Effect of Carica Papaya seeds, seeds and leaves … of Carica Papaya seeds...Ebeed Abd Elatty Saleh 1, kadry Mohamed sadek 2, Heba Farouk Elaarag 3 1 Prof. of meat hygiene , technology

International Food Safety Conference th5

Damanhour University

October 2018 th, 13Saturday

337

Effect of Carica Papaya seeds, seeds and leaves extract on Staphylococcus aureus and

Escherichia coli in minced meat and on meat quality

Ebeed Abd Elatty Saleh 1, kadry Mohamed sadek 2, Heba Farouk Elaarag 3

1 Prof. of meat hygiene , technology and food safety Faculty of Veterinary Medicine,

Damanhur university

2 Assistant prof. of bio-chemistry safety Faculty of Veterinary Medicine Damanhur

University.

3 Bachelor of veterinary medicine Kafr El Sheikh University

Abstract

Meat is considered an excellent source of nutrient for human through its contents of protein,

mineral and vitamins. In addition, it considered a suitable media for multiplication of

spoilage microorganisms and food borne pathogens. Papaya contains powerful antioxidant as

vitamin C, E and flavonoids; the B vitamins, folate and pantothenic acid. Papaya and its

extracts have antibacterial activity against Staphylococcus aureus, Pseudomonas, E-coli,

Proteus mirabilis, Klebsiella pneumonia and Salmonella typhi. The aim of this work was to

replace the use of chemical preservatives by a natural preservative. We studied the possibility

of the use of papaya seeds, seeds and leaves extract in preservation of raw minced meat

through evaluation of antibacterial and antioxidant capacities. The work was done by

dividing meat into 4 groups; one as a control group and the other three groups each divided

into three subgroups to test the effect of papaya seeds, papaya seeds and leaves extracts with

(1%, 2% and 3%). Each concentration was added to the meat groups and preserved at 3:5oC.

A sample from each group was taken every day from day zero till signs of spoilage were

appeared. We also compared their effect on Staph aureus and E. coli and on meat quality

through measuring total antioxidant capacity and lipid peroxidation. The results revealed that

papaya leaves extract (3%) gave the best result and the longest period of preservation with

lowest bacterial count followed by seeds extract at concentration 3%. Dried powdered papaya

seeds without extraction nearly had no effect. In conclusion, preservation of meat with leaves

extract 3% is preferred than other tested methods of preservation.

1- Introduction

Meat is considered an excellent source of nutrients for human through its contents of

protein, mineral and vitamins . also a suitable media for multiplication of spoilage

microorganisms and food born pathogen through its content of moisture, nitrogenous

compounds, mineral, growth factors, fermentable carbohydrates (glycogen) and favourable

pH (Aymerich et al., 2008).




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Contamination of fresh meat starts from live stock rearing environment,

transportation to slaughter house, improper processing practices, usage of dirty contaminated

tools, unhygienic slaughter house environment, ill workers and contamination during

transportation from slaughter house to shops which result to spread of microorganisms from

gastrointestinal track to muscle till improper handling in retail shops (Afshin et al., 2011).

Food born diseases and microbial spoilage result from failure to control

microorganisms at one or more stages from production to consumption (Normann et al.,

2005). These food born infections result in economic and health losses (Adeyemo, 2002).

Staphylococcus aureus is one of most important food born pathogen as it produces

thermostable enterotoxin (Tang and Stratton, 2010), this toxin cause vomiting and diarrhea

(Hennekinne et al., 2012) .

E. coli is the most dominant aerobic commensal flora in gastrointestinal track of

animal and human, it is considered an indicator as a source of fecal contamination if presents

in meat and some serotypes of E.coli lead to death of young children and cause cholera like

syndrome (Gyles, 2007).

Structure of meat is a complex physical structure and chemical composition that is

very susceptible to oxidation . Appearance, color, texture and flavor are the main sensory

quality affect consumer acceptance of meat, and lipid peroxidation is the primary cause of

these quality deteriorations in meat (wood et al., 2008). Lipid peroxidation primarily occurs

through a free radical chain reaction, and oxygen is the most important factor on the

development of lipid peroxidation in meat (Decker, 1992)

Carica papaya belonges to the family of Caricacae, and several species of this family

have active compounds which are valuable in the treatment and control of many diseases.

(Alabi et al., 2012). Papaya contains powerful antioxidant as vitamin C and flavonoids; the B

vitamins, folate and pantothenic acid; and minerals , potassium and magnesium; which

protect against colon cancer and keep function of cardiovascular system (Arvind et al.,

2013). Papaya also has anti-helminthic and anti- amoebic (Arvind et al.,2013) and anti-

inflammatory activity (Oladunmoye and Osho, 2007). Papaya has properties to act as

analgesic, cardiotonic, cholagogue, hypotensive, digestive, laxative and

stomachic(Anibijuwon and Udeze, 2009).

Papaya seeds protect from kidney failure through their effect on kidney toxins and also

have antimicrobial activity against Trichomonas vaginalis trophozoites so it can be used for

urogenital disorder with care to avoid toxicity and chewing of seedes used for treatment of

intestinal worm (Peter et al.,2008). Also Leaves of Papaya tea or its extract have renown to

be used as anti-tumer agent (Baskaran et al.,2012). Papaya seeds and its extract and papaw

leaf extract have antibacterial activity against Staphylococcus aureus , Pseudomonas

aeuginosa, E-coli, Proteus mirabilis, Klebsiella pneumonia and Salmonella typhi (Peter et




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al., 2014). Papaya seeds contain active ingredients which have antibacterial and antioxidant

effects (Peter et al.,2008). Also leaves contain phenolic compounds which have powerful

antioxidant effects and antimicrobial effects (Romasiet al.,2011). The digestive properties of

papaya is related to papain which treat dyspepsia. Papain also has clotting properties to milk

(rennet) and it is useful for tenderization of meat (Udohet al., 2005).

Therefore, the aim of this work was planned for studying the effect of carica papaya

seeds, leaves and seeds extract on count of staphylococcus aureus and E-coli and on total

antioxidant capacity and lipid peroxidation in minced meat.

2- Materials and Methods

2.1.Collection of samples :

Sample of 10 kg of minced meat collected from different butcher shops in kafr El Zayat city.

Carica Papaya fruit and leaves collected from trees in kafr El Zayat and surrounding villages

2.2. Preparation of leaves and seeds extracts :

2.2.1. Preparation of hot aqueous seed extract : (Oyagade et al., 1999).

2.2.2. Preparation of aqueous leaves extract : (Oyagade et al., 1999)

2.3. Dividing of minced meat into parts and addition of of papaya extracts and seeds :

10 Kg of minced meat were divided into 100 part which divided into 4 groups :

Group 1 : 10 parts consist of 1 kg used as control sample

Group 2 : 30 parts consist of 3 Kg used for addition of leaves extract

This group divided into 3 sub groups with 3 concentrations of leaves extract each one

consists of 10 part of 1 kg weight

Sub-group 1 : add concentration 1% of leaves extract (10 gm extract / 1 kg meat)

Sub-group 2 : add concentration 2% of leaves extract ( 20 gm extract / 1 kg meat)

Sub group 3 : add concentration 3 % of leaves extract ( 30 gm extract / 1 kg meat )

Group 3 : 30 parts consists of 3 kg for addition of seeds extract and divided into 3 sub-

groups with different concentration of extract as described before in group 2

Group 4 : also consists of 3o part of 3 kg for addition of seeds and also divided into 3 sub

groups as in group 2 and 3.

preservation by refrigeration at temperature 3:5oC and take every day sample from each

group started from 1st day of addition of herbs until spoilage signs begin to appear on

samples. And make counting to staph. aureus and E.coli and compare count in all groups .

And also studying the antioxidant effect of papaya extract by :

2.4.determination of total antioxidant capacity and lipid peroxidation.

Preparation of samples (ISO,2007)




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2.5.Microbiological examination :

A) Determination of Staphylococcus count (ICMSF, 1996)

B) Identification of suspected Staphylococcus aureus

Morphological identification

1- Gram’s stain (cruickshank et al.,1978)

2- Motility test (ICMSF,1996)

Biochemical identification of S.aureus (ISO,2007)

1- Coagulase test (tube technique)(APHA,2001)

2- Oxidation-fermentation of glucose(O-F test)

3- Catalase test (slide technique)(MACFADDIN, 1976)

4- Mannitol fermentation test (Baily and Scott,1978)

5- Urease test

6- Thermo-stable nuclease production

7- Detection of haemolysis (Baily and Scott,1978)

C)Isolation , identification and counting of E coli :

Morphological examination

1- Staining: (Cruickshank et al.,1975)

2- Motility test (ICMSF,1978)

biochemical identification (FDA,1998)

1- Indole production test

2- Methyl red test

3- Voges – praskauer test

4- Citrate utilization test

2.6.Determination of lipid peroxidation and total antioxidant capacity (Satoh, 1978)

1-Determination of lipid peroxidation

2-Determination of total antioxidant capacity

3- Results and Discusion:

Meat and meat products are considered the major vehicle of most food borne microorganism.

Contamination of meat and meat products starts from abattoir, utensils, environments,

transportation, food handlers and manufacturing. Unhygienic practices during meat handling




341

result to growth and multiplication of spoilage and pathogenic microorganisms which lead to

public health hazards. ( Isis et al., 2009).

Staphylococcal count:

Staphylococci commonly present on skin and upper respiratory tract of human and

animals so, staphylococci can easily contaminate meat and meat products if the control of

hazards is not occur during any step from farm to fork and occur contamination by bacterial

or its toxins lead to gastroenteritis when occur consumption of food.

In our study we found that the mean value of count of Staphylococcus aureus in minced meat

was 8.8×103 CFU/g. as shown in table (1). This result similar to the range reported by (Reda,

2010) , (Marwa, 2017) and (Shimaa, 2016)

According Egyptian standard (E.O.S.Q.C, 2005) the permissible staphylococcal count is 102

CFU/g.

Table (1) The effect of different concentrations of herbs on count of Staph. aureus

(CFU/g) in minced meat during storage under refrigeration.

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8

Control 8.8×103 12.3×103 4.8×104 6.0×104 Decomposed

Leaves Extract 1% 8.0×103 7.8×103 6.3×103 10.8×103 4.3×104 Decomposed

Leaves Extract 2% 7.6×103 6.4×103 5.1×103 9.4×103 16.8×103 4.3×104 Decomposed

Leaves Extract 3% 6.9×103 5.8×103 4.7×103 7.2×103 9.7×103 13.4×103 16.1×103 3.1×104

Seeds 1% 8.8×103 2.3×103 4.7×104 5.9×104 Decomposed


Seeds 3% 8.1×103 7.3×103 6.3×103 10.2×103 4.4×104 Decomposed

Seeds Extract 1% 8.8×103 2.3×103 4.6×104 5.9×104 Decomposed

E. coli count :

(Marwa, 2017) and (Barka and kihal, 2010) found the range of E. coli count which is

similar to our result of mean value of count of E. coli in minced meat that was equal 2.9×10

CFU/g. as present in table (2)

The presence of E. coli mainly due to fecal contamination during evisceration in abattoir or

from food handlers during food processing and marketing. (CDC, 2011).

According Egyptian standard (E.O.S.Q.C, 2005) E. coli must be absent in minced meat.




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Table (2) The effect of different concentrations of herbs on count of E. coli (CFU/g) in

minced meat during storage under refrigeration.

Table (3) The average of total antioxidant capacity (TAC) (mmol/gm) per day in each

group


Control 1.4 1 0.9 0.5 Decomposed

Leaves Extract 1% 1.7 1.5 1.2 0.9 0.6 Decomposed

Leaves Extract 2% 1.7 1.6 1.2 1 0.6 0.5 Decomposed

Leaves Extract 3% 2.4 2 1.9 1.7 1.6 1.5 1.1 1

Seeds 1% 1.5 1.1 1 0.7 Decomposed

Seeds 2% 1.6 1.2 1 0.8 Decomposed

Seeds 3% 1.6 1.5 1.2 0.9 0.5 Decomposed

Seeds Extract 1% 1.5 1.2 1.1 0.8 Decomposed

Seeds Extract 2% 1.6 1.4 1.1 0.8 0.5 Decomposed

Seeds Extract 3% 1.7 1.5 1.4 1.1 1 0.7 0.6 Decomposed

The results revealed that the count of E. coli and Staph. aureus is higher than the

permissible limit in Egyptian standard so meat is susceptible to be a source of food poisoning

and intoxication. So we need a safe preservative material to prevent multiplication and

decrease count of bacteria to limit food poisoning and on the other hand prolong shelf life of

product. So in this work we study the effect of Carica Papaya seeds, seeds and leaves

extracts on Staphylococcus aureus, E. coli and on oxidation of lipid and therefore on shelf

life.


Control 2.9×10 4.0×10 9.2×10 12.9×10 Decomposed

Leaves Extract 1% 2.5×10 2.0×10 5.5×10 8.5×10 11.0×10 Decomposed

Leaves Extract 2% 2.4×10 2.1×10 1.8×10 3.5×10 7.4×10 11.9×10 Decomposed

Leaves Extract 3% 2.1×10 1.7×10 1.5×10 1.9×10 2.8×10 4.1×10 5.2×10 7.9×10



Seeds 3% 2.4×10 2.1×10 5.5×10 8.6×10 11.3×10 Decomposed

Seeds Extract 1% 2.8×10 3.9×10 9.1×10 12.7×10 Decomposed

Seeds Extract 2% 2.5×10 2.3×10 5.6×10 8.6×10 11.8×10 Decomposed

Seeds Extract 3% 2.3×10 1.9×10 1.7×10 1.7×10 3.9×10 5.6×10 7.7×10 Decomposed




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Table (4) The average of Lipid Peroxidation (MDA) (nmol/gm) per day in each studied

group


Control 26.1 27.5 30.7 36.9 Decomposed

Leaves Extract 1% 23.1 24.6 28 32 34.3 Decomposed

Leaves Extract 2% 20.8 22.7 25.4 26.8 29.9 33.4 Decomposed

Leaves Extract 3% 16.9 18.9 19.8 21.4 22.3 24.2 26.6 29.7

Seeds 1% 26 27.3 30.1 36.2 Decomposed

Seeds 2% 25.9 26.8 29.3 35.6 Decomposed

Seeds 3% 23.3 25 28.1 32.4 34.9 Decomposed

Seeds Extract 1% 24.3 25.7 28.6 35.8 Decomposed

Seeds Extract 2% 24 25.2 29.3 33.1 36.2 Decomposed

Seeds Extract 3% 19.2 21.3 22.8 23.9 24.7 27.5 30.2 Decomposed

The results revealed that leaves extract gave better effect than seeds extract and the the best

effect on bacteria appeared at concentration 3%

The result nearly same as the result reported by (Jyotsna et al., 2014).

The MIC of leaves extract appeared at concentration 1% ( 10 gm / kg) and MIC of seeds

extract at concentration 2% (20 gm / kg).

These MICs are higher than MICs reported by (Muhammad et al., 2015) and (Anibijuwon

and Udeze, 2009). This may be due to the extracts were put directly on bacteria and

meassure the inhibition zone using agar diffusion method. but in this work the extracts were

put on meat contain bacteria not in direct contact with bacterial cells.

The total antioxidant capacities and lipid peroxidation results in table (3&4) revealed that

leave extract 3% had the highest antioxidant capacity and the lowest lipid peroxidation

The same result reported by Maisarah el al. (2013)

Dried powdered seeds its effect started to appear at concentration 3% and concentrations 2%

and 1% had no effect may be due to the active ingredients not concentrated by extraction.

The previous table shows that all group were significant in Staph and E-coli except Seeds

1%, Seeds 2% and Seeds extract 1%. In addition to, the most significant group was “Leaves

Extract 3%”.

The leaves extract at concentration 3% gave the longest period of preservation due to its

powerful antibacterial and antioxidant effect. As shown in table (1&2).




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The odor of meat after spoilage in samples with extracts was more acceptable than odor in

control group.

Table (5) Mann-Whitney test results on bacteria types

Bacteria

types

Ranks Mann-

Whitney

P-Value Significance

Group N Mean

rank

Sum of

ranks

Staph.

aureus

Control 40 61.54 2461.5 358.5 0.00 Sig.

Leaves Extract 1% 50 32.67 1633.5

Control 40 67.98 2719 501 0.00 Sig.

Leaves Extract 2% 60 38.85 2331

Control 40 84.26 3370.5 649.5 0.00 Sig.

Leaves Extract 3% 80 48.62 3889.5

Control 40 40.85 1634 786 0.893 Not Sig.

Seeds 1% 40 40.15 1606

Control 40 41.76 1670.5 749.5 0.627 Not Sig.

Seeds 2% 40 39.24 1569.5

Control 40 62.04 2481.5 338.5 0.00 Sig.

Seeds 3% 50 32.27 1613.5

Control 40 40.98 1639 781 0.855 Not Sig.

Seeds Extract 1% 40 40.03 1601

Control 40 59.95 2398 422 0.00 Sig.


Control 40 77.14 3085.5 534.5 0.00 Sig.

Seeds Extract 3% 70 43.14 3019.5

E-coli Control 40 52.24 2089.5 730.5 0.029 Sig.

Leaves Extract 1% 50 40.11 2005.5

Control 40 64.96 2598.5 621.5 0.00 Sig.

Leaves Extract 2% 60 40.86 2451.5

Control 40 85.68 3427 593 0.00 Sig.


Control 40 40.98 1639 781 0.855 Not Sig.

Seeds 1% 40 40.03 1601

Control 40 43.5 1740 680 0.248 Not Sig.

Seeds 2% 40 37.5 1500

Control 40 52.35 2094 726 0.026 Sig.

Seeds 3% 50 40.02 2001

Control 40 41.3 1652 768 0.758 Not Sig.


Control 40 51.74 2069.5 750.5 0.043 Sig.

Seeds Extract 2% 50 40.51 2025.5

Control 40 76.78 3071 549 0.00 Sig.





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Table (6) Mann-Whitney test results on chemical tests

Chemical Ranks Mann-

Whitney

P-

Value

Significance

Group N Mean

rank

Sum of

ranks

Total

Antioxidant

Capacity

(TAC)

Control 4 4.25 17 7 0.462 Not Sig.


Control 4 4.88 19.5 9.5 0.593 Not Sig.

Leaves Extract 2% 6 5.92 35.5

Control 4 3.25 13 3 0.027 Sig.


Control 4 4 16 6 0.564 Not Sig.

Seeds 1% 4 5 20

Control 4 3.88 15.5 5.5 0.468 Not Sig.

Seeds 2% 4 5.13 20.5

Control 4 4.25 17 7 0.462 Not Sig.

Seeds 3% 5 5.6 28

Control 4 3.75 15 5 0.386 Not Sig.


Control 4 4.5 18 8 0.624 Not Sig.


Control 4 4.75 19 9 0.345 Not Sig.


Lipid

Peroxidation

(MDA)

Control 4 5.5 22 8 0.624 Not Sig.




Control 4 9.75 39 3 0.027 Sig.



Seeds 1% 4 4 16


Seeds 2% 4 4 16

Control 4 5.5 22 8 0.624 Not Sig.

Seeds 3% 5 4.6 23

Control 4 5.25 21 5 0.386 Not Sig.


Control 4 5.5 22 8 0.624 Not Sig.


Control 4 8.5 34 4 0.059 Not Sig.


The previous table shows that all group were not significant except the group “Leaves

Extract 3%”.

4-Conclusion and recommendations

The previous results revealed that using of leaves extract at concentration 3% gave the

best result the longest period of preservation , lowest bacterial count and higher antioxidant

capacity and as a result the lowest oxidation of lipid. The seed extract 3% comes after leaves

extract 3% in bacterial count and preservation period.




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Seeds without extraction specially at concentration 1 & 2 % nearly had no effect.

So the use of leaves extract 3% is the best and its use with reduce food intoxication and

prolong shelf life of minced meat.

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الملخص العربي

ة واإلشريشيا كوالي في البكتيريا الكروية العنقودية الذهبيتأثير بذور نبات الباباظ ومستخلص البذور واالوراق على

وعلى جودتها اللحوم المفرومة

هبه فاروق األعرج 3, قدرى محمد صادق2 ,عبيد عبد العاطي صالح1

دمنهور.استاذ ورئيس قسم الرقابة الصحية على اللحوم جامعة 1

استاذ مساعد الكيمياء الحيوية كلية الطب البيطرى جامعة دمنهور. 2

بكالوريوس العلوم الطبية البيطرية جامعة كفر الشيخ. 3

اصر غذائية كالبروتينات واالمالحتعتبر اللحوم المفرومة مصدر مهم لغذاء االنسان لما تحتويه من عن

والفيتامينات وكذلك تعتبر وسط مناسب لتكاثر البكتريا المسببة للتعفن و التسمم الغذائى. نبات الباباظ يحتوي على عناصر

قوية مضادة لالكسدة كفيتامين )ج ـ هـ( الى جانب فيتامين ب وحمض الفوليك و حمض البانتوثينيك و كذلك يحتوى على

وية العنقودية الذهبية و االشريشيا كوالى و السمونيال. يهدف البحث الى استبدال مضادات للبكتريا تؤثر على البكتريا الكر

تم دراسة امكانية استخدام بذور نبات الباباظ وكذلك مستخلص البذور واالوراق فى ;المواد الحافظة الكيماوية بمواد طبيعية

ة األولى "مجموعة التحكم" والمجموعات مجموعات: المجموع 4حفظ اللحوم المفرومة. تم تقسيم اللحم المفروم الى

مجموعات فرعيه كل منها تحتوى على تركيزات مختلفه من البذور 3الثالثة االخرى تم تقسيم كل مجموعة منهم الى

%(. واضافة التركيزات المختلفة الى مجموعات اللحم المفروم و 3و %2, %1ومستخلص البذور و مستخلص االوراق )

م و اخذ عينة كل يوم ابتداء من اليوم االول الى ان بدأت عالمات التعفن فى الظهور و 05م :03ارة حفظها فى درجة حر

المقارنة بينهم من حيث التأثير على البكتريا الكروية العنقودية الذهبية و االشريشيا كوالى و كذلك على اكسدة الدهون.

اقل عدد بكتيريا واطول مدة حفظ و يليه ;االفضل % اعطى النتيجة3كانت النتيجة ان مستخلص االوراق بتركيز

% 3% بينما كانت البذور المجففة المطحونة تقريبا ليس لها تأثير و لذلك مستخلص االوراق بتركيز 3مستخلص البذور

هو االفضل بين باقى المجموعات.

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Effect of Carica Papaya seeds, seeds and leaves … of Carica Papaya seeds...Ebeed Abd Elatty Saleh 1, kadry Mohamed sadek 2, Heba Farouk Elaarag 3 1 Prof. of meat hygiene , technology