بسم هللا الرحمن الرحيم

)و قل رب زدنى علما (

صدق هللا العظيم

القاهرة جامعة البيطرى الطب كلية

الميكروبيولوجيا قسم

.دراسات عن الفطريات الملوثة للحوم سالة مقدمة منر

دعاء عبدهللا محمد العيدروس/ ب . ط

جامعة القاهرة– ١٩٩٥بكالوريوس العلوم الطبية البيطرية للحصول على

ميكروبيولوجيا -درجة الماجستير فى العلوم الطبية البيطرية

(ميكولوجيا –امينولوجيا –بكتريولوجيا )

تحت اشراف

محمد كمال رفاعى/ االستاذ الدكتور استاذ الميكروبيولوجى

جامعة القاهرة –كلية الطب البيطرى

عاطف عبدالعزيز حسن/ د. أ ندى خليفة محمد منصور / د . أ قسم الرقابة الصحية على االغذية رئيس بحوث قسم الفطريات و السموم الفطرية –استاذ صحة اللحوم

–الدقى –جامعة القاهرة معهد بحوث صحة الحيوان –كلية الطب البيطرى الجيزة

٢٠٠٦

Cairo University Faculty of Veterinary Medicine Department of Microbiology

STUDIES ON FUNGAL CONTAMINATION OF MEAT

A thesis presented by Doaa Abdalla Mohamed El Aidrose

( B.V.Sc. 1995- Cairo University) for degree of M.V.Sc.

In Microbiology (Bacteriology, Immunology and Mycology )

Under supervision of

Prof. Dr. Mohamed Kamal Refai Prof . of Microbiology.

Faculty of Veterinary Medicine, Cairo University

Prof. Dr Nada Kh.M.MansourProf. of Meat HygieneFaculty of Veterinary Medicine

Cairo university

Prof. Dr.Atef Abdel Aziz HassanChief Researcher of Mycology&Mycotoxins -

Aminal HealthInstitute, Dokki, Giza

2006

INTRODUCION AND AIM OF WORk

• Fungi are widely distributed in nature and affect human and animal through various ways due to lack of hygienic measures during handling, processing, transportation and storage of food.

• Fungi pollute meat and other animal tissues at the time of slaughter as a result of environmental factors contamination which favour growth of fungi and mycotoxins production as; high temperature, water activity or relative humidity, carbon dioxide, PH, oxidation potential and unclean animal houses.

• Mycosis and allergies due to fungal infection are probably a major concern in the consumption of meat and there is a potential for mycotoxins to enter meat product either by direct fungal growth on meat consumed by human or by indirect carry over from animal feed to edible tissues. Also indirect way may resulte from consumption of animal product as meat and milk which contain residues of mycotoxins resulting from feeding mouldy food to food producing animal.

• The contamination of meat with mould leads to either food poisoning or spoilage and render it to be of inferior quality.

• Also, the consumption of the contaminated meat by mould and their mycotoxins induces haemorrhage with hepatotoxic, nephrotoxic, neurotoxic, dermatotoxic, genotoxic, teratotoxic, carcinogenic or hormonal effects and immunosuppression.

• The mould and yeast were responsible for contamination of meat and its products which may lead to food poisoning and\ or spoilage including stickness, whiskers, black spots, green spots or patches, fat decomposition and off odour.

• Therefore, this study was carried out for : • 1- Evaluation of the fungal flora that may contaminate slaughtered

animal carcasses ( cattle, bufallo calves and sheep ) as well as in the surrounding environment of slaughter halls of Cairo abattoir (air, floor, walls, equipment (knives blades), washing water and worker hands). 2- Studying the mycotoxgenicity of isolated moulds.

• 3-Testing the proteolytic and lipolytic activeties of the recovered fungi.

• 4- Recommendation for prevention of fungal contamination.

Material and method • Material

• Samples • Media and reagent • Sabouraud,s dextrose agar medium (SDA) • Sabouraud,s dextrose broth • 1% Peptone water • Nitrogen-base medium devoid of sugar • 2% sugar solution • Bromocresol purole 5% • Lacophenol aniline blue • Yeast extract (2%) and sucrose medium (YES) • Skim milk agar medium • Egg yolk medium • Equipment and glasses • Methods • Techniues used in collection of samples • Meat samples • Air • Floors and walls • Knives blades • Worker hands • Water

• Colony count and isolation of fungi from meat samples • Identification of isolated fungi • Identification of moulds • Macroscopic identification • Microscopic morphology of the mould colonies • The culture mount technique • Micro-slide technique • Identification of yeasts • Direct microscopic examination • Sugar fermintation • Sugar assimilation • Screening of the isolated Aspergillus flavus and Aspergillus ochraceus

isolates for production of aflatoxins and ochratoxins. • Cultivation and extraction of aflatoxins and ochratoxins • Chromatographic analysis of chloroform extract • Proteolytic and lipolytic activity of the isolated fungi from meat. • Proteolytic activity of the isolated fungi • Determination of lipolytic activity of isolated fungi by egg yolk agar method.

Results

Samples No of samples examined No of fungal isolates

Meat surface 130 295

Environment 295 490

Total 425 785

Prevalence of fungi on meat surfaces and the environment in slaughter houses. Table 1: Results of mycological examination of meat and its environment.

Table 2: Fungi isolated from meat surfaces and the environment.

Fungal genera Meat (130) Environment(295) Total (425)

No % No % No %

Aspergillus 125 42.40 208 42.40 333 42.40

Pencillium 59 20.00 84 17.20 143 18.20

Rhizopus 30 10.16 68 13.87 98 12.48

Cladosporium 26 8.81 30 6.12 56 7.20

Fusarium - - 5 1.02 5 0.63

Candida

Rhodotorula

30

25

10.16

8.47

47

48

9.60

9.79

77

73

9.80

9.29

Total 295 100.00 490 100.00 785 100.00

Prevalence of fungi on meat surfaces.

Table 3: Prevalence of fungal genera on meat surfaces.

Fungal genera Cattle (50) Sheep (40) Buffalo (40) Total (130)

No % No % No % No %

Aspergillus 40 80 36 90 24 60 125 96.1

Penicillium 25 50 14 35 20 50 59 45.38

Rhizopus 18 36 4 10 8 20 30 23.07

Cladosporiu

m 4 8 8 20 14 35 26 20

Rhodotorula 3 6 6 15 16 40 25 19.23

Candida 4 8 14 35 12 30 30 23.07

Total 124 85 86 295

Prevalence of Aspergillus species on meat surfaces.

Table 4: Prevalence of Aspergillus species on meat

Fungi Cattle Sheep Buffalo

Total No % No % No %

A. Niger 25 50 12 30 6 15 43

A. Flavus 19 38 15 37.5 10 25 44

A. Ochraceus 13 26 4 10 0 0 17

A.Terreus 3 6 0 0 0 0 3

A. fumigatus 6 12 0 0 0 0 6

A. candidus 4 8 8 20 0 0 12

Total 70 39 16 125

4.5. Prevalence of fungi in the environment. Table 5: Results of mycological examination of meat environment.

Source of sample

Air Water Worker’s hands Floor Wall Knives Total

No % No % No % No % No % No % No %

Cattle 94 53.4 39 54.2 30 51.7 30 46.2 21 38.2 37 57.8 251 51.2

Sheep 42 23.9 20 27.8 16 27.6 18 27.7 18 32.7 15 23.4 129 26.3

Buffaloes 40 22.7 13 18.0 12 20.7 17 26.1 16 29.1 12 18.8 110 22.5

Total 176 100.0 72 100.0 58 100.0 65 100.0 55 100.0 64 490 100.0

Table 6: Fungal genera isolated from meat environment.

Cattle Sheep Buffalo Total

No %

Aspergillus 151 32 25 208 42.45

Penicillium 17 43 24 84 17.14

Rhizopus 41 12 15 68 13.88

Cladosporium 6 9 15 30 6.12

Fusarium 2 3 - 5 9.59

Candida 21 12 14 47 9.79

Rhodotorula 13 18 17 48 1.03

Total 251 129 110 490 100.0

Prevalence of fungi in the environment of cattle slaughter halls.

Table 7: Prevalence of fungi in the environment of slaughter halls (cattle).

Fungi Air (50) Water(25) Worker(25) Floor(25) Wall(25) Knives(25)

Total

(175) No % No % No % No % No % No %

Aspergillus 57 114 24 96 20 80 16 64 11 44 23 92 151

Rhizopus 5 10 10 40 7 28 7 28 6 24 6 24 41

Penicillium 10 20 2 8 1 4 1 4 1 4 2 8 17

Cladosporium 6 12 0 0 0 0 0 0 0 0 0 0 6

Fusarium 0 0 1 4 0 0 0 0 0 0 1 4 2

Candida 10 20 0 0 1 4 5 20 1 4 4 16 21

Rhodotorula 6 12 2 8 1 4 1 4 2 8 1 4 13

Total 94 39 30 30 21 37 251

Table 8: Prevalence of Aspergillus species in the environment of slaughter house (cattle).

Aspergillus spp

Air(50) Water(25) Worker(25) Floor(25) Wall(25) Knives(25) Total

(175) No % No % No % No % No % No %

A. niger 25 50 13 52 11 44 10 40 4 16 5 20 68

A. flavus 20 40 5 24 7 28 5 20 5 20 10 40 52

A. candidus 7 14 3 12 1 4 1 4 1 4 2 8 15

A. ochraceus 5 10 2 8 1 4 0 0 1 4 5 20 14

A. parasiticus 0 0 1 4 0 0 0 0 0 0 1 4 2

Total 57 24 20 16 11 23 151

Prevalence of fungi in the environment of sheep slaughter halls.

Table 9: Prevalence of fungi in the environment of slaughter halls (sheep).

Fungi

Air(10) Water(10) Worker(10) Floor(10) Wall(10) Knives(10) Total

No % No % No % No % No % No %

Penicillium 7 70 8 80 9 90 6 60 9 90 4 40 43

Aspergillus 8 80 7 70 2 20 9 90 4 40 2 20 32

Rhizopus 6 60 0 0 3 30 2 20 1 10 0 0 12

Cladosporium 9 90 0 0 0 0 0 0 0 0 0 0 9

Fusarium 0 0 0 0 0 0 0 0 0 0 3 3 3

Rhodotorula 6 60 5 50 1 10 1 10 3 30 2 20 18

Candida 6 60 0 0 1 10 0 0 1 10 4 40 12

Total

42 20 16 18 18 15 129

Table 10: Prevalence of Aspergillus species in the environment of slaughter halls (sheep).

Aspergillus sp

Air (10) Water(10) Worker(10) Floor(10) Wall(10) Knives Total

No % No % No % No % No % No %

A. niger 4 40 6 60 2 20 8 80 4 40 2 20 26

A. flavus 3 30 1 10 0 0 1 10 0 0 0 0 5

A. ochraceus 1 10 0 0 0 0 0 0 0 0 0 0 1

Total 8 7 2 9 4 2 32

Prevalence of fungi in the

environment of buffalo slaughter hall.

Table 11: Prevalence of fungi in the environment of slaughter halls (buffalo calves).

Fungi

Air(10) Water (10) Worker(10) Floor(10) Wall(10) Knives(10) Total

No % No % No % No % No % No % No %

Aspergillus 5 50 3 30 6 60 6 60 2 20 3 30 25 41.6

Penicillium 6 60 4 40 1 10 2 20 6 60 5 50 24 40.0

Rhizopus 7 70 0 0 1 10 4 40 3 30 0 0 15 25

Cladosporium 10 100 0 0 0 0 2 20 3 30 0 0 15 25

Rhodotorula 6 60 4 40 3 30 1 10 0 0 0 0 17 23.3

Candida 6 60 2 20 1 10 2 20 2 20 4 40 14 28.3

Total 40 13 12 17 16 12 110

Table 12: Prevalence of Aspergillus species in the environment of slaughter halls

(buffalo).

Aspergillus spp

Air(10) Water(10) Worker(10) Floor(10) Wall(10) Knives(10)

Total

No % No % No % No % No % No %

A.flavus 4 40 1 10 1 10 3 30 0 0 2 20 11

A. niger 0 0 1 10 2 20 2 20 2 20 1 10 8

A. ochraceus 1 10 0 0 1 10 1 10 0 0 0 0 3

A. terreus 0 0 0 0 2 20 0 0 0 0 0 0 2

A. candidus 0 0 1 10 0 0 0 0 0 0 0 0 1

Total 5 3 6 6 2 3 25

Determination of fungal colony count in meat. Table 13: Mean fungal colony count of meat.

Fungal colony count

Maximum Minimum Mean S.E

Cattle meat 6.4 x 103 1.75 x 102 1.1 x 103 76.75 x 102

Buffalo meat 1.2 x 103 6.5 x 101 2.9 x 102 13.40 x 102

Sheep meat 8.8 x 102 6.0 x 101 4.0 x 102 15.55 x102

Table 14: Mean fungal colony count of meat.

Fungal genera Mean fungal colony count cfu/g

Cattle meat Buffalo meat Sheep meat

A. niger 8 x 101 1.2 x 101 8.2 x 101

A. flavus 82 x 101 4.3 x 101 9.9 x 101

A. ochraceus 1.3 x 102 - 2.9 x 101

A. terreus 8.7 x 101 - -

A. fumigatus 3.3 x101 - -

A. candidus 2.9 x 101 - 7.5

Penicillium sp 7.3 x 101 5.0 x 101 2.6 x 101

Rhizopus sp 3.5 x 101 1.1 x 101 1.0 x 101

Cladosporium sp 1.0 x 101 1.5 x 101 1.6 x 101

Rhodotorula sp 1.32 x 102 1.00 x 102 2.0 x 101

Candida albicans 3.69 x 102 7.5 x 101 3.6 x 101

S.E 0.33-8.45 0.82-8.22 0.38-4.0

The macro and micro morphology of the isolated fungi are

demonstrated in photo 1, 2, 3, 4, 5, 6, 7, 8.

Photo I: Macromorphology of A. niger. Photo 2: Micromorphology of A. niger.

Photo 3: Macromorphology of A. flavus. Photo 4: Micromorphology of A. flavus.

Photo 5: Macromorphology of A. ochraceus. Photo 6: Micromorphology of A. ochraceus.

Photo 7: macromorphology of Penicillium spp.

Photo 8: Micromorphology Penicillium spp.

Enzymatic activities of fungi isolated from meat. Proteolytic activity.

Table 15: Proleolytic activity of the isolated mould from meat.

Mould No of tested isolates No of +ve %

A.niger 4 4 100

A.flavus 6 5 83.3

A.ochraceus 5 4 80

A.Parasiticus 5 3 60

Penicillium spp 6 5 83.3

Fusarium spp 4 2 50

Cladosporium spp 4 3 75

Candida albicans 2 1 50

Photo 9: Positive proteolytic activity of A. flavus.

Photo 10: Positive proteolytic activity of Penicillium spp.

Photo 11: Positive proteolytic activity of Fusarium spp.

Lipolytic activity. Table 16: Lipolytic activity of isolated fungi from meat.

Mould No of tested isolates No of +ve %

A.flavus 6 4 66.6

A.niger 4 3 75

A.ochraceus 5 3 60

A.Parasiticus 5 3 60

Penicillium spp 6 5 83.3

Fusarium spp 4 2 50

Cladosporium spp 4 2 50

Candida albicans 2 2 100

Photo 12: positive lipolytic Candida albicans.

Photo 13: positive lipolytic A. flavus.

Photo 14: positive lipolytic A. niger.

Photo 14: positive lipolytic A. niger.

Photo 15: positive lipolytic A. ochraceus.

Photo 16: Positive lipolytic Penicillium spp.

Mycotoxin production by A. flavus and A. ochraceus isolated from meat.

Table 17: Mycotoxins production by A.flavus&A.ochraceus isolated from meat.

Tested isolates No of

tested

isolate

No % Level of produced toxin μg / μl Type

Max Min Mean S.E

A.flavus 12 10 83.3 20 3 7.5 0.3 B1,B2

G1,G2

A.ochraceus 12 8 66.6 18 4 5.3 0.5 Ochratoxin A

Photo 17: Mycotoxin spots extracted from A. flavus.

Photo 18: mycotoxins spots extracted from A. flavus.

• In conclusion, meat and meat product must be protected away from contamination. All hygienic measures must be adapted during different steps of meat production including care of living animal health and clear environmental condition of the slaughtered carcasses hall (air, water, floor, worker,s hands and clothes and knives blades. The quality and hygeinic status of meat is associated with the care of previous measures. All ways are taken to keep food free from causes of the diseases for saving the human health.