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

Production of bacteriocins by Enterococcus spp. isolated from traditional, Iranian, raw milk cheeses, and detection of their encoding genes

Strong bacteriocins, or bacteriocins with a wide range of activity against pathogens and spoilage microorganisms, are actively sought for use as natural food preservatives. This work reports the inhibitory activity of 96 enterococcal isolates from two Iranian, raw milk cheeses against five indicator organisms (including Listeria innocua). Forty-eight isolates inhibited at least one indicator in spot agar assays. Of these, 20 isolates corresponding to 15 different strains were shown to produce bacteriocin-like substances in liquid cultures. PCR analysis revealed the genes coding for enterocins (enterococcal bacteriocins) A, B, P or X, or their combinations, in all but one of these 15 strains. In addition, the gene coding for enterocin 31 was detected in two strains. No amplification was obtained in one strain when using specific primers for all 13 bacteriocin genes sought. Three different enterocin genes were identified in most strains and four in one strain. Although the concomitant production of bacteriocins is still to be verified, producers of multiple enterocins could be of great technological potential as protective cultures in the cheese industry. © 2012 Springer-Verlag.This research was partially supported by a project from the Spanish Ministry of Science and Innovation (MICINN) (AGL2007-61869-ALI). A.A. was awarded a scholarship of the Severo Ochoa program from FICYT (BP08-053). S. Delgado was sup- ported by a research contract from MICINN under Juan de la Cierva program (JCI-2008-02391). The authors wish to thank the Iranian Ministry of Industries and Mines, as well as Razavi Dairy Industry (Mash- had, Iran) and the O Y ce of Industrial Relationships (OIR) of Ferdowsi University of Mashhad (FUM).Peer Reviewe

Microbial diversity of the traditional Iranian cheeses Lighvan and Koozeh, as revealed by polyphasic culturing and culture-independent approaches

The microbiota of two traditional Iranian cheeses (Lighvan and Koozeh) made of raw ewe's milk or mixtures of ewe's and goat's milk without starter addition was explored by culture-independent and culture-dependent approaches. Three batches of Lighvan and one of Koozeh were subjected to culture-independent polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of dominant bands to assess the microbial structure and dynamics through manufacturing and ripening. In addition, culturing in elective media for lactic acid bacteria (M17, MRS and KAA), isolation of single colonies (n=130), molecular identification by PCR-amplified ribosomal DNA restriction analysis and sequencing, and differentiation at the strain level by repetitive extragenic palindromic PCR was also performed. DGGE analysis showed that the dominant amplicons in all four cheese batches belonged to Lactococcus lactis and Streptococcus parauberis. In addition, Escherichia coli and Lactococcus garvieae were frequently identified in both Lighvan and Koozeh, while Streptococcus thermophilus was found occasionally. In contrast, Enterococcus faecium and Enterococcus faecalis were found to be dominant among the isolates in all batches. These species showed a high genetic diversity. The discrepancy between culturing and DGGE results suggested that dominant populations were in a nonrecoverable state in the used media. This reinforces the idea that culture-dependent and cultureindependent techniques provide complementary data, ultimately affording a better description of cheese ecosystems. These data could be of help in the selection of commercial starters for industrial-scale manufacture of Lighvan and Koozeh cheeses using pasteurised milk. Alternatively, microbial analysis would allow the selection of appropriate strains for designing of specific starters for traditional cheese manufacture. © 2011 Springer-Verlag.This research was partially supported by a project from the Spanish Ministry of Science and Innovation (MICINN) to BM (Ref. AGL2007-61869-ALI). AA was awarded a scholarship of the Severo Ochoa programme from FICYT (Ref. BP08-053)Peer Reviewe

Evaluation of antioxidant, antibacterial and cytotoxicity activities of exopolysaccharide from Enterococcus strains isolated from traditional Iranian Kishk

In this study, the antimicrobial effect of exopolysaccharide (EPS) extracted from Enterococcus strains [E. durans K48 (MT437,248), E. faecium R114 (MT437,249) and E. faecium T52 (MT437,250)] isolated from Kishk was applied against some foodborne pathogenic bacteria using well diffusion and microdilution methods. The antioxidant activity of EPS was also evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay and ferric reducing antioxidant power (FRAP) method. The cytotoxicity effect of EPS on human Gingival Fibroblast (HGF) cell line was also assessed. The results obtained by antimicrobial test showed that the most resistant bacteria to the examined EPS was Listeria monocytogenes, and the most susceptible were Staphylococcus aureus and E. faecalis. The results showed that the DPPH inhibitory percentage of EPS (25 mg/mL) from E. durans K48, E. faecium R114, and E. faecium T52 was 53%, 58% and 64%, respectively. EPS from E. faecium T52 displayed the highest reducing power, but statistically, there was no significant difference between the reducing power of EPS T52 and EPS R114 (P ≥ 0.05). The lowest toxicity percentage of EPS k48, EPS T52, and EPS R114 on normal human cell line at a concentration of 0.2 mg/mL was 10%, 15%, and 13%, respectively, which was statistically significant (P < 0.05). The obtained results in the present study indicate that EPS from the examined LAB strains with no in vitro cytotoxicity can be a potential source of natural antioxidant and antibacterial agent to be used in food and pharmaceutical industries.This work was financially supported by a Grant (No. 47333) from Ferdowsi University of Mashhad (Research affairs), Iran

Iranian kishk as a source of lactic acid bacteria producing exopolysaccharide

Trabajo presentado en el XIII International Scientific Agriculture Symposium AGROSYM 2022, celebrado en Sarajevo (Bosnia y Herzegovina), del 6 al 9 de octubre de 2022Exopolysaccharides are high molecular weight polymers composed of sugar subunits. Produced exopolysaccharides by lactic acid bacteria (LAB) play a significant role in improvement of organoleptic properties of fermented dairy products such as yogurt. Diversely, the probiotic function of these bacteria and the prebiotic properties of their produced biopolymers promote consumer¿s health. For this purpose, a traditional dairy product known as ¿Kishk¿ was selected. 143 strains of lactic acid bacteria were isolated from Iranian Kishk in Khorasan Province and cultured in formulated MRS mediums with different sugars such as glucose, fructose, sucrose and, lactose (40 g/L) and incubated in anaerobic conditions at 30 and 37°C for 48 hours. The microscopic features of the isolates were assessed and the production of exopolysaccharide in the culture medium was evaluated by disk and ruthenium red methods. The phenol-sulfuric and weight method were used to quantify exopolysaccharide production. Results showed pH of Kishk samples ranged from 3.60 to 4.08 and the average of total mesophilic count and LAB count of samples were 6.50 and 5.89 log CFU/g, respectively. Analysis of data exhibited 79 out of 143 lactic acid bacteria isolates were exopolysaccharide producer and 70% of them were cocci. The average of maximum and minimum production by weight method were 2.61 g/L and 0.08 g/L, respectively. The average of highest and the lowest amount of exopolysaccharide by phenol sulfuric method were measured 1.87 g/L and 0.06 g/L, respectively. This study indicates the potential of exopolysaccharide production by Iranian native species from dairy products

Biodiversity of exopolysaccharide-producing lactic acid bacteria from Iranian traditional Kishk and optimization of EPS yield by Enterococcus spp

The production of polysaccharides derived from lactic acid bacteria (LAB) can be a valuable alternative to current polysaccharides. In this study, eight samples of Kishk (traditional dairy product) were collected in sterile conditions and directly cultured on MRS agar medium. Following purification and examination of microscopic, macroscopic characteristics and doing biochemical tests, 143 isolates were selected, and the production of exopolysaccharide (EPS) was investigated by the ruthenium red and disc methods. The EPS production of 79 isolates was confirmed. Total carbohydrate of EPS was determined by the phenol sulfuric acid method. Finally, Enterococcus durans K48, Enterococcus faecium R114, and Enterococcus faecium T52 strains were selected as the best EPS producers. The optimization of EPS production was then performed using Central Composite Design (CCD) and Response Surface Methodology (RSM) approaches. Optimization plots showed the highest EPS yield for E. durans K48, E. faecium R114, and E. faecium T52 occurred at 38.4, 37.4 and 36.7 °C and pH of 5.9, 5.6 and 5.8, respectively, cultured in a Sucrose-MRS medium. Under optimal conditions, the maximum predicted and actual production of EPSs for the examined isolates were 3.18, 3.21, 2.99 and 3.02, 3.15, 3.15, g L, respectively. As determined by Gel Permeation Chromatography, the EPSs molecular weights were in the range of 2.93 × 10 to 3.52 × 10 Da. Analysis of the component monosaccharides by HPLC showed that all three tested EPSs were heteropolysaccharides. After appropriate evaluation, the EPSs produced by native species of LAB isolated from Iranian Kishk could be of interest for industrial applications or as functional food ingredients.This work was financially supported by a grant (No. 3/47333) from the Ferdowsi University of Mashhad (Research Affairs), Iran

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

Identification, typing and functional characterization of dominant lactic acid bacteria strains from Iranian traditional yoghurt

Interest in the microbiota of traditional fermented dairy products, such as yoghurt, continues due to the need for new lactic acid bacteria (LAB) strains that can complement or replace currently-in-use starters. In this work, five traditional yoghurts and one milk sample (for making yoghurt) from different areas of the Khorasan-e-Razavi region, Iran, were subjected to microbial characterization. Among the 102 isolates recovered, which included 29 from milk, the thermophilic LAB species Streptococcus thermophilus (34) and Lactobacillus delbrueckii (36) (with similar numbers of the subsp. bulgaricus and lactis) were the majority bacteria in all samples. Wide intra-species phenotypic and genetic diversity was encountered among the isolates. The safety and technological features of 29 strains belonging to the species S. thermophilus, L. delbrueckii and Lactobacillus helveticus, all candidates for use in yoghurt-making, were examined. In culture, two S. thermophilus strains produced the biogenic amine histamine from histidine (its precursor amino acid), and five L. delbrueckii strains showed atypical resistance to tetracycline (MIC ≥ 8 μg mL). The remaining strains coagulated milk, producing a coagulum of good appearance and pleasant acidic aroma. None of them produced appreciable amounts of exopolysaccharides in milk under the incubation conditions. After fermentation of milk with individual and mixed strains, major yoghurt aroma volatile compounds (i.e. acetaldehyde, diacetyl and acetic acid) were detected. Also as being used in the design of commercial starters, the thermophilic LAB strains characterized in this study might be used to help improve the safety of traditional yoghurts, while maintaining their original qualitative and sensorial characteristics.This research was partially funded by projects from INIA (Ref. RM2011-00005-00-00) and MINECO (Ref. AGL2014-57820-R), and. A.B. Flórez was supported by a research contract under JAE-Doc Program (CSIC) with funding from the European Social Funds. The authors would also like to thank the Iran National Science Foundation (INSF) for financial support (Project No. 91059662).Peer Reviewe