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.

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.

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.

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.

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.

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.

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).

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.

Significance of Limosilactobacillus fermentum and Saccharomyces cerevisiae on the Growth Performance, Haematological Traits, Serum Biochemistry, Faecal and Caeca Microbiota of Broiler Chickens.

OBJECTIVE: The aim of the study was to supplement Lactobacillus and yeast in broiler feed by replacing immunomodulators to develop antibiotic free meat and egg production by analyzing broiler performance, haematological traits, serum biochemistry, histopathology, fecal bacterial count, and metagenomic analysis of broiler ceca. METHOD: Two cultures i.e. KGL4 (Limosilactobacillus fermentum MTCC 25515) and WBS2A (Saccharomyces cerevisiae GI: MG101828) were considered for the evaluation of Broiler chicken's health and growth during 42 days study without supplementing immunomodulators and commercial probiotics in poultry feeds. The 96-day-old broiler chickens were grouped into: T1 [Control: basal diet + immunomodulatory factor and commercial probiotic], T2 [Basal diet without immunomodulatory factor and commercial probiotic + KGL4 (108 CFU/mL), T3 [Basal diet without immunomodulatory factor and commercial probiotic + WBS2A (107 CFU/mL), and T4 [Basal diet without immunomodulatory factor and commercial probiotic + KGL4 + WBS2A in a 1:1 ratio] (Institutional Animal Ethics Committee (IAEC) No. 365/PRS/2022). The following parameters, i.e., body weight gain, feed consumption ratio (FCR), white blood cell count (WBC), red blood cell count (RBC), hemoglobin content, platelet count, cholesterol content, triglycerides, high density lipoprotein (HDL), very low-density lipoprotein (VLDL), fecal counts and metagenomic analysis of broiler ceca samples, were measured. RESULTS: In the study, amongst various traits, the overall performance of the group treated along with Limosilactobacillus fermentum (KGL4) showed improved results as compared to control group. Limosilactobacillus fermentum (KGL4) treated group had higher body weight gain (2583.04 ± 35.421 g), FCR (1.60 ± 0.019), WBC (235.60 ± 2.562 × 103/µL), hemoglobin content (14.10 ± 0.442 g/dl), and HDL (131.40 ± 11.400 mg/dl). The investigation did not show significant variations in the relative proportions of genus or phylum among various groups during metagenomic analysis of ceca samples. There was also an improvement in haematological traits; no evidence of necrosis in heart, intestine and liver tissues. CONCLUSIONS: The present study conclude that it is safe to feed Limosilactobacillus fermentum and Saccharomyces cerevisiae to broilers as feed supplements and also supports the current knowledge regarding the use of yeast and lactic acid bacteria as an effective alternative stimulant for maintaining health and growth of broiler chickens.

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.

Purification and Characterization of Novel Antihypertensive and Antioxidative Peptides From Whey Protein Fermentate: In Vitro, In Silico, and Molecular Interactions Studies.

OBJECTIVE: The goal of this research was to purify and characterize the novel angiotensin-converting enzyme (ACE)-inhibitory and antioxidant peptides from fermented whey protein concentrate produced by Lactobacillus paracasei and Saccharomyces cerevisiae in a co-fermentation system. METHOD: Whey protein fermented with lactic acid bacteria and yeast culture was analyzed for antioxidative, ACE inhibition, as well as anti-inflammatory activity followed by SDS-PAGE, isoelectric focusing, and 2-dimensional (2D) analysis. Anti-inflammatory activity of whey protein fermentate was also studied on the RAW 264.7 cell line. The bioactive peptides were separated from the whey protein fermentate using reverse-phase high-performance liquid chromatography (RP-HPLC) and reverse-phase liquid chromatography mass spectrometry (RPLC/MS), and thus identification and characterization of purified bioactive peptide was performed. RESULTS: Whey protein fermentate samples' bioactivity was analyzed at specific time intervals at 12, 24, 36, and 48 hours at 37 °C for M11 and at 25 °C for WBS2A. The development settings (incubation time [12, 24, 36, and 48 hours) and inoculation rates [1.5%, 2.0%, and 2.5%]) were optimized for peptide synthesis via the o-phthaldialdehyde (OPA) method (proteolytic activity). Maximum proteolytic activity was observed at 37 °C for M11 (6.50 mg/mL) and at 25 °C for WBS2A (8.59 mg/mL) for 48 hours of incubation. Protein profiling was carried out using SDS-PAGE and 2D gel electrophoresis, in which Sodium dodecyl-sulfate (SDS) exhibited protein bands in the 10- to 55-kDa range, while 2D showed protein bands varying from 10 to 70 kDa. Every spot from 2D was digested by trypsin and identified by RPLC/MS. Protein fractionations (3- and 10-kDa permeates) were carried out employing RP-HPLC. Whey protein fermentate has anti-inflammatory action in RAW 264.7 macrophages that have been exposed to lipopolysaccharide. A molecular docking system was also used to investigate the interactions of peptides (AFLDSRTR, ILGAFIQIITFR) with human myeloperoxidase enzyme. CONCLUSIONS: The antihypertensive and antioxidative peptides discovered from whey protein fermentate may be helpful in the design of pharmacologically active healthy ingredients in the upcoming years.

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.

Exploring the potential of Lacticaseibacillus paracasei M11 on antidiabetic, anti-inflammatory, and ACE inhibitory effects of fermented dromedary camel milk (Camelus dromedaries) and the release of antidiabetic and anti-hypertensive peptides.

The goal of this investigation was to find antidiabetic peptides and inhibit angiotensin converting enzyme (ACE) in Lacticaseibacillus paracasei (M11) fermented dromedary camel milk (Camelus dromedaries). According to the findings, the rate of antidiabetic activity increased along with the incubation periods and reached its peak after 48 hr of fermentation. The inhibitions of α-amylase, α-glucosidase, and lipase were 80.75, 59.62, and 65.46%, respectively. The inhibitory activity of ACE was 78.33%, and the proteolytic activity was 8.90 mg/mL. M11 at 0.25 mg/mL effectively suppressed LPS-induced pro-inflammatory cytokines and their mediators such as NO, TNF-α, IL-6, and IL-1ß in RAW 264.7 cells. The rate of inoculum in the optimization phase was 1.5-2.5%, and the greatest proteolytic activity was observed after 48 hr of fermentation. The investigation of the above property in the ultrafiltered fermented milk exhibited the highest antidiabetic and ACE inhibition activities in the 3 kDa than 10 kDa fractions. The molecular weight was determined employing SDS-PAGE, and the six-peptide sequences were identified using 2D gel electrophoresis. Due to its high proteolytic activity, the L. paracasei strain has been reported to be useful in the production of ACE-inhibitory and antidiabetic peptides. Amino acid sequences such from ɑ1, ɑ2, and ß-caseins have been identified within fermented camel milk by searching on online databases, including BIOPEP (for antidiabetic peptides) and AHTPDB (for hypertension peptides) to validate the antidiabetic and ACE-inhibitory actions of several peptides. PRACTICAL APPLICATIONS: The study aims to identify antidiabetic peptides and inhibit ACE in dromedary camel milk fermented with Lacticaseibacillus paracasei M11. Maximum antidiabetic and ACE-inhibitory actions of the fermented camel milk were observed in 3 kDa permeate fractions. Fermented camel milk significantly reduced the excessive TNF-α, IL-6, and IL-1ß production in LPS-activated RAW 264.7 cells. RP-LC/MS was used to identify 6 bioactive peptides from dromedary fermented camel milk. This fermented camel milk could be used for the management of hypertension and diabetic related problems.

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.

Smartphone-Based Image Analysis for Rapid Evaluation of Kiwifruit Quality during Cold Storage.

As a vitamin C-rich fruit, choosing the eating time for kiwifruit with the best quality during the shelf period is still a problem for consumers. This paper mainly focuses on the correlation between cold storage time, quality indexes, volatile flavor compounds of postharvest kiwifruit and RGB value readouts from photos taken by mobile phone. Results indicated that the R to B ratio values (Central R/B) and B to G ratio values (Central B/G) of the central site of kiwifruit were strongly associated with storage time and all quality indicators. The central R/B was negatively correlated with titratable acidity, vitamin C and 2,6-Nonadienal contents and firmness and positively correlated with storage time, weight loss, soluble solids content, total soluble sugars, total plate counts and 1,3-Cyclooctadiene. We provide a novel and smart strategy to predict the shelf life and quality parameters of kiwifruit by capturing and calculating RGB values using a smartphone.

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.

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.

Current Trends and Applications of Food-derived Antihypertensive Peptides for the Management of Cardiovascular Disease.

Food-derived antihypertensive peptides are considered a natural supplement for controlling hypertension. Food protein serves as a macronutrient and acts as a raw material for the biosynthesis of physiologically active peptides. Food sources, like milk and milk products, animal proteins such as meat, chicken, fish, eggs, and plant-derived proteins from food products like soy, rice, wheat, mushroom, and pumpkins contain higher quantities of antihypertensive peptides. The food-derived antihypertensive peptides can suppress the action of renin and the angiotensinconverting enzyme (ACE), which are mainly involved in the regulation of blood pressure by RAS. ACE inhibitory peptides enhance endothelial nitric oxide's biosynthesis, which increases nitric oxide production in vascular walls and encourages vasodilation. The peptides also inhibit the interaction between angiotensin II and its receptor, which helps reduce hypertension. This review explores the novel sources and applications of food-derived peptides for the management of hypertension.