Production and characterization of anti-hypertensive and anti-diabetic peptides from fermented sheep milk with anti-inflammatory activity: in vitro and molecular docking studies.

BACKGROUND: The present study aimed to evaluate the anti-hypertensive and anti-diabetic activities from biologically active peptides produced by fermented sheep milk with Lacticaseibacillus paracasei M11 (MG027695), as well as to purify and characterize the angiotensin-converting enzyme (ACE) inhibitory and anti-diabetic peptides produced from fermented sheep milk. RESULTS: After 48 h of fermentation at 37 °C, sheep milk demonstrated significant changes in anti-diabetic effects and ACE-I effects, with inhibition percentages observed for ACE inhibition (76.32%), α-amylase (70.13%), α-glucosidase (70.11%) and lipase inhibition (68.22%). The highest level of peptides (9.77 mg mL-1) was produced by optimizing the growth conditions, which included an inoculation rate of 2.5% and a 48 h of incubation period. The comparison of molecular weight distributions among protein fractions was conducted through sodium dodecyl-sulfate polyacrylamide gel electrophoresis analysis, whereas spots were separated using 2D gel electrophoresis according to both the molecular weight and pH. Peptide characterization with ultra-filtration membranes at 3 and 10 kDa allowed the study to assess molecular weight-based separation. Nitric oxide generated by lipopolysaccharide and the secretion of pro-inflammatory cytokines in RAW 264.7 immune cells were both inhibited by sheep milk fermented with M11. Fourier-transform infrared spectroscopy was employed to assess changes in functional groups after fermentation, providing insights into the structural changes occurring during fermentation. CONCLUSION: The present study demonstrates that fermentation with L. paracasei (M11) led to significant changes in fermented sheep milk, enhancing its bioactive properties, notably in terms of ACE inhibition and anti-diabetic activities, and the generation of peptides with bioactive properties has potential health benefits. © 2024 Society of Chemical Industry.

Peptidomics-based identification of antihypertensive and antidiabetic peptides from sheep milk fermented using Limosilactobacillus fermentum KGL4 MTCC 25515 with anti-inflammatory activity: in silico, in vitro, and molecular docking studies.

This study investigated the synthesis of bioactive peptides from sheep milk through fermentation with Limosilactobacillus fermentum KGL4 MTCC 25515 strain and assessed lipase inhibition, ACE inhibition, α-glucosidase inhibition, and α-amylase inhibition activities during the fermentation process. The study observed the highest activities, reaching 74.82%, 70.02%, 72.19%, and 67.08% (lipase inhibition, ACE inhibition, α-glucosidase inhibition, and α-amylase inhibition) after 48 h at 37°C, respectively. Growth optimization experiments revealed that a 2.5% inoculation rate after 48 h of fermentation time resulted in the highest proteolytic activity at 9.88 mg/mL. Additionally, fractions with less than 3 kDa of molecular weight exhibited superior ACE-inhibition and anti-diabetic activities compared to other fractions. Fermentation of sheep milk with KGL4 led to a significant reduction in the excessive production of NO, TNF-α, IL-6, and IL-1ß produced in RAW 267.4 cells upon treatment with LPS. Peptides were purified utilizing SDS-PAGE and electrophoresis on 2D gels, identifying a maximum number of proteins bands ranging 10-70 kDa. Peptide sequences were cross-referenced with AHTPDB and BIOPEP databases, confirming potential antihypertensive and antidiabetic properties. Notably, the peptide (GPFPILV) exhibited the highest HPEPDOCK score against both α-amylase and ACE.

Sex-specific effects of ketogenic diet on anxiety-like behavior and neuroimmune response in C57Bl/6J mice.

The ketogenic diet (KD) has been shown to reduce anxiety and enhance cognitive functions in neurological diseases. However, the sex-specific effects of KD on anxiety-like behavior in healthy individuals and the underlying molecular mechanisms contributing to these effects, including neuroinflammation, are unelucidated. This study investigated the sex-specific effects of KD on anxiety-like behavior and the neuroimmune response in the prefrontal cortex (PFC) and hippocampus of healthy C57BL/6J male and female mice. Animals were fed either a control diet (CD- 17% fat, 65% carb, 18% protein) or a KD (80% fat, 5% carb, 15% protein) for 4 weeks. KD increased the levels of circulating ß-hydroxybutyrate (BHB) both in males and females. However, PFC BHB levels were found to be elevated only in KD males. Moreover, KD did not affect the behavior of females but improved motor abilities and reduced anxiety levels in males. KD suppressed the mRNA expression of the pan microglial markers (Cd68, P2ry12) and induced morphological changes in the male PFC microglia. A sex-specific decrease in IL1ß and an increase in IL-10 levels was found in the PFC of KD males. A similar trend was observed in the hippocampus of males where KD reduced the mRNA expression of P2ry12, Il1ß, and cFos. Additionally, BHB increased the production of IL-10 whereas it decreased the production of IL1ß from human microglia in in-vitro conditions. In summary, these results demonstrate that the anxiolytic and motor function enhancement abilities of KD are male-specific. Reduced pro-inflammatory and improved anti-inflammatory factors in the male PFC and hippocampus may underlie these effects.

Sex-specific effects of sucrose withdrawal on anxiety-like behavior and neuroimmune response.

Sugar bingeing induces maladaptive neuroadaptations to decrease dietary control and promote withdrawal symptoms. This study investigated sex differences in sucrose bingeing, sucrose withdrawal-induced negative mood effects and underlying neuroimmune response in the prefrontal cortex (PFC) and nucleus accumbens (NAc) of C57BL/6J male and female mice. Two-bottle sucrose choice paradigm was used to develop sucrose dependence in mice. Female mice consumed more sucrose than male mice when given free access to water and 10% sucrose for four weeks. A significant increase in the mRNA expression of neuroinflammatory markers (Il1ß, Tnfα) was found in the PFC of males exposed to sucrose withdrawal. Sucrose bingeing and subsequent sucrose withdrawal showed elevated protein levels of pro-inflammatory cytokines/chemokines/growth factors in the PFC (IL-1ß, IL-6, TNFα, IFN-γ, IL-10, CCL5, VEGF) and NAc (IL-1ß, IL-6, IL-10, VEGF) of male mice as compared to their water controls. These effects were concurrent with reduced mRNA expression of neuronal activation marker (cFos) in the PFC of sucrose withdrawal males. One week of sucrose withdrawal after prolonged sucrose consumption showed anxiety-like behavior in male mice, not in females. In conclusion, this study demonstrates that repeated access to sucrose induces anxiety-like behavior when the sugar is no longer available in the diet and these effects are male-specific. Elevated neuroinflammation in reward neurocircuitry may underlie these sex-specific effects.

Supplementation of probiotic Bifidobacterium breve Bif11 reverses neurobehavioural deficits, inflammatory changes and oxidative stress in Parkinson's disease model.

Human gut microbiota are thought to affect different physiological processes in the body, including brain functions. Gut dysbiosis has been linked to the progression of Parkinson's disease (PD) and thus, restoring the healthy gut microbiota with supplementation of putative probiotic strains can confer some benefits in PD. In the current study, we explored the neuroprotective potential of Bifidobacterium breve Bif11 supplementation in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) treated female Sprague Dawley rats. This study investigated the behavioural, molecular and biochemical parameters in the MPTP rat model. A pharmacological intervention of Bif11 at doses of 1 × 1010 CFU and 2 × 1010 CFU for 21 days was found to attenuate the cognitive and motor changes in the MPTP rat model. Furthermore, it also increased the tyrosine hydroxylase levels, reduced pro-inflammatory markers and decreased oxidative and nitrosative stress in the mid brain of MPTP-lesioned rats. Bif11 supplementation even restored the levels of short-chain fatty acids and decreased intestinal epithelial permeability in MPTP-induced PD model rats. In summary, these findings demonstrate that B. breve Bif11 has the potential to ameliorate symptoms of PD. However, this therapy needs to be further investigated with in-depth mechanistic insights in the future for the treatment of PD.

GABA synthesizing lactic acid bacteria and genomic analysis of Levilactobacillus brevis LAB6.

This study was conducted to investigate the γ-aminobutyric acid (GABA) production ability of 20 Lactobacillus and 25 Bifidobacterium strains which were previously isolated in our laboratory. Effect of initial pH, incubation time, monosodium glutamate (MSG), and pyridoxal-5'-phosphate (PLP) concentration for highest GABA production by two potent bacterial strains, Levilactobacillus brevis LAB6 and Limosilactobacillus fermentum LAB19 were optimized in the MRS media. A threefold increase in GABA production at an initial pH 4.0, incubation time of 120 h in medium supplemented with 3% MSG and 400 µM of PLP for LAB6 and 300 µM for LAB19 lead to the production of 19.67 ± 0.28 and 20.77 ± 0.14 g/L of GABA, respectively. Coculturing both strains under optimized conditions led to a GABA yield of 20.02 ± 0.17 g/L. Owing to potent anti-inflammatory activity in-vitro, as reported previously, and highest GABA production ability of LAB6 (MTCC 25662), its whole-genome sequencing and bioinformatics analysis was carried out for mining genes related to GABA metabolism. LAB6 harbored a complete glutamate decarboxylase (GAD) gene system comprising gadA, gadB, and gadC as well as genes responsible for the beneficial probiotic traits, such as for acid and bile tolerance and host adhesion. Comparative genomic analysis of LAB6 with 28 completely sequenced Levilactobacillus brevis strains revealed the presence of 95 strain-specific genes-families that was significantly higher than most other L. brevis strains. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03918-7.

Allyl isothiocyanate, a TRPA1 agonist, protects against olanzapine-induced hypothalamic and hepatic metabolic aberrations in female mice.

Olanzapine, a widely prescribed atypical antipsychotic, poses a great risk to the patient's health by fabricating a plethora of severe metabolic and cardiovascular adverse effects eventually reducing life expectancy and patient compliance. Its heterogenous receptor binding profile has made it difficult to point out a specific cause or treatment for the related side effects. Growing body of evidence suggest that transient receptor potential (TRP) channel subfamily Ankyrin 1 (TRPA1) has pivotal role in pathogenesis of type 2 diabetes and obesity. With this background, we aimed to investigate the role of pharmacological manipulations of TRPA1 channels in antipsychotic (olanzapine)-induced metabolic alterations in female mice using allyl isothiocyanate (AITC) and HC-030031 (TRPA1 agonist and antagonist, respectively). It was found that after 6 weeks of treatment, AITC prevented olanzapine-induced alterations in body weight and adiposity; serum, and liver inflammatory markers; glucose and lipid metabolism; and hypothalamic appetite regulation, nutrient sensing, inflammatory and TRPA1 channel signaling regulating genes. Furthermore, several of these effects were absent in the presence of HC-030031 (TRPA1 antagonist) indicating protective role of TRPA1 agonism in attenuating olanzapine-induced metabolic alterations. Supplementary in-depth studies are required to study TRPA1 channel effect on other aspects of olanzapine-induced metabolic alterations.

Non-carcinogenic and carcinogenic health risk assessment of heavy metals in cooked beans and vegetables in Punjab, North India.

Beans and vegetables are consumed with cereals in India on daily basis. The aim of the study was to assess carcinogenic and non-carcinogenic risk of heavy metals in cooked beans and cooked vegetables consumed by adults (18-59 years) and elderly (≥60 years) subjects from two districts (Ludhiana and Bathinda) of Punjab. A total of 150 households were selected from 30 different locations covering both rural and urban areas. The mean daily consumption of beans and vegetables in Ludhiana was recorded as 35.09 and 215.93 g, respectively. The corresponding figures in Bathinda were observed as 26.85 and 230.54 g. The average amounts of arsenic, cadmium, lead, and mercury were 1.44 × 10-5, 8.21 × 10-5, 1.30 × 10-3, and 2.61 × 10-7 mg/kg for cooked vegetables in urban households of Ludhiana district, respectively. The corresponding values for rural households were 1.53 × 10-5, 5.58 × 10-5, and 2.98 × 10-4 mg/kg while mercury was not detected. The mean chronic daily intake (CDI) of arsenic from cooked beans was significantly (p ≤ .001) higher in urban adult males of Ludhiana (7.74 × 10-9 mg/kg/day) and Bathinda (5.31 × 10-9 mg/kg/day) compared to their rural counterparts. Similar trend was observed in CDI of heavy metals from vegetables. The mean CDI of cadmium from cooked vegetables in urban adult females of Ludhiana (3.76 × 10-7 mg/kg/day) was significantly (p ≤ .001) higher than their rural counterparts and both urban and rural adult females of Bathinda. The study concluded that the subjects of both districts were found safe from non-carcinogenic and carcinogenic risk associated with heavy metals present in cooked beans and vegetables, except for urban subjects and rural adult subjects of Ludhiana district who had cancer risk due to cadmium present in cooked vegetable samples.

Immunomodulatory action of synbiotic comprising of newly isolated lactic acid producing bacterial strains against allergic asthma in mice.

Given the reported role of gut-microbiota in asthma pathogenesis, the present work was carried to evaluate immunomodulatory action of newly isolated lactic acid producing bacterial strains Bifidobacterium breve Bif11 and Lactiplantibacillus plantarum LAB31 against asthma using ovalbumin (OVA) based mouse model. Our results show that both strains modulate Th2 immune response potentially through production of short chain fatty acids (SCFAs), resulting in suppression of OVA-induced airway inflammation. Furthermore, synbiotic comprising of both strains and prebiotic, Isomaltooligosaccharide exhibited superior potential in amelioration of OVA-induced airway inflammation through improved modulation of Th2 immune response. Further, synbiotic protects against OVA-induced mucus hyper-production and airway-hyperresponsiveness. Such protection was associated with normalization of gut microbiome and enhanced production of SCFAs in cecum which correlates closely with population of T-regulatory cells in spleen. Overall, our novel synbiotic possesses the ability to fine-tune the immune response for providing protection against allergic asthma.

Superior Photophysical and Photosensitizing Properties of Nanoaggregates of Weakly Emissive Dyes for Use in Bioimaging and Photodynamic Therapy.

The development of luminescent dyes based on 1,1,4,4-tetracyanobuta-1,3-dienes (TCBDs) is an active research area, and a quantum yield (ΦF) of 7.8% has been achieved so far in cyclohexane by appending a fluorophore. Our novel method radically refines weakly emissive 2,3-disubstituted TCBD (phenyl-TCBD 1) (ΦF = 2.3% in CH3CN) into a water-soluble, biocompatible nanoformulation as highly emissive aggregates 1NPs ⊂ PF-127 with ΦF = 7.9% in H2O and without fluorophore conjugation. Characterization of 1NPs ⊂ PF-127 was carried out using various spectroscopic techniques, and its predominant size was found to be 80-100 nm according to transmission electron microscopy and dynamic light scattering techniques. Spectroscopic studies including Fourier transform infrared spectroscopy revealed that aggregated phenyl-TCBD particles were encapsulated in a nonluminescent triblock copolymer (PF-127)-based nanomicelles with the TCBD entrapment efficiency of 77%. With increasing water fraction, the phenyl-TCBD nanoaggregates exhibited a 3-fold higher quantum yield, a greater lifetime, and a red shift (155 nm). This remarkable enhancement in red emissivity enabled them to be used as a bioprobe for bioimaging applications and in photodynamic therapy to selectively target cancer cell lines with singlet oxygen generation capability (ΦΔ = 0.25). According to the MTT assay, compared to the native molecular form (1229 nM), the aggregated 1NPs ⊂ PF-127 (13.51 nM) exhibited dose-dependent cell death when exposed to light with 91-fold increased activity. The histoarchitectures of various vital organs (liver, kidneys, heart, lungs, and spleen) were intact when tested for in vivo biocompatibility. This study has significant implications for developing nonplanar push-pull chromophore-based dyes as biosensors and with potential applications beyond bioimaging.

Molecular binding mechanism and novel antidiabetic and anti-hypertensive bioactive peptides from fermented camel milk with anti-inflammatory activity in raw macrophages cell lines.

The investigation was to determine the effect of camel milk fermented with Limosilactobacillus fermentum KGL4 (MTCC 25515) on ACE-inhibiting, anti-inflammatory, and diabetes-preventing properties and also to release the novel peptides with antidiabetic and anti-hypertensive attributes with molecular interaction studies. Growth conditions were optimised on the basis of total peptide production by inoculating the culture in camel milk at different rates (1.5, 2.0, and 2.5%) along with different incubation periods (12, 24, 36, and 48 h). However, after 48 h of fermentation with a 2.5% rate of inoculum, the highest proteolytic activity was obtained. Reverse phase high-pressure liquid chromatography (RP-HPLC) was used to calculate the % Rpa from permeates of 3 kDa and 10 kDa fractions. Molecular weight distributions of fermented and unfermented camel milk protein fractions were compared using SDS-PAGE. Spots obtained from 2D gel electrophoresis were separated on the basis of pH and molecular weight. Spots obtained from 2D gel were digested with trypsin, and the digested samples were subjected to RP-LC/MS for the generation of peptide sequences. The inhibition of tumour necrosis factor alpha, interleukin-6, and interleukin-1 during fermentation was studied using RAW 264.7 macrophages. In the study, fermented camel milk with KGL4 (CMKGL4) inhibited LPS-induced nitric oxide (NO) production and pro-inflammatory cytokine production (TNF-α, IL-6, and IL-1ß) by the murine macrophages. The results showed that the peptide structures (YLEELHRLNK and YLQELYPHSSLKVRPILK) exhibited considerable binding affinity against hPAM and hMGA during molecular interaction studies.

Safety aspects, probiotic potentials of yeast and lactobacillus isolated from fermented foods in North-Eastern India, and its anti-inflammatory activity.

Lactobacillus and yeast obtained from fermented foods in North-East India were tested for safety and probiotic properties. All the lactobacilli and yeast tested negative for the catalase, indole, urease, phenylalanine, hemolysis, gelatin hydrolysis, and biogenic amine production tests, indicating that they are safe to use as probiotics in food supplements. Lactiplantibacillus plantarum KGL3A (accession no. MG722814) was capable of resisting the replicated gastric fluid (pH 2) till 2 h of exposure, whereas both KGL3A and Lacticaseibacillus rhamnosus K4E (accession no. KX950834.1) strains were able to resist pH 3 till 2 h of exposure with a reduction in overall viable cell count from 7.48 log CFU/mL to 1.09 log CFU/mL and 7.77 log CFU/mL to 0.83 log CFU/mL, respectively. In vitro gastric juice simulation conditions were tolerated by the yeast Saccharomyces cerevisiae WBS2A. The cell surface hydrophobicity (CSH) towards hydrocarbons (n-hexadecane) was seen highest in L. plantarum KGL3A (77.16± 0.84%) and Limosilactobacillus fermentum KGL4 accession no. MF951099 (72.60 ± 2.33%). The percentage auto-aggregation ranged from 8.70 to 25.53 after 2 h, which significantly increased to 10.50 to 26.94 during the fifth hour for cultures. Also, a higher percentage of co-aggregation was found for the culture L. rhamnosus K4E with S. typhi (34.18 ± 0.03%), E. coli (32.97 ± 0.02 %) and S. aureus (26.33 ± 0.06 %) and for the yeast S. cerevisiae WBS2A, a higher percentage of co-aggregation was found with Listeria monocytogenes (25.77 ± 0.22%). The antioxidant activity and proteolytic activity were found to be higher for Lactobacillus helveticus K14 and L. rhamnosus K4E. The proportion of decreased cholesterol was noticeably higher in KGL4 (29.65 ± 4.30%). ß glucosidase activity was significantly higher in the L. fermentum KGL4 strain (0.359 ± 0.002), and α galactosidase activity was significantly higher in the L. rhamnosus K4E strain (0.415 ± 0.016). MTT assays suggested that KGL4 and WBS2A at a lower dose did not exhibit cytotoxicity.

Production and Characterization of ACE Inhibitory and Anti-Diabetic Peptides from Buffalo and Camel Milk Fermented with Lactobacillus and Yeast: A Comparative Analysis with In Vitro, In Silico, and Molecular Interaction Study.

The investigation aimed at assessing a comparative study on the production and characterization of ACE inhibitory, anti-diabetic, and anti-inflammatory activities, along with the production of ACE inhibitory and anti-diabetic peptides through the fermentation of buffalo and camel milk by Limosilactobacillus fermentum (KGL4) and Saccharomyces cerevisiae (WBS2A). The angiotensin-converting enzyme (ACE) inhibitory and anti-diabetic properties were evaluated at particular time intervals (12, 24, 36, and 48 h) at 37 °C, and we discovered maximum activity at 37 °C after 48 h of incubation. The maximum ACE inhibitory, lipase inhibitory activities, alpha-glucosidase inhibitory, and alpha-amylase inhibitory activities were found in the fermented camel milk (77.96 ± 2.61, 73.85 ± 1.19, 85.37 ± 2.15, and 70.86 ± 1.02), as compared to the fermented buffalo milk (FBM) (75.25 ± 1.72, 61.79 ± 2.14, 80.09 ± 0.51, and 67.29 ± 1.75). Proteolytic activity was measured with different inoculation rates (1.5%, 2.0%, and 2.5%) and incubation times (12, 24, 36, and 48 h) to optimize the growth conditions. Maximum proteolysis was found at a 2.5% inoculation rate and at a 48 h incubation period in both fermented buffalo (9.14 ± 0.06) and camel milk (9.10 ± 0.17). SDS-PAGE and 2D gel electrophoresis were conducted for protein purification. The camel and buffalo milk that had not been fermented revealed protein bands ranging from 10 to 100 kDa and 10 to 75 kDa, respectively, whereas all the fermented samples showed bands ranging from 10 to 75 kDa. There were no visible protein bands in the permeates on SDS-PAGE. When fermented buffalo and camel milk were electrophoresed in 2D gel, 15 and 20 protein spots were detected, respectively. The protein spots in the 2D gel electrophoresis ranged in size from 20 to 75 kDa. To distinguish between different peptide fractions, water-soluble extract (WSE) fractions of ultrafiltration (3 and 10 kDa retentate and permeate) of fermented camel and buffalo milk were employed in RP-HPLC (reversed-phase high-performance liquid chromatography). The impact of fermented buffalo and camel milk on inflammation induced by LPS (lipopolysaccharide) was also investigated in the RAW 264.7 cell line. Novel peptide sequences with ACE inhibitory and anti-diabetic properties were also analyzed on the anti-hypertensive database (AHTDB) and bioactive peptide (BIOPEP) database. We found the sequences SCQAQPTTMTR, EMPFPK, TTMPLW, HPHPHLSFMAIPPK, FFNDKIAK, ALPMHIR, IPAVFK, LDQWLCEK, and AVPYPQR from the fermented buffalo milk and the sequences TDVMPQWW, EKTFLLYSCPHR, SSHPYLEQLY, IDSGLYLGSNYITAIR, and FDEFLSQSCAPGSDPR from the fermented camel milk.

Health risk assessment of arsenic, lead and cadmium from milk consumption in Punjab, India.

Milk is an integral part of the human diet and its contamination with heavy metals may alter the health of its consumers. The study was conducted to assess the health risk associated with the heavy metals in milk samples collected from urban and rural households of Ludhiana and Bathinda districts of Punjab, India. One hundred and fifty milk samples were analyzed for heavy metals i.e. arsenic, cadmium, lead and mercury using Inductively Coupled Plasma Mass Spectrometry ICP-MS. The health risks, such as non-carcinogenic and carcinogenic risks from heavy metals in milk samples, were calculated for selected males and females of adults, children and elderly subjects. The results indicated that the arsenic, cadmium and lead content in milk samples were within permissible limit whereas mercury was not detected in any sample. The mean values showed that the selected urban and rural population of both districts was safe from non-carcinogenic risk associated with heavy metal content of milk. However, urban (50% males and 86% females) and rural (25% males) children of Bathinda district were at risk of cancer from arsenic and cadmium present in milk samples, respectively. It was also observed that the selected population of both districts were safe from carcinogenic risk due to the combined effects of heavy metals. It was concluded that even with a small amount of heavy metal in milk samples, the rural adults, rural male children and urban female children of Bathinda district had carcinogenic risk due to milk consumption. Hence, regular monitoring and testing of milk samples must be done as a public health measure to prevent heavy metal contamination in milk to safeguard the health of consumers.

Capsazepine-Induced Altered Colonic Mucosal Health Limits Isomalto-oligosaccharide Action in High-Fat Diet-Fed C57BL/6J Mice.

The present study sought to understand the effects of a combination of altered colonic mucosal health (intrarectal capsazepine administration) and high-fat diet (HFD) administration in mice. Furthermore, we also studied whether this combination prevents protective actions of dietary prebiotic, isomaltooligosaccharides. We studied the alterations in intestinal permeability, histological and transcriptional changes, short-chain fatty acid (SCFA) concentrations, and gut microbial abundance. Capsazepine (CPZ) was administered rectally twice a day along with HFD feeding. Following confirmation of CPZ action (loss of TRPA1 and TRPV1-associated nocifensive behavior), the intrarectal dose of CPZ was reduced to once in 2 days up to 8 weeks. Simultaneous intrarectal administration of CPZ exacerbated the HFD (8 weeks feeding)-induced damage to mucosal lining, intestinal permeability, tight junction protein expression, SCFA levels, and gut bacterial abundances. This higher degree of mucosal damage and pathological alteration in colonic mucosa prevented the previously reported protective actions of isomaltooligosaccharides as a prebiotic in HFD-fed mice. Overall, we present evidence that colonic precondition (gut permeability and mucosal lining) is an important factor in determination of HFD-induced changes in the colon, and success of diet-associated interventions (dietary fibers, pre/probiotics, etc.) is dependent on it.

Transient Receptor Potential (TRP) based polypharmacological combination stimulates energy expending phenotype to reverse HFD-induced obesity in mice.

BACKGROUND & AIM: Obesity is a worldwide epidemic leading to decreased quality of life, higher medical expenses and significant morbidity. Enhancing energy expenditure and substrate utilization in adipose tissues through dietary constituents and polypharmacological approaches is gaining importance for the prevention and therapeutics of obesity. An important factor in this regard is Transient Receptor Potential (TRP) channel modulation and resultant activation of "brite" phenotype. Various dietary TRP channel agonists like capsaicin (TRPV1), cinnamaldehyde (TRPA1), and menthol (TRPM8) have shown anti-obesity effects, individually and in combination. We aimed to determine the therapeutic potential of such combination of sub-effective doses of these agents against diet-induced obesity, and explore the involved cellular processes. KEY FINDINGS: The combination of sub-effective doses of capsaicin, cinnamaldehyde and menthol induced "brite" phenotype in differentiating 3T3-L1 cells and subcutaneous white adipose tissue of HFD-fed obese mice. The intervention prevented adipose tissue hypertrophy and weight gain, enhanced the thermogenic potential, mitochondrial biogenesis and overall activation of brown adipose tissue. These changes observed in vitro as well as in vivo, were linked to increased phosphorylation of kinases, AMPK and ERK. In the liver, the combination treatment enhanced insulin sensitivity, improved gluconeogenic potential and lipolysis, prevented fatty acid accumulation and enhanced glucose utilization. SIGNIFICANCE: We report on the discovery of therapeutic potential of TRP-based dietary triagonist combination against HFD-induced abnormalities in metabolic tissues. Our findings indicate that a common central mechanism may affect multiple peripheral tissues. This study opens up avenues of development of therapeutic functional foods for obesity.

Bifidobacterium breve Bif11 supplementation improves depression-related neurobehavioural and neuroinflammatory changes in the mouse.

Gut dysbiosis has been closely linked to the onset and progression of several brain-related disorders such as depression. The administration of microbiota-based formulations such as probiotics helps restore healthy gut flora and plays a role in preventing and treating depression-like behavior. Therefore, we evaluated the efficacy of probiotic supplementation using our recently isolated putative probiotic Bifidobacterium breve Bif11 in ameliorating lipopolysaccharide (LPS)-induced depression-like behavior in male Swiss albino mice. Mice were fed orally with B. breve Bif11 (1 × 1010 CFU and 2 × 1010 CFU) for 21 days before being challenged with a single intraperitoneal LPS injection (0.83 mg/kg). Behavioral, biochemical, histological and molecular analysis were done with an emphasis on inflammatory pathways linked to depression-like behavior. Daily supplementation with B. breve Bif11 for 21 days prevented the onset of depression-like behavior induced by LPS injection, besides reducing the levels of inflammatory cytokines such as matrix metalloproteinase-2, c-reactive protein, interleukin-6, tumor necrosis factor-alpha and nuclear factor kappa-light-chain-enhancer of activated B cells. It also prevented the decrease of the brain-derived neurotrophic factor levels and neuronal cell viability in the prefrontal cortex of LPS-treated mice. Furthermore, we observed that gut permeability was reduced, there was an improved short-chain fatty acid profile and reduced gut dysbiosis in the LPS mice fed with B. breve Bif11. Similarly, we observed a decrease in behavioural deficits and restoration of gut permeability in chronic mild stress. Together, these results would help in deciphering the role of probiotics in the management of neurological disorders where depression, anxiety and inflammation are prominent clinical features.

Modified cereal bran (MCB) from finger millet, kodo millet, and rice bran prevents high-fat diet-induced metabolic derangements.

Cereal bran consumption improves gastrointestinal and metabolic health. Unprocessed cereal brans have a limited shelf-life and contain anti-nutrient phytochemicals. In the present study, lipids and antinutrients (flavonoids, tannin, and polyphenol) were removed from finger millet, kodo millet and rice bran using chemo-enzymatic processing. The thus-obtained modified cereal brans (MCBs) were evaluated for their potential in preventing high fat diet (HFD)-induced obesity. C57BL/6 mice were fed a HFD or a HFD supplemented with 10% w/w modified finger millet bran (mFMB), modified kodo millet bran (mKMB), modified rice bran (mRB), or a combination of the modified brans (1 : 1 : 1) for twelve weeks. The MCBs reduced HFD-induced body weight gain, improved glucose homeostasis, decreased the Firmicutes/Bacteroidetes ratio, and increased the short chain fatty acid (SCFA) levels in the cecum. Liver dyslipidemia, oxidative stress, inflammation, visceral white adipose tissue (vWAT) hypertrophy, and lipolysis were also prevented by the MCBs. Among the individual MCBs, mRB showed a greater effect in preventing HFD-induced increase in the inflammatory cytokines (IL-6, TNF-α, and LPS) than mFMB and mKMB. mFMB and mKMB supplementation more significantly restored the relative abundance of Akkermansia muciniphila and butyrate-producing genera such as Lachnospiraceae, Eubacterium, and Ruminococcus than mRB. Ex vivo gut permeability assay, immunohistochemistry of tight junction proteins, and gene expression analysis in the colon revealed that the combination of three brans was better in preventing HFD-induced leaky gut in comparison to the individual brans. Hierarchical clustering analysis showed that the combination group was clustered closest to the NPD group, suggesting an additive effect. Our study implies that a combination of mFMB, mKMB, and mRB could be used as a nutraceutical or functional food ingredient for preventing HFD-induced gut derangements and associated metabolic complications.

A synbiotic combination of Bifidobacterium longum Bif10 and Bifidobacterium breve Bif11, isomaltooligosaccharides and finger millet arabinoxylan prevents dextran sodium sulphate induced ulcerative colitis in mice.

Decreased bifidobacterial abundance, disrupted gut barrier function, dysregulated immune response and ulceration have been reported in the gut microbiota of IBD patients. Non-digestible carbohydrates with bifidogenic effect enrich the gut microbiota with Bifidobacterium spp. and could help in overcoming inflammatory gut conditions. In this study, the protective effect of Bifidobacterium longum Bif10 and Bifidobacterium breve Bif11; isomaltooligosaccharides (IMOS); Finger millet arabinoxylan (FM-AX) and their Synbiotic mix were evaluated against dextran sodium sulphate (DSS) induced UC in male Balb/c mice for 25 days. All the interventions ameliorated symptoms of colitis such as disease activity index (DAI), histological damage to the colon, gut-bacterial dysbiosis and inflammation. However, the synbiotic mix was more potent in amelioration of some of the parameters such as decreased TNF-α and lipocalin levels; increased anti-inflammatory markers (IL-10 and IL-22), and improved short chain fatty acids (SCFAs) levels in the cecum content. Furthermore, mouse colitis histological scoring (MCHI) also suggested the preventive role of synbiotic mix. All the dietary interventions aid in improving the DAI and immune parameters; restoration or regeneration of the altered selected gut bacteria, enhances the SCFA production, strengthens gut barrier, prevents gut inflammation and decreases the colonic MCHI score in DSS fed mice.

Significance of Lactobacillus fermentum on Antioxidative and Anti-Inflammatory Activities and Ultrafiltration Peptide Fractions as Potential Sources of Antioxidative Peptides from Fermented Camel Milk (Indian Breed).

OBJECTIVE: The present study aimed to assess the bio-functional analysis of camel milk viz. anti-oxidative, anti-inflammatory activities using potent Lactobacillus fermentum (KGL4) strain through fermentation and also to release the bioactive peptides during fermentation. METHOD: The antioxidant and proteolytic activities of the fermented camel milk were studied followed by SDS-PAGE analysis and 2 D PAGE. The separations of the bioactive peptides of water-soluble extract (WSE) of 3 and 10 kDa (Permeates & Retentates) were achieved by RP-HPLC. The purified bioactive peptides were identified and characterized using RPLC/MS and the effect of WSE of camel milk fermented with KGL4 on lipopolysaccharide (LPS)/endotoxin-induced inflammation in RAW 264.7 macrophages were also studied. RESULTS: The maximal activity was observed in ABTS assay (64.03%), then in hydroxyl free radical scavenging assay, and minimal activity was observed in superoxide free radical assay (57.75%). ABTS assay was significantly (P < 0.05) higher than other assays. MTT assay was performed on WSE of camel milk fermented with KGL4 using treated macrophage cells with different concentrations and found the decreasing range of cell viability at 0.25 mg/mL treatment which was non-significant. 7.80 mg/ml peptide production was found after 48 h of fermentation using the OPA method. Further, WSE of fermented camel milk was separated and analyzed their protein profiles using SDS-PAGE and 2 D-PAGE techniques. Here, many new peptides were found in camel milk when fermented with KGL4 strain. Each protein sequence was characterized through bioinformatic tools, including SWISS-PROT & PIR protein databases. Novel bioactive anti-oxidative peptides were found by searching in the BIOPEP database. CONCLUSIONS: The present study suggests that the L. fermentum KGL4 strain could be explored to produce novel antioxidative peptides from fermented camel milk (Indian breed).