In Vitro Fermentation Characteristics Of Mulberry Leaf Albumin And Enzymatic Hydrolysates And The Alleviating Effect Of Enzymatic Hydrolysates On DSS-induced Ulcerative Colitis

Mulberry(Morus alba L.)is a medicinal and edible plant.Mulberry leaf has a long-time history being consumed as foods and functional foods.Mulberry leaf albumin(MP),as the main component of mulberry leaf protein,has good antioxidant activity.Mulberry leaf protein hydrolysate(HMP)prepared by controlled enzymatic hydrolysis technology also showed high antioxidant activity.This article aims to explore the functional activities of mulberry leaf albumin and protein hydrolysate and their effects on the gut microbiota.Firstly,the static in vitro simulated digestion was used to simulate the gastrointestinal digestion of mulberry leaf albumin and protein hydrolysate,and the molecular size changes after digestion were analyzed by SDS-PAGE.Then,the albumin and protein hydrolysate before and after digestion were simulated in vitro to analyze the influence of the protein and hydrolysate on the intestinal flora and the production of metabolite organic acid.Secondly,FBS(fetal bovine serum)-induced steatosis cell model was used to preliminary study the lipid-lowering activity of albumin and protein hydrolysate before and after digestion.The anti-inflammatory activity of protein hydrolysate was explored through an in vitro inflammatory cell model.Finally,a mouse model of colitis induced by DSS(Dextran Sodium Sulfate)was used to evaluate the anti-inflammatory activity of protein hydrolysate intervention.The main research conclusions are as follows:1.By using static in vitro simulated digestion experiments,it was found that the two subunits with a molecular size of 245 and 70 k Da were degraded after gastric digestion,and only the two subunits with molecular weights of 55 and 10 k Da were retained.After digestion in the small intestine,some of the subunits with molecular weights of 55 and 10 k Da in mulberry leaf protein remained.SDS-PAGE electrophoresis found that the molecular size of the protein hydrolysate was less than 10 k Da.These results indicate that mulberry leaf albumin is only partially degraded after simulated gastrointestinal digestion.2.The mulberry leaf albumin and protein hydrolysate were fermented in vitro using the stool samples of volunteers as inoculum.The results showed that: compared with the blank group,the content of SCFAs(acetic acid,propionic acid,butyric acid)and BCFAs(isobutyric acid,isovaleric acid)increased significantly(p <0.05).After fermentation for 24 h,it was found that except for the DMP group(simulated digested mulberry leaf albumin group),the butyric acid production of the other groups was not significantly different from that of the inulin group.The main reason is that glutamic acid,the highest content of mulberry leaf albumin and protein hydrolysate,is fermented to produce butyric acid.Due to the presence of branched-chain amino acids(Leu,Val,Ile)in mulberry leaf albumin and protein hydrolysate,the content of isobutyric acid and isovaleric acid is higher than that in blank group and inulin group,and the content of branched-chain amino acid in protein hydrolysate is higher than that in mulberry leaf albumin,so the BCFA content produced after fermentation of protein hydrolysate is higher.3.The results of intestinal flora analysis in in vitro fermentation showed that:(1)albumin and protein hydrolysate can reduce the index F/B(the ratio of Firmicutes to Bacteroidetes)associated with obesity,indicating that proteins and peptides may have the effect of improving obesity;(2)Albumin and protein hydrolysate can promote the increase of the relative abundance of anti-inflammatory-associated bacteria(Phascolarctobacterium,Parabacteroides,Oscillospira),which demonstrated that proteins and protein hydrolysate may be able to regulate the intestine by gut microbiota to achieve the anti-inflammatory effect.Also,the absolute quantitation of Akkermansia muciniphila using q PCR found that mulberry leaf protein hydrolysate without simulated digestion can promote its growth.These results indicate that mulberry leaf albumin and mulberry leaf protein hydrolysate may have the potential to improve obesity and anti-inflammatory effects.4.Fetal bovine serum(FBS)?induced steatosis in human liver cell line L02 was used to study the lipid-lowering activity of the mulberry protein and mulberry peptide before and after digestion in the stomach and small intestine.Studies have shown that 1000 μg/m L MP(mulberry leaf albumin),HMP(mulberry leaf protein hydrolysate)and DHMP(mulberry leaf protein hydrolysate after gastrointestinal digestion)can significantly improve fat accumulation and reduce TG/TC content,of which MP and DHMP were the most significant,TG/TC content showed no significant difference from the blank group.It shows that MP and DHMP have good lipid-lowering activity at this concentration.In the LPS-induced macrophage inflammation model experiment,it was found that different concentrations of HMP(mulberry leaf protein hydrolysate)have different anti-inflammatory effects,and the concentration of 125 μg/m L HMP has the best anti-inflammatory activity.5.An in vivo mouse model of colitis was used to explore the alleviating effect of the protein hydrolysate on colitis.Mulberry leaf protein hydrolysate(HMP)has antioxidant and anti-inflammatory activities in vitro.However,there is no relevant research on the preventive effect of the protein hydrolysate in inflammatory bowel disease in vivo.To evaluate the anti-inflammatory capacity and mechanism of protein hydrolysate,male C57BL/ 6 mice were given intragastric PH for 14 days(1-14 d,PHD group,preventive intervention group)and 7 days(8-14 d,DPH group,therapeutic intervention group),and using 3% dextran sodium sulfate(DSS)to induce acute colitis through drinking water(8-14 d).The results showed that both the PHD group and the DPH group could increase feed intake,body weight,and colon length,reduce the DAI score and histological score of IBD mice,reduce goblet cell depletion and mucus destruction,inhibit proinflammatory cytokines,and improve the structure of intestinal flora,such as improving the alpha diversity of intestinal flora,alleviating the imbalance of F/B value,increasing the relative abundance of beneficial bacteria Lactobacillus,reducing the relative abundance of harmful bacteria Escherichia-Shigella and increasing the production of SCFA.In addition,during the onset of inflammation,PHD is more effective in promoting goblet cell maturation and increasing mucin secretion than DPH.However,DPH increases the alpha diversity of intestinal flora.In conclusion,the intervention of mulberry peptide can alleviate DSS-induced colitis by inhibiting proinflammatory cytokines,improving the mucosal barrier,and regulating the intestinal flora.In addition,preventive intervention is superior to therapeutic intervention.These results suggest that protein hydrolysate may be a novel and attractive dietary intervention that can alleviate the condition of patients with colitis.