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  • Title: Purification and Characterization of Novel Antihypertensive and Antioxidative Peptides From Whey Protein Fermentate: In Vitro, In Silico, and Molecular Interactions Studies.
    Author: Chopada K, Basaiawmoit B, Sakure AA, Maurya R, Bishnoi M, Kondepudi KK, Solanki D, Singh BP, Padhi S, Rai AK, Liu Z, Mishra BK, Hati S.
    Journal: J Am Nutr Assoc; 2023 Aug; 42(6):598-617. PubMed ID: 36416542.
    Abstract:
    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.
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