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.

Exploring the biofunctionalities of lactic fermented cactus pear (Opuntia elatior Mill.) fruit beverage: an exotic superfood.

Cactus pear fruit is known with many health benefits in ethnomedicine of countries like Mexico, Portugal, Chine, India etc. The study was aimed to develop biofunctional lactic fermented cactus pear fruit beverage to add values to the medicinal fruit. The processing parameters such as quantity of freeze dried cactus pear fruit powder, sucrose and incubation time were optimised using response surface methodology. The optimized product was then subjected to proximate compositional, physicochemical, biofunctional and microbial analysis. The lactic fermented cactus pear fruit beverage was prepared by mixing 12% [w/v] freeze dried cactus pear fruit powder and 3% sucrose in water, then pasteurised and inoculated with 3% Lactobacillus fermentum MTCC 25515 and Lactobacillus rhamnosus M9, then incubated at 37 °C for 6 h. The moisture content of the beverage was 87.77% and major constituent was carbohydrate (9.58% per wet matter basis). The 100 mL beverage contains 89.84 mg GAE phenolic compounds, 5.86 mg QE flavonoids, 71.82 mg betacyanin, 28.08 mg betaxanthin, 10.59 mg ascorbic acid. The beverage also exhibited 58% ABTS antioxidant activity. The beverage was shelf stable for 20 days at 7 ± 1 °C. Such a biofunctional beverage loaded with antioxidant potential can be consumed as refreshing drink.

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.

Genomic and probiotic attributes of Lactobacillus strains from rice-based fermented foods of North Eastern India.

The probiotic attributes and genomic profiles of amylase-producing Lactobacillus strains from rice-based fermented foods of Meghalaya in the North-Eastern India were evaluated in the study. A preliminary screening of 17 lactic acid bacteria strains was performed based on their starch hydrolysis and glucoamylase activities. Out of 17 strains, 5 strains (L. fermentum KGL4, L. rhamnosus RNS4, L. fermentum WTS4, L. fermentum KGL2, and L. rhamnosus KGL3A) were selected for further characterization of different probiotic attributes. Whole-genome sequencing of two of the best strains was carried out using a shotgun sequencing platform based on their rich probiotic attributes. The EPS production was in the range of 2.89-3.92 mg/mL. KGL2 (41.5%) and KGL3A (41%) showed the highest antioxidant activity. The highest antibiotic susceptibility was exhibited by all the five Lactobacillus strains against ampicillin, ranging from 24.66 to 27.33 mm. The lactobacilli isolates used in the study could survive the simulated gastric/intestinal juices. Genomic characterization of KGL4 and KGL3A illustrated their possible adherence to the intestinal wall, specialized metabolic patterns, and possible role in boosting host immunity. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05633-8.

Bioactive peptides in the management of lifestyle-related diseases: Current trends and future perspectives.

Lifestyle-related diseases constitute a major concern in the twenty-first century, with millions dying worldwide each year due to chosen lifestyles and associated complications such as obesity, type 2 diabetes, hypertension, and hypercholesterolemia. Although synthetic drugs have been shown to be quite effective in the treatment of these conditions, safety of these compounds remains a concern. Natural alternatives to drugs include food-derived peptides are now being explored for the prevention and treatment of lifestyle-related complications. Peptides are fragments nascent in the primary protein sequences and could impart health benefits beyond basic nutritional advantages. Evidence suggests that by controlling adipocyte differentiation and lipase activities, bioactive peptides may be able to prevent obesity. Bioactive peptides act as agents against type 2 diabetes because of their ability to inhibit enzymatic activities of DPP-IV, α-amylase, and α-glucosidase. Moreover, bioactive peptides can act as competitive inhibitors of angiotensin-converting enzyme, thus eliciting an antihypertensive effect. Bioactive peptides may have a hypocholesterolemic effect by inhibiting cholesterol metabolism pathways and cholesterol synthesis. This review addresses current knowledge of the impact of food-derived bioactive peptides on lifestyle diseases. In addition, future insights on the clinical trials, allergenicity, cytotoxicity, gastrointestinal stability, and regulatory approvals have also been considered.

Peptidomic profiling of fermented goat milk: considering the fermentation-time dependent proteolysis by Lactobacillus and characterization of novel peptides with Antioxidative activity.

In this study, antioxidant activities were evaluated for goat milk fermented with Lactobacillus helveticus MTCC 5463. The fermentation conditions (inoculation rate and incubation time) were optimized by estimating proteolytic action of Lactobacillus. SDS-PAGE and 2D gel electrophoresis were carried out for identification of molecular weight and purification of identified peptides. 3 and 10 kDa peptides fractions were obtained through ultrafiltration and also by using RP-HPLC. Then, spots from 2D and fractions from RP-HPLC were also evaluated in RP-LC/MS for identification and characterization of peptides. Identified peptides were matched with online database of goat milk i.e. BLASTp (NCBI) and Protein information resource database (PIR) and subsequently, antioxidant activity of these peptides were also confirmed with BIOPEP database. However, antioxidative peptides from fermented goat milk with Lactobacillus helveticus MTCC 5463 could be produced in developing functional goat milk yoghurt. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-021-05243-w.

Characterization of Angiotensin I-Converting Enzyme (ACE) inhibitory peptides produced in fermented camel milk (Indian breed) by Lactobacillus acidophilus NCDC-15.

Fermented camel milk provides many health benefits like antidiabetic activity, anti-hypertensive activity etc. Fermented camel milk contains IPP or VPP rich ACE inhibitory peptides. The aim of this study was to spot the novel Angiotensin I-Converting Enzyme inhibitory peptides liberated by the potent proteolytic Lactobacillus acidophilus NCDC-15 from camel milk (Indian breed). NCDC-15 had exhibited maximum PepX activity (0.655) and ACE-inhibitory activity (78.33%) at 12 and 48 h of incubation at 37 °C respectively. Proteolytic activity was measured using o-phthaldialdehyde method and observed maximum (0.976 OD) at 2% of inoculation for 12 h of incubation at 37 °C. Water soluble extracts derived from fermented camel milk were ultrafiltered through 3 kDa, 5 kDa and 10 kDa membrane filters from which 3 kDa permeates (48.01% peptides production & 49.46% ACE-inhibition) and 10 kDa permeates (55.04% peptides production & 42.40% ACE-inhibition) had shown maximum peptides production and ACE-inhibitory activity. Overall, 24 peptides were identified from the samples of 3 kDa permeates [6 fractions (K1, L1, M1, N1, O1 and P1)] and 10 permeates [5 fractions (S, T, U, V and W)]. Novel peptide (AIGPVADLHI) was matched with k-casein in AHTPDB database and other peptides were also found matched with α and ß-caseins of camel milk. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05357-9.

Significance of whey protein hydrolysate on anti-oxidative, ACE-inhibitory and anti-inflammatory activities and release of peptides with biofunctionality: an in vitro and in silico approach.

The study aimed to investigate potent antioxidant activities (ABTS assay, Hydroxyl free radical scavenging assay, and Superoxide free radical assay), ACE inhibitory activity, and anti-inflammatory activity in the WPC (whey protein concentrate) hydrolysate using Alcalase. The hydrolysis conditions (addition rate and incubation times) for peptide synthesis were also optimized using proteolytic activity. The generation of proinflammatory cytokines by lipopolysaccharide-treated murine macrophages was reduced when the protein hydrolysate concentration was low. In comparison to unhydrolyzed WPC, SDS-PAGE examination revealed no protein bands in WPC hydrolysates. Two-Dimensional (2D) gel electrophoresis did not show any protein spots. Using the 'In-solution trypsin digestion' approach, the trypsin digested protein samples were put into RPLC/MS for amino acid sequencing. Peptides were also identified using RPLC/MS on fractions of 3 and 10 kDa permeates and retentates. The MASCOT database was used to look up the raw masses of LC/MS. By comparing hydrolyzed whey protein to the BLASTp (NCBI), PIR, BIOPEP, and AHTPDB databases, novel antioxidative and ACE inhibitory peptides were reported. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-021-05282-3.

Purification and characterization of antioxidative and antimicrobial peptides from lactic-fermented sheep milk.

This study aims to identify antioxidant and antimicrobial peptides from sheep milk produced using Lactobacillus plantarum (KGL3A). It was inferred that antioxidative and antimicrobial activities increased with increasing incubation time, and antioxidative properties (ABTS assay, superoxide free radical & hydroxyl free radical scavenging activity were 34.5, 34.7, and 29.2% respectively) and antimicrobial properties against Escherichia coli, S. typhimurium, E. faecalis, & B. cereus were 11.3, 12.7, 13.3, & 12.3 mm. However, inoculation of culture at a level of 2.5% and 48 h fermentation give the highest proteolysis activities. Fermented sheep milk fractions of 3 & 10 kDa were analysed for antioxidative and antimicrobial activity, and the 10 kDa permeate showed the highest ABTS assay. The hydroxyl free radical scavenging activity was greatest in 10 kDa retentate and superoxide free radical scavenging activity was observed in 3 kDa permeate (34.7, 43.4, and 34.6%, respectively). Antimicrobial activity of 10 kDa retentate against B. cereus & E. coli (13.3 mm) was greater than 3 and 10 kDa retentate against S. typhimurium (13 mm) and 3 kDa retentate against E. faecalis (13.7 mm). The molecular weight of the protein was estimated using SDS-PAGE. On electrophoresis on a 2-D gel, 6 peptides were identified using RP-LC/MS. BIOPEP, a database for antioxidative and antimicrobial peptides, validated the antioxidative & antimicrobial activities of several peptides in sheep's milk that has been fermented. Sheep milk fermented using Lactobacillus could be considered a novel source of antioxidative and antimicrobial proteins. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05493-2.

Biofunctional Attributes and Storage Study of Soy Milk Fermented by Lactobacillus rhamnosus and Lactobacillus helveticus.

Different soybean products are considered as traditional functional food among the Mongolian population in Northeast India. In the present study, the effect of different flavours (mango, orange, vanilla and white rose), inoculation rates of Lactobacillus rhamnosus K4E (KX950834) and Lactobacillus helveticus K14 (KU644578), and mass fractions of skimmed milk and sugar on the acceptability of soy yoghurts was studied. Physicochemical (pH, titratable acidity) and microbial analyses (total bacterial, total coliform, yeast and mould count) were conducted, and organoleptic (aroma, taste, colour, mouthfeel, texture and overall acceptability) and biofunctional properties (angiotensin-converting enzyme (ACE) inhibitory activity, antioxidant and antimicrobial activities and biotransformation of isoflavones) were evaluated during storage up to 10 days under refrigeration conditions (6-8 °C). Panellists preferred white rose soy yoghurt more than other flavours. The pH was from 5.65 to 4.20, the titratable acidity (expressed as mass fraction of lactic acid) was from 0.33 to 0.51% and total Lactobacillus count ranged from 6.81 to 8.69 log CFU/mL during storage. The ACE inhibitory activity increased from 21.17% on day 0 to 81.03% on day 5, followed by a decrease of the activity after 10 days (38.85%). The antioxidant activity was the highest on day 5 (87%). White rose soy yoghurt had the highest antimicrobial activity against Listeria monocytogenes, followed by Bacillus subtilis, Staphylococcus aureus, Salmonella typhi and Escherichia coli. RP-HPLC analysis showed that after 18 h, the production of soy isoflavone aglycones genistein and daidzein in yoghurt was 87.3 and 58.4%, respectively.

ß-Glucosidase activity and bioconversion of isoflavones during fermentation of soymilk.

BACKGROUND: Lactobacillus rhamnosus C6 strain showed higher ß-glucosidase activity as well as biotransformation of isoflavones from glycones (daidzin and genistin) to aglycones (daidzein and genistein) in soymilk. However, L. rhamnosus C2 and Lactobacillus casei NCDC297 also exhibited similar activity during soymilk fermentation. These three strains can be selected for the development of functional fermented soy foods enriched with aglycone forms of isoflavones, such as soy yoghurt, soy cheese, soy beverages and soy dahi. RESULTS: The study determined ß-glucosidase activity of probiotic Lactobacillus cultures for bioconversion of isoflavones to aglycones in fermenting soymilk medium. Soymilk was fermented with six strains (L. rhamnosus C6 and C2, L. rhamnosus NCDC19 and NCDC24 and L. casei NCDC17 and NCDC297) at 37 °C for 12 h. The highest ß-glucosidase activity and isoflavone bioconversion after 12 h occurred by L. rhamnosus C6 culture during fermentation in soymilk. Increased isoflavone aglycone content in fermented soymilk is likely to improve the biological functionality of soymilk (e.g. antioxidant activity, alleviation of hormonal disorders in postmenopausal women, etc.). CONCLUSION: Lactobacillus rhamnosus C6 culture can be used for the development of functional fermented soy-based products.

Enhancement of survival of alginate-encapsulated Lactobacillus casei NCDC 298.

BACKGROUND: Micro-encapsulation of hydrocolloids improves the survival of sensitive probiotic bacteria in the harsh conditions that prevail in foods and during gastrointestinal passage by segregating them from environments. Incorporation of additives in encapsulating hydrocolloids and coatings of microcapsules further improves the survival of the probiotics. In this study, the effect of incorporation of resistant-maize starch in alginate for micro-encapsulation and coating of microcapsules with poly-l-lysine, stearic acid and bees wax on the survival of encapsulated Lactobacillus casei NCDC 298 at pH 1.5, 2% high bile salt, 65 °C for 20 min and release of viable lactobacilli cells from the capsule matrix in simulated aqueous solutions of colonic pH were assessed. RESULTS: Addition of resistant maize starch (2%) improved the survival of encapsulated L. casei NCDC 298. Coating of microcapsules with poly-L-lysine did not further improve the protection of encapsulated cells from the harsh conditions; however, bees wax and stearic acid (2%) improved the survival under similar conditions. Incorporation of maize starch (2%) in alginate followed by coating of beads with stearic acid (2%) led to better protection and complete release of entrapped lactobacilli in simulated colonic pH solution was observed. CONCLUSION: Additional treatments improve the survival of alginate-encapsulated lactobacilli cells without hindering the release of active cells from the capsule matrix and hence, the resulting encapsulated probiotics can be exploited in the development of probiotic functional foods with better survival of sensitive probiotic organisms.

Incorporation of carbon dots into polyvinyl alcohol/corn starch based film and its application on shiitake mushroom preservation.

Developing eco-friendly edible packaging films with multi-functional properties is highly required. This study involved synthesizing carbon dots (CDs) from dragon fruit, then incorporating them into a composite film based on polyvinyl alcohol (PVA)/corn starch (CS) to create a functional package to extend the shelf life of fresh shiitake mushrooms. Functional composite films with varying levels of CDs were formulated. The films' characteristics of morphology, mechanical properties, antioxidant properties, etc. were then determined, as well as their preservation effect on the fresh shiitake mushrooms. The results showed that the PVA/CS/CDs composite film showed excellent mechanical property, Ultraviolet (UV) barrier capability, antioxidant and antimicrobial properties. Specifically, addition of 8 mg/mL CDs in the composite films reduced weight loss of shiitake mushrooms by 30.74 %, decreased the decline in soluble solids content by 10.48 %, and halved the reduction in total sugar content after a 7-day storage period compared to films without added CDs. Furthermore, the films effectively lowered the respiratory intensity and browning of the mushrooms. This research demonstrates that CDs can serve as an effective component for the development of eco-friendly edible packaging films, as well as for their application in food preservation.

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.

Understanding the Probiotic Bacterial Responses Against Various Stresses in Food Matrix and Gastrointestinal Tract: A Review.

Probiotic bacteria are known to have ability to tolerate inhospitable conditions experienced during food preparation, food storage, and gastrointestinal tract of consumer. As probiotics are living cells, they are adversely affected by the harsh environment of the carrier matrix as well as low pH, bile salts, oxidative stress, osmotic pressure, and commensal microflora of the host. To overcome the unfavorable environments, many probiotics switch on the cell-mediated protection mechanisms, which helps them to survive, acclimatize and remain operational in the harsh circumstances. In this review, we provide comprehensive understanding on the different stresses experienced by the probiotic when added in carrier food as well as during human gastrointestinal tract transit. Under such situation how these health beneficial bacteria protect themselves by activation of several defense systems and get adapted to the lethal environments.

Incorporation of probiotics into 3D printed Pickering emulsion gel stabilized by tea protein/xanthan gum.

Different xanthan gum (XG) concentrations on the rheological/texture properties of Pickering emulsion (PE) gel stabilized by tea protein/xanthan gum (TP/XG) were studied to achieve an ink feasible for 3D printing. Afterwards, the effects of 3D printing and digestion process on the viability of probiotics were studied when encapsulated in the PE gel. Results indicated that gel strength, stability, storage modulus (G') and loss modulus (G″) increased as XG concentration increased. Nozzle diameter and printing temperature of 45 and 55℃ had no significant effect on probiotic's viability, but printing temperature of 65℃ reduced viable probiotics from 8.07 to 6.59 log CFU/g. No significant change of probiotics viability in 3D printed samples was observed during 11-day storage at 4℃. PE gel encapsulated probiotic's viability was significantly improved under heat treatment and simulated gastrointestinal environment. This study gives insights on the production of 3D printed foods using PE gel incorporating probiotics.

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).

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.

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.

Anti-Inflammatory, ACE Inhibitory, Antioxidative Activities and Release of Novel Antihypertensive and Antioxidative Peptides from Whey Protein Hydrolysate with Molecular Interactions.

OBJECTIVE: The aim of the study was to evaluate the whey protein hydrolysate with bio-functional attributes viz. antioxidative, anti-inflammatory and ACE inhibition efficacy and release of bioactive peptides with antioxidative and ACE-inhibitory activity by employing Pepsin. METHOD: The antioxidant, Anti-inflammatory, ACE inhibitory and proteolytic activities of the whey protein hydrolysates were studied followed by SDS-PAGE analysis and IEF. Anti-inflammatory activity of whey protein hydrolysate was also studied on RAW 264.7 cell line. The separation of the bioactive peptides from whey protein hydrolysate was achieved by RP-HPLC. The purified bioactive peptides were identified and characterized using RPLC/MS. RESULTS: WPC (Whey protein concentrate) hydrolysate with pepsin showed proteolytic activity ranging between 14.46 and 18.87 mg/ml. Using the ABTS assay, the highest antioxidative activity was observed in 10 kDa retentate (84.50%) and 3 kDa retentate (85.96%), followed by the highest proteolytic activity (13.83 mg/ml) and ACE inhibitory activity (58.37%) in a 5% WPC solution at 65 °C after 8 h of pepsin hydrolysis. When the protein hydrolysate concentration was low, the production of proinflammatory cytokines by lipopolysaccharide-treated murine macrophages (RAW 264.7) was reduced. SDS-PAGE results exhibited very little protein bands when comparing with WPC hydrolysates to insoluble WPC. There were no protein spots on 2 D gel electrophoresis and "in-solution trypsin digestion" technique have been utilized to digest protein samples directly from WPC hydrolysates. Novel antioxidative peptides and ACE inhibitory peptides were also observed by comparing two databases, i.e., BIOPEP and AHTPDB respectively. The peptide sequences used in this study were found to have excellent potential to be used as inhibitors of hACE as all of them were able to show substantial interactions against the enzyme's active site. CONCLUSIONS: The antihypertensive and antioxidative peptides from whey protein hydrolysates may be beneficial for the future development of physiologically active functional foods. Further, in vivo investigations are required to establish the health claim for each individual bioactive peptide from whey protein hydrolysate.Supplemental data for this article is available online at.