennett et al 2013) Rice and pasta contaminated with B cereus are the causes for emetic
syndrome while the causes for diarrheal illness are desserts meat dishes and dairy products
(Granum 2005) Endospores of B cereus were able to survive in improperly cooked rice and
they caused Fried Rice Syndrome (Sandra et al 2012) In Malaysia B cereus was fIrst
4
Pusat Khidmat Maklumat Ak d Ut lVERSTTJ MA AYSIA SARAWAK
suspected as the cause for food poisoning which affected 114 female students in Klang
(Rampal et al 1984) In order to confIrm that gastroenteritis is caused by B cereus the
number of B cereus iso lated from suspected food need to exceed 105 co lony- forming unit per
gram (CFUg) (Tortora et al 20[0)
213 Isolation and identification of B cereus
Mannitol egg yolk polymyxin agar (MYF) Kim-Geopfert agar Columbia base-blood
polymyxin B agar (CBA-P) and Polymyxin B-egg yolk-mannitol bromothymol blue agar
(PEMBA) are some of the selective media suitable for isoIation and identifIcation of B cereus
(van Netten and Kramer 1992) The inability to ferment mannitol causes B cereus colonies to
appear as pink on MYP and lecithinase activity enables the observation of precipitated zone
around colonies (Pacheco-Sanchez and Massaguer 2007) Food and Drug Administration
(FDA) recommended MYP agar as the stapdard media for identifIcation of B cereus
214 Production of enterotoxins
The two forms of illness associated with B cereus enterotoxins are diarrheal and emetic
syndromes Onset of watery diarrhea and abdominal pain which resemble Clostridium
pefringens intoxications occur 8 hours to 16 hours after ingestion of contaminated food are
the symptoms of diarrheal outbreak (Shinagawa 1990 Tortora et ai 2010) This diarrheal
syndrome occurs after the ingestion of vegetattve cells which later produce a protein
enterotoxin in the gastrointinal tract (Bottone 2010) This type of enterotoxin is heat-labile
(Bello et al 2014) Haemolysin BL (HBL) non-haemolytic enterotoxin and cytotoxin K are
the three types of enterotoxins produced by this bacterium (Samarzija et al 2012 Food
Standards Australia New Zealand 2013) Reis et al (2013) concluded that hblD was the
5
common HBL gene of B cereus detected in milk and dairy products leading to diarrheal form
ofB cereus foodborne disease
The symptoms of emetic outbreaks are similar to Staphylococcus aureus food
intoxication and occur 05 hour to 6 hours upon ingestion of contaminated food
(Messelhausser et al 2014) The ingestion of food contaminated with preformed cereulide
which is also known as the emetic toxin leads to emetic syndrome (Bottone 2010) This heatshy
stable toxin causes nausea and vomiting in the infected patients (Bello et al2014)
215 Effect of temperature and pH on growth and survival of B cereus
Generally B cereus grows between 4 C and 55 C but exhibit optimum growth between 30 C
and 40 C (Schraft and Griffiths 2006 Food Standards Australia New Zealand 2013) Growth
of B cereus had been detected at temperature ranging from 7 C to 45 C and significant
growth was observed at 45 c in steril ized raw milk samples (Bellow et al 2007) In the
absence of oxygen or at temperature below 10 C emetic B cereus are believed to show
incapability in growth and production of toxin (EFSA 2005) At temperature below 10 C
with the limitation of other factors the lag time and generation times of B cereus are longer
(EFSA 2005)
As mentioned by Food Standards Australia New Zealand (2013) B cereus is able to
grow at pH between pH 49 and pH 100 and the optimum pH is between pH 60 and pH 70
Bello et al (2014) concluded that B cereus was unable to grow at pH 63 to pH 68 for 24
hour in pasteurized whole cow milk and the growth was inhibited at pH 68 for a period of 48
hours Byaruhanga et af (1999) studied that mague base inoculated with B cereus for 24
hours at 3S C was able to grow when pH decreases from pH 65 to pH 50 Brain Heart
6
--~~~----~----~----------
Infusion (BHI) medium at pH 88 inhibits growth of emetic and diarrheal strains of B cereus
better than pH 50 of the same medium (Garcia-Arribas and Kramer 1990)
22 UHT milk
Milk treated with UHT pasteurization is heated to 138 middotC for 2 seconds and aseptically placed
into a sterile substantial container which can be stored without refrigeration for six months to
a year (Ledenbach and Marshall 2009) UHT treatment minimized heat denaturation and
produces long shelf life milk that has similar taste and color to pasteurized milk (Harding
1995) UHT milk is generally contaminated during technological processing because food
pathogens are destroyed by the UHT temperature (Vyletelova et al 2002) Longer storage
period causes the drop of UHT milk pH and enzymes may be the reason for changes in UHT
milk (Hassan et al 2009)
Among the 110 UHT milk samples tested by Vidal-Martins et al (2005) 118
(1 3110) samples were positive for B cereus Researchers Rezende-Lago et al (2007)
identified B cereus in four (13 3) of the UHT milk samples tested In the study performed
by Reis et al (2013) among the 260 samples of UHT milk pasteurized milk and powdered
milk tested in Brazil a total of 63 isolates of B cereus were found and 12 of them were from
UHT milk
23 Infant milk
Cows milk and soy protein are the major component of infant milk or infant formulas For the
production of infant formula from milk the milk is heat-treated concentrated and dried while
infant fonnula from other ingredients is dry-blended wetted and dried again or only dry-
blended (Becker et al 1994) The sublethally injured pathogens can recover after the process
7
of drying because this process caused only little damage to the cell (Miller et ai 2012) During
the process of drying components such as lactose protein and milk fat that are present in
milk-based infant formula have the potential to provide protection to the bacteria (Mi1er et ai
20 12) Infant formulas are given to newborns and the content of nutrition is similar to breast
milk (Becker et al 1994) Currently infant formulas in the market are available in powder
liquid concentration and ready-to-feed After rehydration and inappropriate storage lowshy
moisture foods such as infant milk powder should be consumed within a short time because
growth of microorganisms may occur (lLSI Europe 2011)
Becker et al (1994) tested 92 infant milk formula samples and 52 of the samples
were B cereus positive Another study by Rowan et al (1997) tested 100 samples of infant
milk formula from United Kingdom and 17 of the samples were B cereus positive In the
study done by Haughton et al (20 I 0) B cereus were detected in 59 out of 100 infant milk
formula samples from Ireland These studies showed that B cereus is commonly found in
most of the infant milk formula and this poses threat to infants who are highly susceptible
24 Polymerase Chain Reaction (PCR)
As ment ioned by Sandra et al (2012) PCR allows rapid detection of pathogens Schraft and
Griffiths (2006) mentioned that the detection of B cereus targetting B cereus per se and
specific sub-groups have been performed by PCR Yamada et al (1999) designed PCR primer
set for gyrB fragment of B cereus and used this primer set in the detection of B cereus in
boiled rice Confirmation of B cereus in typical colonies are performed using PCR techniques
(Rahimi el al 2013) Thousands to millions copies of specific DNA sequence are produced
from the amplification of a single or a few copies of a piece of DNA by PCR (Joshi and
8
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
UNIVERSITI MALAYSIA SARAWAK
Grade
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Final Year Project Report IV I Masters 0 PhD D
DECLARATION OF ORIGINAL WORK
This declaration is made on the ~ day of-~~2015
Students Declaration
I Kong Wan Wei 36541 of Faculty of Resource Science and Technology hereby declare that the work entitled Survival of Bacillus cereus in Artificially Contaminated UHT Milk and Infant Milk is my original work I have not copied from any other students work or from any other sources except where due reference or acknowledgement is made explicitly in the text nor has any part been written for me by another person
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Lesley Mamce Bilunl (PhD) Senior Lecturer
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Universiti Malaysia Sarawak Date 1
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P sat Khidmat Maklumat Akademik UN VERSITI MALAYSIA ARAWAK
Table of Contents
Acknowledgement
Declaration II
Table of Contents IV
List of Abbreviations VI
List ofTables VII
List of Figures IX
Abstract XI
10 Introduction
20 Literature Review 4
21 Bacillus cereus 4
211 Morphological and biochemical characteristics 4
21 2 B cereus food borne disease 4
213 Isolation and identification of Bcereus 5
214 Production of enterotoxins 5
215 Effect of temperature and pH on growth and survival of B 6
cereus
22 UHT milk 7
23 Infant rriilk 7
24 Polymerase Chain Reaction (PCR) nd agarose gel electrophoresis 8
IV
30 Materials and Methods 10
31 The milk 10
32 Inocula preparation 10
33 Survival and growth of B cereus in UHT milk and infant milk 10
331 Effect of temperature 10
332 Effect ofpH 11
34 Determination of growth and survival of B cereus in UHT milk and 11
infant milk samples
35 Genomic Deoxyribonucleic acid (DNA) extraction 12
36 PCR 12
37 Statistical analysis 12
40 Results 14
50 Discussion 42
60 Conclusion 51
70 References 52
v
I
List of Abbreviations
ANOVA Analysis of Variance
B cereus Bacillus cereus
BHI Brain Heart Infusion
CBA-P Columbia base-blood polymyxin B agar
CFU colony-forming unit
FDA Food and Drug Administration
gyrB gyrase B
HCl Hydrogen chloride
MYP Mannitol Egg Yolk agar
microlitre
ml millilitre
NaOH Sodium hydroxide
PEMBA Polymyxin B-egg yolk-mannitol bromothymol blue agar
PCR Polymerase Chain Reaction
UHT ultra-high temperature
VP Voges-Proskauer
VI
List of Tables
Table 1 Mixture volumes ofPCR for detection of B cereus 13
Table 2 Thermal cycling profile ofPCR for detection ofB cereus 13
Table 3 B cereus colony count from day 0 to day 7 in UHT milk stored at 14
temperature range of4 middotC to 7 middotC
Table 4 B cereus colony count from day 0 to day 7 in infant milk stored at 15
temperature range of 4 middotC to 7 c
Table 5 B cereus colony count from day 0 to day 7 in UHT milk stored at 16
temperature range of 10 middotC to 13 middotC
Table 6 B cereus colony count from day 0 to day 7 in infant milk stored at 17
temperature range of 10 middotC to 13 middotC
Table 7 B cereus colony count from day 0 to day 7 in UHT milk stored at 25degC 18
Table 8 B cereus colony count from day 0 to day 7 in infant milk stored at 25 19
DC
Table 9 B cereus colony count from day 0 to day 7 in UHT milk stored at 35degC 20
Table 10 B cereus colony count from day 0 to day 7 in infant milk stored at 35 20
DC
Table 11 B cereus colony count from day 0 to day 7 in UHT milk stored at 45degC 22
Table 12 B cereus colony count from day 0 to day 7 in infant milk stored at 45 22
DC
Table 13 B cereus colony count from day 0 to day 7 in UHT milk stored at 55degC 24
VII
Table 14 B cereus colony count from day 0 to day 7 in infant milk stored at 55 24
0c
Table 15 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 29
ofpH 620 stored at refrigerated temperature
Table 16 B cereus colony count from day 0 to day 7 in infant milk with initial pH 30
of pH 620 stored at refrigerated temperature
Table 17 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 31
of pH 640 stored at refrigerated temperature
Table 18 B cereus colony count from day 0 to day 7 in infant milk with initial pH 32
of pH 640 stored at refrigerated temperature
Table 19 B cereus colony count from day 0 to day 7 in UHT milk with initia l pH 33
of pH 660 stored at refrigerated temperature
Table 20 B cereus colony count from d-ay 0 to day 7 in infant milk with initial pH 34
of pH 660 stored at refrigerated temperature
Table 21 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 35
of pH 680 stored at refrigerated temperature
Table 22 B cereus colony count from day 0 to day 7 in infant milk with initial pH 36
of pH 680 stored at refrigerated temperature
VIII
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
List of Figures
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 4 middotC to 7 c
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 10 middotC to 13 middotC
15
17
The survival curve of B cereus
infant milk stored at 25 c
The survival curve of B cereus
infant milk stored at 35 c
The survival curve of B cereus
infant milk stored at 45 c
The survival curve of B cereus
infant milk stored at 55 middotC
in artificially contaminated UHT and 19
in artificially contaminated UHT and 21
in artificially contaminated UHT and 23
in artificially contaminated UHT and 25
The survival curve of B cereus in aiiifIcially contaminated UHT milk 27
stored at 4 middotC -7 middotc 10 middotC-13 middotC 25 middotc 35 middotC 45 middotC and 55 middotC
The survival curve of B cereus in artificially contaminated infant milk 28
stored at 4 middotC -7 middotC 10 middotC-13 middotC 25 middotC 35 middotC 45 middotC and 55 c
The survival curve of B cereus in artifIcially contaminated UHT and 30
infant milk with initial pH of pH 620 stored at refrigerated temperature
(4 QC-7 QC)
IX
Figure 10 The survival curve of B cereus in artificially contaminated UHT and 32
infant milk with initial pH of pH 640 stored at refrigerated temperature
(4 middotC-7 C)
Figure 11 The survival curve of B cereus in artificially contaminated UHT and 34
infant milk with initial pH of pH 660 stored at refrigerated temperature
(4 middotC-7 C)
Figure 12 The survival curve of B cereus in artificially contaminated UHT and 36
infant milk with initial pH of pH 680 stored at refrigerated temperature
(4 middotC-7 C)
Figure 13 The survival curve of B cereus in artificially contaminated UHT milk 38
with different initial pH stored at refrigerated temperature (4 middotC-7 C)
Figure 14 The survival curve of B cereus in artificially contaminated infant milk 39
with different initial pH stored- at refrigerated temperature (4 middotC-7 middotC)
Figure 15 B cereus colonies grown on MYP Agar 40
Figure 16 Morphology of B cereus isolated from UHT milk and infant milk 40
samples
Figure 17 Amplification of the gyrB gene (475 bp) for the confirmation of B 41
cereus in UHT milk and infant milk samples
x
Survival ofBacillus cereus in Artificially Contaminated UHT Milk and Infant Milk
Kong Wan Wei
Resource Bioteclmology Department of Molecular Biology
Faculty of Resource Science and Teclmology Universiti Malaysia Sarawak
Abstract
The inappropriate storage temperature and pH enable the survival of Bacillus cereus a known food pathogen in processed milk products resulting in foodborne diseases In the present study UHT milk and infant milk purchased from local supermarkets in Kuching Sarawak were inoculated with 103 CFUml of B cereus Selected storage temperatures of 4 C-7 C 10 C-13 C 25 C 35 C 45 C and 55 C were used to investigate the effects of temperature on the survival of B cereus Initial pH of 620640 660 and 680 at refrigerated temperature (4 C-7 C) were used to study the effects of pH on the survival of B cereus Enumeration of colony counts were investigated daily for the duration of seven days Gram staining and Polymerase Chain Reaction (PCR) targeting gyrB gene were performed for the confirmation of B cereus isolates The present findings revealed that the survival of B cereus in UHT milk and infant mil k showed significant differences (pltO05) in different temperatures and pH At 25 C there were significant difference (plt005) between the survival of B cereus in UHT milk and infant milk B cereus were able to survive under all the experimental temperatures However growth was detected at all storage temperatures except 55 c Survival of B cereus in artificially contaminated UHT milk and infant milk with different initial pH stored at referigerated temperature were also recorded The results from this study can be treated as background data for the implementation of safety measure to avoid food poisoning caused by B cere liS
Key words Bacillus cereus survival milk temperature pH
Abstrak
Suhll penyimpanan dan pH yang tidak sesllai membolehkall kehidupan Bacillus cereus sejenis pathogen makanan dalam produk susu diproses dan mellyebabkan penyakit bawaan makanall Dalam kajian ini SIISII UHT dan SIISU bayi yang dibeli daripada pasar raya tempatan di Kuching Sarawak telah diinoklliasi dengan B cerells sebanyak J(i CFUlml Suhll penyimpanan 4 C-7 c 10 C-3 c 25 c 35 middotc 45 c dan 55 c telah digllnakan IIntllk mengkaji kesan slIhll terhadap kehidupan B cerells Pada suhll peti sejuk (4 C-7 C) pH 620 pH 640 pH 660 dan pH 680 telah digunakan sebagai pH permulaal IIntuk mengkaji kesan pH terhadap keflidupan B cereus Penggiraall kololli telah dijalankan setiap hari selama IIIjuh hari Pewarnaan Gram dan penggllnaan reaksi rantai polimerase (PCR) yang menyasarkan gen gyrB telah mengesahkan kewujlldan koloni B cereus Kajian ini menllnjukkan buhawa kehidupan B cereus dalam SIISU UHT dan susu bayi menunjukkan perbezaan yang ketara (plt005) di suhu penyimpanan dan pH yang berbeza Perbezaan yang ketara (plt005) berlaku anlara kehidupan B cereus dalam susu UHT dengan kehidllpan B cereus dalam SIISII bayi pada sllhu 25 c B cereus dapal menunjllkkan kehidupan dalam SIISU UHT dan SIISU bayi yang telah disimpan pada semua suhll eksperimen Walau bagaimanapun pertumbllhan dapat dikesan pada semua sllhll eksperimen keclali 55 c Selain itll kehidllpan B cerells dalam SUSII UHT dan SUSII bayi yang tercemar secara bllalan pada slIhll peli sejuk telah dicatat pada peJbagai pH Hasil daripadu kajian ini boleh dianggap sebagai dala lalar belakallg tllltllk pelaksanuan langkah keselamatan bagi mengelakkan keracunall makanall daripada B cereus
Kata kunci Bacillus cerellS keflidllpan SIISU sllltl pH
XI
10 Introduction
Population growth and increase in consciousness about health are some of the factors that
increase demand for milk and milk products in Malaysia In Malaysian market ultra-high
temperature (UHT) milk and infant milk powders come under a variety of brands and are
either locally produced or imported Milk provides calcium protein vitamin A vitamin B 12
riboflavin and zinc to the human diet Consumption of milk helps to build and maintain bone
health as well as to reduce the risk of osteoporosis Infant milk provides major nutrients to
infants and it is essential for the growth of newborns
Milk is a high moisture and high nutritious medium which is also suitable for the
growth of mircoorganisms Most of the people believed that processed milk samples are
totally safe for consumption but actually there are many ways that leads to milk
contamination Contamination happened duripg milk processing as Bacillus cereus (B cereus)
strains which were not found in raw milk samples was present in the processed milk samples
(Banyk6 and Vyletelova 2009) Microorganisms contaminate the milk and affects the milk
quality by infecting the mammary gland udder and teat surfaces milking equipment and
storage tank (Gleeson et al 2013) Moreover improper processing and the usage of raw milk
that did not meet the microbiology and physio-chemical standards lead to the defects in raw
and UHT milk (Abdul-Hadi et al 2014) Inappropriate food storage and handling of
contaminated food encourage the growth of B cereus to the levels which can cause food
poisoning (Granum 2005) In order to maintain the quality and safety of processed milk
products consumers are suggested to store these products at temperature of 4 middotC or [less (Boor
and Fromm 2006)
Food pOlsonmg which may even have lethal effect on human is due to the
inappropriate food preparation and food exposure to 30 middotC (Sandra et at 2012) From year
1998 until 2008 there were 235 cases of foodbome disease outbreaks associated with B
cereus in United States (Bennett et at 2013) In 2011 the number of B cereus foodborne
diseases in Europe showed an increase of 1222 (MesseJhausser et ai 2014) The number
reported shows that B cereus poses a threat to the public health as it is often found in
contaminated food However the number of B cereus foodbome poisoning is believed to be
under reported because this type of food poisoning is mild and reporting these cases is not an
compulsory act (EFSA 2005)
B cereus was recognized as foodbome pathogen since 1955 and it is the cause for
about 2 of all foodbome disease worldwide (Lesley et at 2013) Garcia-Arribas and Kramer
(1990) mentioned that B cereus contribute to economic loss by causing contamination
spoilage and food-poisoning B cereus are found in dairy products meats spices and cereals
(Browne and Dowds 2002) as well as in infant foods and dried milk products (Becker et aI
1994)
Most of the patients involved in B cereus outbreaks were infants or elders (Becker et
ai 1994) The immune system of infants are still developing so they are highly susceptible to
B cereus food poisoning after consuming contaminated infant milk Although infant milk
basically present in low moisture powder form the vegetative cells and spores of
microorganisms are able to survive after drying but with their metabolism reduced (I LSI
Europe 2011) This poses threat to infants because some consumers keep the reconstituted
infant formula at room temperature for a long period of time as they wrongly believed that
infant formula are sterilised and are completely free of B cereus (ILSI Europe 2011)
2
B cereus is conunonly related to dairy products because they are able to survive at
different environmental stresses such as pasteurization (Foltys and Kirchnerova 2006 Sadek
et (II 2006) Schelderman et al (2006) discussed that aerobic spore former such as B cereus
has increased tolerance towards low temperature low pH sterilization and UHT treatments
and these conditions increase the concern in food industry In year 2011 there were food
poisoning associated with UHT milk provided from 1 Malaysia Milk Progranune and B cereus
were detected in these UHT milk (Yusof 2011) In 2013 infant milk formula product from
Fonterra were tested positive for Clostridium botulinum which is known as an endospore
former (Frediansyah 2014) The botulism scare was later confirmed as false alarm and the
actual organism was Clostridium 5porogenes However these pathogens have alarmed public
on the safety ofUHT milk and infant milk Currently there are no known studies in Malaysia
examining the factors influencing the survival of B cereus in UHT milk and infant milk
Hence the objectives of this study were
I To study the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk stored at different temperature for seven days
II To examine the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk with different initial pH stored at refrigerated temperature for seven
days
3
20 Literature Review
21 Bacillus cereus
211 Morphological and biochemical characteristics
Bacillus cereus is a Gram positive motile rod-shaped aerobic or facultative anaerobe and
aerobic spore forming foodborne pathogen belonging to Bacillus genus that is normally found
in soil and vegetation (Tortora et al 2010 Food Standards Australia New Zealand 2013
Gleeson et al 2013) This bacterium is mesophile or psychrotroph This bacterium is closely
related to B thuringiensis B mycoides B weihenstephanensis B pseudomycoides and B
anthracis (Gleeson et al 2013) The ability of B cereus to produce endospores which are heat
resistant increases the survivality of B cereus at unfavourable conditions such as high
temperatures extreme pH values or envirorunents with insufficient nutrients compared to
other bacterial enteropathogens (Di Pinto et al 2013 Gleeson et al 2013) For the
biochemical characteristics B cereus are positive for motility Voges-Proskauer (VP)
reaction starch hydrolysis citrate catalase and negative for oxidase tests (Shinagawa 1990
Choma ef al 2000) B cereus is shown as slender bacilli with square end singly or in short
chains under Gram-staining (Bottone 2010)
212 B cereus food borne disease
Rice dishes and meat or poultry dishes were the common sources for B cereus outbreaks
(Bennett et al 2013) Rice and pasta contaminated with B cereus are the causes for emetic
syndrome while the causes for diarrheal illness are desserts meat dishes and dairy products
(Granum 2005) Endospores of B cereus were able to survive in improperly cooked rice and
they caused Fried Rice Syndrome (Sandra et al 2012) In Malaysia B cereus was fIrst
4
Pusat Khidmat Maklumat Ak d Ut lVERSTTJ MA AYSIA SARAWAK
suspected as the cause for food poisoning which affected 114 female students in Klang
(Rampal et al 1984) In order to confIrm that gastroenteritis is caused by B cereus the
number of B cereus iso lated from suspected food need to exceed 105 co lony- forming unit per
gram (CFUg) (Tortora et al 20[0)
213 Isolation and identification of B cereus
Mannitol egg yolk polymyxin agar (MYF) Kim-Geopfert agar Columbia base-blood
polymyxin B agar (CBA-P) and Polymyxin B-egg yolk-mannitol bromothymol blue agar
(PEMBA) are some of the selective media suitable for isoIation and identifIcation of B cereus
(van Netten and Kramer 1992) The inability to ferment mannitol causes B cereus colonies to
appear as pink on MYP and lecithinase activity enables the observation of precipitated zone
around colonies (Pacheco-Sanchez and Massaguer 2007) Food and Drug Administration
(FDA) recommended MYP agar as the stapdard media for identifIcation of B cereus
214 Production of enterotoxins
The two forms of illness associated with B cereus enterotoxins are diarrheal and emetic
syndromes Onset of watery diarrhea and abdominal pain which resemble Clostridium
pefringens intoxications occur 8 hours to 16 hours after ingestion of contaminated food are
the symptoms of diarrheal outbreak (Shinagawa 1990 Tortora et ai 2010) This diarrheal
syndrome occurs after the ingestion of vegetattve cells which later produce a protein
enterotoxin in the gastrointinal tract (Bottone 2010) This type of enterotoxin is heat-labile
(Bello et al 2014) Haemolysin BL (HBL) non-haemolytic enterotoxin and cytotoxin K are
the three types of enterotoxins produced by this bacterium (Samarzija et al 2012 Food
Standards Australia New Zealand 2013) Reis et al (2013) concluded that hblD was the
5
common HBL gene of B cereus detected in milk and dairy products leading to diarrheal form
ofB cereus foodborne disease
The symptoms of emetic outbreaks are similar to Staphylococcus aureus food
intoxication and occur 05 hour to 6 hours upon ingestion of contaminated food
(Messelhausser et al 2014) The ingestion of food contaminated with preformed cereulide
which is also known as the emetic toxin leads to emetic syndrome (Bottone 2010) This heatshy
stable toxin causes nausea and vomiting in the infected patients (Bello et al2014)
215 Effect of temperature and pH on growth and survival of B cereus
Generally B cereus grows between 4 C and 55 C but exhibit optimum growth between 30 C
and 40 C (Schraft and Griffiths 2006 Food Standards Australia New Zealand 2013) Growth
of B cereus had been detected at temperature ranging from 7 C to 45 C and significant
growth was observed at 45 c in steril ized raw milk samples (Bellow et al 2007) In the
absence of oxygen or at temperature below 10 C emetic B cereus are believed to show
incapability in growth and production of toxin (EFSA 2005) At temperature below 10 C
with the limitation of other factors the lag time and generation times of B cereus are longer
(EFSA 2005)
As mentioned by Food Standards Australia New Zealand (2013) B cereus is able to
grow at pH between pH 49 and pH 100 and the optimum pH is between pH 60 and pH 70
Bello et al (2014) concluded that B cereus was unable to grow at pH 63 to pH 68 for 24
hour in pasteurized whole cow milk and the growth was inhibited at pH 68 for a period of 48
hours Byaruhanga et af (1999) studied that mague base inoculated with B cereus for 24
hours at 3S C was able to grow when pH decreases from pH 65 to pH 50 Brain Heart
6
--~~~----~----~----------
Infusion (BHI) medium at pH 88 inhibits growth of emetic and diarrheal strains of B cereus
better than pH 50 of the same medium (Garcia-Arribas and Kramer 1990)
22 UHT milk
Milk treated with UHT pasteurization is heated to 138 middotC for 2 seconds and aseptically placed
into a sterile substantial container which can be stored without refrigeration for six months to
a year (Ledenbach and Marshall 2009) UHT treatment minimized heat denaturation and
produces long shelf life milk that has similar taste and color to pasteurized milk (Harding
1995) UHT milk is generally contaminated during technological processing because food
pathogens are destroyed by the UHT temperature (Vyletelova et al 2002) Longer storage
period causes the drop of UHT milk pH and enzymes may be the reason for changes in UHT
milk (Hassan et al 2009)
Among the 110 UHT milk samples tested by Vidal-Martins et al (2005) 118
(1 3110) samples were positive for B cereus Researchers Rezende-Lago et al (2007)
identified B cereus in four (13 3) of the UHT milk samples tested In the study performed
by Reis et al (2013) among the 260 samples of UHT milk pasteurized milk and powdered
milk tested in Brazil a total of 63 isolates of B cereus were found and 12 of them were from
UHT milk
23 Infant milk
Cows milk and soy protein are the major component of infant milk or infant formulas For the
production of infant formula from milk the milk is heat-treated concentrated and dried while
infant fonnula from other ingredients is dry-blended wetted and dried again or only dry-
blended (Becker et al 1994) The sublethally injured pathogens can recover after the process
7
of drying because this process caused only little damage to the cell (Miller et ai 2012) During
the process of drying components such as lactose protein and milk fat that are present in
milk-based infant formula have the potential to provide protection to the bacteria (Mi1er et ai
20 12) Infant formulas are given to newborns and the content of nutrition is similar to breast
milk (Becker et al 1994) Currently infant formulas in the market are available in powder
liquid concentration and ready-to-feed After rehydration and inappropriate storage lowshy
moisture foods such as infant milk powder should be consumed within a short time because
growth of microorganisms may occur (lLSI Europe 2011)
Becker et al (1994) tested 92 infant milk formula samples and 52 of the samples
were B cereus positive Another study by Rowan et al (1997) tested 100 samples of infant
milk formula from United Kingdom and 17 of the samples were B cereus positive In the
study done by Haughton et al (20 I 0) B cereus were detected in 59 out of 100 infant milk
formula samples from Ireland These studies showed that B cereus is commonly found in
most of the infant milk formula and this poses threat to infants who are highly susceptible
24 Polymerase Chain Reaction (PCR)
As ment ioned by Sandra et al (2012) PCR allows rapid detection of pathogens Schraft and
Griffiths (2006) mentioned that the detection of B cereus targetting B cereus per se and
specific sub-groups have been performed by PCR Yamada et al (1999) designed PCR primer
set for gyrB fragment of B cereus and used this primer set in the detection of B cereus in
boiled rice Confirmation of B cereus in typical colonies are performed using PCR techniques
(Rahimi el al 2013) Thousands to millions copies of specific DNA sequence are produced
from the amplification of a single or a few copies of a piece of DNA by PCR (Joshi and
8
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
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P sat Khidmat Maklumat Akademik UN VERSITI MALAYSIA ARAWAK
Table of Contents
Acknowledgement
Declaration II
Table of Contents IV
List of Abbreviations VI
List ofTables VII
List of Figures IX
Abstract XI
10 Introduction
20 Literature Review 4
21 Bacillus cereus 4
211 Morphological and biochemical characteristics 4
21 2 B cereus food borne disease 4
213 Isolation and identification of Bcereus 5
214 Production of enterotoxins 5
215 Effect of temperature and pH on growth and survival of B 6
cereus
22 UHT milk 7
23 Infant rriilk 7
24 Polymerase Chain Reaction (PCR) nd agarose gel electrophoresis 8
IV
30 Materials and Methods 10
31 The milk 10
32 Inocula preparation 10
33 Survival and growth of B cereus in UHT milk and infant milk 10
331 Effect of temperature 10
332 Effect ofpH 11
34 Determination of growth and survival of B cereus in UHT milk and 11
infant milk samples
35 Genomic Deoxyribonucleic acid (DNA) extraction 12
36 PCR 12
37 Statistical analysis 12
40 Results 14
50 Discussion 42
60 Conclusion 51
70 References 52
v
I
List of Abbreviations
ANOVA Analysis of Variance
B cereus Bacillus cereus
BHI Brain Heart Infusion
CBA-P Columbia base-blood polymyxin B agar
CFU colony-forming unit
FDA Food and Drug Administration
gyrB gyrase B
HCl Hydrogen chloride
MYP Mannitol Egg Yolk agar
microlitre
ml millilitre
NaOH Sodium hydroxide
PEMBA Polymyxin B-egg yolk-mannitol bromothymol blue agar
PCR Polymerase Chain Reaction
UHT ultra-high temperature
VP Voges-Proskauer
VI
List of Tables
Table 1 Mixture volumes ofPCR for detection of B cereus 13
Table 2 Thermal cycling profile ofPCR for detection ofB cereus 13
Table 3 B cereus colony count from day 0 to day 7 in UHT milk stored at 14
temperature range of4 middotC to 7 middotC
Table 4 B cereus colony count from day 0 to day 7 in infant milk stored at 15
temperature range of 4 middotC to 7 c
Table 5 B cereus colony count from day 0 to day 7 in UHT milk stored at 16
temperature range of 10 middotC to 13 middotC
Table 6 B cereus colony count from day 0 to day 7 in infant milk stored at 17
temperature range of 10 middotC to 13 middotC
Table 7 B cereus colony count from day 0 to day 7 in UHT milk stored at 25degC 18
Table 8 B cereus colony count from day 0 to day 7 in infant milk stored at 25 19
DC
Table 9 B cereus colony count from day 0 to day 7 in UHT milk stored at 35degC 20
Table 10 B cereus colony count from day 0 to day 7 in infant milk stored at 35 20
DC
Table 11 B cereus colony count from day 0 to day 7 in UHT milk stored at 45degC 22
Table 12 B cereus colony count from day 0 to day 7 in infant milk stored at 45 22
DC
Table 13 B cereus colony count from day 0 to day 7 in UHT milk stored at 55degC 24
VII
Table 14 B cereus colony count from day 0 to day 7 in infant milk stored at 55 24
0c
Table 15 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 29
ofpH 620 stored at refrigerated temperature
Table 16 B cereus colony count from day 0 to day 7 in infant milk with initial pH 30
of pH 620 stored at refrigerated temperature
Table 17 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 31
of pH 640 stored at refrigerated temperature
Table 18 B cereus colony count from day 0 to day 7 in infant milk with initial pH 32
of pH 640 stored at refrigerated temperature
Table 19 B cereus colony count from day 0 to day 7 in UHT milk with initia l pH 33
of pH 660 stored at refrigerated temperature
Table 20 B cereus colony count from d-ay 0 to day 7 in infant milk with initial pH 34
of pH 660 stored at refrigerated temperature
Table 21 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 35
of pH 680 stored at refrigerated temperature
Table 22 B cereus colony count from day 0 to day 7 in infant milk with initial pH 36
of pH 680 stored at refrigerated temperature
VIII
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
List of Figures
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 4 middotC to 7 c
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 10 middotC to 13 middotC
15
17
The survival curve of B cereus
infant milk stored at 25 c
The survival curve of B cereus
infant milk stored at 35 c
The survival curve of B cereus
infant milk stored at 45 c
The survival curve of B cereus
infant milk stored at 55 middotC
in artificially contaminated UHT and 19
in artificially contaminated UHT and 21
in artificially contaminated UHT and 23
in artificially contaminated UHT and 25
The survival curve of B cereus in aiiifIcially contaminated UHT milk 27
stored at 4 middotC -7 middotc 10 middotC-13 middotC 25 middotc 35 middotC 45 middotC and 55 middotC
The survival curve of B cereus in artificially contaminated infant milk 28
stored at 4 middotC -7 middotC 10 middotC-13 middotC 25 middotC 35 middotC 45 middotC and 55 c
The survival curve of B cereus in artifIcially contaminated UHT and 30
infant milk with initial pH of pH 620 stored at refrigerated temperature
(4 QC-7 QC)
IX
Figure 10 The survival curve of B cereus in artificially contaminated UHT and 32
infant milk with initial pH of pH 640 stored at refrigerated temperature
(4 middotC-7 C)
Figure 11 The survival curve of B cereus in artificially contaminated UHT and 34
infant milk with initial pH of pH 660 stored at refrigerated temperature
(4 middotC-7 C)
Figure 12 The survival curve of B cereus in artificially contaminated UHT and 36
infant milk with initial pH of pH 680 stored at refrigerated temperature
(4 middotC-7 C)
Figure 13 The survival curve of B cereus in artificially contaminated UHT milk 38
with different initial pH stored at refrigerated temperature (4 middotC-7 C)
Figure 14 The survival curve of B cereus in artificially contaminated infant milk 39
with different initial pH stored- at refrigerated temperature (4 middotC-7 middotC)
Figure 15 B cereus colonies grown on MYP Agar 40
Figure 16 Morphology of B cereus isolated from UHT milk and infant milk 40
samples
Figure 17 Amplification of the gyrB gene (475 bp) for the confirmation of B 41
cereus in UHT milk and infant milk samples
x
Survival ofBacillus cereus in Artificially Contaminated UHT Milk and Infant Milk
Kong Wan Wei
Resource Bioteclmology Department of Molecular Biology
Faculty of Resource Science and Teclmology Universiti Malaysia Sarawak
Abstract
The inappropriate storage temperature and pH enable the survival of Bacillus cereus a known food pathogen in processed milk products resulting in foodborne diseases In the present study UHT milk and infant milk purchased from local supermarkets in Kuching Sarawak were inoculated with 103 CFUml of B cereus Selected storage temperatures of 4 C-7 C 10 C-13 C 25 C 35 C 45 C and 55 C were used to investigate the effects of temperature on the survival of B cereus Initial pH of 620640 660 and 680 at refrigerated temperature (4 C-7 C) were used to study the effects of pH on the survival of B cereus Enumeration of colony counts were investigated daily for the duration of seven days Gram staining and Polymerase Chain Reaction (PCR) targeting gyrB gene were performed for the confirmation of B cereus isolates The present findings revealed that the survival of B cereus in UHT milk and infant mil k showed significant differences (pltO05) in different temperatures and pH At 25 C there were significant difference (plt005) between the survival of B cereus in UHT milk and infant milk B cereus were able to survive under all the experimental temperatures However growth was detected at all storage temperatures except 55 c Survival of B cereus in artificially contaminated UHT milk and infant milk with different initial pH stored at referigerated temperature were also recorded The results from this study can be treated as background data for the implementation of safety measure to avoid food poisoning caused by B cere liS
Key words Bacillus cereus survival milk temperature pH
Abstrak
Suhll penyimpanan dan pH yang tidak sesllai membolehkall kehidupan Bacillus cereus sejenis pathogen makanan dalam produk susu diproses dan mellyebabkan penyakit bawaan makanall Dalam kajian ini SIISII UHT dan SIISU bayi yang dibeli daripada pasar raya tempatan di Kuching Sarawak telah diinoklliasi dengan B cerells sebanyak J(i CFUlml Suhll penyimpanan 4 C-7 c 10 C-3 c 25 c 35 middotc 45 c dan 55 c telah digllnakan IIntllk mengkaji kesan slIhll terhadap kehidupan B cerells Pada suhll peti sejuk (4 C-7 C) pH 620 pH 640 pH 660 dan pH 680 telah digunakan sebagai pH permulaal IIntuk mengkaji kesan pH terhadap keflidupan B cereus Penggiraall kololli telah dijalankan setiap hari selama IIIjuh hari Pewarnaan Gram dan penggllnaan reaksi rantai polimerase (PCR) yang menyasarkan gen gyrB telah mengesahkan kewujlldan koloni B cereus Kajian ini menllnjukkan buhawa kehidupan B cereus dalam SIISU UHT dan susu bayi menunjukkan perbezaan yang ketara (plt005) di suhu penyimpanan dan pH yang berbeza Perbezaan yang ketara (plt005) berlaku anlara kehidupan B cereus dalam susu UHT dengan kehidllpan B cereus dalam SIISII bayi pada sllhu 25 c B cereus dapal menunjllkkan kehidupan dalam SIISU UHT dan SIISU bayi yang telah disimpan pada semua suhll eksperimen Walau bagaimanapun pertumbllhan dapat dikesan pada semua sllhll eksperimen keclali 55 c Selain itll kehidllpan B cerells dalam SUSII UHT dan SUSII bayi yang tercemar secara bllalan pada slIhll peli sejuk telah dicatat pada peJbagai pH Hasil daripadu kajian ini boleh dianggap sebagai dala lalar belakallg tllltllk pelaksanuan langkah keselamatan bagi mengelakkan keracunall makanall daripada B cereus
Kata kunci Bacillus cerellS keflidllpan SIISU sllltl pH
XI
10 Introduction
Population growth and increase in consciousness about health are some of the factors that
increase demand for milk and milk products in Malaysia In Malaysian market ultra-high
temperature (UHT) milk and infant milk powders come under a variety of brands and are
either locally produced or imported Milk provides calcium protein vitamin A vitamin B 12
riboflavin and zinc to the human diet Consumption of milk helps to build and maintain bone
health as well as to reduce the risk of osteoporosis Infant milk provides major nutrients to
infants and it is essential for the growth of newborns
Milk is a high moisture and high nutritious medium which is also suitable for the
growth of mircoorganisms Most of the people believed that processed milk samples are
totally safe for consumption but actually there are many ways that leads to milk
contamination Contamination happened duripg milk processing as Bacillus cereus (B cereus)
strains which were not found in raw milk samples was present in the processed milk samples
(Banyk6 and Vyletelova 2009) Microorganisms contaminate the milk and affects the milk
quality by infecting the mammary gland udder and teat surfaces milking equipment and
storage tank (Gleeson et al 2013) Moreover improper processing and the usage of raw milk
that did not meet the microbiology and physio-chemical standards lead to the defects in raw
and UHT milk (Abdul-Hadi et al 2014) Inappropriate food storage and handling of
contaminated food encourage the growth of B cereus to the levels which can cause food
poisoning (Granum 2005) In order to maintain the quality and safety of processed milk
products consumers are suggested to store these products at temperature of 4 middotC or [less (Boor
and Fromm 2006)
Food pOlsonmg which may even have lethal effect on human is due to the
inappropriate food preparation and food exposure to 30 middotC (Sandra et at 2012) From year
1998 until 2008 there were 235 cases of foodbome disease outbreaks associated with B
cereus in United States (Bennett et at 2013) In 2011 the number of B cereus foodborne
diseases in Europe showed an increase of 1222 (MesseJhausser et ai 2014) The number
reported shows that B cereus poses a threat to the public health as it is often found in
contaminated food However the number of B cereus foodbome poisoning is believed to be
under reported because this type of food poisoning is mild and reporting these cases is not an
compulsory act (EFSA 2005)
B cereus was recognized as foodbome pathogen since 1955 and it is the cause for
about 2 of all foodbome disease worldwide (Lesley et at 2013) Garcia-Arribas and Kramer
(1990) mentioned that B cereus contribute to economic loss by causing contamination
spoilage and food-poisoning B cereus are found in dairy products meats spices and cereals
(Browne and Dowds 2002) as well as in infant foods and dried milk products (Becker et aI
1994)
Most of the patients involved in B cereus outbreaks were infants or elders (Becker et
ai 1994) The immune system of infants are still developing so they are highly susceptible to
B cereus food poisoning after consuming contaminated infant milk Although infant milk
basically present in low moisture powder form the vegetative cells and spores of
microorganisms are able to survive after drying but with their metabolism reduced (I LSI
Europe 2011) This poses threat to infants because some consumers keep the reconstituted
infant formula at room temperature for a long period of time as they wrongly believed that
infant formula are sterilised and are completely free of B cereus (ILSI Europe 2011)
2
B cereus is conunonly related to dairy products because they are able to survive at
different environmental stresses such as pasteurization (Foltys and Kirchnerova 2006 Sadek
et (II 2006) Schelderman et al (2006) discussed that aerobic spore former such as B cereus
has increased tolerance towards low temperature low pH sterilization and UHT treatments
and these conditions increase the concern in food industry In year 2011 there were food
poisoning associated with UHT milk provided from 1 Malaysia Milk Progranune and B cereus
were detected in these UHT milk (Yusof 2011) In 2013 infant milk formula product from
Fonterra were tested positive for Clostridium botulinum which is known as an endospore
former (Frediansyah 2014) The botulism scare was later confirmed as false alarm and the
actual organism was Clostridium 5porogenes However these pathogens have alarmed public
on the safety ofUHT milk and infant milk Currently there are no known studies in Malaysia
examining the factors influencing the survival of B cereus in UHT milk and infant milk
Hence the objectives of this study were
I To study the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk stored at different temperature for seven days
II To examine the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk with different initial pH stored at refrigerated temperature for seven
days
3
20 Literature Review
21 Bacillus cereus
211 Morphological and biochemical characteristics
Bacillus cereus is a Gram positive motile rod-shaped aerobic or facultative anaerobe and
aerobic spore forming foodborne pathogen belonging to Bacillus genus that is normally found
in soil and vegetation (Tortora et al 2010 Food Standards Australia New Zealand 2013
Gleeson et al 2013) This bacterium is mesophile or psychrotroph This bacterium is closely
related to B thuringiensis B mycoides B weihenstephanensis B pseudomycoides and B
anthracis (Gleeson et al 2013) The ability of B cereus to produce endospores which are heat
resistant increases the survivality of B cereus at unfavourable conditions such as high
temperatures extreme pH values or envirorunents with insufficient nutrients compared to
other bacterial enteropathogens (Di Pinto et al 2013 Gleeson et al 2013) For the
biochemical characteristics B cereus are positive for motility Voges-Proskauer (VP)
reaction starch hydrolysis citrate catalase and negative for oxidase tests (Shinagawa 1990
Choma ef al 2000) B cereus is shown as slender bacilli with square end singly or in short
chains under Gram-staining (Bottone 2010)
212 B cereus food borne disease
Rice dishes and meat or poultry dishes were the common sources for B cereus outbreaks
(Bennett et al 2013) Rice and pasta contaminated with B cereus are the causes for emetic
syndrome while the causes for diarrheal illness are desserts meat dishes and dairy products
(Granum 2005) Endospores of B cereus were able to survive in improperly cooked rice and
they caused Fried Rice Syndrome (Sandra et al 2012) In Malaysia B cereus was fIrst
4
Pusat Khidmat Maklumat Ak d Ut lVERSTTJ MA AYSIA SARAWAK
suspected as the cause for food poisoning which affected 114 female students in Klang
(Rampal et al 1984) In order to confIrm that gastroenteritis is caused by B cereus the
number of B cereus iso lated from suspected food need to exceed 105 co lony- forming unit per
gram (CFUg) (Tortora et al 20[0)
213 Isolation and identification of B cereus
Mannitol egg yolk polymyxin agar (MYF) Kim-Geopfert agar Columbia base-blood
polymyxin B agar (CBA-P) and Polymyxin B-egg yolk-mannitol bromothymol blue agar
(PEMBA) are some of the selective media suitable for isoIation and identifIcation of B cereus
(van Netten and Kramer 1992) The inability to ferment mannitol causes B cereus colonies to
appear as pink on MYP and lecithinase activity enables the observation of precipitated zone
around colonies (Pacheco-Sanchez and Massaguer 2007) Food and Drug Administration
(FDA) recommended MYP agar as the stapdard media for identifIcation of B cereus
214 Production of enterotoxins
The two forms of illness associated with B cereus enterotoxins are diarrheal and emetic
syndromes Onset of watery diarrhea and abdominal pain which resemble Clostridium
pefringens intoxications occur 8 hours to 16 hours after ingestion of contaminated food are
the symptoms of diarrheal outbreak (Shinagawa 1990 Tortora et ai 2010) This diarrheal
syndrome occurs after the ingestion of vegetattve cells which later produce a protein
enterotoxin in the gastrointinal tract (Bottone 2010) This type of enterotoxin is heat-labile
(Bello et al 2014) Haemolysin BL (HBL) non-haemolytic enterotoxin and cytotoxin K are
the three types of enterotoxins produced by this bacterium (Samarzija et al 2012 Food
Standards Australia New Zealand 2013) Reis et al (2013) concluded that hblD was the
5
common HBL gene of B cereus detected in milk and dairy products leading to diarrheal form
ofB cereus foodborne disease
The symptoms of emetic outbreaks are similar to Staphylococcus aureus food
intoxication and occur 05 hour to 6 hours upon ingestion of contaminated food
(Messelhausser et al 2014) The ingestion of food contaminated with preformed cereulide
which is also known as the emetic toxin leads to emetic syndrome (Bottone 2010) This heatshy
stable toxin causes nausea and vomiting in the infected patients (Bello et al2014)
215 Effect of temperature and pH on growth and survival of B cereus
Generally B cereus grows between 4 C and 55 C but exhibit optimum growth between 30 C
and 40 C (Schraft and Griffiths 2006 Food Standards Australia New Zealand 2013) Growth
of B cereus had been detected at temperature ranging from 7 C to 45 C and significant
growth was observed at 45 c in steril ized raw milk samples (Bellow et al 2007) In the
absence of oxygen or at temperature below 10 C emetic B cereus are believed to show
incapability in growth and production of toxin (EFSA 2005) At temperature below 10 C
with the limitation of other factors the lag time and generation times of B cereus are longer
(EFSA 2005)
As mentioned by Food Standards Australia New Zealand (2013) B cereus is able to
grow at pH between pH 49 and pH 100 and the optimum pH is between pH 60 and pH 70
Bello et al (2014) concluded that B cereus was unable to grow at pH 63 to pH 68 for 24
hour in pasteurized whole cow milk and the growth was inhibited at pH 68 for a period of 48
hours Byaruhanga et af (1999) studied that mague base inoculated with B cereus for 24
hours at 3S C was able to grow when pH decreases from pH 65 to pH 50 Brain Heart
6
--~~~----~----~----------
Infusion (BHI) medium at pH 88 inhibits growth of emetic and diarrheal strains of B cereus
better than pH 50 of the same medium (Garcia-Arribas and Kramer 1990)
22 UHT milk
Milk treated with UHT pasteurization is heated to 138 middotC for 2 seconds and aseptically placed
into a sterile substantial container which can be stored without refrigeration for six months to
a year (Ledenbach and Marshall 2009) UHT treatment minimized heat denaturation and
produces long shelf life milk that has similar taste and color to pasteurized milk (Harding
1995) UHT milk is generally contaminated during technological processing because food
pathogens are destroyed by the UHT temperature (Vyletelova et al 2002) Longer storage
period causes the drop of UHT milk pH and enzymes may be the reason for changes in UHT
milk (Hassan et al 2009)
Among the 110 UHT milk samples tested by Vidal-Martins et al (2005) 118
(1 3110) samples were positive for B cereus Researchers Rezende-Lago et al (2007)
identified B cereus in four (13 3) of the UHT milk samples tested In the study performed
by Reis et al (2013) among the 260 samples of UHT milk pasteurized milk and powdered
milk tested in Brazil a total of 63 isolates of B cereus were found and 12 of them were from
UHT milk
23 Infant milk
Cows milk and soy protein are the major component of infant milk or infant formulas For the
production of infant formula from milk the milk is heat-treated concentrated and dried while
infant fonnula from other ingredients is dry-blended wetted and dried again or only dry-
blended (Becker et al 1994) The sublethally injured pathogens can recover after the process
7
of drying because this process caused only little damage to the cell (Miller et ai 2012) During
the process of drying components such as lactose protein and milk fat that are present in
milk-based infant formula have the potential to provide protection to the bacteria (Mi1er et ai
20 12) Infant formulas are given to newborns and the content of nutrition is similar to breast
milk (Becker et al 1994) Currently infant formulas in the market are available in powder
liquid concentration and ready-to-feed After rehydration and inappropriate storage lowshy
moisture foods such as infant milk powder should be consumed within a short time because
growth of microorganisms may occur (lLSI Europe 2011)
Becker et al (1994) tested 92 infant milk formula samples and 52 of the samples
were B cereus positive Another study by Rowan et al (1997) tested 100 samples of infant
milk formula from United Kingdom and 17 of the samples were B cereus positive In the
study done by Haughton et al (20 I 0) B cereus were detected in 59 out of 100 infant milk
formula samples from Ireland These studies showed that B cereus is commonly found in
most of the infant milk formula and this poses threat to infants who are highly susceptible
24 Polymerase Chain Reaction (PCR)
As ment ioned by Sandra et al (2012) PCR allows rapid detection of pathogens Schraft and
Griffiths (2006) mentioned that the detection of B cereus targetting B cereus per se and
specific sub-groups have been performed by PCR Yamada et al (1999) designed PCR primer
set for gyrB fragment of B cereus and used this primer set in the detection of B cereus in
boiled rice Confirmation of B cereus in typical colonies are performed using PCR techniques
(Rahimi el al 2013) Thousands to millions copies of specific DNA sequence are produced
from the amplification of a single or a few copies of a piece of DNA by PCR (Joshi and
8
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
P sat Khidmat Maklumat Akademik UN VERSITI MALAYSIA ARAWAK
Table of Contents
Acknowledgement
Declaration II
Table of Contents IV
List of Abbreviations VI
List ofTables VII
List of Figures IX
Abstract XI
10 Introduction
20 Literature Review 4
21 Bacillus cereus 4
211 Morphological and biochemical characteristics 4
21 2 B cereus food borne disease 4
213 Isolation and identification of Bcereus 5
214 Production of enterotoxins 5
215 Effect of temperature and pH on growth and survival of B 6
cereus
22 UHT milk 7
23 Infant rriilk 7
24 Polymerase Chain Reaction (PCR) nd agarose gel electrophoresis 8
IV
30 Materials and Methods 10
31 The milk 10
32 Inocula preparation 10
33 Survival and growth of B cereus in UHT milk and infant milk 10
331 Effect of temperature 10
332 Effect ofpH 11
34 Determination of growth and survival of B cereus in UHT milk and 11
infant milk samples
35 Genomic Deoxyribonucleic acid (DNA) extraction 12
36 PCR 12
37 Statistical analysis 12
40 Results 14
50 Discussion 42
60 Conclusion 51
70 References 52
v
I
List of Abbreviations
ANOVA Analysis of Variance
B cereus Bacillus cereus
BHI Brain Heart Infusion
CBA-P Columbia base-blood polymyxin B agar
CFU colony-forming unit
FDA Food and Drug Administration
gyrB gyrase B
HCl Hydrogen chloride
MYP Mannitol Egg Yolk agar
microlitre
ml millilitre
NaOH Sodium hydroxide
PEMBA Polymyxin B-egg yolk-mannitol bromothymol blue agar
PCR Polymerase Chain Reaction
UHT ultra-high temperature
VP Voges-Proskauer
VI
List of Tables
Table 1 Mixture volumes ofPCR for detection of B cereus 13
Table 2 Thermal cycling profile ofPCR for detection ofB cereus 13
Table 3 B cereus colony count from day 0 to day 7 in UHT milk stored at 14
temperature range of4 middotC to 7 middotC
Table 4 B cereus colony count from day 0 to day 7 in infant milk stored at 15
temperature range of 4 middotC to 7 c
Table 5 B cereus colony count from day 0 to day 7 in UHT milk stored at 16
temperature range of 10 middotC to 13 middotC
Table 6 B cereus colony count from day 0 to day 7 in infant milk stored at 17
temperature range of 10 middotC to 13 middotC
Table 7 B cereus colony count from day 0 to day 7 in UHT milk stored at 25degC 18
Table 8 B cereus colony count from day 0 to day 7 in infant milk stored at 25 19
DC
Table 9 B cereus colony count from day 0 to day 7 in UHT milk stored at 35degC 20
Table 10 B cereus colony count from day 0 to day 7 in infant milk stored at 35 20
DC
Table 11 B cereus colony count from day 0 to day 7 in UHT milk stored at 45degC 22
Table 12 B cereus colony count from day 0 to day 7 in infant milk stored at 45 22
DC
Table 13 B cereus colony count from day 0 to day 7 in UHT milk stored at 55degC 24
VII
Table 14 B cereus colony count from day 0 to day 7 in infant milk stored at 55 24
0c
Table 15 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 29
ofpH 620 stored at refrigerated temperature
Table 16 B cereus colony count from day 0 to day 7 in infant milk with initial pH 30
of pH 620 stored at refrigerated temperature
Table 17 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 31
of pH 640 stored at refrigerated temperature
Table 18 B cereus colony count from day 0 to day 7 in infant milk with initial pH 32
of pH 640 stored at refrigerated temperature
Table 19 B cereus colony count from day 0 to day 7 in UHT milk with initia l pH 33
of pH 660 stored at refrigerated temperature
Table 20 B cereus colony count from d-ay 0 to day 7 in infant milk with initial pH 34
of pH 660 stored at refrigerated temperature
Table 21 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 35
of pH 680 stored at refrigerated temperature
Table 22 B cereus colony count from day 0 to day 7 in infant milk with initial pH 36
of pH 680 stored at refrigerated temperature
VIII
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
List of Figures
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 4 middotC to 7 c
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 10 middotC to 13 middotC
15
17
The survival curve of B cereus
infant milk stored at 25 c
The survival curve of B cereus
infant milk stored at 35 c
The survival curve of B cereus
infant milk stored at 45 c
The survival curve of B cereus
infant milk stored at 55 middotC
in artificially contaminated UHT and 19
in artificially contaminated UHT and 21
in artificially contaminated UHT and 23
in artificially contaminated UHT and 25
The survival curve of B cereus in aiiifIcially contaminated UHT milk 27
stored at 4 middotC -7 middotc 10 middotC-13 middotC 25 middotc 35 middotC 45 middotC and 55 middotC
The survival curve of B cereus in artificially contaminated infant milk 28
stored at 4 middotC -7 middotC 10 middotC-13 middotC 25 middotC 35 middotC 45 middotC and 55 c
The survival curve of B cereus in artifIcially contaminated UHT and 30
infant milk with initial pH of pH 620 stored at refrigerated temperature
(4 QC-7 QC)
IX
Figure 10 The survival curve of B cereus in artificially contaminated UHT and 32
infant milk with initial pH of pH 640 stored at refrigerated temperature
(4 middotC-7 C)
Figure 11 The survival curve of B cereus in artificially contaminated UHT and 34
infant milk with initial pH of pH 660 stored at refrigerated temperature
(4 middotC-7 C)
Figure 12 The survival curve of B cereus in artificially contaminated UHT and 36
infant milk with initial pH of pH 680 stored at refrigerated temperature
(4 middotC-7 C)
Figure 13 The survival curve of B cereus in artificially contaminated UHT milk 38
with different initial pH stored at refrigerated temperature (4 middotC-7 C)
Figure 14 The survival curve of B cereus in artificially contaminated infant milk 39
with different initial pH stored- at refrigerated temperature (4 middotC-7 middotC)
Figure 15 B cereus colonies grown on MYP Agar 40
Figure 16 Morphology of B cereus isolated from UHT milk and infant milk 40
samples
Figure 17 Amplification of the gyrB gene (475 bp) for the confirmation of B 41
cereus in UHT milk and infant milk samples
x
Survival ofBacillus cereus in Artificially Contaminated UHT Milk and Infant Milk
Kong Wan Wei
Resource Bioteclmology Department of Molecular Biology
Faculty of Resource Science and Teclmology Universiti Malaysia Sarawak
Abstract
The inappropriate storage temperature and pH enable the survival of Bacillus cereus a known food pathogen in processed milk products resulting in foodborne diseases In the present study UHT milk and infant milk purchased from local supermarkets in Kuching Sarawak were inoculated with 103 CFUml of B cereus Selected storage temperatures of 4 C-7 C 10 C-13 C 25 C 35 C 45 C and 55 C were used to investigate the effects of temperature on the survival of B cereus Initial pH of 620640 660 and 680 at refrigerated temperature (4 C-7 C) were used to study the effects of pH on the survival of B cereus Enumeration of colony counts were investigated daily for the duration of seven days Gram staining and Polymerase Chain Reaction (PCR) targeting gyrB gene were performed for the confirmation of B cereus isolates The present findings revealed that the survival of B cereus in UHT milk and infant mil k showed significant differences (pltO05) in different temperatures and pH At 25 C there were significant difference (plt005) between the survival of B cereus in UHT milk and infant milk B cereus were able to survive under all the experimental temperatures However growth was detected at all storage temperatures except 55 c Survival of B cereus in artificially contaminated UHT milk and infant milk with different initial pH stored at referigerated temperature were also recorded The results from this study can be treated as background data for the implementation of safety measure to avoid food poisoning caused by B cere liS
Key words Bacillus cereus survival milk temperature pH
Abstrak
Suhll penyimpanan dan pH yang tidak sesllai membolehkall kehidupan Bacillus cereus sejenis pathogen makanan dalam produk susu diproses dan mellyebabkan penyakit bawaan makanall Dalam kajian ini SIISII UHT dan SIISU bayi yang dibeli daripada pasar raya tempatan di Kuching Sarawak telah diinoklliasi dengan B cerells sebanyak J(i CFUlml Suhll penyimpanan 4 C-7 c 10 C-3 c 25 c 35 middotc 45 c dan 55 c telah digllnakan IIntllk mengkaji kesan slIhll terhadap kehidupan B cerells Pada suhll peti sejuk (4 C-7 C) pH 620 pH 640 pH 660 dan pH 680 telah digunakan sebagai pH permulaal IIntuk mengkaji kesan pH terhadap keflidupan B cereus Penggiraall kololli telah dijalankan setiap hari selama IIIjuh hari Pewarnaan Gram dan penggllnaan reaksi rantai polimerase (PCR) yang menyasarkan gen gyrB telah mengesahkan kewujlldan koloni B cereus Kajian ini menllnjukkan buhawa kehidupan B cereus dalam SIISU UHT dan susu bayi menunjukkan perbezaan yang ketara (plt005) di suhu penyimpanan dan pH yang berbeza Perbezaan yang ketara (plt005) berlaku anlara kehidupan B cereus dalam susu UHT dengan kehidllpan B cereus dalam SIISII bayi pada sllhu 25 c B cereus dapal menunjllkkan kehidupan dalam SIISU UHT dan SIISU bayi yang telah disimpan pada semua suhll eksperimen Walau bagaimanapun pertumbllhan dapat dikesan pada semua sllhll eksperimen keclali 55 c Selain itll kehidllpan B cerells dalam SUSII UHT dan SUSII bayi yang tercemar secara bllalan pada slIhll peli sejuk telah dicatat pada peJbagai pH Hasil daripadu kajian ini boleh dianggap sebagai dala lalar belakallg tllltllk pelaksanuan langkah keselamatan bagi mengelakkan keracunall makanall daripada B cereus
Kata kunci Bacillus cerellS keflidllpan SIISU sllltl pH
XI
10 Introduction
Population growth and increase in consciousness about health are some of the factors that
increase demand for milk and milk products in Malaysia In Malaysian market ultra-high
temperature (UHT) milk and infant milk powders come under a variety of brands and are
either locally produced or imported Milk provides calcium protein vitamin A vitamin B 12
riboflavin and zinc to the human diet Consumption of milk helps to build and maintain bone
health as well as to reduce the risk of osteoporosis Infant milk provides major nutrients to
infants and it is essential for the growth of newborns
Milk is a high moisture and high nutritious medium which is also suitable for the
growth of mircoorganisms Most of the people believed that processed milk samples are
totally safe for consumption but actually there are many ways that leads to milk
contamination Contamination happened duripg milk processing as Bacillus cereus (B cereus)
strains which were not found in raw milk samples was present in the processed milk samples
(Banyk6 and Vyletelova 2009) Microorganisms contaminate the milk and affects the milk
quality by infecting the mammary gland udder and teat surfaces milking equipment and
storage tank (Gleeson et al 2013) Moreover improper processing and the usage of raw milk
that did not meet the microbiology and physio-chemical standards lead to the defects in raw
and UHT milk (Abdul-Hadi et al 2014) Inappropriate food storage and handling of
contaminated food encourage the growth of B cereus to the levels which can cause food
poisoning (Granum 2005) In order to maintain the quality and safety of processed milk
products consumers are suggested to store these products at temperature of 4 middotC or [less (Boor
and Fromm 2006)
Food pOlsonmg which may even have lethal effect on human is due to the
inappropriate food preparation and food exposure to 30 middotC (Sandra et at 2012) From year
1998 until 2008 there were 235 cases of foodbome disease outbreaks associated with B
cereus in United States (Bennett et at 2013) In 2011 the number of B cereus foodborne
diseases in Europe showed an increase of 1222 (MesseJhausser et ai 2014) The number
reported shows that B cereus poses a threat to the public health as it is often found in
contaminated food However the number of B cereus foodbome poisoning is believed to be
under reported because this type of food poisoning is mild and reporting these cases is not an
compulsory act (EFSA 2005)
B cereus was recognized as foodbome pathogen since 1955 and it is the cause for
about 2 of all foodbome disease worldwide (Lesley et at 2013) Garcia-Arribas and Kramer
(1990) mentioned that B cereus contribute to economic loss by causing contamination
spoilage and food-poisoning B cereus are found in dairy products meats spices and cereals
(Browne and Dowds 2002) as well as in infant foods and dried milk products (Becker et aI
1994)
Most of the patients involved in B cereus outbreaks were infants or elders (Becker et
ai 1994) The immune system of infants are still developing so they are highly susceptible to
B cereus food poisoning after consuming contaminated infant milk Although infant milk
basically present in low moisture powder form the vegetative cells and spores of
microorganisms are able to survive after drying but with their metabolism reduced (I LSI
Europe 2011) This poses threat to infants because some consumers keep the reconstituted
infant formula at room temperature for a long period of time as they wrongly believed that
infant formula are sterilised and are completely free of B cereus (ILSI Europe 2011)
2
B cereus is conunonly related to dairy products because they are able to survive at
different environmental stresses such as pasteurization (Foltys and Kirchnerova 2006 Sadek
et (II 2006) Schelderman et al (2006) discussed that aerobic spore former such as B cereus
has increased tolerance towards low temperature low pH sterilization and UHT treatments
and these conditions increase the concern in food industry In year 2011 there were food
poisoning associated with UHT milk provided from 1 Malaysia Milk Progranune and B cereus
were detected in these UHT milk (Yusof 2011) In 2013 infant milk formula product from
Fonterra were tested positive for Clostridium botulinum which is known as an endospore
former (Frediansyah 2014) The botulism scare was later confirmed as false alarm and the
actual organism was Clostridium 5porogenes However these pathogens have alarmed public
on the safety ofUHT milk and infant milk Currently there are no known studies in Malaysia
examining the factors influencing the survival of B cereus in UHT milk and infant milk
Hence the objectives of this study were
I To study the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk stored at different temperature for seven days
II To examine the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk with different initial pH stored at refrigerated temperature for seven
days
3
20 Literature Review
21 Bacillus cereus
211 Morphological and biochemical characteristics
Bacillus cereus is a Gram positive motile rod-shaped aerobic or facultative anaerobe and
aerobic spore forming foodborne pathogen belonging to Bacillus genus that is normally found
in soil and vegetation (Tortora et al 2010 Food Standards Australia New Zealand 2013
Gleeson et al 2013) This bacterium is mesophile or psychrotroph This bacterium is closely
related to B thuringiensis B mycoides B weihenstephanensis B pseudomycoides and B
anthracis (Gleeson et al 2013) The ability of B cereus to produce endospores which are heat
resistant increases the survivality of B cereus at unfavourable conditions such as high
temperatures extreme pH values or envirorunents with insufficient nutrients compared to
other bacterial enteropathogens (Di Pinto et al 2013 Gleeson et al 2013) For the
biochemical characteristics B cereus are positive for motility Voges-Proskauer (VP)
reaction starch hydrolysis citrate catalase and negative for oxidase tests (Shinagawa 1990
Choma ef al 2000) B cereus is shown as slender bacilli with square end singly or in short
chains under Gram-staining (Bottone 2010)
212 B cereus food borne disease
Rice dishes and meat or poultry dishes were the common sources for B cereus outbreaks
(Bennett et al 2013) Rice and pasta contaminated with B cereus are the causes for emetic
syndrome while the causes for diarrheal illness are desserts meat dishes and dairy products
(Granum 2005) Endospores of B cereus were able to survive in improperly cooked rice and
they caused Fried Rice Syndrome (Sandra et al 2012) In Malaysia B cereus was fIrst
4
Pusat Khidmat Maklumat Ak d Ut lVERSTTJ MA AYSIA SARAWAK
suspected as the cause for food poisoning which affected 114 female students in Klang
(Rampal et al 1984) In order to confIrm that gastroenteritis is caused by B cereus the
number of B cereus iso lated from suspected food need to exceed 105 co lony- forming unit per
gram (CFUg) (Tortora et al 20[0)
213 Isolation and identification of B cereus
Mannitol egg yolk polymyxin agar (MYF) Kim-Geopfert agar Columbia base-blood
polymyxin B agar (CBA-P) and Polymyxin B-egg yolk-mannitol bromothymol blue agar
(PEMBA) are some of the selective media suitable for isoIation and identifIcation of B cereus
(van Netten and Kramer 1992) The inability to ferment mannitol causes B cereus colonies to
appear as pink on MYP and lecithinase activity enables the observation of precipitated zone
around colonies (Pacheco-Sanchez and Massaguer 2007) Food and Drug Administration
(FDA) recommended MYP agar as the stapdard media for identifIcation of B cereus
214 Production of enterotoxins
The two forms of illness associated with B cereus enterotoxins are diarrheal and emetic
syndromes Onset of watery diarrhea and abdominal pain which resemble Clostridium
pefringens intoxications occur 8 hours to 16 hours after ingestion of contaminated food are
the symptoms of diarrheal outbreak (Shinagawa 1990 Tortora et ai 2010) This diarrheal
syndrome occurs after the ingestion of vegetattve cells which later produce a protein
enterotoxin in the gastrointinal tract (Bottone 2010) This type of enterotoxin is heat-labile
(Bello et al 2014) Haemolysin BL (HBL) non-haemolytic enterotoxin and cytotoxin K are
the three types of enterotoxins produced by this bacterium (Samarzija et al 2012 Food
Standards Australia New Zealand 2013) Reis et al (2013) concluded that hblD was the
5
common HBL gene of B cereus detected in milk and dairy products leading to diarrheal form
ofB cereus foodborne disease
The symptoms of emetic outbreaks are similar to Staphylococcus aureus food
intoxication and occur 05 hour to 6 hours upon ingestion of contaminated food
(Messelhausser et al 2014) The ingestion of food contaminated with preformed cereulide
which is also known as the emetic toxin leads to emetic syndrome (Bottone 2010) This heatshy
stable toxin causes nausea and vomiting in the infected patients (Bello et al2014)
215 Effect of temperature and pH on growth and survival of B cereus
Generally B cereus grows between 4 C and 55 C but exhibit optimum growth between 30 C
and 40 C (Schraft and Griffiths 2006 Food Standards Australia New Zealand 2013) Growth
of B cereus had been detected at temperature ranging from 7 C to 45 C and significant
growth was observed at 45 c in steril ized raw milk samples (Bellow et al 2007) In the
absence of oxygen or at temperature below 10 C emetic B cereus are believed to show
incapability in growth and production of toxin (EFSA 2005) At temperature below 10 C
with the limitation of other factors the lag time and generation times of B cereus are longer
(EFSA 2005)
As mentioned by Food Standards Australia New Zealand (2013) B cereus is able to
grow at pH between pH 49 and pH 100 and the optimum pH is between pH 60 and pH 70
Bello et al (2014) concluded that B cereus was unable to grow at pH 63 to pH 68 for 24
hour in pasteurized whole cow milk and the growth was inhibited at pH 68 for a period of 48
hours Byaruhanga et af (1999) studied that mague base inoculated with B cereus for 24
hours at 3S C was able to grow when pH decreases from pH 65 to pH 50 Brain Heart
6
--~~~----~----~----------
Infusion (BHI) medium at pH 88 inhibits growth of emetic and diarrheal strains of B cereus
better than pH 50 of the same medium (Garcia-Arribas and Kramer 1990)
22 UHT milk
Milk treated with UHT pasteurization is heated to 138 middotC for 2 seconds and aseptically placed
into a sterile substantial container which can be stored without refrigeration for six months to
a year (Ledenbach and Marshall 2009) UHT treatment minimized heat denaturation and
produces long shelf life milk that has similar taste and color to pasteurized milk (Harding
1995) UHT milk is generally contaminated during technological processing because food
pathogens are destroyed by the UHT temperature (Vyletelova et al 2002) Longer storage
period causes the drop of UHT milk pH and enzymes may be the reason for changes in UHT
milk (Hassan et al 2009)
Among the 110 UHT milk samples tested by Vidal-Martins et al (2005) 118
(1 3110) samples were positive for B cereus Researchers Rezende-Lago et al (2007)
identified B cereus in four (13 3) of the UHT milk samples tested In the study performed
by Reis et al (2013) among the 260 samples of UHT milk pasteurized milk and powdered
milk tested in Brazil a total of 63 isolates of B cereus were found and 12 of them were from
UHT milk
23 Infant milk
Cows milk and soy protein are the major component of infant milk or infant formulas For the
production of infant formula from milk the milk is heat-treated concentrated and dried while
infant fonnula from other ingredients is dry-blended wetted and dried again or only dry-
blended (Becker et al 1994) The sublethally injured pathogens can recover after the process
7
of drying because this process caused only little damage to the cell (Miller et ai 2012) During
the process of drying components such as lactose protein and milk fat that are present in
milk-based infant formula have the potential to provide protection to the bacteria (Mi1er et ai
20 12) Infant formulas are given to newborns and the content of nutrition is similar to breast
milk (Becker et al 1994) Currently infant formulas in the market are available in powder
liquid concentration and ready-to-feed After rehydration and inappropriate storage lowshy
moisture foods such as infant milk powder should be consumed within a short time because
growth of microorganisms may occur (lLSI Europe 2011)
Becker et al (1994) tested 92 infant milk formula samples and 52 of the samples
were B cereus positive Another study by Rowan et al (1997) tested 100 samples of infant
milk formula from United Kingdom and 17 of the samples were B cereus positive In the
study done by Haughton et al (20 I 0) B cereus were detected in 59 out of 100 infant milk
formula samples from Ireland These studies showed that B cereus is commonly found in
most of the infant milk formula and this poses threat to infants who are highly susceptible
24 Polymerase Chain Reaction (PCR)
As ment ioned by Sandra et al (2012) PCR allows rapid detection of pathogens Schraft and
Griffiths (2006) mentioned that the detection of B cereus targetting B cereus per se and
specific sub-groups have been performed by PCR Yamada et al (1999) designed PCR primer
set for gyrB fragment of B cereus and used this primer set in the detection of B cereus in
boiled rice Confirmation of B cereus in typical colonies are performed using PCR techniques
(Rahimi el al 2013) Thousands to millions copies of specific DNA sequence are produced
from the amplification of a single or a few copies of a piece of DNA by PCR (Joshi and
8
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
30 Materials and Methods 10
31 The milk 10
32 Inocula preparation 10
33 Survival and growth of B cereus in UHT milk and infant milk 10
331 Effect of temperature 10
332 Effect ofpH 11
34 Determination of growth and survival of B cereus in UHT milk and 11
infant milk samples
35 Genomic Deoxyribonucleic acid (DNA) extraction 12
36 PCR 12
37 Statistical analysis 12
40 Results 14
50 Discussion 42
60 Conclusion 51
70 References 52
v
I
List of Abbreviations
ANOVA Analysis of Variance
B cereus Bacillus cereus
BHI Brain Heart Infusion
CBA-P Columbia base-blood polymyxin B agar
CFU colony-forming unit
FDA Food and Drug Administration
gyrB gyrase B
HCl Hydrogen chloride
MYP Mannitol Egg Yolk agar
microlitre
ml millilitre
NaOH Sodium hydroxide
PEMBA Polymyxin B-egg yolk-mannitol bromothymol blue agar
PCR Polymerase Chain Reaction
UHT ultra-high temperature
VP Voges-Proskauer
VI
List of Tables
Table 1 Mixture volumes ofPCR for detection of B cereus 13
Table 2 Thermal cycling profile ofPCR for detection ofB cereus 13
Table 3 B cereus colony count from day 0 to day 7 in UHT milk stored at 14
temperature range of4 middotC to 7 middotC
Table 4 B cereus colony count from day 0 to day 7 in infant milk stored at 15
temperature range of 4 middotC to 7 c
Table 5 B cereus colony count from day 0 to day 7 in UHT milk stored at 16
temperature range of 10 middotC to 13 middotC
Table 6 B cereus colony count from day 0 to day 7 in infant milk stored at 17
temperature range of 10 middotC to 13 middotC
Table 7 B cereus colony count from day 0 to day 7 in UHT milk stored at 25degC 18
Table 8 B cereus colony count from day 0 to day 7 in infant milk stored at 25 19
DC
Table 9 B cereus colony count from day 0 to day 7 in UHT milk stored at 35degC 20
Table 10 B cereus colony count from day 0 to day 7 in infant milk stored at 35 20
DC
Table 11 B cereus colony count from day 0 to day 7 in UHT milk stored at 45degC 22
Table 12 B cereus colony count from day 0 to day 7 in infant milk stored at 45 22
DC
Table 13 B cereus colony count from day 0 to day 7 in UHT milk stored at 55degC 24
VII
Table 14 B cereus colony count from day 0 to day 7 in infant milk stored at 55 24
0c
Table 15 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 29
ofpH 620 stored at refrigerated temperature
Table 16 B cereus colony count from day 0 to day 7 in infant milk with initial pH 30
of pH 620 stored at refrigerated temperature
Table 17 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 31
of pH 640 stored at refrigerated temperature
Table 18 B cereus colony count from day 0 to day 7 in infant milk with initial pH 32
of pH 640 stored at refrigerated temperature
Table 19 B cereus colony count from day 0 to day 7 in UHT milk with initia l pH 33
of pH 660 stored at refrigerated temperature
Table 20 B cereus colony count from d-ay 0 to day 7 in infant milk with initial pH 34
of pH 660 stored at refrigerated temperature
Table 21 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 35
of pH 680 stored at refrigerated temperature
Table 22 B cereus colony count from day 0 to day 7 in infant milk with initial pH 36
of pH 680 stored at refrigerated temperature
VIII
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
List of Figures
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 4 middotC to 7 c
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 10 middotC to 13 middotC
15
17
The survival curve of B cereus
infant milk stored at 25 c
The survival curve of B cereus
infant milk stored at 35 c
The survival curve of B cereus
infant milk stored at 45 c
The survival curve of B cereus
infant milk stored at 55 middotC
in artificially contaminated UHT and 19
in artificially contaminated UHT and 21
in artificially contaminated UHT and 23
in artificially contaminated UHT and 25
The survival curve of B cereus in aiiifIcially contaminated UHT milk 27
stored at 4 middotC -7 middotc 10 middotC-13 middotC 25 middotc 35 middotC 45 middotC and 55 middotC
The survival curve of B cereus in artificially contaminated infant milk 28
stored at 4 middotC -7 middotC 10 middotC-13 middotC 25 middotC 35 middotC 45 middotC and 55 c
The survival curve of B cereus in artifIcially contaminated UHT and 30
infant milk with initial pH of pH 620 stored at refrigerated temperature
(4 QC-7 QC)
IX
Figure 10 The survival curve of B cereus in artificially contaminated UHT and 32
infant milk with initial pH of pH 640 stored at refrigerated temperature
(4 middotC-7 C)
Figure 11 The survival curve of B cereus in artificially contaminated UHT and 34
infant milk with initial pH of pH 660 stored at refrigerated temperature
(4 middotC-7 C)
Figure 12 The survival curve of B cereus in artificially contaminated UHT and 36
infant milk with initial pH of pH 680 stored at refrigerated temperature
(4 middotC-7 C)
Figure 13 The survival curve of B cereus in artificially contaminated UHT milk 38
with different initial pH stored at refrigerated temperature (4 middotC-7 C)
Figure 14 The survival curve of B cereus in artificially contaminated infant milk 39
with different initial pH stored- at refrigerated temperature (4 middotC-7 middotC)
Figure 15 B cereus colonies grown on MYP Agar 40
Figure 16 Morphology of B cereus isolated from UHT milk and infant milk 40
samples
Figure 17 Amplification of the gyrB gene (475 bp) for the confirmation of B 41
cereus in UHT milk and infant milk samples
x
Survival ofBacillus cereus in Artificially Contaminated UHT Milk and Infant Milk
Kong Wan Wei
Resource Bioteclmology Department of Molecular Biology
Faculty of Resource Science and Teclmology Universiti Malaysia Sarawak
Abstract
The inappropriate storage temperature and pH enable the survival of Bacillus cereus a known food pathogen in processed milk products resulting in foodborne diseases In the present study UHT milk and infant milk purchased from local supermarkets in Kuching Sarawak were inoculated with 103 CFUml of B cereus Selected storage temperatures of 4 C-7 C 10 C-13 C 25 C 35 C 45 C and 55 C were used to investigate the effects of temperature on the survival of B cereus Initial pH of 620640 660 and 680 at refrigerated temperature (4 C-7 C) were used to study the effects of pH on the survival of B cereus Enumeration of colony counts were investigated daily for the duration of seven days Gram staining and Polymerase Chain Reaction (PCR) targeting gyrB gene were performed for the confirmation of B cereus isolates The present findings revealed that the survival of B cereus in UHT milk and infant mil k showed significant differences (pltO05) in different temperatures and pH At 25 C there were significant difference (plt005) between the survival of B cereus in UHT milk and infant milk B cereus were able to survive under all the experimental temperatures However growth was detected at all storage temperatures except 55 c Survival of B cereus in artificially contaminated UHT milk and infant milk with different initial pH stored at referigerated temperature were also recorded The results from this study can be treated as background data for the implementation of safety measure to avoid food poisoning caused by B cere liS
Key words Bacillus cereus survival milk temperature pH
Abstrak
Suhll penyimpanan dan pH yang tidak sesllai membolehkall kehidupan Bacillus cereus sejenis pathogen makanan dalam produk susu diproses dan mellyebabkan penyakit bawaan makanall Dalam kajian ini SIISII UHT dan SIISU bayi yang dibeli daripada pasar raya tempatan di Kuching Sarawak telah diinoklliasi dengan B cerells sebanyak J(i CFUlml Suhll penyimpanan 4 C-7 c 10 C-3 c 25 c 35 middotc 45 c dan 55 c telah digllnakan IIntllk mengkaji kesan slIhll terhadap kehidupan B cerells Pada suhll peti sejuk (4 C-7 C) pH 620 pH 640 pH 660 dan pH 680 telah digunakan sebagai pH permulaal IIntuk mengkaji kesan pH terhadap keflidupan B cereus Penggiraall kololli telah dijalankan setiap hari selama IIIjuh hari Pewarnaan Gram dan penggllnaan reaksi rantai polimerase (PCR) yang menyasarkan gen gyrB telah mengesahkan kewujlldan koloni B cereus Kajian ini menllnjukkan buhawa kehidupan B cereus dalam SIISU UHT dan susu bayi menunjukkan perbezaan yang ketara (plt005) di suhu penyimpanan dan pH yang berbeza Perbezaan yang ketara (plt005) berlaku anlara kehidupan B cereus dalam susu UHT dengan kehidllpan B cereus dalam SIISII bayi pada sllhu 25 c B cereus dapal menunjllkkan kehidupan dalam SIISU UHT dan SIISU bayi yang telah disimpan pada semua suhll eksperimen Walau bagaimanapun pertumbllhan dapat dikesan pada semua sllhll eksperimen keclali 55 c Selain itll kehidllpan B cerells dalam SUSII UHT dan SUSII bayi yang tercemar secara bllalan pada slIhll peli sejuk telah dicatat pada peJbagai pH Hasil daripadu kajian ini boleh dianggap sebagai dala lalar belakallg tllltllk pelaksanuan langkah keselamatan bagi mengelakkan keracunall makanall daripada B cereus
Kata kunci Bacillus cerellS keflidllpan SIISU sllltl pH
XI
10 Introduction
Population growth and increase in consciousness about health are some of the factors that
increase demand for milk and milk products in Malaysia In Malaysian market ultra-high
temperature (UHT) milk and infant milk powders come under a variety of brands and are
either locally produced or imported Milk provides calcium protein vitamin A vitamin B 12
riboflavin and zinc to the human diet Consumption of milk helps to build and maintain bone
health as well as to reduce the risk of osteoporosis Infant milk provides major nutrients to
infants and it is essential for the growth of newborns
Milk is a high moisture and high nutritious medium which is also suitable for the
growth of mircoorganisms Most of the people believed that processed milk samples are
totally safe for consumption but actually there are many ways that leads to milk
contamination Contamination happened duripg milk processing as Bacillus cereus (B cereus)
strains which were not found in raw milk samples was present in the processed milk samples
(Banyk6 and Vyletelova 2009) Microorganisms contaminate the milk and affects the milk
quality by infecting the mammary gland udder and teat surfaces milking equipment and
storage tank (Gleeson et al 2013) Moreover improper processing and the usage of raw milk
that did not meet the microbiology and physio-chemical standards lead to the defects in raw
and UHT milk (Abdul-Hadi et al 2014) Inappropriate food storage and handling of
contaminated food encourage the growth of B cereus to the levels which can cause food
poisoning (Granum 2005) In order to maintain the quality and safety of processed milk
products consumers are suggested to store these products at temperature of 4 middotC or [less (Boor
and Fromm 2006)
Food pOlsonmg which may even have lethal effect on human is due to the
inappropriate food preparation and food exposure to 30 middotC (Sandra et at 2012) From year
1998 until 2008 there were 235 cases of foodbome disease outbreaks associated with B
cereus in United States (Bennett et at 2013) In 2011 the number of B cereus foodborne
diseases in Europe showed an increase of 1222 (MesseJhausser et ai 2014) The number
reported shows that B cereus poses a threat to the public health as it is often found in
contaminated food However the number of B cereus foodbome poisoning is believed to be
under reported because this type of food poisoning is mild and reporting these cases is not an
compulsory act (EFSA 2005)
B cereus was recognized as foodbome pathogen since 1955 and it is the cause for
about 2 of all foodbome disease worldwide (Lesley et at 2013) Garcia-Arribas and Kramer
(1990) mentioned that B cereus contribute to economic loss by causing contamination
spoilage and food-poisoning B cereus are found in dairy products meats spices and cereals
(Browne and Dowds 2002) as well as in infant foods and dried milk products (Becker et aI
1994)
Most of the patients involved in B cereus outbreaks were infants or elders (Becker et
ai 1994) The immune system of infants are still developing so they are highly susceptible to
B cereus food poisoning after consuming contaminated infant milk Although infant milk
basically present in low moisture powder form the vegetative cells and spores of
microorganisms are able to survive after drying but with their metabolism reduced (I LSI
Europe 2011) This poses threat to infants because some consumers keep the reconstituted
infant formula at room temperature for a long period of time as they wrongly believed that
infant formula are sterilised and are completely free of B cereus (ILSI Europe 2011)
2
B cereus is conunonly related to dairy products because they are able to survive at
different environmental stresses such as pasteurization (Foltys and Kirchnerova 2006 Sadek
et (II 2006) Schelderman et al (2006) discussed that aerobic spore former such as B cereus
has increased tolerance towards low temperature low pH sterilization and UHT treatments
and these conditions increase the concern in food industry In year 2011 there were food
poisoning associated with UHT milk provided from 1 Malaysia Milk Progranune and B cereus
were detected in these UHT milk (Yusof 2011) In 2013 infant milk formula product from
Fonterra were tested positive for Clostridium botulinum which is known as an endospore
former (Frediansyah 2014) The botulism scare was later confirmed as false alarm and the
actual organism was Clostridium 5porogenes However these pathogens have alarmed public
on the safety ofUHT milk and infant milk Currently there are no known studies in Malaysia
examining the factors influencing the survival of B cereus in UHT milk and infant milk
Hence the objectives of this study were
I To study the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk stored at different temperature for seven days
II To examine the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk with different initial pH stored at refrigerated temperature for seven
days
3
20 Literature Review
21 Bacillus cereus
211 Morphological and biochemical characteristics
Bacillus cereus is a Gram positive motile rod-shaped aerobic or facultative anaerobe and
aerobic spore forming foodborne pathogen belonging to Bacillus genus that is normally found
in soil and vegetation (Tortora et al 2010 Food Standards Australia New Zealand 2013
Gleeson et al 2013) This bacterium is mesophile or psychrotroph This bacterium is closely
related to B thuringiensis B mycoides B weihenstephanensis B pseudomycoides and B
anthracis (Gleeson et al 2013) The ability of B cereus to produce endospores which are heat
resistant increases the survivality of B cereus at unfavourable conditions such as high
temperatures extreme pH values or envirorunents with insufficient nutrients compared to
other bacterial enteropathogens (Di Pinto et al 2013 Gleeson et al 2013) For the
biochemical characteristics B cereus are positive for motility Voges-Proskauer (VP)
reaction starch hydrolysis citrate catalase and negative for oxidase tests (Shinagawa 1990
Choma ef al 2000) B cereus is shown as slender bacilli with square end singly or in short
chains under Gram-staining (Bottone 2010)
212 B cereus food borne disease
Rice dishes and meat or poultry dishes were the common sources for B cereus outbreaks
(Bennett et al 2013) Rice and pasta contaminated with B cereus are the causes for emetic
syndrome while the causes for diarrheal illness are desserts meat dishes and dairy products
(Granum 2005) Endospores of B cereus were able to survive in improperly cooked rice and
they caused Fried Rice Syndrome (Sandra et al 2012) In Malaysia B cereus was fIrst
4
Pusat Khidmat Maklumat Ak d Ut lVERSTTJ MA AYSIA SARAWAK
suspected as the cause for food poisoning which affected 114 female students in Klang
(Rampal et al 1984) In order to confIrm that gastroenteritis is caused by B cereus the
number of B cereus iso lated from suspected food need to exceed 105 co lony- forming unit per
gram (CFUg) (Tortora et al 20[0)
213 Isolation and identification of B cereus
Mannitol egg yolk polymyxin agar (MYF) Kim-Geopfert agar Columbia base-blood
polymyxin B agar (CBA-P) and Polymyxin B-egg yolk-mannitol bromothymol blue agar
(PEMBA) are some of the selective media suitable for isoIation and identifIcation of B cereus
(van Netten and Kramer 1992) The inability to ferment mannitol causes B cereus colonies to
appear as pink on MYP and lecithinase activity enables the observation of precipitated zone
around colonies (Pacheco-Sanchez and Massaguer 2007) Food and Drug Administration
(FDA) recommended MYP agar as the stapdard media for identifIcation of B cereus
214 Production of enterotoxins
The two forms of illness associated with B cereus enterotoxins are diarrheal and emetic
syndromes Onset of watery diarrhea and abdominal pain which resemble Clostridium
pefringens intoxications occur 8 hours to 16 hours after ingestion of contaminated food are
the symptoms of diarrheal outbreak (Shinagawa 1990 Tortora et ai 2010) This diarrheal
syndrome occurs after the ingestion of vegetattve cells which later produce a protein
enterotoxin in the gastrointinal tract (Bottone 2010) This type of enterotoxin is heat-labile
(Bello et al 2014) Haemolysin BL (HBL) non-haemolytic enterotoxin and cytotoxin K are
the three types of enterotoxins produced by this bacterium (Samarzija et al 2012 Food
Standards Australia New Zealand 2013) Reis et al (2013) concluded that hblD was the
5
common HBL gene of B cereus detected in milk and dairy products leading to diarrheal form
ofB cereus foodborne disease
The symptoms of emetic outbreaks are similar to Staphylococcus aureus food
intoxication and occur 05 hour to 6 hours upon ingestion of contaminated food
(Messelhausser et al 2014) The ingestion of food contaminated with preformed cereulide
which is also known as the emetic toxin leads to emetic syndrome (Bottone 2010) This heatshy
stable toxin causes nausea and vomiting in the infected patients (Bello et al2014)
215 Effect of temperature and pH on growth and survival of B cereus
Generally B cereus grows between 4 C and 55 C but exhibit optimum growth between 30 C
and 40 C (Schraft and Griffiths 2006 Food Standards Australia New Zealand 2013) Growth
of B cereus had been detected at temperature ranging from 7 C to 45 C and significant
growth was observed at 45 c in steril ized raw milk samples (Bellow et al 2007) In the
absence of oxygen or at temperature below 10 C emetic B cereus are believed to show
incapability in growth and production of toxin (EFSA 2005) At temperature below 10 C
with the limitation of other factors the lag time and generation times of B cereus are longer
(EFSA 2005)
As mentioned by Food Standards Australia New Zealand (2013) B cereus is able to
grow at pH between pH 49 and pH 100 and the optimum pH is between pH 60 and pH 70
Bello et al (2014) concluded that B cereus was unable to grow at pH 63 to pH 68 for 24
hour in pasteurized whole cow milk and the growth was inhibited at pH 68 for a period of 48
hours Byaruhanga et af (1999) studied that mague base inoculated with B cereus for 24
hours at 3S C was able to grow when pH decreases from pH 65 to pH 50 Brain Heart
6
--~~~----~----~----------
Infusion (BHI) medium at pH 88 inhibits growth of emetic and diarrheal strains of B cereus
better than pH 50 of the same medium (Garcia-Arribas and Kramer 1990)
22 UHT milk
Milk treated with UHT pasteurization is heated to 138 middotC for 2 seconds and aseptically placed
into a sterile substantial container which can be stored without refrigeration for six months to
a year (Ledenbach and Marshall 2009) UHT treatment minimized heat denaturation and
produces long shelf life milk that has similar taste and color to pasteurized milk (Harding
1995) UHT milk is generally contaminated during technological processing because food
pathogens are destroyed by the UHT temperature (Vyletelova et al 2002) Longer storage
period causes the drop of UHT milk pH and enzymes may be the reason for changes in UHT
milk (Hassan et al 2009)
Among the 110 UHT milk samples tested by Vidal-Martins et al (2005) 118
(1 3110) samples were positive for B cereus Researchers Rezende-Lago et al (2007)
identified B cereus in four (13 3) of the UHT milk samples tested In the study performed
by Reis et al (2013) among the 260 samples of UHT milk pasteurized milk and powdered
milk tested in Brazil a total of 63 isolates of B cereus were found and 12 of them were from
UHT milk
23 Infant milk
Cows milk and soy protein are the major component of infant milk or infant formulas For the
production of infant formula from milk the milk is heat-treated concentrated and dried while
infant fonnula from other ingredients is dry-blended wetted and dried again or only dry-
blended (Becker et al 1994) The sublethally injured pathogens can recover after the process
7
of drying because this process caused only little damage to the cell (Miller et ai 2012) During
the process of drying components such as lactose protein and milk fat that are present in
milk-based infant formula have the potential to provide protection to the bacteria (Mi1er et ai
20 12) Infant formulas are given to newborns and the content of nutrition is similar to breast
milk (Becker et al 1994) Currently infant formulas in the market are available in powder
liquid concentration and ready-to-feed After rehydration and inappropriate storage lowshy
moisture foods such as infant milk powder should be consumed within a short time because
growth of microorganisms may occur (lLSI Europe 2011)
Becker et al (1994) tested 92 infant milk formula samples and 52 of the samples
were B cereus positive Another study by Rowan et al (1997) tested 100 samples of infant
milk formula from United Kingdom and 17 of the samples were B cereus positive In the
study done by Haughton et al (20 I 0) B cereus were detected in 59 out of 100 infant milk
formula samples from Ireland These studies showed that B cereus is commonly found in
most of the infant milk formula and this poses threat to infants who are highly susceptible
24 Polymerase Chain Reaction (PCR)
As ment ioned by Sandra et al (2012) PCR allows rapid detection of pathogens Schraft and
Griffiths (2006) mentioned that the detection of B cereus targetting B cereus per se and
specific sub-groups have been performed by PCR Yamada et al (1999) designed PCR primer
set for gyrB fragment of B cereus and used this primer set in the detection of B cereus in
boiled rice Confirmation of B cereus in typical colonies are performed using PCR techniques
(Rahimi el al 2013) Thousands to millions copies of specific DNA sequence are produced
from the amplification of a single or a few copies of a piece of DNA by PCR (Joshi and
8
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
List of Abbreviations
ANOVA Analysis of Variance
B cereus Bacillus cereus
BHI Brain Heart Infusion
CBA-P Columbia base-blood polymyxin B agar
CFU colony-forming unit
FDA Food and Drug Administration
gyrB gyrase B
HCl Hydrogen chloride
MYP Mannitol Egg Yolk agar
microlitre
ml millilitre
NaOH Sodium hydroxide
PEMBA Polymyxin B-egg yolk-mannitol bromothymol blue agar
PCR Polymerase Chain Reaction
UHT ultra-high temperature
VP Voges-Proskauer
VI
List of Tables
Table 1 Mixture volumes ofPCR for detection of B cereus 13
Table 2 Thermal cycling profile ofPCR for detection ofB cereus 13
Table 3 B cereus colony count from day 0 to day 7 in UHT milk stored at 14
temperature range of4 middotC to 7 middotC
Table 4 B cereus colony count from day 0 to day 7 in infant milk stored at 15
temperature range of 4 middotC to 7 c
Table 5 B cereus colony count from day 0 to day 7 in UHT milk stored at 16
temperature range of 10 middotC to 13 middotC
Table 6 B cereus colony count from day 0 to day 7 in infant milk stored at 17
temperature range of 10 middotC to 13 middotC
Table 7 B cereus colony count from day 0 to day 7 in UHT milk stored at 25degC 18
Table 8 B cereus colony count from day 0 to day 7 in infant milk stored at 25 19
DC
Table 9 B cereus colony count from day 0 to day 7 in UHT milk stored at 35degC 20
Table 10 B cereus colony count from day 0 to day 7 in infant milk stored at 35 20
DC
Table 11 B cereus colony count from day 0 to day 7 in UHT milk stored at 45degC 22
Table 12 B cereus colony count from day 0 to day 7 in infant milk stored at 45 22
DC
Table 13 B cereus colony count from day 0 to day 7 in UHT milk stored at 55degC 24
VII
Table 14 B cereus colony count from day 0 to day 7 in infant milk stored at 55 24
0c
Table 15 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 29
ofpH 620 stored at refrigerated temperature
Table 16 B cereus colony count from day 0 to day 7 in infant milk with initial pH 30
of pH 620 stored at refrigerated temperature
Table 17 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 31
of pH 640 stored at refrigerated temperature
Table 18 B cereus colony count from day 0 to day 7 in infant milk with initial pH 32
of pH 640 stored at refrigerated temperature
Table 19 B cereus colony count from day 0 to day 7 in UHT milk with initia l pH 33
of pH 660 stored at refrigerated temperature
Table 20 B cereus colony count from d-ay 0 to day 7 in infant milk with initial pH 34
of pH 660 stored at refrigerated temperature
Table 21 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 35
of pH 680 stored at refrigerated temperature
Table 22 B cereus colony count from day 0 to day 7 in infant milk with initial pH 36
of pH 680 stored at refrigerated temperature
VIII
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
List of Figures
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 4 middotC to 7 c
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 10 middotC to 13 middotC
15
17
The survival curve of B cereus
infant milk stored at 25 c
The survival curve of B cereus
infant milk stored at 35 c
The survival curve of B cereus
infant milk stored at 45 c
The survival curve of B cereus
infant milk stored at 55 middotC
in artificially contaminated UHT and 19
in artificially contaminated UHT and 21
in artificially contaminated UHT and 23
in artificially contaminated UHT and 25
The survival curve of B cereus in aiiifIcially contaminated UHT milk 27
stored at 4 middotC -7 middotc 10 middotC-13 middotC 25 middotc 35 middotC 45 middotC and 55 middotC
The survival curve of B cereus in artificially contaminated infant milk 28
stored at 4 middotC -7 middotC 10 middotC-13 middotC 25 middotC 35 middotC 45 middotC and 55 c
The survival curve of B cereus in artifIcially contaminated UHT and 30
infant milk with initial pH of pH 620 stored at refrigerated temperature
(4 QC-7 QC)
IX
Figure 10 The survival curve of B cereus in artificially contaminated UHT and 32
infant milk with initial pH of pH 640 stored at refrigerated temperature
(4 middotC-7 C)
Figure 11 The survival curve of B cereus in artificially contaminated UHT and 34
infant milk with initial pH of pH 660 stored at refrigerated temperature
(4 middotC-7 C)
Figure 12 The survival curve of B cereus in artificially contaminated UHT and 36
infant milk with initial pH of pH 680 stored at refrigerated temperature
(4 middotC-7 C)
Figure 13 The survival curve of B cereus in artificially contaminated UHT milk 38
with different initial pH stored at refrigerated temperature (4 middotC-7 C)
Figure 14 The survival curve of B cereus in artificially contaminated infant milk 39
with different initial pH stored- at refrigerated temperature (4 middotC-7 middotC)
Figure 15 B cereus colonies grown on MYP Agar 40
Figure 16 Morphology of B cereus isolated from UHT milk and infant milk 40
samples
Figure 17 Amplification of the gyrB gene (475 bp) for the confirmation of B 41
cereus in UHT milk and infant milk samples
x
Survival ofBacillus cereus in Artificially Contaminated UHT Milk and Infant Milk
Kong Wan Wei
Resource Bioteclmology Department of Molecular Biology
Faculty of Resource Science and Teclmology Universiti Malaysia Sarawak
Abstract
The inappropriate storage temperature and pH enable the survival of Bacillus cereus a known food pathogen in processed milk products resulting in foodborne diseases In the present study UHT milk and infant milk purchased from local supermarkets in Kuching Sarawak were inoculated with 103 CFUml of B cereus Selected storage temperatures of 4 C-7 C 10 C-13 C 25 C 35 C 45 C and 55 C were used to investigate the effects of temperature on the survival of B cereus Initial pH of 620640 660 and 680 at refrigerated temperature (4 C-7 C) were used to study the effects of pH on the survival of B cereus Enumeration of colony counts were investigated daily for the duration of seven days Gram staining and Polymerase Chain Reaction (PCR) targeting gyrB gene were performed for the confirmation of B cereus isolates The present findings revealed that the survival of B cereus in UHT milk and infant mil k showed significant differences (pltO05) in different temperatures and pH At 25 C there were significant difference (plt005) between the survival of B cereus in UHT milk and infant milk B cereus were able to survive under all the experimental temperatures However growth was detected at all storage temperatures except 55 c Survival of B cereus in artificially contaminated UHT milk and infant milk with different initial pH stored at referigerated temperature were also recorded The results from this study can be treated as background data for the implementation of safety measure to avoid food poisoning caused by B cere liS
Key words Bacillus cereus survival milk temperature pH
Abstrak
Suhll penyimpanan dan pH yang tidak sesllai membolehkall kehidupan Bacillus cereus sejenis pathogen makanan dalam produk susu diproses dan mellyebabkan penyakit bawaan makanall Dalam kajian ini SIISII UHT dan SIISU bayi yang dibeli daripada pasar raya tempatan di Kuching Sarawak telah diinoklliasi dengan B cerells sebanyak J(i CFUlml Suhll penyimpanan 4 C-7 c 10 C-3 c 25 c 35 middotc 45 c dan 55 c telah digllnakan IIntllk mengkaji kesan slIhll terhadap kehidupan B cerells Pada suhll peti sejuk (4 C-7 C) pH 620 pH 640 pH 660 dan pH 680 telah digunakan sebagai pH permulaal IIntuk mengkaji kesan pH terhadap keflidupan B cereus Penggiraall kololli telah dijalankan setiap hari selama IIIjuh hari Pewarnaan Gram dan penggllnaan reaksi rantai polimerase (PCR) yang menyasarkan gen gyrB telah mengesahkan kewujlldan koloni B cereus Kajian ini menllnjukkan buhawa kehidupan B cereus dalam SIISU UHT dan susu bayi menunjukkan perbezaan yang ketara (plt005) di suhu penyimpanan dan pH yang berbeza Perbezaan yang ketara (plt005) berlaku anlara kehidupan B cereus dalam susu UHT dengan kehidllpan B cereus dalam SIISII bayi pada sllhu 25 c B cereus dapal menunjllkkan kehidupan dalam SIISU UHT dan SIISU bayi yang telah disimpan pada semua suhll eksperimen Walau bagaimanapun pertumbllhan dapat dikesan pada semua sllhll eksperimen keclali 55 c Selain itll kehidllpan B cerells dalam SUSII UHT dan SUSII bayi yang tercemar secara bllalan pada slIhll peli sejuk telah dicatat pada peJbagai pH Hasil daripadu kajian ini boleh dianggap sebagai dala lalar belakallg tllltllk pelaksanuan langkah keselamatan bagi mengelakkan keracunall makanall daripada B cereus
Kata kunci Bacillus cerellS keflidllpan SIISU sllltl pH
XI
10 Introduction
Population growth and increase in consciousness about health are some of the factors that
increase demand for milk and milk products in Malaysia In Malaysian market ultra-high
temperature (UHT) milk and infant milk powders come under a variety of brands and are
either locally produced or imported Milk provides calcium protein vitamin A vitamin B 12
riboflavin and zinc to the human diet Consumption of milk helps to build and maintain bone
health as well as to reduce the risk of osteoporosis Infant milk provides major nutrients to
infants and it is essential for the growth of newborns
Milk is a high moisture and high nutritious medium which is also suitable for the
growth of mircoorganisms Most of the people believed that processed milk samples are
totally safe for consumption but actually there are many ways that leads to milk
contamination Contamination happened duripg milk processing as Bacillus cereus (B cereus)
strains which were not found in raw milk samples was present in the processed milk samples
(Banyk6 and Vyletelova 2009) Microorganisms contaminate the milk and affects the milk
quality by infecting the mammary gland udder and teat surfaces milking equipment and
storage tank (Gleeson et al 2013) Moreover improper processing and the usage of raw milk
that did not meet the microbiology and physio-chemical standards lead to the defects in raw
and UHT milk (Abdul-Hadi et al 2014) Inappropriate food storage and handling of
contaminated food encourage the growth of B cereus to the levels which can cause food
poisoning (Granum 2005) In order to maintain the quality and safety of processed milk
products consumers are suggested to store these products at temperature of 4 middotC or [less (Boor
and Fromm 2006)
Food pOlsonmg which may even have lethal effect on human is due to the
inappropriate food preparation and food exposure to 30 middotC (Sandra et at 2012) From year
1998 until 2008 there were 235 cases of foodbome disease outbreaks associated with B
cereus in United States (Bennett et at 2013) In 2011 the number of B cereus foodborne
diseases in Europe showed an increase of 1222 (MesseJhausser et ai 2014) The number
reported shows that B cereus poses a threat to the public health as it is often found in
contaminated food However the number of B cereus foodbome poisoning is believed to be
under reported because this type of food poisoning is mild and reporting these cases is not an
compulsory act (EFSA 2005)
B cereus was recognized as foodbome pathogen since 1955 and it is the cause for
about 2 of all foodbome disease worldwide (Lesley et at 2013) Garcia-Arribas and Kramer
(1990) mentioned that B cereus contribute to economic loss by causing contamination
spoilage and food-poisoning B cereus are found in dairy products meats spices and cereals
(Browne and Dowds 2002) as well as in infant foods and dried milk products (Becker et aI
1994)
Most of the patients involved in B cereus outbreaks were infants or elders (Becker et
ai 1994) The immune system of infants are still developing so they are highly susceptible to
B cereus food poisoning after consuming contaminated infant milk Although infant milk
basically present in low moisture powder form the vegetative cells and spores of
microorganisms are able to survive after drying but with their metabolism reduced (I LSI
Europe 2011) This poses threat to infants because some consumers keep the reconstituted
infant formula at room temperature for a long period of time as they wrongly believed that
infant formula are sterilised and are completely free of B cereus (ILSI Europe 2011)
2
B cereus is conunonly related to dairy products because they are able to survive at
different environmental stresses such as pasteurization (Foltys and Kirchnerova 2006 Sadek
et (II 2006) Schelderman et al (2006) discussed that aerobic spore former such as B cereus
has increased tolerance towards low temperature low pH sterilization and UHT treatments
and these conditions increase the concern in food industry In year 2011 there were food
poisoning associated with UHT milk provided from 1 Malaysia Milk Progranune and B cereus
were detected in these UHT milk (Yusof 2011) In 2013 infant milk formula product from
Fonterra were tested positive for Clostridium botulinum which is known as an endospore
former (Frediansyah 2014) The botulism scare was later confirmed as false alarm and the
actual organism was Clostridium 5porogenes However these pathogens have alarmed public
on the safety ofUHT milk and infant milk Currently there are no known studies in Malaysia
examining the factors influencing the survival of B cereus in UHT milk and infant milk
Hence the objectives of this study were
I To study the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk stored at different temperature for seven days
II To examine the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk with different initial pH stored at refrigerated temperature for seven
days
3
20 Literature Review
21 Bacillus cereus
211 Morphological and biochemical characteristics
Bacillus cereus is a Gram positive motile rod-shaped aerobic or facultative anaerobe and
aerobic spore forming foodborne pathogen belonging to Bacillus genus that is normally found
in soil and vegetation (Tortora et al 2010 Food Standards Australia New Zealand 2013
Gleeson et al 2013) This bacterium is mesophile or psychrotroph This bacterium is closely
related to B thuringiensis B mycoides B weihenstephanensis B pseudomycoides and B
anthracis (Gleeson et al 2013) The ability of B cereus to produce endospores which are heat
resistant increases the survivality of B cereus at unfavourable conditions such as high
temperatures extreme pH values or envirorunents with insufficient nutrients compared to
other bacterial enteropathogens (Di Pinto et al 2013 Gleeson et al 2013) For the
biochemical characteristics B cereus are positive for motility Voges-Proskauer (VP)
reaction starch hydrolysis citrate catalase and negative for oxidase tests (Shinagawa 1990
Choma ef al 2000) B cereus is shown as slender bacilli with square end singly or in short
chains under Gram-staining (Bottone 2010)
212 B cereus food borne disease
Rice dishes and meat or poultry dishes were the common sources for B cereus outbreaks
(Bennett et al 2013) Rice and pasta contaminated with B cereus are the causes for emetic
syndrome while the causes for diarrheal illness are desserts meat dishes and dairy products
(Granum 2005) Endospores of B cereus were able to survive in improperly cooked rice and
they caused Fried Rice Syndrome (Sandra et al 2012) In Malaysia B cereus was fIrst
4
Pusat Khidmat Maklumat Ak d Ut lVERSTTJ MA AYSIA SARAWAK
suspected as the cause for food poisoning which affected 114 female students in Klang
(Rampal et al 1984) In order to confIrm that gastroenteritis is caused by B cereus the
number of B cereus iso lated from suspected food need to exceed 105 co lony- forming unit per
gram (CFUg) (Tortora et al 20[0)
213 Isolation and identification of B cereus
Mannitol egg yolk polymyxin agar (MYF) Kim-Geopfert agar Columbia base-blood
polymyxin B agar (CBA-P) and Polymyxin B-egg yolk-mannitol bromothymol blue agar
(PEMBA) are some of the selective media suitable for isoIation and identifIcation of B cereus
(van Netten and Kramer 1992) The inability to ferment mannitol causes B cereus colonies to
appear as pink on MYP and lecithinase activity enables the observation of precipitated zone
around colonies (Pacheco-Sanchez and Massaguer 2007) Food and Drug Administration
(FDA) recommended MYP agar as the stapdard media for identifIcation of B cereus
214 Production of enterotoxins
The two forms of illness associated with B cereus enterotoxins are diarrheal and emetic
syndromes Onset of watery diarrhea and abdominal pain which resemble Clostridium
pefringens intoxications occur 8 hours to 16 hours after ingestion of contaminated food are
the symptoms of diarrheal outbreak (Shinagawa 1990 Tortora et ai 2010) This diarrheal
syndrome occurs after the ingestion of vegetattve cells which later produce a protein
enterotoxin in the gastrointinal tract (Bottone 2010) This type of enterotoxin is heat-labile
(Bello et al 2014) Haemolysin BL (HBL) non-haemolytic enterotoxin and cytotoxin K are
the three types of enterotoxins produced by this bacterium (Samarzija et al 2012 Food
Standards Australia New Zealand 2013) Reis et al (2013) concluded that hblD was the
5
common HBL gene of B cereus detected in milk and dairy products leading to diarrheal form
ofB cereus foodborne disease
The symptoms of emetic outbreaks are similar to Staphylococcus aureus food
intoxication and occur 05 hour to 6 hours upon ingestion of contaminated food
(Messelhausser et al 2014) The ingestion of food contaminated with preformed cereulide
which is also known as the emetic toxin leads to emetic syndrome (Bottone 2010) This heatshy
stable toxin causes nausea and vomiting in the infected patients (Bello et al2014)
215 Effect of temperature and pH on growth and survival of B cereus
Generally B cereus grows between 4 C and 55 C but exhibit optimum growth between 30 C
and 40 C (Schraft and Griffiths 2006 Food Standards Australia New Zealand 2013) Growth
of B cereus had been detected at temperature ranging from 7 C to 45 C and significant
growth was observed at 45 c in steril ized raw milk samples (Bellow et al 2007) In the
absence of oxygen or at temperature below 10 C emetic B cereus are believed to show
incapability in growth and production of toxin (EFSA 2005) At temperature below 10 C
with the limitation of other factors the lag time and generation times of B cereus are longer
(EFSA 2005)
As mentioned by Food Standards Australia New Zealand (2013) B cereus is able to
grow at pH between pH 49 and pH 100 and the optimum pH is between pH 60 and pH 70
Bello et al (2014) concluded that B cereus was unable to grow at pH 63 to pH 68 for 24
hour in pasteurized whole cow milk and the growth was inhibited at pH 68 for a period of 48
hours Byaruhanga et af (1999) studied that mague base inoculated with B cereus for 24
hours at 3S C was able to grow when pH decreases from pH 65 to pH 50 Brain Heart
6
--~~~----~----~----------
Infusion (BHI) medium at pH 88 inhibits growth of emetic and diarrheal strains of B cereus
better than pH 50 of the same medium (Garcia-Arribas and Kramer 1990)
22 UHT milk
Milk treated with UHT pasteurization is heated to 138 middotC for 2 seconds and aseptically placed
into a sterile substantial container which can be stored without refrigeration for six months to
a year (Ledenbach and Marshall 2009) UHT treatment minimized heat denaturation and
produces long shelf life milk that has similar taste and color to pasteurized milk (Harding
1995) UHT milk is generally contaminated during technological processing because food
pathogens are destroyed by the UHT temperature (Vyletelova et al 2002) Longer storage
period causes the drop of UHT milk pH and enzymes may be the reason for changes in UHT
milk (Hassan et al 2009)
Among the 110 UHT milk samples tested by Vidal-Martins et al (2005) 118
(1 3110) samples were positive for B cereus Researchers Rezende-Lago et al (2007)
identified B cereus in four (13 3) of the UHT milk samples tested In the study performed
by Reis et al (2013) among the 260 samples of UHT milk pasteurized milk and powdered
milk tested in Brazil a total of 63 isolates of B cereus were found and 12 of them were from
UHT milk
23 Infant milk
Cows milk and soy protein are the major component of infant milk or infant formulas For the
production of infant formula from milk the milk is heat-treated concentrated and dried while
infant fonnula from other ingredients is dry-blended wetted and dried again or only dry-
blended (Becker et al 1994) The sublethally injured pathogens can recover after the process
7
of drying because this process caused only little damage to the cell (Miller et ai 2012) During
the process of drying components such as lactose protein and milk fat that are present in
milk-based infant formula have the potential to provide protection to the bacteria (Mi1er et ai
20 12) Infant formulas are given to newborns and the content of nutrition is similar to breast
milk (Becker et al 1994) Currently infant formulas in the market are available in powder
liquid concentration and ready-to-feed After rehydration and inappropriate storage lowshy
moisture foods such as infant milk powder should be consumed within a short time because
growth of microorganisms may occur (lLSI Europe 2011)
Becker et al (1994) tested 92 infant milk formula samples and 52 of the samples
were B cereus positive Another study by Rowan et al (1997) tested 100 samples of infant
milk formula from United Kingdom and 17 of the samples were B cereus positive In the
study done by Haughton et al (20 I 0) B cereus were detected in 59 out of 100 infant milk
formula samples from Ireland These studies showed that B cereus is commonly found in
most of the infant milk formula and this poses threat to infants who are highly susceptible
24 Polymerase Chain Reaction (PCR)
As ment ioned by Sandra et al (2012) PCR allows rapid detection of pathogens Schraft and
Griffiths (2006) mentioned that the detection of B cereus targetting B cereus per se and
specific sub-groups have been performed by PCR Yamada et al (1999) designed PCR primer
set for gyrB fragment of B cereus and used this primer set in the detection of B cereus in
boiled rice Confirmation of B cereus in typical colonies are performed using PCR techniques
(Rahimi el al 2013) Thousands to millions copies of specific DNA sequence are produced
from the amplification of a single or a few copies of a piece of DNA by PCR (Joshi and
8
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
List of Tables
Table 1 Mixture volumes ofPCR for detection of B cereus 13
Table 2 Thermal cycling profile ofPCR for detection ofB cereus 13
Table 3 B cereus colony count from day 0 to day 7 in UHT milk stored at 14
temperature range of4 middotC to 7 middotC
Table 4 B cereus colony count from day 0 to day 7 in infant milk stored at 15
temperature range of 4 middotC to 7 c
Table 5 B cereus colony count from day 0 to day 7 in UHT milk stored at 16
temperature range of 10 middotC to 13 middotC
Table 6 B cereus colony count from day 0 to day 7 in infant milk stored at 17
temperature range of 10 middotC to 13 middotC
Table 7 B cereus colony count from day 0 to day 7 in UHT milk stored at 25degC 18
Table 8 B cereus colony count from day 0 to day 7 in infant milk stored at 25 19
DC
Table 9 B cereus colony count from day 0 to day 7 in UHT milk stored at 35degC 20
Table 10 B cereus colony count from day 0 to day 7 in infant milk stored at 35 20
DC
Table 11 B cereus colony count from day 0 to day 7 in UHT milk stored at 45degC 22
Table 12 B cereus colony count from day 0 to day 7 in infant milk stored at 45 22
DC
Table 13 B cereus colony count from day 0 to day 7 in UHT milk stored at 55degC 24
VII
Table 14 B cereus colony count from day 0 to day 7 in infant milk stored at 55 24
0c
Table 15 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 29
ofpH 620 stored at refrigerated temperature
Table 16 B cereus colony count from day 0 to day 7 in infant milk with initial pH 30
of pH 620 stored at refrigerated temperature
Table 17 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 31
of pH 640 stored at refrigerated temperature
Table 18 B cereus colony count from day 0 to day 7 in infant milk with initial pH 32
of pH 640 stored at refrigerated temperature
Table 19 B cereus colony count from day 0 to day 7 in UHT milk with initia l pH 33
of pH 660 stored at refrigerated temperature
Table 20 B cereus colony count from d-ay 0 to day 7 in infant milk with initial pH 34
of pH 660 stored at refrigerated temperature
Table 21 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 35
of pH 680 stored at refrigerated temperature
Table 22 B cereus colony count from day 0 to day 7 in infant milk with initial pH 36
of pH 680 stored at refrigerated temperature
VIII
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
List of Figures
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 4 middotC to 7 c
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 10 middotC to 13 middotC
15
17
The survival curve of B cereus
infant milk stored at 25 c
The survival curve of B cereus
infant milk stored at 35 c
The survival curve of B cereus
infant milk stored at 45 c
The survival curve of B cereus
infant milk stored at 55 middotC
in artificially contaminated UHT and 19
in artificially contaminated UHT and 21
in artificially contaminated UHT and 23
in artificially contaminated UHT and 25
The survival curve of B cereus in aiiifIcially contaminated UHT milk 27
stored at 4 middotC -7 middotc 10 middotC-13 middotC 25 middotc 35 middotC 45 middotC and 55 middotC
The survival curve of B cereus in artificially contaminated infant milk 28
stored at 4 middotC -7 middotC 10 middotC-13 middotC 25 middotC 35 middotC 45 middotC and 55 c
The survival curve of B cereus in artifIcially contaminated UHT and 30
infant milk with initial pH of pH 620 stored at refrigerated temperature
(4 QC-7 QC)
IX
Figure 10 The survival curve of B cereus in artificially contaminated UHT and 32
infant milk with initial pH of pH 640 stored at refrigerated temperature
(4 middotC-7 C)
Figure 11 The survival curve of B cereus in artificially contaminated UHT and 34
infant milk with initial pH of pH 660 stored at refrigerated temperature
(4 middotC-7 C)
Figure 12 The survival curve of B cereus in artificially contaminated UHT and 36
infant milk with initial pH of pH 680 stored at refrigerated temperature
(4 middotC-7 C)
Figure 13 The survival curve of B cereus in artificially contaminated UHT milk 38
with different initial pH stored at refrigerated temperature (4 middotC-7 C)
Figure 14 The survival curve of B cereus in artificially contaminated infant milk 39
with different initial pH stored- at refrigerated temperature (4 middotC-7 middotC)
Figure 15 B cereus colonies grown on MYP Agar 40
Figure 16 Morphology of B cereus isolated from UHT milk and infant milk 40
samples
Figure 17 Amplification of the gyrB gene (475 bp) for the confirmation of B 41
cereus in UHT milk and infant milk samples
x
Survival ofBacillus cereus in Artificially Contaminated UHT Milk and Infant Milk
Kong Wan Wei
Resource Bioteclmology Department of Molecular Biology
Faculty of Resource Science and Teclmology Universiti Malaysia Sarawak
Abstract
The inappropriate storage temperature and pH enable the survival of Bacillus cereus a known food pathogen in processed milk products resulting in foodborne diseases In the present study UHT milk and infant milk purchased from local supermarkets in Kuching Sarawak were inoculated with 103 CFUml of B cereus Selected storage temperatures of 4 C-7 C 10 C-13 C 25 C 35 C 45 C and 55 C were used to investigate the effects of temperature on the survival of B cereus Initial pH of 620640 660 and 680 at refrigerated temperature (4 C-7 C) were used to study the effects of pH on the survival of B cereus Enumeration of colony counts were investigated daily for the duration of seven days Gram staining and Polymerase Chain Reaction (PCR) targeting gyrB gene were performed for the confirmation of B cereus isolates The present findings revealed that the survival of B cereus in UHT milk and infant mil k showed significant differences (pltO05) in different temperatures and pH At 25 C there were significant difference (plt005) between the survival of B cereus in UHT milk and infant milk B cereus were able to survive under all the experimental temperatures However growth was detected at all storage temperatures except 55 c Survival of B cereus in artificially contaminated UHT milk and infant milk with different initial pH stored at referigerated temperature were also recorded The results from this study can be treated as background data for the implementation of safety measure to avoid food poisoning caused by B cere liS
Key words Bacillus cereus survival milk temperature pH
Abstrak
Suhll penyimpanan dan pH yang tidak sesllai membolehkall kehidupan Bacillus cereus sejenis pathogen makanan dalam produk susu diproses dan mellyebabkan penyakit bawaan makanall Dalam kajian ini SIISII UHT dan SIISU bayi yang dibeli daripada pasar raya tempatan di Kuching Sarawak telah diinoklliasi dengan B cerells sebanyak J(i CFUlml Suhll penyimpanan 4 C-7 c 10 C-3 c 25 c 35 middotc 45 c dan 55 c telah digllnakan IIntllk mengkaji kesan slIhll terhadap kehidupan B cerells Pada suhll peti sejuk (4 C-7 C) pH 620 pH 640 pH 660 dan pH 680 telah digunakan sebagai pH permulaal IIntuk mengkaji kesan pH terhadap keflidupan B cereus Penggiraall kololli telah dijalankan setiap hari selama IIIjuh hari Pewarnaan Gram dan penggllnaan reaksi rantai polimerase (PCR) yang menyasarkan gen gyrB telah mengesahkan kewujlldan koloni B cereus Kajian ini menllnjukkan buhawa kehidupan B cereus dalam SIISU UHT dan susu bayi menunjukkan perbezaan yang ketara (plt005) di suhu penyimpanan dan pH yang berbeza Perbezaan yang ketara (plt005) berlaku anlara kehidupan B cereus dalam susu UHT dengan kehidllpan B cereus dalam SIISII bayi pada sllhu 25 c B cereus dapal menunjllkkan kehidupan dalam SIISU UHT dan SIISU bayi yang telah disimpan pada semua suhll eksperimen Walau bagaimanapun pertumbllhan dapat dikesan pada semua sllhll eksperimen keclali 55 c Selain itll kehidllpan B cerells dalam SUSII UHT dan SUSII bayi yang tercemar secara bllalan pada slIhll peli sejuk telah dicatat pada peJbagai pH Hasil daripadu kajian ini boleh dianggap sebagai dala lalar belakallg tllltllk pelaksanuan langkah keselamatan bagi mengelakkan keracunall makanall daripada B cereus
Kata kunci Bacillus cerellS keflidllpan SIISU sllltl pH
XI
10 Introduction
Population growth and increase in consciousness about health are some of the factors that
increase demand for milk and milk products in Malaysia In Malaysian market ultra-high
temperature (UHT) milk and infant milk powders come under a variety of brands and are
either locally produced or imported Milk provides calcium protein vitamin A vitamin B 12
riboflavin and zinc to the human diet Consumption of milk helps to build and maintain bone
health as well as to reduce the risk of osteoporosis Infant milk provides major nutrients to
infants and it is essential for the growth of newborns
Milk is a high moisture and high nutritious medium which is also suitable for the
growth of mircoorganisms Most of the people believed that processed milk samples are
totally safe for consumption but actually there are many ways that leads to milk
contamination Contamination happened duripg milk processing as Bacillus cereus (B cereus)
strains which were not found in raw milk samples was present in the processed milk samples
(Banyk6 and Vyletelova 2009) Microorganisms contaminate the milk and affects the milk
quality by infecting the mammary gland udder and teat surfaces milking equipment and
storage tank (Gleeson et al 2013) Moreover improper processing and the usage of raw milk
that did not meet the microbiology and physio-chemical standards lead to the defects in raw
and UHT milk (Abdul-Hadi et al 2014) Inappropriate food storage and handling of
contaminated food encourage the growth of B cereus to the levels which can cause food
poisoning (Granum 2005) In order to maintain the quality and safety of processed milk
products consumers are suggested to store these products at temperature of 4 middotC or [less (Boor
and Fromm 2006)
Food pOlsonmg which may even have lethal effect on human is due to the
inappropriate food preparation and food exposure to 30 middotC (Sandra et at 2012) From year
1998 until 2008 there were 235 cases of foodbome disease outbreaks associated with B
cereus in United States (Bennett et at 2013) In 2011 the number of B cereus foodborne
diseases in Europe showed an increase of 1222 (MesseJhausser et ai 2014) The number
reported shows that B cereus poses a threat to the public health as it is often found in
contaminated food However the number of B cereus foodbome poisoning is believed to be
under reported because this type of food poisoning is mild and reporting these cases is not an
compulsory act (EFSA 2005)
B cereus was recognized as foodbome pathogen since 1955 and it is the cause for
about 2 of all foodbome disease worldwide (Lesley et at 2013) Garcia-Arribas and Kramer
(1990) mentioned that B cereus contribute to economic loss by causing contamination
spoilage and food-poisoning B cereus are found in dairy products meats spices and cereals
(Browne and Dowds 2002) as well as in infant foods and dried milk products (Becker et aI
1994)
Most of the patients involved in B cereus outbreaks were infants or elders (Becker et
ai 1994) The immune system of infants are still developing so they are highly susceptible to
B cereus food poisoning after consuming contaminated infant milk Although infant milk
basically present in low moisture powder form the vegetative cells and spores of
microorganisms are able to survive after drying but with their metabolism reduced (I LSI
Europe 2011) This poses threat to infants because some consumers keep the reconstituted
infant formula at room temperature for a long period of time as they wrongly believed that
infant formula are sterilised and are completely free of B cereus (ILSI Europe 2011)
2
B cereus is conunonly related to dairy products because they are able to survive at
different environmental stresses such as pasteurization (Foltys and Kirchnerova 2006 Sadek
et (II 2006) Schelderman et al (2006) discussed that aerobic spore former such as B cereus
has increased tolerance towards low temperature low pH sterilization and UHT treatments
and these conditions increase the concern in food industry In year 2011 there were food
poisoning associated with UHT milk provided from 1 Malaysia Milk Progranune and B cereus
were detected in these UHT milk (Yusof 2011) In 2013 infant milk formula product from
Fonterra were tested positive for Clostridium botulinum which is known as an endospore
former (Frediansyah 2014) The botulism scare was later confirmed as false alarm and the
actual organism was Clostridium 5porogenes However these pathogens have alarmed public
on the safety ofUHT milk and infant milk Currently there are no known studies in Malaysia
examining the factors influencing the survival of B cereus in UHT milk and infant milk
Hence the objectives of this study were
I To study the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk stored at different temperature for seven days
II To examine the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk with different initial pH stored at refrigerated temperature for seven
days
3
20 Literature Review
21 Bacillus cereus
211 Morphological and biochemical characteristics
Bacillus cereus is a Gram positive motile rod-shaped aerobic or facultative anaerobe and
aerobic spore forming foodborne pathogen belonging to Bacillus genus that is normally found
in soil and vegetation (Tortora et al 2010 Food Standards Australia New Zealand 2013
Gleeson et al 2013) This bacterium is mesophile or psychrotroph This bacterium is closely
related to B thuringiensis B mycoides B weihenstephanensis B pseudomycoides and B
anthracis (Gleeson et al 2013) The ability of B cereus to produce endospores which are heat
resistant increases the survivality of B cereus at unfavourable conditions such as high
temperatures extreme pH values or envirorunents with insufficient nutrients compared to
other bacterial enteropathogens (Di Pinto et al 2013 Gleeson et al 2013) For the
biochemical characteristics B cereus are positive for motility Voges-Proskauer (VP)
reaction starch hydrolysis citrate catalase and negative for oxidase tests (Shinagawa 1990
Choma ef al 2000) B cereus is shown as slender bacilli with square end singly or in short
chains under Gram-staining (Bottone 2010)
212 B cereus food borne disease
Rice dishes and meat or poultry dishes were the common sources for B cereus outbreaks
(Bennett et al 2013) Rice and pasta contaminated with B cereus are the causes for emetic
syndrome while the causes for diarrheal illness are desserts meat dishes and dairy products
(Granum 2005) Endospores of B cereus were able to survive in improperly cooked rice and
they caused Fried Rice Syndrome (Sandra et al 2012) In Malaysia B cereus was fIrst
4
Pusat Khidmat Maklumat Ak d Ut lVERSTTJ MA AYSIA SARAWAK
suspected as the cause for food poisoning which affected 114 female students in Klang
(Rampal et al 1984) In order to confIrm that gastroenteritis is caused by B cereus the
number of B cereus iso lated from suspected food need to exceed 105 co lony- forming unit per
gram (CFUg) (Tortora et al 20[0)
213 Isolation and identification of B cereus
Mannitol egg yolk polymyxin agar (MYF) Kim-Geopfert agar Columbia base-blood
polymyxin B agar (CBA-P) and Polymyxin B-egg yolk-mannitol bromothymol blue agar
(PEMBA) are some of the selective media suitable for isoIation and identifIcation of B cereus
(van Netten and Kramer 1992) The inability to ferment mannitol causes B cereus colonies to
appear as pink on MYP and lecithinase activity enables the observation of precipitated zone
around colonies (Pacheco-Sanchez and Massaguer 2007) Food and Drug Administration
(FDA) recommended MYP agar as the stapdard media for identifIcation of B cereus
214 Production of enterotoxins
The two forms of illness associated with B cereus enterotoxins are diarrheal and emetic
syndromes Onset of watery diarrhea and abdominal pain which resemble Clostridium
pefringens intoxications occur 8 hours to 16 hours after ingestion of contaminated food are
the symptoms of diarrheal outbreak (Shinagawa 1990 Tortora et ai 2010) This diarrheal
syndrome occurs after the ingestion of vegetattve cells which later produce a protein
enterotoxin in the gastrointinal tract (Bottone 2010) This type of enterotoxin is heat-labile
(Bello et al 2014) Haemolysin BL (HBL) non-haemolytic enterotoxin and cytotoxin K are
the three types of enterotoxins produced by this bacterium (Samarzija et al 2012 Food
Standards Australia New Zealand 2013) Reis et al (2013) concluded that hblD was the
5
common HBL gene of B cereus detected in milk and dairy products leading to diarrheal form
ofB cereus foodborne disease
The symptoms of emetic outbreaks are similar to Staphylococcus aureus food
intoxication and occur 05 hour to 6 hours upon ingestion of contaminated food
(Messelhausser et al 2014) The ingestion of food contaminated with preformed cereulide
which is also known as the emetic toxin leads to emetic syndrome (Bottone 2010) This heatshy
stable toxin causes nausea and vomiting in the infected patients (Bello et al2014)
215 Effect of temperature and pH on growth and survival of B cereus
Generally B cereus grows between 4 C and 55 C but exhibit optimum growth between 30 C
and 40 C (Schraft and Griffiths 2006 Food Standards Australia New Zealand 2013) Growth
of B cereus had been detected at temperature ranging from 7 C to 45 C and significant
growth was observed at 45 c in steril ized raw milk samples (Bellow et al 2007) In the
absence of oxygen or at temperature below 10 C emetic B cereus are believed to show
incapability in growth and production of toxin (EFSA 2005) At temperature below 10 C
with the limitation of other factors the lag time and generation times of B cereus are longer
(EFSA 2005)
As mentioned by Food Standards Australia New Zealand (2013) B cereus is able to
grow at pH between pH 49 and pH 100 and the optimum pH is between pH 60 and pH 70
Bello et al (2014) concluded that B cereus was unable to grow at pH 63 to pH 68 for 24
hour in pasteurized whole cow milk and the growth was inhibited at pH 68 for a period of 48
hours Byaruhanga et af (1999) studied that mague base inoculated with B cereus for 24
hours at 3S C was able to grow when pH decreases from pH 65 to pH 50 Brain Heart
6
--~~~----~----~----------
Infusion (BHI) medium at pH 88 inhibits growth of emetic and diarrheal strains of B cereus
better than pH 50 of the same medium (Garcia-Arribas and Kramer 1990)
22 UHT milk
Milk treated with UHT pasteurization is heated to 138 middotC for 2 seconds and aseptically placed
into a sterile substantial container which can be stored without refrigeration for six months to
a year (Ledenbach and Marshall 2009) UHT treatment minimized heat denaturation and
produces long shelf life milk that has similar taste and color to pasteurized milk (Harding
1995) UHT milk is generally contaminated during technological processing because food
pathogens are destroyed by the UHT temperature (Vyletelova et al 2002) Longer storage
period causes the drop of UHT milk pH and enzymes may be the reason for changes in UHT
milk (Hassan et al 2009)
Among the 110 UHT milk samples tested by Vidal-Martins et al (2005) 118
(1 3110) samples were positive for B cereus Researchers Rezende-Lago et al (2007)
identified B cereus in four (13 3) of the UHT milk samples tested In the study performed
by Reis et al (2013) among the 260 samples of UHT milk pasteurized milk and powdered
milk tested in Brazil a total of 63 isolates of B cereus were found and 12 of them were from
UHT milk
23 Infant milk
Cows milk and soy protein are the major component of infant milk or infant formulas For the
production of infant formula from milk the milk is heat-treated concentrated and dried while
infant fonnula from other ingredients is dry-blended wetted and dried again or only dry-
blended (Becker et al 1994) The sublethally injured pathogens can recover after the process
7
of drying because this process caused only little damage to the cell (Miller et ai 2012) During
the process of drying components such as lactose protein and milk fat that are present in
milk-based infant formula have the potential to provide protection to the bacteria (Mi1er et ai
20 12) Infant formulas are given to newborns and the content of nutrition is similar to breast
milk (Becker et al 1994) Currently infant formulas in the market are available in powder
liquid concentration and ready-to-feed After rehydration and inappropriate storage lowshy
moisture foods such as infant milk powder should be consumed within a short time because
growth of microorganisms may occur (lLSI Europe 2011)
Becker et al (1994) tested 92 infant milk formula samples and 52 of the samples
were B cereus positive Another study by Rowan et al (1997) tested 100 samples of infant
milk formula from United Kingdom and 17 of the samples were B cereus positive In the
study done by Haughton et al (20 I 0) B cereus were detected in 59 out of 100 infant milk
formula samples from Ireland These studies showed that B cereus is commonly found in
most of the infant milk formula and this poses threat to infants who are highly susceptible
24 Polymerase Chain Reaction (PCR)
As ment ioned by Sandra et al (2012) PCR allows rapid detection of pathogens Schraft and
Griffiths (2006) mentioned that the detection of B cereus targetting B cereus per se and
specific sub-groups have been performed by PCR Yamada et al (1999) designed PCR primer
set for gyrB fragment of B cereus and used this primer set in the detection of B cereus in
boiled rice Confirmation of B cereus in typical colonies are performed using PCR techniques
(Rahimi el al 2013) Thousands to millions copies of specific DNA sequence are produced
from the amplification of a single or a few copies of a piece of DNA by PCR (Joshi and
8
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
Table 14 B cereus colony count from day 0 to day 7 in infant milk stored at 55 24
0c
Table 15 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 29
ofpH 620 stored at refrigerated temperature
Table 16 B cereus colony count from day 0 to day 7 in infant milk with initial pH 30
of pH 620 stored at refrigerated temperature
Table 17 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 31
of pH 640 stored at refrigerated temperature
Table 18 B cereus colony count from day 0 to day 7 in infant milk with initial pH 32
of pH 640 stored at refrigerated temperature
Table 19 B cereus colony count from day 0 to day 7 in UHT milk with initia l pH 33
of pH 660 stored at refrigerated temperature
Table 20 B cereus colony count from d-ay 0 to day 7 in infant milk with initial pH 34
of pH 660 stored at refrigerated temperature
Table 21 B cereus colony count from day 0 to day 7 in UHT milk with initial pH 35
of pH 680 stored at refrigerated temperature
Table 22 B cereus colony count from day 0 to day 7 in infant milk with initial pH 36
of pH 680 stored at refrigerated temperature
VIII
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
List of Figures
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 4 middotC to 7 c
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 10 middotC to 13 middotC
15
17
The survival curve of B cereus
infant milk stored at 25 c
The survival curve of B cereus
infant milk stored at 35 c
The survival curve of B cereus
infant milk stored at 45 c
The survival curve of B cereus
infant milk stored at 55 middotC
in artificially contaminated UHT and 19
in artificially contaminated UHT and 21
in artificially contaminated UHT and 23
in artificially contaminated UHT and 25
The survival curve of B cereus in aiiifIcially contaminated UHT milk 27
stored at 4 middotC -7 middotc 10 middotC-13 middotC 25 middotc 35 middotC 45 middotC and 55 middotC
The survival curve of B cereus in artificially contaminated infant milk 28
stored at 4 middotC -7 middotC 10 middotC-13 middotC 25 middotC 35 middotC 45 middotC and 55 c
The survival curve of B cereus in artifIcially contaminated UHT and 30
infant milk with initial pH of pH 620 stored at refrigerated temperature
(4 QC-7 QC)
IX
Figure 10 The survival curve of B cereus in artificially contaminated UHT and 32
infant milk with initial pH of pH 640 stored at refrigerated temperature
(4 middotC-7 C)
Figure 11 The survival curve of B cereus in artificially contaminated UHT and 34
infant milk with initial pH of pH 660 stored at refrigerated temperature
(4 middotC-7 C)
Figure 12 The survival curve of B cereus in artificially contaminated UHT and 36
infant milk with initial pH of pH 680 stored at refrigerated temperature
(4 middotC-7 C)
Figure 13 The survival curve of B cereus in artificially contaminated UHT milk 38
with different initial pH stored at refrigerated temperature (4 middotC-7 C)
Figure 14 The survival curve of B cereus in artificially contaminated infant milk 39
with different initial pH stored- at refrigerated temperature (4 middotC-7 middotC)
Figure 15 B cereus colonies grown on MYP Agar 40
Figure 16 Morphology of B cereus isolated from UHT milk and infant milk 40
samples
Figure 17 Amplification of the gyrB gene (475 bp) for the confirmation of B 41
cereus in UHT milk and infant milk samples
x
Survival ofBacillus cereus in Artificially Contaminated UHT Milk and Infant Milk
Kong Wan Wei
Resource Bioteclmology Department of Molecular Biology
Faculty of Resource Science and Teclmology Universiti Malaysia Sarawak
Abstract
The inappropriate storage temperature and pH enable the survival of Bacillus cereus a known food pathogen in processed milk products resulting in foodborne diseases In the present study UHT milk and infant milk purchased from local supermarkets in Kuching Sarawak were inoculated with 103 CFUml of B cereus Selected storage temperatures of 4 C-7 C 10 C-13 C 25 C 35 C 45 C and 55 C were used to investigate the effects of temperature on the survival of B cereus Initial pH of 620640 660 and 680 at refrigerated temperature (4 C-7 C) were used to study the effects of pH on the survival of B cereus Enumeration of colony counts were investigated daily for the duration of seven days Gram staining and Polymerase Chain Reaction (PCR) targeting gyrB gene were performed for the confirmation of B cereus isolates The present findings revealed that the survival of B cereus in UHT milk and infant mil k showed significant differences (pltO05) in different temperatures and pH At 25 C there were significant difference (plt005) between the survival of B cereus in UHT milk and infant milk B cereus were able to survive under all the experimental temperatures However growth was detected at all storage temperatures except 55 c Survival of B cereus in artificially contaminated UHT milk and infant milk with different initial pH stored at referigerated temperature were also recorded The results from this study can be treated as background data for the implementation of safety measure to avoid food poisoning caused by B cere liS
Key words Bacillus cereus survival milk temperature pH
Abstrak
Suhll penyimpanan dan pH yang tidak sesllai membolehkall kehidupan Bacillus cereus sejenis pathogen makanan dalam produk susu diproses dan mellyebabkan penyakit bawaan makanall Dalam kajian ini SIISII UHT dan SIISU bayi yang dibeli daripada pasar raya tempatan di Kuching Sarawak telah diinoklliasi dengan B cerells sebanyak J(i CFUlml Suhll penyimpanan 4 C-7 c 10 C-3 c 25 c 35 middotc 45 c dan 55 c telah digllnakan IIntllk mengkaji kesan slIhll terhadap kehidupan B cerells Pada suhll peti sejuk (4 C-7 C) pH 620 pH 640 pH 660 dan pH 680 telah digunakan sebagai pH permulaal IIntuk mengkaji kesan pH terhadap keflidupan B cereus Penggiraall kololli telah dijalankan setiap hari selama IIIjuh hari Pewarnaan Gram dan penggllnaan reaksi rantai polimerase (PCR) yang menyasarkan gen gyrB telah mengesahkan kewujlldan koloni B cereus Kajian ini menllnjukkan buhawa kehidupan B cereus dalam SIISU UHT dan susu bayi menunjukkan perbezaan yang ketara (plt005) di suhu penyimpanan dan pH yang berbeza Perbezaan yang ketara (plt005) berlaku anlara kehidupan B cereus dalam susu UHT dengan kehidllpan B cereus dalam SIISII bayi pada sllhu 25 c B cereus dapal menunjllkkan kehidupan dalam SIISU UHT dan SIISU bayi yang telah disimpan pada semua suhll eksperimen Walau bagaimanapun pertumbllhan dapat dikesan pada semua sllhll eksperimen keclali 55 c Selain itll kehidllpan B cerells dalam SUSII UHT dan SUSII bayi yang tercemar secara bllalan pada slIhll peli sejuk telah dicatat pada peJbagai pH Hasil daripadu kajian ini boleh dianggap sebagai dala lalar belakallg tllltllk pelaksanuan langkah keselamatan bagi mengelakkan keracunall makanall daripada B cereus
Kata kunci Bacillus cerellS keflidllpan SIISU sllltl pH
XI
10 Introduction
Population growth and increase in consciousness about health are some of the factors that
increase demand for milk and milk products in Malaysia In Malaysian market ultra-high
temperature (UHT) milk and infant milk powders come under a variety of brands and are
either locally produced or imported Milk provides calcium protein vitamin A vitamin B 12
riboflavin and zinc to the human diet Consumption of milk helps to build and maintain bone
health as well as to reduce the risk of osteoporosis Infant milk provides major nutrients to
infants and it is essential for the growth of newborns
Milk is a high moisture and high nutritious medium which is also suitable for the
growth of mircoorganisms Most of the people believed that processed milk samples are
totally safe for consumption but actually there are many ways that leads to milk
contamination Contamination happened duripg milk processing as Bacillus cereus (B cereus)
strains which were not found in raw milk samples was present in the processed milk samples
(Banyk6 and Vyletelova 2009) Microorganisms contaminate the milk and affects the milk
quality by infecting the mammary gland udder and teat surfaces milking equipment and
storage tank (Gleeson et al 2013) Moreover improper processing and the usage of raw milk
that did not meet the microbiology and physio-chemical standards lead to the defects in raw
and UHT milk (Abdul-Hadi et al 2014) Inappropriate food storage and handling of
contaminated food encourage the growth of B cereus to the levels which can cause food
poisoning (Granum 2005) In order to maintain the quality and safety of processed milk
products consumers are suggested to store these products at temperature of 4 middotC or [less (Boor
and Fromm 2006)
Food pOlsonmg which may even have lethal effect on human is due to the
inappropriate food preparation and food exposure to 30 middotC (Sandra et at 2012) From year
1998 until 2008 there were 235 cases of foodbome disease outbreaks associated with B
cereus in United States (Bennett et at 2013) In 2011 the number of B cereus foodborne
diseases in Europe showed an increase of 1222 (MesseJhausser et ai 2014) The number
reported shows that B cereus poses a threat to the public health as it is often found in
contaminated food However the number of B cereus foodbome poisoning is believed to be
under reported because this type of food poisoning is mild and reporting these cases is not an
compulsory act (EFSA 2005)
B cereus was recognized as foodbome pathogen since 1955 and it is the cause for
about 2 of all foodbome disease worldwide (Lesley et at 2013) Garcia-Arribas and Kramer
(1990) mentioned that B cereus contribute to economic loss by causing contamination
spoilage and food-poisoning B cereus are found in dairy products meats spices and cereals
(Browne and Dowds 2002) as well as in infant foods and dried milk products (Becker et aI
1994)
Most of the patients involved in B cereus outbreaks were infants or elders (Becker et
ai 1994) The immune system of infants are still developing so they are highly susceptible to
B cereus food poisoning after consuming contaminated infant milk Although infant milk
basically present in low moisture powder form the vegetative cells and spores of
microorganisms are able to survive after drying but with their metabolism reduced (I LSI
Europe 2011) This poses threat to infants because some consumers keep the reconstituted
infant formula at room temperature for a long period of time as they wrongly believed that
infant formula are sterilised and are completely free of B cereus (ILSI Europe 2011)
2
B cereus is conunonly related to dairy products because they are able to survive at
different environmental stresses such as pasteurization (Foltys and Kirchnerova 2006 Sadek
et (II 2006) Schelderman et al (2006) discussed that aerobic spore former such as B cereus
has increased tolerance towards low temperature low pH sterilization and UHT treatments
and these conditions increase the concern in food industry In year 2011 there were food
poisoning associated with UHT milk provided from 1 Malaysia Milk Progranune and B cereus
were detected in these UHT milk (Yusof 2011) In 2013 infant milk formula product from
Fonterra were tested positive for Clostridium botulinum which is known as an endospore
former (Frediansyah 2014) The botulism scare was later confirmed as false alarm and the
actual organism was Clostridium 5porogenes However these pathogens have alarmed public
on the safety ofUHT milk and infant milk Currently there are no known studies in Malaysia
examining the factors influencing the survival of B cereus in UHT milk and infant milk
Hence the objectives of this study were
I To study the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk stored at different temperature for seven days
II To examine the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk with different initial pH stored at refrigerated temperature for seven
days
3
20 Literature Review
21 Bacillus cereus
211 Morphological and biochemical characteristics
Bacillus cereus is a Gram positive motile rod-shaped aerobic or facultative anaerobe and
aerobic spore forming foodborne pathogen belonging to Bacillus genus that is normally found
in soil and vegetation (Tortora et al 2010 Food Standards Australia New Zealand 2013
Gleeson et al 2013) This bacterium is mesophile or psychrotroph This bacterium is closely
related to B thuringiensis B mycoides B weihenstephanensis B pseudomycoides and B
anthracis (Gleeson et al 2013) The ability of B cereus to produce endospores which are heat
resistant increases the survivality of B cereus at unfavourable conditions such as high
temperatures extreme pH values or envirorunents with insufficient nutrients compared to
other bacterial enteropathogens (Di Pinto et al 2013 Gleeson et al 2013) For the
biochemical characteristics B cereus are positive for motility Voges-Proskauer (VP)
reaction starch hydrolysis citrate catalase and negative for oxidase tests (Shinagawa 1990
Choma ef al 2000) B cereus is shown as slender bacilli with square end singly or in short
chains under Gram-staining (Bottone 2010)
212 B cereus food borne disease
Rice dishes and meat or poultry dishes were the common sources for B cereus outbreaks
(Bennett et al 2013) Rice and pasta contaminated with B cereus are the causes for emetic
syndrome while the causes for diarrheal illness are desserts meat dishes and dairy products
(Granum 2005) Endospores of B cereus were able to survive in improperly cooked rice and
they caused Fried Rice Syndrome (Sandra et al 2012) In Malaysia B cereus was fIrst
4
Pusat Khidmat Maklumat Ak d Ut lVERSTTJ MA AYSIA SARAWAK
suspected as the cause for food poisoning which affected 114 female students in Klang
(Rampal et al 1984) In order to confIrm that gastroenteritis is caused by B cereus the
number of B cereus iso lated from suspected food need to exceed 105 co lony- forming unit per
gram (CFUg) (Tortora et al 20[0)
213 Isolation and identification of B cereus
Mannitol egg yolk polymyxin agar (MYF) Kim-Geopfert agar Columbia base-blood
polymyxin B agar (CBA-P) and Polymyxin B-egg yolk-mannitol bromothymol blue agar
(PEMBA) are some of the selective media suitable for isoIation and identifIcation of B cereus
(van Netten and Kramer 1992) The inability to ferment mannitol causes B cereus colonies to
appear as pink on MYP and lecithinase activity enables the observation of precipitated zone
around colonies (Pacheco-Sanchez and Massaguer 2007) Food and Drug Administration
(FDA) recommended MYP agar as the stapdard media for identifIcation of B cereus
214 Production of enterotoxins
The two forms of illness associated with B cereus enterotoxins are diarrheal and emetic
syndromes Onset of watery diarrhea and abdominal pain which resemble Clostridium
pefringens intoxications occur 8 hours to 16 hours after ingestion of contaminated food are
the symptoms of diarrheal outbreak (Shinagawa 1990 Tortora et ai 2010) This diarrheal
syndrome occurs after the ingestion of vegetattve cells which later produce a protein
enterotoxin in the gastrointinal tract (Bottone 2010) This type of enterotoxin is heat-labile
(Bello et al 2014) Haemolysin BL (HBL) non-haemolytic enterotoxin and cytotoxin K are
the three types of enterotoxins produced by this bacterium (Samarzija et al 2012 Food
Standards Australia New Zealand 2013) Reis et al (2013) concluded that hblD was the
5
common HBL gene of B cereus detected in milk and dairy products leading to diarrheal form
ofB cereus foodborne disease
The symptoms of emetic outbreaks are similar to Staphylococcus aureus food
intoxication and occur 05 hour to 6 hours upon ingestion of contaminated food
(Messelhausser et al 2014) The ingestion of food contaminated with preformed cereulide
which is also known as the emetic toxin leads to emetic syndrome (Bottone 2010) This heatshy
stable toxin causes nausea and vomiting in the infected patients (Bello et al2014)
215 Effect of temperature and pH on growth and survival of B cereus
Generally B cereus grows between 4 C and 55 C but exhibit optimum growth between 30 C
and 40 C (Schraft and Griffiths 2006 Food Standards Australia New Zealand 2013) Growth
of B cereus had been detected at temperature ranging from 7 C to 45 C and significant
growth was observed at 45 c in steril ized raw milk samples (Bellow et al 2007) In the
absence of oxygen or at temperature below 10 C emetic B cereus are believed to show
incapability in growth and production of toxin (EFSA 2005) At temperature below 10 C
with the limitation of other factors the lag time and generation times of B cereus are longer
(EFSA 2005)
As mentioned by Food Standards Australia New Zealand (2013) B cereus is able to
grow at pH between pH 49 and pH 100 and the optimum pH is between pH 60 and pH 70
Bello et al (2014) concluded that B cereus was unable to grow at pH 63 to pH 68 for 24
hour in pasteurized whole cow milk and the growth was inhibited at pH 68 for a period of 48
hours Byaruhanga et af (1999) studied that mague base inoculated with B cereus for 24
hours at 3S C was able to grow when pH decreases from pH 65 to pH 50 Brain Heart
6
--~~~----~----~----------
Infusion (BHI) medium at pH 88 inhibits growth of emetic and diarrheal strains of B cereus
better than pH 50 of the same medium (Garcia-Arribas and Kramer 1990)
22 UHT milk
Milk treated with UHT pasteurization is heated to 138 middotC for 2 seconds and aseptically placed
into a sterile substantial container which can be stored without refrigeration for six months to
a year (Ledenbach and Marshall 2009) UHT treatment minimized heat denaturation and
produces long shelf life milk that has similar taste and color to pasteurized milk (Harding
1995) UHT milk is generally contaminated during technological processing because food
pathogens are destroyed by the UHT temperature (Vyletelova et al 2002) Longer storage
period causes the drop of UHT milk pH and enzymes may be the reason for changes in UHT
milk (Hassan et al 2009)
Among the 110 UHT milk samples tested by Vidal-Martins et al (2005) 118
(1 3110) samples were positive for B cereus Researchers Rezende-Lago et al (2007)
identified B cereus in four (13 3) of the UHT milk samples tested In the study performed
by Reis et al (2013) among the 260 samples of UHT milk pasteurized milk and powdered
milk tested in Brazil a total of 63 isolates of B cereus were found and 12 of them were from
UHT milk
23 Infant milk
Cows milk and soy protein are the major component of infant milk or infant formulas For the
production of infant formula from milk the milk is heat-treated concentrated and dried while
infant fonnula from other ingredients is dry-blended wetted and dried again or only dry-
blended (Becker et al 1994) The sublethally injured pathogens can recover after the process
7
of drying because this process caused only little damage to the cell (Miller et ai 2012) During
the process of drying components such as lactose protein and milk fat that are present in
milk-based infant formula have the potential to provide protection to the bacteria (Mi1er et ai
20 12) Infant formulas are given to newborns and the content of nutrition is similar to breast
milk (Becker et al 1994) Currently infant formulas in the market are available in powder
liquid concentration and ready-to-feed After rehydration and inappropriate storage lowshy
moisture foods such as infant milk powder should be consumed within a short time because
growth of microorganisms may occur (lLSI Europe 2011)
Becker et al (1994) tested 92 infant milk formula samples and 52 of the samples
were B cereus positive Another study by Rowan et al (1997) tested 100 samples of infant
milk formula from United Kingdom and 17 of the samples were B cereus positive In the
study done by Haughton et al (20 I 0) B cereus were detected in 59 out of 100 infant milk
formula samples from Ireland These studies showed that B cereus is commonly found in
most of the infant milk formula and this poses threat to infants who are highly susceptible
24 Polymerase Chain Reaction (PCR)
As ment ioned by Sandra et al (2012) PCR allows rapid detection of pathogens Schraft and
Griffiths (2006) mentioned that the detection of B cereus targetting B cereus per se and
specific sub-groups have been performed by PCR Yamada et al (1999) designed PCR primer
set for gyrB fragment of B cereus and used this primer set in the detection of B cereus in
boiled rice Confirmation of B cereus in typical colonies are performed using PCR techniques
(Rahimi el al 2013) Thousands to millions copies of specific DNA sequence are produced
from the amplification of a single or a few copies of a piece of DNA by PCR (Joshi and
8
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
List of Figures
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 4 middotC to 7 c
The survival curve of B cereus in artificially contaminated UHT and
infant milk stored at temperature range of 10 middotC to 13 middotC
15
17
The survival curve of B cereus
infant milk stored at 25 c
The survival curve of B cereus
infant milk stored at 35 c
The survival curve of B cereus
infant milk stored at 45 c
The survival curve of B cereus
infant milk stored at 55 middotC
in artificially contaminated UHT and 19
in artificially contaminated UHT and 21
in artificially contaminated UHT and 23
in artificially contaminated UHT and 25
The survival curve of B cereus in aiiifIcially contaminated UHT milk 27
stored at 4 middotC -7 middotc 10 middotC-13 middotC 25 middotc 35 middotC 45 middotC and 55 middotC
The survival curve of B cereus in artificially contaminated infant milk 28
stored at 4 middotC -7 middotC 10 middotC-13 middotC 25 middotC 35 middotC 45 middotC and 55 c
The survival curve of B cereus in artifIcially contaminated UHT and 30
infant milk with initial pH of pH 620 stored at refrigerated temperature
(4 QC-7 QC)
IX
Figure 10 The survival curve of B cereus in artificially contaminated UHT and 32
infant milk with initial pH of pH 640 stored at refrigerated temperature
(4 middotC-7 C)
Figure 11 The survival curve of B cereus in artificially contaminated UHT and 34
infant milk with initial pH of pH 660 stored at refrigerated temperature
(4 middotC-7 C)
Figure 12 The survival curve of B cereus in artificially contaminated UHT and 36
infant milk with initial pH of pH 680 stored at refrigerated temperature
(4 middotC-7 C)
Figure 13 The survival curve of B cereus in artificially contaminated UHT milk 38
with different initial pH stored at refrigerated temperature (4 middotC-7 C)
Figure 14 The survival curve of B cereus in artificially contaminated infant milk 39
with different initial pH stored- at refrigerated temperature (4 middotC-7 middotC)
Figure 15 B cereus colonies grown on MYP Agar 40
Figure 16 Morphology of B cereus isolated from UHT milk and infant milk 40
samples
Figure 17 Amplification of the gyrB gene (475 bp) for the confirmation of B 41
cereus in UHT milk and infant milk samples
x
Survival ofBacillus cereus in Artificially Contaminated UHT Milk and Infant Milk
Kong Wan Wei
Resource Bioteclmology Department of Molecular Biology
Faculty of Resource Science and Teclmology Universiti Malaysia Sarawak
Abstract
The inappropriate storage temperature and pH enable the survival of Bacillus cereus a known food pathogen in processed milk products resulting in foodborne diseases In the present study UHT milk and infant milk purchased from local supermarkets in Kuching Sarawak were inoculated with 103 CFUml of B cereus Selected storage temperatures of 4 C-7 C 10 C-13 C 25 C 35 C 45 C and 55 C were used to investigate the effects of temperature on the survival of B cereus Initial pH of 620640 660 and 680 at refrigerated temperature (4 C-7 C) were used to study the effects of pH on the survival of B cereus Enumeration of colony counts were investigated daily for the duration of seven days Gram staining and Polymerase Chain Reaction (PCR) targeting gyrB gene were performed for the confirmation of B cereus isolates The present findings revealed that the survival of B cereus in UHT milk and infant mil k showed significant differences (pltO05) in different temperatures and pH At 25 C there were significant difference (plt005) between the survival of B cereus in UHT milk and infant milk B cereus were able to survive under all the experimental temperatures However growth was detected at all storage temperatures except 55 c Survival of B cereus in artificially contaminated UHT milk and infant milk with different initial pH stored at referigerated temperature were also recorded The results from this study can be treated as background data for the implementation of safety measure to avoid food poisoning caused by B cere liS
Key words Bacillus cereus survival milk temperature pH
Abstrak
Suhll penyimpanan dan pH yang tidak sesllai membolehkall kehidupan Bacillus cereus sejenis pathogen makanan dalam produk susu diproses dan mellyebabkan penyakit bawaan makanall Dalam kajian ini SIISII UHT dan SIISU bayi yang dibeli daripada pasar raya tempatan di Kuching Sarawak telah diinoklliasi dengan B cerells sebanyak J(i CFUlml Suhll penyimpanan 4 C-7 c 10 C-3 c 25 c 35 middotc 45 c dan 55 c telah digllnakan IIntllk mengkaji kesan slIhll terhadap kehidupan B cerells Pada suhll peti sejuk (4 C-7 C) pH 620 pH 640 pH 660 dan pH 680 telah digunakan sebagai pH permulaal IIntuk mengkaji kesan pH terhadap keflidupan B cereus Penggiraall kololli telah dijalankan setiap hari selama IIIjuh hari Pewarnaan Gram dan penggllnaan reaksi rantai polimerase (PCR) yang menyasarkan gen gyrB telah mengesahkan kewujlldan koloni B cereus Kajian ini menllnjukkan buhawa kehidupan B cereus dalam SIISU UHT dan susu bayi menunjukkan perbezaan yang ketara (plt005) di suhu penyimpanan dan pH yang berbeza Perbezaan yang ketara (plt005) berlaku anlara kehidupan B cereus dalam susu UHT dengan kehidllpan B cereus dalam SIISII bayi pada sllhu 25 c B cereus dapal menunjllkkan kehidupan dalam SIISU UHT dan SIISU bayi yang telah disimpan pada semua suhll eksperimen Walau bagaimanapun pertumbllhan dapat dikesan pada semua sllhll eksperimen keclali 55 c Selain itll kehidllpan B cerells dalam SUSII UHT dan SUSII bayi yang tercemar secara bllalan pada slIhll peli sejuk telah dicatat pada peJbagai pH Hasil daripadu kajian ini boleh dianggap sebagai dala lalar belakallg tllltllk pelaksanuan langkah keselamatan bagi mengelakkan keracunall makanall daripada B cereus
Kata kunci Bacillus cerellS keflidllpan SIISU sllltl pH
XI
10 Introduction
Population growth and increase in consciousness about health are some of the factors that
increase demand for milk and milk products in Malaysia In Malaysian market ultra-high
temperature (UHT) milk and infant milk powders come under a variety of brands and are
either locally produced or imported Milk provides calcium protein vitamin A vitamin B 12
riboflavin and zinc to the human diet Consumption of milk helps to build and maintain bone
health as well as to reduce the risk of osteoporosis Infant milk provides major nutrients to
infants and it is essential for the growth of newborns
Milk is a high moisture and high nutritious medium which is also suitable for the
growth of mircoorganisms Most of the people believed that processed milk samples are
totally safe for consumption but actually there are many ways that leads to milk
contamination Contamination happened duripg milk processing as Bacillus cereus (B cereus)
strains which were not found in raw milk samples was present in the processed milk samples
(Banyk6 and Vyletelova 2009) Microorganisms contaminate the milk and affects the milk
quality by infecting the mammary gland udder and teat surfaces milking equipment and
storage tank (Gleeson et al 2013) Moreover improper processing and the usage of raw milk
that did not meet the microbiology and physio-chemical standards lead to the defects in raw
and UHT milk (Abdul-Hadi et al 2014) Inappropriate food storage and handling of
contaminated food encourage the growth of B cereus to the levels which can cause food
poisoning (Granum 2005) In order to maintain the quality and safety of processed milk
products consumers are suggested to store these products at temperature of 4 middotC or [less (Boor
and Fromm 2006)
Food pOlsonmg which may even have lethal effect on human is due to the
inappropriate food preparation and food exposure to 30 middotC (Sandra et at 2012) From year
1998 until 2008 there were 235 cases of foodbome disease outbreaks associated with B
cereus in United States (Bennett et at 2013) In 2011 the number of B cereus foodborne
diseases in Europe showed an increase of 1222 (MesseJhausser et ai 2014) The number
reported shows that B cereus poses a threat to the public health as it is often found in
contaminated food However the number of B cereus foodbome poisoning is believed to be
under reported because this type of food poisoning is mild and reporting these cases is not an
compulsory act (EFSA 2005)
B cereus was recognized as foodbome pathogen since 1955 and it is the cause for
about 2 of all foodbome disease worldwide (Lesley et at 2013) Garcia-Arribas and Kramer
(1990) mentioned that B cereus contribute to economic loss by causing contamination
spoilage and food-poisoning B cereus are found in dairy products meats spices and cereals
(Browne and Dowds 2002) as well as in infant foods and dried milk products (Becker et aI
1994)
Most of the patients involved in B cereus outbreaks were infants or elders (Becker et
ai 1994) The immune system of infants are still developing so they are highly susceptible to
B cereus food poisoning after consuming contaminated infant milk Although infant milk
basically present in low moisture powder form the vegetative cells and spores of
microorganisms are able to survive after drying but with their metabolism reduced (I LSI
Europe 2011) This poses threat to infants because some consumers keep the reconstituted
infant formula at room temperature for a long period of time as they wrongly believed that
infant formula are sterilised and are completely free of B cereus (ILSI Europe 2011)
2
B cereus is conunonly related to dairy products because they are able to survive at
different environmental stresses such as pasteurization (Foltys and Kirchnerova 2006 Sadek
et (II 2006) Schelderman et al (2006) discussed that aerobic spore former such as B cereus
has increased tolerance towards low temperature low pH sterilization and UHT treatments
and these conditions increase the concern in food industry In year 2011 there were food
poisoning associated with UHT milk provided from 1 Malaysia Milk Progranune and B cereus
were detected in these UHT milk (Yusof 2011) In 2013 infant milk formula product from
Fonterra were tested positive for Clostridium botulinum which is known as an endospore
former (Frediansyah 2014) The botulism scare was later confirmed as false alarm and the
actual organism was Clostridium 5porogenes However these pathogens have alarmed public
on the safety ofUHT milk and infant milk Currently there are no known studies in Malaysia
examining the factors influencing the survival of B cereus in UHT milk and infant milk
Hence the objectives of this study were
I To study the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk stored at different temperature for seven days
II To examine the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk with different initial pH stored at refrigerated temperature for seven
days
3
20 Literature Review
21 Bacillus cereus
211 Morphological and biochemical characteristics
Bacillus cereus is a Gram positive motile rod-shaped aerobic or facultative anaerobe and
aerobic spore forming foodborne pathogen belonging to Bacillus genus that is normally found
in soil and vegetation (Tortora et al 2010 Food Standards Australia New Zealand 2013
Gleeson et al 2013) This bacterium is mesophile or psychrotroph This bacterium is closely
related to B thuringiensis B mycoides B weihenstephanensis B pseudomycoides and B
anthracis (Gleeson et al 2013) The ability of B cereus to produce endospores which are heat
resistant increases the survivality of B cereus at unfavourable conditions such as high
temperatures extreme pH values or envirorunents with insufficient nutrients compared to
other bacterial enteropathogens (Di Pinto et al 2013 Gleeson et al 2013) For the
biochemical characteristics B cereus are positive for motility Voges-Proskauer (VP)
reaction starch hydrolysis citrate catalase and negative for oxidase tests (Shinagawa 1990
Choma ef al 2000) B cereus is shown as slender bacilli with square end singly or in short
chains under Gram-staining (Bottone 2010)
212 B cereus food borne disease
Rice dishes and meat or poultry dishes were the common sources for B cereus outbreaks
(Bennett et al 2013) Rice and pasta contaminated with B cereus are the causes for emetic
syndrome while the causes for diarrheal illness are desserts meat dishes and dairy products
(Granum 2005) Endospores of B cereus were able to survive in improperly cooked rice and
they caused Fried Rice Syndrome (Sandra et al 2012) In Malaysia B cereus was fIrst
4
Pusat Khidmat Maklumat Ak d Ut lVERSTTJ MA AYSIA SARAWAK
suspected as the cause for food poisoning which affected 114 female students in Klang
(Rampal et al 1984) In order to confIrm that gastroenteritis is caused by B cereus the
number of B cereus iso lated from suspected food need to exceed 105 co lony- forming unit per
gram (CFUg) (Tortora et al 20[0)
213 Isolation and identification of B cereus
Mannitol egg yolk polymyxin agar (MYF) Kim-Geopfert agar Columbia base-blood
polymyxin B agar (CBA-P) and Polymyxin B-egg yolk-mannitol bromothymol blue agar
(PEMBA) are some of the selective media suitable for isoIation and identifIcation of B cereus
(van Netten and Kramer 1992) The inability to ferment mannitol causes B cereus colonies to
appear as pink on MYP and lecithinase activity enables the observation of precipitated zone
around colonies (Pacheco-Sanchez and Massaguer 2007) Food and Drug Administration
(FDA) recommended MYP agar as the stapdard media for identifIcation of B cereus
214 Production of enterotoxins
The two forms of illness associated with B cereus enterotoxins are diarrheal and emetic
syndromes Onset of watery diarrhea and abdominal pain which resemble Clostridium
pefringens intoxications occur 8 hours to 16 hours after ingestion of contaminated food are
the symptoms of diarrheal outbreak (Shinagawa 1990 Tortora et ai 2010) This diarrheal
syndrome occurs after the ingestion of vegetattve cells which later produce a protein
enterotoxin in the gastrointinal tract (Bottone 2010) This type of enterotoxin is heat-labile
(Bello et al 2014) Haemolysin BL (HBL) non-haemolytic enterotoxin and cytotoxin K are
the three types of enterotoxins produced by this bacterium (Samarzija et al 2012 Food
Standards Australia New Zealand 2013) Reis et al (2013) concluded that hblD was the
5
common HBL gene of B cereus detected in milk and dairy products leading to diarrheal form
ofB cereus foodborne disease
The symptoms of emetic outbreaks are similar to Staphylococcus aureus food
intoxication and occur 05 hour to 6 hours upon ingestion of contaminated food
(Messelhausser et al 2014) The ingestion of food contaminated with preformed cereulide
which is also known as the emetic toxin leads to emetic syndrome (Bottone 2010) This heatshy
stable toxin causes nausea and vomiting in the infected patients (Bello et al2014)
215 Effect of temperature and pH on growth and survival of B cereus
Generally B cereus grows between 4 C and 55 C but exhibit optimum growth between 30 C
and 40 C (Schraft and Griffiths 2006 Food Standards Australia New Zealand 2013) Growth
of B cereus had been detected at temperature ranging from 7 C to 45 C and significant
growth was observed at 45 c in steril ized raw milk samples (Bellow et al 2007) In the
absence of oxygen or at temperature below 10 C emetic B cereus are believed to show
incapability in growth and production of toxin (EFSA 2005) At temperature below 10 C
with the limitation of other factors the lag time and generation times of B cereus are longer
(EFSA 2005)
As mentioned by Food Standards Australia New Zealand (2013) B cereus is able to
grow at pH between pH 49 and pH 100 and the optimum pH is between pH 60 and pH 70
Bello et al (2014) concluded that B cereus was unable to grow at pH 63 to pH 68 for 24
hour in pasteurized whole cow milk and the growth was inhibited at pH 68 for a period of 48
hours Byaruhanga et af (1999) studied that mague base inoculated with B cereus for 24
hours at 3S C was able to grow when pH decreases from pH 65 to pH 50 Brain Heart
6
--~~~----~----~----------
Infusion (BHI) medium at pH 88 inhibits growth of emetic and diarrheal strains of B cereus
better than pH 50 of the same medium (Garcia-Arribas and Kramer 1990)
22 UHT milk
Milk treated with UHT pasteurization is heated to 138 middotC for 2 seconds and aseptically placed
into a sterile substantial container which can be stored without refrigeration for six months to
a year (Ledenbach and Marshall 2009) UHT treatment minimized heat denaturation and
produces long shelf life milk that has similar taste and color to pasteurized milk (Harding
1995) UHT milk is generally contaminated during technological processing because food
pathogens are destroyed by the UHT temperature (Vyletelova et al 2002) Longer storage
period causes the drop of UHT milk pH and enzymes may be the reason for changes in UHT
milk (Hassan et al 2009)
Among the 110 UHT milk samples tested by Vidal-Martins et al (2005) 118
(1 3110) samples were positive for B cereus Researchers Rezende-Lago et al (2007)
identified B cereus in four (13 3) of the UHT milk samples tested In the study performed
by Reis et al (2013) among the 260 samples of UHT milk pasteurized milk and powdered
milk tested in Brazil a total of 63 isolates of B cereus were found and 12 of them were from
UHT milk
23 Infant milk
Cows milk and soy protein are the major component of infant milk or infant formulas For the
production of infant formula from milk the milk is heat-treated concentrated and dried while
infant fonnula from other ingredients is dry-blended wetted and dried again or only dry-
blended (Becker et al 1994) The sublethally injured pathogens can recover after the process
7
of drying because this process caused only little damage to the cell (Miller et ai 2012) During
the process of drying components such as lactose protein and milk fat that are present in
milk-based infant formula have the potential to provide protection to the bacteria (Mi1er et ai
20 12) Infant formulas are given to newborns and the content of nutrition is similar to breast
milk (Becker et al 1994) Currently infant formulas in the market are available in powder
liquid concentration and ready-to-feed After rehydration and inappropriate storage lowshy
moisture foods such as infant milk powder should be consumed within a short time because
growth of microorganisms may occur (lLSI Europe 2011)
Becker et al (1994) tested 92 infant milk formula samples and 52 of the samples
were B cereus positive Another study by Rowan et al (1997) tested 100 samples of infant
milk formula from United Kingdom and 17 of the samples were B cereus positive In the
study done by Haughton et al (20 I 0) B cereus were detected in 59 out of 100 infant milk
formula samples from Ireland These studies showed that B cereus is commonly found in
most of the infant milk formula and this poses threat to infants who are highly susceptible
24 Polymerase Chain Reaction (PCR)
As ment ioned by Sandra et al (2012) PCR allows rapid detection of pathogens Schraft and
Griffiths (2006) mentioned that the detection of B cereus targetting B cereus per se and
specific sub-groups have been performed by PCR Yamada et al (1999) designed PCR primer
set for gyrB fragment of B cereus and used this primer set in the detection of B cereus in
boiled rice Confirmation of B cereus in typical colonies are performed using PCR techniques
(Rahimi el al 2013) Thousands to millions copies of specific DNA sequence are produced
from the amplification of a single or a few copies of a piece of DNA by PCR (Joshi and
8
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
Figure 10 The survival curve of B cereus in artificially contaminated UHT and 32
infant milk with initial pH of pH 640 stored at refrigerated temperature
(4 middotC-7 C)
Figure 11 The survival curve of B cereus in artificially contaminated UHT and 34
infant milk with initial pH of pH 660 stored at refrigerated temperature
(4 middotC-7 C)
Figure 12 The survival curve of B cereus in artificially contaminated UHT and 36
infant milk with initial pH of pH 680 stored at refrigerated temperature
(4 middotC-7 C)
Figure 13 The survival curve of B cereus in artificially contaminated UHT milk 38
with different initial pH stored at refrigerated temperature (4 middotC-7 C)
Figure 14 The survival curve of B cereus in artificially contaminated infant milk 39
with different initial pH stored- at refrigerated temperature (4 middotC-7 middotC)
Figure 15 B cereus colonies grown on MYP Agar 40
Figure 16 Morphology of B cereus isolated from UHT milk and infant milk 40
samples
Figure 17 Amplification of the gyrB gene (475 bp) for the confirmation of B 41
cereus in UHT milk and infant milk samples
x
Survival ofBacillus cereus in Artificially Contaminated UHT Milk and Infant Milk
Kong Wan Wei
Resource Bioteclmology Department of Molecular Biology
Faculty of Resource Science and Teclmology Universiti Malaysia Sarawak
Abstract
The inappropriate storage temperature and pH enable the survival of Bacillus cereus a known food pathogen in processed milk products resulting in foodborne diseases In the present study UHT milk and infant milk purchased from local supermarkets in Kuching Sarawak were inoculated with 103 CFUml of B cereus Selected storage temperatures of 4 C-7 C 10 C-13 C 25 C 35 C 45 C and 55 C were used to investigate the effects of temperature on the survival of B cereus Initial pH of 620640 660 and 680 at refrigerated temperature (4 C-7 C) were used to study the effects of pH on the survival of B cereus Enumeration of colony counts were investigated daily for the duration of seven days Gram staining and Polymerase Chain Reaction (PCR) targeting gyrB gene were performed for the confirmation of B cereus isolates The present findings revealed that the survival of B cereus in UHT milk and infant mil k showed significant differences (pltO05) in different temperatures and pH At 25 C there were significant difference (plt005) between the survival of B cereus in UHT milk and infant milk B cereus were able to survive under all the experimental temperatures However growth was detected at all storage temperatures except 55 c Survival of B cereus in artificially contaminated UHT milk and infant milk with different initial pH stored at referigerated temperature were also recorded The results from this study can be treated as background data for the implementation of safety measure to avoid food poisoning caused by B cere liS
Key words Bacillus cereus survival milk temperature pH
Abstrak
Suhll penyimpanan dan pH yang tidak sesllai membolehkall kehidupan Bacillus cereus sejenis pathogen makanan dalam produk susu diproses dan mellyebabkan penyakit bawaan makanall Dalam kajian ini SIISII UHT dan SIISU bayi yang dibeli daripada pasar raya tempatan di Kuching Sarawak telah diinoklliasi dengan B cerells sebanyak J(i CFUlml Suhll penyimpanan 4 C-7 c 10 C-3 c 25 c 35 middotc 45 c dan 55 c telah digllnakan IIntllk mengkaji kesan slIhll terhadap kehidupan B cerells Pada suhll peti sejuk (4 C-7 C) pH 620 pH 640 pH 660 dan pH 680 telah digunakan sebagai pH permulaal IIntuk mengkaji kesan pH terhadap keflidupan B cereus Penggiraall kololli telah dijalankan setiap hari selama IIIjuh hari Pewarnaan Gram dan penggllnaan reaksi rantai polimerase (PCR) yang menyasarkan gen gyrB telah mengesahkan kewujlldan koloni B cereus Kajian ini menllnjukkan buhawa kehidupan B cereus dalam SIISU UHT dan susu bayi menunjukkan perbezaan yang ketara (plt005) di suhu penyimpanan dan pH yang berbeza Perbezaan yang ketara (plt005) berlaku anlara kehidupan B cereus dalam susu UHT dengan kehidllpan B cereus dalam SIISII bayi pada sllhu 25 c B cereus dapal menunjllkkan kehidupan dalam SIISU UHT dan SIISU bayi yang telah disimpan pada semua suhll eksperimen Walau bagaimanapun pertumbllhan dapat dikesan pada semua sllhll eksperimen keclali 55 c Selain itll kehidllpan B cerells dalam SUSII UHT dan SUSII bayi yang tercemar secara bllalan pada slIhll peli sejuk telah dicatat pada peJbagai pH Hasil daripadu kajian ini boleh dianggap sebagai dala lalar belakallg tllltllk pelaksanuan langkah keselamatan bagi mengelakkan keracunall makanall daripada B cereus
Kata kunci Bacillus cerellS keflidllpan SIISU sllltl pH
XI
10 Introduction
Population growth and increase in consciousness about health are some of the factors that
increase demand for milk and milk products in Malaysia In Malaysian market ultra-high
temperature (UHT) milk and infant milk powders come under a variety of brands and are
either locally produced or imported Milk provides calcium protein vitamin A vitamin B 12
riboflavin and zinc to the human diet Consumption of milk helps to build and maintain bone
health as well as to reduce the risk of osteoporosis Infant milk provides major nutrients to
infants and it is essential for the growth of newborns
Milk is a high moisture and high nutritious medium which is also suitable for the
growth of mircoorganisms Most of the people believed that processed milk samples are
totally safe for consumption but actually there are many ways that leads to milk
contamination Contamination happened duripg milk processing as Bacillus cereus (B cereus)
strains which were not found in raw milk samples was present in the processed milk samples
(Banyk6 and Vyletelova 2009) Microorganisms contaminate the milk and affects the milk
quality by infecting the mammary gland udder and teat surfaces milking equipment and
storage tank (Gleeson et al 2013) Moreover improper processing and the usage of raw milk
that did not meet the microbiology and physio-chemical standards lead to the defects in raw
and UHT milk (Abdul-Hadi et al 2014) Inappropriate food storage and handling of
contaminated food encourage the growth of B cereus to the levels which can cause food
poisoning (Granum 2005) In order to maintain the quality and safety of processed milk
products consumers are suggested to store these products at temperature of 4 middotC or [less (Boor
and Fromm 2006)
Food pOlsonmg which may even have lethal effect on human is due to the
inappropriate food preparation and food exposure to 30 middotC (Sandra et at 2012) From year
1998 until 2008 there were 235 cases of foodbome disease outbreaks associated with B
cereus in United States (Bennett et at 2013) In 2011 the number of B cereus foodborne
diseases in Europe showed an increase of 1222 (MesseJhausser et ai 2014) The number
reported shows that B cereus poses a threat to the public health as it is often found in
contaminated food However the number of B cereus foodbome poisoning is believed to be
under reported because this type of food poisoning is mild and reporting these cases is not an
compulsory act (EFSA 2005)
B cereus was recognized as foodbome pathogen since 1955 and it is the cause for
about 2 of all foodbome disease worldwide (Lesley et at 2013) Garcia-Arribas and Kramer
(1990) mentioned that B cereus contribute to economic loss by causing contamination
spoilage and food-poisoning B cereus are found in dairy products meats spices and cereals
(Browne and Dowds 2002) as well as in infant foods and dried milk products (Becker et aI
1994)
Most of the patients involved in B cereus outbreaks were infants or elders (Becker et
ai 1994) The immune system of infants are still developing so they are highly susceptible to
B cereus food poisoning after consuming contaminated infant milk Although infant milk
basically present in low moisture powder form the vegetative cells and spores of
microorganisms are able to survive after drying but with their metabolism reduced (I LSI
Europe 2011) This poses threat to infants because some consumers keep the reconstituted
infant formula at room temperature for a long period of time as they wrongly believed that
infant formula are sterilised and are completely free of B cereus (ILSI Europe 2011)
2
B cereus is conunonly related to dairy products because they are able to survive at
different environmental stresses such as pasteurization (Foltys and Kirchnerova 2006 Sadek
et (II 2006) Schelderman et al (2006) discussed that aerobic spore former such as B cereus
has increased tolerance towards low temperature low pH sterilization and UHT treatments
and these conditions increase the concern in food industry In year 2011 there were food
poisoning associated with UHT milk provided from 1 Malaysia Milk Progranune and B cereus
were detected in these UHT milk (Yusof 2011) In 2013 infant milk formula product from
Fonterra were tested positive for Clostridium botulinum which is known as an endospore
former (Frediansyah 2014) The botulism scare was later confirmed as false alarm and the
actual organism was Clostridium 5porogenes However these pathogens have alarmed public
on the safety ofUHT milk and infant milk Currently there are no known studies in Malaysia
examining the factors influencing the survival of B cereus in UHT milk and infant milk
Hence the objectives of this study were
I To study the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk stored at different temperature for seven days
II To examine the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk with different initial pH stored at refrigerated temperature for seven
days
3
20 Literature Review
21 Bacillus cereus
211 Morphological and biochemical characteristics
Bacillus cereus is a Gram positive motile rod-shaped aerobic or facultative anaerobe and
aerobic spore forming foodborne pathogen belonging to Bacillus genus that is normally found
in soil and vegetation (Tortora et al 2010 Food Standards Australia New Zealand 2013
Gleeson et al 2013) This bacterium is mesophile or psychrotroph This bacterium is closely
related to B thuringiensis B mycoides B weihenstephanensis B pseudomycoides and B
anthracis (Gleeson et al 2013) The ability of B cereus to produce endospores which are heat
resistant increases the survivality of B cereus at unfavourable conditions such as high
temperatures extreme pH values or envirorunents with insufficient nutrients compared to
other bacterial enteropathogens (Di Pinto et al 2013 Gleeson et al 2013) For the
biochemical characteristics B cereus are positive for motility Voges-Proskauer (VP)
reaction starch hydrolysis citrate catalase and negative for oxidase tests (Shinagawa 1990
Choma ef al 2000) B cereus is shown as slender bacilli with square end singly or in short
chains under Gram-staining (Bottone 2010)
212 B cereus food borne disease
Rice dishes and meat or poultry dishes were the common sources for B cereus outbreaks
(Bennett et al 2013) Rice and pasta contaminated with B cereus are the causes for emetic
syndrome while the causes for diarrheal illness are desserts meat dishes and dairy products
(Granum 2005) Endospores of B cereus were able to survive in improperly cooked rice and
they caused Fried Rice Syndrome (Sandra et al 2012) In Malaysia B cereus was fIrst
4
Pusat Khidmat Maklumat Ak d Ut lVERSTTJ MA AYSIA SARAWAK
suspected as the cause for food poisoning which affected 114 female students in Klang
(Rampal et al 1984) In order to confIrm that gastroenteritis is caused by B cereus the
number of B cereus iso lated from suspected food need to exceed 105 co lony- forming unit per
gram (CFUg) (Tortora et al 20[0)
213 Isolation and identification of B cereus
Mannitol egg yolk polymyxin agar (MYF) Kim-Geopfert agar Columbia base-blood
polymyxin B agar (CBA-P) and Polymyxin B-egg yolk-mannitol bromothymol blue agar
(PEMBA) are some of the selective media suitable for isoIation and identifIcation of B cereus
(van Netten and Kramer 1992) The inability to ferment mannitol causes B cereus colonies to
appear as pink on MYP and lecithinase activity enables the observation of precipitated zone
around colonies (Pacheco-Sanchez and Massaguer 2007) Food and Drug Administration
(FDA) recommended MYP agar as the stapdard media for identifIcation of B cereus
214 Production of enterotoxins
The two forms of illness associated with B cereus enterotoxins are diarrheal and emetic
syndromes Onset of watery diarrhea and abdominal pain which resemble Clostridium
pefringens intoxications occur 8 hours to 16 hours after ingestion of contaminated food are
the symptoms of diarrheal outbreak (Shinagawa 1990 Tortora et ai 2010) This diarrheal
syndrome occurs after the ingestion of vegetattve cells which later produce a protein
enterotoxin in the gastrointinal tract (Bottone 2010) This type of enterotoxin is heat-labile
(Bello et al 2014) Haemolysin BL (HBL) non-haemolytic enterotoxin and cytotoxin K are
the three types of enterotoxins produced by this bacterium (Samarzija et al 2012 Food
Standards Australia New Zealand 2013) Reis et al (2013) concluded that hblD was the
5
common HBL gene of B cereus detected in milk and dairy products leading to diarrheal form
ofB cereus foodborne disease
The symptoms of emetic outbreaks are similar to Staphylococcus aureus food
intoxication and occur 05 hour to 6 hours upon ingestion of contaminated food
(Messelhausser et al 2014) The ingestion of food contaminated with preformed cereulide
which is also known as the emetic toxin leads to emetic syndrome (Bottone 2010) This heatshy
stable toxin causes nausea and vomiting in the infected patients (Bello et al2014)
215 Effect of temperature and pH on growth and survival of B cereus
Generally B cereus grows between 4 C and 55 C but exhibit optimum growth between 30 C
and 40 C (Schraft and Griffiths 2006 Food Standards Australia New Zealand 2013) Growth
of B cereus had been detected at temperature ranging from 7 C to 45 C and significant
growth was observed at 45 c in steril ized raw milk samples (Bellow et al 2007) In the
absence of oxygen or at temperature below 10 C emetic B cereus are believed to show
incapability in growth and production of toxin (EFSA 2005) At temperature below 10 C
with the limitation of other factors the lag time and generation times of B cereus are longer
(EFSA 2005)
As mentioned by Food Standards Australia New Zealand (2013) B cereus is able to
grow at pH between pH 49 and pH 100 and the optimum pH is between pH 60 and pH 70
Bello et al (2014) concluded that B cereus was unable to grow at pH 63 to pH 68 for 24
hour in pasteurized whole cow milk and the growth was inhibited at pH 68 for a period of 48
hours Byaruhanga et af (1999) studied that mague base inoculated with B cereus for 24
hours at 3S C was able to grow when pH decreases from pH 65 to pH 50 Brain Heart
6
--~~~----~----~----------
Infusion (BHI) medium at pH 88 inhibits growth of emetic and diarrheal strains of B cereus
better than pH 50 of the same medium (Garcia-Arribas and Kramer 1990)
22 UHT milk
Milk treated with UHT pasteurization is heated to 138 middotC for 2 seconds and aseptically placed
into a sterile substantial container which can be stored without refrigeration for six months to
a year (Ledenbach and Marshall 2009) UHT treatment minimized heat denaturation and
produces long shelf life milk that has similar taste and color to pasteurized milk (Harding
1995) UHT milk is generally contaminated during technological processing because food
pathogens are destroyed by the UHT temperature (Vyletelova et al 2002) Longer storage
period causes the drop of UHT milk pH and enzymes may be the reason for changes in UHT
milk (Hassan et al 2009)
Among the 110 UHT milk samples tested by Vidal-Martins et al (2005) 118
(1 3110) samples were positive for B cereus Researchers Rezende-Lago et al (2007)
identified B cereus in four (13 3) of the UHT milk samples tested In the study performed
by Reis et al (2013) among the 260 samples of UHT milk pasteurized milk and powdered
milk tested in Brazil a total of 63 isolates of B cereus were found and 12 of them were from
UHT milk
23 Infant milk
Cows milk and soy protein are the major component of infant milk or infant formulas For the
production of infant formula from milk the milk is heat-treated concentrated and dried while
infant fonnula from other ingredients is dry-blended wetted and dried again or only dry-
blended (Becker et al 1994) The sublethally injured pathogens can recover after the process
7
of drying because this process caused only little damage to the cell (Miller et ai 2012) During
the process of drying components such as lactose protein and milk fat that are present in
milk-based infant formula have the potential to provide protection to the bacteria (Mi1er et ai
20 12) Infant formulas are given to newborns and the content of nutrition is similar to breast
milk (Becker et al 1994) Currently infant formulas in the market are available in powder
liquid concentration and ready-to-feed After rehydration and inappropriate storage lowshy
moisture foods such as infant milk powder should be consumed within a short time because
growth of microorganisms may occur (lLSI Europe 2011)
Becker et al (1994) tested 92 infant milk formula samples and 52 of the samples
were B cereus positive Another study by Rowan et al (1997) tested 100 samples of infant
milk formula from United Kingdom and 17 of the samples were B cereus positive In the
study done by Haughton et al (20 I 0) B cereus were detected in 59 out of 100 infant milk
formula samples from Ireland These studies showed that B cereus is commonly found in
most of the infant milk formula and this poses threat to infants who are highly susceptible
24 Polymerase Chain Reaction (PCR)
As ment ioned by Sandra et al (2012) PCR allows rapid detection of pathogens Schraft and
Griffiths (2006) mentioned that the detection of B cereus targetting B cereus per se and
specific sub-groups have been performed by PCR Yamada et al (1999) designed PCR primer
set for gyrB fragment of B cereus and used this primer set in the detection of B cereus in
boiled rice Confirmation of B cereus in typical colonies are performed using PCR techniques
(Rahimi el al 2013) Thousands to millions copies of specific DNA sequence are produced
from the amplification of a single or a few copies of a piece of DNA by PCR (Joshi and
8
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
Survival ofBacillus cereus in Artificially Contaminated UHT Milk and Infant Milk
Kong Wan Wei
Resource Bioteclmology Department of Molecular Biology
Faculty of Resource Science and Teclmology Universiti Malaysia Sarawak
Abstract
The inappropriate storage temperature and pH enable the survival of Bacillus cereus a known food pathogen in processed milk products resulting in foodborne diseases In the present study UHT milk and infant milk purchased from local supermarkets in Kuching Sarawak were inoculated with 103 CFUml of B cereus Selected storage temperatures of 4 C-7 C 10 C-13 C 25 C 35 C 45 C and 55 C were used to investigate the effects of temperature on the survival of B cereus Initial pH of 620640 660 and 680 at refrigerated temperature (4 C-7 C) were used to study the effects of pH on the survival of B cereus Enumeration of colony counts were investigated daily for the duration of seven days Gram staining and Polymerase Chain Reaction (PCR) targeting gyrB gene were performed for the confirmation of B cereus isolates The present findings revealed that the survival of B cereus in UHT milk and infant mil k showed significant differences (pltO05) in different temperatures and pH At 25 C there were significant difference (plt005) between the survival of B cereus in UHT milk and infant milk B cereus were able to survive under all the experimental temperatures However growth was detected at all storage temperatures except 55 c Survival of B cereus in artificially contaminated UHT milk and infant milk with different initial pH stored at referigerated temperature were also recorded The results from this study can be treated as background data for the implementation of safety measure to avoid food poisoning caused by B cere liS
Key words Bacillus cereus survival milk temperature pH
Abstrak
Suhll penyimpanan dan pH yang tidak sesllai membolehkall kehidupan Bacillus cereus sejenis pathogen makanan dalam produk susu diproses dan mellyebabkan penyakit bawaan makanall Dalam kajian ini SIISII UHT dan SIISU bayi yang dibeli daripada pasar raya tempatan di Kuching Sarawak telah diinoklliasi dengan B cerells sebanyak J(i CFUlml Suhll penyimpanan 4 C-7 c 10 C-3 c 25 c 35 middotc 45 c dan 55 c telah digllnakan IIntllk mengkaji kesan slIhll terhadap kehidupan B cerells Pada suhll peti sejuk (4 C-7 C) pH 620 pH 640 pH 660 dan pH 680 telah digunakan sebagai pH permulaal IIntuk mengkaji kesan pH terhadap keflidupan B cereus Penggiraall kololli telah dijalankan setiap hari selama IIIjuh hari Pewarnaan Gram dan penggllnaan reaksi rantai polimerase (PCR) yang menyasarkan gen gyrB telah mengesahkan kewujlldan koloni B cereus Kajian ini menllnjukkan buhawa kehidupan B cereus dalam SIISU UHT dan susu bayi menunjukkan perbezaan yang ketara (plt005) di suhu penyimpanan dan pH yang berbeza Perbezaan yang ketara (plt005) berlaku anlara kehidupan B cereus dalam susu UHT dengan kehidllpan B cereus dalam SIISII bayi pada sllhu 25 c B cereus dapal menunjllkkan kehidupan dalam SIISU UHT dan SIISU bayi yang telah disimpan pada semua suhll eksperimen Walau bagaimanapun pertumbllhan dapat dikesan pada semua sllhll eksperimen keclali 55 c Selain itll kehidllpan B cerells dalam SUSII UHT dan SUSII bayi yang tercemar secara bllalan pada slIhll peli sejuk telah dicatat pada peJbagai pH Hasil daripadu kajian ini boleh dianggap sebagai dala lalar belakallg tllltllk pelaksanuan langkah keselamatan bagi mengelakkan keracunall makanall daripada B cereus
Kata kunci Bacillus cerellS keflidllpan SIISU sllltl pH
XI
10 Introduction
Population growth and increase in consciousness about health are some of the factors that
increase demand for milk and milk products in Malaysia In Malaysian market ultra-high
temperature (UHT) milk and infant milk powders come under a variety of brands and are
either locally produced or imported Milk provides calcium protein vitamin A vitamin B 12
riboflavin and zinc to the human diet Consumption of milk helps to build and maintain bone
health as well as to reduce the risk of osteoporosis Infant milk provides major nutrients to
infants and it is essential for the growth of newborns
Milk is a high moisture and high nutritious medium which is also suitable for the
growth of mircoorganisms Most of the people believed that processed milk samples are
totally safe for consumption but actually there are many ways that leads to milk
contamination Contamination happened duripg milk processing as Bacillus cereus (B cereus)
strains which were not found in raw milk samples was present in the processed milk samples
(Banyk6 and Vyletelova 2009) Microorganisms contaminate the milk and affects the milk
quality by infecting the mammary gland udder and teat surfaces milking equipment and
storage tank (Gleeson et al 2013) Moreover improper processing and the usage of raw milk
that did not meet the microbiology and physio-chemical standards lead to the defects in raw
and UHT milk (Abdul-Hadi et al 2014) Inappropriate food storage and handling of
contaminated food encourage the growth of B cereus to the levels which can cause food
poisoning (Granum 2005) In order to maintain the quality and safety of processed milk
products consumers are suggested to store these products at temperature of 4 middotC or [less (Boor
and Fromm 2006)
Food pOlsonmg which may even have lethal effect on human is due to the
inappropriate food preparation and food exposure to 30 middotC (Sandra et at 2012) From year
1998 until 2008 there were 235 cases of foodbome disease outbreaks associated with B
cereus in United States (Bennett et at 2013) In 2011 the number of B cereus foodborne
diseases in Europe showed an increase of 1222 (MesseJhausser et ai 2014) The number
reported shows that B cereus poses a threat to the public health as it is often found in
contaminated food However the number of B cereus foodbome poisoning is believed to be
under reported because this type of food poisoning is mild and reporting these cases is not an
compulsory act (EFSA 2005)
B cereus was recognized as foodbome pathogen since 1955 and it is the cause for
about 2 of all foodbome disease worldwide (Lesley et at 2013) Garcia-Arribas and Kramer
(1990) mentioned that B cereus contribute to economic loss by causing contamination
spoilage and food-poisoning B cereus are found in dairy products meats spices and cereals
(Browne and Dowds 2002) as well as in infant foods and dried milk products (Becker et aI
1994)
Most of the patients involved in B cereus outbreaks were infants or elders (Becker et
ai 1994) The immune system of infants are still developing so they are highly susceptible to
B cereus food poisoning after consuming contaminated infant milk Although infant milk
basically present in low moisture powder form the vegetative cells and spores of
microorganisms are able to survive after drying but with their metabolism reduced (I LSI
Europe 2011) This poses threat to infants because some consumers keep the reconstituted
infant formula at room temperature for a long period of time as they wrongly believed that
infant formula are sterilised and are completely free of B cereus (ILSI Europe 2011)
2
B cereus is conunonly related to dairy products because they are able to survive at
different environmental stresses such as pasteurization (Foltys and Kirchnerova 2006 Sadek
et (II 2006) Schelderman et al (2006) discussed that aerobic spore former such as B cereus
has increased tolerance towards low temperature low pH sterilization and UHT treatments
and these conditions increase the concern in food industry In year 2011 there were food
poisoning associated with UHT milk provided from 1 Malaysia Milk Progranune and B cereus
were detected in these UHT milk (Yusof 2011) In 2013 infant milk formula product from
Fonterra were tested positive for Clostridium botulinum which is known as an endospore
former (Frediansyah 2014) The botulism scare was later confirmed as false alarm and the
actual organism was Clostridium 5porogenes However these pathogens have alarmed public
on the safety ofUHT milk and infant milk Currently there are no known studies in Malaysia
examining the factors influencing the survival of B cereus in UHT milk and infant milk
Hence the objectives of this study were
I To study the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk stored at different temperature for seven days
II To examine the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk with different initial pH stored at refrigerated temperature for seven
days
3
20 Literature Review
21 Bacillus cereus
211 Morphological and biochemical characteristics
Bacillus cereus is a Gram positive motile rod-shaped aerobic or facultative anaerobe and
aerobic spore forming foodborne pathogen belonging to Bacillus genus that is normally found
in soil and vegetation (Tortora et al 2010 Food Standards Australia New Zealand 2013
Gleeson et al 2013) This bacterium is mesophile or psychrotroph This bacterium is closely
related to B thuringiensis B mycoides B weihenstephanensis B pseudomycoides and B
anthracis (Gleeson et al 2013) The ability of B cereus to produce endospores which are heat
resistant increases the survivality of B cereus at unfavourable conditions such as high
temperatures extreme pH values or envirorunents with insufficient nutrients compared to
other bacterial enteropathogens (Di Pinto et al 2013 Gleeson et al 2013) For the
biochemical characteristics B cereus are positive for motility Voges-Proskauer (VP)
reaction starch hydrolysis citrate catalase and negative for oxidase tests (Shinagawa 1990
Choma ef al 2000) B cereus is shown as slender bacilli with square end singly or in short
chains under Gram-staining (Bottone 2010)
212 B cereus food borne disease
Rice dishes and meat or poultry dishes were the common sources for B cereus outbreaks
(Bennett et al 2013) Rice and pasta contaminated with B cereus are the causes for emetic
syndrome while the causes for diarrheal illness are desserts meat dishes and dairy products
(Granum 2005) Endospores of B cereus were able to survive in improperly cooked rice and
they caused Fried Rice Syndrome (Sandra et al 2012) In Malaysia B cereus was fIrst
4
Pusat Khidmat Maklumat Ak d Ut lVERSTTJ MA AYSIA SARAWAK
suspected as the cause for food poisoning which affected 114 female students in Klang
(Rampal et al 1984) In order to confIrm that gastroenteritis is caused by B cereus the
number of B cereus iso lated from suspected food need to exceed 105 co lony- forming unit per
gram (CFUg) (Tortora et al 20[0)
213 Isolation and identification of B cereus
Mannitol egg yolk polymyxin agar (MYF) Kim-Geopfert agar Columbia base-blood
polymyxin B agar (CBA-P) and Polymyxin B-egg yolk-mannitol bromothymol blue agar
(PEMBA) are some of the selective media suitable for isoIation and identifIcation of B cereus
(van Netten and Kramer 1992) The inability to ferment mannitol causes B cereus colonies to
appear as pink on MYP and lecithinase activity enables the observation of precipitated zone
around colonies (Pacheco-Sanchez and Massaguer 2007) Food and Drug Administration
(FDA) recommended MYP agar as the stapdard media for identifIcation of B cereus
214 Production of enterotoxins
The two forms of illness associated with B cereus enterotoxins are diarrheal and emetic
syndromes Onset of watery diarrhea and abdominal pain which resemble Clostridium
pefringens intoxications occur 8 hours to 16 hours after ingestion of contaminated food are
the symptoms of diarrheal outbreak (Shinagawa 1990 Tortora et ai 2010) This diarrheal
syndrome occurs after the ingestion of vegetattve cells which later produce a protein
enterotoxin in the gastrointinal tract (Bottone 2010) This type of enterotoxin is heat-labile
(Bello et al 2014) Haemolysin BL (HBL) non-haemolytic enterotoxin and cytotoxin K are
the three types of enterotoxins produced by this bacterium (Samarzija et al 2012 Food
Standards Australia New Zealand 2013) Reis et al (2013) concluded that hblD was the
5
common HBL gene of B cereus detected in milk and dairy products leading to diarrheal form
ofB cereus foodborne disease
The symptoms of emetic outbreaks are similar to Staphylococcus aureus food
intoxication and occur 05 hour to 6 hours upon ingestion of contaminated food
(Messelhausser et al 2014) The ingestion of food contaminated with preformed cereulide
which is also known as the emetic toxin leads to emetic syndrome (Bottone 2010) This heatshy
stable toxin causes nausea and vomiting in the infected patients (Bello et al2014)
215 Effect of temperature and pH on growth and survival of B cereus
Generally B cereus grows between 4 C and 55 C but exhibit optimum growth between 30 C
and 40 C (Schraft and Griffiths 2006 Food Standards Australia New Zealand 2013) Growth
of B cereus had been detected at temperature ranging from 7 C to 45 C and significant
growth was observed at 45 c in steril ized raw milk samples (Bellow et al 2007) In the
absence of oxygen or at temperature below 10 C emetic B cereus are believed to show
incapability in growth and production of toxin (EFSA 2005) At temperature below 10 C
with the limitation of other factors the lag time and generation times of B cereus are longer
(EFSA 2005)
As mentioned by Food Standards Australia New Zealand (2013) B cereus is able to
grow at pH between pH 49 and pH 100 and the optimum pH is between pH 60 and pH 70
Bello et al (2014) concluded that B cereus was unable to grow at pH 63 to pH 68 for 24
hour in pasteurized whole cow milk and the growth was inhibited at pH 68 for a period of 48
hours Byaruhanga et af (1999) studied that mague base inoculated with B cereus for 24
hours at 3S C was able to grow when pH decreases from pH 65 to pH 50 Brain Heart
6
--~~~----~----~----------
Infusion (BHI) medium at pH 88 inhibits growth of emetic and diarrheal strains of B cereus
better than pH 50 of the same medium (Garcia-Arribas and Kramer 1990)
22 UHT milk
Milk treated with UHT pasteurization is heated to 138 middotC for 2 seconds and aseptically placed
into a sterile substantial container which can be stored without refrigeration for six months to
a year (Ledenbach and Marshall 2009) UHT treatment minimized heat denaturation and
produces long shelf life milk that has similar taste and color to pasteurized milk (Harding
1995) UHT milk is generally contaminated during technological processing because food
pathogens are destroyed by the UHT temperature (Vyletelova et al 2002) Longer storage
period causes the drop of UHT milk pH and enzymes may be the reason for changes in UHT
milk (Hassan et al 2009)
Among the 110 UHT milk samples tested by Vidal-Martins et al (2005) 118
(1 3110) samples were positive for B cereus Researchers Rezende-Lago et al (2007)
identified B cereus in four (13 3) of the UHT milk samples tested In the study performed
by Reis et al (2013) among the 260 samples of UHT milk pasteurized milk and powdered
milk tested in Brazil a total of 63 isolates of B cereus were found and 12 of them were from
UHT milk
23 Infant milk
Cows milk and soy protein are the major component of infant milk or infant formulas For the
production of infant formula from milk the milk is heat-treated concentrated and dried while
infant fonnula from other ingredients is dry-blended wetted and dried again or only dry-
blended (Becker et al 1994) The sublethally injured pathogens can recover after the process
7
of drying because this process caused only little damage to the cell (Miller et ai 2012) During
the process of drying components such as lactose protein and milk fat that are present in
milk-based infant formula have the potential to provide protection to the bacteria (Mi1er et ai
20 12) Infant formulas are given to newborns and the content of nutrition is similar to breast
milk (Becker et al 1994) Currently infant formulas in the market are available in powder
liquid concentration and ready-to-feed After rehydration and inappropriate storage lowshy
moisture foods such as infant milk powder should be consumed within a short time because
growth of microorganisms may occur (lLSI Europe 2011)
Becker et al (1994) tested 92 infant milk formula samples and 52 of the samples
were B cereus positive Another study by Rowan et al (1997) tested 100 samples of infant
milk formula from United Kingdom and 17 of the samples were B cereus positive In the
study done by Haughton et al (20 I 0) B cereus were detected in 59 out of 100 infant milk
formula samples from Ireland These studies showed that B cereus is commonly found in
most of the infant milk formula and this poses threat to infants who are highly susceptible
24 Polymerase Chain Reaction (PCR)
As ment ioned by Sandra et al (2012) PCR allows rapid detection of pathogens Schraft and
Griffiths (2006) mentioned that the detection of B cereus targetting B cereus per se and
specific sub-groups have been performed by PCR Yamada et al (1999) designed PCR primer
set for gyrB fragment of B cereus and used this primer set in the detection of B cereus in
boiled rice Confirmation of B cereus in typical colonies are performed using PCR techniques
(Rahimi el al 2013) Thousands to millions copies of specific DNA sequence are produced
from the amplification of a single or a few copies of a piece of DNA by PCR (Joshi and
8
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
10 Introduction
Population growth and increase in consciousness about health are some of the factors that
increase demand for milk and milk products in Malaysia In Malaysian market ultra-high
temperature (UHT) milk and infant milk powders come under a variety of brands and are
either locally produced or imported Milk provides calcium protein vitamin A vitamin B 12
riboflavin and zinc to the human diet Consumption of milk helps to build and maintain bone
health as well as to reduce the risk of osteoporosis Infant milk provides major nutrients to
infants and it is essential for the growth of newborns
Milk is a high moisture and high nutritious medium which is also suitable for the
growth of mircoorganisms Most of the people believed that processed milk samples are
totally safe for consumption but actually there are many ways that leads to milk
contamination Contamination happened duripg milk processing as Bacillus cereus (B cereus)
strains which were not found in raw milk samples was present in the processed milk samples
(Banyk6 and Vyletelova 2009) Microorganisms contaminate the milk and affects the milk
quality by infecting the mammary gland udder and teat surfaces milking equipment and
storage tank (Gleeson et al 2013) Moreover improper processing and the usage of raw milk
that did not meet the microbiology and physio-chemical standards lead to the defects in raw
and UHT milk (Abdul-Hadi et al 2014) Inappropriate food storage and handling of
contaminated food encourage the growth of B cereus to the levels which can cause food
poisoning (Granum 2005) In order to maintain the quality and safety of processed milk
products consumers are suggested to store these products at temperature of 4 middotC or [less (Boor
and Fromm 2006)
Food pOlsonmg which may even have lethal effect on human is due to the
inappropriate food preparation and food exposure to 30 middotC (Sandra et at 2012) From year
1998 until 2008 there were 235 cases of foodbome disease outbreaks associated with B
cereus in United States (Bennett et at 2013) In 2011 the number of B cereus foodborne
diseases in Europe showed an increase of 1222 (MesseJhausser et ai 2014) The number
reported shows that B cereus poses a threat to the public health as it is often found in
contaminated food However the number of B cereus foodbome poisoning is believed to be
under reported because this type of food poisoning is mild and reporting these cases is not an
compulsory act (EFSA 2005)
B cereus was recognized as foodbome pathogen since 1955 and it is the cause for
about 2 of all foodbome disease worldwide (Lesley et at 2013) Garcia-Arribas and Kramer
(1990) mentioned that B cereus contribute to economic loss by causing contamination
spoilage and food-poisoning B cereus are found in dairy products meats spices and cereals
(Browne and Dowds 2002) as well as in infant foods and dried milk products (Becker et aI
1994)
Most of the patients involved in B cereus outbreaks were infants or elders (Becker et
ai 1994) The immune system of infants are still developing so they are highly susceptible to
B cereus food poisoning after consuming contaminated infant milk Although infant milk
basically present in low moisture powder form the vegetative cells and spores of
microorganisms are able to survive after drying but with their metabolism reduced (I LSI
Europe 2011) This poses threat to infants because some consumers keep the reconstituted
infant formula at room temperature for a long period of time as they wrongly believed that
infant formula are sterilised and are completely free of B cereus (ILSI Europe 2011)
2
B cereus is conunonly related to dairy products because they are able to survive at
different environmental stresses such as pasteurization (Foltys and Kirchnerova 2006 Sadek
et (II 2006) Schelderman et al (2006) discussed that aerobic spore former such as B cereus
has increased tolerance towards low temperature low pH sterilization and UHT treatments
and these conditions increase the concern in food industry In year 2011 there were food
poisoning associated with UHT milk provided from 1 Malaysia Milk Progranune and B cereus
were detected in these UHT milk (Yusof 2011) In 2013 infant milk formula product from
Fonterra were tested positive for Clostridium botulinum which is known as an endospore
former (Frediansyah 2014) The botulism scare was later confirmed as false alarm and the
actual organism was Clostridium 5porogenes However these pathogens have alarmed public
on the safety ofUHT milk and infant milk Currently there are no known studies in Malaysia
examining the factors influencing the survival of B cereus in UHT milk and infant milk
Hence the objectives of this study were
I To study the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk stored at different temperature for seven days
II To examine the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk with different initial pH stored at refrigerated temperature for seven
days
3
20 Literature Review
21 Bacillus cereus
211 Morphological and biochemical characteristics
Bacillus cereus is a Gram positive motile rod-shaped aerobic or facultative anaerobe and
aerobic spore forming foodborne pathogen belonging to Bacillus genus that is normally found
in soil and vegetation (Tortora et al 2010 Food Standards Australia New Zealand 2013
Gleeson et al 2013) This bacterium is mesophile or psychrotroph This bacterium is closely
related to B thuringiensis B mycoides B weihenstephanensis B pseudomycoides and B
anthracis (Gleeson et al 2013) The ability of B cereus to produce endospores which are heat
resistant increases the survivality of B cereus at unfavourable conditions such as high
temperatures extreme pH values or envirorunents with insufficient nutrients compared to
other bacterial enteropathogens (Di Pinto et al 2013 Gleeson et al 2013) For the
biochemical characteristics B cereus are positive for motility Voges-Proskauer (VP)
reaction starch hydrolysis citrate catalase and negative for oxidase tests (Shinagawa 1990
Choma ef al 2000) B cereus is shown as slender bacilli with square end singly or in short
chains under Gram-staining (Bottone 2010)
212 B cereus food borne disease
Rice dishes and meat or poultry dishes were the common sources for B cereus outbreaks
(Bennett et al 2013) Rice and pasta contaminated with B cereus are the causes for emetic
syndrome while the causes for diarrheal illness are desserts meat dishes and dairy products
(Granum 2005) Endospores of B cereus were able to survive in improperly cooked rice and
they caused Fried Rice Syndrome (Sandra et al 2012) In Malaysia B cereus was fIrst
4
Pusat Khidmat Maklumat Ak d Ut lVERSTTJ MA AYSIA SARAWAK
suspected as the cause for food poisoning which affected 114 female students in Klang
(Rampal et al 1984) In order to confIrm that gastroenteritis is caused by B cereus the
number of B cereus iso lated from suspected food need to exceed 105 co lony- forming unit per
gram (CFUg) (Tortora et al 20[0)
213 Isolation and identification of B cereus
Mannitol egg yolk polymyxin agar (MYF) Kim-Geopfert agar Columbia base-blood
polymyxin B agar (CBA-P) and Polymyxin B-egg yolk-mannitol bromothymol blue agar
(PEMBA) are some of the selective media suitable for isoIation and identifIcation of B cereus
(van Netten and Kramer 1992) The inability to ferment mannitol causes B cereus colonies to
appear as pink on MYP and lecithinase activity enables the observation of precipitated zone
around colonies (Pacheco-Sanchez and Massaguer 2007) Food and Drug Administration
(FDA) recommended MYP agar as the stapdard media for identifIcation of B cereus
214 Production of enterotoxins
The two forms of illness associated with B cereus enterotoxins are diarrheal and emetic
syndromes Onset of watery diarrhea and abdominal pain which resemble Clostridium
pefringens intoxications occur 8 hours to 16 hours after ingestion of contaminated food are
the symptoms of diarrheal outbreak (Shinagawa 1990 Tortora et ai 2010) This diarrheal
syndrome occurs after the ingestion of vegetattve cells which later produce a protein
enterotoxin in the gastrointinal tract (Bottone 2010) This type of enterotoxin is heat-labile
(Bello et al 2014) Haemolysin BL (HBL) non-haemolytic enterotoxin and cytotoxin K are
the three types of enterotoxins produced by this bacterium (Samarzija et al 2012 Food
Standards Australia New Zealand 2013) Reis et al (2013) concluded that hblD was the
5
common HBL gene of B cereus detected in milk and dairy products leading to diarrheal form
ofB cereus foodborne disease
The symptoms of emetic outbreaks are similar to Staphylococcus aureus food
intoxication and occur 05 hour to 6 hours upon ingestion of contaminated food
(Messelhausser et al 2014) The ingestion of food contaminated with preformed cereulide
which is also known as the emetic toxin leads to emetic syndrome (Bottone 2010) This heatshy
stable toxin causes nausea and vomiting in the infected patients (Bello et al2014)
215 Effect of temperature and pH on growth and survival of B cereus
Generally B cereus grows between 4 C and 55 C but exhibit optimum growth between 30 C
and 40 C (Schraft and Griffiths 2006 Food Standards Australia New Zealand 2013) Growth
of B cereus had been detected at temperature ranging from 7 C to 45 C and significant
growth was observed at 45 c in steril ized raw milk samples (Bellow et al 2007) In the
absence of oxygen or at temperature below 10 C emetic B cereus are believed to show
incapability in growth and production of toxin (EFSA 2005) At temperature below 10 C
with the limitation of other factors the lag time and generation times of B cereus are longer
(EFSA 2005)
As mentioned by Food Standards Australia New Zealand (2013) B cereus is able to
grow at pH between pH 49 and pH 100 and the optimum pH is between pH 60 and pH 70
Bello et al (2014) concluded that B cereus was unable to grow at pH 63 to pH 68 for 24
hour in pasteurized whole cow milk and the growth was inhibited at pH 68 for a period of 48
hours Byaruhanga et af (1999) studied that mague base inoculated with B cereus for 24
hours at 3S C was able to grow when pH decreases from pH 65 to pH 50 Brain Heart
6
--~~~----~----~----------
Infusion (BHI) medium at pH 88 inhibits growth of emetic and diarrheal strains of B cereus
better than pH 50 of the same medium (Garcia-Arribas and Kramer 1990)
22 UHT milk
Milk treated with UHT pasteurization is heated to 138 middotC for 2 seconds and aseptically placed
into a sterile substantial container which can be stored without refrigeration for six months to
a year (Ledenbach and Marshall 2009) UHT treatment minimized heat denaturation and
produces long shelf life milk that has similar taste and color to pasteurized milk (Harding
1995) UHT milk is generally contaminated during technological processing because food
pathogens are destroyed by the UHT temperature (Vyletelova et al 2002) Longer storage
period causes the drop of UHT milk pH and enzymes may be the reason for changes in UHT
milk (Hassan et al 2009)
Among the 110 UHT milk samples tested by Vidal-Martins et al (2005) 118
(1 3110) samples were positive for B cereus Researchers Rezende-Lago et al (2007)
identified B cereus in four (13 3) of the UHT milk samples tested In the study performed
by Reis et al (2013) among the 260 samples of UHT milk pasteurized milk and powdered
milk tested in Brazil a total of 63 isolates of B cereus were found and 12 of them were from
UHT milk
23 Infant milk
Cows milk and soy protein are the major component of infant milk or infant formulas For the
production of infant formula from milk the milk is heat-treated concentrated and dried while
infant fonnula from other ingredients is dry-blended wetted and dried again or only dry-
blended (Becker et al 1994) The sublethally injured pathogens can recover after the process
7
of drying because this process caused only little damage to the cell (Miller et ai 2012) During
the process of drying components such as lactose protein and milk fat that are present in
milk-based infant formula have the potential to provide protection to the bacteria (Mi1er et ai
20 12) Infant formulas are given to newborns and the content of nutrition is similar to breast
milk (Becker et al 1994) Currently infant formulas in the market are available in powder
liquid concentration and ready-to-feed After rehydration and inappropriate storage lowshy
moisture foods such as infant milk powder should be consumed within a short time because
growth of microorganisms may occur (lLSI Europe 2011)
Becker et al (1994) tested 92 infant milk formula samples and 52 of the samples
were B cereus positive Another study by Rowan et al (1997) tested 100 samples of infant
milk formula from United Kingdom and 17 of the samples were B cereus positive In the
study done by Haughton et al (20 I 0) B cereus were detected in 59 out of 100 infant milk
formula samples from Ireland These studies showed that B cereus is commonly found in
most of the infant milk formula and this poses threat to infants who are highly susceptible
24 Polymerase Chain Reaction (PCR)
As ment ioned by Sandra et al (2012) PCR allows rapid detection of pathogens Schraft and
Griffiths (2006) mentioned that the detection of B cereus targetting B cereus per se and
specific sub-groups have been performed by PCR Yamada et al (1999) designed PCR primer
set for gyrB fragment of B cereus and used this primer set in the detection of B cereus in
boiled rice Confirmation of B cereus in typical colonies are performed using PCR techniques
(Rahimi el al 2013) Thousands to millions copies of specific DNA sequence are produced
from the amplification of a single or a few copies of a piece of DNA by PCR (Joshi and
8
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
Food pOlsonmg which may even have lethal effect on human is due to the
inappropriate food preparation and food exposure to 30 middotC (Sandra et at 2012) From year
1998 until 2008 there were 235 cases of foodbome disease outbreaks associated with B
cereus in United States (Bennett et at 2013) In 2011 the number of B cereus foodborne
diseases in Europe showed an increase of 1222 (MesseJhausser et ai 2014) The number
reported shows that B cereus poses a threat to the public health as it is often found in
contaminated food However the number of B cereus foodbome poisoning is believed to be
under reported because this type of food poisoning is mild and reporting these cases is not an
compulsory act (EFSA 2005)
B cereus was recognized as foodbome pathogen since 1955 and it is the cause for
about 2 of all foodbome disease worldwide (Lesley et at 2013) Garcia-Arribas and Kramer
(1990) mentioned that B cereus contribute to economic loss by causing contamination
spoilage and food-poisoning B cereus are found in dairy products meats spices and cereals
(Browne and Dowds 2002) as well as in infant foods and dried milk products (Becker et aI
1994)
Most of the patients involved in B cereus outbreaks were infants or elders (Becker et
ai 1994) The immune system of infants are still developing so they are highly susceptible to
B cereus food poisoning after consuming contaminated infant milk Although infant milk
basically present in low moisture powder form the vegetative cells and spores of
microorganisms are able to survive after drying but with their metabolism reduced (I LSI
Europe 2011) This poses threat to infants because some consumers keep the reconstituted
infant formula at room temperature for a long period of time as they wrongly believed that
infant formula are sterilised and are completely free of B cereus (ILSI Europe 2011)
2
B cereus is conunonly related to dairy products because they are able to survive at
different environmental stresses such as pasteurization (Foltys and Kirchnerova 2006 Sadek
et (II 2006) Schelderman et al (2006) discussed that aerobic spore former such as B cereus
has increased tolerance towards low temperature low pH sterilization and UHT treatments
and these conditions increase the concern in food industry In year 2011 there were food
poisoning associated with UHT milk provided from 1 Malaysia Milk Progranune and B cereus
were detected in these UHT milk (Yusof 2011) In 2013 infant milk formula product from
Fonterra were tested positive for Clostridium botulinum which is known as an endospore
former (Frediansyah 2014) The botulism scare was later confirmed as false alarm and the
actual organism was Clostridium 5porogenes However these pathogens have alarmed public
on the safety ofUHT milk and infant milk Currently there are no known studies in Malaysia
examining the factors influencing the survival of B cereus in UHT milk and infant milk
Hence the objectives of this study were
I To study the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk stored at different temperature for seven days
II To examine the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk with different initial pH stored at refrigerated temperature for seven
days
3
20 Literature Review
21 Bacillus cereus
211 Morphological and biochemical characteristics
Bacillus cereus is a Gram positive motile rod-shaped aerobic or facultative anaerobe and
aerobic spore forming foodborne pathogen belonging to Bacillus genus that is normally found
in soil and vegetation (Tortora et al 2010 Food Standards Australia New Zealand 2013
Gleeson et al 2013) This bacterium is mesophile or psychrotroph This bacterium is closely
related to B thuringiensis B mycoides B weihenstephanensis B pseudomycoides and B
anthracis (Gleeson et al 2013) The ability of B cereus to produce endospores which are heat
resistant increases the survivality of B cereus at unfavourable conditions such as high
temperatures extreme pH values or envirorunents with insufficient nutrients compared to
other bacterial enteropathogens (Di Pinto et al 2013 Gleeson et al 2013) For the
biochemical characteristics B cereus are positive for motility Voges-Proskauer (VP)
reaction starch hydrolysis citrate catalase and negative for oxidase tests (Shinagawa 1990
Choma ef al 2000) B cereus is shown as slender bacilli with square end singly or in short
chains under Gram-staining (Bottone 2010)
212 B cereus food borne disease
Rice dishes and meat or poultry dishes were the common sources for B cereus outbreaks
(Bennett et al 2013) Rice and pasta contaminated with B cereus are the causes for emetic
syndrome while the causes for diarrheal illness are desserts meat dishes and dairy products
(Granum 2005) Endospores of B cereus were able to survive in improperly cooked rice and
they caused Fried Rice Syndrome (Sandra et al 2012) In Malaysia B cereus was fIrst
4
Pusat Khidmat Maklumat Ak d Ut lVERSTTJ MA AYSIA SARAWAK
suspected as the cause for food poisoning which affected 114 female students in Klang
(Rampal et al 1984) In order to confIrm that gastroenteritis is caused by B cereus the
number of B cereus iso lated from suspected food need to exceed 105 co lony- forming unit per
gram (CFUg) (Tortora et al 20[0)
213 Isolation and identification of B cereus
Mannitol egg yolk polymyxin agar (MYF) Kim-Geopfert agar Columbia base-blood
polymyxin B agar (CBA-P) and Polymyxin B-egg yolk-mannitol bromothymol blue agar
(PEMBA) are some of the selective media suitable for isoIation and identifIcation of B cereus
(van Netten and Kramer 1992) The inability to ferment mannitol causes B cereus colonies to
appear as pink on MYP and lecithinase activity enables the observation of precipitated zone
around colonies (Pacheco-Sanchez and Massaguer 2007) Food and Drug Administration
(FDA) recommended MYP agar as the stapdard media for identifIcation of B cereus
214 Production of enterotoxins
The two forms of illness associated with B cereus enterotoxins are diarrheal and emetic
syndromes Onset of watery diarrhea and abdominal pain which resemble Clostridium
pefringens intoxications occur 8 hours to 16 hours after ingestion of contaminated food are
the symptoms of diarrheal outbreak (Shinagawa 1990 Tortora et ai 2010) This diarrheal
syndrome occurs after the ingestion of vegetattve cells which later produce a protein
enterotoxin in the gastrointinal tract (Bottone 2010) This type of enterotoxin is heat-labile
(Bello et al 2014) Haemolysin BL (HBL) non-haemolytic enterotoxin and cytotoxin K are
the three types of enterotoxins produced by this bacterium (Samarzija et al 2012 Food
Standards Australia New Zealand 2013) Reis et al (2013) concluded that hblD was the
5
common HBL gene of B cereus detected in milk and dairy products leading to diarrheal form
ofB cereus foodborne disease
The symptoms of emetic outbreaks are similar to Staphylococcus aureus food
intoxication and occur 05 hour to 6 hours upon ingestion of contaminated food
(Messelhausser et al 2014) The ingestion of food contaminated with preformed cereulide
which is also known as the emetic toxin leads to emetic syndrome (Bottone 2010) This heatshy
stable toxin causes nausea and vomiting in the infected patients (Bello et al2014)
215 Effect of temperature and pH on growth and survival of B cereus
Generally B cereus grows between 4 C and 55 C but exhibit optimum growth between 30 C
and 40 C (Schraft and Griffiths 2006 Food Standards Australia New Zealand 2013) Growth
of B cereus had been detected at temperature ranging from 7 C to 45 C and significant
growth was observed at 45 c in steril ized raw milk samples (Bellow et al 2007) In the
absence of oxygen or at temperature below 10 C emetic B cereus are believed to show
incapability in growth and production of toxin (EFSA 2005) At temperature below 10 C
with the limitation of other factors the lag time and generation times of B cereus are longer
(EFSA 2005)
As mentioned by Food Standards Australia New Zealand (2013) B cereus is able to
grow at pH between pH 49 and pH 100 and the optimum pH is between pH 60 and pH 70
Bello et al (2014) concluded that B cereus was unable to grow at pH 63 to pH 68 for 24
hour in pasteurized whole cow milk and the growth was inhibited at pH 68 for a period of 48
hours Byaruhanga et af (1999) studied that mague base inoculated with B cereus for 24
hours at 3S C was able to grow when pH decreases from pH 65 to pH 50 Brain Heart
6
--~~~----~----~----------
Infusion (BHI) medium at pH 88 inhibits growth of emetic and diarrheal strains of B cereus
better than pH 50 of the same medium (Garcia-Arribas and Kramer 1990)
22 UHT milk
Milk treated with UHT pasteurization is heated to 138 middotC for 2 seconds and aseptically placed
into a sterile substantial container which can be stored without refrigeration for six months to
a year (Ledenbach and Marshall 2009) UHT treatment minimized heat denaturation and
produces long shelf life milk that has similar taste and color to pasteurized milk (Harding
1995) UHT milk is generally contaminated during technological processing because food
pathogens are destroyed by the UHT temperature (Vyletelova et al 2002) Longer storage
period causes the drop of UHT milk pH and enzymes may be the reason for changes in UHT
milk (Hassan et al 2009)
Among the 110 UHT milk samples tested by Vidal-Martins et al (2005) 118
(1 3110) samples were positive for B cereus Researchers Rezende-Lago et al (2007)
identified B cereus in four (13 3) of the UHT milk samples tested In the study performed
by Reis et al (2013) among the 260 samples of UHT milk pasteurized milk and powdered
milk tested in Brazil a total of 63 isolates of B cereus were found and 12 of them were from
UHT milk
23 Infant milk
Cows milk and soy protein are the major component of infant milk or infant formulas For the
production of infant formula from milk the milk is heat-treated concentrated and dried while
infant fonnula from other ingredients is dry-blended wetted and dried again or only dry-
blended (Becker et al 1994) The sublethally injured pathogens can recover after the process
7
of drying because this process caused only little damage to the cell (Miller et ai 2012) During
the process of drying components such as lactose protein and milk fat that are present in
milk-based infant formula have the potential to provide protection to the bacteria (Mi1er et ai
20 12) Infant formulas are given to newborns and the content of nutrition is similar to breast
milk (Becker et al 1994) Currently infant formulas in the market are available in powder
liquid concentration and ready-to-feed After rehydration and inappropriate storage lowshy
moisture foods such as infant milk powder should be consumed within a short time because
growth of microorganisms may occur (lLSI Europe 2011)
Becker et al (1994) tested 92 infant milk formula samples and 52 of the samples
were B cereus positive Another study by Rowan et al (1997) tested 100 samples of infant
milk formula from United Kingdom and 17 of the samples were B cereus positive In the
study done by Haughton et al (20 I 0) B cereus were detected in 59 out of 100 infant milk
formula samples from Ireland These studies showed that B cereus is commonly found in
most of the infant milk formula and this poses threat to infants who are highly susceptible
24 Polymerase Chain Reaction (PCR)
As ment ioned by Sandra et al (2012) PCR allows rapid detection of pathogens Schraft and
Griffiths (2006) mentioned that the detection of B cereus targetting B cereus per se and
specific sub-groups have been performed by PCR Yamada et al (1999) designed PCR primer
set for gyrB fragment of B cereus and used this primer set in the detection of B cereus in
boiled rice Confirmation of B cereus in typical colonies are performed using PCR techniques
(Rahimi el al 2013) Thousands to millions copies of specific DNA sequence are produced
from the amplification of a single or a few copies of a piece of DNA by PCR (Joshi and
8
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
B cereus is conunonly related to dairy products because they are able to survive at
different environmental stresses such as pasteurization (Foltys and Kirchnerova 2006 Sadek
et (II 2006) Schelderman et al (2006) discussed that aerobic spore former such as B cereus
has increased tolerance towards low temperature low pH sterilization and UHT treatments
and these conditions increase the concern in food industry In year 2011 there were food
poisoning associated with UHT milk provided from 1 Malaysia Milk Progranune and B cereus
were detected in these UHT milk (Yusof 2011) In 2013 infant milk formula product from
Fonterra were tested positive for Clostridium botulinum which is known as an endospore
former (Frediansyah 2014) The botulism scare was later confirmed as false alarm and the
actual organism was Clostridium 5porogenes However these pathogens have alarmed public
on the safety ofUHT milk and infant milk Currently there are no known studies in Malaysia
examining the factors influencing the survival of B cereus in UHT milk and infant milk
Hence the objectives of this study were
I To study the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk stored at different temperature for seven days
II To examine the growth and survival of B cereus in artificially contaminated UHT milk
and infant milk with different initial pH stored at refrigerated temperature for seven
days
3
20 Literature Review
21 Bacillus cereus
211 Morphological and biochemical characteristics
Bacillus cereus is a Gram positive motile rod-shaped aerobic or facultative anaerobe and
aerobic spore forming foodborne pathogen belonging to Bacillus genus that is normally found
in soil and vegetation (Tortora et al 2010 Food Standards Australia New Zealand 2013
Gleeson et al 2013) This bacterium is mesophile or psychrotroph This bacterium is closely
related to B thuringiensis B mycoides B weihenstephanensis B pseudomycoides and B
anthracis (Gleeson et al 2013) The ability of B cereus to produce endospores which are heat
resistant increases the survivality of B cereus at unfavourable conditions such as high
temperatures extreme pH values or envirorunents with insufficient nutrients compared to
other bacterial enteropathogens (Di Pinto et al 2013 Gleeson et al 2013) For the
biochemical characteristics B cereus are positive for motility Voges-Proskauer (VP)
reaction starch hydrolysis citrate catalase and negative for oxidase tests (Shinagawa 1990
Choma ef al 2000) B cereus is shown as slender bacilli with square end singly or in short
chains under Gram-staining (Bottone 2010)
212 B cereus food borne disease
Rice dishes and meat or poultry dishes were the common sources for B cereus outbreaks
(Bennett et al 2013) Rice and pasta contaminated with B cereus are the causes for emetic
syndrome while the causes for diarrheal illness are desserts meat dishes and dairy products
(Granum 2005) Endospores of B cereus were able to survive in improperly cooked rice and
they caused Fried Rice Syndrome (Sandra et al 2012) In Malaysia B cereus was fIrst
4
Pusat Khidmat Maklumat Ak d Ut lVERSTTJ MA AYSIA SARAWAK
suspected as the cause for food poisoning which affected 114 female students in Klang
(Rampal et al 1984) In order to confIrm that gastroenteritis is caused by B cereus the
number of B cereus iso lated from suspected food need to exceed 105 co lony- forming unit per
gram (CFUg) (Tortora et al 20[0)
213 Isolation and identification of B cereus
Mannitol egg yolk polymyxin agar (MYF) Kim-Geopfert agar Columbia base-blood
polymyxin B agar (CBA-P) and Polymyxin B-egg yolk-mannitol bromothymol blue agar
(PEMBA) are some of the selective media suitable for isoIation and identifIcation of B cereus
(van Netten and Kramer 1992) The inability to ferment mannitol causes B cereus colonies to
appear as pink on MYP and lecithinase activity enables the observation of precipitated zone
around colonies (Pacheco-Sanchez and Massaguer 2007) Food and Drug Administration
(FDA) recommended MYP agar as the stapdard media for identifIcation of B cereus
214 Production of enterotoxins
The two forms of illness associated with B cereus enterotoxins are diarrheal and emetic
syndromes Onset of watery diarrhea and abdominal pain which resemble Clostridium
pefringens intoxications occur 8 hours to 16 hours after ingestion of contaminated food are
the symptoms of diarrheal outbreak (Shinagawa 1990 Tortora et ai 2010) This diarrheal
syndrome occurs after the ingestion of vegetattve cells which later produce a protein
enterotoxin in the gastrointinal tract (Bottone 2010) This type of enterotoxin is heat-labile
(Bello et al 2014) Haemolysin BL (HBL) non-haemolytic enterotoxin and cytotoxin K are
the three types of enterotoxins produced by this bacterium (Samarzija et al 2012 Food
Standards Australia New Zealand 2013) Reis et al (2013) concluded that hblD was the
5
common HBL gene of B cereus detected in milk and dairy products leading to diarrheal form
ofB cereus foodborne disease
The symptoms of emetic outbreaks are similar to Staphylococcus aureus food
intoxication and occur 05 hour to 6 hours upon ingestion of contaminated food
(Messelhausser et al 2014) The ingestion of food contaminated with preformed cereulide
which is also known as the emetic toxin leads to emetic syndrome (Bottone 2010) This heatshy
stable toxin causes nausea and vomiting in the infected patients (Bello et al2014)
215 Effect of temperature and pH on growth and survival of B cereus
Generally B cereus grows between 4 C and 55 C but exhibit optimum growth between 30 C
and 40 C (Schraft and Griffiths 2006 Food Standards Australia New Zealand 2013) Growth
of B cereus had been detected at temperature ranging from 7 C to 45 C and significant
growth was observed at 45 c in steril ized raw milk samples (Bellow et al 2007) In the
absence of oxygen or at temperature below 10 C emetic B cereus are believed to show
incapability in growth and production of toxin (EFSA 2005) At temperature below 10 C
with the limitation of other factors the lag time and generation times of B cereus are longer
(EFSA 2005)
As mentioned by Food Standards Australia New Zealand (2013) B cereus is able to
grow at pH between pH 49 and pH 100 and the optimum pH is between pH 60 and pH 70
Bello et al (2014) concluded that B cereus was unable to grow at pH 63 to pH 68 for 24
hour in pasteurized whole cow milk and the growth was inhibited at pH 68 for a period of 48
hours Byaruhanga et af (1999) studied that mague base inoculated with B cereus for 24
hours at 3S C was able to grow when pH decreases from pH 65 to pH 50 Brain Heart
6
--~~~----~----~----------
Infusion (BHI) medium at pH 88 inhibits growth of emetic and diarrheal strains of B cereus
better than pH 50 of the same medium (Garcia-Arribas and Kramer 1990)
22 UHT milk
Milk treated with UHT pasteurization is heated to 138 middotC for 2 seconds and aseptically placed
into a sterile substantial container which can be stored without refrigeration for six months to
a year (Ledenbach and Marshall 2009) UHT treatment minimized heat denaturation and
produces long shelf life milk that has similar taste and color to pasteurized milk (Harding
1995) UHT milk is generally contaminated during technological processing because food
pathogens are destroyed by the UHT temperature (Vyletelova et al 2002) Longer storage
period causes the drop of UHT milk pH and enzymes may be the reason for changes in UHT
milk (Hassan et al 2009)
Among the 110 UHT milk samples tested by Vidal-Martins et al (2005) 118
(1 3110) samples were positive for B cereus Researchers Rezende-Lago et al (2007)
identified B cereus in four (13 3) of the UHT milk samples tested In the study performed
by Reis et al (2013) among the 260 samples of UHT milk pasteurized milk and powdered
milk tested in Brazil a total of 63 isolates of B cereus were found and 12 of them were from
UHT milk
23 Infant milk
Cows milk and soy protein are the major component of infant milk or infant formulas For the
production of infant formula from milk the milk is heat-treated concentrated and dried while
infant fonnula from other ingredients is dry-blended wetted and dried again or only dry-
blended (Becker et al 1994) The sublethally injured pathogens can recover after the process
7
of drying because this process caused only little damage to the cell (Miller et ai 2012) During
the process of drying components such as lactose protein and milk fat that are present in
milk-based infant formula have the potential to provide protection to the bacteria (Mi1er et ai
20 12) Infant formulas are given to newborns and the content of nutrition is similar to breast
milk (Becker et al 1994) Currently infant formulas in the market are available in powder
liquid concentration and ready-to-feed After rehydration and inappropriate storage lowshy
moisture foods such as infant milk powder should be consumed within a short time because
growth of microorganisms may occur (lLSI Europe 2011)
Becker et al (1994) tested 92 infant milk formula samples and 52 of the samples
were B cereus positive Another study by Rowan et al (1997) tested 100 samples of infant
milk formula from United Kingdom and 17 of the samples were B cereus positive In the
study done by Haughton et al (20 I 0) B cereus were detected in 59 out of 100 infant milk
formula samples from Ireland These studies showed that B cereus is commonly found in
most of the infant milk formula and this poses threat to infants who are highly susceptible
24 Polymerase Chain Reaction (PCR)
As ment ioned by Sandra et al (2012) PCR allows rapid detection of pathogens Schraft and
Griffiths (2006) mentioned that the detection of B cereus targetting B cereus per se and
specific sub-groups have been performed by PCR Yamada et al (1999) designed PCR primer
set for gyrB fragment of B cereus and used this primer set in the detection of B cereus in
boiled rice Confirmation of B cereus in typical colonies are performed using PCR techniques
(Rahimi el al 2013) Thousands to millions copies of specific DNA sequence are produced
from the amplification of a single or a few copies of a piece of DNA by PCR (Joshi and
8
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
20 Literature Review
21 Bacillus cereus
211 Morphological and biochemical characteristics
Bacillus cereus is a Gram positive motile rod-shaped aerobic or facultative anaerobe and
aerobic spore forming foodborne pathogen belonging to Bacillus genus that is normally found
in soil and vegetation (Tortora et al 2010 Food Standards Australia New Zealand 2013
Gleeson et al 2013) This bacterium is mesophile or psychrotroph This bacterium is closely
related to B thuringiensis B mycoides B weihenstephanensis B pseudomycoides and B
anthracis (Gleeson et al 2013) The ability of B cereus to produce endospores which are heat
resistant increases the survivality of B cereus at unfavourable conditions such as high
temperatures extreme pH values or envirorunents with insufficient nutrients compared to
other bacterial enteropathogens (Di Pinto et al 2013 Gleeson et al 2013) For the
biochemical characteristics B cereus are positive for motility Voges-Proskauer (VP)
reaction starch hydrolysis citrate catalase and negative for oxidase tests (Shinagawa 1990
Choma ef al 2000) B cereus is shown as slender bacilli with square end singly or in short
chains under Gram-staining (Bottone 2010)
212 B cereus food borne disease
Rice dishes and meat or poultry dishes were the common sources for B cereus outbreaks
(Bennett et al 2013) Rice and pasta contaminated with B cereus are the causes for emetic
syndrome while the causes for diarrheal illness are desserts meat dishes and dairy products
(Granum 2005) Endospores of B cereus were able to survive in improperly cooked rice and
they caused Fried Rice Syndrome (Sandra et al 2012) In Malaysia B cereus was fIrst
4
Pusat Khidmat Maklumat Ak d Ut lVERSTTJ MA AYSIA SARAWAK
suspected as the cause for food poisoning which affected 114 female students in Klang
(Rampal et al 1984) In order to confIrm that gastroenteritis is caused by B cereus the
number of B cereus iso lated from suspected food need to exceed 105 co lony- forming unit per
gram (CFUg) (Tortora et al 20[0)
213 Isolation and identification of B cereus
Mannitol egg yolk polymyxin agar (MYF) Kim-Geopfert agar Columbia base-blood
polymyxin B agar (CBA-P) and Polymyxin B-egg yolk-mannitol bromothymol blue agar
(PEMBA) are some of the selective media suitable for isoIation and identifIcation of B cereus
(van Netten and Kramer 1992) The inability to ferment mannitol causes B cereus colonies to
appear as pink on MYP and lecithinase activity enables the observation of precipitated zone
around colonies (Pacheco-Sanchez and Massaguer 2007) Food and Drug Administration
(FDA) recommended MYP agar as the stapdard media for identifIcation of B cereus
214 Production of enterotoxins
The two forms of illness associated with B cereus enterotoxins are diarrheal and emetic
syndromes Onset of watery diarrhea and abdominal pain which resemble Clostridium
pefringens intoxications occur 8 hours to 16 hours after ingestion of contaminated food are
the symptoms of diarrheal outbreak (Shinagawa 1990 Tortora et ai 2010) This diarrheal
syndrome occurs after the ingestion of vegetattve cells which later produce a protein
enterotoxin in the gastrointinal tract (Bottone 2010) This type of enterotoxin is heat-labile
(Bello et al 2014) Haemolysin BL (HBL) non-haemolytic enterotoxin and cytotoxin K are
the three types of enterotoxins produced by this bacterium (Samarzija et al 2012 Food
Standards Australia New Zealand 2013) Reis et al (2013) concluded that hblD was the
5
common HBL gene of B cereus detected in milk and dairy products leading to diarrheal form
ofB cereus foodborne disease
The symptoms of emetic outbreaks are similar to Staphylococcus aureus food
intoxication and occur 05 hour to 6 hours upon ingestion of contaminated food
(Messelhausser et al 2014) The ingestion of food contaminated with preformed cereulide
which is also known as the emetic toxin leads to emetic syndrome (Bottone 2010) This heatshy
stable toxin causes nausea and vomiting in the infected patients (Bello et al2014)
215 Effect of temperature and pH on growth and survival of B cereus
Generally B cereus grows between 4 C and 55 C but exhibit optimum growth between 30 C
and 40 C (Schraft and Griffiths 2006 Food Standards Australia New Zealand 2013) Growth
of B cereus had been detected at temperature ranging from 7 C to 45 C and significant
growth was observed at 45 c in steril ized raw milk samples (Bellow et al 2007) In the
absence of oxygen or at temperature below 10 C emetic B cereus are believed to show
incapability in growth and production of toxin (EFSA 2005) At temperature below 10 C
with the limitation of other factors the lag time and generation times of B cereus are longer
(EFSA 2005)
As mentioned by Food Standards Australia New Zealand (2013) B cereus is able to
grow at pH between pH 49 and pH 100 and the optimum pH is between pH 60 and pH 70
Bello et al (2014) concluded that B cereus was unable to grow at pH 63 to pH 68 for 24
hour in pasteurized whole cow milk and the growth was inhibited at pH 68 for a period of 48
hours Byaruhanga et af (1999) studied that mague base inoculated with B cereus for 24
hours at 3S C was able to grow when pH decreases from pH 65 to pH 50 Brain Heart
6
--~~~----~----~----------
Infusion (BHI) medium at pH 88 inhibits growth of emetic and diarrheal strains of B cereus
better than pH 50 of the same medium (Garcia-Arribas and Kramer 1990)
22 UHT milk
Milk treated with UHT pasteurization is heated to 138 middotC for 2 seconds and aseptically placed
into a sterile substantial container which can be stored without refrigeration for six months to
a year (Ledenbach and Marshall 2009) UHT treatment minimized heat denaturation and
produces long shelf life milk that has similar taste and color to pasteurized milk (Harding
1995) UHT milk is generally contaminated during technological processing because food
pathogens are destroyed by the UHT temperature (Vyletelova et al 2002) Longer storage
period causes the drop of UHT milk pH and enzymes may be the reason for changes in UHT
milk (Hassan et al 2009)
Among the 110 UHT milk samples tested by Vidal-Martins et al (2005) 118
(1 3110) samples were positive for B cereus Researchers Rezende-Lago et al (2007)
identified B cereus in four (13 3) of the UHT milk samples tested In the study performed
by Reis et al (2013) among the 260 samples of UHT milk pasteurized milk and powdered
milk tested in Brazil a total of 63 isolates of B cereus were found and 12 of them were from
UHT milk
23 Infant milk
Cows milk and soy protein are the major component of infant milk or infant formulas For the
production of infant formula from milk the milk is heat-treated concentrated and dried while
infant fonnula from other ingredients is dry-blended wetted and dried again or only dry-
blended (Becker et al 1994) The sublethally injured pathogens can recover after the process
7
of drying because this process caused only little damage to the cell (Miller et ai 2012) During
the process of drying components such as lactose protein and milk fat that are present in
milk-based infant formula have the potential to provide protection to the bacteria (Mi1er et ai
20 12) Infant formulas are given to newborns and the content of nutrition is similar to breast
milk (Becker et al 1994) Currently infant formulas in the market are available in powder
liquid concentration and ready-to-feed After rehydration and inappropriate storage lowshy
moisture foods such as infant milk powder should be consumed within a short time because
growth of microorganisms may occur (lLSI Europe 2011)
Becker et al (1994) tested 92 infant milk formula samples and 52 of the samples
were B cereus positive Another study by Rowan et al (1997) tested 100 samples of infant
milk formula from United Kingdom and 17 of the samples were B cereus positive In the
study done by Haughton et al (20 I 0) B cereus were detected in 59 out of 100 infant milk
formula samples from Ireland These studies showed that B cereus is commonly found in
most of the infant milk formula and this poses threat to infants who are highly susceptible
24 Polymerase Chain Reaction (PCR)
As ment ioned by Sandra et al (2012) PCR allows rapid detection of pathogens Schraft and
Griffiths (2006) mentioned that the detection of B cereus targetting B cereus per se and
specific sub-groups have been performed by PCR Yamada et al (1999) designed PCR primer
set for gyrB fragment of B cereus and used this primer set in the detection of B cereus in
boiled rice Confirmation of B cereus in typical colonies are performed using PCR techniques
(Rahimi el al 2013) Thousands to millions copies of specific DNA sequence are produced
from the amplification of a single or a few copies of a piece of DNA by PCR (Joshi and
8
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
Pusat Khidmat Maklumat Ak d Ut lVERSTTJ MA AYSIA SARAWAK
suspected as the cause for food poisoning which affected 114 female students in Klang
(Rampal et al 1984) In order to confIrm that gastroenteritis is caused by B cereus the
number of B cereus iso lated from suspected food need to exceed 105 co lony- forming unit per
gram (CFUg) (Tortora et al 20[0)
213 Isolation and identification of B cereus
Mannitol egg yolk polymyxin agar (MYF) Kim-Geopfert agar Columbia base-blood
polymyxin B agar (CBA-P) and Polymyxin B-egg yolk-mannitol bromothymol blue agar
(PEMBA) are some of the selective media suitable for isoIation and identifIcation of B cereus
(van Netten and Kramer 1992) The inability to ferment mannitol causes B cereus colonies to
appear as pink on MYP and lecithinase activity enables the observation of precipitated zone
around colonies (Pacheco-Sanchez and Massaguer 2007) Food and Drug Administration
(FDA) recommended MYP agar as the stapdard media for identifIcation of B cereus
214 Production of enterotoxins
The two forms of illness associated with B cereus enterotoxins are diarrheal and emetic
syndromes Onset of watery diarrhea and abdominal pain which resemble Clostridium
pefringens intoxications occur 8 hours to 16 hours after ingestion of contaminated food are
the symptoms of diarrheal outbreak (Shinagawa 1990 Tortora et ai 2010) This diarrheal
syndrome occurs after the ingestion of vegetattve cells which later produce a protein
enterotoxin in the gastrointinal tract (Bottone 2010) This type of enterotoxin is heat-labile
(Bello et al 2014) Haemolysin BL (HBL) non-haemolytic enterotoxin and cytotoxin K are
the three types of enterotoxins produced by this bacterium (Samarzija et al 2012 Food
Standards Australia New Zealand 2013) Reis et al (2013) concluded that hblD was the
5
common HBL gene of B cereus detected in milk and dairy products leading to diarrheal form
ofB cereus foodborne disease
The symptoms of emetic outbreaks are similar to Staphylococcus aureus food
intoxication and occur 05 hour to 6 hours upon ingestion of contaminated food
(Messelhausser et al 2014) The ingestion of food contaminated with preformed cereulide
which is also known as the emetic toxin leads to emetic syndrome (Bottone 2010) This heatshy
stable toxin causes nausea and vomiting in the infected patients (Bello et al2014)
215 Effect of temperature and pH on growth and survival of B cereus
Generally B cereus grows between 4 C and 55 C but exhibit optimum growth between 30 C
and 40 C (Schraft and Griffiths 2006 Food Standards Australia New Zealand 2013) Growth
of B cereus had been detected at temperature ranging from 7 C to 45 C and significant
growth was observed at 45 c in steril ized raw milk samples (Bellow et al 2007) In the
absence of oxygen or at temperature below 10 C emetic B cereus are believed to show
incapability in growth and production of toxin (EFSA 2005) At temperature below 10 C
with the limitation of other factors the lag time and generation times of B cereus are longer
(EFSA 2005)
As mentioned by Food Standards Australia New Zealand (2013) B cereus is able to
grow at pH between pH 49 and pH 100 and the optimum pH is between pH 60 and pH 70
Bello et al (2014) concluded that B cereus was unable to grow at pH 63 to pH 68 for 24
hour in pasteurized whole cow milk and the growth was inhibited at pH 68 for a period of 48
hours Byaruhanga et af (1999) studied that mague base inoculated with B cereus for 24
hours at 3S C was able to grow when pH decreases from pH 65 to pH 50 Brain Heart
6
--~~~----~----~----------
Infusion (BHI) medium at pH 88 inhibits growth of emetic and diarrheal strains of B cereus
better than pH 50 of the same medium (Garcia-Arribas and Kramer 1990)
22 UHT milk
Milk treated with UHT pasteurization is heated to 138 middotC for 2 seconds and aseptically placed
into a sterile substantial container which can be stored without refrigeration for six months to
a year (Ledenbach and Marshall 2009) UHT treatment minimized heat denaturation and
produces long shelf life milk that has similar taste and color to pasteurized milk (Harding
1995) UHT milk is generally contaminated during technological processing because food
pathogens are destroyed by the UHT temperature (Vyletelova et al 2002) Longer storage
period causes the drop of UHT milk pH and enzymes may be the reason for changes in UHT
milk (Hassan et al 2009)
Among the 110 UHT milk samples tested by Vidal-Martins et al (2005) 118
(1 3110) samples were positive for B cereus Researchers Rezende-Lago et al (2007)
identified B cereus in four (13 3) of the UHT milk samples tested In the study performed
by Reis et al (2013) among the 260 samples of UHT milk pasteurized milk and powdered
milk tested in Brazil a total of 63 isolates of B cereus were found and 12 of them were from
UHT milk
23 Infant milk
Cows milk and soy protein are the major component of infant milk or infant formulas For the
production of infant formula from milk the milk is heat-treated concentrated and dried while
infant fonnula from other ingredients is dry-blended wetted and dried again or only dry-
blended (Becker et al 1994) The sublethally injured pathogens can recover after the process
7
of drying because this process caused only little damage to the cell (Miller et ai 2012) During
the process of drying components such as lactose protein and milk fat that are present in
milk-based infant formula have the potential to provide protection to the bacteria (Mi1er et ai
20 12) Infant formulas are given to newborns and the content of nutrition is similar to breast
milk (Becker et al 1994) Currently infant formulas in the market are available in powder
liquid concentration and ready-to-feed After rehydration and inappropriate storage lowshy
moisture foods such as infant milk powder should be consumed within a short time because
growth of microorganisms may occur (lLSI Europe 2011)
Becker et al (1994) tested 92 infant milk formula samples and 52 of the samples
were B cereus positive Another study by Rowan et al (1997) tested 100 samples of infant
milk formula from United Kingdom and 17 of the samples were B cereus positive In the
study done by Haughton et al (20 I 0) B cereus were detected in 59 out of 100 infant milk
formula samples from Ireland These studies showed that B cereus is commonly found in
most of the infant milk formula and this poses threat to infants who are highly susceptible
24 Polymerase Chain Reaction (PCR)
As ment ioned by Sandra et al (2012) PCR allows rapid detection of pathogens Schraft and
Griffiths (2006) mentioned that the detection of B cereus targetting B cereus per se and
specific sub-groups have been performed by PCR Yamada et al (1999) designed PCR primer
set for gyrB fragment of B cereus and used this primer set in the detection of B cereus in
boiled rice Confirmation of B cereus in typical colonies are performed using PCR techniques
(Rahimi el al 2013) Thousands to millions copies of specific DNA sequence are produced
from the amplification of a single or a few copies of a piece of DNA by PCR (Joshi and
8
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
common HBL gene of B cereus detected in milk and dairy products leading to diarrheal form
ofB cereus foodborne disease
The symptoms of emetic outbreaks are similar to Staphylococcus aureus food
intoxication and occur 05 hour to 6 hours upon ingestion of contaminated food
(Messelhausser et al 2014) The ingestion of food contaminated with preformed cereulide
which is also known as the emetic toxin leads to emetic syndrome (Bottone 2010) This heatshy
stable toxin causes nausea and vomiting in the infected patients (Bello et al2014)
215 Effect of temperature and pH on growth and survival of B cereus
Generally B cereus grows between 4 C and 55 C but exhibit optimum growth between 30 C
and 40 C (Schraft and Griffiths 2006 Food Standards Australia New Zealand 2013) Growth
of B cereus had been detected at temperature ranging from 7 C to 45 C and significant
growth was observed at 45 c in steril ized raw milk samples (Bellow et al 2007) In the
absence of oxygen or at temperature below 10 C emetic B cereus are believed to show
incapability in growth and production of toxin (EFSA 2005) At temperature below 10 C
with the limitation of other factors the lag time and generation times of B cereus are longer
(EFSA 2005)
As mentioned by Food Standards Australia New Zealand (2013) B cereus is able to
grow at pH between pH 49 and pH 100 and the optimum pH is between pH 60 and pH 70
Bello et al (2014) concluded that B cereus was unable to grow at pH 63 to pH 68 for 24
hour in pasteurized whole cow milk and the growth was inhibited at pH 68 for a period of 48
hours Byaruhanga et af (1999) studied that mague base inoculated with B cereus for 24
hours at 3S C was able to grow when pH decreases from pH 65 to pH 50 Brain Heart
6
--~~~----~----~----------
Infusion (BHI) medium at pH 88 inhibits growth of emetic and diarrheal strains of B cereus
better than pH 50 of the same medium (Garcia-Arribas and Kramer 1990)
22 UHT milk
Milk treated with UHT pasteurization is heated to 138 middotC for 2 seconds and aseptically placed
into a sterile substantial container which can be stored without refrigeration for six months to
a year (Ledenbach and Marshall 2009) UHT treatment minimized heat denaturation and
produces long shelf life milk that has similar taste and color to pasteurized milk (Harding
1995) UHT milk is generally contaminated during technological processing because food
pathogens are destroyed by the UHT temperature (Vyletelova et al 2002) Longer storage
period causes the drop of UHT milk pH and enzymes may be the reason for changes in UHT
milk (Hassan et al 2009)
Among the 110 UHT milk samples tested by Vidal-Martins et al (2005) 118
(1 3110) samples were positive for B cereus Researchers Rezende-Lago et al (2007)
identified B cereus in four (13 3) of the UHT milk samples tested In the study performed
by Reis et al (2013) among the 260 samples of UHT milk pasteurized milk and powdered
milk tested in Brazil a total of 63 isolates of B cereus were found and 12 of them were from
UHT milk
23 Infant milk
Cows milk and soy protein are the major component of infant milk or infant formulas For the
production of infant formula from milk the milk is heat-treated concentrated and dried while
infant fonnula from other ingredients is dry-blended wetted and dried again or only dry-
blended (Becker et al 1994) The sublethally injured pathogens can recover after the process
7
of drying because this process caused only little damage to the cell (Miller et ai 2012) During
the process of drying components such as lactose protein and milk fat that are present in
milk-based infant formula have the potential to provide protection to the bacteria (Mi1er et ai
20 12) Infant formulas are given to newborns and the content of nutrition is similar to breast
milk (Becker et al 1994) Currently infant formulas in the market are available in powder
liquid concentration and ready-to-feed After rehydration and inappropriate storage lowshy
moisture foods such as infant milk powder should be consumed within a short time because
growth of microorganisms may occur (lLSI Europe 2011)
Becker et al (1994) tested 92 infant milk formula samples and 52 of the samples
were B cereus positive Another study by Rowan et al (1997) tested 100 samples of infant
milk formula from United Kingdom and 17 of the samples were B cereus positive In the
study done by Haughton et al (20 I 0) B cereus were detected in 59 out of 100 infant milk
formula samples from Ireland These studies showed that B cereus is commonly found in
most of the infant milk formula and this poses threat to infants who are highly susceptible
24 Polymerase Chain Reaction (PCR)
As ment ioned by Sandra et al (2012) PCR allows rapid detection of pathogens Schraft and
Griffiths (2006) mentioned that the detection of B cereus targetting B cereus per se and
specific sub-groups have been performed by PCR Yamada et al (1999) designed PCR primer
set for gyrB fragment of B cereus and used this primer set in the detection of B cereus in
boiled rice Confirmation of B cereus in typical colonies are performed using PCR techniques
(Rahimi el al 2013) Thousands to millions copies of specific DNA sequence are produced
from the amplification of a single or a few copies of a piece of DNA by PCR (Joshi and
8
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
--~~~----~----~----------
Infusion (BHI) medium at pH 88 inhibits growth of emetic and diarrheal strains of B cereus
better than pH 50 of the same medium (Garcia-Arribas and Kramer 1990)
22 UHT milk
Milk treated with UHT pasteurization is heated to 138 middotC for 2 seconds and aseptically placed
into a sterile substantial container which can be stored without refrigeration for six months to
a year (Ledenbach and Marshall 2009) UHT treatment minimized heat denaturation and
produces long shelf life milk that has similar taste and color to pasteurized milk (Harding
1995) UHT milk is generally contaminated during technological processing because food
pathogens are destroyed by the UHT temperature (Vyletelova et al 2002) Longer storage
period causes the drop of UHT milk pH and enzymes may be the reason for changes in UHT
milk (Hassan et al 2009)
Among the 110 UHT milk samples tested by Vidal-Martins et al (2005) 118
(1 3110) samples were positive for B cereus Researchers Rezende-Lago et al (2007)
identified B cereus in four (13 3) of the UHT milk samples tested In the study performed
by Reis et al (2013) among the 260 samples of UHT milk pasteurized milk and powdered
milk tested in Brazil a total of 63 isolates of B cereus were found and 12 of them were from
UHT milk
23 Infant milk
Cows milk and soy protein are the major component of infant milk or infant formulas For the
production of infant formula from milk the milk is heat-treated concentrated and dried while
infant fonnula from other ingredients is dry-blended wetted and dried again or only dry-
blended (Becker et al 1994) The sublethally injured pathogens can recover after the process
7
of drying because this process caused only little damage to the cell (Miller et ai 2012) During
the process of drying components such as lactose protein and milk fat that are present in
milk-based infant formula have the potential to provide protection to the bacteria (Mi1er et ai
20 12) Infant formulas are given to newborns and the content of nutrition is similar to breast
milk (Becker et al 1994) Currently infant formulas in the market are available in powder
liquid concentration and ready-to-feed After rehydration and inappropriate storage lowshy
moisture foods such as infant milk powder should be consumed within a short time because
growth of microorganisms may occur (lLSI Europe 2011)
Becker et al (1994) tested 92 infant milk formula samples and 52 of the samples
were B cereus positive Another study by Rowan et al (1997) tested 100 samples of infant
milk formula from United Kingdom and 17 of the samples were B cereus positive In the
study done by Haughton et al (20 I 0) B cereus were detected in 59 out of 100 infant milk
formula samples from Ireland These studies showed that B cereus is commonly found in
most of the infant milk formula and this poses threat to infants who are highly susceptible
24 Polymerase Chain Reaction (PCR)
As ment ioned by Sandra et al (2012) PCR allows rapid detection of pathogens Schraft and
Griffiths (2006) mentioned that the detection of B cereus targetting B cereus per se and
specific sub-groups have been performed by PCR Yamada et al (1999) designed PCR primer
set for gyrB fragment of B cereus and used this primer set in the detection of B cereus in
boiled rice Confirmation of B cereus in typical colonies are performed using PCR techniques
(Rahimi el al 2013) Thousands to millions copies of specific DNA sequence are produced
from the amplification of a single or a few copies of a piece of DNA by PCR (Joshi and
8
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
of drying because this process caused only little damage to the cell (Miller et ai 2012) During
the process of drying components such as lactose protein and milk fat that are present in
milk-based infant formula have the potential to provide protection to the bacteria (Mi1er et ai
20 12) Infant formulas are given to newborns and the content of nutrition is similar to breast
milk (Becker et al 1994) Currently infant formulas in the market are available in powder
liquid concentration and ready-to-feed After rehydration and inappropriate storage lowshy
moisture foods such as infant milk powder should be consumed within a short time because
growth of microorganisms may occur (lLSI Europe 2011)
Becker et al (1994) tested 92 infant milk formula samples and 52 of the samples
were B cereus positive Another study by Rowan et al (1997) tested 100 samples of infant
milk formula from United Kingdom and 17 of the samples were B cereus positive In the
study done by Haughton et al (20 I 0) B cereus were detected in 59 out of 100 infant milk
formula samples from Ireland These studies showed that B cereus is commonly found in
most of the infant milk formula and this poses threat to infants who are highly susceptible
24 Polymerase Chain Reaction (PCR)
As ment ioned by Sandra et al (2012) PCR allows rapid detection of pathogens Schraft and
Griffiths (2006) mentioned that the detection of B cereus targetting B cereus per se and
specific sub-groups have been performed by PCR Yamada et al (1999) designed PCR primer
set for gyrB fragment of B cereus and used this primer set in the detection of B cereus in
boiled rice Confirmation of B cereus in typical colonies are performed using PCR techniques
(Rahimi el al 2013) Thousands to millions copies of specific DNA sequence are produced
from the amplification of a single or a few copies of a piece of DNA by PCR (Joshi and
8
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
Deshpande 2010) Garibyan and Avashia (2013) mentioned that template DNA primers
nucleotides and DNA polymerase are essential for every peR assay During peR the
part icular DNA sequence first undergo denaturation followed by aIU1ealing and extension to
generate large amount of copies of the specific DNA fragment (Garibyan and Avashia 2013)
The peR product is mostly analyzed using agarose gel electrophoresis which separates the
DNA based on size and charge (Lee and Bahaman 2012 Garibyan and Avashia 2013)
9
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
30 Materials and Mjethods
The materials and apparatus used in this study is as listed in Appendix A
31 The milk
UHT milk (n=l) and infant milk powder (n=l) were purchased from local supermarkets and
tored at room temperature In order to reconstitute the infant milk powder 25 grams of infant
milk powder was dissolved in 225 millilitres (ml) of sterile water at 56 middotC by shaking until it
was mixed homogenously (Rowan and Anderson 1998) In this experiment 9 m1 of UHT
milk and infant milk samples were transferred into sterilized falcon tubes
32 Inocula preparation
Modified inocula preparation from Little and Kn0chel (1994) was performed by transferring
one loopful of pure culture of Bacillus cereus ATCC 33019 into 5 ml of BHI broth The
culture was incubated at 37 middotC for 18 hours to late exponential phase (Little and Kn0chel
1994) Enumeration of B cereus was performed using the modified method described by
Little and Kn0chel (1994) where I ml of BHI broth containing B cereus was serially diluted
in 01 peptone water before plating on Cereus selective agar base acc to MOSSEL (MYP
agar) spread plates at 37 middotC for 24 hours
33 urvival and growth of B cereus in UHT milk and infant milk
331 Effect of Temperature
UHT mi lk and infant milk with a volume of 9 ml were inoculated with 1 ml of appropriately
diluted B cereus inoculum to give population of approximately 103 colony-forming units per
10
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
millilitre (CFUml) Negative control and inoculated milk samples were subsequently
incubated at 4 middotC-7 middotC 10 middotC- 3 middotC 25 middotC 35 middotC 45 middotC and 55middotC Analysis on the samples
incubated at different temperatures were performed daily for seven days
332 Effect of pH
UHT milk and infant milk with a volume of 9 ml were inoculated with 1 rnl of diluted B
cereus inoculum to give population of approximately 103 CFUml Prior to inoculation the
original pH of UHT milk and infant milk samples were measured using pH meter (CyberScan
pH 510 Eutech Instruments Singapore) According to the modified method described by
Bello et aJ (2014) the initial pH 0 f inoculated and non-inoculated milk samples were adjusted
with either hydrochloric acid (HCI) or sodium hydroxide (NaOH) to pH 620 pH 640 pH
660 and pH 680 respectively The survival of B cereus in inoculated milk samples incubated
at refrigerated temperature (4 middotC-7 C) for seen days were analyzed daily
34 Determination of growth and survival of B cereus in UHT milk and infant milk
samples
Using the modified method described by Beuchat et al (1997) 01 rnl of homogenized
samples were serially diluted in sterile 01 peptone and spread plated onto MYP agar plates
in triplicate using steriled hockey stick Enumeration of B cereus was performed using the
modified method described by Lesley et al (2013) The spread plates were incubated at 37 middotC
for 24 hours Observations on MYP agar plates were performed to inspect for pinkish white
colonies and the colony count were expressed in CFUml Gram staining and Polymerase
Chain Reaction (PCR) assay were performed for the confirmation of B cereus isolates
11
