Efficacy of Reuterin and Bacteriocins Nisin and Pediocin in the Preservation of Raw Milk from Dairy Farms.

RESEARCH BACKGROUND: In the current scenario of milk production in developing and developed countries, several factors influence the shelf-life of raw milk and add significant numbers of microbial contaminants that drastically lower the initial microbial quality leading to milk spoilage by the time it reaches the processing units. EXPERIMENTAL APPROACH: The present study was undertaken to investigate the biopreservative efficacy of reuterin system along with different combinations of bacteriocins in controlling the initial microflora of raw milk at farm level. Lactobacillus reuteri strain LR47, having effective antimicrobial activity, was shortlisted from our previous study and further characterized for reuterin production and tested in raw milk system. RESULTS AND CONCLUSIONS: Preliminary testing of the cell-free supernatant from L. reuteri LR47 demonstrated significant growth inhibition of the majority of the tested bacterial indicators of milk spoilage. Further genetic analysis of the L. reuteri LR47 revealed the presence of two genes (pduC and dhaB) involved in the utilization of glycerol to produce reuterin via two different pathways. The strain LR47 was also found to possess comparatively higher capacity to convert glycerol into reuterin when checked through colorimetric assay. In the raw milk biopreservation experiment with reuterin alone or in combination with bacteriocins, the highest level of growth suppression in the total bacterial load and coliform counts was observed in the sample that was treated with a combination of reuterin, nisin and pediocin. The treatment combining these three natural biopreservatives at specific concentrations was able to maintain the initial microbial quality and extend the shelf-life of raw milk by 6 h at 37 °C based on the microbial counts and physicochemical properties, viz. pH and titratable acidity. In conclusion, the results confirm that the use of reuterin in combination with bacteriocins is a promising approach for temporary control of the raw milk microflora and extension of its shelf-life until further processing. NOVELTY AND SCIENTIFIC CONTRIBUTION: This study demonstrates for the first time the use of reuterin for the extension of shelf-life of raw milk as an alternative treatment method.

Assessing safety of Lactobacillus plantarum MTCC 5690 and Lactobacillus fermentum MTCC 5689 using in vitro approaches and an in vivo murine model.

Lactobacilli have a long history of safe use in human nutrition, however, inclusion of any new strain, despite its safe usage evidence, warrants proper analysis of its safety and toxicity under the purview of existing regulations. In the present investigation, Lactobacillus plantarum MTCC 5690 and Lactobacillus fermentum MTCC 5689 were evaluated for their safety and toxicity using both in vitro and in vivo approaches. The in vitro assays included mucin degradation, hemolytic activity, biogenic amine production and platelet aggregation assay. The safety was also assessed using acute, subacute and subchronic assays, bacterial translocation studies, intravenous and intravenous administration and genotoxicity assay in murine model. The outcome of this toxicological safety assessment indicated that both the test strains lacked any harmful metabolic activity or any genotoxic effects. Furthermore, the results of oral toxicity studies in mice revealed that short term administration of high cell mass concentration of 1012 cfu/animal as well as long term feeding of the probiotic strains did not alter any hematological, general health parameters or cause any organ specific disorder. Based upon these scientific assessments and supported by long history of safe use, both MTCC 5690 and MTCC 5689 may be considered safe for human consumption.

Purification and characterization of a broad spectrum bacteriocin produced by a selected Lactococcus lactis strain 63 isolated from Indian dairy products.

Lactococcus lactis subsp. lactis strain 63 was isolated from Indian dairy products, produced a bacteriocin with broad spectrum inhibition against several of food pathogens like Listeria monocytogenes and Bacillus cereus as well as Gram negative bacteria viz., E. coli, Yersinia, Citrobacter, Proteus, Enterobacter, Klebsiella and Serratia strains. Bacteriocin production was higher in GM-17 and MRS as compared to TYGE broth and enriched skim milk broth and reached the maximum level during the early stationary phase. The bacteriocin was purified by performing ammonium sulfate precipitation. The bacteriocin was able to survive 90 °C/10 min but not 100 °C/10 min. Complete inactivation of bacteriocin was observed after autoclaving. The bacteriocin maintained its activity over a wide range of pH (3-9). The antimicrobial compound produced by the isolate 63, was sensitive to papain, pepsin, trypsin and amylase but was resistant to detergents like SDS and urea. Tween 20, Tween-80 as well as Triton X-100 enhanced its activity. Since the treatment with proteolytic enzymes resulted in loss of activity, this shows that the proteinaceous nature of the antimicrobial substance. Tentative molecular weight of the bacteriocin was found to be between 3.5 and 5 kDa by Tricine SDS-PAGE. Finally, we confirmed the presence of gene for nisin, and the sequence thus obtained, was identical to the sequences previously described for nisin Z. Lactococcus lactis subsp. lactis 63 or its bacteriocin, which has a wide inhibitory spectrum, has the potential for use as a starter or protective culture in the manufacture of fermented products.

Postbiotic Lipoteichoic acid of probiotic Lactobacillus origin ameliorates inflammation in HT-29 cells and colitis mice.

Lipoteichoic acid (LTA) is a key surface component of probiotic lactobacilli that is involved in important cellular functions including cross talk with the host immune cells. In this study, the anti-inflammatory and ameliorative properties of LTA from probiotic lactobacilli strains were assessed in in vitro HT-29 cells and in vivo colitis mice. The LTA was extracted with n-butanol and its safety was confirmed based on its endotoxin content and cytotoxicity in HT-29 cells. In the Lipopolysaccharide stimulated HT-29 cells, the LTA from the test probiotics evoked a visible but non-significant increase in IL-10 and decrease in TNF-α levels. During the colitis mice study, probiotic LTA treated mice showed substantial improvement in external colitis symptoms, disease activity score and weight gain. The treated mice also showed improvements in key inflammatory markers such as the gut permeability, myeloperoxidase activity and histopathological damages in colon, although non-significant improvements were recorded for the inflammatory cytokines. Furthermore, structural studies by NMR and FTIR revealed increased level of D-alanine substitution in the LTA of LGG strain over MTCC5690. The present study demonstrates the ameliorative effect of LTA as a postbiotic component from probiotics which can be helpful in building effective strategies for combating gut inflammatory disorders.

Ameliorative Effect of Surface Proteins of Probiotic Lactobacilli in Colitis Mouse Models.

The increase in concern from viable cells of probiotics specifically in acute inflammatory conditions has led to the emergence of the concept of postbiotics as a safer alternative therapy in the field of health and wellness. The aim of the present study was to evaluate the efficacy of surface proteins from three probiotic strains in dextran sodium sulfate and trinitrobenzenesulphonic acid = induced colitis mouse models. The molecular weight of total surface proteins extracted from the three probiotic strains ranged from ∼25 to ∼250 kDa with the presence of negligible levels of endotoxins. Surface layer proteins (SLPs) (∼45 kDa) were found to be present only in the Lactobacillus acidophilus NCFM strain. In the in vivo study, significant differences were not observed in the weight loss and general appetite, however, the decrease in colon length was apparent in TNBS colitis control mice. Further, the administration of these surface proteins significantly reversed the histopathological damages induced by the colitogens and improved the overall histological score. The oral ingestion of these surface proteins also led to a decrease in myeloperoxidase activity and TNF-α expression while the IL-10 levels significantly increased for the strain NCFM followed by MTCC 5690 and MTCC 5689. Overall, the present study signifies the ameliorative role of probiotic surface proteins in colitis mice, thereby, offering a potential and safer alternative for the management of inflammatory bowel disorders.

Assessing the Safety and Efficacy of Lactobacillus plantarum MTCC 5690 and Lactobacillus fermentum MTCC 5689 in Colitis Mouse Model.

Probiotic lactobacilli have an unprecedented history of safe use, although some cases of infections have raised concerns about their safety, and hence, a rigorous screening of any new strain even of Lactobacillus is a must in order to study possible adverse interactions with the host, particularly under unhealthy conditions. The present study was, therefore, undertaken to investigate the safety as well as therapeutic efficacy of probiotic Lactobacillus plantarum MTCC 5690 and L. fermentum MTCC 5689 strains in dextran sodium sulfate (DSS)-induced colitis mouse model. Both MTCC 5690 and MTCC 5689 did not induce any detrimental effect on the colitic mice, as was reflected by normal colon and caecum length, blood biochemistry, hematology, and absence of inflammation. Although translocation of both the strains was observed in extraintestinal organs, probiotic-fed mice had significantly improved intestinal permeability and decreased myeloperoxidase (MPO) activity. Probiotic interventions also led to an improved health index and better growth of colitis mice compared to colitis animals with no probiotic intervention. These results point towards the safe use of L. plantarum MTCC 5690 and L. fermentum MTCC 5689 as biotherapeutics for amelioration of inflammatory conditions after establishing their efficacy in human clinical trials.