الفهرس 
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Abstract
Sterile skimmed bovine milk samples, collected from supermarkets in Japan, were separately inoculated with Lactobacillus and pediococcus strains. The proteolytic and fermentative activities were studied after separation of supernatants (whey). The antimicrobial efficacy of inhibitory substances isolated from whey especially water soluble peptides was determined against H. pylori.
1. Preliminary screening the proteolytic activity of lactic acid bacteria (LAB), Lactobacillus fermentum (GBF), Lactobacillus plantarum (MMY) and Pediococcus pentosous (MKH6-3):
i. All examined (LAB) strains possessed proteolytic activity. Lb. plantarum and Lb. fermentum were strong protease producers (hydrolytic zone ≥ 6 mm) by spot cultivation and overlay agar assays while P. pentosous showed moderate activity. All strains showed lower proteolysis when examined by well diffusion assay with average hydrolytic zone values of ≥ 4 mm except Lb. fermentum with hydrolytic zone diameter of > 2 mm.
2. Preliminary screening the antimicrobial activity of (LAB), Lactobacillus fermentum (GBF), Lactobacillus plantarum (MMY) and Pediococcus pentosous (MKH6-3) against Staphylococcus aureus:
i. All tested (LAB) strains showed strong antimicrobial activity against S. aureus as an indicator strain with average inhibition zone diameter was ≥ 9 mm by well diffusion assay. Lb. fermentum was the most active one.
3. Monitoring the fermentation and pH performance of (LAB), Lactobacillus fermentum (GBF), Lactobacillus plantarum (MMY) and Pediococcus pentosous (MKH6-3) in skimmed bovine milk:
i. All tested (LAB) strains exhibited glycolytic and proteolytic systems enabling them to ferment lactose and produce amino acids for growth in skimmed bovine milk.
ii. Lb. plantarum MMY was the most active and rapid acidifier strain with a pronounced decrease in pH value to be 5, 4, 3 at 4th, 8th and 12th day, respectively. Typical curd was formed at 4th day during fermentation.
iii. P. pentosous MKH6-3 was a medium acidifier strain with a moderate decrease in pH values 5.5, 4 and 4 at 4th, 8th and 12th day of fermentation, respectively, Typical curd was formed at 6th day during fermentation.
iv. Finally, Lb. fermentum GBF was a slow acidifier strain with a slow decrease in pH values 6, 5 and 4.5 at 4th, 8th and 12th day of fermentation, respectively. Typical curd was formed at 10th day during fermentation.
4. Screening anti H. pylori activity of Lactobacillus fermentum (GBF) and Lactobacillus plantarum (MMY) harvesting supernatants (whey) after fermentation of skimmed bovine milk:
i. Lb. fermentum GBF and Lb. plantarum MMY in whey of the skimmed bovine milk and fermented MRS supernatants had > 99 to100% inhibition of H. pylori within 24 h and they grew on Brucella blood agar plates when compared with control unfermented skimmed bovine milk. The Lb. fermentum GBF count values were 2.6 × 106 and 5.9 × 106 CFU/ml in whey of the skimmed bovine milk and fermented MRS supernatant while Lb. plantarum MMY count values were 6.3 × 107 and 8.1 × 107 CFU/ml, respectively.
ii. Microscopic examination revealed the differences in shape and cell morphology between H. pylori colonies grown on control BBA plates and Lb. fermentum GBF or Lb. plantarum MMY colonies on plates inoculated with H. pylori and whey of skimmed bovine milk or MRS supernatants.
5. Monitoring the presence of total water soluble peptides in whey fraction of (LAB) fermented skimmed bovine milk by Lactobacillus fermentum (GBF), Lactobacillus plantarum (MMY) and Pediococcus pentosous (MKH6-3) using RP-HPLC:
i. RP-HPLC analysis showed an increase in the released peptides by the developing proteolytic system of the selected (LAB) strains.
ii. Three peptide peaks were detected for Lb. plantarum MMY at 24, 36 and 74 min which were absent in unfermented skimmed bovine milk (control). Similarly, three peptide peaks obtained at 64, 68 and 74 min for Lb. fermentum GBF. Finally, peptide profile of P. pentosous MKH6-3 was similar to Lb. plantarum MMY in peak eluted at 24 min with the exception of two peaks were observed at 60 and 72 minutes which were absent in the control.
6. Screening anti H. pylori activity of RP-HPLC analyzed total water soluble peptides of Lactobacillus fermentum (GBF) and Lactobacillus plantarum (MMY) in whey fraction of fermented skimmed bovine milk:
i. Analyzed RP-HPLC water soluble peptides of Lb. fermentum GBF and Lb. plantarum MMY in the whey of fermented skimmed bovine milk exhibited anti H. pylori activity at different dilutions when compared with fermented MRS supernatants and unfermented skimmed bovine milk. The minimum bactericidal concentrations (MBC) which achieved complete inhibition of H. pylori organisms were at peptide dilutions 1:8 and 1:4 for Lb. fermentum GBF and Lb. plantarum MMY, respectively.
ii. The result investigated that Lb. fermentum organisms produced more strong antimicrobial peptides from bovine milk which were more active against H. pylori than Lb. plantarum MMY released peptides in milk.
7. Isolation of antimicrobial peaks of sub-peptide fractions in whey of Lactobacillus fermentum (GBF) fermented skimmed bovine milk by RP-HPLC:
i. The result of RP-HPLC analysis revealed that the presence of four sub-fraction peaks, designated as P1, P2, P3 and P4 derived from Lb. fermentum GBF fermented skimmed bovine milk as well as unfermented skimmed bovine milk supernatants.
ii. Peaks of sub-peptide fractions P1, P2 and P3 for the examined supernatants were fast-eluting hydrophilic peptides while, P4 observed at the end of analysis and was different from other obtained peptide peaks as it might represent organic acids.
iii. Peaks of sub- fractions P1 and P4 were observed at 17.5 to 29 and 120 to 144 min of analysis, respectively for the examined supernatants. In contrast, there were obvious differences in P2 and P3 between the examined supernatants which referred to strong proteolytic nature of Lb. fermentum and formation of powerful antimicrobial peptides with anti H. pylori activity.
8. Screening anti H. pylori activity of ”RP-HPLC” analyzed antimicrobial peaks of sub- peptide fractions in whey of Lactobacillus fermentum (GBF) at different concentrations:
i. All peaks of Lb. fermentum GBF fermented skimmed bovine milk were active against H. pylori at different peptide concentrations, 215, 107.5, 53.75, 26.87 and 10 µg/ml with bactericidal or bacteriostatic effects. P1 of unfermented bovine milk (control) enhanced H. pylori growth in a concentration dependent manner. The most active H. pylori cidal peptides were in P2 and P3 with MBC values 53.75 and 26.87 µg/ml, respectively while MBC of P4 (non-peptides agent) was at 53.75 µg/ml.
ii. In addition, all peaks of Lb. fermentum GBF fermented skimmed bovine milk were more active against H. pylori than peaks obtained from control except P1 Lb. fermentum GBF was less active peptide peak.
9. Broth microdilution assay for determining minimum inhibitory concentration (MIC) of antimicrobial peaks of sub- peptide fractions in whey of Lactobacillus fermentum (GBF):
i. Peak of sub-peptide fractions P3 of Lb. fermentum GBF was the most potent antimicrobial agent against H. pylori with the same MIC and MBC value at peptides concentration 26.87 and MIC index value (1), while P2 and P4 showed similar MBC value at concentration 53.75 μg/mL and MIC value at 26.87 μg/mL and MIC index value was 2 for both peaks.
iii. Finally, all peaks of Lb. fermentum GBF fermented skimmed bovine milk showed higher antimicrobial activity against H. pylori than control.
The in vitro experiments in the present study revealed the excellent role of glycolytic and proteolytic systems of Lb. plantaram, Lb. fermentum and P. pentosous in bovine milk fermentation and in turn production of a plethora of peptides with vulnerable bioactivities. In addition, skimmed bovine milk fermented by Lb. fermentum cultures were efficacious for inhibiting H. pylori in vitro owing to their engorgement with various inhibitory substances such as bioactive peptides and organic acids in addition to the direct inhibitory effect of Lb. fermentum organisms on H. pylori. In addition, fermented milks containing (LAB) and their antimicrobial products can be used for bio-preservation for extending storage life and enhancing human health.