بسم هللا الرحمن الرحيم

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1 بسم هللا الرحمن الرحيم

2 Evaluation of environmental factors affecting degradation of mycotoxins with special reference to laser A Thesis presented by Rania Momtaz Abd ElLatif Azab Under supervision of Prof. Dr. Mohamed Kamal Refai Professor of Microbiology Faculty Veterinary Medicine Cairo University Prof. Dr. Hasan Mostafa ElAgrab Professor of Vet. Hygiene and Management, Faculty of Veterinary Medicine, Cairo University Dr. Amira Abdou M. ElAdly Lecturer of Microbiology National Institute of Laser Enhanced Sciences, Cairo Univ

3 . INTRODUCTION

4 The field of antimicrobial fight is one of the constant challenges, particularly in view of rapid evolutionary changes and plethora of new pathogens encountered. Unfortunately, pathogenic and harmful microbes are widely spread every where, as mycotoxigenic fungi which are considered unavoidable contaminants in foods and feeds. The most important mycotoxigenic fungi are those producing aflatoxins. The toxins occur naturally on several key animal feeds, including corn, cottonseeds, wheat and others. Occurrence of aflatoxins on some field crops tends to spike in years when drought and insect damage facilitate invasion by the causative organisms; Aspergillus flavus and Aspergillus parasiticus which abound in the crop environment.

5 Aflatoxins have received greater attention than any of the other mycotoxins because of their demonstrated carcinogenic effects in susceptible animals. Decontamination of foods and feeds containing aflatoxins is an objective for improving the food and feed supply. Data on the loss of productivity in farm animals consuming contaminated feeds and carcinogenicity in experimental animals provide sufficient evidence on the hazardous nature of aflatoxins. Recent trends in global and feed production, processing, distribution, preparation and saving are inducing a growing demand for food safety research in order to ensure a safer global food and feed supply.

6 A new approach to decontaminate feed is cost effective and an environmentally friendly way is still a problem One of possible approach is to use photodynamic therapy (PDT) which is a novel and promising biophotonic technology. Photodynamic therapy is an entirely new modality and its development can likened to that of the discovery of antibiotics. Raab in 900 was first to observe the death of Paramecium caudatum after exposure to light when acridine orange was present. It was impossible to understand the mechanism of the death hundred years ago, recently it has been accepted world wide that this phenomenon might be named "photosensitization. In 9, this phenomenon was applied to cure skin cancer.

7 It is important to note that really major advances have been made in photosensitized antimicrobial chemotherapy in particular disinfection of blood, blood products, treating locally infected wounds or even different oral infection. It is important to make an effort to develop safe and practical detoxification methods using different radiation types, one of them is laser which is now just over thirty six years since its action was first successfully demonstrated using crystal of ruby as the laser medium. In this time the laser has grown from the status of applications to that of being one of most important inventions of our time. It is now a vital tool for areas as diverse as manufacturing, industries, medicine, communications, halo graphic system and the basis of many scientific measurements and research programs.

8 AIM OF WORK

9 . Reporting the prevalence of fungi in different feed stuffs.. Evaluation of the isolated strains of Aspergillus flavus and Aspergillus parasiticus for aflatoxin B production.. Determination the level of aflatoxin B contamination in different feed stuffs.. Testing the effect of gamma irradiation and photodynamic inactivation (PDI) on fungal growth, aflatoxin B production and its degradation.

10 . MATERIAL AND METHODS

11 Samples Type of samples bran Barley Soya bean Bone and blood meal Layer's concentrate Broiler's concentrate Poultry ration Processed animal feed Total of samples

12 Culture media. Sabouraud's dexstrose agar medium (SDA):. CzapekDox agar medium (Al Doory, 90). Malt extract agar medium (Cruickshank et al., 97). Fresh potato dexstrose agar medium (Shotwell et al., 966). Yeast extract sucrose liquid medium (Bauer et al., 9)

13 Standard aflatoxins : Standards of aflatoxin B, B, G and G were purchased from Sigma (USA). Toxigenic strain of Aspergillus flavus : from M.R.C Stains : Lactophenol cotton blue stain Photo sensitizer : Phloxine B (D & C ) photo sensitizer with absorption spectrum 7 nm was purchased from Sigma (USA). Chemicals and reagents : Buffered peptone water (BPW).

14 Thin Layer Chromatographic (T.L.C.): Apparatus, Chromatographic column, Glass chromatographic plates 0 x 0 cm coated with 0. mm silica gel ( type G), Glass developing tank with cover, Ultraviolet lamp (U.V. ) long wave 6 nm, Short wave nm ). Quantitatve automatic micropipettes with disposable glass tips Fluorometer: Vicam series flurometer (Vicom, USA) with the following accessories: specific FG is Afla test standards. Source of gamma irradiation: Cobalt 60 gamma cell ORIEL Solar simulator: Green light Emitting Diodes : Thermostatic controlled water bath

15 Methods: Mycological examination:. Isolation of fungi (Refai, 979):.. Preparation of sample homogenate:.. Dilution:.. Plate pouring:.. Incubation:. Identification of moulds:.. Macroscopic examination:.. Microscopic examination:... Direct Microscopic examination:... Slide culture technique (Ajello et al., 96):

16 Screening of isolated strains of Aspergillus flavus and Aspergillus parasiticus for aflatoxin B production (Bauer et al., 9). Extraction of aflatoxin B from feed samples: Preparation of aflatoxin B standard solutions: Estimation of aflatoxin B:. Qualitative estimation of aflatoxin B by thin layer chromatography (T.L.C.), (AOAC, 97).. Quantitative estimation of aflatoxin B by a fluorometeric method according to AOAC (990):

17 Fungal culture filtrate: Detoxification of aflatoxin B contaminated feed samples by different radiation types:. Gama radiation (Aziz and Youssef, 00):. Photodynamic inactivation (PDI):.. By solar simulator:.. By LED: The effect of different types of radiation on spore germination mycelium growth and aflatoxin B production:

18 . RESULTS

19 Table (): Prevalence of main fungal species in feed samples. Total Processed animal feed Poultry ration Broiler's concentrate Layer's concentrate Bone and blood meal Soyabean Barley bran Type of sample of examined samples +ve sample Isolates Aspergillus spp Penicillium spp Fusarium spp Mucor spp Rhizopus spp Cladosporium spp.. 0 Yeast.7. Alternaria spp Curvularia spp Scopulariopsis spp..7 Rhodotorula spp Chaetomium glabosum

20 Table (): Prevalence of members of Aspergillus, Penicillium and Fusarium species in feed samples. Total Processed animal feed Poultry ration Broiler's concentrate Layer's concentrate Bone and blood meal Soyabean Barley bran Type of sample of examined samples +ve sample Isolates Aspergillus spp A. candidus A. fumigatus A. flavus A. niger A. ochraceus A. parasiticus A. terreus Penicillum spp P. chrysogenum P. digitatum P. funicolosum P. oryzee P.sclerotigenum P. thomii P. viridicatum P. vercossum Fusarium spp F. oxysporium F. solani F. tabacinum F. tricinactum F. moniliforme

21 Table (): Total and incidence of different members of Aspergillus, penicillium and Fusarium species in feed samples. Type of sample bran Barley Soyabean Bone and blood meal Layer's Broiler's concentrate concentrate Processed Poultry animal ration feed Total of examined samples Aspergillus spp Total Incidence/sample Penicillum spp Total Incidence/sample Fusarium spp. 7 Total 0 7 Incidence/sample

22 Table (6): Aflatoxin B production by isolated strains of Aspergillus flavus and Aspergillus parasiticus. Type of isolate of tested isolates Zero μg AFB / ml YES broth Amount of AFB / ml YES broth 0 μg AFB 00 μg AFB > 00 μg AFB Aspergil lus flavus Aspergil lus parasitic us

23 Table (7): Aflatoxin B detection in the examined feed samples. Types of examined samples of samples Negative sample Total ppb Positive sample 6 ppb 0 ppb 00 ppb 0000 ppb bran Barley 0 0 Soya bean 6 Bone and blood meal 9 6 Layer's concentrate Broiler's concentrate Poultry ration 6 Processed animal feed 6 6 Total

24 AFB (ppb) Detox. Fig. (): Effect of gamma irradiation on ppb AFB contamination level bran Barley Soybean Bone & Blood meal Layer's concentrates Broiler's con. 0 Poultry ration Processed Animal Feed Radiation dose (kgy) bran Barley

25 Fig. (9): Effect of gamma irradiation on 0 ppb AFB contamination level.

26 AFB (ppb). Detox. Fig. (): Effect of gamma irradiation on 0 ppb AFB contamination level bran Barley Soybean Bone & Blood meal Layer's concentrates Broiler's con. 6 0 Poultry ration Processed Animal Feed Radiation dose (kgy) bran Barley

27 AFB (ppb) Detox. Fig. (): Effect of gamma irradiation on ppb AFB contamination level bran Barley Soybean Bone & Blood meal Layer's concentrates Broiler's con. Poultry ration Processed Animal Feed Radiation dose (kgy) bran Barley

28 AFB (ppb) Detox. Soybean Fig. (): Effect of gamma irradiation on ppb AFB contamination level bran Barley Soybean Bone & Blood meal Layer's concentrates Broiler's con. 0 Poultry ration Processed Animal Feed Radiation dose (kgy) bran Barley

29 AFB (ppb). Detox. Fig. (): Photodynamic effect of Phloxine B on ppb AFB contamination level after exposure to solar simulator bran 6 60 Barley 0 Soyabean Bone&Bl.meal Layer's concentrates Broiler's con. 0 Poultry ration Processed Animal Feed Concentartion of dye (mg) bran

30 AFB Detox. Fig. (): Photodynamic effect of Phloxine B on 0 ppb AFB contamination level after exposure to solar simulator bran Barley Soyabean Bone&Bl.meal Layer's concentrates Broiler's con. Poultry ration Processed Animal Feed Concentartion of dye (mg) 0 bran

31 AFB (ppb). Detox. Fig. (): Photodynamic effect of Phloxine B on 0 ppb AFB contamination level after exposure to solar simulator bran Barley Soyabean Bone&Bl.meal Layer's concentrates Broiler's con. Poultry ration Processed Animal Feed Concentration of dye (mg) 0 bran Barley

32 Fig. (6): Photodynamic effect of Phloxine B on ppb AFB contamination level after exposure to solar simulator. AFB (ppb). Detoxification bran 60 Barley 0 6 Soyabean Bone&Bl.meal Layer's concentrates 0 Broiler's con. 0 Poultry ration Processed Animal Feed Concentration of dye (mg) bran

33 AFB (ppb) Detoxification Fig. (7): Photodynamic effect of Phloxine B on ppb AFB contamination level after exposure to solar simulator bran 0 60 Barley 0 0 Soyabean Bone&Bl.meal Layer's concentrates Broiler's con. 0 Poultry ration Processed Animal Feed Concentartion of dye (mg) bran Barley

34 Fig. (): Photodynamic effect of phloxine B on ppb AFB contamination level after exposure to LED.

35 Fig. (9): Photodynamic effect of phloxine B on 0 ppb AFB contamination level after exposure to LED.

36 AFB (ppb). Detox. Fig. (0): Photodynamic effect of phloxine B on 0 ppb AFB contamination level after exposure to LED Concentartion of dye (mg) bran Barley Soybean Bone & Blood meal Layer's concentrates Broiler's con. Poultry ration Processed Animal Feed bran

37 AFB (ppb). Detox. bran Fig. (): Photodynamic effect of phloxine B on ppb AFB contamination level after exposure to LED Concentartion of dye (mg) bran Barley Soybean Bone & Blood meal Layer's concentrates Broiler's con. Poultry ration Processed Animal Feed

38 Fig. (): Photodynamic effect of phloxine B on ppb AFB contamination level after exposure to LED.

39 Mean Log Surviving count / ml Fig. (): Effect of gamma radiation on spores of germination of toxigenic strains of A. flavus and A. parasiticus A. flavus A. parasitcus Radiation doses (kgy)

40 Fig. (6): Effect of gamma radiation on growth of toxigenic strains of A. flavus and A. parasiticus and AFB production in synthetic medium. Mean Log Surviving count / ml A.flavus Mycelium dry weight (g/) A.flavus Aflatoxin B concentartion (ug/) A. parasitcus Mycelium dry weight (g/) A. parasitcus Aflatoxin B concentartion (ug/) Radiation doses (kgy)

41 Fig. (): Photodynamic effect of phloxine B on spore germination of toxigenic strains of Aspergillus flavus and Aspergillus parasiticus after exposure to solar simulator. Mean Log Surviving count / ml A. flavus A. parasitcus Concentration of dye mg

42 Fig. (0) Photodynamic effect of phoxine B on growth toxigenic strains of Aspergillus flavus and Aspergillus parasiticus and AFB production after exposure to solar simulator A. flavus (Mycelium) g/l A. flavus (Aflatoxin) ug/l A. parasitcus (Mycelium) g/l A. parasitcus (Aflatoxin) ug/l Concentration of dye (mg)

43 Fig. (): Photodynamic effect of Phloxine B on spore germination of toxigenic strains of A. flavus and A. parasitcus after exposure to LED. Mean Log Surviving count / ml A. flavus A. parasitcus Concentration of dye mg

44 Fig. (): Photodynamic effect of Phloxine B on growth of toxigenic strains of A. flavus and A. parasitcus and aflatoxin B production after exposure to LED A. flavus Mycelium dry weight (g/l) A. flavus Aflatoxin B concentartion (ug/l) A. paraiticus Mycelium dry weight (g/l) A. paraiticus Aflatoxin B concentartion (ug/l) Concetration of dye (mg)

45 . Conclusion

49 THANK YOU

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

بسم هللا الرحمن الرحيم (و قل رب زدنى علما ( صدق هللا العظيم جامعة القاهرة كلية الطب البيطرى قسم الميكروبيولوجيا دراسات عن الفطريات الملوثة للحوم. رسالة مقدمة من ط. ب / دعاء عبدهللا محمد العيدروس بكالوريوس