الفهرس 
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Abstract
Aflatoxins are a group of mycotoxins which are considered as the most potent carcinogens. Aflatoxins can not only found as contaminates in the stable diet (cereal grains) but also found in milk when the dairy animals ingest contaminated feed with aflatoxin B1 and B2.Some of this aflatoxin B1 (AFB1) is converted by the normal metabolism process to aflatoxin M1 and aflatoxin B2 (AFB2) is converted to aflatoxin M2 and then aflatoxin M1 and aflatoxin M2 are excreted and occurred in milk so aflatoxin M1 (AFM1) and aflatoxin M2 (AFM2) are considered as hepatic hydroxlated metabolites of aflatoxin B1 and aflatoxin B2.
Milk and milk products are important contribution to the diet of human especially children. The quality of these milk and milk products according to their aflatoxins presence beginning from the good dairy feed and also, depend on the proper hygienic conditions during production of dairy products, storage and distribution conditions. However, the presence of aflatoxins (AFM1, AFB1, AFB2, AFG1 and AFG2) in our milk and milk product are considered undesirables due to their health risks in consumer’s body.
This study aimed to:
1- To determine the existenceof aflatoxins (AFM1, AFB1, AFB2, AFG1 and AFG2) in milk and milk products.
2- To assess the ability of some natural viable strains (Lactobacillusplantarum, Lactobacillus acidophilus and Bifidobacterium bifidum) on reduction ofAFM1.
3- To assess the potential of some nonviable strains (Lactobacillusplantarum, Lactobacillus acidophilus, Bifidobacterium bifidum, kluyveromyces lactis and Saccharomyces cerevisiae) on sequestration ofAFM1.
4- To evaluate the best efficient of the combination of strains on AFM1 sequestration to applied in milk.
5- To investigate the sensory evaluation of nonviable bacterial and yeast strains to be applied in different dairy products.
To fulfill this aim the following methods were used:
1. In this study,a total of103 samples of milk and different types of commercial dairy products (milk, cheese, fermented milk and ice cream) were collected randomly from different supermarkets, retail markets and shops from different districts in Alexandria in 2016 to analyze for the existence of aflatoxins(AFM1, AFB1, AFB2, AFG1 and AFG2) by HPLC to determine the quantification of aflatoxins in samples.
2. The present experimental study was assessed the ability of some natural viable probiotic bacterial strains (Lactobacillusplantarum, Lactobacillus acidophilus and Bifidobacterium bifidum) on reduction ofAFM1 by determination of AFM1 residues by HPLC after different incubation period (12h, 24h, 48h and 72h) in PBS media at 37 ºC.
3. The study also, was assessed the potential of some nonviable strains probiotic bacterial and yeast strains (Lactobacillusplantarum, Lactobacillus acidophilus, Bifidobacterium bifidum, kluyveromyces lactis and Saccharomyces cerevisiae) on sequestration ofAFM1 by determination of AFM1 residues by HPLC after different incubation period (12h, 24h, 48h and 72h) in PBS media at 37 ºC.
4. The best removal result of AFM1 (50 ng/ml) by the combination of nonviable probiotic bacterial and yeast strains in PBS media was applied in milk instead of PBS media and detected the effect of these combination on AFM1 reduction in milk by determination of AFM1 residues by HPLC after different incubation period (12h, 24h, 48h and 72h) in milk as experimental media at 37 ºC.
5. The combination of nonviable probiotic bacterial and yeast strains were used in preparation of some milk products and detected the organoleptic properties (appearance, consistency, tenderness and flavour) of these milk products with nonviable probiotic bacterial and yeast strains.
Results of the present study can be summarized as follows:
1. Existence ofaflatoxins (AFM1, AFB1, AFB2, AFG1 and AFG2) existence in milk and milk products
1.1. Milk samples
1.1.1. Pasteurized milk
The positive samples in the examined pasteurized milkregarding toAFM1, AFB1, AFB2 and AFG1 were30%, 10%, 10% and30%, respectively with mean value at 21.86±13.08, 3.41±0.00, 1.96±0.00 and 9.01±5.22 ng/L, respectively. On the other hand, AFG2was not determined in any pasteurized milk samples. These all positive samples did not accepted by Egyptian standard specification and European standard (free fromAFB1, AFB2, AFG1and AFG2). However, all pasteurized milk samples complied with standards acceptable level (≤ 50 ng/L).
We also, distributed the source of aflatoxins in milk as 30% from the metabolic toxin (AFM1) which reached to pasteurized milk samples from the feeding of dairy animal with contaminated feed with AFB1 and also, about 30% from the contaminates toxins (AFB1, AFB2, AFG1and AFG2) which reached to pasteurized milk samples from the external contamination of milk by aflatoxigenic moulds that produced their toxins before or during packaging.
Also, we found that 6(60%) of samples contained total aflatoxins (AFM1, AFB1, AFB2, AFG1and AFG2) with maximum concentration at 30.65 ng/L and mean at 16.32±11.51 ng/L which might be reached to consumers who consumed pasteurized.
1.1.2. Powder milk
The negative results detected from powder milk samples were 100%, 100%, 80%, 100% and 80% for AFM1, AFB1, AFB2, AFG1 and AFG2, respectively while the positive samples determined in AFB2 and AFG2 in 20% of powder milk samples with mean 0.80±0.00 and 1.76±0.00 ng/kg, respectively which exceeded than Egyptian standard limit (free from AFB2 and AFG2). On the other hand, AFM1, AFB1 and AFG1 did not determine in any powder milk samples which complied Egyptianand European standard limit (free from AFB1 and AFG1) and withAFM1 ≤ 50 ng/kg.
Also, the aflatoxins found in powder milk samples due to the external contamination source (contaminates toxins) (B2 and G2) as 20% of the tested powder milk samples while the samples free from M1. These negative samples were baby formula milk powder.
1.1.3. Raw milk
The positive samples in the examined raw milk which was purchased in bottle from farmers in street (Raw milk A) according toAFM1, AFB1, AFB2, AFG1 and AFG2 were25%, 50%, 16.66%, 33.44% and 25%, respectively with mean value at 311.64±342.13, 24.16±41.37, 19.95±18.28, 36.32±28.59 and 29.63±32.94 ng/L, respectively.
Concerning to the examined raw milk which was purchased in plastic bag from supermarket tanks (Raw milkB) collected from different markets in Alexandria, the results were 25%, 16.66%, 41.66% and 8.33% for AFM1, AFB1, AFG1 and AFG2, respectively with mean value at 20.73±25.59, 0.85±0.56, 10.59±10.49 and 11.98±0.00 ng/L, respectively. These positive samples did not accepted by Egyptian standard specificationand European commission (free fromAFB1, AFB2, AFG1and AFG2). On the other hand AFB2did not detect in any tested raw milkB.
However, we distributed the source of aflatoxins in all raw milk samples as 25% from the metabolic toxin (AFM1) and also, 58.33% from contaminates toxins (AFB1, AFB2, AFG1and AFG2) which reached to raw milk samples from the external contamination during milking, transportation or storage.
Moreover, we calculated the total aflatoxins (AFM1, AFB1, AFB2, AFG1and AFG2) in all raw milk samples to explain that consumers might be exposed to 78.04±191.50 ng/L in the positive raw milk samples (58.33%). Total aflatoxins mean was 150.44±266.55 ng/L in 9 (75%) of raw milk A. However, in raw milk B samples determined at 12.88±15.33 ng/L in 10 (83.33%) of samples so the consumers who consumed raw milk A were exposing for higher total aflatoxins than raw milk B.
1.2. Fermented milk
1.2.1. Raib samples
20% of examined raib samples had AFM1 at mean value 38.37±45.99 ng/Kg while AFB1, AFB2, AFG1and AFG2 were not appeared in any examined raib samples. These all negative samples accepted by Egyptian standard specification and European commission (free fromAFB1, AFB2, AFG1and AFG2).
1.2.2. Yoghurtsamples
The positive samples in the examined yoghurt according toAFM1, AFB1, AFB2 and AFG1 were10%, 10%, 20% and 30%, respectively with mean value at 257.81±0.00, 3.03±0.00, 6.89±0.24 and 66.53±55.22 ng/Kg, respectively. The metabolic toxin (AFM1) in 10% of samples reached yoghurt samples from using milk with AFM1 while, 50% of samples had contaminates toxins from the contaminated equipment during the yoghurt processing. On the other hand, the consumers might be exposed to 50% of positive yoghurt samples according to total aflatoxins (AFM1, AFB1, AFB2, AFG1and AFG2) with mean level 83.56±119.74 ng/Kg.
1.3. Cheese samples
1.3.1.Feta cheese samples
The positive samples in the examined feta cheese samples forAFM1, AFB1 and AFB2 were20%, 10% and 10%, respectively with mean value at 54.59±27.71, 3.23±0.00 and 0.83±0.00ng/Kg, respectively. On the other hand, AFG1and AFG2 were not detected in any feta cheese sample. The sources of these aflatoxins in feta cheese samples were distributed as 20% of samples with metabolic toxin and also 20% of samples withcontaminates toxins.
Moreover, the consumers might be exposing to 30% of positive feta cheese samples according to total aflatoxins (AFM1, AFB1, AFB2, AFG1and AFG2) with mean value 37.75±35.97 ng/Kg.
1.3.2.Karieshcheese samples
The positive samples in the tested karieshcheese samples according toAFM1, AFB1 and AFG2 were33.33%, 8.33% and 8.33%, respectively with mean value at 250.90±278.75, 6.64±0.00 and 3.22±0.00 ng/Kg, respectively. On the other hand, AFB2and AFG1 were not detected in any karieshcheese sample.33.33% of samples had metabolic toxin (AFM1) while, contaminates toxins appeared in 16.66% of samples due to moulds contamination with any tools during kariesh cheese processing.
Moreover, the consumers might be exposing to 50% of positive karieshcheese samples according to total aflatoxins (AFM1, AFB1, AFB2, AFG1and AFG2) with mean value 168.91±250.50 ng/Kg.
1.3.3.Domiati cheese samples
The positive samples in the tested domiati cheese samples according toAFM1, AFB1, AFB2, AFG1 and AFG2 were16.66%, 16.66%, 8.33%, 8.33% and 16.66%, respectively with mean value at 46.90±7.92, 32.25±27.93, 4.00±0.00, 208.00±0.00 and 44.25±55.51 ng/Kg, respectively. The aflatoxins in domiati cheese samples were metabolic toxin in 20% of domiati cheese samples while,contaminates toxins in 30% of samples.
Moreover, the consumers might be exposing to 41.66% of positive domiati cheese samples according to total aflatoxins (AFM1, AFB1, AFB2, AFG1and AFG2) with mean value 91.76±113.15 ng/Kg.
1.4. Ice cream samples
The positive samples in the tested domiati cheese samples according toAFM1, AFB1, AFB2, AFG1 and AFG2 were10%, 30%, 10%, 30% and 30%, respectively with mean value at 60.00±0.00, 84.16±93.18, 250.00±0.00, 64.66±52.72 and 31.83±23.51ng/Kg, respectively. The sources of these aflatoxins in ice cream samples were distributed as 10% of samples with metabolic toxin while,60% of samples with contaminates toxins.
Moreover, the consumers might be exposing to 60% of positive domiati cheese samples according to total aflatoxins (AFM1, AFB1, AFB2, AFG1and AFG2) with mean value 142.00±236.96 ng/Kg.
2. Evaluation the ability of some natural viable probiotic bacterial dairy strains onaflatoxin M1reduction
The highest concentration (5×109 CFU/ml) from each viable probiotic bacterial strains (LactobacillusplantarumLactobacillus acidophilus and Bifidobacterium bifidum) reduced AFM1 from 50 to 31.55±1.22, 25.65±1.76 and 21.16±0.87 ng/ml, respectively with removal percent to AFM1 in PBS media as 36.90%, 48.70% and 57.68%. On the other hand, the combination of these viable probiotic bacterial strains (LactobacillusplantarumLactobacillus acidophilus and Bifidobacterium bifidum) at 5×109 CFU/ml had higher removal effect (64.62%)than each individual strain which reduced AFM1(50 ng/ml) to 17.69±1.24 ng/ml after 72h of the incubation time.
3. Assessment the potential of some nonviable probiotic bacterial and yeast strains (by heat treatment on sequestration of aflatoxin M1
3.1.Evaluation the ability of some nonviable probiotic bacterial dairy strains onaflatoxin M1reduction
The highest concentration (5×109 CFU/ml) from each nonviable probiotic bacterial strains (LactobacillusplantarumLactobacillus acidophilus and Bifidobacterium bifidum) reduced AFM1 from 50 to 24.13±0.95, 16.04±1.00 and 14.69±0.93 ng/ml, respectively with removal percent to AFM1 in PBS media as 51.74%, 67.92% and 70.62%. On the other hand, the combination of these nonviable probiotic bacterial strains (LactobacillusplantarumLactobacillus acidophilus and Bifidobacterium bifidum) at 5×109 CFU/ml had higher removal effect (79.66%) than each individual strain which reduced AFM1(50 ng/ml) to 10.17±1.03 ng/ml after 72h of the incubation time.
3.2. Evaluation the efficiency of some nonviable yeast strains onaflatoxin M1reduction
The highest concentration (5×109 CFU/ml) from each nonviable yeast strains (kluyveromyces lactis and Saccharomyces cerevisiae) reduced AFM1 from 50 to 15.43±1.15 and 10.63±1.01 ng/ml, respectively with removal percent to AFM1 in PBS media as 69.14% and 78.74%. On the other hand, the combination of these nonviable probiotic bacterial strains (kluyveromyces lactis and Saccharomyces cerevisiae) at 5×109 CFU/ml had higher removal effect (85.68%) than each individual strain which reduced AFM1 (50 ng/ml) to 7.16±0.90 ng/ml after 72h of the incubation time so the removal effect of the combination of nonviable yeast strains was higher than the combination of nonviable probiotic bacterial strains.
3.3. Evaluation the efficiency of some nonviable bacterial and yeast strains on reduction of aflatoxin M1
The removal results of AFM1 (from 50 to 6.04±0.15 ng/ml) by the combination of nonviablebacterial and yeast strains (Lactobacillusplantarum, Lactobacillus acidophilus, Bifidobacterium bifidum, kluyveromyces lactis and Saccharomyces cerevisiae) in PBS media were87.92% after 72h of the incubation time. The combination of nonviable bacterial and yeast strains was considered the highest removal effect between the combination of nonviablebacterial strains and the combination of nonviableyeast strains so we used this combination in reduction of aflatoxin M1 in milk as experimental media.
4. Evaluation potential of the combination of nonviable probiotic bacterial and yeast strainson aflatoxin M1 reduction in milk
The combination of nonviable probioticbacterial and yeast strains (Lactobacillusplantarum, Lactobacillus acidophilus, Bifidobacterium bifidum, kluyveromyces lactis and Saccharomyces cerevisiae) reduced AFM1 (50 ng/ml) during different times (12h, 24h, 24h, 48h and 72h) to 9.72±1.31, 6.68±0.55, 5.70±0.33 and 4.56±0.15 ng/ml, respectively and with high removal % of AFM1 to 80.56%, 86.64%, 88.60% and 90.88%, respectively in milk sample.
5. Sensory evaluationofthe prepared treatment yoghurt sample was inoculated with nonviable combination of probiotic bacterial and yeast strains.
Treatment yoghurt sample was inoculated with nonviable combination of probiotic bacterial and yeast strains (Lactobacillusplantarum, Lactobacillus acidophilus, Bifidobacterium bifidum, kluyveromyces lactis and Saccharomyces cerevisiae)for sensory evaluation by 32 members according to appearance, texture, tenderness, flavour and overall acceptance which was gives scores as 5.84, 5.75, 5.84, 5.96 and 5.96 (good score), respectively.
It is recommended to:
1- Use good quality dairy feed for the dairy animals without AFB1 or aflatoxigenic fungi to produce milk without AFM1.
2- Store the animal feed in proper environmental conditions to prevent the growth of moulds and production of aflatoxins in animal feed during storage.
3- Clean and sanitize all the equipment, utensils and tools contacting with milk and milk products during milking, distribution, processing and storage.
4- Use biological method for aflatoxins removal from milk and milk products as probioticbacterial and yeast strains (Lactobacillusplantarum, Lactobacillus acidophilus, Bifidobacterium bifidum, kluyveromyces lactis and Saccharomyces cerevisiae).
5- Inspected continuously our food for the existence of different types of aflatoxins (AFM1, AFB1, AFB2, AFG1 and AFG2) to detect the incidence of these toxins.