Production of Bioactive Peptides in Milk Using Two Native Strains of Levilactobacillus brevis

Background and Objective: Milk proteins are precursors of several biologically active peptides. One of the methods of producing these peptides is fermentation using lactic acid bacteria. The aim of this study was to investigate production of antioxidant and angiotensin-I converting enzyme inhibitory bioactive peptides in cow milk fermented by two strains of Levilactobacillus brevis. Material and Methods: Two strains of Levilactobacillus brevis KX572376 (M2) and Levilactobacillus brevis KX572382 (M8) were used in fermentation of low-fat cow milk. Moreover, pH changes, proteolytic activity, water-soluble extract biological activity (antioxidant activity and angiotensin-I converting enzyme inhibition) of the samples and peptide fraction less than 3 kDa were investigated at 24 and 48 h of fermentation (30 °C). Peptide profile of the superior sample was analyzed as well. Statistical analysis was carried out using one-way of variance, Tukey test and SPSS software v.25. Results and Conclusion: The two strains decreased milk pH to a similar level in the first 24 h. Quantities of free amine groups in the samples treated with M2 and M8 strains within 24 and 48 h of fermentation were significantly different (p≤0.05), compared to the control sample. In the first 24 h of fermentation, no difference was observed in the quantity of free amines of M2 and M8 samples. In the second 24 h, further free amine groups were produced due to the activity of M8 strain in milk. Antioxidant activity of the water-soluble extracts of M2 and M8 samples was significantly (p≤0.05) higher than that of the control sample during fermentation. Antioxidant activity in fractions less than 3 kDa did not show significant differences in M2 and M8 samples at 24 and 48 h of fermentation. In the control sample, no antioxidant activity was observed in fractions less than 3 kDa. The highest ACE inhibitory activity in fractions less than 3 kDa of M8 was observed after 48 h. No angiotensin-I converting enzyme inhibition was seen in fractions less than 3 kDa of M2 and control sample. The RP-HPLC peptide patterns of the fraction less than 3 kDa of M8 and control sample were different, which was a justification for the biological activity in this sample. Conflict of interest: The authors declare no conflict of interest

The biodiversity of Lactobacillus spp. from Iranian raw milk Motal cheese and antibacterial evaluation based on bacteriocin-encoding genes

Abstract Lactobacilli, as the largest group of lactic acid bacteria, produce large amounts of antimicrobial metabolites such as organic acids, fatty acids, ammonia, hydrogen peroxide, diacetyl and bacteriocin, which inhibit the growth of pathogenic bacteria and increase shelf life of food. The aim of this study was to identify the Lactobacillus spp. isolated from Iranian raw milk Motal cheese and to detect the presence of bacteriocin genes in the isolated Lactobacillus strains exhibiting antimicrobial activity. For this purpose, 6 Motal cheese samples from Dasht-e-Moghan region, Iran, were subjected to microbial characterization. Nineteen Lactobacillus spp. were isolated and subsequently identified based on biochemical and molecular methods. According to the sequencing of isolates, Lactobacillus spp. consisted primarily of Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus casei and Lactobacillus buchneri. The identified isolates were then evaluated for antimicrobial activity against Escherichia coli ATCC 25922, Listeria innocua ATCC 33090 and Staphylococcus aureus ATCC 25923. The results of PCR analysis using specific primers of genes encoding Bacteriocin, revealed the presence of Plantaricin A and Plantaricin EF in all Lactobacillus plantarum isolates and Brevicin 174A in 5 of Lactobacillus brevis isolates, whereas the gene encoding Pediocin PA-1 was not observed in any of examined isolates. It is therefore concluded that bacteriocinogenic isolates could be recommended as suitable candidates to be used as starter, adjunct-starter or antimicrobial agents for production of fermented and non-fermented products

Evaluation of Inhibitory and Lethal Effects of Aqueous, Ethanolic and Hydroalcoholic Extracts of Aerial Parts of Salvia chorassanica against Some Gram-negative and Gram-positive Bacteria in Vitro

Abstract Background and Objectives: Development of bacterial resistance to antibiotics has led to an increased tendency to development of new more effective and non-toxic antimicrobial compounds. In this study, the inhibitory and lethal effects of aqueous, ethanolic, and hydroalcoholic extracts of aerial parts of Salvia chorassanica were evaluated against Listeria monocytogenes, Bacillus cereus, Salmonella typhi, and Escherichia coli O:157. Methods: In this study, Kirby&ndash;Bauer disk diffusion method was used to evaluate antimicrobial activity. In this method, bacteria were cultivated as grass culture in Mueller Hinton Agar (MHA) media. To determine the minimum inhibitory concentration and minimum bactericidal concentration, micro-dilution method with ELISA and addition of phenyl tetrazolium chloride reagent, were used. Data were analyzed using one-way ANOVA and Duncan&rsquo;s test at the significance level of p<0.05. Results: The highest diameter of inhibition in agar diffusion method was related to hydroalcoholic extract of aerial parts of Salvia chorassanica against Gram-positive bacteria Bacillus cereus. The amount of calculated MIC of hydro-alcoholic extract for Gram-positive bacteria was 30mg/ml. This amount was the lowest among other measured MIC. Conclusion: Based on the results of this study, Gram-negative bacteria showed more resistance to different extracts of aerial parts of Salvia chorassanica as compared to Gram-positive bacteria, so that Salmonella typhi was found to be the most resistant bacterium among the tested bacteria

Effect of probiotic and vinegar on growth performance, meat yields, immune responses, and small intestine morphology of broiler chickens

The present study was conducted to investigate the effect of dietary supplementation of probiotic and drinking water (DW) supplemented with different levels of vinegar on performance, meat yields, immune responses, and small intestine histology of broiler chickens. Three hundred thirty day-old Ross 308 male broiler chicks assigned to six treatments in a completely randomised design experiment with a factorial arrangement (2 × 3) and five replicates of 11 chicks each. The treatments included two levels of dietary probiotic supplementation (0 and 1 × 1010 CFU lactic acid/kg of diet) and three levels (0%, 1%, and 2%) of DW supplemented with vinegar (5% acetic acid concentration). The study lasted from 1 to 42 d. Growth performance, meat yields and lymphoid organs relative weight, humoral and cellular immune responses, and small intestine histomorphometry were measured. The average daily feed intake (ADFI) and feed conversion ratio (FCR) during 1–10 days of age significantly decreased in the birds that fed supplemented diet with probiotic and drunk supplemented water with vinegar than the birds fed and drunk free of any additive. Experimental treatments did not have a significant effect on performance during other growth periods, carcase yields, and lymphoid organs relative weight. DW supplemented with vinegar significantly increased villus height (VH), crypt depth (CD) and decreased small intestine muscular thickness (MT) and abdominal fat. Dietary supplementation of probiotic significantly improved immune response to sheep red blood cell (SRBC) inoculation. In conclusion, this study confirms beneficial effects of probiotic and vinegar on 1–10 d performance, immune and intestine health of broiler chickens

Screening of lactic acid bacteria strains isolated from Iranian traditional dairy products for GABA production and optimization by response surface methodology

Abstract A total of 50 lactic acid bacteria (LAB) isolates from Iranian traditional dairy products (Motal and Lighvan cheeses, and artisanal yogurt) were screened for gamma-aminobutyric acid (GABA) production. Firstly, a rapid colorimetric test was performed to evaluate the glutamate decarboxylase (GAD) activity among the LAB isolates examined. Thin layer chromatography (TLC) was then performed on selected strains to identify isolates with high/moderate GABA producing capacity, and a GABase micro-titer plate assay was employed to quantify GABA. Finally, two Lactococcus (Lac.) lactis strains were selected for GABA production optimization via Response Surface Methodology (RSM) following Central Composite Design (CCD). Forty-one out of the 50 isolates showed GAD activity according to the colorimetric assay. Eight isolates displayed strong GAD activity, while nine showed no activity; low to moderate GAD activity was scored for all other isolates. GABA production was confirmed by TLC in all isolates with high GAD activity and in four selected among isoaltes with moderate activity. Among the Lactococcus strains tested, Lac. lactis 311 and Lac. lactis 491 were the strongest GABA producers with amounts of 3.3 and 1.26 mM, respectively. These two strains were subjected to GABA production optimization applying RSM and CCD on three key variables: Monosodium glutamate concentration (MSG) (between 25 and 150 mM), incubation temperature (between 25 and 37 °C), and pH (between 4.0 and 5.0). Optimal conditions for GABA production by Lac. lactis 311 and Lac. lactis 491 of temperature, pH and MSG concentration were, respectively, 35.4 and 30 °C, pH 4.5 and 4.6, and MSG concentration of 89 and 147.4 mM, respectively. Under the above conditions, the amount of GABA produced by Lac. lactis 311 and Lac. lactis 491 was 0.395 and 0.179 mg/mL, respectively. These strains and the optimal culture conditions determined in this study could be used for the biotechnological production of GABA or applied in food fermentations for the development of naturally GABA-enriched foods