Study On Isolation And Purification,Enzymolysis And Digestion Of Phycocyanin

As a natural plant pigment protein with high bioavailability,phycocyanin is powerful and widely used.Starch is an important part of the human diet,and its energy distribution is dense,often causing postprandial blood sugar levels to fluctuate,thereby increasing the risk of chronic diseases.Studies have shown that protein-starch interactions can slow down starch digestion and lower postprandial blood sugar levels.Therefore,in this paper,the crude phycocyanin product was isolated,purified and enzymatically hydrolyzed,the effect of phycocyanin and its enzymatic hydrolysis peptides on the digestion characteristics of wheat starch was systematically studied,and the target peptides were gradually screened,which broadened the research and development ideas of anti-digestion functional foods and the application range of phycocyanin.Firstly,a set of low-cost and high-efficiency purification procedures was established,and phycocyanin was isolated and purified only by step-by-step salting out method and hydroxyapatite column chromatography,so as to effectively improve the purity while simplifying the purification procedure,and then simulating the structure of the α and βsubunits according to the phycocyanin sequence sequence in the nucleotide sequence database.The experimental results showed that after a series of purification procedures,the purity of phycocyanin increased from 1.79 of crude to 4.37 of the reagent grade,an increase of 2.58,the ultraviolet scanning spectrum showed that the absorption peaks at 280 nm and 340 nm decreased significantly,and the SDS-PAGE diagram showed clear bands of α and β subunits,with molecular weights of 17.2Ku and 19.7Ku,respectively,without tailing,indicating that the purification effect was better.Observing the subunit structure simulation map shows that the phycocyanin bound on the β subunit may be more susceptible to destruction due to environmental changes,and the other two chromophores bound to the α and β subunits are involved in energy transfer within the polymer.Secondly,four enzymatic hydrolysis peptides were obtained by enzymatic hydrolysis of reagent-grade phycocyanin using alkaline protease,pepsin,trypsin and papain,and the effects of phycocyanin and its enzymatic hydrolysis peptides on wheat starch hydrolysis index and glycemic index were studied,and its inhibitory effect and inhibition type on starch digestive enzyme activity were determined.The experimental results showed that phycocyanin and its enzymatic hydrolyzed peptides could inhibit the activities of α-amylase and α-glucosidase,and the alkaline protease-hydrolyzed peptide and pepsin-hydrolyzed peptide had a good inhibitory effect on α-amylase and α-glucosidase,and the inhibition methods were mixed inhibition,and the ability of phycocyanin to bind directly to the active sites of the two digestive enzymes was stronger than its ability to bind to enzymes and substrate complexes,and in the enzymatic kinetic system,phycocyanin had a stronger affinity with α-glucosidase.Therefore,phycocyanin,alkaline proteolytic peptide and pepsin-hydrolyzed peptide were screened for follow-up experiments.Phycocyanin and its enzymatic hydrolysis peptides reduced the hydrolysis rate of starch,decreased the content of RDS,increased the content of RS,and with the addition of phycocyanin and its enzymatic hydrolytic peptides,the p GI value of wheat starch decreased significantly,among which the effect of alkaline protease peptide was the strongest.Therefore,alkaline protease-hydrolyzed peptides were screened for follow-up experiments.Finally,the spectral characteristics and molecular weight distribution of enzymatic hydrolyzed peptides were explored by ultraviolet spectroscopy,SDS-PAGE and gel permeation chromatography,the peptides were separated by ultrafiltration technology,the inhibition effect of each peptide was detected by the inhibition rate of starch digesting enzyme,and then amino acid determination and structural characterization were carried out on the peptide with the best inhibition effect.The experimental results showed that the absorbance value at 275 nm and 348 nm in the ultraviolet spectrum scanning pattern increased,and the absorbance value at 615 nm decreased,which may be caused by aromatic amino acid residue exposure,phycocyanin deprostoprotein and tetrapyrrole color group,enzymatic hydrolysis peptide contains three main components,J1(molecular weight range<1000u)accounted for75.90%,J2(molecular weight range of 1000-3000u)accounted for 13.97%,J3(molecular weight range>3000u)accounted for 10.13%.At a protein concentration of 30mg/m L,the inhibition rates of α-amylase by J1,J2 and J3 components were 43.08%,32.65% and 12.99%,respectively,and the inhibition rates of α-glucosidase were 31.36%,23.13% and 14.51%,respectively,that is,the J1 component had the best inhibitory effect on both amylases,so J1 was screened for subsequent research.A total of 17 amino acids were detected in the J1 component,of which glutamic acid content was the highest(12.98%),and hydrophobic amino acids accounted for the largest proportion(45.80%),and the calculation of amino acid content(AAS)and amino acid chemical content(CS)showed that the first limiting amino acid was phenylalanine + tyrosine,and the second limiting amino acid was methionine + cystine.LCMS/MS results showed that the molecular weight of the first 10 peptides with high scores in the J1 component was between 400-800 u,and the database comparison was from the genus spirulina,and the amino acid species at both ends of the peptide conformed to the action site of alkaline protease.