Enzymatic Preparation,Characterization And Application Of Leaf Peptide Derived From Millettia Speciosa Champ.

Millettia Speciosa Champ.(M.speciosa)is resource rich,and primarily distributed in subtropical regions,such as Guangdong and Guangxi Provinces.Its dried roots can be used in medicine because it can tonify deficiencies,moisten lungs,strengthen tendons,and activate collateral.In addition,the leaves of M.speciosa can also be used for steeping in water or making soup,which dissipates internal heat,removes toxins,relieves inflammation,and invigorates blood circulation.As a traditional Chinese medicine that can be used as both food and medicine,M.speciosa,has high nutritional and healthcare values,but research into its active components is still not thorough.In this study,the leaf protein of Millettia Speciosa Champ.(MLP)were extracted by the alkali extraction and acid precipitation method,and its physicochemical and functional properties were investigated.Then,M.speciosa leaf peptide(MLPP)was prepared by enzymatic hydrolysis,and the deep structure-activity relationships of the bioactive peptides were explored.by separation and purification,structure identification,as well as study of antioxidant activity and ACE inhibition activity.Finally,the biological activity of nano-selenium was enhanced,and the peptide application range was widened by using MLPP as the template regulator for the nano-selenium.The experimental results from this study are:1.Alkali extraction and acid precipitation were used to extract MLP.Through the single factor test and response surface test,the optimal extraction conditions were determined to be:solid-liquid ratio of 1:40(w/v),extraction time of 2.0 h,and extraction temperature of 50?.The results from the Fourier transform infrared spectroscopy(FTIR)and Circular dichroism(CD)spectra analysis exhibited that the main structure of MLP contained ?-fold and ?-corner.By SDS-PAGE analysis,molecular weight of MLP was in the range of 14.4-18.4 k Da.In addition,the amino acid composition and functional properties of MLP suggested that it is superior and could be considered a kind of high-quality natural protein.Meanwhile,the amino acid composition of MLP indicated that it was a high-quality natural protein with abundant amino acids,and the ratio of essential amino acids to non-essential was 65.37%,which was higher than the 60% established by the WHO/FAO ideal protein standards.Among them,the relative content of glutamic acid was the highest,reaching 13.72%,while the relative content of methionine was the lowest,reaching 1.20%,which was the first restricted amino acid.The water holding capacity and oil absorption capacity are 4.85±0.19% and 7.52±0.22 g/g,respectively.The precipitation point is about p H 3.0,where the solubility is at a minimum,and the foaming stability and emulsification stability are excellent.The foaming and emulsifying stability of MLP were better when the p H was 7.0.2.Four proteases,Trypsin,Alcalase,Neutrase,and Papain,were selected to hydrolyze MLP under their respective suitable conditions.The results indicated that Alcalase was the best enzymatic protease because its hydrolysis speed was relatively quick,while the antioxidant activity of hydrolyzed peptides was relatively strong.Then,response surface methodology was used to explore the effects of different enzymatic hydrolysis temperatures,enzyme dosages,and hydrolysis times on the antioxidant activity of MLPP to optimize enzymatic hydrolysis conditions,which were finally determined to be: the enzymatic hydrolysis temperature 55?,enzyme dosage 3%,and enzymatic hydrolysis time 5 h.Under these conditions,MLPP exhibited strong antioxidant activity,even exceeding ascorbic acid in a certain concentration range,and also possessed great ACE inhibitory activity.The main secondary structure of MLPP is ?-fold,and which is obviously different from MLP in a lamellar structure.MLPP is primarily tubular and spherical.Additionally,according to the molecular weight distribution,small peptides(below 5 k Da)accounted for 93.52%.G1 and G2 components were isolated and purified by gel chromatography,and the latter exhibited considerable antioxidant and ACE inhibitory activities.G2 was isolated and identified by UPLC-MS/MS,and the four peptides were obtained,with amino acid sequences that were Gly-Asp,Asp-Pro,Glu-Ala,and Glu-Met,and the molecular weights were 195.1 Da,225.1Da,229.1 Da,and 279.2 Da,respectively.MLPP and its two separated components possessed complete amino acid compositions,and the essential amino acids accounted for 32.38%,39.36%,and 47.62%,respectively.Glutamic acid(Glu),aspartic acid(Asp),leucine(Leu),and valine(Val)are the most abundant amino acids.3.Using MLPP as the regulation template for nano-selenium,MLPP-Se NPs were prepared in a reduction system of sodium selenite and ascorbic acid.The effects of different volume ratios,substrates,and concentrations on the particle sizes and distributions of nano-selenium were studied.When the volume ratio of sodium selenite,ascorbic acid: and bovidine leaf protein peptide was 1:1:2,and the substrate concentration was 1.0 mg/m L,the MLPP-Se NPs with ideal morphology,an average particle size of 80-100 nm,uniform distribution.And stable 20-day storage could be prepared under these conditions.Additionally,the results suggested that in the reaction process,MLPP and nano-selenium may be through the interaction of Se-O,Se-N and physical adsorption.MLPP-Se NPs has considerable antioxidant activity and has certain inhibitory effects on Escherichia coli and Staphylococcus aureus.Moreover,the cytotoxicity test indicated that MLPP,MLPP-Se NPs,and sodium selenite all possessed dose-dependent inhibitory effects on Hep G2 cells.The toxicity of the leaf protein peptide was the lowest,followed by MLPP-Se NPs and sodium selenite.