بسم هللا الرحمن الرحيم (و قل رب زدنى علما ( صدق هللا العظيم

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1 بسم هللا الرحمن الرحيم (و قل رب زدنى علما ( صدق هللا العظيم

2 جامعة القاهرة كلية الطب البيطرى قسم الميكروبيولوجيا دراسات عن الفطريات الملوثة للحوم. رسالة مقدمة من ط. ب / دعاء عبدهللا محمد العيدروس بكالوريوس العلوم الطبية البيطرية ١٩٩٥ جامعة القاهرة للحصول على درجة الماجستير فى العلوم الطبية البيطرية- ميكروبيولوجيا )بكتريولوجيا امينولوجيا ميكولوجيا ) تحت اشراف االستاذ الدكتور / محمد كمال رفاعى استاذ الميكروبيولوجى كلية الطب البيطرى جامعة القاهرة أ. د/ عاطف عبدالعزيز حسن أ. د / ندى خليفة محمد منصور رئيس بحوث قسم الفطريات و السموم الفطرية استاذ صحة اللحوم قسم الرقابة الصحية على االغذية معهد بحوث صحة الحيوان الدقى الجيزة كلية الطب البيطرى جامعة القاهرة ٢٠٠٦

3 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 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 2006 Prof. Dr.Atef Abdel Aziz HassanChief Researcher of Mycology&Mycotoxins - Aminal HealthInstitute, Dokki, Giza

4 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.

5 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.

6 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

7 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.

8 Results Prevalence of fungi on meat surfaces and the environment in slaughter houses. Table 1: Results of mycological examination of meat and its environment. Samples No of samples examined No of fungal isolates Meat surface Environment Total

9 Table 2: Fungi isolated from meat surfaces and the environment. Fungal genera Meat (130) Environment(295) Total (425) No % No % No % Aspergillus Pencillium Rhizopus Cladosporium Fusarium Candida Rhodotorula Total

10 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 Penicillium Rhizopus Cladosporiu m Rhodotorula Candida Total

11 Prevalence of Aspergillus species on meat surfaces. Table 4: Prevalence of Aspergillus species on meat Fungi Cattle Sheep Buffalo No % No % No % Total A. Niger A. Flavus A. Ochraceus A.Terreus A. fumigatus A. candidus Total

12 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 Sheep Buffaloes Total

13 Table 6: Fungal genera isolated from meat environment. Cattle Sheep Buffalo Total No % Aspergillus Penicillium Rhizopus Cladosporium Fusarium Candida Rhodotorula Total

14 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) No % No % No % No % No % No % Total (175) Aspergillus Rhizopus Penicillium Cladosporium Fusarium Candida Rhodotorula Total

15 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) No % No % No % No % No % No % Total (175) A. niger A. flavus A. candidus A. ochraceus A. parasiticus Total

16 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 Aspergillus Rhizopus Cladosporium Fusarium Rhodotorula Candida Total

17 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 A. flavus A. ochraceus Total

18 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 Penicillium Rhizopus Cladosporium Rhodotorula Candida Total

19 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) No % No % No % No % No % No % Total A.flavus A. niger A. ochraceus A. terreus A. candidus Total

20 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 x x x 10 2 Buffalo meat 1.2 x x x x 10 2 Sheep meat 8.8 x x x x10 2

21 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 x x 10 1 A. flavus 82 x x x 10 1 A. ochraceus 1.3 x x 10 1 A. terreus 8.7 x A. fumigatus 3.3 x A. candidus 2.9 x Penicillium sp 7.3 x x x 10 1 Rhizopus sp 3.5 x x x 10 1 Cladosporium sp 1.0 x x x 10 1 Rhodotorula sp 1.32 x x x 10 1 Candida albicans 3.69 x x x 10 1 S.E

22 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.

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

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

25 Photo 7: macromorphology of Penicillium spp. Photo 8: Micromorphology Penicillium spp.

26 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 A.flavus A.ochraceus A.Parasiticus Penicillium spp Fusarium spp Cladosporium spp Candida albicans

27 Photo 9: Positive proteolytic activity of A. flavus.

28 Photo 10: Positive proteolytic activity of Penicillium spp.

29 Photo 11: Positive proteolytic activity of Fusarium spp.

30 Lipolytic activity. Table 16: Lipolytic activity of isolated fungi from meat. Mould No of tested isolates No of +ve % A.flavus A.niger A.ochraceus A.Parasiticus Penicillium spp Fusarium spp Cladosporium spp Candida albicans

31 Photo 12: positive lipolytic Candida albicans.

32 Photo 13: positive lipolytic A. flavus.

33 Photo 14: positive lipolytic A. niger.

34 Photo 14: positive lipolytic A. niger.

35 Photo 15: positive lipolytic A. ochraceus.

36 Photo 16: Positive lipolytic Penicillium spp.

37 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 B1,B2 G1,G2 A.ochraceus Ochratoxin A

38 Photo 17: Mycotoxin spots extracted from A. flavus. Photo 18: mycotoxins spots extracted from A. flavus.

39 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.