الفهرس 
INTRODUCTION AND AIM OF THE WORKOnly 14 pages are availabe for public view
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Abstract
The growing mandate for fresh-like food products and the possible hazards of chemically preserved foods necessitate the search for natural, efficient, and safe alternatives to meet the needs of the market. For several decades, lactic acid bacteria (LAB) have been incorporated extensively all over the world in the fermentation of a wide range of food products due to their limited health risk to humans, their contribution to food biopreservation, and the enhancement of the texture and flavor in these products. LAB can inhibit spoilage and pathogenic microorganisms through the production of different antimicrobial compounds such as organic acids, hydrogen peroxide, and antimicrobial peptides, including bacteriocins, while maintaining the nutritional value and the sensory attributes unchanged. Bacteriocins and other antimicrobial compounds produced by LAB are generally considered to be narrow-spectrum, however, some recent studies have reported their potential activities against Gram-negative bacteria.
Poultry industry is one of the biggest food industries worldwide besides being one of the most frequently consumed type of meat. One of the biggest dilemmas that faces this industry is the high perishability and the limited shelf life of poultry meats regardless of refrigerated storage. Chicken meat acts as a perfect matrix for the survival and growth of spoilage and/or pathogenic microorganisms due to its readily available nutrients. Moreover, poultry and poultry products are frequently incorporated in foodborne outbreaks. Although many chemical and physical treatments are being used, natural alternatives for enhancing the poultry products’ safety without harming their sensory attributes are under investigation. Several studies have reported that application of LAB cultures or their antimicrobials on raw poultry meat seemed to be a promising natural preservative alternative and would not affect the meat`s organoleptic properties
The valuable effects of LAB and their antimicrobials is not restricted to food preservation, but they can also be used as an alternative to the traditionally available antibiotics. Thus, their usage can help in controlling the global problem of antimicrobial resistance among pathogenic and food spoilage bacteria. Bacteriocins and bacteriocin-like substances can be used in a synergistic combination with antibiotics.
The results obtained in this current work are summarized as follows:
1. This study was carried over 18 months to assess the capability of 562 LAB strains, previously isolated from Egyptian raw milk and dairy products, for the production of antimicrobial compounds to be used in food biopreservation or to be used in the treatment for foodborne diseases.
2. Out of the 562 LAB isolates, only 101 LAB isolates showed a promising antimicrobial potential against one or more of the pathogenic and spoilage indicators when tested using the agar spot assay. Most of the potential LAB strains (73) showed inhibition against B. cereus, followed in order by E. coli (50), Ps. aeruginosa (39), C. albicanis (37), S. Typhimurium (40), L. monocytogenes (31), and Staph. aureus (18).
3. Of 101 LAB isolates, only 13 isolates were selected after being screened by the micro-dilution assay as these potential LAB strains reduced the visible growth of Gram-negative indicators by 90% or more compared with control trials.
4. Results of the agar well diffusion assay confirmed the results of the micro-dilution assay. Where, the neutralized CFS produced by the 13 LAB isolates showed the largest inhibition diameters against the Gram-negative indicators (18±0.9-27±2.0 mm against S. Typhimurium ATCC 14028, 17±2.0- 25±1.0 mm against E. coli ATCC 8739, and 13.5±1.0- 24±1.0 against Ps. aeruginosa ATCC 9027).
5. The antimicrobial activity of the neutralized CFS of the potential 13 LAB strains showed thermal stability at different temperatures (90˚C/10min, 100˚C/10min, 121˚C/10min, and 121˚C/20min). Furthermore, they preserve their residual inhibitory activity against the Gram-negative indicators after storage for 3 months at -20˚C and 4˚C.
6. Most of the isolates’ neutralized CFS had a stable antibacterial activity against the Gram-negative indicators over a narrow pH range from 3.0 to 5.0. However, few isolates were able to keep their residual antibacterial effects at pH 6.0 and 7.0.
7. Generally, the residual antimicrobial activity of the potential LAB isolates was partially lost after enzymatic digestion by proteinase K, pepsin, and trypsin.
8. Molecular biology identification of the 13 LAB strains by 16S rDNA gene sequence and their similarity search revealed 99% sequence homology. These isolates were identified as Lc. lactis FFNL 1940, Lb. plantarum FFNL 1553, Lb. plantarum FFNL 635, Lb. plantarum FFNL 796, FFNL 1812, Lb. plantarum FFNL 606, Lb. plantarum FFNL 1381, Lb. plantarum FFNL 2711, Lb. plantarum FFBL 2651, Lb. rhamnosus FFNL 1308 and Lb. pentosus FFNL 2885.
9. The auto-aggregation abilities of the 13 LAB strains ranged from 21.48 to 67.49% after 24 h incubation at 37◦C. Lb. plantarum FFNL 606 had the highest auto-aggregation rate (67.49±0.24%), while Lb. plantarum FFNL1381 showed the least rate (21.48±0.91%).
10. The highest co-aggregation against S. Typhimurium (89.71%) was found with Lb. plantarum FFNL2711, however, Lb. plantarum FFNL1381 had the lowest (58.36%) capability. However, Lb. rhamnosus FFNL1308 had the highest co-aggregation % with both E. coli (78.57%) and Ps. aeruginosa (85.5%), while Lb. plantarum FFNL1381 had the least co-aggregation % with both E. coli (52.11%) and Ps. aeruginosa (48.6%).
11. All the 13 LAB isolates were sensitive to the clinically important antibiotics, except for isolate FFNL 1812 that showed a resistance against all the tested antibiotics. All the 13 LAB isolates were phenotypically resistant to vancomycin, kanamycin and nalidixic acid. Moreover, all potential LAB strains showed a negative response to the production of gelatinase, DNase and histamine, besides the production of γ-hemolysis.
12. Five LAB isolates (Lc. lactis FFNL1940, Lb. plantarum FFNL606, Lb. plantarum FFNL2711, Lb. rhamnosus FFNL1308 and Lb. pentosus FFNL2885) were finally chosen after showing the highest inhibition results against Gram-negative indicators, highest auto-aggregation and co-aggregation capabilities, stability to different treatments (temperature, proteolytic enzymes and pH), besides being safe. These 5 LAB isolates did not show antagonism against each other, and they were expected to work together without interference.
13. The highest activity of the antimicrobial compounds of the selected strains (Lc. lactis FFNL1940, Lb. plantarum FFNL606, Lb. plantarum FFNL2711, Lb. rhamnosus FFNL1308 and Lb. pentosus FFNL2885) against Gram-negative bacteria were precipitated at 60% ammonium sulphate concentration.
14. The titer activity increased by precipitation and ranged from 128 to 512 AU/ml. Lb. pentosus FFNL 2885 had the highest specific activity (26,947.4 AU/mg), while Lc. lactis FFNL 1940 showed the lowest specific activity (2,285.71 AU/mg).
15. The initial MIC values of the individual antimicrobial compounds from each isolate after ammonium sulfate precipitation and dialysis against S. Typhimurium ranged from 64 to 128 AU/ml, which significantly increased to 4,096 AU/ml after mixing an equal volume of the partially purified antimicrobial compounds.
16. Generally, the application of the protective culture mixture at a final concentration of 106 CFU/ml or 1 × MIC of their PPACs cocktails on the surfaces of raw chicken fillets during chilled storage for 12 days resulted in improving the hygienic quality of the product and prolonging its shelf life. After 12 days of storage, the APC of the control fillets reached (8.32±0.06 log10 CFU/g), while the counts in raw fillets treated with the protective LAB cultures and their PPACs cocktail were only 5.24±0.03 and 6.68±0.03 log10 CFU/g, respectively.
17. After 12 days of storage, a 2.15 and 1.54 log10 CFU/g reduction in the PTC after treatment with the protective LAB mixture and PPACs mixture, respectively. While the treatment of the raw fillets with the live mixture and their PPACs cocktail led to a decrease in the Enterobacteriaceae counts by 2.32 and 1.83 log10 CFU/g, respectively, in comparison to the untreated fillets.
18. On the last day of storage period, the mean pH values of chicken fillets treated with protective LAB mixture (5.79±0.02) and PPACs mixture (6.07±0.04) were significantly lower (P<0.05) than the control fillets (6.81±0.06).
19. On day 9 of storage, the TVB-N values of chicken fillets treated with protective LAB mixture and PPACs mixture were 10.8±0.02 mg/100g and 17.9±0.02 mg/100g, respectively. However, the TBARS values were 0.46±0.01 mg/kg and 0.69±0.02 mg/kg for chicken fillets treated with the protective LAB mixture and PPACs mixture, respectively.
20. The application of LAB mixtures or 1 × MIC of PPACs cocktail did not affect (P≥0.05) the sensory attributes of fresh chicken fillets, besides remaining acceptable till day 9 of storage compared to the untreated control fillets during chilled storage.
21. Challenging the raw chicken fillets with S. Typhimurium, E. coli, Ps. aeruginosa, L. monocytogenes, and Staph. aureus after being treated with the protective LAB mixture or 1 × MIC of their PPACs cocktail led to a significant decrease (P<0.05) in these pathogens’ counts in comparison to their controls. Interestingly, the inhibitory effect of the protective LAB cultures on the raw chicken fillets was better than the results produced by the PPACs cocktail.
22. The in vivo administration of protective LAB cultures at a concentration of 1× 109CFU or PPACs mixture at a final concentration of 1 × MIC to mice led to insignificant differences in their general health scores, body weights, and food intake, in comparison to the mice of the control group that received only PBS.
23. The in vivo treatment of Salmonella-infected mice with either the protective LAB cultures at a concentration of 1× 109CFU, 1 × MIC of PPACs, ciprofloxacin, or a mixture of ½ × MIC PPACs-½ × MIC ciprofloxacin resulted in a significant improvement in their general health scores, body weights and food intake in comparison to the un-treated infected group.
24. Moreover, mice that received either the protective LAB cultures at a concentration of 1× 109CFU, 1 × MIC of PPACs mixture, ciprofloxacin, or a mixture of ½ × MIC PPACs-½ × MIC ciprofloxacin as treatments showed a significant reduction (P<0.05) in the Salmonella counts in their feces, besides a decreased translocation rate to internal organs (liver and spleen).
25. Histopathological analyses of the ileum of mice that received different treatments suggested that these treatments could significantly ameliorate the damage of villi and inflammatory cell infiltration, thus reducing the ileal tissue damages.
It can be concluded from the present study that:
1. The Egyptian raw milk and artisanal dairy products are rich in a wide range of LAB strains that could inhibit a broad range of spoilage and pathogenic foodborne bacteria including Gram-negative bacteria.
2. The application of protective LAB cultures or 1 × MIC of PPACs mixture on raw chicken fillets resulted in a significant improvement in the microbiological quality, physicochemical and sensory attributes, besides increasing the products’ shelf life.
3. The pre-treatment of challenged raw chicken fillets with protective LAB cultures or 1 × MIC of PPACs resulted in a significant reduction in the pathogens counts. Noticeably, the protective LAB mixture was more effective on raw chicken fillets than the PPPACs mixture.
4. The administration of protective LAB cultures, PPACs mixture, ciprofloxacin, or a mixture of PPACs-ciprofloxacin to Salmonella-infected mice alleviated the symptoms, damages and negative impact of the S. Typhimurium infection.
Recommendations and future merits
1. Screening more indigenous fermented products for the presence of LAB strains with potent antimicrobial activities.
2. There is a crucial need to determine the exact nature of the molecules that produce the antimicrobial activity against the Gram-negative bacteria and to fully purify and characterize them.
3. The application of LAB or their fully/partially purified antimicrobial compounds on more raw products, especially meat products, and the evaluation of their efficiency in improving the microbiological and physicochemical should be evaluated.
4. Moreover, the research should expand and includes consumers and industry and evaluate their acceptability of these products.
5. The re-evaluation of the Egyptian legal requirements and standards regarding the adding of live cultures or their antimicrobial compounds to fresh raw products.
6. Further in vivo research should be conducted to evaluate the efficiency of more LAB strains and their antimicrobial compounds as treatments to infections that resulted from foodborne pathogens.
7. More studies should be done to evaluate the toxicity that might result from the long-term feeding of laboratory animals by live LAB strains or their antimicrobial compounds.