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 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.