Comparative evaluation of milk proteins and oil-seed-cake-derived proteins extracted by chemical and biological methods for obesity management.

BACKGROUND: In light of the exponential rise in global population, there is a critical requirement to reduce food waste on a global scale. According to studies, agricultural wastes such as oil-seed cakes offer great nutritional value. Acid precipitation (A) and alkaline extraction methods (traditional methods) were used to extract protein from oil-seed cakes; however, both procedures are linked to decreased protein quality and quantity, which prompted the development of a novel strategy known as the biological/microbial/probiotic (B) method. Therefore, the present study aimed to highlight the optimal way of protein extraction from oil-seed cakes and the effect of extraction methods on protein efficacy against obesity. The outcomes were also compared with milk proteins. RESULTS: In vitro study provided evidence that proteins from both sources (plant and milk) suppressed adipogenesis and stimulated adipolysis in 3T3L-1 cells. For the in vivo study, mice were fed with different protein extracts: soya protein preparation (SPP), ground protein preparation (GPP), whey protein (WP) and casein protein (CP) containing 40% of their calories as fat. Body weight decreased significantly in all the rats except CP-fed rats. Body mass index, atherogenic index, plasma triglyceride and very-low-density lipoprotein cholesterol level decreased significantly in all the groups in comparison to the model group (high-fat-diet group), but the decrease was more pronounced in plant proteins than milk proteins. In hepatocytes, the expression of fasting-induced adipose factor, carnitine palmitoyltransferase I and peroxisome proliferator-activated receptor α genes was increased significantly in SPP-fed groups. Adiponectin gene expression was upregulated significantly in visceral fat tissue in groups fed SPP-B, GPP-A and CP, whereas leptin gene was downregulated significantly in all groups except SPP-A. CONCLUSION: This study demonstrates that SPP-B showed the most effective anti-obesity property, followed by WP. Additionally, we found that the biological precipitation approach produced better outcomes for plant proteins isolated from oil-seed cakes than the acid precipitation method. © 2023 Society of Chemical Industry.

Influence of exopolysaccharide EPSKar1-iron complexation on iron bioavailability and alleviating iron deficiency anaemia in Wistar rats.

The prevalence of iron deficiency anaemia is a significant issue worldwide, affecting individuals of all ages and often associated with inadequate iron bioavailability. Despite the use of ferrous salt supplements to address anaemia, their limited bioaccessibility and bioavailability in human GIT and adverse impact on food properties remain significant challenges. Hence, this study aims to explore the iron chelation mechanism of an exopolysaccharide EPSKar1 to enhance iron bioaccessibility, bioavailability, and anti-anaemic effects using cell culture and an anaemic rat model. EPSKar1 was extracted from Lacticaseibacillus rhamnosus Kar1 and complexed with FeSO4 to form "EPSKar1-iron". This novel complex, besides being bio-accessible after in vitro gastric digestion, demonstrated 61.27 ± 1.96% iron bioavailability to the Caco-2 cells. In line with these in vitro findings, intragastric administration of the EPSKar1-iron complex to anaemic Wistar rats at 25 and 50 mg per kg body weight significantly restored blood haemoglobin levels and re-established the morphological features of red blood cells. Furthermore, the apparent digestibility co-efficient and iron uptake improved significantly without adversely affecting the serum biochemical parameters in these anaemic rats. The levels of iron-transport proteins including serum transferrin and ferritin in tissue and plasma have increased remarkably upon oral administration of EPSKar1-iron at a higher dose of 50 mg per kg body weight. Oral supplementation of EPSKar1-iron did not foster adverse histological changes in the liver, kidneys, and spleen. In fact, the treatment with the EPSKar1-iron complex had a restitution effect on the tissue architecture, thereby ameliorating the tissue lesions. These findings collectively indicate that the EPSKar1-iron complex shows nutraceutical potential in enhancing the bioavailability of iron and could be a promising approach to tackle iron deficiency anaemia.

Strain-specific effects of probiotic Lactobacilli on mRNA expression of epigenetic modifiers in intestinal epithelial cells.

The epigenome of an organism is as important as the genome for the normal development and functioning of an individual. The human epigenome can be affected by various environmental factors including nutrients, microbiota and probiotics through epigenetic modifiers and mediates various health-promoting effects. The present study was aimed to explore the temporal changes in DNA and histone modifiers (DNMT1, TET2, p300, HDAC1, KMT2A, KDM5B, EzH2 and JMJD3) in intestinal epithelial cells (Caco-2) by probiotic lactobacilli (Limosilactobacillus fermentum MTCC 5898 and Lacticaseibacillus rhamnosus MTCC 5897) in comparison to opportunistic commensal pathogen Escherichia coli (ATCC 14849). Cells were treated separately with probiotic strains and E. coli for different durations and temporal changes in gene expression among DNA and histone modifiers were measured. Time-dependent studies showed that L. fermentum enhanced the transcription of epigenetic modifiers at 12 h of treatment (P < 0.05) contrary to E. coli which reduced the expression of these genes during the same duration of treatment. On the other hand, probiotic L. rhamnosus was not able to induce any significant changes in gene expression of these modifiers. Furthermore, during the exclusion of E. coli by L. fermentum, the probiotic was found to resist the changes made by E. coli in the transcription of some of the epigenetic modifiers. Thus, it is concluded that the probiotics modulated the mRNA expression of DNA and histone modifiers contrarily to E. coli in a strain-specific manner.

Health-promoting role of dietary bioactive compounds through epigenetic modulations: a novel prophylactic and therapeutic approach.

The epigenome is an overall epigenetic state of an organism, which is as important as that of the genome for normal development and functioning of an individual. Epigenetics involves heritable but reversible changes in gene expression through alterations in DNA methylation, histone modifications and regulation of non-coding RNAs in cells, without any change in the DNA sequence. Epigenetic changes are owned by various environmental factors including pollution, microbiota and diet, which have profound effects on epigenetic modifiers. The bioactive compounds present in the diet mainly include curcumin, resveratrol, catechins, quercetin, genistein, sulforaphane, epigallocatechin-3-gallate, alkaloids, vitamins, and peptides. Bioactive compounds released during fermentation by the action of microbes also have a significant effect on the host epigenome. Besides, recent studies have explored the new insights in vitamin's functions through epigenetic regulation. These bioactive compounds exert synergistic, preventive and therapeutic effects when combined as well as when used with chemotherapeutic agents. Therefore, these compounds have potential of therapeutic agents that could be used as "Epidrug" to treat many inflammatory diseases and various cancers where chemotherapy results have many side effects. In this review, the effect of diet derived bioactive compounds through epigenetic modulations on in vitro and in vivo models is discussed.

Protective effects of potential probiotic Lactobacillus rhamnosus (MTCC-5897) fermented whey on reinforcement of intestinal epithelial barrier function in a colitis-induced murine model.

Fermented foods provide essential nutritional components and bioactive molecules that have beneficial effects on several gastrointestinal disorders. In the present investigation, the potential protective effects of whey fermented with probiotic Lactobacillus rhamnosus MTCC-5897 on gastrointestinal health in a murine ulcerative colitis model induced by dextran sulfate sodium (DSS) were evaluated. Pre-consumption of whey fermented with probiotic L. rhamnosus (PFW) before colitis induction significantly reduced (p < 0.01) the disease activity index and improved (p < 0.05) the hematological parameters and histological scores. The considerably diminished levels (p < 0.01) of pro-inflammatory markers (IL-4, TNF-α, CRP and MPO activity) and the enhanced (p < 0.05) levels of the anti-inflammatory cytokine TGF-ß with IgA in the intestine upon feeding PFW appeared to prevent inflammation on colitis induction. Transcriptional modulations in pathogen recognition receptors (TLR-2/4) and tight junctional genes (ZO-1, occludin, claudin-1) along with localized distribution of junctional (claudin-1, occludin and ZO-1) and cytoskeleton (actin) proteins improved immune homeostasis and intestinal barrier integrity. Besides, significantly reduced (p < 0.05) levels of the FITC-dextran marker in serum upon consumption of PFW directly confirmed the healthy status of the host gut.

miR300 intervenes Smad3/ß-catenin/RunX2 crosstalk for therapy with an alternate function as indicative biomarker in osteoporosis.

The study reports a theranostic nature of rno-miR-300 (miR300) in the osteoblast functioning, by influencing the signaling pathway(s), associated with osteoblast differentiation. Excessive expression of miR300 suppresses osteoblast functions. Smad3 served as a validated target for miR300, on homology-based computational analysis and experimental testimony, which activates ß-catenin, and subsequently potentiates Runx2. The impact of miR300 on the Smad3/ß-catenin/Runx2 signaling interactions in the induction of osteoblast differentiation was scrutinized by immunoblotting and in vivo miRNA antagonism. Overexpression of miR300 in the rat calvarial osteoblasts decreases the protein levels of Smad3, ß-catenin and Runx2. Besides, in vivo silencing of miR300 in the neonatal pups and adult rats by AntimiR300 abolishes the suppressing action of miR300 on the osteoblast differentiation and expressions of Smad3/ß-catenin/Runx2 axis. MicroCT studies showed improved trabecular microarchitecture in the AntimiR300 transfected ovariectomised rat model compared to sham and negative control. Furthermore, expression levels of miR300 were evaluated in serum samples from an independent set of 30 osteoporotic patients followed by a Receiver Operating Characteristic Curve (ROC) based analysis for the diagnostic efficiency of miR300. Interestingly, the results exhibited high levels of miR300 (p < 0.0001) in the serum samples from osteoporotic patients relative to non-osteoporotic subjects (AUC = 0.9689). Thus, miR300 negatively regulates the differentiation of osteoblasts by targeting crosstalk among Smad3, ß-catenin and Runx2, unveiling an enormous ability to serve as a therapeutic target for bone-related disorder management strategies. Besides, miR300 may potentially function for the diagnosis of osteoporosis as a non-invasive biomarker.

Repertoire of Structure-Activity-Based Novel Modified Peptides Elicits Enhanced Osteogenic Potential.

Biologically active peptides in milk proteins can be used as effective dietary supplements for management of bone-associated issues including osteoporosis. A bioactive peptide derived from milk, viz. VLPVPQK/PepC, has been validated previously from our lab for its osteoanabolic action. In this study, we report 14 novel variants of PepC, designed in silico, based on the structure-activity relationship, aiming to enhance its osteogenic effect that holds tremendous therapeutic utility for bone-related injuries. PepC was computationally modified at seven positions of its original sequence, resulting in 14 modified synthetic peptides for functional predictions and in vitro assessment by comparative analysis of modified peptides by PepC for improved ability in osteogenic functional assays (proliferation potential, antioxidant ability, gene and protein expression, cytotoxic effect, bone mineralization) using calvarial osteoblasts. For most peptides with the highest Peptide7 response relative to PepC (p < 0.05), enhanced osteoanabolic response was observed. Further observations on Peptide7 have therefore been investigated in depth (qPCR, immunoblotting, LCMS/MS, and PCA analysis). Peptide7 displayed a rise in the expression of osteogenes (Osterix, Opg, Bmp2, and Runx2, p < 0.05) and protein (Runx2 and Bmp2, p < 0.05). Besides, LCMS/MS findings suggest Peptide7 escapes intestinal peptidases degradation. Experimental evidence supports an improved osteological reaction to newly modified peptides and hence exploitation in the preparation of functional foods or supplements.

Bioavailability assessment of zinc enriched lactobacillus biomass in a human colon carcinoma cell line (Caco-2).

The present study utilizes lactobacilli strains having the potential to accumulate a significant amount of Zinc (Zn) in their biomass and ability to deliver the same mineral in a highly bioavailable form. A human origin Lactobacillus fermentum SR4 and Lactobacillus rhamnosus GG (LGG) were studied for their ability to accumulate Zn by growing them in the medium containing Zn salt. Further, Zn enriched cell lysates were prepared by Ultrasonication, as an organic Zn source. Various functional groups involved in bacterial Zn binding were identified by FT-IR spectroscopy and elemental Zn in bio-chelated cell lysate complex was confirmed by SEM and Energy Dispersive X-ray Spectrometry (EDX). Experimental data demonstrated a significantly higher (P < 0.05) bioavailability of Zn chelated by SR4 followed by LGG i.e., 57% and 48%, as compared to the commercially available inorganic (ZnSo4) and even organic (Zinc gluconate) forms tested which has 15.6% and 21.7% respectively.

Escherichia coli K12: An evolving opportunistic commensal gut microbe distorts barrier integrity in human intestinal cells.

Commensal enteric microbes under specific conditions viz. immunocompromised system, altered microbiota or uncompetitive niche induce their otherwise dormant pathogenic phenotype to distort host cellular functioning. Here we investigate how under in vitro environment established by using Caco-2 cells, commensal gut microbe E. coli K12 (ATCC 14849) disrupt intestinal epithelial barrier function. Caco-2 cells exposed to E. coli showed the time dependent significant (P < 0.01) decrease in transepithelial electrical resistance (TEER) and concomitantly increased phenol red flux across cell monolayer in contrast to non infected control cells. E. coli infected intestinal cells were observed with suppressed (p < 0.05) mRNA levels of ZO-1, Claudin-1, Occludin and Cingulin-1 in contrast to significantly (p < 0.05) higher PIgR and hbd-2 mRNA fold changes. Immunofluorescent and electron micrographs revealed the disrupted distribution and localisation of specific tight junction proteins (Zo-1 and Claudin-1) and actin filament in E. coli infected Caco-2 cells that ultimately resulted in deformed cellular morphology. Taken together, E. coli K12 under compromised in vitro milieu disrupted the intestinal barrier functions by decreasing the expression of important tight junction genes along with the altered distribution of associated proteins that increased the intestinal permeability as reflected by phenol red flux and TEER values.

Adherence capability and safety assessment of an indigenous probiotic strain Lactobacillus rhamnosus MTCC-5897.

With the growing interest in probiotic microorganisms based on their well established immense health benefits, the present investigation was aimed to assess the adhesion potential and safety of probiotic Lactobacillus rhamnosus MTCC- 5897 (LR) before it can be put into a probiotic formulations. L. rhamnosus showed an adhesion index of 166.7 ±â€¯11, which was further confirmed by scanning electron microscopy and relative expression of mucus binding protein (Mub) and mucus adhesion promoting protein (Map-A) genes. In vitro safety assessment by tetrazolium dye reduction, neutral red and lactate dehydrogenase (LDH) release assays revealed unchanged metabolic activity of Caco-2 cells even when incubated with L. rhamnosus ranged between 106-1010 cfu/mL for 24 h. Similarly, a moderate increase in bile salt hydrolase (bsh) expression (6.84 ±â€¯0.73 and 3.42 ±â€¯0.39 folds in 1% and 3% bile medium respectively) further proved its safety towards normal lipid digestion and absorption. Moreover, L. rhamnosus feeding to mice (107, 109, 1011 and 1013 cfu/animal/d) repetitively for 28 days revealed no adverse effects on parameters of general animal health status including body weight, organ indices, plasma glucose, liver malondialdehyde (MDA), serum aspartate amino transaminase (AST), cholesterol, triglycerides, high-density lipoprotein (HDL). Similarly, significant (p ≤ 0.05) reduced activities of serum alanine amino transaminase (ALT) and LDH on continuous probiotic feeding were also indicative of normal liver/kidney functions as they were in normal range for mice. Further, insignificant changes in macrophage chemoattractant protein (MCP-1) in intestinal fluid irrespective of bacterial dose fed along with significant reduction (p ≤ 0.05) of tumor necrosis factor-α (TNF-α) at much higher dose (1013 cfu/animal/d) also confirmed safe response of probotic L.rhamnosus against inflammation. To conclude, the results obtained under in vitro and in vivo studies has established the Lactobacillus rhamnosus as safe and non-toxic to weaning mice as well as human epithelial cells and thus may be used as a safe food additive.

Nrf2 dependent antiaging effect of milk-derived bioactive peptide in old fibroblasts.

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 ß-galactosidase (SA-ß-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.

Bio-accessible milk casein derived tripeptide (LLY) mediates overlapping anti- inflammatory and anti-oxidative effects under cellular (Caco-2) and in vivo milieu.

Inflammation and oxidative stress are closely linked patho-physiological processes which occur concurrently in many diseased conditions. Recently, interdependence between these two processes explains the antioxidant paradox associated with failure to select appropriate agents required for prevention of diseases known to be induced by oxidative stress. Present study established the overlapping anti-inflammatory and anti-oxidative potential along with bio-accessibility of milk casein derived tripeptide (LLY). Tripeptide exhibited anti-inflammatory response under ex vivo conditions by suppressing (P<.01) mice splenocytes proliferation and modulating their cytokines (IFN-γ, IL-10 and TGF-ß) with improved phagocytosis of peritoneal macrophages. Conversely, tripeptide displayed extraordinary radical scavenging ability and cellular anti-oxidative potential using chemical assays and H2O2 induced oxidative stress model on Caco-2 cells. Under cellular assessment, on one hand tripeptide inhibited (P<.01) intracellular ROS generation and reduced MDA and protein carbonyls but on the other also increased (P<.01) the activity of anti-oxidative enzyme, catalase without much effect on SOD and GPx. This anti-oxidative potential was further established by studying relative expression of genes (Nrf-2 and Keap1) and Nrf-2 nuclear translocation associated with anti-oxidative signaling in Caco-2 cells. Bio-accessibility of tripeptide and its intact transport across Caco-2 cell monolayer was also found to be 1.72±0.22% through PepT1 mediated transport mechanism. Besides, tripeptide displayed strong anti-oxidative and anti-inflammatory potential under in vivo conditions in mice against ethanol induced oxidative stress by elevating (P<.01) liver GSH content and by decreasing (P<.01) the activities of anti-oxidative enzymes, MDA along with reduced expression of CYP2E1, PPAR-α, TNF-α and COX-2 genes than ethanol control.

Evaluation of anti-diabetic attributes of Lactobacillus rhamnosus MTCC: 5957, Lactobacillus rhamnosus MTCC: 5897 and Lactobacillus fermentum MTCC: 5898 in streptozotocin induced diabetic rats.

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.

Interferon-tau stimulated gene expression: A proxy to predict embryonic mortality in dairy cows.

The embryonic mortality in cows is a growing concern for an ever-expanding dairy industry. The current study was an attempt to shorten the open period of dairy cows having suffered embryonic loss by diagnosing them at an earlier stage. The blood samples were collected from the Karan Fries (KF) cows on days 0 (day of AI/estrus), 4, 8, 12, 14, 16, 18, 21, 24, 28, 35 and 42 post insemination. The experimental animals were then categorized into pregnant (P), conception failure/early embryonic mortality (EEM) and late embryonic mortality cows (LEM), based on progesterone assay, ultrasonography and per-rectal palpation. There were 6 animals in each group. The plasma progesterone was higher in pregnant than EEM and LEM cows. Plasma Interferon-tau concentration was significantly (p < 0.05) lower in LEM than pregnant cows where it could be detected from day 14-21 but was non-detectable in EEM cows. The mRNA expression of ISG15, OAS1, MX1 and MX2 in blood neutrophils was significantly (p < 0.05) higher from day 8-42 as against day 0 in pregnant cows. The highest expression was observed around day 18-21 in pregnant cows. The ISG15, OAS1, MX1 and MX2 mRNA expression was significantly (p < 0.05) higher from day 4-42 as compared to day 0 in LEM cows, whereas in EEM cows the expression stayed close to that of day 0 (1.00 ±â€¯0.00). The mRNA expression of ISG15, OAS1, MX1 and MX2 started to decline from day 24 onwards. The degree of expression of Interferon-tau stimulated genes was higher in pregnant and LEM cows than EEM cows. The study reveals that the Interferon tau stimulated gene expression in neutrophils can act as peripheral biomarkers for detecting the embryonic mortality in dairy cows.

Physicochemical characterization of mineral (iron/zinc) bound caseinate and their mineral uptake in Caco-2 cells.

Milk proteins (especially caseins) are widely accepted as good vehicle for the delivery of various bioactive compounds including minerals. Succinylation is one of the most acceptable chemical modification techniques to enhance the mineral binding ability of caseins. Addition of minerals to succinylated proteins may alter their physicochemical and biochemical properties. Physicochemical characteristics of succinylated sodium caseinate (S.NaCN)-mineral (iron/zinc) complexes were elucidated. Chromatographic behaviour and fluorescence intensity confirmed the structural modification of S.NaCN upon binding with minerals. The bound mineral from protein complexes showed significantly higher (P < 0.05) in vitro bioavailability (mineral uptake) than mineral salts in Caco-2 cells. Also, iron bound S.NaCN showed higher cellular ferritin formation than iron in its free form. These mineral bound protein complexes with improved bioavailability could safely replace inorganic fortificants in various functional food formulations.

Osteoanabolic activity of whey-derived anti-oxidative (MHIRL and YVEEL) and angiotensin-converting enzyme inhibitory (YLLF, ALPMHIR, IPA and WLAHK) bioactive peptides.

Exploring bone rebuilding anabolic agents has been gaining much attention due to their potential therapeutic effects in treating several bone disorders including osteoporosis. Whey protein has been reported to affect bone health osteoanabolically, in terms of proliferation and differentiation of primary osteoblast cells. This study investigates whether whey derived anti-oxidative (AO) (P1- MHIRL, P2- YVEEL) and angiotensin converting enzyme inhibitory (ACE inhibitory) (P3- YLLF, P4-ALPMHIR, P5-IPA, P6- WLAHK) bioactive peptides affect the proliferation and differentiation of primary osteoblast cells isolated from rat calvaria. The proliferation and osteogenic activity of osteoblast cells in presence of these peptides were determined by MTT assay, DNA quantification study, Alkaline phosphatase activity (ALP) and ALP staining, Alizarin red activity and staining, and secretory osteocalcin measurement. The expression of osteogenesis-related genes (COLI-α, ALP, OCN and RUNX2) were determined by real-time quantitative PCR (RT-PCR) analysis over a period of 21days. The peptide treated osteoblasts showed a significant increase in viable cell density and proliferation in the order of P2>P6>P3 at optimised concentration. Furthermore, the osteoblastic differentiation markers in response to these peptides were found to be significantly up regulated in the order of P2>P6>P3 when compared to the controls. These results demonstrated that bioactive whey-derived AO and ACE inhibitory peptides can play a potential therapeutic role in osteoporosis by activating osteoblasts anabolically.

Antioxidative peptide from milk exhibits antiosteopenic effects through inhibition of oxidative damage and bone-resorbing cytokines in ovariectomized rats.

OBJECTIVES: Oxidative stress has been implicated as a crucial pathogenic factor in the development of postmenopausal osteoporosis. Milk-derived antioxidative peptides are gaining much attention toward the development of prodrugs for alleviating several human diseases, including osteoporosis. The aim of the present study was to determine whether antioxidant peptides are good candidates for alleviating postmenopausal osteoporosis. METHODS: In the present study, an ovariectomized (OVX) osteoporotic rat model was used to investigate the protective effects of buffalo milk casein-derived novel peptide VLPVPQK (PEP) against OVX-induced bone loss and the related mechanisms. RESULTS: Results of the present study indicated that daily administration of antioxidative peptide PEP at 50 and 100 µg/kg for 8 wk prevents body weight gain, uterine weight loss, and atrophy of endometrial lumen. Moreover, PEP increased femur dry weight, ash weight, bone ash calcium, and serum calcium and phosphorus level. Interestingly, PEP increased bone mineral density and improved trabecular microarchitecture in both femur and tibia of OVX rats. Additionally, PEP increased bone strength, reduced serum bone turnover markers, inhibited bone resorbing cytokines and decreased malondialdehyde level in OVX rat. Furthermore, PEP-elevated serum transforming growth factor-ß, increased, reduced glutathione levels, superoxide dismutase, and catalase activity altered by OVX. CONCLUSION: We demonstrated that PEP exhibits antiosteopenic effects via enhancement of antioxidant activity and reduction of bone-resorbing cytokines expression.

Buffalo casein derived peptide can alleviates H2O2 induced cellular damage and necrosis in fibroblast cells.

Oxidative stress is one of a critical pathogenic factor in the progression of aging and chronic diseases such as cancer, myocardial inflammation and diabetes. In the present scenario, peptides with short half life and more biological specificities are gaining much attention as prodrugs. Thus, the present investigation carried out to screen potential antioxidative peptide, VLPVPQK to cope with the cellular oxidative damage. Our results showed that treatment of rat fibroblast cells with 0.2mM H2O2 for 6h significantly declined different oxidative stress biomarkers such as SOD, CAT, GSH, and promoted LDH activity. In addition, ROS and TNF-α levels were also increased upon H2O2 exposure for 6h and thereby, it induced cell death. Amazingly, pretreatment of the peptide (VLPVPQK) significantly elevated cell survivability, by reversing all H2O2 induced alterations in fibroblast cells. Therefore, our results indicated that, the peptide (VLPVPQK) acted as a potential cytoprotective agent, who restored redox balance and cell homeostasis in cultured fibroblast cells, even after H2O2 exposure, suggesting that the peptide can be valuable as an effective remedy in treatment of oxidative stress related diseases and skin inflammation related disorders.

Akt drives buffalo casein-derived novel peptide-mediated osteoblast differentiation.

Milk is a potential nutraceutical with wide range of bioactive compounds that are antioxidative, antimicrobial, antithrombotic, immunomodulatory, opioid and antihypertensive. Various intervention studies with milk reflect its stupendous role in elevating bone mineral density. Milk and milk products have shown a preventive effect in bone loss during pre- and postmenopausal women. Since, milk is proved to have a vital role in bone health promotion, there is a need to identify bioactive compounds within it. Recently we have reported four novel peptides from milk casein for their osteoblast proliferation activity. Their role in differentiation and the signaling cascade evoked by them have not been studied. Thus, the present study has been designed to investigate the differentiation potential and signaling cascade of one of the novel peptides, that is, NAVPITPTL by analyzing osteoblast differentiation markers such as alkaline phosphatase activity, osteocalcin and mineral deposition. All the experimentations suggested a significant role of this peptide in osteoblast differentiation. The inhibitor studies, immunocytochemistry and immunoblotting have proven that the peptide-induced differentiation through pAkt signaling cascade as pAkt was observed in nucleus. Moreover, the peptide was found to be bioaccessible up to 1%.

Consumption of probiotic Lactobacillus rhamnosus (MTCC: 5897) containing fermented milk plays a key role in development of the immune system in newborn mice during the suckling-weaning transition.

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 (ß-galactosidase, ß-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.