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Aims: To elucidate the antibiotic resistance and virulence genes of nisin-resistant Enterococcus faecalis isolated from raw buffalo milk and to study the effect of nisin-sensitive and -resistant E. faecalis on the innate immunity of rats., Methods and Results: Slanetz-Bartley agar plates containing nisin were used to isolate nisin-resistant E. faecalis. The virulence factors were ascertained using quantitative real-time polymerase chain reaction. Cell viability, phagocytosis, intracellular survival and enzyme assays were performed to investigate the interaction of E. faecalis with rat macrophages. Nisin-resistant E. faecalis was less prone to phagocytosis and survived longer inside the macrophages, due to reduced production of reactive oxygen species and nitric oxide. The viability and activation of macrophages was also reduced in the presence of resistant E. faecalis, as observed by enhanced lactate dehydrogenase production and reduced β-galactosidase., Conclusions: Nisin-resistant E. faecalis and its virulence factors were reported in raw buffalo milk. This study shows that nisin-resistant variants exhibited cross resistance to antibiotics and suppressed the innate immune responses of rats by directly affecting macrophage activity., Significance and Impact of the Study: This study elucidated the contamination of raw buffalo milk by nisin-resistant E. faecalis, which may pose food safety risk., (© 2019 The Society for Applied Microbiology.)

Prolonged passaging of primary fibroblast cells totally shapes the natural biological phenomena and leads to the appearance of features related to senescence. As a result, it is a good natural tool to delineate the molecular mechanism of cellular aging. The present investigation revealed the antiaging effect of milk-derived novel bioactive peptide (VLPVPQK). The peptide played an important role in downregulating apoptosis-related markers in late passages of cultured fibroblast cells. The peptide treatment to aged fibroblasts caused enhancement in cell migration, DNA integrity, and decrease in the lipid peroxidation, reactive oxygen species, nitric oxide production as well as pro-inflammatory cytokines, TNF-α and IL-6. Moreover, the peptide decreased the expression of apoptotic caspases, Bax, and senescence-associated β-galactosidase (SA-β-gal) proteins. The peptide pretreatment also enhanced the extracellular collagen protein and antiapoptotic, Bcl-xL. In addition, the peptide treatment reversed the senescence-related activity in fibroblasts by stimulating Nrf2 mediated antioxidative defense system and inhibiting the action of NFkB/p38MAPK signaling, similar to the commercially available inhibitor (SB203580) of p38MAPK. Thus, the peptide exhibits the antiaging effect in dermal fibroblast cells., (© 2018 Wiley Periodicals, Inc.)

There is a growing and alarming prevalence that increased serum cholesterol is closely related to increased cardiovascular disease risk. Probiotic consumption could be a safe and natural strategy to combat. Therefore, we sought to examine the cholesterol-lowering potential of co-supplementation of probiotic bacteria Lactobacillus fermentum MTCC: 5898-fermented buffalo milk (2.5% fat) in rats fed cholesterol-enriched diet. Male Wistar rats were divided into three groups on the basis of feed, viz. group 1, fed standard diet (SD); group 2, fed cholesterol-enriched diet (CED); and group 3, fed cholesterol-enriched diet along with L. fermentum MTCC: 5898-fermented milk (CED+LF) for 90 days. At the endpoint, significantly higher levels of serum total cholesterol, low-density lipoprotein cholesterol, triacylglycerols, very low density lipoprotein cholesterol, atherogenic index, coronary artery risk index, hepatic lipids, lipid peroxidation, and mRNA expression of inflammatory cytokines (TNF-α and IL-6) in the liver while significantly lower levels of serum high-density lipoprotein cholesterol and anti-oxidative enzyme activities, catalase, superoxide dismutase, and glutathione peroxidase in the liver and kidney were observed in the CED group compared to the SD group. Compared to the CED group, these adverse physiological alterations were found significantly improved in the CED+LF group. Hence, this study proposes that L. fermentum MTCC: 5898 is a potential probiotic bacteria that can be consumed to tackle hypercholesterolemia. Graphical Abstract ᅟ.

Interest in probiotics has grown significantly in the last decades due to their reported nutritional and health promoting effects. The aim of this study is to investigate the therapeutic potential of probiotic fermented milk (PFM) prepared using three different probiotic strains i.e. Lactobacillus rhamnosus MTCC: 5957, Lactobacillus rhamnosus MTCC: 5897 and Lactobacillus fermentum MTCC: 5898; independently or in combination, for treating streptozotocin induced type-1 diabetes in male Wistar rats. Diabetic rats were fed with PFM preparations for 6 weeks and then analyzed for the various biochemical parameters associated. The results indicated that feeding of PFM significantly improved glucose metabolism (fasting blood glucose, glycated hemoglobin, serum insulin), serum inflammation status (tumor necrosis factor-α, and serum interleukin-6), oxidative stress (thiobarbituric acid reactive substance, catalase, superoxide dismutase and glutathione peroxidase activities in liver and kidney), serum lipid profile (total cholesterol, low density lipoprotein-cholesterol, very low density lipoprotein-cholesterol, triglycerides) in diabetic rats. In addition, feeding of PFM has significantly reduced mRNA expression of pepck and g6pase genes that code the key enzymes of gluconeogenesis pathway. The results of this study showed that daily consumption of PFM can be effective in combating of type -1 diabetes and its complications., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Early infancy, the period when offspring rely not only on their own immunity to combat food-borne antigens but also acquire immunity through maternal sources (via transplacental routes and breast milk), is critical for immune system development Hence the present study was designed to evaluate the effect on offspring of administration of probiotic-containing fermented milk (PFM) either to mothers during the suckling period or to their offspring after weaning either separately or sequentially. PFM-fed mice showed enhanced leukocyte functionality in offspring as evidenced by significantly (P < 0.05) increased release of lysosomal enzymes (β-galactosidase, β-glucuronidase) in peritoneal fluid and nitric oxide production in culture supernatants of activated macrophages. Further, remarkably reduced levels (P < 0.01) of inflammatory markers (TNF-α, monocyte chemotactic protein-1) and allergic antibodies (total and milk specific IgE) were observed in offspring where PFM was fed either to them or to their mothers. However, considerably increased levels (P < 0.05) of SIgA were found in the guts of control and experimental groups animals irrespective of their exposure to PFM. Restoration of Th1/Th2 homeostasis further confirmed the useful effects of PFM supplementation by shifting the cytokine profile (IL-4, IFN-γ and IL-10) with increased IFN-γ/IL-4 and reduced IgE/Ig2Ga ratios. Hence, it is logical to conclude that administration of Lactobacillus rhamnosus-containing (MTCC:5897) fermented milk to mothers during the suckling period and to their offspring after weaning has beneficial effects on the development of newborns immune systems; this effect appears to be more pronounced when mothers are fed with it., (© 2015 The Societies and John Wiley & Sons Australia, Ltd.)

We investigated the mechanism by which an Indian indigenous probiotic culture, Lactobacillus rhamnosus S1K3, could overcome the pathogenic strain Salmonella enterica with an emphasis on the response at the intestinal mucosal level after long-term (30days) consumption. S1K3 was able to produce antimicrobial compounds against the pathogens. The probiotic adhered strongly to intestinal epithelium and maintained its integrity in presence of Salmonella through stimulation of tight junction and antimicrobial peptide genes in vitro. Mice prefed for 30days with S1K3-fermented milk exhibited low incidence of pathogenic Salmonella at mucosal and systemic levels. The probiotic induced TLRs transcripts at the Peyer's patches, followed by an increase in the Secretory-IgA in intestinal fluid, the IgA-secreting cells in lamina propria of small intestine and the IgA level in serum. Moreover, S1K3 maintained the protein level of IL-12, increased the IL-4 and reduced the TGF-β level in intestinal fluid/serum at the later stage of infection. All these actions concurred to lower the count of Salmonella in feces, its invasion in spleen, liver and intestine tissues and improved the health status of probiotic-fed group. In view of this performance, S1K3 appears to be a suitable candidate for the development of nutraceutical food., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Atherosclerosis is associated with coronary artery disease and occurs in developing as well as developed countries. In the present investigation, hypolipidaemic and anti-oxidative properties of encapsulated herb (Terminalia arjuna, 1.8%) added vanilla chocolate dairy drink was evaluated in high cholesterol fed Wistar rats for 60 days., Results: At the end of the experimental period, a significant decrease in the body weight gain by rats receiving the encapsulated herb extract was noted as compared to high cholesterol fed rats. Administration of microencapsulated herb showed a statistically significant decrease in organ weights (epididymal fat and liver). Moreover, a significant decrease in serum lipids such as triglycerides, total cholesterol, low-density lipoprotein cholesterol, very-low-density lipoprotein cholesterol and atherogenic index was observed with encapsulated Terminalia arjuna extract in high cholesterol fed group. Increases in reduced glutathione and decreases in TBARS levels were also reported in both liver and red blood cell lysates with encapsulated herb supplementation., Conclusion: The results demonstrated that the bioactive components (phytosterols, flavanoids, saponins and tannins etc.) which are present in the encapsulated T. arjuna not only withstand the processing conditions but also are effectively released in the intestine and show their effects, such as hypolipidaemic and antioxidant activities, for better treating cardiovascular disease., (© 2015 Society of Chemical Industry.)

The transepithelial transport of an antioxidative and ACE inhibitory peptide, VLPVPQK (named peptide C) derived from casein hydrolysates was investigated along with extensively studied opioid peptide β-casomorphin using a human intestinal cell (Caco-2) monolayer. The susceptibility to the brush-border peptidases and route of transepithelial transport were observed to be the primary factors influencing the transport of these peptides. The apical to basal transport mechanism was studied using bradykinin as control as it shows resistance to cellular peptidases and its route of transepithelial transport had been established. VLPVPQK and BCM 5 were hydrolyzed by cellular peptidases while bradykinin was found intact. The transport of VLPVPQK (1.0%) was found to be relatively much higher than BCM 5 (0.03%) and bradykinin (0.1%). Interestingly the effect of some inhibitors on the transport of VLPVPQK suggested involvement of PepT1 like transporters/SOPT2 while BCM 5, its hydrolytic product and bradykinin were suggested to be transported mainly via the intracellular transcytosis pathway., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The neonatal period is often polarised to T helper (Th2) response at the time of birth, predisposing offspring to allergic disorders. Passive immunity through the mother's milk is critical for immune system development of newborns. Probiotics have been proposed to harmonise Th1/Th2 imbalance in allergic conditions in adults. In the present study, the anti-allergic effects of feeding probiotic Lactobacillus rhamnosus-fermented milk (PFM) either to dams during the suckling period or to their offspring after weaning individually or else in successive periods against ovalbumin (OVA)-induced allergy in newborns was analysed. After allergen sensitisation, physical symptoms of allergy, gut immune response, humoral immune response and cell-mediated response through interleukins were detected. Consumption of PFM by mothers and offspring showed a reduction (P<0·01) in physical allergic symptoms in newborns with an increase (P<0·01) in the numbers of goblet and IgA+ cells in the small intestine. Similarly, considerable (P<0·001) decreases in OVA-specific antibodies (IgE, IgG, IgG1) and ratios of IgE/IgG2a and IgG1/IgG2a in the sera of newborn mice were recorded. A decrease in IL-4 and an increase in interferon-γ levels further confirmed the shift from Th2 to Th1 pathway in PFM-fed mice. It is logical to conclude that the timing of PFM intervention in alleviating allergic symptoms is critical, which was found to be most effective when mothers were fed during the suckling period.

Crossbred Karan Fries (KF) cows, among the best yielders of milk in India are carriers of A1 and A2 alleles. These genetic variants have been established as the source of β-casomorphins (BCMs) bioactive peptides that are implicated with various physiological and health issues. Therefore, the present study was aimed to investigate the release of BCM-7/5 from β-casein variants of KF by simulated gastrointestinal digestion (SGID) performed with proteolytic enzymes, in vitro. β-Casein variants (A1A1, A1A2 and A2A2) were isolated from milk samples of genotyped Karan Fries animals and subjected to hydrolysis by SGID using proteolytic enzymes (pepsin, trypsin, chymotrypsin and pancreatin), in vitro. Detection of BCMs were carried out in two peptide fractions (A and B) of RP-HPLC collected at retention time (RT) 24 and 28min respectively corresponding to standard BCM-5 and BCM-7 by MS-MS and competitive ELISA. One of the RP-HPLC fractions (B) showed the presence of 14 amino acid peptide (VYPFPGPIHNSLPQ) having encrypted internal BCMs sequence while no such peptide or precursor was observed in fraction A by MS-MS analysis. Further hydrolysis of fraction B of A1A1 and A1A2 variants of β-casein with elastase and leucine aminopeptidase revealed the release of BCM-7 by competitive ELISA. The yield of BCM-7 (0.20±0.02mg/g β-casein) from A1A1 variant was observed to be almost 3.2 times more than A1A2 variant of β-casein. However, release of BCM-7/5 could not be detected from A2A2 variant of β-casein. The biological activity of released peptides on rat ileum by isolated organ bath from A1A1 (IC50=0.534-0.595μM) and A1A2 (IC50=0.410-0.420μM) hydrolysates further confirmed the presence of opioid peptide BCM-7., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Although probiotics are known to enhance the host immune response, their roles in modulating immunosenescence, resisting infection, and improving redox homeostasis during aging remain unclear. Therefore, the present study was devised in aging mice to assess the antiimmunosenescence potential from the consumption of milk that is fermented with probiotic Lactobacillus fermentum MTCC 5898 (LF). We hypothesized that probiotic supplementation would boost immunity, improve antioxidant capacity, and resist severity of pathogenic infection in aging mice. To test this hypothesis, during a trial period of 2 months, 16-month-old male Swiss mice were kept on 3 experimental diets: basal diet (BD), BD supplemented with skim milk, and BD supplemented with probiotic LF-fermented milk. A concurrent analysis of several immunosenescence markers that include neutrophil functions, interleukins profile, inflammation and antibody responses in the intestine as well as analysis of antioxidant enzymes in the liver and red blood cells was performed. Neutrophil respiratory burst enzymes and phagocytosis increased significantly in probiotic LF-fed groups, whereas no exacerbation in plasma levels of monocyte chemotactic protein 1 and tumor necrosis factor α was observed. Splenocytes registered increased interferon-γ but decreased interleukin 4 and interleukin 10 production, whereas humoral antibodies registered decreases in immunoglobulin G1 (IgG1)/IgG2a ratio and IgE levels in the probiotic-fed groups. Antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) in LF-fed groups showed increased activities, which were more pronounced in the liver than in red blood cell. An Escherichia coli-based infection model in aging mice was also designed to validate the protective attributes of LF. Administration of probiotic LF significantly reduced E coli population in organs (intestine, liver, spleen, and peritoneal fluid), as compared with control groups, by enhancing E coli-specific antibodies and inflammatory proteins. Based on these results, it appears that LF supplementation alleviated immunosenescence, enhanced antioxidant enzyme activities, and resisted E coli infection in aging mice; thereby, signifying its potential in augmenting healthy aging., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Purpose: Recently, apprehension has been raised regarding "A1/A2 hypothesis" suggesting relationship between consumption of A1 "like" variants of cow β-casein and various physiological disorders. The information available is based on either the human epidemiological data of milk consumption or in vitro trials on cell lines with β-casomorphin peptides. The direct scientific evidence establishing the link between consumption of A1/A2 "like" milk and health is scanty. Thus, under present investigation, in vivo trials in mice were undertaken to study the effect of feeding three genetic variants (A1A1, A1A2 and A2A2) of cow β-casein milk on gastrointestinal immune system as it is the first and foremost site of immunological interactions., Methods: Animals were divided into four groups for feeding with basal diet (control) and β-casein isolated from milk of genotyped (A1A1, A1A2 and A2A2) dairy animals, respectively. Gut immune response was analyzed by spectrophotometric assessment of MPO activity, quantitative sandwich ELISA of inflammatory cytokines (MCP-1 and IL-4), antibodies (total IgE, IgG, sIgA, IgG1 and IgG2a) and qRT-PCR of mRNA expression for toll-like receptors (TLR-2 and TLR-4). Histological enumeration of goblet cells, total leukocytes and IgA(+) cells was also carried out., Results: It was observed that consumption of A1 "like" variants (A1A1 and A1A2) significantly increased (p < 0.01) the levels of MPO, MCP-1, IL-4, total IgE, IgG, IgG1, IgG2a and leukocyte infiltration in intestine. TLR-2 and TLR-4 mRNA expression was also up-regulated (p < 0.01) on administration of A1 "like" variants. However, no changes in sIgA, IgA(+) and goblet cell numbers were recorded on consumption of any of the β-casein variants., Conclusion: It is reasonable to conclude that consumption of A1 "like" variants of β-casein induced inflammatory response in gut by activating Th2 pathway as compared to A2A2 variants. The present study thus supports the purported deleterious impacts of consumption of A1 "like" variants of β-casein and suggests possible aggravation of inflammatory response for etiology of various health disorders.

The objective of the present investigation was to determine bioavailability of calcium and vitamin D₂ from milk fortified with either calcium or vitamin D₂ alone or when both were used for preparation of multiple micronutrient fortified milk and also to study its interaction with iron and zinc bioavailability. 32 weanling male rats (aged 21-28 days) were assigned into four groups and were fed milk and milk fortified with calcium, vitamin D₂ and calcium+vitamin D₂. Vitamin D₂ increased calcium bioavailability. In multiple micronutrient fortified milk, the bioavailability of both calcium+vitamin D₂ increased in comparison to single fortification. Calcium fortification decreased, whereas vitamin D₂ increased the absorption of iron and zinc. However, calcium and vitamin D₂ when fortified in combination, the iron and zinc bioavailability was similar to control group. There was positive association between bioavailability of calcium and vitamin D₂., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The present investigation aimed at identifying the abilities of three different species of probiotic lactobacilli to modulate cellular immune responses in mouse neutrophils and macrophages in vivo over a study period of 60 days. Neutrophil respiratory burst enzymes (cytochrome c reductase and MPO) showed remarkable increased activity (P ≤ 0.01) after consumption of milks fermented by different species of probiotics over 30 and 60 days of feeding trials. Enzyme activities (β-galactosidase and β-glucuronidase) and nitric oxide production also increased considerably (P ≤ 0.01) in macrophages, both in peritoneal fluid and in enriched cell cultures. The effects of enhanced enzyme activities were corroborated by simultaneous increases in the phagocytic activities of neutrophils and macrophages. The increases in cellular functions were invariably maximal during the first 30 days of study and were maintained, but did not increase, over the next 30 days. Further, Lactobacillus helveticus-fed groups were most effective at modulating neutrophil functions whereas Lactobacillus paracasei-fed groups were more potent at enhancing macrophage functions. Together, our results indicate that probiotics have strain specific effects on stimulating cellular functions while not causing excessive stimulation of the immune system over longer feeding periods, thereby resulting in maximum and stable health benefits., (© 2013 The Societies and Wiley Publishing Asia Pty Ltd.)

Background: Heat treatment is the most common method for reducing pathogen load, but it remains controversial in reducing the incidence of hyperimmune reactions. The aim of this study was to compare the allergenicity of caseins (CSN) and whey proteins (WP) of thermally processed cow and buffalo milk in a mouse model. Swiss albino mice were sensitised by intraperitoneal injections (administered in three doses at weekly intervals) of CSN or WP from cow or buffalo milk for the evaluation of humoral response and splenocyte stimulation index., Results: After 3 weeks of intraperitoneal stimulation of mice with milk proteins, the sterilised milk protein group displayed significantly lowered (P ≤ 0.05) serum IgG and IgE levels, while considerably increased cow milk protein-specific responses (IgE) were shown by proteins of pasteurised milk compared with those of raw milk. The stimulation index of splenocytes induced by CSN or WP of boiled and sterilised milk was also lower (P ≤ 0.05) than that of raw milk of both cow and buffalo., Conclusion: The experiment showed that boiling and sterilisation of cow and buffalo milk clearly affect the allergenicity by decreasing the humoral and cell-mediated responses in mice. All results indicated that CSN and WP of sterilised milk are less allergenic than those of raw milk in mice., (© 2013 Society of Chemical Industry.)

Milk samples were collected from 34 lactating Murrah buffaloes on days 0, 5, 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270 and day 300 after calving. Milk somatic cell counts (SCC) were highest in multiparous buffaloes. Milk SCC were significantly lower in buffaloes of third and fourth parity during early lactation and than increased significantly (p < 0.01) by the end of lactation. Milk neutrophils were significantly lower in all the buffaloes during early lactation, but increased significantly (p < 0.01) afterwards. Milk lymphocytes were significantly higher during early lactation, but decreased significantly (p < 0.01) by the end of lactation. Phagocytic activity (PA) was highest in day 1 colostrum and then decreased significantly (p < 0.01) by the fourth milking in buffaloes of second, third and fourth parity. Phagocytic index (PI) was also highest in colostrums of primiparous buffaloes. Irrespective of parity, maximum PA and PI was observed during mid lactation. In terms of in vitro phagocytic activity, early lactation is the most critical period followed by late and mid lactation., (© 2010 Blackwell Verlag GmbH.)