Preparation, Isolation, Purification And Identification Of Intestinal Mucosal Antioxidant Active Peptides

Intestinal mucosal protein was a kind of nutritious animal protein material by intestinal mucosal hydrolysis,not only rich in protein,fat,fiber and calcium and other minerals,a variety of amino acids,peptides and nucleotides,if the direct discharge of sewage,may cause the protein resource waste and environmental pollution in large amount.Preparation of active polypeptide by using natural casings processing by-products of intestinal mucosa,improved the utilization of animal by-products recycling,recycling can not only realize the animal by-products resources,enhanced the protein ratio of resources,and also reduced environmental pollution from the source,to promote the sustainable development of natural casings.In current study,raw pig sausage processing by-product intestinal mucosa was used as raw material.Preparation of intestinal mucosal polypeptide by enzymatic hydrolysis.The antioxidation activity of intestinal mucosa peptides was evaluated by the method of in vitro chemistry and animal model.Then used ultrafiltration and gel filtration chromatography separation technology on intestinal mucosa peptide purification step by step,and by capillary high performance liquid chromatography(HPLC)coupled to the Q-Exactive mass spectrum of the amino acid sequence of target peptide identification.The main results were as follows:1.Intestinal mucous membrane protein content was 57.14%,and contained 17 kinds of free amino acid,the main antioxidative amino acids(Tyr,Met,ihs,Cys,Phe)content was 15.22%,the amino acid was an important material of intestinal mucosa with antioxidant activity.Papain,trypsin and pepsin hydrolysis of intestinal mucosa,degree of hydrolysis as evaluation standard,the result showed that papain hydrolysis effect was significantly better than that of pepsin and trypsin hydrolysis effect(P<0.05).Through the single factor experiment and response surface method optimization in intestinal mucosa enzyme hydrolysis conditions,the optimum condition for papain enzyme quantity was 4500 U/g,hydrolysis time 6.6 h,hydrolysis temperature 47?,solid-liquid ratio 1:5,the resulting peptides content was 50.17%.Best condition for trypsin enzyme quantity was 4800 U/g,hydrolysis time 7.2 h,hydrolysis temperature 54 ?,solid-liquid ratio 1:5,the resulting peptide content was 45.25%,actual value and the model prediction of two protease was no significant difference(P>0.05).2.The antioxidant activity of intestinal mucosa peptides was evaluated by four methods of external chemistry and animal model.In vitro antioxidant research results showed that intestinal mucosa polypeptide was clearing DPPH free radicals,reducing power,and ability of chelating metal ion and the ability to inhibit lipid peroxidation,papain hydrolysis of intestinal mucosa polypeptide showed better antioxidant activity in vitro.Set up feeding papain preparation of intestinal mucosa polypeptide of the subacute aging model mice induced by D-galactose,aging model group and positive control group compared with intestinal mucosa peptide treatment group,the results showed that injection of D-galactose of heart,liver and serum MDA content in mice were 60.10 nmol/mg protein,107.24 nmol/mg protein,46.59 nmol/mL serum was significantly higher than other groups(P<0.05),and reduced the body's antioxidant enzyme activity,proves the validity of this old model.Antioxidant enzymes activity of mice aging model group was significantly lower than the blank control group,peptide treatment group had a significantly higher antioxidant enzyme activity of mice blank control group(P<0.05),while the MDA content was significantly lower than the blank control group(P<0.05),suggesting papain preparation of intestinal mucosa polypeptide has antioxidant activity in the body.In vitro experiments and in vivo experiment fully demonstrated that intestinal mucosa peptides have antioxidant activity.3.A series of separation techniques of ultrafiltration,gel filtration chromatography and capillary high performance liquid chromatography were used to isolate and purify intestinal mucosa polypeptide.The ultrafiltration of intestinal mucosa was separated into two components:RSH-I(MW>3.0 kDa),RSH-II(MW<3.0 kDa),the molecular weight was less than 3.0 kDa peptide intestinal mucosa antioxidant activity were significantly higher(P<0.05)in intestinal mucosa of molecular weight greater than 3.0 kDa peptide antioxidant activity.The components A1 and A2 were obtained after the composition of the components RSH-II by gel-25 gel chromatography,and the group A2 with the lower molecular weight showed better anti-oxidative activity.The two amino acid sequences of target peptides were obtained by using capillary high performance liquid chromatography tandem q-exactive MS to identify the intestinal mucosal antioxidant peptides,and the two amino acid sequences of target peptides were respectively Asp-Gly-Leu-Lys-Asn-Asn-Leu-Asn-Leu-Lys(1152.64 Da)and Asn-Asp-Trp-His-Leu-Ala-Val-Lys(1226.58 Da).Antioxidant activity of the purified peptide and synthetic peptide that there was no significant difference(P>0.05),and that proved the Asp-Gly-Leu-Lys-His-Leu-Asp-Asn-Leu-Lys and Asn-Asp-Trp-Glu-Asp-His-Leu-Ala-Val-Lys were major antioxidant peptides in the intestinal mucosa.