Extraction Of Active Substances Inhibiting Xanthine Oxidase From Kidney Beans And Preparation Of Dietary Supplements

Hyperuricemia(HUA)is a common metabolic disorder whose incidence is increasing worldwide.But most of the drugs have certain side effects.Xanthine oxidase(XO)is related to purine metabolism in the body and can regulate the production of uric acid.Previous studies in our laboratory have shown that the protein hydrolysates derived from kidney bean has good XO inhibitory activity.Based on the fact that anthocyanin also has XO inhibitory activity,this project used purple-flowered kidney beans as raw material to make full use of kidney bean anthocyanin and peptides resources.The structure of kidney bean peptides was identified by liquid-mass spectrometry,and then the mechanism of action with XO was explored by molecular docking technology.Then,the alcohol extract containing anthocyanins was compounded with the protease hydrolysates,and the stability of the compound was investigated.Finally,a dietary supplement for HUA inhibition was designed and developed.The main findings of the study were as follows:1 Preparation of kidney bean alcohol extracts and kidney bean proteinThe extraction conditions of the alcoholic extract of kidney beans were determined by single-factor and orthogonal tests using the acid-alcoholic extraction method with the content of anthocyanin as the index.The extraction conditions were as follows:the concentration of ethanol was 65%,the liquid-solid ratio was 20:1,the extraction temperature was 60℃,the extraction time was 55 min,the concentration of hydrochloric acid was 1.0%.Under these conditions,the anthocyanins content in the alcohol extracts were 10.29 mg/100g.Then,the kidney bean protein was extracted by the alkali.The optimal extraction scheme of protein was determined:the concentration of alkali was 0.05 mol/L,the liquid-solid ratio was 15:1,the extraction temperature was 55℃,and the extraction time was 1 h.Under these conditions,the protein extraction rate was 86.71±0.06%.Amino acid analysis of kidney bean protein showed that its essential amino acid content was about 43.15%,and the ratio of essential amino acids and non-essential amino acids was about 0.759,which was a high-quality protein source.It was rich in aromatic amino acids and hydrophobic amino acids,and had a potential active basis for inhibiting XO.2 Preparation and molecular docking study of kidney bean protease hydrolysatesThe hydrolysis degree of kidney bean protein and the inhibitory activity of XO were taken as indicators.The optimal conditions for the proteolysis of kidney bean were determined as follows:the protein substrate concentration was 3%,the amount of enzyme added was 4%,and the time was 1.5 h.Under the conditions,the XO inhibition rate was 35.14%.HPLC-ESI-MS/MS technology was used to identify the structure of kidney bean protease hydrolysate,and the peptides with the top 100 Abundances values were molecularly docked to the MOS and FAD active sites of XO.It was found that peptides could inhibit XO activity by competitive and non-competitive inhibition.There are 58 peptides that bind to the MOS active site,and 100 peptides that bind well to the FAD active site.The two peptides with the highest scores for molecular docking,ISSTE and VLVKPDDRRE,were selected for visual analysis.Both peptides form stable complexes by forming hydrogen bonds and hydrophobic interactions with amino acid residues surrounding the active site of XO.3 Compounding of alcoholic and enzymatic extracts and their product developmentA compounding ratio of 1:0.8 was determined based on the yield of the raffinose alcoholic extract and proteolytic extract,and at this compounding ratio the XO inhibition was 66.80±0.62%.The stability of the complexes under temperature,pH,artificial gastrointestinal fluid,oxidant,light and storage conditions was further investigated.The results showed that the activity of the complexes increased after simulated digestion in vitro.Light and short-term heat treatment had little effect on the XO inhibitory activity of the complexes.It is suitable for storage at low temperatures and should be avoided in contact with oxidants.After that,a dietary supplement to inhibit HUA was designed.The formula was:whey protein powder added 6.00 g,casein added 1.16 g,maltodextrin added 3.20 g,oat flour added 2.00 g,vegetable fat powder added 3.17 g,skimmed milk powder added 5.00 g,corn flour added 6.40 g,rice flour added 10.26 g,oligosaccharide maltose added 2.81 g,1.60 g for multivitamins and minerals,1.20 g for complexes and 0.43 g for sodium carboxymethylcellulose(CMC-Na).This formula is rich in protein and has a high nutritional value.The dietary supplements supplemented with different concentrations of CMC-Na were subjected to rheological measurements.All experimental groups showed non-Newtonian shear thinning behavior and different degrees of lag.Combining the results of time-dependent tests and frequency scans,the dietary supplement was stable at 1.0%CMC-Na addition.The dissolvability and powder characteristics of dietary supplements showed that dietary supplements should be eaten with hot water with a temperature higher than 60℃,the solubility of the dietary supplement was good on this condition,the wetting and sinking property of the dietary supplement is 145 s,the dispersion time is 23.67 s.The angle of repose,Carr index,Hausner ratio and bulk density of the dietary supplement were 39.13°,30.63%,1.44 and 0.48 g/mL,respectively,which could meet the production needs of factory applications.