| The development of traditional bioactive peptides production technology is mainly based on the theory that the small molecule peptides can be directly absorbed after entering the body,without considering the function of the human gastrointestinal tract,resulting in the possibility of the excessive enzymolysis and the significant increase of the production costs.If the digestive function of the human body is considered,the activity detection of enzymatically hydrolysed products in the development of peptide production technology should be carried out after the simulated gastrointestinal digestion in vitro.In this way,it is possible to obtain the desired functions without enzymatically converting the protein to small molecules,or omitting the step of enzymolysis.The digestive enzymes in the human gastrointestinal tract have specific enzyme digestion sites and digestive properties.Pepsin is an endopeptidase,which mainly cleaves the peptide bonds of the carboxyl terminal of the acidic amino acids,especially the peptide bonds which carboxyl group is an aromatic amino acid;trypsin specifically cleaves the peptide bonds next to lysine and arginine;chymotrypsin breaks down the peptide bonds adjacent to hydrophobic and aromatic amino acids.Hydrophobic and acidic amino acids dominated the amino acid composition in walnut protein and the contents of glutamic acid,arginine and aspartic acid are relatively high,which is more suitable for the digestion of digestive enzymes in gastrointestinal tract.Therefore,in this work,new ideas of enzymatic hydrolysis evaluation were established and the antioxidant and antihypertensive activities were selected as the assessment indicators,the necessity of walnut protein to produce peptides with these two activities through enzymatic hydrolysis was studied in-depth and systematically.The research contents and the main conclusions are as follows:(1)The amino acid composition and nutritional value of walnut protein were evaluated,and a comparative study of the effect of in vitro simulated gastrointestinal digestion on walnut protein and its alcalase hydrolysate was conducted with the peptide and free amino acid contents,ACE inhibitory activity,DPPH radical-scavenging activity and molecular weight distributions as the evaluation indexes.Results showed that the walnut protein had a reasonable amino acid composition.The essential amino acid content(26.98%),the predicted value of protein efficiency ratio(1.55,1.58)and the biological value(87.96%)were high.After simulated digestion in vitro,the peptide content of the walnut protein digests was 21.66 mg/mL,which was higher than that of the digests of alcalase hydrolysate(19.84~21.16 mg/mL);the free amino acid content(8.09 mg/mL)and the ACE inhibitory activity(44.85%)of walnut protein digests were not significantly different(p>0.05)from those of walnut protein hydrolysate digests;the DPPH radical-scavenging activity(66.53%)was higher than that of the hydrolysate digest(p>0.05).Results of molecular weight distribution showed that both walnut protein and hydrolysate could be hydrolyzed into small peptides with molecular weights <3000 Da after the simulated digestion in vitro.According to preliminary judgment,hydrolysis with alcalase was unnecessary to produce walnut peptides with potent ACE inhibitory and DPPH radical-scavenging activities and it is sufficient to eat walnut protein directly.(2)The walnut protein hydrolysates with different degrees of hydrolysis were produced by enzymatic hydrolysis with different proteases.The antioxidant activity of walnut protein and its hydrolysate before and after the simulated gastrointestinal digestion in vitro were studied,and were verified in vivo by the D-galactose-induced oxidative damage model in SD rats.Results showed that after gastrointestinal digestion in vitro,the DPPH radical-scavenging activity and the reducing power of walnut protein digests reached 66.53% and 8.55 ?mol TE/mL,respectively,which were all showed no significant difference(p>0.05)with the walnut protein hydrolysate digests with low degree of hydrolysis(?8%),and slightly higher than those of the walnut protein hydrolysate digests with high hydrolysis degree(>8%).In the in vivo verification experiment,walnut protein and its hydrolysate could significantly alleviate the slow weight gain of rats induced by the damage of D-galactose-induced oxidative,and significantly reduce the oxidative damage to liver,heart,and spleen(p<0.05).Additionally,it could significantly relieve the increase in MDA content(p<0.01),the decrease in GSH(p<0.05)content and T-AOC(p<0.01)caused by oxidative damage.Both walnut protein and its hydrolysates had good antioxidant effect and it seemed unnecessary to produce walnut peptide by enzymatic hydrolysis in terms of the antioxidant activity.(3)The walnut protein hydrolysates with different degrees of hydrolysis were produced by enzymatic hydrolysis with different proteases.The ACE inhibitory activity of walnut protein and its hydrolysates before and after the simulated gastrointestinal digestion in vitro was studied.Besides,the in vivo antihypertensive effect of walnut protein and its hydrolysates and the potential mechanism were studied by an acute oral administration and a long-term intragastric administration test in SHRs.Results showed that after the simulated digestion in vitro,the ACE inhibition rate of walnut protein digests reached 44.85%,which was not significantly different(p>0.05)from the walnut protein hydrolysate digests with different degrees of hydrolysis.After the oral administration of walnut protein and its hydrolysate for 3 h in the acute oral administration test,the systolic blood pressure reached the maximum reduction of 21.65 mmHg and 15.22 mmHg,respectively.In the long-term administration of walnut protein and its hydrolysate,the systolic blood pressure of SHRs all began to decrease at the 2nd week,with the maximum reduction reached 18.33 mmHg and 24.42 mmHg,respectively.It suggested that the walnut protein and its hydrolysate all had significant effect on lowering the blood pressure.Results of tissue ACE activities and serum biochemical indicators showed that the walnut protein could reduce the levels of ACE activities in aorta(p<0.05),kidney(p<0.01)and lung(p<0.01)of SHRs,and reduce the endothelin-1(p<0.01)and tumor necrosis factor-?(p<0.01)levels,while increase the bradykinin(p<0.01)and nitric oxide(p<0.01)levels in serum to lower the blood pressure.Walnut protein hydrolysate could reduce the levels of ACE activities in aorta(p<0.05)and lung(p<0.01)of SHRs,and reduce the tumor necrosis factor-?(p<0.01)levels,while increase the bradykinin(p<0.01)and nitric oxide synthase(p<0.01)levels in serum to lower the blood pressure.In general,walnut protein showed a more stable and longer-lasting antihypertensive effect than its hydrolysate and it seemed unnecessary to produce walnut peptide by enzymatic hydrolysis in terms of the antihypertensive activity. |
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