الفهرس 
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Abstract
Probiotics are the major discussion topic of health today due to their biomedical benefits. Probiotics are defined as living microorganisms, which once administered in appropriate amounts; confer a health profit on the host. Probiotics are applied in many different products worldwide. In addition to food products, probiotics are also used in pharmaceuticals and animal feed. Isolation of Egyptian probiotic strains and evaluation of their properties in order to select promising ones that can replace the imported probiotics in the Egyptian market was the main target of this study.
In the present study, fourteen bacterial isolates and four yeast isolates were isolated from eight different sources (Cow milk, baby stool, pickle, breast milk, natural and commercial yogurt, sausage and Rayeb) on MRS agar medium. The preliminary selection was carried out in order to select the most promising probiotic isolates. The criteria used for in vitro selection of probiotics, which allow them to be functioning in the intestinal tract, included pH tolerance and bile tolerance, antibiotic resistance and antimicrobial activity.
Among the selected isolates, five bacterial isolates (M, St3, Sa2, Sa3 and Sa4) exhibited tolerance to pH 3 for 3 hours and survive at bile (0.3%) for 3 hours, were selected for further studies. While other isolates showed sensitivity to pH 3. Successive gradual growth of the isolates which failed to grow at pH 3 was carried out at pH 6, 5, 4 and 3 in order to obtain adapted isolates able to grow at lower pH to be used as probiotics. Only yeast isolates (P1, P2, P3 and SaY) were able to tolerate pH 3 after adaptation.
All the selected bacterial isolates showed resistance toward AK, VA, CIP and B while showed sensitivity to sensitive to RA, SXT, E, IPM, C and AMC antibiotics. The four tested yeast isolates were able to resist all the studied antibiotics. Stool consortium (C) showed antibiotic resistance against AK, VA, SXT, S, GM and CIP antibiotics. Also, all the selected probiotic isolates were tested for their resistance toward anti-yeast antifungal drugs. Isolates M, Sa3, Sa4 and P3 were resistant to Nystatin, while P1 and P2 were resistant to Flagyl. On the other hand St3, Sa2 and SaY were sensitive to both Flagyl and Nystatin. Stool consortium showed resistance against both drugs.
The selected probiotic bacterial isolates (M, St3, Sa2, Sa3 and Sa4) in addition to stool consortium (C) showed antibacterial activity against seven pathogens; Staphylococcus aureus, Streptococcus sp., Pseudomonas sp., Salmonella sp., Proteus sp., Escherichia coli and Klebsiella pneumonia while, yeasts exhibit antibacterial activity against two pathogenic bacteria (Staphylococcus aureus and Pseudomonas).
The ability to adhere to epithelial cells and mucosal surfaces has been suggested to be an important property of many strains used as probiotics. Isolate M was able to adhere to solvents such as toluene (80.43 ± 0.34%) and xylene (78.2 ± 0.26%) indicating hydrophobic property, while stool consortium (C) and St3 isolate showed hydrophobicity with xylene 56.87 ± 0.4 % and 44.77 ± 0.27 % respectively.
The ability of the selected probiotic isolates to remove lead (Pb) from aqueous solution containing 1000 ppm of lead was evaluated using ICP. Lead removal was tested after 5 minutes and 1 h from lead containing solution. The removal percentage of lead recorded by isolates after 5 minutes was 71.28 ± 0.4, 75.79 ± 0.5, 86.44 ± 0.3, 54.82 ± 0.3, 84.18 ± 0.4, 78.61 ± 0.2, 79.78 ± 0.3, 76.96 ± 0.2, 72.86 ± 0.5 and 61.83 ± 0.3 % for M, St3, Sa2, Sa3, Sa4, P1, P2, P3, SaY and C respectively. The highest removal percentage of lead was recorded by isolate P2 after 1 h and reached 87.79 ± 0.4%.
The probiotic isolates (M, St3 and Sa2) and stool consortium (C) were selected as promising probiotic isolates for further studies. The antioxidant activity of the selected isolates was evaluated using DPPH scavenging method. Maximum antioxidant activity was recorded by M isolate (51.85 ± 0.2 %) while the lowest percentage was achieved by Sa2 isolate (47.15 ± 0.1 %). Also, the ability of the probiotics to survive in food products was studied. The selected probiotic isolates were used for manufacturing yoghurt. The count of each probiotic isolate in each produced yoghurt was followed up separately over 0, 7, 14 and 21 days to test the shelf time of each. All the selected isolates showed stability of the count up to 21 days of refrigeration.
The most promising probiotic isolates (M and St3) were identified by 16S rRNA gene sequence as Lactobacillus plantarum strain M (KY508300) and Lactobacillus plantarum strain St3 (KY508301) respectively.
The effects of environmental conditions on the growth of the selected probiotics was examined including; inoculum size, temperature, pH, type and concentration of carbon and nitrogen sources as well as tween 80 concentration and radiation. The maximum growth of both isolates was achieved when grown at pH 6, 1% inoculum size, incubation temperature 37oC, 15 g/l glucose or lactose, 8 g/l of yeast extract and 15 g/l of peptone. Increasing concentration of glucose or lactose, yeast extract and peptone revealed no remarkable difference in the count of both isolates. On the other hand, no remarkable difference in the growth of both isolates was observed either by removing or adding different concentrations of tween 80. L. plantarum strain M and L. plantarum strain St3 were evaluated for their tolerance to microwave irradiation for 0, 30, 60 and 90 seconds. Both isolates showed high tolerance after exposure to microwave irradiation till 90 seconds. The promising probiotic isolates, L. plantarum strain M and L. plantarum strain St3 were exposed to different doses of gamma irradiation 1, 2, 3, 4 and 5 KGy. Both probiotic isolates were found to tolerate gamma radiation until 4 KGy. The calculated D10-value of L. plantarum strain M and L. plantarum strain St3 were found to be 0.546 and 0.55 KGy, respectively.
On studying the effect of small therapeutic doses of gamma irradiation 45 and 60 Gray on antibiotic resistance property of both isolates. Both isolates showed slight resistance against most antibiotics after exposure to the model therapeutic doses (45 and 60 Gray).
On studying the sensitivity of both probiotics toward some daily beverages including; tea, anise, mint, coffee, Nescafe and Pepsi, the results showed that anise, mint, Coffee and Pepsi nearly has no effect on the growth of both isolates compared to controls. The same result was achieved on studying the effect of honey and molasses, while black tea and Nescafe decreased the growth of both isolates.
On evaluating the drug resistance of the both probiotic isolates toward different drugs including; Abimol, Brufen, Catafly, one two three, Apidone, Claritin, Cyrinol, Neo minophylline, Visceralgine, Antinal, Mucosol and Smecta, it was observed that only two drugs (Brufen and Smecta) inhibited the growth of both probiotic isolates.
In a comparative study, the growth of L. plantarum strain M and L. plantarum strain St3 on MRS medium was compared with the use of whey dairy waste as a growth medium. The count of L. plantarum strain M and L. plantarum strain St3 was (3.4 ± 0.7) x109, (3± 0.7) x 109 and (0.5 ± 0.2) x 109, (0.2 ± 0.09) x 109 on MRS medium and whey respectively. The results showed nearly the same growth of both isolates on whey comparing to their growth on MRS medium.
According to the promising criteria of the selected probiotics, in vivo studies are highly recommended to test their safety to be used as food supplement.