A comparative study of fermented buffalo and camel milk with anti-inflammatory, ACE-inhibitory and anti-diabetic properties and release of bio active peptides with molecular interactions: In vitro, in silico and molecular study

The aim of this study is to compare ACE inhibitory,anti-diabetic and Anti-inflammatory activity of buffalo and camel milk using Lacticaseibacillus paracasei(M11)in combination with yeast Saccharomyces cerevisiae(WBS2A)and to release peptides with antihypertensive and anti-diabetic properties.Fermented buffalo and camel milk were considered for the evaluation of Angiotensin-converting enzyme(ACE)inhibitory and anti-diabetic activities at specific time intervals(12,24,36 and 48 h)at 37℃.For the growth condition optimization,proteolytic activity was evaluated with specific inoculation rates(1.5%,2.0%,and 2.5%)and at specific incubation periods(12,24,36,and 48 h).The highest peptides were produced at 2.5%inoculation rate and at 48 h of incubation in both buffalo and camel milk at 37℃.Further,camel and buffalo milk fermented with M11+WBS2A significantly lower the overproduction of TNF-α,IL-6,IL-1βand Nitric oxide(NO),that was induced by lipopolysaccharide(LPS)stimulation in RAW 267.4 cells.For protein purification,2D gel electrophoresis and SDS-PAGE were used.Camel and buffalo milk that had not been fermented displayed protein bands between 10 and 100 kDa and 10 and 75 kDa respectively,whereas all the fermented samples showed bands between 10 and 75 kDa.On SDS-PAGE,in permeates,there were no apparent protein bands.When fermented buffalo and camel milk were electrophoresed in 2D gel,a total of 26 and 25 protein spots were found.In 2D gel electrophoresis,protein spots had sizes between 10 and 37 kDa.In RP-HPLC,water-soluble extracts(WSEs)of ultra-filtered fractions were used to distinguish different fractions of a peptide.Peakview software was used to characterize the LC/MS results.Fourier transform-infrared spectroscopy(FTIR)was used to evaluate various functional groups of fermented milks.In molecular docking study,the peptide sequences identified from the fermented buffalo milk(FBM)and camel milk(FCM)were examined for their binding affinity against the binding sites of hPAM and hMGA.

Exploring the potential of Lactobacillus and Saccharomyces for biofunctionalities and the release of bioactive peptides from whey protein fermentate

Whey protein concentrate-80(WPC-80)fermented with L.fermentum(KGL4)(37℃)and S.cerevisiae(WBS2A)(25℃)was tested for ACE-inhibitory and antioxidant activities over different periods(12,24,36 and 48 h).Proteolytic activity(OPA method)was used to optimize the growth conditions(inoculation rate,i.e.at 1.5%,2.0%,and 2.5%and incubation time,i.e.12,24,36,and 48 h)for peptide production.Results indicated that the highest amount of peptides was obtained at 7.24 mg/mL for KGL4(37℃,48 h)and 8.59 mg/mL for WBS2A(25℃,48 h).The whey protein fermentate inhibited the LPS-induced NO production,while enhanced production concentrations of TNF-α,IL-6,and IL-1β.Subsequently,SDS-PAGE,as well as Two-Dimensional(2D)gel electrophoresis methods,were applied for protein purification using water-soluble extracts(WSEs)of WPC-80 fermented by a combination of L.fermentum and S.cerevisiae.On SDS-PAGE,protein bands were observed in the range of 10-55 kDa,whereas on the 2D page,protein spots were in the range of 10-70 kDa.All the 2D spots were analyzed using RPLC/MS.WSEs of 3 kDa and 10 kDa permeates were used in RP-HPLC to identify distinct peptide fractions.The data from LC/MS was also characterized by utilizing ProteinPilot software.Further,different functional groups were also analyzed using FTIR investigation.The research aims to isolate and characterize novel ACE-inhibitory and antioxidative peptides from fermented WPC-80 produced by Lactobacillus fermentum and S.cerevisiae.