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.

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.

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.