INSTANT NOODLES OF PROPER CRITICAL BI …mulinet11.li.mahidol.ac.th/e-thesis/scan/4236133.pdfPoints at the processing line of instant noodle seasoning were identified. ... 5.1 Questionnaire

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EF'F'ECT OF INSTANT NOODLES PREPARATION ANDIDENTIFICATION OF THE PROPER CRITICAL CONTROL

POINTS FOR CONTROLLING BI CILLUS CEREUSCONTAMINATION IN INSTANT NOODLE SEASONING

JIRAPORN SONYIM=

A THESIS SUBMITTED IN PARTIAL FULFILLMENTOF THE REQUIREMENTS FOR

THE DEGREE OF MASTER OF SCIENCE(FOOD AND NUTRITIONAL TOXICOLOGD

FACT]LTY OF GRADUATE STIJ'DTESMAHIDOL UNIYERSITY

2003

ISBN 974-04-3154-2COPYRIGHT OF MAHIDOL UNIVERSITY

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Copyright by Mahidol University

Thesisentitled

EFFECT OF INSTANT NOODLES PREPARATION ANDIDENTIFICATION OF THE PROPER CzuTICAL CONTROL POINTS

FOR CONTROLLING BACILLUS CEREUS CONTAMINATION ININSTANT NOODLE SEASONING

4t/ l{n 761n ?6 Yiyl.'"""""i"""'

Miss Jirapom SonfmCandidate

4r,P*,' /h";,"" ""'f"""'.Assoc. Prof. Prapasri Puwastien,Ph.D. (Food Technology)Major-Advisor

O-"S\"--L.,,..^€..I..............Lect. Chittima Shinghavanich,M.Sc. (Food Science and Technology)Co-Advisor

P^,.7,1"/-f*,kr"krll;; ili;i;;;#"y,k iM.Sc. (Food Science)Co-Advisor

Master of Science Programme in FoodAnd Nutritional Toxicologylnstitute of Nukition

Ph.D.DeanFaculty of Graduate Studies


,ffii::EFFECT OF INSTANT NOODLES PREPARATION AND

IDENTIFICATION OF THE PROPER CRITICAL CONTROL POINTSFOR CONTROLLING BACILL{JS CEREUS CONTAMINATION IN

INSTANT NOODLE SEASONING

was submitted to the Faculty_of Graduate Studies, Mahidol UniversityFor the degree of Master of Science (Food and Nutrition Toxicologyj

4 April, 2003

,(.)i{ro,',, aeni,u...,.r..r.1...:........,./.....,.....

Miss Jirapom SonyiirCandidate

4ry*, ,'(r^-rtr-Assoc. Prof. Prapasri puwastien,Ph.D. (Food Technology)Chair

O - \', *1\^.o-.,".; "/-t.'....'.'....'.Lect. Chittima Shinghavanich,M.Sc- (Food Science and Technology)Member

A T,i.l,l^lhnukul...'...r........Lect. Renu Twichatw"r tayakul,M.Sc. (Food Science)Member

Assoc. Prof. Rassmidara Hoonsawat, Ass'oc. Prof. Songsak Srianujaia. Ph.D.Ph.D.DeanFaculty of Graduate StudiesMahidol University

DirecterInstitute of NutritionMahidol University


ACKNOWLEDGEMENT

I wish to attribute the success of this thesis to the extensive support and

assistance from my major advisor, Assoc. prof. prapasri puwastien. My deepest

gatitude and sincere appreciation are credited to her, who kindly adviced instructed

and guided me throughout out this research.

Grateful acknowledgements are likewise extended to my co_advisor, lect.

chittima Singhavanich and Lect. Renu Twichatwitayakul for their valuable advices

and constructive criticisms.

Special appreciation is also offered to Asst. prof Apinya Assavanig for her

kindness in serving as the thesis committee and the extemals examine of the thesis

defense. Her valuable comments and suggestions are much appreciated.

Most sincerely, I would like to affirm my gratefulness to all staff in Food

Micro lab of INMU for their facilitation in this research.

My thanks are also extened to lect. Nipa and lect. Wanpen who guided me

for statistical techniques used in this study.

I deeply grateful to three noodle manufactures for their concem in food safty

for consumers. All samples explored in this thesis were kindly supported by them.

My thanks is due to all friends for their love, care, and encouragement.

Finally, my family, my father and mother, deserve my utmost gatitude for their love,

mental support and all assistance during my entire study period.

Jirapom Sonyim


Fac. OfGrad. Studies, Mahidol Univ. Thesis / iv

EFFECT OF INSTANT NOODLES PREPARATION AND IDENTIFICATION OFTHE PROPER CRITICAL CONTROL POINTS FOR CONTROLLING BACILLUSC'REUS CONTAMINATION IN INSTANT NOODLE SEASONING

JIRAPORN SONYIM 4236133 NUFT/M

M.Sc. (FOOD AND NUTRITION TOXICOLOGY)

THESIS ADVISOR : PRAPASRI PUWASTIEN,.Ph.D., CHITTIMASINGHAVANICH, M.Sc., RENU TWICHATWITAYAKUL, M.Sc.

ABSTRACTInstant noodle are a food consumed worldwide, especially in ASEAN.

Bacillus cereus conlamination was detected in various brands of instant noodleseasonings (abott 20%o of the examined samples) during the last seven years. In thisstudy the eating habit of instant noodle were surveyed. The status of Bacillus cereuscontamination in the three most popular instant noodles with the most favouriteflavours (minced pork, hot and sour-shrimp and chicken soup) were determined. Theelfect of cooking methods on the log reduction of Bacillus cereus in the inoculatedseasoning was evaluated. The contaminated sources of B. cereus and critical controlPoints at the processing line of instant noodle seasoning were identified.

B. cereus contamination was found in all studied brands of instant noodles,with a slightly higher percentage (about 26%) than the previous report. The averagepercentage of B. cereus contaminated seasoning of minced pork and chicken flavourswas twice (about 35 %) as much of the hot and sour-shrimp ilavolr (17o/o) with thehighest level of B. cereus found in the minced pork flavour at 23 MpN/g. Cookingmethods could not eliminate the spore of -8. cereus in the contaminated instant noodle.Cooking by mixing instant noodles with its seasoning, then pouring boiling water andleaving covered for 3 min, as instructed on most of the label of instant noodles,showed the highest reduction (25%), reducing Bacillus cereus by 1.37 log CFU/g.This effect was similar to cooking noodles in boiling water for 2 mins aad then addingthe seasoning. Dried spring onion showed the highest risk ofB. cereus contarrinationamong other ingredients, with 100% detection and at the highest level, 460 MpN/g.critical control Points for the processing lines of instant noodle ingredients - chili,garlic and spring onion - were identified. Modifications ofpractices were proposed insome steps to increase the effectiveness in preventing the contamination, sporegermination and outgrowth and the enterotoxin production ofBa cillus cereus.

KEY WORDS : BACILLUS CEREUS / INSTANT NOODLES / SEASONING/

CRTTICAL CONTROL POINT

106 P. ISBN 974-04-31s4-2Copyright by Mahidol University

Fac. Of Grad. Studies, Mahidol Univ. Thesis / v

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EFFECT OF INSTANT NOODLES PREPARATION AND IDENTIFICATION OFTHE PROPER CRITICAL CONTROL POINTS FOR CONTROLLING BACILLUSCER''US CONTAMINATION IN INSTANT NOODLE SEASONING

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CONTENTS

ACKNOWLEDGEMENTS

ABSTRACT

LIST OF TABLES

LIST OF FIGURES

CHAPTER

Page

iii

iv

x

xii

1

3

4

4

4

6

6

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7

7

7

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o

10

11

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t2

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t4

I INTRODUCTION

II OBJECTIVES

III LITERATIIRE REVIEW

3.1 Instant noodle products

3.1.1 Process of instant noodle

3.2 Seasoning

3.2.1 Seasoningproduction

3.3 Bacillus cereus

3.3. I Morphology

3.3.2 Growth factor

3.3.3 Clinical manifestation

3.3.3. 1 Gastrointestinal disease

3.3.3.2 Non-Gastrointestinal disease

3.3.4 Foodbomeoutbreaks

3.4 HACCP assurance system

3.4.1 The advantage of HACCP program

3.4.2 History of HACCP system

3.5 HACCP implementation in Thai lood manufacturer

3.6 Prerequisite progam for HACCP

3.6.1 Good Manufacturing Practices (GMP)

3.6.2 Standard Sanitation Operating Procedures (SSOps)


vll

CONTENTS (Cont.)

IV

3.7 HACCP principle

3.8 Application of HACCP

MATERIALS AND METHODS

4.1 Questionnairesurvey

4.2 Determination of Bacillus cereus in seasoning of

instant noodle

4.2. 1 Selection of three seasoning brand and flavour 20

4.2.2 Sampling plan 2l

4.2.3 Sample preparation 2l

4.2.4 Determination of -B. cereus in seasoning 2l

of instant noodle

4.2.4.1 Stock culture preparation 21

4.2.4.2 MPN technique 22

4.3 Eflect ofhome preparation ofinstant noodle on 22

B ac i I lus cer eus r eduction

4.3.1 Test sample 22

4.3.2 Preparation ofspore suspension of B. cereus 22

4.3.3 Cooking ofinstant noodle 234.3.4 Enumeration and identification ofsurvival of 24

Bacillus cereus

4.35 Statistical analysis 25

4.4 Identihcation ofproper critical control points 25

for B. cereus in the production line of instant noodle

seasoning

4.4.1 Communication with the manufacturers. 25

4.4.2 Development of HACCP model, followin1 26

Principle 1 and 2, for determination

of Critical Control Points

Page

15

16

20

20

20


CONTENTS (Cont.)

4.4.2.1 B. cereus analysis of ingredients

4.4.2.2 Development of HACCP model

RESULTS

5.1 Questionnaire survey

5. i.1 Popular brands and flavours ofinstant noodle

5.1 .2 Preparation and eating habit of instant noodle

5.2 B. cereus contamination in the seasoning olinstant noodle

5.3 Effect of different methods of home cooking on

B. cereus reduction

5.4 Proper critical control points for.B. cereus in

the production line of instant noodle seasoning

5.4.1 B. cereus contamination in ingredient and the

seasoning of instant noodle

5..4.2 Development of HACCP model for identification 40

oI Critical Control Point

5.4.2.1 Establishment and description ofprocess 40

flow diagram of minced pork flavour

seasoning

5.4.2.2 Product description

5.4.2.3 Ingredient description

5.4.2.4 Process description

5.4.2.5 Hazard analysis and control measure

5.4.2.6 Identifi cation of Critical Control Point

Page

26

Zh

32

32

32

35

35

37

39

39

42

42

51

64


lx

CONTENTS (Cont..1

VI DISCUSSION

6.1 Popular brands and flavours of instant noodle and

characteristics of the consumers

6.2 The status of the B. cereus contamination

in the seasoning ofinstant noodle in finished product

6.3 Effect of different home cooking on the survival

of B. cereus in the most contaminated instant noodles.

6.4 Development of HACCP model for identification

of critical control points

VII CONCLUSION

REFERENCES

APPENDIX

BIOGRAPHY

Page

7l

7t

72

73

76

8l

84

90

106


LIST OF TABLES

Table

1. Comparison of Bacillus cereas ofEnterotoxins

Page

2. The list olfactories were qualified with HACCP system 13

in Thailand in 1998

3. Characteristicsofrespondentconsumers

4. Three most popular brands of instant noodles and

JJ

34

the degree of preference

5. Order olpreference ofseasoning flavours of different brands 34

of instant noodles

6. Per cent of B. cereus contaminated instant noodle seasoning of 36

and range detected level

7. Average percentage B. cereus contuninated seasoning 36

of each flavour

8. Effects oldifferent home cooking methods on the reduction 38

ofinoculated B. cereus in instant noodles

9. Bacillus cereus contamination in 10 lots of lour ingredients 40

before mixing, in mixed products before and after packaging


xl

LIST OF TABLES (Cont.)

Table Page

10. Form 1. Product description 45

11. Form 2. lngredient description 46

12 Form 3. Process description 47

13 Form 4. Hazard analysis 52

14 Form 5. Control measure 59

15 Form 6. Critical Control Point 65

16 Differential characteristics of large-celled Grotp I Bacillus species 103


Figure

1.

2.

J

4

6.

7.

8.

LIST OF FIGURES

Flow diagram of instant noodle production

The CCP Decision Tree

Experimental design

Flow diagram ofinstant noodle seasoning production

flavor: minced pork

Flow diagram of instant noodle seasoning production

flavor: hot and sour-shrimp

Flow diagram of instant noodle seasoning production

flavor: chicken soup

Flow chart of Most Probable Number flow chart

(MPN technique)

Flow chart of confirm biochemical test for Bacillus cereus

Page

5

29

30

44

935.

104

105


Fac. Of Grad. Studies, Mahidol Univ. M.Sc. (Food and Nutritional Toxicology) / I

CHAPTERI

INTRODUCTION

Instant noodle was first consumed in Japan as a national food (r). It becomes

an intemational popular food, and is consumed in more than g0 countries in the world(1). In Thailand, the instant noodle was introduced and later produced in 1970 (2). Itis consumed among people ofall socioeconomic levels in both urban and rural areas ofthe country. The product can be eaten as breakfast, lunch, dinner, or anytime. Theyare sold in ready-to-prepare packages, easy and take a short time to cook, have morechoice of flavour, and good taste. A package of instant noodle (costs 5 BahV55-60 gpackage) is cheaper than other one serving of rhai dishes.(3) In addition of low price,the product has acceptable sensory characteristic and good shelf stability for at least

six months at room temperature. Some protein sources, i.e., egg or meat andvegetables are suggested to be added to increase the balance of the nutrients in theinstant noodles. According to the above characteristics of the instant noodles, itbecomes one of the popular food products. Thailand is one of the top five instantnoodle producers in the world, with annual production of 1.9 billion packages per year(a). six million packages of instant noodle are produced daily and over 90 % are thefried type. Most are consumed domestically. Market growth is 5_10 yo per year (2, 5).

Four food-bome pathogens, Baci w cereus, Salmonella, Clostridiumperfringens and Staphylococcus aureus were determined in several types of foodsamples by Food Microbiorogy Laboratory at Institute of Nutrition Mahidol University(INMU) during 1995-2002. It was found that B. cereus was the most commonorganism found in 20 yo of the examined (213 samples) samples of seasoning but rarein the fried noodles (6, 7).

Bacillus cereus has been recognised for many years as a ubiquitouscontarninant of the environment. Moreover, its role as a foodbome pathogenresponsible for food poisoning has been suspected since the 1950s (g). Two types ofillness are caused by B. cereus. One is a dianheal+ype syndrome, which resulted inCopyright by Mahidol University

Jiraporn Sonyim Introduction / 2

diarrheal and abdominal pain within 8-16 h after the ingestion of contaminated food.

The other is a vomiting-type syndrome, which is characterized by nausea and vomiting

v/ithin 1-5 h of ingestion. B. cereus is an opportunistic pathogen that can cause more

or less severe infection by toxin production (9). It is the spore former and the spore is

heat resistant. The normal cooking temperature cannot kill its spore, so it is important

to control B. cereus in the seasoning of instant noodle. Although food poisoning fiom

B. cereus has not yet been reported fiom consuming of instant noodles, for the safety

assurance of the consumers, especially children, the products are controlled by the

Thai Notification of Public Health, number two hundred and tenth (2000) (10). They

are not allowed to contain any foodbome pathogens, including B. cereus. Therefore, a

systematic research for controlling.B. cereus in the seasoning ol instant noodle should

be carried out.


Fac. OfGrad. Studies, Mahidol Univ. M.Sc. (Food and Nutritional Toxicology) / 3

CHAPTER IIOBJECTIVES

2.1 General objective

The objective of this study was to determine the status of Bacillus cereus

contamination in various brands of instant noodles. Effect of common cooking

methods of instant noodles on survival of B. cereus were determined to study the

feasibility to reduce or eliminate the contaminated organisms. Analysis of potential

hazards for B. cereus contamination and control measures in the production line ofinstant noodle seasoning were also conducted and proper critical control points were

assigrred for controlling Bacillus cereus contamination. Hazard analysis of the

production line of instant noodle seasoning was also studied to indicate proper critical

control points for controlling Bacillus cereus conlamination possible hazard zones ofB. cereus contamination and assign the proper critical control points in order toprevent ard control the contamination of B. cereus.

2.2 Specific Objectives

2'2.1 To survey three most popular brands and flavours of instant noodles and

eating habit ofthe consumers.

2-2.2 To determine the status of B. cereus contamination in the seasonings ofinstant noodles in finished products.

2.2.3. To study the effect of different common home cooking methods on the

redrrction of B. cereus.

2.2.4. To identify potenti al hazards and control measures of B. cereus

contamination and to assign the proper critical control points at the

processing line ofinstant noodle seasoning.


Jirapom Sonyim Literature review / 4

CHAPTER IIILITERATT]RES REVIEWS

3.1 Instant noodle products

The basic ingredients in instant noodles are wheat flour, buck wheat flour, starch

water and salt or kansui (alkaline salt mixtures of sodium carbonate, and sodium

phosphate). ln addition, other ingredients can be added to improve the structures,

textures, and flavours (1). Generally, instant noodles are prepared by adding boiling

water and ready to consume within 3-5 min. The main steps of instant noodle

production are shown in Figure 1.

3.1.1 Process of instant noodle (l t, 12, 13)

Ingredients other than flour are pre-dissolved in water and mixed with

wheat flour. The flour is kneaded in the mixer. The mixing time for common noodles

is about 15-20 min. During mixing the ingredients are uniformly distributed and the

flour particles are hydrated. After mixing, the dough pieces are compressed into a

continuous sheet by repeated passage through pairs of rolls and folded. The folded

sheet is successively reduced to the desired thickness and cut into noodles. The unique

waves of noodles are made by conveying the noodles on a system in which the

traveling conveyor moves more slowly than the cutting rolls above it. Then the wavy

noodle strands pass through a steam machine, exposing temperature around 100 oC for

1-5 min. The steamed noodles are showered with its flavor soup and cut and molded

into one serving size. Next, they are fried at 150- 170 oC for 1-2 min. The noodles are

then quickly cooled and checked for weigh, color, shape, moisture content, and

general quality. The instant noodles are packaged with seasoning and other

ingredients, using films that cannot be permeated by moisture and gases. In drying

noodles, the water activity must be less than 0.7 at 20 oC and salt content less than

14.9 %.



5. Kansui (KrCO3, Na2CO3, ...)

8. Resting for 15min

10. Cutting and molding

I L Streamingat 100 "C, 10 min

13. Forming single serving

14. Frying at 150 -170 "Cfor l-2 min

18. Packing with seasoningand oil package

Figure l. Flow diagram of instant noodle

12. Showenng with soup


Jiraporn Sonyim Literature review / 6

3.2 Seasoning

Seasoning are compounds, containing one or more spices, or spice extract, and

other condiments such as sugar, salt, monosodium glutamate which added to foodduring its manufacture or in its preparation, before it is served. They enhance the

,ature flavors ofthe food and thereby increase their acceptance by the consumer ( ).

The seasonings are used directly or diluted, as in soup. They can be liquid, paste, orpowder. lngredients depending on the product type or flavor (1). Seasoning powder isusually added before a food is ready for serving. Importance factors to consider theproduct quality are the sheen, stability characteristic, freedom from lumping, ease ofdispersion, freedom from off taste and odor. it must have the right texture, an

appealing appearance to the eye and it should be nutritious; it must have the rightviscosity upon the addition ofhot water; it must have at least six months shelf life; itmust be easily packaged (11). There are thousands ofpossible flavour combinations

lor each type of seasoning powder. The flavour of the product itself varies by region.

Hydrolyzed plant proteins and monosodium glutamate are normally used as basic

ingredients for most seasoning powders. It wiI be noted that spring onions appear in

each of the formulas. while other spices are used to improve the flavour, round it out,eliminate blandness, and build up flavour uniqueness. Instant noodles in Thailand

come in several flavors based on the Thai eating culture (2). The sour shrimp and

minced po.k are the most favorite flavor, which share at least 60 % of the market.

3.2.1 Seasoning production

The seasonings of instant noodles are prepared without heating process.

The first step is weighing all of dried ingredients according as its flavor formura,

dividing each ingredient into small pack for mixing. Then the ingredients based on its

flavor formula and quantity are mixed in a big chamber mixer until ho,rogenized, one

batch of mixed seasoning is 300 kilograms each. Finally the mixed seasonings are

transfered to packing room ror single serving packages. The steps of seasoning

production are shown in Appendix 2.


Fac. OfGrad. Srudies. Mahidol Univ. M.Sc. (Food and Nutritional Toxicology) / 7

3.3 Bacillas cereus

Bacillus cereus is an aerobic spore-forming bacterium that is commonly found in

soil, dust, on vegetable, in many raw and processed foods. consumption olfood that

contain more than 10 58. cereus lgmay result in food poisoning. Food incriminated inpast outbreaks include cooked meat and vegetables, boiled or fried rice, vanilla sauce,

custards, soups, spice, spice mixes and raw vegetable spouts (14).

3.3.1 Morphology

Bacillus cereus ate gram-positive rods, square ended, vegetative cell size

1.0 pm x 3.0-5.0 pm. They are facultative aerobic spore-former which spores are oval

or cylinder shapes, central position, do not swell the sporangium. They are active

motile with peritrichous flagella.(14)

3.3.2 Growth factors

The optimum conditions for Bacillus cereus growth is 35 to 40 oC. Some

strains have the ability to grow at low temperatures and can be regarded as

psychrotrophs with a minimal growth temperature of 4 oC, the maximum at 55oC ( 15).

The range of pH is 4.5 to 9.3 (15), and the minimum water activity for growth is 0.92.

Bacillus cereus are sporulated and germinated easily, they have short generation time

about 20 to 30 min. The sporcs of Bacillus cereus are high heat and drfng resistance,

at i00 oC, D-values of there arc 6.7 to 8.3 min in water (14). This bacterium can

produce acid but not gas when they ferment sugar except mannitol . Bacillus cereus

are mildly proteolytic and tolerate high salt condition. The maximum concentration ofsodium chloride for bacterium groMh is 18 %.

3.3.3 Clinical manifestation

3.3.3. 1 Gastrointestinal disease

B. cereus is a fiequent cause of gastroenteritis worldwide. They

can produce up to five different enterotoxins three of which are likely to be involved

in food-bome illnesses. The wellknown association of B. cereus foodborne illness are

the diarrheal and the emetic type (16).



1) The diarrheal syndrome

The dianheal type ofillness is caused by a large molecular weight protein of diarrhea-

genic toxin, which can be inactivated by heat, low pH and proteases. The diarrheal

enterotoxin is produced in the small intestine and elaborate several toxins during the

exponential growth phase, but maximum toxin is lound in the earl1, stationary phase

(17). The diarheal tlpe is characterized by diarrhea and abdominal pain. The onset

of watery diarrhea, abdominal cramps, and pain occurs 6-15 hours after consumption

of contaminated food. Nausea may accompany diarrhea, but vomiting (emesis) rarely

occurs. SlT nptoms persist for 24 hours in most instances. The organism is associated

with a variety of floods, including meat and vegetable dishes, sauces, pastas, dessefis,

and dairy products. The symptoms of B. cereus dianheal type lood poisoning mimic

those of Clostridiurn perfringens food poisoning.(16)

2) The emetic sl,ndrome

The vomiting (emetic) type of illness has been named cereulide. The emetic toxin has

a low molecular weight and consists of a ring structure comprising three repeats offour amino and oxy acids (t 8). The emetic toxin is quite thermostable and remains

active from pH 2 to pH 11. It is thought to be produced by enzymatic synthesis rather

than translation of a gene product. cereulide has been shown to bind to 5-HT3

receptors and thereby activate the vagus nerve, leading to vomiting. It also

deleteriously affects mitochondrial transmembrane potential. The emetic toxin is

released when spore formation during the stationary phase of replication, which has

been associated with a particular flagella serotype called H-1 (19). The victim ingests

food co,taining the preformed toxin and quickly develops nausea, malaise, and

vomiting within 1 to 5 hours, duration of illness is 6 to 24 hours. while the emetic

illness is almost always self-limited and resolves within 24 hours, fulminant liverfailure and death have been reported. The strnptoms of this type of fborl poisoning

parallel those caused by staphylococcus aureus foodbome intoxication. The

implicated food closely associated with rice especially boiled rice that is held forprolonged periods at ambient temperature and then quick-fried before serving.

Although dairy products or other foods such as cereal product are occasionally

responsible. Spores found in uncooked rice germinate and replicate rapidly in cooked


Fac. OfGrad. Studies, Mahidol Univ. M.Sc. (Food and Nutritional Toxicology) ./ 9

rice particularly when it is allowed to cool to room temperature for a period of time

(14, 16, 19). The comparison of B. cereus Enterotoxins are shown in Table l.

Table l. Comparison of Bacillus cereus enterotoxins (15, 20)

3.3.3.2 Non-gastrointestinal disease

B. cereus produces a variety of toxins including enterotoxin,

hemolysin, phospholipase C, and the emetic toxin (21). The necrotizing enterotoxin

has been proposed as the major virulence factor in non-gastrointestinal lesions. The

hemolysins and phospholipases are thought to contribute to tissue injury. various host

factors are thought to play a role in infections by the organism. This organism is the

cause of serious infections of various types, usually, but not always, in immuno-

compromised hosts. Other patient background factors include surgery and other

trauma, bums, intravenous drug abuse, implantation of catheters and various prosthetic

Property Diarrheal Toxin Emetic toxin

Nature Protein, - 40 kDa Peptide, - 5 kDa

Heat stabilities Stable 45 oC, 30 minUnstable 56 oC, 5 min

Stable 126 oC, 90 minHighly stable toxin

pH Most stable pH 4-1 1 Stable pH 2-11

Enrymes Sensitive to pronase, trypsin Resistant to pepsin, trypsin

Storage Unstable Stable

Toxin production Toxin preformed in smallintestine of digester

Toxin preformed in foods

Optimum temp. 32-37'C 25-30 "C

Growth Late exponential phaseMaximum at stationary phase

Spore formation at 35-45 oC

give lesser emetic activityrelated spore formation atstationary phase

Incubation period Diarrhea in 8-16 hr Vomiting in 0.5-5 hr

Infective dose >10s CFU/g > lo3cFuig


Jirapom Sonyim Literature review / l0

devices (including heart valves), and use of hemodialysis and peritoneal dialysis.

These include immunosuppression, indwelling catheters or implants and intravenous

drug use. Types of infections described include septicemia, endocarditis, necrotizing

pneumonia with or without empyema, meningitis, rapidly destructive ophthalmitis,

peritonitis, wound infection, myonecrosis, and osteomyelitis. Myonecrosis has

followed surgery and trauma and maysimulate clostridial myonecrosis (gas gangrene).

In animal test, injection of B. cereus exotoxin into the skin of rabbits causes

increased vascular permeability and necrosis; production of this toxin seems to

correlate with the severity of clinical infection. Rabbit skin necrosis is attributed to

two factors, an enterotoxin and a hemolysin. phospholipase C (lecithinase) isproduced by almost all strains of B. cereus; its pathogenic role is doubtful (22).

3.3.4 Foodborne outbreaks (16)

The dominating type of disease caused by B. cereus differs from

country. In Japan the emetic tlpe is reported about 10 times more frequently than the

diarrheal type (16), while in Europe and North America the diarrheal type is the most

frequently reported (16). since B. cereus food, poisoning is not a reportable disease in

any country, there are very few figures given of the total number of these kinds offood poisoning. Even if the two sytdromes were reportable one would expect

dramatic underreporting, since few seek medical help during the active phase of the

disease, and the patients recover quickly thereafter. only a few countries in Europe

have published convincing data in recent years. B. cereus is the cause of 33%of the

total cases of food poisoning (excluding virus) in Norway (1999-1993) (16), 47%in

iceland (1985-1992),22% in Finland (1992), 8.5% in The Netherlands (1991), and 5%

in Denmark (1990-1992) (16). Much lower numbers have been reported before from

other countries, such as England and Wales (0.7%), Japan (0.8%), USA (1.3%) and,

Canada(2.2%) (16).


Fac. OfGrad. Studies, Mahidol Univ.

3.4 HACCP assurance system

M.Sc. (Food and Nutritional Toxicology) / I I

HACCP is a abbreviation for Hazard Analysis Critical Control point and has

become a btyzword in recent years. It is frequently heard and talked about at

conferences and within companies, but is also often misunderstood and poorly applied

in real situations. The HACCP concept has been around in food industry for some

time but only recently has undergone some major updates. These developments mean

that the HACCP techniques have progressed considerably since their early beginnings,

and have meant that some companies have found their systems to be out ofdate (23).

3.4.1 The advantage of HACCP program

The HACCP concept is a systematic approach to the identification and

assessment of the risk of biological, chemical, and physical hazards from a particular

food production process or practice and the control of those hazards. HACCp is a

preventive strategy for food safety. Under it, the food producer develops a plan that

anticipates and identifies the critical control points (CCP's), under HACCp, identified

CCP's are systematically monitored to ensue that critical limits (CL's) are not

exceeded, and records are kept ofthat monitoring. Corrective actions are taken when

control of a CCP is lost, including proper disposition ofthe food produced during that

period, and these actions are documented. The effectiveness of HACCp is also

systematically verified by the processor (23).

3.4.2 History of HACCP system

The concept and reduction to practice of the Hazard Analysis and Critical

Control Point (HACCP) system was directly related to the pillsbury Company,s

projects in food production and research for the space program. The basics were

developed by the Pillsbury Company with the cooperation and participation of The

National Aeronautics and Space Agency (NASA), the Natick Laboratories of the U.S.

Amy, and the U.S. Air Force Space Laboratory Project Group. The pathway to the

HACCP system started in 1959 when Pillsbury was asked to produce the first foods to

be used in space. Pillsbury realized that no one fully understood how foods and

especially particulate would behave in zero gravity. To address this problem,

Pillsbury developed bite-sized foods covered with a flexible edible coating to preventCopyright by Mahidol University

Jiraporn Sonyim Literature review / l2

crumbling and consequently atmospheric contaminated. The most dilficult part of the

program was to come as close as possible to 100 % assurance that the food products

used for the space program would not be contaminated with bacterial or viral

pathogens, toxins, chemicals, or physical hazards that could cause an illness or injury.

Such hazard might result in an aborted or catastrophic mission (24). HACCp is the

most effective method of maximizing product safety. It is a cosleffective system,

which targets resource to critical areas of processing, and in doing so reduces the risk

of manufacturing and selling unsafe products (25).

3.5 HACCP Implementation in Thai food matrufacturer

In the survey by the Thai nation food institute in March, 1998. It was found that

even factories that are already on the process of implementing. The HACCP have sill

expressed there need to understand system better. In that survey, 96 manufactures

responded of which 37 o/o were seafood manufactures, 21 % fiuits and vegetables, g %

milk products processors, 6 % poultry and poultry products and 27 oh other lood

products producers. Their responses indicated that majority believe that HACCp is a

necessary for product safety. 50 yo of the respondents indicated that they were on the

process ol implementing if they have not implement the HACCP system yet in their

factories whlle 7 Yo indicated that they have never heard about HACCP system.

Of 50 % respondents who showed interest in implementing the HACCp plan,

they also expressed their needs for help in understanding further the basic concept,

design and steps in implementing the system and in knowing the technology to assist

them the verification and improvement of their own quality control system. Most

manufacturers also expressed their willingness to pay for the services rendered to set

up their HACCP scheme. Many manufacturers have also chosen to be audited and

certified by recognized intemational organization. In addition, 20 more type

manufacturers in Thailand are now on the HACCP implement process which includes

sealood products, fruit and vegetable, rice cracker, frozen foodlfrozen ready to eat

food, poultry product, flour product, bakery product and tomato/chili sauce. The

growing numbers of food and beverage factories evidently depicts the significance ofthis sector in Thailand. As of 1999, the numbers of factories was 12,080. This sector

currently structures by 85 % of small-scale, 12 oh and 3 % of medium and large-scaleCopyright by Mahidol University

Fac. Of Grad. Srudres, Mahidol Univ. M.Sc. (Food and Nutritional Toxicology) / t3

factories. of the totality, only 155 factories were qualiiied with HACCP systems.

The certified factories are under the following catego nes e6,27).

3,6 Prerequisite Program for HACCP

The production of safe food products requires that the HACCp system be builtupon a solid foundation of prerequisite progam. Each segment of the food industrymust provide the conditions necessary to protect food wh e it is under control. Thishas traditionally been accomplished through the application GMps. These conditions

and practice are now considered to be prerequisite to the development and

implementation of effective HACCp plans.

3.6.1 Good manufacturing practices (GMp)

wHo defines Good manufacturing practices (GMp) as "that part of qualityassuriulce which ensures that products are consistently produced and controlled to thequality standards appropriate to their intended use and as required by the marketingauthorization". GMP covers all aspects of the manufacturing process: defined

Table 2. The list of factories were qualified with HACCp system in Thailand in 199g

F actories No. of food factories%

Total HACCP qualilied

Vegetable and fruit 586 l5 2.56

Meat and poultry 621 12 2.74

Milk 101 5 4.95

Fisherv 547 90 16.4s

Seed and fiber 4639 J 0.06

Starch and flour t645 4 0.24

Coffee, cocoa, chocolate 556 4 0.72

Seasoning 468 6 1.28



manufacturing process; validated critical manufacturing steps; suitable premises,

storage, transport, qualified and trained production and quality personnel; adequate

laboratory facilities; approved written procedures and instructions; record to show all

steps of defined procedures taken; full tractability ofproduct through batch processing

records and distribution records; and systems for recall and investigation of

complaints.

The guiding principle of GMP is quality that built into a product, and not

just test into a finished product. Therefore, the assurance is the product not only meets

the final specifications, but that it has been made by the same procedures under the

same condition each and every time that it is made. There are many ways, this is

controlled-controlling the quality of the facility and its systems, the quality of the

starting materials, the quality of production at all stages, the quality of the testing of

the product, the identity of materials by adequate labeling and segregation, the quality

of materials and product by adequate storage, etc. A1l of these controls must follow

prescribed, formal, approved procedures, written as protocols, SSOPs, or Master

Formulae, describing all the tasks carried out in an entire manulacturing and control

process (28).

3.6.2 Standard sanitation operating procedures (SSOPS)

Standard sanitation operating procedures (SSOPs) are the detailed written

instructions that specify how a test or administrative procedure is to be performed, or

how a piece of equipment is operated, maintained and calibrated. SSOPS describe the

" standard" approved sanitation procedures that are routinely carried out in a GMP

facility. They indicated exactly how things are done, and kept curent by review and

approved revision on a predetermined schedule, or when planned changes are made to

the procedure or equipment and reagents used in the procedure. The original of a

current version of a SSOPs is maintained in a central file, and copies are distributed to

the locations where the procedure is performed. The procedure for describing the

writing, revising and approving of SSOPs and the control of distribution of SSOPs is

one ofthe important quality assurance procedures.



SSoPs follows a scientific format, and are written with the view that theywill be used by persons trained in the procedure. They shourd be specific instructionsfor each step in sequential order including the preparatory work which must be done

before starting the main procedure, as well as instructions for recording and reportingthe results (28).

3.7 HACCP principles

The concept of HACCp was consist of 7 principles as follows:

o Principle 1. List alt potential hazards associated with each step, conduct

cotrtrol identifieda hazard analysis, and consider any measures to

hazards.

The HACCp team should next conduct a hazard. analysis toidentify for the HACCp plan, which hazards are of such a nature that theirelimination or reduction to acceptable levels is essential to the production ofa safe food (29).

r Principle 2. Determine Critical Control points

There many be more than one CCp at which control is applied

to address the same hazard. The determination of a ccp in the HACCp system

can be facilitated by the application of a decision tree, which indicates a logicreasoning approach. It should be used for guidance when determining CCps(2e).

o Principle 3, Establish Critical Limits for each CCp

Critical limits must be specified and validated if possible foreach critical contror point. In some cases more than one critical limit will beelaborated at a particular step (30).

r Principle 4. Estabtish a Monitoring System for Each CCp

Monitoring is the scheduled measurement or observation of a

ccP relative to its critical limits. The monitoring procedures must be able todetect loss ofcontrol at the CCp (31).



o Principle 5. Establish Corrective Actions

Specific corrective actions must be developed for each CCP

in the HACCP system in order to deal with deviations when they occur. The

actions must ensure that the CCP has been brought under control (32).

. Principle 6. Establish Verification Procedures

Verification and auditing methods, procedures and tests,

including random sampling and analysis, can be used to determine if the

HACCP system is working correctly. The frequency of verification should be

sufficient to confirm that the HACCP system is working effectively (33)'

o Principle 7. Establish Documentation and Record Keeping

Efficient and accurate record keeping is essential to the

application of a HACCP procedures should be documented. Documentation

and record keeping should be appropriate to the nature and size of the

operation (34).

3.8 Application of HACCP

The application of HACCP principles consists of the following tasks as identified

in the Logic Sequence for Application of HACCP.

3.8.1 Assemble HACCP team

The food operation should assure that the appropriate product specific

knowledge and expertise is available for the development of an effective HACCP

plan. Optimally, this may be accomplished by assembling a multidisciplinary team.

Where such expertise is not available on site, expert advice should be obtained fiom

other sources. The scope ofthe HACCP plan should be identified (30).

3.8.2 Describe product

A full description of the product should be drawn up, including relevant

safety information such as: composition, physical structure (including Aw, pH, etc.),

microcidal/static treatments (e.g. healtreatment, freezing, brining, smoking, etc.),

packaging, durability and storage conditions and method ofdistribution (30).



3.8.3 Identify intended use

M.Sc. (Food and Nutritional Toxicology) / 17

The intended use should be based on the expected uses of the product by

the end user or consurner. In specific cases, lulnerable groups of the population, e. g.

institutional feeding may have to be considered (31).

3.8.4 Construct flow diagram

The flow diagram should be constructed by the HACCp team. The flow

diagram should cover all steps in the operation. When applyrng HACCP to a given

operation, consideration should be given to steps preceding and following the

specified operation (32).

3.8.5 On-site confirmation of flow diagram

The HACCP team should confirm the processing operation against the

flow diagram during all stages and hours of operation and amend the flow diagram

where appropriate (33).

3.8.6 List all potential hazards associated with each step, conduct a hazard

analysis, and consider any measures to control identified hazards

The HACCP team should list all of the hazards that may be reasonably

expected to occur at each step, processing, manufacture, and distribution until the

point of consumption. ln conducting the hazard analysis, wherever possible the

following should be included the likely occrrrrence of hazards and severity of their

adverse health effects, the qualitative and /or quantitative evaluation of the presence ofhazards, survival or multiplication of microorganisms of concern, production or

persistence in foods oftoxins, chemicals, or physical agents, conditions leading to the

above (33).

3.8.7 Determine Critical Control Points

The determination of a CCp in the HACCp system can be facilitated by

the application of a decision tree, which indicates a logic reasoning approach. Itshould be used for guidance when determining ccps. This example of decision tree

may not be applicable to all situations. other approaches may be used. Training inthe application of the decision tree is recommended (34).


Jirapom Sonyim Literahrre revie* / l8

3.8.8 Establish Critical Limits for each CCp

critical limits must be specified and validated if possible for each critical

control point. ln some cases more than one criticat limit will be elaborated at a

particular step (3 5).

3.8.9 Establish a Monitoring System for Each CCp

Monitoring is the scheduled measurement or observation or a ccp relative

to its critical limits. The monitoring procedures must be able to detect loss of control

at the ccP. Further, monitoring should ideally provide this information in time tomake adjustments to ensure control of the process to prevent violating the critical

limits. where possible, process adjustments should be made when monitoring results

indicate a trend toward loss of control at a ccp. The adjustments should be made

when monitoring must be evaluated by a designated person with knowledge and

authority to carry out corrective actions when indicated. If monitoring is not

continuous, then the amount of frequency of monitoring must be sufficient toguarantee the CCP is in control (36).

3.8.10 Establish Corrective Actions

Specific corrective actions must be developed for each CCp in the

HACCP system in order to deal with deviations when they occur. The actions must

ensure that the ccP has been brought under control. Actions taken must also include

proper disposition of the affected product. Deviation and product disposition

procedures must be documented in the HACCP record keeping (37).

3.8.11 Establish Verification Procedures

Verification and auditing methods, procedures and test, including random

sampling and analysis, can be used to determine if the HACCP system is working

correctly. The frequency of verification should be sufficient to confim that the

HACCP system is working effectively. Examples of verification activities include

review of the HACCP system and its records, review of deviations and product

dispositions, confirmation that CCPs and kept under control (3g).

3.8.12 Establish Documentation and Record Keeping

Efficient and accurate record keeping is essential to the application of a

HACCP procedures should be documented. Documentation and record keeping

should be appropriate to the nature and size ofthe operation.Copyright by Mahidol University


Documentation examples are hazard analysis, ccp determination, critical limitdetermination and record examples are ccp monitoring activities, deviations and

associated corrective actions modifications to the HACCp system (3g).


Jirapom Sonyim Material and method / 20

CHAPTER IV

MATERIALS AND METHODS


Questionnaire suwey was carried out with the objectives to select of three most

popular brands and flavours of instant noodles and to study the instant noodle

preparation and eating habit ofthe consumers. The designed questionnaire is shown

in Appendix 1. The survey was aimed to carry out in 350 consumers, age ranged 15-

30 years. The consumers were students at a secondary school (Mahidol

Vitayanusorn), two universities (Thammasart and Mahidol) and people at two

department stores (PATA and Central Plaza - Pinklao branch).

In addition to questioruraire survey, the information on three most popular

brands of instant noodles fiom market share reported in The Nation Newspaper in

2001 was checked (39). Three manufacturers were recorded as the main market shares

of instant noodle in Thailand. Noodle-l has been the leader of the domestic instant-

noodle market, with 56 percent of sales, followed by Noodle-2 at 26 percent and

Noodle-3 at 13 percent.

4.2 Determination ofB cereus in seasoning of instant noodles

4.2.1 Selection of three seasoning brands and flavours

The three popular brands were selected by using data from market share

and questionnaire survey. They were Noodle-l, Noodle-2, and Noodle-3. The three

flavours of the instant noodle were chosen fiom the most favorite flavour obtained

from questionnaire data, two of them were hot and sour, shrimp and minced pork.

The third one was chicken flavour, selected from the reported data at Food

Microbiology laboratory, lnstitute of Nutrition Malidol University (6, 7) for the most

finding B. cereru* contamination.

* During 1995 to 2002, 213 seasoning samples were examined at INMU. Forty thrce out of the totalsamples were found positive for -8. cereas. The most often B. cereus detected seasoning was chickenflavour (19 %). Copyright by Mahidol University


For the brand that does not have chicken flavour, it was replaced by the readyto-eat

flavour, which has been the highest sale, recorded at the manufacturer. After

selection of the flavours and the brands of instant noodles to be studied, the

manufacturers of the three brands were approached for cooperation. Experimental

desigr and sampling plan of the study were informed and the permission for visiting,

cooperation and support were requested.

4.2.2 Sampling plan

The samples of seasonings, hot and sour-shrimp, minced pork and chicken

flavours, of finished products from tkee manufacturers were sampling by the staff of

manufacturers under detailed instruction in the experimental desigrr. Ten individual

lots per flavour were collected fiom each manufacturer. Each lot composed of 30

single packages of instant noodles (in a box), collected on one day per week from

different processing lines. One box ofeach flavour from one company was collected

every week. Each brand was collected for 10 weeks. Therefore, in total, 30 single

packages x 10 lotsi flavor/brand were collected.

4.2.2 Samplepreparation

Ten single packages of seasoning per flavour were randomly sampling

from each lot of sample (10 single packages x 3 flavours x 3 brands). The packages

of each flavour were pooled (about 90 g) and thoroughly mixed in a plastic bag to

prepared a single composite sample. Therefore, the total number of 30 single samples

per brand (3 flavours x 10 single composite samples) were determined for B. cereus.

4.2.4 Determination ofB. cereus in seasoning of instant noodle

4.2.4.1 Stock culture preparation

Bacillus cereus (ATCC 1 1778, DMST 5040) was used as

positive control. It was provided by Division of Clinical Pathology, Department of

Medical Science, Ministry of Public Health, Thailand. The culture was grown and

ma-intained on nutrient agar slant (NA, Merck, Darmstadt, Gerrnany), incubated at

35oC for 24 h, kept in a refrigerator at 4oC and the fresh culture was prepared every

month. Copyright by Mahidol University


4.2.4.2 Q\artitative determination of Bacillus cereas by Most probable

Number (MPN) technique

B. cereus was determined by MpN technique and the organism

was gonfirmg{ by biochemical . test following the methods in Bacteriological

Analytical Manual (BAM), 8th edition, AOAC Intemational Gaithersburg, MD;

chapter 14.1995. The detailed method is shown in Appendix 3.(40)

4.3 Effect of home preparation of instant noodl e on B. cereus reduction

4.3.1 Test sample

Minced pork flavour seasoning powder, the mostly found, B. cereus

seasonings flavour, was used as test material in this experiment. Sixty single packages

from the same lot number were collected from a supermarket. Seasonings from each

package were pooled, and aseptically prepared by blending in a steriled electric

blender, and kept in a plastic bag. Aliquot samples of 6 g were prepared when

required for cooking.

4.3.2 Preparation of spore suspension ofB. cereus (9)

Since most the seasoning in the package of instant noodles contained low

level of B. cereus. Therefore, spores suspensiorts of B. cereus were inoculated into

the seasoning during preparation ofinstant noodles.

B. cereus spores were prepared by plating 1.5 mL of 24-h culture in

trypticase soy broth (TSB, Difco, Detroit, MI, USA) onto nutrient agar (NA, Difoo)

plate. After incubation at 30 oC for 3 days, the spores were harvested by flooding the

agar surface with steriled distilled water. The spore suspension was centrifuged at

3500 r.p.m. for 15 min using a centrifuge (KOKUSAN H-103 N-series). The spores

was then washed five times with steriled distilled water. After the last centrifugation,

the spores were heated in hot water bath at 80'c for 10 min to kill vegetative cells.

The spores were resuspended in steriled distilled water to obtain a final concentration

of 108-10e spores/ml and checked for the absence of vegetative cells by spore staining

method (41). The spores suspension was kept at 4oc for further use and the number

of spore was checked daily by spreaded-plate count technique (40).


Fac. OfCrrad. Studies, Mahidol Unir. M.Sc. (Food and Nutritional Toxicology) / 23

Spore suspension of B. cereus was inoculated to the seasoning during

instant noodle preparation in section 4.3.3 at the final concenkation of 10s cFU/g ofsample.

4.3.3 Cooking of instant noodle

Four methods of instant noodle preparation, in triplicate, were designed in

this study. The first one followed instruction by the manufacturers on the label, the

other two selected methods were obtained from the questionnaire survey of the

consumers in Section 4.1 and the last method was proposed in this study. Water and

utensil used for soaking or boiling were steriled. Each method was described as

follows.

Method 1. Instruction on the label ofthe instant noodles

Place fried noodles in a bowl

Add all seasonings*

o Add 360 mL of boiling water, cover the lid for 3 min

Method 2. Selection by the consumers: 3 sets each, with different soaking time

(soaking time was 3 min for method 2.1, 4 min for method 2.2, and 5 min for

method 2.3)

r Place fried noodles in a bowl

o Add 360 mL of boiling water, cover the lid for 3 or 4 or 5 min

r Add all seasonings*

Method 3. Selection by the consumers, 3 sets each, with different boiling time

(boiling period was 2 min for method 3.1, 3 min for method 3.2, and,5 min for

method 3.3)

Boil noodles in 360 mL of water for 2 or 3 or 5 min

Add all seasonings+

a

o

* Inoculated with spore suspension ofB. cereus at the concentration of l0s CFU/g of sample.


J iraporn SonyimMaterial and method / 24

Method 4. Proposed method, 3 sets each, with different boiling time(boiling period was 2 min for method 4. 1, 3 min for method 4.2, and 5 min formethod 4.3)

Boil all seasonings* in 360 mL water for 2 or 3 or 5 minAdd fried noodles and continued boiling for 1 min.

* Inoculated with spore suspension of B. cereus ar the concenhation of l0s CFU/g of sample

Three independent sets of instant noodles were prepared for each methodofcooking' Instant noodre with seasoning and without adding spore suspension wasused as a negative control. A positive control - instant noodle inoculated with thespores without heating - was prepared in each set ofthe experiment.

4.3.4 Enumeration and identification of survival of Bacillus cereus

The samples of inoculated cooked instant noodle prepared in Section4'3 3 were homogenised at room temperature for 1 min using a stomacher 400(Colworth Larme'ris) and immediately determined for B. cereus.

The survival of B. cereus in the heated noodles was determined byspreaded-plate count technique for B. cereus, following the methods in BacteriologicalAnalyical Manual (BAM, 8'h edition, AOAC Intemational Gaithersburg, MD; chapter14.1995) as lollows.

The homogenised sample was weighed 25 g in a plastic bag, addedwith 225 mL of steriled 0. 1 % Butterfield's phosphate buffer and homogenised for 2min by a Stomacher 400. one milriliter of the homogenised sample was transferredinto 9 mL of phosphate buffer, which was then serially lO-fotd diluted to providecountable numbers on plates. A 0.1 mL of the last three appropriate diluted sampleswere spreaded on the Myp agar plate in duplicate (39). The plates were incubated at35"Cfor24h. Number oltypical colony (eosin pink, lecithinase_postive) ofB. cereuswere counted from the plate containing 15 tol50 colonies. Five of typical colonieswere picked and confirmed by biochemistry tests for identification of B. cereusaccording to the rnethod described in Appendix 3, flow chart 2. The number ofB.cereus, cFUrg of sample, was calculated based on percentage of tested coronies thatwere confirmed as B. cereus.Copyright by Mahidol University

Fac. OlCrad. Studies, Maludol Unir. M.Sc. (Food and Nutritional Toxicology) / 25

The reduction number ofB. cereus was calculated from the logarithmic

number of B. cerers in the positive control sample (sample with added B. cereus

spores but without heat treatment) minus the logarithmic number of B. cereus in the

sample after heat treatment. The per cent reduction of B. cerets was calculated by

comparing with the total number of B. cereus in the positive control.

4.3.5 Statistical analysis (42)

The effect of 4 methods with 10 conditions of home_cooking on the

reduction of B. cereus were statistically evaluated using SpSS for wINDow Release

10.0 software. Nonparametric tests with K-independent samples were applied.

Kruskal-wallis H test type was used for indication of any significant difference in

these treatments. For each pairwise comparison, Mann-Whitney U test was used to

indicate two independent heated samples which showed significant difference in the

log reduction number of .B cereus. The significant degree of difference was set at p-

value <0.05. The Holm's sequential Bonferroni procedure was used to control for

Type I error. This procedure was used only when the pairwise comparison showed

signifi cant difference.

4,4 Identification of proper critical control points for B. cereus in the production

line of instant noodle seasoning

The identification of potential hazards ofB. cereus and control measures in the

processing line of instant noodle seasoning was performed and the proper critical

control points were identified according to the concept ofHACCP, Principle I and 2.

4.4.1 Communication with the mannfacturers.

Three top manufacturers (obtained from Section 4.1 euestionnaire

survey) were contacted for visiting and studlng the product processing line. The

detailed plan of study, i.e., the number ofvisits, activities during the visit, information

needed, sampling plan, number of samples to be analysed, were explained. Finally,

only one manufacturer was agreed to be a model of the study. The production line ofminced pork seasoning which mostly found ,B. cereus contaminatioq was chosen as

the study model. Copyright by Mahidol University

Jiraporn SonyimMaterial and method / 26

4.4.2 Development of HACCP model, following principle I and 2, fordetermination of Critical Control points (29)..

4.4.2.1 B. cereas analysis of ingredients

Four ingredients of minced pork flavour seasoning, i.e., garlicpowder, dned spring onion, dried ground chili, and pepper powder, which were thehigh-risk of B. cereus contamination, were sampling to check ror the presence of B.cereus. Ten lots ol each ingredient (100 g, each) were randomly sampling fromproduction lines. The mixed seasonings prepared {iom the same lots of theingredients, before and after packaging, were also collected and analysed for B.

cereus. The samples were then homogenised by mixing vigorously in a plastic bag,

and individually determined for the number of B. cereus following the method shownin Section 4.2.4.2.

4.4.2.2 Development of HACCp model

The concept of HACCp, principle I and 2, was conducted to

develop inspection models for HACCp implementation in seasonings of instantnoodle. A flow diagramme of over all production of minced pork seasoning was

constructed. Then six forms ol HACCP implementation were developed. Theactivities in each step and the forms were as follows

o Reviewing the product description and determining how this

information could influence hazard analysis (F,orm l)r Listing product's ingredients and incoming material.( Form 2)

o Describing each processing step (Form 3) according to flow diagram

Determining and describing biological, chemical and physical

hazards that could exist at each step (Form 4).

Determining of control measures at each step (Form 5)

Identilying ofcritical control points (Form 6)

I ) Construction ol flow diagram of minced pork seasoning production

The flow diagram was constructed (Figure 4). It covered all steps in theoperation of minced pork seasoning. When applying HACCP to the process,

consideration was given to steps preceding and following the specified operation.


Fac. OfCrad. Studies, Mahidol Univ. M.Sc. (Food and Nutritional Toxicology) / 27

On-site confirmation of flow diagram of all stages and period of operation was

conducted by QC manager of the manufacturer and the flow diagram was amended

where appropriate.

2) Development ofForm 1: product description

A full description of mixed seasoning, pork flavour, was listed. It

included product's name and relevant safety information, i.e., physical/chemical

properties (including pH, Aw, moisture, characteristics), how is product to be used,

packaging, process, durability and storage conditions, method of distribution and

target group.

3) Development ofForm 2: ingredient description

The ingredients composition of mixed seasoning, pork flavour were

listed. Detailed information of each ingredient, which included supplier site,

characteristics, packaging, net content and storage condition were described.

4) Development of Form 3: process description

For each step of minced pork seasoning production, raw material,

processes and the equipment used were described.

5) Development ofForm 4: hazard analysis and 5: control measure

The description of the product, ingredients and process as presented

in Form I to 3 were used in hazard analysis to determine the Critical Control Points of

B. cereus contamination at the processing line of minced pork flavour seasoning. All

potential biological, chemical and physical hazards associated with each step including

ingredients and raw materials of minced pork seasoning products were listed, and

hazard analyses were conducted following the protocol in Principle 1. The severity

and their likely occurrence were evaluated for level of hazards. Consideration of the

"likely occurrence" was usually based upon a combination of experience,

epidemiological data and information in the technical literature. Each hazard was

classified into two levels of severity - major or minor - or satisfactory and the

underlying reasons were noted. ln this HACCP model, the biological hazard was

emphasised and defined only for -8. cereus which was the most finding contaminated

organism in the seasoning. Chemical and physical hazards were considered

satisfactory or hazard with minor level of severity for seasoning processing because

each ingredient was inspected and controlled by the process of GMP at the purchasedCopyright by Mahidol University


points. All information were presented in Form 4. when all potential hazards have

been identified and analysed, the next step should go on to list the associated

preventative measures. These are the control mechanisms for each hazard and are

normally defined as those factors which are required to eliminate or reduce the

occulrence of hazards to an acceptable level Any measures to control each identified

hazard (principle l) at each step was listed in Form 5 Then the raw materials and

processing steps which were identified as significant hazards with major level and

their control measures were considered whether they were ccp, using Decision Tree.

6) ldenrification ol Crirical Conrrol points (CCp)

The Principle 2 of HACCP concept, using standard Decision

Tree, was applied to identify CCp at each I

by answering the following lour questions.

to identify CCP at each step ol minced pork seasoning production

Figure 2.

Question 1. Do preventive measures exist for the identified hazard?

Ifthe answer is yes, process to the next step.

If the answer is no, asks the question " Is control at this step necessary

for saflety?"

Ifcontrol is not necessary this step is not CCp.

Question 2. Does this step eliminate or reduce the likely occurrence ofthe hazard to an acceptable level?

If the answer is yes, this step is CCP.

If the answer is no, proceed to the next question.

Question 3. Could contamination with identified hazard occur in exceed

of acceptable levels or could these increase to unacceptable levels?

If the answer is yes, proceed to the next question.

Ilthe answer is no, the step is not CCP.

Question 4. Will a subsequent step eliminate identified hazard or reduce

the likely occurence to an acceptable level.

Ilthe answer is yes, the step is not CCP.

If the answer is no, this step is CCP.

The Decision Tree was summarised in


Q. I Do preventative measures exist for the identified hazard? <--lModified step, process or product

NoI

Y

- Is control necessary at this step for safety? ---->Ii

No ----| Not a CCp -----) Stop

Q. 2 Is the step specifically designed to eliminate or reduce the likely occurrence of

the hazard to an acceptable level?

Yes -------------* Not a CCP

-|'

Stop

Figure 2. The CCP Decision Tree (25)

4,5 Experimentaldesign

Summary of the over-all study was presented in Figure 3.

Fac. OfGrad. Studies, Mahidol Univ. M.Sc. (Food and Nutritional Toxicology) i 29

JYes

1Yes

I

-

No ---* Yesi

Q.3 Could contamination occur at or increase to unacceptable levels? I

I \ I

* \"o ---------) Not a ccp -------+ srop I

llQ.4 Will a subsequent step or action eliminate or reduce the hazard to an acceptable

I

rever? 1 I )+ No-LLPa'

IIt

Q.3 Could contamination occur at or increase to unacceptable levels?


Jirapom Sonyim

Brand I

Flavourl-10lots

Flavour2-10lots

Flavour3-10lots

Brand 2

Flavour l - 10lots

Flavour2 - 10lots

Flavour3-10lotsIv

Brand 3

Flavourl-10lots

Flavour2 - i0lots

Flavour3-10lots

*-'

I

41t 2

\.]

1

qzl z

\,

1

q.tl z

\,

Material and method / 30

1. Questionnaire survey (350 consumer)I

Marketshare data I a""O Microbiological reporled data\l-,.Selected instant noodles (3 flavours, 3 brands)

\2. Determination of B. cereus contamination in finished products (MpN)

ISelected flavour with mostly found of B. cereus contamination

I

t3. Study the effect of different methods of home cooking on B. cereus

reduction

Method 3 Method 4Method 2

l,,r'.,| -'\ -I t.r

t,[" (il,l*r.t (,

\3

Cooking Method 1

/N,/l\123(triplicate sets)

1

ztlz\.

1

t.zl z

\,

1

nlz\,

+B. cereus determination (Spread -plate count )Copyright by Mahidol University

Fac. Of Grad. Studies, Mahidol Univ. M.Sc. (Food and Nutritional Toxicology) / 31

Mixed seasoning10 lots

Form 4

Hazard analysis sheet

Form 5

Control measure sheetI

V

Determine and identify B. cereas contamination

IiConstruction of flow diagram of seasoning

I

Y

Perform HACCP Model Forml-6

Form 2

lngredient description

+Aralyses step by step follow as HACCP principle 1IJ

Form 6. Critical Control Points sheet

(HACCP principle 2)

I

+The proper Critical Control Points of B. cereus

Figure 3, Experimental design

4. Identification ofproper critical control points

Ingredient 3

10 lotslngredient 4

10 lots


Jirapoln Sonyim Resuit / 32

CHAPTERV

RESULTS


The characteristics (sex, age) of consumers were shown in Table 3. The

percentage of females was about fwo times more than males. The highest percentage

ol respondents' age ranged from 19-25 year (56%). About 98% of the surveyed

consumers (342 out oftotal of350 subjects) returned completed questionnaires.

5.1.1 Popular brands and flavors of instant noodle

Three most favorite brands and flavours of the instant noodles were

identified from the questionnaire survey. The three most favorite brands shown in

Table 4 in descending order of preference were Noodle-I, Noodle-2 and Noodle-3

with 81, 13 and 6 percent of majority votes, respectively. The order of preference was

in accordance with the data from market shares obtained from The Nation Newspaper.

The three most lavorite flavours of instant noodle seasonings (Table 5)

were Noodle-l, Noodle-2 and Noodle-3 either the first or second or any order olchoices. It was found that the 2 most favorite flavours among these three favorite

brands were the same. Hot and sour-shrimp flavour and minced pork were the most

and the second from the most favorite flavours, respectively. The third lavorite

flavour for Noodle-l was Moo-num-tok (pork with dried blood), for Noodle-2 was

Quick-chili paste and for Noodle-3 was roasted pork.


Table 3, Characteristics olrespondent consumers

Characteristics of respondent consumers (n:342)

L Sex

. Male

. Fcn]ale

l. Ageo I4-18 v

o 26-10 vJ. rrruari|ltoll melnoo. By boiling only. By soakrng rn borled water onlr -- =----r Eatins raw onlv

No. ofrespondentconsumers (%)

lr0 (ll)242 (69)

ll0 (32)

r93 (56)

39 ( l2)

l0 (3)

r2 (4)

r (0.1). By soaking in boiled water or boiling or eatingJaw 264177). Boiling or soaking in boiled water. Boiling or eating raw

23 \6.'7 )14 (4)

Soaking in boiled water or eating raw r8 (5)4. Preparation period. Boiling time (min)

<lnrin r(0.3)I-2 min3-5 min

57 (t',t.'7)

236 (74)>5< l0 min 24 (8)

. Soaking time (min)<l min 3 (il-2 min l't (12)l-5 min 260 {83)>5<10 min t2 (4)

5, Adding seasoning (asked only consumers who prepared the noodles by soakjnq). Bcfore soaking 196 (60)

After adding hot water 2 (0.6). BefoJe consumption 128 (19). Not adding r(0.4)6. Adding ofother food ingrgdients * (n=150). Nothing 24 (t6). Egg r (7.). N4eat 2l (r5). Vegetables r3 (9). Egg + meat + vegetables 23 t t5). N4cat + vesetables 28 ( l9). Egg + vegetables l5 (10). Egg + meat r4 (9)7. Time of eating after preparqtion. Immediately 258 (7()). l-5min 68 (20)

>5<l0min 8 (2)r I0-10 min 7 (2)8. Frequency of consumption of ilstant noodle. >4 times/week 13 (4). 3-4 times/week 48 ( r4). l-2 times/week r78 (52). I -3 times/month 103 (30)

r\3Ltl)t\


Table 4. Three most popular brands ofinstant noodles and the degree ofpreference

Most popular brands No. ofconsumers' vote (Yo) (n=342\

1. Noodle-1 276 (81)

2. Noodle-2 46 (13)

3. Noodle-3 20 (6)

Jiraporn Sonyim Result / 34

Table 5. Order ofpreference ofseasoning flavours ofdifferent brands ofinstantnoodles

Brands Flavours No. of respondents (%)

1 . Noodle- I Hot and sour-shrimp flavour 307 (4s)

Minced pork 257 (37)

Moo num tok 64 (e)

Stew duck soup s0 (7)

Chicken soup t4 (2)

2. Noodle-2 Hot and sour (Tom-yum)-shrimp flavour t6s (42)

Minced pork 124 (31)

Chili paste, tom )um 44 (11)

Ready-to-eat 3e (10)

Tom klong 22 (6)

3. Noodle-3 Hot and sour-shrimp flavour 150 (3e)

Minced pork 111 (2e)

Roasted pork 61(16)

Pad- khee-mow s8 (1s)

Chicken soup 4 (1)Copyright by Mahidol University

Fac. OfGrad. Studies. Mahidol Univ. M.Sc. (Food and Nutritional Toxicology) / 35

5.1.2 Preparation and eating habit of instant noodles

Three most common methods for preparation ol instant noodle were boiling or

soaking in boiled water or eating raw (77% of respondents). Similar period of 3-5 min

boiling or soaking instant noodle in boiled water was practiced by most of the

respondents (74%). About 60% of the respondents added the seasoning powder

before soaking the noodles in boiled water, and about 39% ol them added the

seasoning just before consumption. Most of the consumers added other food

ingredients, especially meat and vegetable (20%) into the instant noodle and eating the

noodle after hnished cooking immediately (76%). The most frequency of consuming

instant noodles was 1-2 time/week (52% ofthe respondents).

5.2 B. cereus contamination in the seasonings of instant noodles

B. cereus was determined in 10 lots (each lot composed of a single composite

sample of 10 single packages) each of three flavours seasoning ol 3 brands instant

noodles by MPN technique. The results are shown in Table 6. For Noodle- 1, 20% of

all three seasoning flavour were contaminated with B. cereus. The highest level ofB.

cereus found was in minced pork flavour (9.2-23 MPN/g). For Noodle-2, no B.

cereus contamination was detected in hot and sour-shrimp flavour and ready{o-eat

flavour. The contamination was detected in 40% of Noodle-2, minced pork flavour,

ranged from 3 to 9.2 MPN/g. For Noodle-3, per cent of products wilh B. cereus

contamination was higher than Noodle-l and Noodle-2 and the organism was detected

in every flavours of seasoning. Thirty per cent of the hot and sour-shrimp flavour

found B. cereus ranged from 3 to 7.4 MPN/g and 50% each of minced pork and

clricken flavour found .8. cereus ranged lrom 3 to 23 MPN/g and 3 to16 MPN/g

sample, respectively.

The status of B. cereus contamination in different flavours (30 samples/flavour

were determined) of instant noodle seasoning is summarised in Table 7. The highest

percentages (37 %) of B. cereus conlamination and at the highest level (23 MPN/g

sample) were found in seasoning of minced pork flavour. The flavour was therefore

selected for studying the effect ofcooking on survival of B. cereus.


Jirapom Sonyim Result / 36

Table 6. Per cent of B. cereus contaminated instant noodle seasoning and range ofdetected level (Number of lots per flavour : 10)

Table 7. Average percentage B. cereus contaminated seasoning of each flavour(No. of sample :30/flavour, except 20 for chicken flavour)

Flavours % positive samples

Hot and sour-shrimp 17

Minced pork

Chicken + 35

* One brand does not produce this flavour.

Brands Flavours No. of lot withpositive

sample (%)

Range of detected

B. cereus (MPN/g)

Mean of detectedB. cereus (MPN/g)

Noodle- I Hot and sour-shdmp

2 (20) 3-9.2 6.1

Minced pork 2 (20) 9.2-23 16.1

Chicken 2 (20) 9.2 9.2

Noodle-2 Hot and sour-shrimp

0 ND* ND

Minced pork 4 (40) 3.0-9.2 4.7

Ready-to-eat 0 ND ND

Nooodle-3 Hot and sour-strimp

3 (30) 3-7.4 5.5

Minced pork s (50) 3-23 13.5

Chicken 5 (50) 3- 16 7.2

* ND (not detected)



5.3 Effect of different methods ofhome cooking on B. cereus reduction

The instant noodle were cooked by various methods with adding spores of B.

cereus, the results are shown in Table 8. Mixing seasonings with instant noodle

before adding boiling water and covered for 3 min (Method 1, as instruction on the

tabel) and the cooking method by boiling the instant noodles in steriled water for 2

min and then added seasonings (Method 3.1) were found to eflectively reduce the

nunrber ofB. cereus by 1.37 and 1.34 log CFU/g, respectively (25 per cent reduction).

These cooking effects on B. cereus were significantly different (at p<0.05) from the

other methods. Method 4, as proposed method for cooking instant noodle seasoning

with different boiling time for 2, 3, 5 min, reduced the number of B. cereus by 0.81-1

log CFU/g. Which equals to 15, 17 and 18 per cent reduction, respectively. Method 2

which soaking of instant noodle in boiling water for 3-5 min and Method 3.2 arld 3.3

which boiling instant noodles for 3 and 5 min reduced the number of B. cereus by

0.84-1.13 log CFU/g (16-21 per cent reduction).


Result / 38Jirapom Sonyim

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Fac. Of Grad. Str.rdies, Mahidol Univ. M.Sc. (Food and Nutritional Toxicology) / 39

5.4 Proper critical control points for B. celeas in the production line of instant

noodle seasoning

5.4,1 B. cereus contamination in four ingredients and the seasoning

of instant noodles.

B. cereus was determined in l0 lots of each 4 high-risk ingredients (garlic

powder, dried spring onion, pepper powder, and chili powder) of minced pork

seasoning flavour, in mixed ingredients (except dried spring onion) before and after

packaging (where spring onion was automatically mixed) by MpN technique. The

results are shown in Table 9. The dried spring onion showed the most frequent

contamination, B. cereus was found in 100 % of determined samples, at the highest

level of B. cereus count (ranged from 3 to 460 MpN/g). After mixing the three

ingredients (except spring onion), B. cereus contamination was found in 60% of the

tested samples and at the level ranged fiom 3.6-23 MPN/g. During packaging ofmixed ingredients, spring onion was added automatically. The contamination was

found in 70Yo of the finished products, at the level ranged fiom 3.0-9.2 MpN/g.


Jiraporn SonyimResult / 40

four ingredients before mixing,Table 9. Bacillus cereus contamination in 10lots ofin mixedrn mrxed products before and after

Ingredients/processed Range of B. cereuscount (MPN/g)

(Mean + SD)

ok of B. cereuscontamination

(n:10)

Garlic powder 9.2-t1

(9.86+0.99)

30

Dried spring onion 3.0-460

(68+ 138.32)

100

White pepper powder 0-3.0

(3+0)

10

Chili powder 0-3.6

(3.6+0)

10

Mixed seasonings before packaging(except spring onion)

3.6-23

(7.46+7.76)

60

Mixed seasonings (included springonion) A fter packaging seasoning

3.0-9.2

(4.6*2.30)

70

5.4.2 Development of HACCP model for identification of critical control

points

The selected manufacturer to be the study model is the large-scale

instant noodle producer, produces about 20,000 boxes (30 single serving size per box)

of minced pork flavour instant noodles per week. The concept ofHACCp, principle 1

and 2, was conducted to develop inspection models lor HACCP implementation in

seasonings of instant noodle. The flow diagramme of the production was followed

throughout the process. The interview and inspection reports were then presented in 6

forms.

5.4.2.1 Establishment and description ofprocess flow diagram ofminced pork flavour seasoning

A simple flow diagramme of overall production olminced pork

flavour seasoning is shown in Figure 4. The ingredients of the minced pork

flavour seasoning were sugar, salt, sodium monoglutarnate, pork powder flavour,Copyright by Mahidol University

Fac. Of Grad. Sh.rdies, Mahidol Univ. M.Sc. (Food and Nutritional Toxicology) / 4t

white pepper powder, dried ground chili, gartic powder and dried spring onion.

Sugar, salt, sodium monoglutamate, and pork powder flavour were purchased as raw

materials. The white pepper powder and dried gound chili were the only 2

ingredients prepared at the manufacturer. The process of white pepper consisted ofroasting the whole seeds at 100 oC for 80 min, cooling for 24 hr, grinding and packing

in plastic bags, 10 kg each. For chili powder processing, the whole pods of dried bird

chili were purchased and inspected. They were first quick rinsed with soft water at the

ratio of 25 kg bird chili to 500 L of soft water, and then steamed under pressure at

temperature of 97-107 "C for 8-12 min. They were dried in an oven at 62 oC for 30

min and then roasted at 170 oC for 5 min, to increase the smell, drying and sterilisation

and cool for 5 - i 0 min. They were ground and packed in plastic bags, 40 kg each.

Garlic powder and dried spring onion were processed at the suppliers, the

processes involved were obtained by interviewing the Quality Control Manager. The

fresh garlic was first dried at 80 oC for 24 h and peeled. The peel and other

contaminant were separated. The peeled garlic were again heated in the oven at 80 oC

for 20 h, cooled to room temperature and ground. The garlic powder was packed in

plastic bags,40 kg each. They were transported to the instant noodle manufacturer to

be used as an ingredient in seasoning.

Dried spring onion was processed as follows. The fresh spring onions with

root were washed by running water and soaked in chlorinated water (300 ppm) for 20

min. They were chopped to be small pieces by machine and soaked again in 50 ppm

chlorinated water for 20 min. They were then drained and heated in an oven at 80

'C lor 5 h and cooled at 25 oC lor about 4 h. The dried spring onions were graded by

sieving and the selected dried pieces ofleaves were re-dried at 60 oC for 40-60 min to

the required moisture content of 5% and packed in plastic bags, 12.5 kg each. They

were transported to the instant noodle manufacturer to be used as an ingredient in

seasoning.

Al[ ingredients were inspected by the Quality Control Manager and kept on the

wood shelves in the storeroom at room temperature (27-35 "C). For each batch of

seasoning, all ingredients were weighed according to the seasoning formula by the

workers. They, except the dried spring onion, were passed through mixing chamber,

and mixed until homogenous powder was obtained. The mixed ingredients was put inCopyright by Mahidol University

Jirapom SonyimResult / 42

a big plastic bag and kept at room temperature, waiting for single packaging. Thisprocess is an option, depending on the requirement of the customers. Some lots wentto single packaging without storage. The studied minced pork flavour seasoning waspassed through this step for 2 days. The mixed ingredients were then packed in smalpackages, 6 g each. During packaging of mixed ingredients, spring onion was added

automatically.

5.4.2.2 prodtct description (Form l)The description of the minced pork seasoning flavour was

reported in Tabre 10. The importance characteristics of minced pork seasoning

flavour were dried powder of red-brown color with some pieces of spring onion, pH5.6, Aw 0.43 and had moisture content of 1.29 gi100g. They are used as seasoning

powder lor instant noodles, and are normally kept at room temperature. 'rhe mincedpork seasoning was packed in a sealed laminated aluminum foil, 6 g each. The

process of seasoning were weighing individual ingredients, mixing, packing intoplastic bag, left at room temperature (an option), single packing, keeping at room

lemperature, and final packing with a serving ofinstant noodle and a package ofgarlicoil. The shelf life of the product is six months. They are sold generally supermarket,

retail shop and convenient store. The labelling instructions consist of brand name,

ingredient information, cooking method, nutrition labelling, net content, address ofmanufacturer, FDA license, and bar code. The products were distributed by truck tothe target shop and the target consumers were all age groups.

5.4.2.3 Ingredient description (Form 2)

The description of each ingredient of minced pork flavourseasoning of instant noodle was shown in Table ll. It included ingredients or raw

materials of the product, i.e. sugar, salt, dried chili, garlic, sodium monoglutamate,

white pepper, spring onion, pork powder flavour, supplier site, characteristics,

packaging, net content, and storage condition. ofeach ingredient.

5.4.2.4 Process description (Form 3)

The flow diagramme in Figure 4 was considered.. Sugar, salt,

sodium monoglutamate and pork powder flavour were thoroughly inspected to reach

the standard by the manufacturer prior to purchasing. Therefore, the details of theingredients in Table 12. included only their packaging and the places and conditionsCopyright by Mahidol University

Fac. OfGrad. Shrdies, Mahidol Univ. M.Sc. (Food and Nutritional Toxicology) / 43

where the raw materials were kept. The process descriptions of other raw materials,white pepper powder, dried chili, garlic powder, and dried spring onion werepresented in Table 12. They included processing steps, condition used, the objectiveof each process, and equipment used at each step thoughout the production of theseasoning (Figure 4.).


Jiraporn SonyimResult / 44

27. Spring onion

Figure 4. Flow diagram of instant noodle seasoningFlavor: minced pork

l.j*",] 2. Salt 10. Dried chilil 18. Garlic

3. MSG I |

4. Pork power flavor I l. Washing withsoft water

19. Drying80 oC 24 hr.

It-..*"*,";--|I too oc for 8o min

I----T-----P**l

I

*,I 8. Grindins II"l

I

12. Steaming + PressureT.91 - ),07

oC for 8 -12 min20. Peeling

21. Sieving13.^ Drying

62 'C l0 min

22. Heating80 oC 20 hr

14. Roastingt70 oC.

5 min 23. Cooling

15. Coolins 24. Grindine

9. Packing inplastic bag

Y

16. Grinding 25. Packing inplastic bag

17. Packing inplastic bag 26. Transfening

by tmck

3 8. Storage (F

39. Weighing 38. Storage

F,"*rr+--->F.P-I pla

--____r

-llrylrlt |J

Ilq{37. Packrng in plastic bag

storing in cold room

+-

| 42. Transfening I

"l

43. Single packaging I ),] o+. s,o.ug. at room temp

3l . Soaking inchlorine solution

33. Dry heating80

oC for 5 hr

36. Drying 60 o C

for 40 - 60 min


Fac. Of Grad. StLrdies, Mahidol Univ. M.Sc. (Food and Nutritional Toxicology) / 45

rble 10. HACCP model lbr large-scale seasoning of instant noodle manufacturer

Company name: N-l Quality manual Page... I

Product description Issue Date

Prepared by..Date....-......

Approved by..Date. . .. .. . .... -

Verified by..Date..... . -...

Product name (Brand.l minced pork flavor

Importance productcharacteristic

2.1 pH 5.6

2.2 Aw 0.43

2.3 moisture I .28 Yo

2.4 characteristics- Small crystal, brown- red

color, have pieces of springonion

2. How is product toBe used

3.1 Type For cooking

3.2 Storage temperature Room Temperature

4. Package4.1 Type Laminated aluminum foil

package

4.2 Net content 6s

5. Process - Weigh ingredient according to it's formula- Mixing- Put into plastic bag in plastic container and packing- Keep at room temp. , contain into small package and pack

with noodle and oil

6. Shelflife 6 months

7. Where will the productsbe sold

Supermarket, retail shop, convenient store

8. Labeling instmction Informati on Ingredient information, cooking method,FDA license

9. Distribution Method Contain in box and carry by truck to targetshop

10. Target group Consumer All age group


Jiraporn Sonyim

Table 11. HA

Result / 46

. HAL T P model lbr large-scale seasonins of instant noodle manufachrer

Company name: N-l Quality manual

Flavor : mined pork Ingredients description Issue Date-.

Prepare by. .

Date.........Approve by. .

Date. ... . .. . .. .

Verified by..Date.........-

Ingredient/Rawmaterial

Suppliersite

Characteristic Packaging

Volume(ks / bas)

Storagecondition

l. Sugar x - solidwhite colorsmall crystal

Plasticbag

50 On shelf, instock room(T.27-35 "C)

2. Salt x solidwhite colorsmall crystal

Plasticbag

25 On shelf, instock room(T.27-35 "C)

3. Sodium

Monoglutamate

x - solidwhite colorsmall crystal

Plasticbag

25 On shelf, instock room(T.27-3s "C\

4. Pork powderFlavor

C - powder- white color

Plasticbug

l5 On shelf, instock room(T.27-3s "C)

5. White pepper Noodle-l - ground- green color

Plasticbag2layer

l0 On shelf, instock room(T.27-35 "C)

6. Dried chili Noodle-l - ground- red color

Plasticbag2layer

40 On shelf, instock room(T.21-3s'C)

7. Garl ic D - powder- yellow color

Plasticbag2layer

40 On shelf, instock room(T.27-3s "C)

8. Dried springOnion

D - pleces

- green colorPlasticbag

t2.5 On shell, instock room(r.27-35 "C)


Fac. OfGrad. Studies. Mahidol Univ.

Table 12. HACCP model for


of instant noodle manufacturer

Step Process/Rawmaterials

Process description Equipment

Sugar The sugars were contained in plastic

lqpon the wood shelf in stock room.- Plastic bag- Wood shelf

2. Salt The salts were contained in plastic bagson the wood shelf in stock room.

- Plastic bag- Wood shelf

3. Sodiummonoglutamate

The MSG were contained in plastic bagson the wood shelf in stock room.


4. Pork powder flavor The pork powder flavour were containedin aluminum foil packages on the woodshelf in stock room.

- Aluminumfoil package

- Wood shelf

5. White pepper The white peppers were contained inplastrc bags on the wood shelfin stockroom_


6. Roasting The white peppers were roasted in bigpot at 100'C for 80 minutes for increasesmell, reduced moisture to 3%anddestroyed the organism.

- Hot pot- Oven

1 Cooling The roasted peppers were cooled inplastic container in room air for 24 h.

- Plasticcontainer

8. Grinding The white peppers were ground by biggrinder until powder.

- Grinder

9. Packing The powder of white peppers werepacked in plastic bags 2 layer with rope

- Plastic bag- Rope

10. Drv chili The dry chilies were contained in plasticbags on the shelf (25Kg / bag)

- Plastic bag

ll Washing The dried chilies were put into softwater and sieved immediately to theline (25 kg chili / 500 lit of soft water)

- Soft water- Sieve


Jiraporn Sonyim

Table 12. HACCP model for

Result / 48

of instant noodle manufacturer (cont.

Step Process/Raw material Process description Equipment

12. Steaming a Pressure The chilies were moved on line tosteaming chamber with pressure for8-12 min, T.97 107 "C for destroyed theorganism.

- Closedsteamingchamber

13. Drying The chilies were moved on line to hotoven for 30 min, T.62 oC for reducedmoisture.

- Hot oven

14. Roasting The chilies were moved on line to big potfor roasting for 5 min, T. 170 "C untilbrown for smelling, reduced moisture to5% and deskoyed the organism.

- Big pot- oven

15. Cooling The roasted chilies were cooled in plasticcontainers in room temperature for 5-10min

- Plasticcontainer

16. Grinding The roasted chilies were put to plasticcontainers and moved to the grindergrinding roasted chilies until sround

-Grinder

t]. Packing The ground ofroasted chilies were packedin plastic bags 2 layer, tied with rope

Plastic bagRope

18. Garlic The garlic were contained in plastic bags Plastrc bag

19. Drying The garlic were dried in hot oven at 80 oC

for 24 h for reduced moisture and easy forpeeling.

- Hot oven

20. Peeling The garlic were peeled with peelerequipment

Peelerequipment

21. Sreving The garlic were sieved for removingforeignness

Sieve

22. Heating The garlic were heated in hot oven at 80"C, 20 h for reduced moisture anddestroyed the organism.

- hot oven


Fac. OfGrad. Studies. Mahidol Univ. (Food and Nutritional Toxicology) / 49

Table 12. HACCP model for -scale seasoninq ol inslant noodle manulaclurer (conl.

Company name : Noodle- I Qualrty manual Page............3........

Flavor : minced pork Process description Issue Date..

Prepare by. .

Date.........Approve by..Date...........

Verified by..Date.. -.. - -...

Step Process/Rawmaterial


23. Cooling The dned garlic were cooled in plastrccontainers in room air for 24hr.

- Plasticcontainer

24. Grindine The dried garlic were ground into Dowder - Grinder

25. Packing The garlic powder were packed intoplastic bas

- Plastic bag

26. Tran sferring Transferring to manufacturer by truck - Truck

27. Spring onions The spring onions were packed with rope - Rope

Washing Cleaned the spring onions with runningwater

- Runningwater

29. Soaking inchlorine solution(300 oom)

The spring onions were put into 300 ppm.of chlonne solution for reduced themicroorganism

- Chlorinesolution(300 ppm)

30. Chopping The spring onions were chopped intosmall pieces with chopper

- Chopper

3r Soaking inchlorine solution(50 ppm)

The small pieces of spring onions wereput into 50 ppm. of chlorine solution forreduced the microorganism

Chlorinesolution(s0 ppm)

32. Draining Drained for remove water and foreignness - Sieve

33. Dry heating The small pieces of spring onions wereheated in hot oven temp.SO oc for 5 h forreduced moisture and destroyed theorganism, .

- Hot oven

34. Cooling The dried spring onions were put in theplastic bags and cooling in air conditionerroom for 4h

- Air conditioner. Plastic bag



able 12. HACCP model fbr -scale seasoning ol'insl.ant noodle manulacturer {cont.

Company name: Noodle-l Quality manual Peoe 4

Flavor : minced pork Process description Issue Date. .

Prepare by..Date.........

Approve by. .

Date...........Verified by..Date-......-.

Step Process/Rawmaterial


35. Grading The dried spring onions were sieved forselected dried pieces of leaves size

Sieve

36. Drying The dned spring onions were dried in hotoven temp. 60 "c, 40-60 min. for reducingmoisture to 5% and long keeping.

Hot oven

37. Packing The dried leaves of spring onions packed inplastic bags, and stored in cold room

Plastrc bagCold room

38. Storage Al1 ofingredients were kept in woodshelves in stock roomT2T-35 "C.

Wood shelvesStock room

39. Weighing Each ofingredients was weighed,according to formula and packing in smallbags on the wood shelfin stock room.

BalancePlastic bagWood shelves

40. Mixing Each of small pack of ingredients (exceptdried spring onion)was poured into mixingchamber

-Mixingchamber

4t. Packing The mixed seasonings were packed intoplastic bag 2 layer, tied with tie in plasticcontainer and kept at room tempareture.formicrobiological checked (depending onthe option of the customers)

Plastic bagTieplasticcontainer

42. Transfemng bylift

Transferred the seasoning package by lift Plastic containerLift

43. Packing Packed into laminated aluminum foil bytrans ferring into package equipment

Aluminum foilPackerequipment

44. Keeping at roomtemp.

Kept package of seasoning in big plasticbags and stored at room temDerature.

stock room

45. Final packing Packed with noodle and oil packagenoodleoil packageCopyright by Mahidol University

Fac. OfGrad. Studies, Mahidol Univ. M.Sc. (Food and Nutritional Toxicology) / 5l

5.3.2.5 Hazard analysis and control measures (Form 4, 5)

The detailed information of the analysis were recorded step bystep in Table 13. The potential significant hazards which divided into biological,

chemical and physical hazard type. chemical and physical hazards were considered

satisfactory or hazard with minor level of severity for seasoning processing because

each ingredient was inspected and controlled by the process olGMp at the purchased

points. The potential hazards - major level of B. cereus were identified through the

whole process of seasoning production as shown in Figure 4. They were recorded inHACCP sheet Table 13. The potential hazards major level - could be 1) raw

materials i.e. sugar, pork powder flavour, white pepper, dried chili, garlic, and spring

onion. 2) risked processes, i.e., washing, grinding, peeling, cooling, soaking in

chlorine solution, transferring by truck, packing, mixing, and keeping at room

temperature. The heating step was considered as the process of critical risk because

they reduced or destroyed the organism. After all potential hazards - major level -were identified, the control measures of each potential hazard of process and raw

material of minced pork seasoning flavour were then listed step by step as shown inTable 14 (Form 5). Then the CCp were identified using Decision Tree.


Jrrapom Sonyim Result / 52

Table 13. HACCP model for large-scale seasoning of instant noodle manufacturer

Company name : Noodle-l HACCP sheet Page............1

Flavor : minced pork Hazard Analysis Issue Date..

Prepare by..Date....... ..

Approve by..Date- - .........

Verified by..Date..........

Step/Rawmaterial

Tlpeof

hazardHazard Hazard

descriptionLevel ofhazard,

Reason

1. Sugar B Microorganismexist

B. cereus Maj orIt is cause of

foodbomedisease

C - Chemicalresidual

- Bleachingagent

Minor Control byGMP

P

2. SaltB


- Bleachingagent

Minor Conkol byGMP

P


B

C Chemicalresidual

Bleachingagent

Minor Control byGMP

P

4. Pork powderflavor

B Microorganismexist

B. cereus Maj or It is cause offoodbome

disease

C

P

5. White pepper B Microorganismexist

B. cereus Maj or It is cause offoodborne

disease

C Pesticideresidual

- pesticideagent

Minor They are smallamount

P ForeignnessSoil, stone,

sand MinorControl by

GMP

6. Roastins B Microorganismsurvival

B. cereus Major It is cause offoodbome

disease

CP



able 13. HACCP model fbr of instant noodle manufacturer (cont.

Company name : Noodle-1 HACCP sheet Page............2..


Prepare by. .

Date..........Approve by..Date.. .. . ... .. .

Verilied by. .

Date..........

Step/Rawmaterial

Typeof

hazard,Hazard Hazard,

descriptionLevel ofhaz.ard

Reason

7. Cooling B Microorganismrecovery

- B. cereus Maj or It is cause offoodbome

diseaseC

P

8. Grinding B Microorganismcontamination


diseaseC

P

9. Packing R Microorganismcontamination

- B. cerett.s Major It is cause olfoodbome

diseaseC

P foreignness Dust MinorConhol by

GMP

10. Dr.r chili B Microorganismexist

- B. cereus Major It is cause offoodbome

disease

C Pesticideresidual

- pestrcideagent

Minor They are smallamount

P Foreignness- Soil,

stone, sand Minor

1 1 . Washing B Microorganismsurvival


diseaseC

P



Tahle 13. HACCP model for large-scale seasoning of insta nt noodle manufacturer (cont.

Company name : Noodle-[ HACCP sheet 1


Prepare by..Date..........

Approve by. .

Date. ... ... . ...Verified by..Date..........

Step/Rawmaterial

Typ.of

hazardHazard Hazard

description

Level ofhazard Reason

12. Streaming +Pressure

B Microorganismsurvival


disease

C

P

13. Drying B Microorganismsurvival


disease

CP

14. Roasting B Microorganismsurvival


disease

CP


- B. cereus Major It is cause offoodborne

disease

CP

16. Gnnding B Microorganismcontamination

- B. cereus MajorIt is cause of

foodbomedisease

C

P

17. Packing B Microorganismcontamination


foodbomedisease

CP loreignness - dust Minor



Table 13. HACCP model lor


mode )r -scale seasoning ofinslant noodle manufaclurer (cont.

Company name : Noodle-1 HACCP sheet Pqap l

Flavor : minced pork Hazard Analysis Issue Date. -


Approve by..Date......--.--

Verified by. .

Date-.... -....

Step/Rawmaterial

Typeof

hazard,Hazard Hazard.

descriptionLevel ofhazard

Reason

18. Garlic D Microorganismexist

- B. cereus Maj orIt is cause of

foodbomedisease

C

P Foreignness-Soil, stone,

sand Minor



foodbomedisease

CP

20. Peehng B Microorganismcontamination


foodbomedisease

CP Foreignness - Soil,

stone, sand

Minor Control byGMP

21. SievingBCP

22. Heating R Microorganismsurvival


foodbomedisease

CP



foodbomedisease

CP



foodbomedisease

CD



Table 13. HACCP model for ol inslant noodle manufacturer (cont.

Company name : Noodle-l HACCP sheet Parle 5

Flavor : minced pork Hazard Analvsis Issue Date. - -


Approve by. .

Date-..--.-....Verified by..Date.-.-......

Step/Rawmaterial

Typeof

hazard.

Hazard Hazarddescription

Level ofhazard, Reason



foodbomedisease

C

P

26. Transferringby tmck

B Microorganismrecovery


foodbomedisease

C

P

27. Spring onion B Microorganismexist


foodbomedisease

CPesticideresidual

- pesticideagent Minor

They are smallamount

P Foreignness - Soil MinorControl by

GMP

28. Washing B Microorganismsuvival


disease

CChemicalresidual

- Chlorinein water Minor

P

29. Soakrng inchlorine solution(300 ppm)



disease


- Chlorinein water

Minor Conhol byGMP

P

30. Chopping B Microorganismcontamination


foodbomedisease

CP Copyright by Mahidol University

Of Grad. Studies. Mahidol Univ. M.Sc. (Food and Nutritional Toxicology) / 57

Table 13. HACCP model for large-scale seasoning of instant noodle manuiacturer (cont.

Company name : Noodle-1 HACCP sheet Page............6...


Prepare by. .

Date.........Approve by..Date. . .. ... . .. .

Verified by. .

Date.. -..... ..

Step/Rawmaterial

Typeof

hazardHazard Hazard

description

Level ofhaz.ard Reason

31. Soaking inchlorine solution(50 ppm)


- B cereus Major It is cause offoodbome

disease

C

Chemicalresidual

- Chlorinein water Minor Control by

GMP

P

32. DrainingBC

P

33. Dry heating B Microorganismsurvival


foodbomedisease

C

P



foodbomedisease

C

P

35. GradingBC

P



foodbomedisease

C

P


- B ce,reus Maj orIt is cause offoodbome

disease

C

P



able 13. HACCP model for of instant noodle manufacturer (cont.

Company name : Noodle-l HACCP sheet Pooe 1


Prepare by. .

Date.. ... .. . .

Approve by. .

Date...........Verified by..Date.... ......

Step/Rawmaterial

Typeof

hazardHazard Hazard

description

Level ofhazard Reason

38. Storage B Microorganismrecovery


foodbomedisease

C

P

39. Weighing B Microorganismcontamination


foodbomedisease

C

P

40. Mixing B Microorganismcontamination


foodbornedisease

C

P



foodbomedisease

C

P

42. Transferringby lift

BC

P



foodbornedisease

C

P

44. Keeping atroom temp.



foodbomedisease

C

P

45. Finalpacking

BC

P


Fac. OfCrad. Studies, Mahidol Univ. M.Sc. (Food and Nutritional Toxicology) / 59

Table 14. HACCP model for of instant noodle manufacturer

Company name : Noodle-l HACCP sheet 1

Flavor : minced pork Control measure Issue Date..

Prepare by..Date... -... -.

Approve by..Date...........

Verified by..Date.. ........

Step/Rawmaterial

Typeof

hazardHaz.ard Hazard

descriptionControl measure

1. Sugar B Microorganismexist

B. cereus - Clean and sanitary- Food-grad material

permitted


B Microorganismexist

B. cereus - Clean and sanitary- Food-grad matenal

pemitted

5. White pepper B Microorganismexist

B. cercus - Clean and sanitary- Food-grad matenal

permitted


B. cereus - Controlled temp. 100 "Cand time for 80 min


- B. cereus - Controlled flowed air inroom and time for 24 hr


- B, cereus - Worker must be washtheir hand with soap andcleaned water and wear capand mask

- cleaned grinder before andafter used


- B. cereus - Worker must be washtheir hand with soap andcleaned water and wear capand mask

- Cleaned plastic containerand used new bag

10. Dry chili B Microorganismexist

- B. cereus - Clean and sanitary- Food-grad material

permitted

I I . Washing B Microorganismsurvival

- B. cereus Controlled volume ofwater / chili (500 L/ 25 ks)Copyright by Mahidol University


able 14. HACCP model lbr large-scale seasoning ofinstant noodle manufacturer (cont.

Company name : Noodle-l HACCP sheet Page............2...


Prepare by. .

Date....... ... .


Step/Rawmaterial

Tlpeof

haz.ard.


Control measure

12. Streaming +pressure


- B. cereus - Controlled Temp. 97-107oC and time for 8-12 min


- B. cereus - Controlled Temp. 62 "Cand time for 30 min


- B. cereus - ControlledTemp 170 "Cand time for 5 min


- B. cereus Confolled air flowed andtime for 5-10 min


- B. cereus- Worker must be wash

their hand with soap andcleaned water and wear capand mask

- cleaning grinder before andafter used


- B. cereus - Worker must be washtheir hand with soap andcleaned water and wear capand mask

- Cleaned plastic containerand use new bag

18. Garlic B Microorganismexist


permitted


- B. cereus - Control temp. 80 "C, for24hr.

20. Peeling B Microorganismcontamination

- B. cereus Cleaned peeling beforeand after used



Table 14. HACCP model for -scale seasonins of instrmt noodle manufacturer (cont.

Company name : Noodle-l HACCP sheet Page............3..


Prepare by..Date....-....

Approve by..Date.. .. .... ..


Step/Rawmaterial

Typeof

hazard,


Control measure

22. Heating B Microorganismsurvival

- B. cereus - Controlled temp 80 "C.and time for 20 hr


- B. cereus Controlled air flowed andtime for 24 hr


- B. cereus- Worker must be wash

their hand with soap andcleaned water and wearcap and mask

- cleaned grinder beforeand after used


- B. cereus - Worker must be washtheir hand with soap andcleaned water and wearcap and mask


26. Transferringby truck



permitted

27. Spnng onion B Microorganismexist

- B. cereus Clean and sanitaryFood-grad matenalpermitted

28. Washing B Microorganismsurvival

- B. cereus Controlled in and out ofrunning water

29. Soaking inchlorine solution


- B. cereus - concentration of chlorine300 ppm. and time for 20min

30. Copping B Microorganismcontamination

- B. cereus Cleaning chopper beforeand after used



Table 14. HACCP model for larqe-scale seasonins ofinsttrnt noodle manutacturer (cont.

Company name : Noodle-1 HACCP sheet 4

Flavor : minced pork Control measure Issue Date...

Prepare by..Date. ........



Step/Rawmaterial

Typeof

hazard


Control measure



- B. cereus - concenftation of chlorine50 ppm. and time for 20

min

33. Dry heating B Microorganismsurvival

- B. cereus - Controlled temp. 80 "C,for 5 hr.


- B- cereus - Control temp.25 oC andtime for 4 hr


- B. cereus - Controlled temp. 60 "C,and time for 40 60 min




38. Storage B Microorganismrecovery

- B. cereus Controlled cleaning, aircirculated and stockrotation (First- ln-First-Out)

39. Weighing R Microorganismcontamination

- B. cereus - Worker must be washtheir hand with soap and

cleaned water and wearcap, mask and gloves

- Cleaned balance beforeand after used

40. Mixing B Microorganismcontamination

- B. cereus Control cleaning ofmixing chamber afterused everydayCopyright by Mahidol University

Fac. OlGrad. Srudies. Mahidol L niv. M.Sc. (Food and Nutritional Toxicology) / 63

ahle 14. HACCP model for -scale seasonins of lnstant noodle manutacturer (cont-

Company name : Noodle-1 HACCP sheet Page............5..

Flavor : minced pork Control measure Issue Date. .

Prepare by..Date........


Step/Rawmaterial

Typeof

hazard


Control measure




- Micrologica'l check

43. SinglePacking

B Microorganismcontamination

- B. cereus - Worker must be washtheir hand with soap andcleaned water and wearcap, mask and gloves

- Cleaned automaticpacking before and afterused and cleaning duringoackins

44. Keepingat room temp.

B Microorganismgrowth

- B. cereus Controlled cleaning, aircirculated and stockrotation (First- In-First-ou0Produced quantiw/day

-a i,@'fy



5.3.2.6 Identifrcation of Critical Control Points (CCPs)

The CCPs (where hazards can be either prevented, eliminated or

reduced to an acceptable level) of the process of minced pork flavour seasoning were

identified using Decision Tree as shown in Tabte 15. (Form 6), Four questions in

Decision Tree (Figure 5) were asked step by step throughout the identified processes

of potential hazards-major level.

After running a Critical Control Point decision tree of the HACCP system,

4 CCPs were identify. The first CCP of B. cereus at the process for minced pork

seasoning flavour production was the steaming with pressure process at 97-107 oC for

8-12 min of dried chili (Step 12). It was because this step was used to eliminate or

reduce the microorganism especially B. cereus and its spore in dried chilli. The

second CCP was the step of heating garlic at 80 oC for 20 h in garlic line (Step 22).

This step controlled the moisture content of garlic to 3 per cent which can reduce the

spore of B. cereus. The third CCP was the soaking the spring onion in 300 ppm

chlorine solution at step 29. It was the step that expected to diminish most of the

organism in the spring onion. The last CCP was drying ol spring onion (Step 36) at

60"C, 40-60 min. This step controlled the moisture of spring onion to the level of 5

per cent and reduced Aw to 0.3. There were no CCPs in the seasoning line at the step

38 to step 45, because they were controlled by GMP.



Table 15. HACCP .odel for large-scale seasoning of instant noodle manufacturer

Company name : Noodle-l HACCP sheet Pase I

Flavor : minced pork Critical Control points Issue Date..


Approve by..l)ate-..-.......

Verified by. ,

Date. .... -....

Step/Rawmaterial

Hazard Control measureDecision Tree

CCP(Y,Dr)Qr Q2 Q3 Q4

l. Sugar B. cereus - Clean and sanitary- Food-grad material

permitted

,( ,( N

2. Salt



B. cereus - Clean and sanitary- Food-gad material

permitted

,( x N

5. White pepper B. cereus

Clean and sanitaryFood-glad materialpelmitted

x N

6. Roasting B. cereus - Controlled temp. 100 "Cand time for 80 min x x N

7. Cooling B. cereus Controlled flowed air inroom and time for 24lr

x N

8. Grinding B. cereus - Worker must be wash

their hand with soap

and cleaned water and

we.[ cap and maskcleaned grinder before

and after used

,( x N

9. Packing B.cereus Worker must be wash

their hand with soap

and cleaned water and

wear cap and maskCleaned plastic container

and used new bag -



Table 15. HACCP model for large-scale seasoning of instant noodle manufacturer (cont.)

Company name : Noodle-l HACCP sheet Page............2..

Flavor : minced pork Critical Control points Issue Date. . .



Verified by. .

Date..........

Step/Rawmaterial

Haz.ard Control measureDecision Tree

CCP(Y,AJ)Q1 Q2 Q3 Q4

10. Dry chili B.cereus - Clean and sanitary- Food-grad matenal

permitted

x N

I 1. Washing B .cereus Control quantity ofwater / chili( 500L/ 25 ks )

x N

12. Steaming+pressure

B . cereus - Controlled Temp. 97-107"C and time for8-12 min

YCCP

1

13. Drying B. cereus - Controlled Temp. 62oC and time Ibr 30 min

x x N

14. Roasting B. cereus - ControlledTemp 170 'Cand time for 5 min

,( ,( N

15. Cooling B. cereus - Controlled air flowedand time for 5-10 min

,( N

16. Grinding B. cereusWorker must be washtheir hand with soapand cleaned water andwear cap and maskCleaning grinder beforeand after used

x x N

17. Packing B. cereus - Worker must be washtheir hand with soap

and cleaned water andwear cap and mask

- Cleaned plasticcontainer and use newbas




Company name : Noodle-1 HACCP sheet Page....... .... .3..


Prepare by..Date....... ..

Approve by. .

Date............Verified by..Date..........

Step/Rawmaterial


CCP(Y,A{)Ql r)) Ql Q4

18. Garlic B. cereus Clean and sanitaryFood-grad materialpermitted

x N

19. Drying B. cereus Controlled temp. 80 "C,and time for 24hr.

,( N

20. Peeling B. cereus Worker must be washtheir hand with soapand cleaned water andwear cap and maskCleaning peelereveryday

x N

21. Sievins

22. Heating B. cereus Controlled temp 80 "C.and time for 20 hr.

x x YCCP

2

23. Cooling B. cereus Controlled air flowedand time for 24 hr

x x N

24. Grinding B. cereus

-Worker must be washtheir hand with soap andcleaned water and wearcap and mask

- Cleaned grinder beforeand after used

x x N

25. Packing B. cereus Worker must be washtheir hand with soapand cleaned water andwear cap and maskCleaned plasticcontainer and use newbag




Company name : Noodle-l HACCP sheet Pase 4





Step/Rawmaterial


CCP(Y,^J)Q1 Q2 Q3 Q4

26. Transferringby truck

B. cereus Clean and sanitaryFood-grad matenalpermitted

x t( N

27. Springonion

B, cereus ' Clean and sanitary- Food-grad matenal

permitted

x N

28. Washing B. cereus - Controlled in and out ofrunning water

x N


B. cereus - Conkolled concentrationof chlorine 300 ppm. andtime for 20 min

YCCP

3

30. Chopping B. cereus - Cleaning chopper beforeand after used

x N

31. Soaking rnchlorine solution

B.cereus Controlled concentrationof chlorine 50 ppm. andtime for 20 min

r N

32. Sievins

33. Dry heating B.cereus Controlled temp 80 "C.and time for 5 hr.

x N

34. Cooling B. cereus Controlled Temp. 25 "Cand time for 4 hr

x x N

35. Grading B. cereu.s

-Worker must be washtheir hand with soap andcleaned water and wearcap and mask

- Cleaned grinder beforeand after used

x N



Table 15, HACCP model for large-scale seasoning of instant noodle manufacturer (cont.)

Company name : Noodle-1 HACCP sheet Paoe 5

Flavor : minced pork Critical Control points Issue Date. -




Step/Rawmaterial

Hazard, Control measureDecision Tree

CCP(Y,/N)Q1 Q2 Q3 Q4

36. Drying B.cereus Controlled temp60 oC.

and time for 40 60min.

x x YCCP

4

37. Packing B. cereus Worker must be washtheir hand with soapand cleaned water andwear cap and maskCleaned plasticcontainer and use newbag

38. Storage B . cereus . Controlled cleaning, aircirculated and stockrotation (First- In-First-Out)

39. Weighing B .cereus Worker must be washtheir hand with soapand cleaned water andwear cap, mask andglovesCleaned balance beforeand after used

x x N

40.. Mixing B. cereus Control cleaning ofmixing chamber afterused everyday

,( x N

41. Packing B. cereus - Worker must be washtheir hand with soapand cleaned water andwear cap and mask

. Cleaned plasticcontainer and use newbag


Company name : Noodle-l HACCP sheet Page........ ....6..

Flavor : minced pork Critical Control points Issue Date.. -

Approve by. .

Date...........Verified by..Date..........

Jirarom Sonyim

Table 15.

Result / 70

HACCP model for large-scale seasoning of instant noodle manufacturer (cont.)

** It was not CCP because that controlled by GMP.

Step/Rawmaterial

Hazard Control measure Decision Tree CCP(Y/N)Ql Q2 Q3 Q4

42- Transferringby lift

43. Singlepacking

B. cereus - Worker must be washtheir hand with soapand cleaned water andwear cap, mask andgloves

-Cleaned automaticpacking before and afterused and cleaningduring packing

44. Storage atroom temp.

B. cereus - Controlled cleaning, aircirculated and stockrotahon (First- In-First-ou0

. Produced quantiMdav45. Final

packins


Fac. OfGrad. Studies. Mahidol Univ. M.Sc. (Food and Nutdtional Toxicology) / 71

CHAPTERVI

DISCUSSION

6.1 Popular brands and flavors of instant noodle, and the characteristics ofthe

consumers

According to the consurners' votes from the questionnaire survey, there were

three popular brands of instant noodle. For confidential purpose, the disguised names

of the three commercial brand as Noodle-l, -2 and -3 were used. The most popular

brand was Noodle-l which was in accordance with the data of market share reported

by The Nation newspaper (39). Most consumers knew Noodle-l for more than 30

years, and became a common name of instant noodles regardless of brands for many

consumers. The most two popular flavours were hot and sour-shrimp and minced

pork.

Different methods for instant noodle preparation were used among the

consumers. Most of them prepared instant noodle by soaking the noodle and its

seasoning in boiled water as shown on the label or boiling or soaking the noodle for 3-

5 min and then added seasoning belore consumption. The methods of preparation

practiced by the consumers were chosen for studying the effect of cooking on sulvival

of B. cereus in the noodles. It was surprised that eating raw instant noodle was one of

the consumers' ways of eating, especially with adding the seasoning. If B. cereus is

contaminated in the instant noodle, it might be harmful for vulnerable consumers like

children and elderly.

Adding of other ingredients such as meat, vegetables, egg, is suggested on most

of the label to increase the nutritive values of the instant noodles. Most of the instant

noodles contain high sodium content, 980-1460 mg/serving, equals to 40-60% Thai

RDI (43). However, the questionnaire survey revealed that instant noodle was not

frequently consumed, on average, the majority of the consumers (52%) consumed 1-2

times per week.


Jiraporn Sonyim f)iscussion / 72

6.2 The status ofB. celerrs contamination in the seasonings of instant noodles in

finished products

During last seven years,B. cereus was the most common organism found in 20 %

of the examined (213 samples) samples of seasoning but not iound in the fried

noodles. The updated status ol the B. cereus contamination in the seasoning of the

three most popular instant noodle was, therefore, determined in this study. B. cereus

contamination was found in every of the studied brands, with a slightly higher

percentage, 25.60/o of the examined samples (23 out of 90 lots). This implied that

although the Ministry of Public Heath Notification Number 2i0 (2000) (10) does not

allow any B. cereus in the instant noodle, the present status of the products have not

reach the Ministry of Public Health Standard. Nevertheless, the highest level of the B.

cereus conlamination that found in minced pork flavour of the instant noodle was, on

average, 23 MPNig (based on the information ftom 3M experiment on E. coli, it is

about 4 to 120 CFU/g (3M personal communication). The MPN determination is

particularly useful for low concentrations of viable organisms (< 100 /g) especially in

milk, water and foods where particulate matters may interlere with accurate colony

counts (40). Owing to the fact that B. cereus has high heat and drying resistance of

spores, they can form spore and germinate easily, have a short generation time, about

20 to 30 min (15). In addition, the optimum temperature for the spores to produce

toxin, especially the diarrheal enterotoxin, is at 25-37oC which is in the range of

body's temperature. Therefore, the consumers who consume the contaminated instant

noodle, even at the low level, could take a risk of B. cereus infection although the

contaminated foods is eaten without delay after cooking. The average percentage of

B. cereus contaminated in minced pork and chicken flavours was twice (about 35 %)

as much of the hot and sour-shrimp flavour (17%). The pH ofhot and sour-shrimp

flavour is about 4.0 which can inhibit the B. cereus growth (14). Although moderate

(26Yo) percenlage of B. cereus contamination in most of instant noodle was found,

with the best literature review, there has been no report on the outbreak after

consuming of the instant noodles. Underreporting of such outbreak is likely due to

the illness associated with B. cereus ts usually self-limiting and not severe. The

infected people may become ill and wait for symptoms to pass. They do not consult aCopyright by Mahidol University

Fac. Of Crad. Srudies. Mahidol Unir . M.Sc. (Food and Nutdtional Toxicology) / 73

doctor and no stool specimens are analysed for the presence of B. cereus. Unless the

instant noodles are controlled to be free from B. cereus, this product should not be

introduced to vulnerable groups of consumers. .8. cereus may play a significant role

as a causative agent of diarrhea in these groups. lnstant noodles are commonly eaten

in Thailand and they are one ofthe hottest food trend around the world, especially in

ASEAN. The good manufacturer practices at all steps of production musr be

strengthened, the elfective control of the products must be concemed and stimulated

for further investigation. The details of a proposed quality control system were

discussed in section 6.4.

6.3 Effect of different home cooking on the survival of B. cercus in the most

contaminated instant noodles.

The natural contamination of B. cereus in commercial instant noodle was not

uniformly distributed. To study the effect of heating on the reduction of B. cereus, the

inoculated seasoning of the instant noodle with B. cereus spores was, therefore,

prepared as the test materials. In this experiment, effect of 10 dilferent cooking

methods (including varying in temperatures and time) of instant noodle which have

been normally practiced by the consumers on the reduction of B. cereus were

demonstrated (Figure 3, Table 8).

The cooking processes of instant noodle applied in this study as instructed by the

manufacturer or practiced by the consumers or even the process which expected to be

an effective method of cooking which included different heating temperature (ranged

from 80 to 100"C) and time (1 to 6 min) cannot completely inactivated high

concentrations of B. cereus spores. This agreed well with the suggestion ofFemandez, 1999 (44,45) that the thermal treatment at 90-95 nC for 6-10 min is not

sufficient to inactivate the sporulated pathogenic flora, i.e., B. cereus or C. botulinum.

Two home cooking methods which showed the highest reduction (25oh) of B. cereus

were Method 1, as instruction on the label (mixing the seasonings with instant noodle

in a bowl belore adding the boiling water, then covered the bowl and left for 3 min)

and Method 3.1, one of the selected methods by the consumers (boiling noodle for 2Copyright by Mahidol University

Jiraporn Sonyim Discussion / 74

min, and then added contaminated seasoning). The temperature of the heated instant

noodle was around 85-95 oC and the noodle v/as heated for at least 4 min (included

during homogenisated time) in Method 1 and around 95-100 "C for 1 min during

homogenisation in Method 3.1. It reduced B. cereus spores by 1.37 and 1.34 log

CFU/g, respectively fiom the original level. This might be because the two processes

included sudden heating (heat shock) of the contaminated seasoning at high

temperature. However, the significant difference in per cent reduction of B. cereus by

heating process of Methods 3.2 and 3.3 which varied in cooking time of instant noodle

but equal time olheating for contaminated seasoning, compared to Method 3.1,cannot

be explained.

The second method of cooking (Method 2.1-2.3) involved soaking noodle in boiling

water for 3,4, 5 min and added contaminated seasoning before consumption. By these

methods, the spores was heat-treated at the lower starting temperature, 80-90oC

(temperature after soaking the noodle for 3 to 5 min) compared to the contacted

temperature of 100"C in Method 1 and 3. They showed lower range of the log

reduction of B. cereus,0.84 to f .i3logCFU/g(16-21 % reduction).

The heat treatment in Method 4.1-4.3 included boiling the seasoning for 2,3, 5

min and then added the instant noodle and continued boiling for 1 min. This was

expected to be the effective method to destroy B. cereus. However, the process

reduced the organism only 15-18 % (0.81-1 log CFU/g) of the original level. The

inoculated seasoning wirh B. cereus spores was first soaked in water at 35 oC, the

temperature of water increased stepwise to boiling at about 100 oC which took about 2

min. The gradually increase of the heating temperature may increase the thermal

resistant of the spores of B. cereus, therefore, showed the least reduction ellect on the

heat resistant B. cereus spores. Femand ez et. al., 2001 (46) reporled that the non-

isothermal step (an increase in the temperature of the substrate in which the

microorganism is suspended in accordance with a temperature program) at heating rate

of 2"C/min from 25 to 80 oC increased the number of germinated spores more than the

isothermal step (80 oC for 10 min) after heating. The non-isothermal step may

increase heat resistance olthe contaminated spores or activate the spore germination at

the later stage. Even though the heating rate in Method 4 was much faster than those

reported by Fernandez et. al., 20Ol (46), starting from 35 and increased to 100 0CCopyright by Mahidol University


within 2 min, but by nature the spores may have evolved adaptive networks to face the

challenges of changing environments and to survive under condition of stress (47).

The mechanism of survival, i.e. the protection of spore DNA by alp-type SASP, spore

core mineralisation, and spore core dehydration, were the natural factors that cause

spore resistant (48). Furthermore, heat resistance of the B. cereus spores is increased

by low water activity due to high salt concentrations in the instant noodle seasoning

and by gradual heating. (49, 50)

Since the spores ofB. cereus are the heat resistant, its D-value in water at 100oC

is 6.7-8.3 min.(14) The ordinary cooking time for the instant noodles (less than 5 min

heating), may destroy the B. cereus vegetative cells but not their spores, and may not

possible to reach even the level at the D-value. If cooking time is extended, the noodle

became too soft and not suitable for consumption.

Since the contaminated spores of Bacillus cereus in instant noodle cannot

be deskoyed by ordinary cooking, therefore, the GMP (51) and an intensive quality

control system of the whole process, starting from the raw materials, is of most

important to prevent B. cereus contamination, spore production and germination.

Usually the instant noodles is consumed immediately after preparation. However, less

thxt l0%o of the consumers left the cooked noodle at rcom temperature for 5-10 min to

be cool or left it for 10-30 min to absorb more iiquid before consumption. These

groups ofconsumers may have more chance to get B. cereus infection

If the contaminated. B. cerens in the instant noodle seasoning is the strain that can

produce emetic toxin. Once the spores have formed on food and the bacteria produced

the toxins, the emetic activity is not reduced through normal food preparation

methods. Thus to prevent the onset of vomiting type food poisoning dle to B. cereus,

it is important to prevent the groMh of bacteria through close temperatue control at

all stages, from the raw ingredients through the preparation and cooking (48, 52, 53).



6.4 Development of HACCP model for identification of critical control points

HACCP is a preventive system of quality conhol. The system when properly applied

can be used to control any area or point in food system that could contribute to a

hazardous situation.(29). HACCP is, therefore, introduced to the process of instant

noodle seasoning. The hazard analysis involves a systematic study of the seasoning

ingredients, their description, the condition of their processing, handing, storage,

packaging, distribution and consumption. B. cereus is a ubiquitous bacterium found

in soil and in many raw and processed food such as rice, milk, dairy products, spices,

vegetable, meat products and iarinaceous (45, 53) and had hecome one of the more

importance causes offood poisoning in the industry world (Granum et. al' 1997)'(16) '

For the last 7 years, the organism was found also in about 20% of the instant

noodle seasoning and in this study abott 25o/o of the products were iound

contaminated with the same organism. Thus, the hazard analysis fot B. cereus was

conducted. The potential hazards and the critical control Points in the overall

processes of instant noodle seasoning included al1 raw materials were indicated.

potential hazard was identified in the high risk ingredients and processes. Since

the ingredients of instant noodle composed of garlic powder, dried spring onion,

pepper powder, and chili powder, all can be considered as high-risk ingredients for B

cereus. The supported information are as follows.

A moderate contamination of B. cereus was found in 30% of the examined dried

garlic in this study. Y alero et. a/.(54) examined B. cereus in fresh garlic, they did not

detect the organism in 6 examined samples which might be because the garlic extract

can inhibit the growth of B. cereus (55). Drying process of garlic may destroy the.B.

cereus inhrbitor in fresh garlic or the whole process for managing dried garlic may

cause B. cereus contamination or spore activation. Thus, dried garlic was considered

to be one of the high risk ingredients.

B. cereus contamination was found in 100% examined dried spring onion and at

the highest tevel. It can be considered as the most high risk ingredient in the instant

noodle seasoning. The results suggested that the treatment process of the dried spring

onion cannot eliminate the contaminated B. cereus and it is the source of the

contamination in finished products ofthe instant noodle seasoning.Copyright by Mahidol University

Fac. OfGrad. Studies, Mahidol Univ. M.Sc. (Food and Nutdtional Toxicology) / 77

B. cereus was detected in 10% of examined samples of white pepper powder.

valero e/. a/.(54) isolated B. cereus from fresh vegetables and detected B. cereus in100% of the examined sample (11 samples) of fresh pepper. It is possible that the

roasting process at high temperature for a long period (100 "c, g0 min) which was one

of the processing step of dried pepper, may reduce the contamination of B. cereus, inthe fiesh pepper, if any.

Minimum level of B. cereus conrarnination was found in l0% of the examined

dried chilli. B. cereus contamination was reported in fresh chili with the aerobic plate

counts of10i CFU/g (55). Other ingredients i.e. sugar, salt , pork powder flavour were

not high risk of B.cereus source. They were controlled with specifications food-

grade materials such as low moisture, Aw and supplier assurance for compliance with

standards (17).

The identification of ccPs in the process of instant noodle seasoning were based

on the findings of any points in the chain of seasoning production where the loss ofcontrol could result in an unacceptable food safety risk (56).

There were no CCP in the existing processes of pepper at the manufacturer

because there was no step in this line that can control B. cereus spore contamination.

Although the roasting step of pepper line was carried out at 100 oC, for 80 min, it was

not considered as the CCP because the spore can tolerance for dry-heating at 100 oC

for almost a month (47,48). However at this step, the moisture and Aw of the pepper

were controlled to be 3-5%o and 0.3 respectively. According to the FDA (1998) (14)

condition, the minimum Aw of -8. cereus is 0.912. Therefore, roasting at the above

conditions may inhibit the growth of B. cereus spores. The recommendation to

reduce or eliminate the spore of the B cereus is as follows. At step 5 when the seeds

reach the manufacturer, they should be cleaned by hot blowing to remove soil and

dust. Then the seeds should be quick washed with water. At this step the seeds willget some moist which increases the effectiveness in elimination of the spore in the

following heat treatment than just dry heating (48). In the grinding step, extensive

washing of the grinder by detergent, and then repeat cleaning with disinfectart i.e.

70o/o alcohol or soaking with high temperatwe water should be done. This is because

B. cereus spore can survive and adhere to the surface ofthe utensils (57).


Jirapom Sonyim Discussion / 78

The wet-heat at 100 oC without pressure, the spores of B. cereus can survive

more than one hour (48). Wet-heating with high pressure may be more effectively for

eliminated the spores (47). For chili, the CCP was identihed at the steaming process

of chili with pressure at step 12 that control the temperature at 97 -107 "C for 8-12

min. At this step, it was expected to destroy most of the microorganism, including B.

ce,,eus spore. After the CCP step, the chili may be cross-contamination so the

roasting step at the temperature of 170 oC lor 5 min should be quarantined for

controlling the low moisture and Aw. To ensure the sterility of the chili, it is

recommended that the temperature at steaming step should be increased to 110-120 "C

and the period of autoclaving can be reduced to 2-5 min which is the condition for

spore inactivation (48).

In the process of garlic, the CCP was identified at the heating step, 80 oC for 20 h,

in step 22. This step controlled the moisture content and the Aw of garlic. Even

though this step involved a long dry heating time, the temperature at 80oC was not

ellectively inactivated the spores of B. cereus (48). Nicholson et. d/., repofied that the

bacilli spore can survive in moist heat (i00 oC atmospheric pressure) with a D-value of

20-30 min and moreover, while spores survive approximately 1,000-fold longer in dry

heat than in moist heat. T he condition of Aw at 0.3 cannot destroy the spore, but

inhibit spores germination and growth (14, 58, 59, 60). The recommendation lor

reduction of B. cereus in Garlic are: the garlic should be first blew to remove soil and

dust and intensively control for low moisture and Aw throughout the processing line.

Two CCPs were identified for spring onion which was found to be the ingredient

with the highest B. cereus contamination. The first CCPs was at the chlorinated

soaking in step 29 which was expected to reduce the contamination of

microorganisms. This can be supported by the finding of Larry et. al. (61). They

found that dipping sprouts in solutions containing 500 ppm chlorine for 2 minutes, the

pathogen was reduced by about 2 log10 CFU/g, compared with the control, and after

treatment with 2,000 ppm, reduced to and under detectable level (<lCFU/g). WHO

(61) also reported that treatment fruits and vegetables in water containing 200-250

ppm chlorine reduced populations of aerobic microorganisms by 90-99% and

psychotrophic microorganisms, yeast and moulds by 50-90%. The second CCP in

spring onion line was the last drying step at 60oC for 40-60 min with similar reasons as

L



the garlic that was to control moisture and Aw (62). Although the CCps in the spring

onion processed line were identified as the chlorinated soaking and the final drying

step. The washing at step 28 should be concemed because this step can substartially

reduce the contaminated of B. cereus from soil and environment. This can increase

the effectiveness in eliminating of the spores in chlorinated soaking at next step.

WHO suggested that the dipping inoculated sporouts in steriled water containing no

chlorine reduced the number of the microorganism by about 10 fold (61).

To reduce the B. cereus contamination in the dried spring onion, the modification

of some processes is recommended. Since only the leaves (the upper part) of the

spring onion are used in the instant noodle seasoning, so at the first step before

washing, this part of the spring onion should be separated from the lower part to

reduce or get rid of the B. cereus from soil contamination. In addition, other

disinfectants such as acid or combined acids, hypochlorite or mixture of acid and

hypochlorite solution may be used (63,64,65). Ellin, 2001 (50) reported acidity (pH

<6.7) and a low water activity (<0.98) inhibit germination and growth of B. cereus

after heating.

There were no CCPs lor the seasoning process at step 38 to 45 in Figure 4

because they have no effective control measure to control the existing B. cereus.. The

best control system should be the intensive GMP. For the storage conditions, the

ingredients and mixed seasoning were kept at room temperature at 27-35oC for a long

time. B. cereus is a mesophilic organism with an optimum temperature for growth at

30-35"C (although some strains are psychrotrophic, growing at temperature down to

5"C ) (17,66,6'/). AI this step, the heat activated B. cereus spores from the previous

process, ifany, can be subsequently outgrowth in the products. . Rocken and Voysey,

1993; Rodriguez et. al., 1992 suggested that the germination and growth of Bacillus

strains can be retarded by avoiding their optimum growth temperature of 37 oC (59,

68, 69). The storage step ofthe instant noodle seasoning should be more concemed.

The intensive quality control system should be set up to limit the opportunity for the

growth of B. cereus, by controlling the temperature, low Aw, sanitation, air flowed,

especially the product management system which the FIFO (First in First Out) (70, 71,

72, 73) system can be applied.


l0 Driedchilil lts carlicl I 27 Snrins onionl-'--"" ""-'--"'hr : _.=__r-_____________7 --:-- -------.\

t. Srga, I

2.S alt

+ j:":1_____,i#,iI with I | 19. Drying I

- I lonon'r,rr-- I

l. MSG | |

4. Pork power flavor l" "Y::lll

k-Blo*ing

[+-wurt'in*v

I o. Roasting II too"c for8ominI

+

F{""r*g-1

T rr^s"lr no-

rick) ;L

a1

Ij

I

' '"" ' ':__lI L_1_VV

"*c;;;-_-1 tr"*tr120

o C for 2-5 min I L___-____l-ttr-"re

.1. Drvins I L--r-;Z.CiO min I -----L ,

| 22. Heating II lsooc2ohr I+ L_-----r-

14. Roastr'rcl70 oc.5 min I lz:.Cooling__l

-_--T- L---------1--

8. GrindingIY

ll5. Coolins I I 24. Grindn;.1

). Packing inplastic bag

V


6. Grinding

17. Packing inplastic bag 26. Transferring

by truck

3 8. Storage <t_

39. Weighing 3 8. Storage

trd--t-tf

-+l41J."ki,u;l37. Packing in plastic bag

storing in cold roomplasl

---T-

.ic bag I

FIFO

+

.*,,'gt.ilr-*l-J 44. Storage at room temp


31 . Soaking inchlorine solution

33. Dry heating80

oC for 5hr

36. Drying 60 o Cfor 40 - 60 min

Figure 7, Recommend flow diagram ofinstant noodle seasoning: minced pork flavor


Fac. OfGrad. Studies, Mahidol Univ. M.Sc. (Food and Nutritional Toxicology) / 8l

CHAPTERVII

CONCLUSION

The objective of this research was to study the feasibility to control Bacillus

cereus corll"arnination in instant noodles seasoning. The study composed the status ofB. cereus contamination, effect of different methods of cooking, identification ofhazard zones ard critical control points of Bacillus cereus inthe processing line of the

instant noodle seasoning. The conclusion of the findings were as follows.

1. The most two popular flavours of the instant noodles were hot and sour-

shrimp and minced pork. The majority of the consumers (52%) consumed the instant

noodles for 1-2 times per week. Most of them prepared instant noodle by soaking the

noodle and its seasoning in boiled water or boiling the noodle for 3-5 min and added

seasoning before consumption. The cornmon cooking methods practiced by the

consnmers were used to designed the study on effect of cooking on B. cereus. Some

of tie consumers (more than 80%) added other ingredients such as meat, vegetables,

egg, mushroom, etc., into the instant noodle. This practice can increase the nutritive

values ofthe instant noodle.

2. B. cereus contamination was found in every of the tkee most popular

brands of the studied instant noodle. The percentage found was slightly higher than

the previous report, 26 o/o (23 ont of 90 lots) compared to 2Oyo of the examined

samples. The minced pork flavour seasoning showed the most B. cereus

contamination but the highest level was only 23 MPN/g. However, the Ministry ofPublic Heath Notification Number 210 (2000) ( 10) does not allow any B. cereus in the

instant noodle because its spores are heat resistant. Spores of B. cereus germinate

easily and have a short generation time (less than 30 min) and can reach the infection

dose at the body temperatufe. Therefore, the consumers who consume the

contaminated instant noodle, even at the low level, could get B. cereus foodbome

illness-Copyright by Mahidol University

Jirapom Sonyim Conclusion / 82

3. The cooking method by mixing instant noodles with its seasoning, ther.r

pouring the boiling water 360 mL and leave covered for 3 min, as instructed on most

of the label of instant noodle, showed the highest reduction (25%), reduced Bacillus

cereus by 1.37 log CFU/g. This eflect was similar to cooking noodle in boiling water

for 2 min and then added the seasoning. For other cooking methods, the B. cereus

spores was reduced by 0.81-1.13 log CFU/g. Similar to other previous studies in

various foods, the normal cooking could not eliminate the spore of B. cereus in the

contaminated instant noodle. Therefore, prevention for contamination, germination.

outgrowth of B. cereus spores and enterotoxin production should be practiced.

4. All ingredients of instant noodles, i.e., garlic powder, dried spring onion,

pepper powder and chilli powder were identified as high risk ingredients for B. cereus.

Its contamination was found in all ingredients. Dried spring onion was identified as

the most high risk ingredient and the source of B. cereus contamination in the instant

noodle seasoning. B. cereus contamination was found in 100% of the examined

sample and at the highest level. The CCPs in the processing lines of the ingredients

- chili, garlic and spring onion - of the instant noodle seasoning were identified.

Modification of practices were proposed in some critical steps to increase the

effectiveness in preventing the contamination, spore germination and outgrowth and

the enterotoxin production of B. cereus, i.e., cleaning raw materials by air blowing,

separation of suspected source of B. cereus contamination, washing raw materials with

running water, adjusting temperature and time for heat treatment. Instant noodles are

commonly eaten in all age groups in Thailand and they are one ol the hottest food

export around the world, especially in ASEAN. To guarantee the quality of the

products, good manufacturer practices at all step of production must be strengthened,

the effective control HACCP system olthe products must be established.


Fac. Of Grad. Str.rdies. Mahidol Univ. M.Sc. (Food and Nutritional Toxicology) / 83

Suggestion for Further Study

1. The proposed CCPs and the recommendation of practices for the effective control

of B. cereus should be disseminated and tested at the instant noodle manufactures.

The elfectiveness of the proposed CCP and the recommendation should be

evaluated and improved, if necessary. The effective GMP and quality control

system should be encouraged at the manulactures of instant noodle ingredients.

The B cereus determination should be included as one of the quality control

criteria lor instant noodle.

2. Although cooking methods applied in this study cannot eliminate the inoculated

infective dose of B. cereus spores in the instant noodle. Other practical methods of

cooking and mechanisms involved should be evaluated using instant noodle with

different level of8. cereas contamination.


l.

2.

Jirapom Sonyim Reference / 84

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chavasit v and Phromyothi D. The Role of Instant noodles in Thailand. In: The

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3. chavasit v aad rontisirin K. Triple fortification of instant noodle in Thailand.

Food and Nutrition Bullentin 1998; l9(2): 164_7 .

4. Jitpleecheep S. Noodles producers to meet in Bangkok. Bangkok post 2001 Mar15; Business.

5. Schlude M. Enriched instant noodles marketing opportunity. J Asia pacific Food

Industry 1992;73-4.

6. Food Microbiology Lab report (SFM6). Institue of Nutrition: Mahidol University.

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1999;10: 213-9.Copyright by Mahidol University

Jirapom Sonyim Appendix A / 90

APPENDIXA



APPENDIX A (uuaornu)

tufr.........................01q..........fl lart......... 01fin..................)).4 ;L --a)"- _

1.F\[u?lrlttyl"]u1ly1r!n.0fl 1trq0 uofl trn Luu 1nr,in001,Ftg utoRnt{noiu

- .].1 s " ,i,. i d - , i2. Lyll i.:tir[5cr! fia'[ofrnnr.lor.t uioil:.]rymrurjotrfi4oc rn!^ntlfios (3dfi0)

1)... . ... a .....3)............

^ t 4v r i )e3.koinyEfio roLl uSofil tyrlurdlrrulJ1nfr4n frfoay 3rn r5fl{crnrolurnt urrioa

1)dio 16 r).................. .. .... 2)...... . .......... . 3)...... . . . ....i"

2)8rro................... ,6 1)..........................2)................. . . .....3). . ...

3)6fi0.. . . ... ,n1)............... ..........2)...... .............. . ...3)..... . .. ..

))" -4.eu ,t6 ujttrvr1uLyx .r,i61ltqr oti1.i'[t.

n. iurl:yvnufil 1. firiryyrluut.t:lnenorrri6iryrudrto{ n.flLllrywturflLfiNrSoriasu4n;{.6u'l rr\............. lnolldrrnn,jr r fio1

5. nlyllUtLllrtvt'tutrulr olr vrlu [6 r.Ato.:1]r.r 56urf] llJ

n. 1d

o.firvirutarflirlr: ^r-irlt-!n,tn v,ru'[drn?atiq.lrnmoutuu

n. riouldr.LSou r. maurduuo.rlfifinioufiqri::rl:lvnu u. du 1 ,=q

z.flrvirutorir r:yr,luLruun,tn yiru'Lfrrarnrnururlryurn:fi urfi

...........u1vr

8. firviru'lorirlryyrluulrufi N viT u1{rqnr fi :ruru ry l run'urfi

,,,,,..,-,,u1}1

,_-.J. .\,:," i da-,9.vr1ufl.l!t:yr1ulryl.l n.i61ttsr!yruyt1111luL?,iL6ts1rto t e1 [tJr{ [iuûRu.1rc.if!lr:yl,tu

x -"-10. yllutnN tuofiEl,t un

n. hi rvr. . . . . . . . . . . . . . ..

viiornia.rrJq.rdu 1 fictLuiolri r:r1

1r tLr Copyright by Mahidol University

Jirapom Sonyim Appendix B / 92

APPENDIX B


1 . Sugar 2. Salt | | :. ci,". *,a n"*a..

" T'""i^S':ffrr'-l4. MSG

8. Driedsalted shrir

_ I . LraYlJ ur rLlru "Irr I

". 1

| 10. Washing I I zo. St------]- tr.97-.IIlYJ lt t. c.inaing, .r,oppirg

I-r+,rf-*l

'ro.,;-l-Jv

Removing oil. cooling I

+

r1r:s-rg I

tr 22. Roasting170oc,5min

23. Cooling

24.Grinding

16. Tkough pipe with pump---T_---125.

Packing in plastic bag

17. Keep in stock tank

38. Storage

r40. Mixing 39. Weighing and packingfor proportion

41. Packing inplastic bag 42. Transferring by lift 38. storag 37. Trans ferring

by truck

43. Single packaging ffiryroomtemp t4. Packing with noodle

ffiFac. Of Grad. Studies, Mahidolurir.;:rr;rt,o: M.sc. (Food and Nutritional roxicology) / 93'r&r,1n,

,,*,,,.r\\\'

APPENDIX B

18. Dried chili

20. Steaming + PressureT.97 - 107

oC for 8 - 12 min

21. Drying62

o c, 30 min

28. Soaking withchlorine solution

(300 ppm)

30.Soaking inchlonne solution

(s0 ppm)

32. Heating 80 o C, 5 hI.

35. Drying 60 o C

, 40 60 min

36. Packing in plastic bagstoring in cold room

Figure 5 Flow diagram of instant noodle seasoning: hot and sour-shrimp flavourCopyright by Mahidol University

Jiraporn SonyimAppendix B /94

T=ll P*,, I8. Garlic

1l\4scll 4. Feed dried egg

II 1. Washing with

soft water19. Drying80oc24hr.

J12. Steaming + Pressure

T.91 107 oC forS -12 min | 20. Peehng-----r-----

I 6. Rn"

I roo ctI'"'^ -,- I21. Sieving

13.^ Drying62 'C 30 min

-"""-'li

f.*,r-lI

+ls. crina,n,, If v

[;;;]I pr*ti. nlre

I

22. Heating80 oC 20 hr

14. Roasting170 oC,

5 min 23. Cooling

15. Coolins 24. Grindine

16. Grinding----_.--r+

Y


17. Packrng inplastic bag 26. Transfemng

by truck

38. Storage

rG

39. Weighing 38. Storage

Ts--t{-il"d;l

.li".kG;-lplastic bag

I---]-

37. Packing in plastic bagstoring in cold room

--->43. Single packaging

I -f44. Storage at room temp

33. Dry heating80 oC for 5 hr

36. Dryrng 60 oC

for 40 60 min

Figure 6 Flow diagram ofinstant noodle seasoning: chicken soup flavourCopyright by Mahidol University


APPENDIX C


JAapom Sonyim Appendix C / 96

APPENDIX C

Enumeration and confirmation oI B. cereus in foods (MpN)

The MPN technique is recommended for enumerating B. cereus in foods that

are expected to contain fewer than 100 B. cereus organism/g.

Materials and equipment

1. Pipettes, 1, 5, and 10 ml, graduated in 0.1 ml units

2. Incubator 35 + 2oC

3. Marking pens

4. Bunsen bumers

5. Wire loops, platinum wire, 2 mm and 3 mm

6. Vortex mixet

7. Microscope, microscope slides

8. Test tubes, 16xl25 mm, 13x 100 mm, sterile

9. Bottles, sterile

10. Anaerobic jar

1 1. Gas tank, with 90%Nz +10%COz

12. Racks

13. Petri dishes, sterile, 15 x 100 mm

Media and reagetrts

1. Selective medium: mannitol-egg yolk-polymyxin (Myp) agar plates

2. Enrichment medium: egg yolk emulsion, 50%, trypticase soy-polymyxin

broth

3. Polyrnyxin B solutions for MYP agar (0.1%) and trypticase soy-poll,rnyxin

broth (0.15%)

4. Media and reagents for biochemical test

4.1 Phenol red glucose broth

4.2 Tyrosine agar

4.3 Lysozyme broth

4.4 Voges-ProskauermediumCopyright by Mahidol University


4.5 Nitrate broth

4.6 Nutrient agar for B. cereus

4.7 Motility medilm (p. cereus)

4.8 Trypticase soy-sheep blood agar

4.9 Nitrite detection reagents

4. 10 Voges-Proskauer test reagents

4.11 Creatine crystals

4.12 Gram stain reagents

4.13 Basic fuchsin staining solution

4.14 Methanol

4.15 Spore stain reagents

4.16 Butterfield's phosphate-buffered dilution water, sterilised in bottles to

yield final volumes of 225 mL and in tubes of 9 mI_

Sampling

If the quantity offood to be examined is large, take representative sample of25g each from different parts of the suspected food because contamination mav be

unevenly distributed.

Sample preparation

Using aseptic technique, weigh 25 g of sample into a steriled plastic bag. Add225 mL butterfield's phosphate-buffered dilutiopn water (1:10 dilution) and blend

with stomacher for 2 min. prepare serial dilution of the l:10 diluted sample until 10-3

dilution is reached.

MPN technique for quantitative determination of B. cereus (Appendix 3 Frowchart 1)

. lnoculate 3 tubes MPN series containing trypticase soy-polymyrin broth

using I ml inoculum of 10-r, 10r, and 10-3 dilutions of sample with 3 tubes

at each dilution.

o lncubate the tubes for 48 h at 35"C and observe for dense growth, which is

typtcal of B. cereus.Copyright by Mahidol University

Jirapom Sonyim Appendix C / 98

. Streak cultures from positive tubes onto separate MYP agar plates and

incubates plates for 24h at35oC.

Pick 5-eosin pink, lecithinase-positive colonies from each MYP agar plate

and transfer each to a nutrient agar slant for confirmation as B. cereus.

Calculate MPN of B. cereus cell/g of sample from MPN table (for 3

fermentation tubes at 0.1, 0.01, 0.001 g inocula) based on the number oftubes at each dilution in which the presence of B. cereus was confirmed.

Con{irmation of B. cereus (Appendix 3, Flow chart 2 )

Incubate nutrient agar slants containing suspected culture for 24 h at 35oC.

Prepare gram-stained smears from slants and examine microscopically. B.

cereus will appear as large gram-positive bacilli in shortto-long chains;

spores are ellipsoidal, central to subterminal, and do not swell the

sporangium.

Transfer 3 mm loopful of culture from each slant to 13 x 100 mm tube

containing 0.5 ml of sterile phosphate-buffered dilution water and suspend

culture in diluent with vortex mixer.

Use suspended cultures to inoculate the following confirmatory media.

The basic characteristics of Bacillus group test

Test 1. Phenol red glucose broth

o Inoculate 3 ml broth with 2 mm loopful of culture. Iacubate tubes

anaerobically 24 h at 35"C in GasPak anaerobic jax.

o Shake tubes vigorously and observe for growth as indicated by increased

turbidity and color change fiom red to yellow, which indicates that acid has

been produced anaerobically from glucose. A partial color change ftom

red to orange/yellow may occur, even in uninocuiated control tubes, due to

a pH reduction upon exposure of media to CO2 formed in GasPak

anaerobic jars. Be sure to use appropriate positive and negative controls so

that a distinction can be made between positive and "false-positive"

reactions. Copyright by Mahidol University


Test 2 Nitrate broth.

. Inoculate 5 ml broth with 3 mm loopful of culture. Incubate tubes 24 h at

35"C.

. To test for nitrite, add 0.25 ml each of nitrite test reagents A and C to each

culture. Al orange color, which develops within 10 min, indicates that

nitrate has been reduced to nitrite.

Test 3. Modified VP medium.

o lnoculate 5 ml medium with 3 mm loopful of culture and incubate tubes 48

+2hat35"C.

o Test for production of acetylmethyl-carbinol by pipetting 1 ml culture into

16 x 125 mm test tube and adding 0.6 ml alpha-naphthol solution (R89) and

0.2 ml 40% potassium hydroxide (R89). Shake, and add a few crystals ofcreatine. Observe results after holding for t h at room temperature. Test is

positive ifpink or violet color develops.

Test 4. Tyrosine agar.

o Inoculate entire surface of tyrosine agar slant with 3 mm loopful of culture.

Incubate slants 48 h at 35"C.

Test 5.

a

Observe for clearing of medium near growth, which indicates that tyrosine

has been decomposed. Examine negative slants for obvious signs of

$owth, and incubate for a total of 7 days before considering as negative.

Lysozyme broth.

Inoculate 2.5 ml of nutrient broth containing 0.001% lysozyme with 2 mm

loopful of culture. Also inoculate 2.5 ml of plain nutrient broth as positive

control. lncubate tubes 24 h at 35'C.

o Examine for growth in lysozyme broth and in nutrient broth control.

Incubate negative tubes for additional 24 h before discarding.

Test 6. Record results obtained with the different confirmatory tests.

Tentatively identify as B. cereus those isolates which

7.1 Produce large Gram-positive rods with spores that do not swell the

sporangium.

Produce lecithinase and do not ferment mannitol on MYP agar.

Grow and produce acid ftom glucose anaerobically.

7.2

7.3Copyright by Mahidol University

!


7.4 Reduce nitrate to nitrite (a few strains may be negative).

7.5 Produce acetylmethylcarbinol (VP-positive).

7.6 Decompose L-tlrosine; and

7.7 Grow in the presence of 0.001% lysozyme.

These basic characteristics are shared with other members of the.B. cereus grc:up,

including the rhizoid shains B. mycoides, the crystalliferous insect pathogen B.

thuringiensis, and the mammalian pathogen B. anthracis. However, these species can

usually be differentiated from B. cereus by determining specific characteristics typical

ofeach species or variety. The tests described in G, below, are useful for this purpose

and can easily be performed in most laboratories. Strains that produce atypical results

from these tests require additional analysis before they can be classified as B. cereus.

Tests for differentiating members of the B. cereus grorup

The following tests are useful for differentiating typical strains of B. cereus fuom

other members of the,B. cereus groop, including B. mycoides, B. thuringiensis, and B.

anthrdcis, the summarized of characteristics are shown in Table 16.

Test 8. Motility test.

o lnoculate BC motility medium by stabbing down the center with 3 mm

loopful of24 h culture suspension. Incubate tubes 18-24 h at 35"C.

o Examine for type of growth along stab line. Motile organisms produce

diffuse growth out into the medium away from the stab. Nonmotile

organisms produce growth only in and along stab. Report whether or not

isolates tested were motile. Most strains of B. cereus and B. thuringiensis

are motile by means of peritrichous flagella. B. anthracis and all except a

few strains of B. mycoides are nonmotile. A few ,8. cereus strains are also

nonmotile.

Test 9. Rhizoid gro*'th.

r Pour 18-20 ml plate count agar into sterile 15 x 100 mm petri dishes and

allow agar to dry at 35"C for 24 h.


Fac. OfGmd. Studies, Mahidol Univ. M.Sc. (Food and Nuhitional Toxicology) / 101

hoculate by gently touching surface of medium near center of each plate

with 2 mm loopful of 24 h culture suspension. Allow inoculum to be

absorbed and incubate plates 7 days at room temperature.

o Examine for development of rhizoid growth, which is characterized by

production of colonies with long hair or root-like structures that may

extend several centimeters from site of inoculation. Rough galaxy-shaped

colonies are often produced by B. cereus strains and should not be

confused with typical rhizoid growth, which is the definitive characteristic

of B. mycoides. Most strains of this species are also nonmotile.

Test 10. Hemolltic activity.

Mark bottom of a plate into 10 equal sections with felt marking pen, and

label each section. lnoculate a premarked 4 cm sq area oftrypticase soy-

sheep blood agar plate by gently touching medium surface with 2 mm

loopful of24 h culture suspension. Incubate plates 24 h at 35oC.

Examine plates for hemolytic activity. B. cereus clltrtr:es usually are

strongly hemolyic and produce 2-4 mm zone of complete hemolysis

surrounding growth. Most B. thuringiensis and, B. mycoides strains are

also hemolytic. B. anthracis strains are usually nonhemolytic after 24 h

incubation.

Tesl ll. Protein toxin crystals.

Inoculate nutrient agar slants with 3 mm loopfuls of 24 h culture

suspensions. Incubate slants 24 h at 30'C and then at room temperature

2-3 days.

Prepare smears with sterile distilled water on microscope slides. Air-dry

and lightly heaffix by passing slide through flame ofBunsen bumer.

Place slide on staining rack and flood with methanol. Let stand 30 s,

pour off methanol, and allow slide to air-dry.

Retum slide to staining rack and flood completely with 0.5% basic

fuchsin or TB carbolfuchsin ZN stain (Difco). Heat slide gently from

below with small Bunsen bumer until steam is seen.

Wait 1-2 min and repeat this step. Let stand 30 s, pour off stain, and

rinse slide thoroughly with clean tap water.Copyright by Mahidol University

Jirapom Sonlm Appendix C / 102

o Dry slide without blotting ajld examine under oil immersion forpresence of free spores and darkly stained tetragonal (diamond_shaped)

toxin crystals.

crystals are usually somewhat smaller than spores. Toxin crystals are usually

abundant in a 3-to4-day-old culture of B. thuringiensrs but cannot be detected by the

staining technique until lysis of the sporangium has occurred. Therefore, unless lree

spores can be seen, cultures should be held at room temperature for a few more days

and re-examined for toxin crystals.

B- thuringiensis usually produces protein toxin crystals that can be detected by the

staining technique either as free crystals or parasporal inclusion bodies within the

exosporium. B. cereus and other members of the -8. cereus gronp do not produce

protein toxin crystals.

Interpreting test results.

on the basis of the test results, identify as B. cereus those isolates which are

actively motile and strongly hemollic and do not produce rhizoid colonies or protein

toxin crystals. Nonmotile B. cereus. strains are also fairly common and a few strains

are weakly hemolyic. These nonpathogenic strains of B. cereus can be differentiated

fiom .8. anthracis by their resistance to penicillin and gamma bacteriophage.

CAUTION: Nonmotile, nonhemolytic isolates that are suspected to be .8. anthracis

should be submitted to a pathology laboratory or destroyed by autoclaving.

Acrystalliferous variants of B. thuringiensis and nonrhizoid strains derived from .8.

mycoides cannot be distinguished from B. cereus by the cultural tests.



,l

3

'+, 90-100o/o ofstrains are positiveb +/-. 50-50% ofsnains are posrtive.

'-, 90-100% of strains are negative.d -, Most strains are negative.

Table 16. Differential characteristics oflarge-celled Group I Bacil/as species

Feature B. ceteus B, thuringiensis B. mycoides B. anthracis B. megaleiurn

Gram reaction + + + +

Catalase + + + + +

Motilitv +/- +l-

Reduction of nitrate + + +

Tyrosine decomposed + + +l- +/-

Lysozyme-resistant + + + +

Egg yolk reaction + + + +

Anaerobic utilization of

glucose+ + + +

VP reaction + + + +

Acid produc€d from

mannitol+

Hemolysis (Sheep RBC) + + +

Known pathogenicity/

characteristic

produces

enterotoxins

endotoxin

crystals

pathogenic to

insects

rhizoidal

gIo\l.th

pathogenic to

animals and

humans



Bacteriological Analysis for Bacillus cereusFigure 7. Most Probable Number (MpN technique)

Sampling 25 g+ 111f1s1225 ml (10-' dilurion)Stomacher 2 min.

I

YTSB + polyrnyxin B 10 ml

(3 tubes)

\l\J1ml/ tube

+TSB + poll,rnyxin B 10 ml

(3 tubes)

1ml

.+buffer 9ml (10-' dilutionl

+1mL/ tube

IiTSB + poli.rrnln<in B 10 mL

(3 tubes)

l loop / tube

IStreak on MYP agar ptatesIncubate 24-48 hr at 35"C

Incubate 48 + 2 hr at 35 o C

Note: Polymyxin B can inhibit gram-negative bacteria

The Microbial World

Pen icillin and other antibiotic

http://helios.ed.ac. uk/bto/microbes/pen icil l. htm

buffer 9 ml (10 2 dilurion)



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{t

Fac. OfGrad. Studies. Mahidol Univ.

NAME

DATE OFBIRTH

PLACE OF BIRTH

INSTITUTIONS ATTENDED

HOME ADDRESS


BIOGRAPHY

iI

Miss.Jirapom Sonyim

30 march, 1971

Petchaburi, Thailand

Mahidol University, 1993 :

Bachelor of Nursing

Mahidol University, 2003 :

Master of Science

(Food and Nutrition toxicology)

393 Dumnuankasam road.

Petchaburi, Thailand

Tel. 032 - 428506-10 Ext. 50319

E-mail: [email protected]


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