| In order to develop safe and non-side effect food-derived antihypertensive peptides and achieve high-value utilization of squid processing by-products,this article uses squid processing by-products as raw materials,respectively using neutral protease digestion and Bacillus natto liquid fermentation The antihypertensive peptides were prepared by technology,and their in vitro ACE inhibitory activity,renin inhibitory activity and stability were studied.The main research methods and conclusions are as follows:?1?Use the national standard composition measurement method to detect crude protein,crude fat,crude ash,amino acids,etc.in the by-products of squid processing to determine its nutritional composition and nutritional evaluation of its amino acids.The results showed that the dry weight contents of crude protein,crude fat and crude ash in by-products of squid processing were 63.18%,12.94%and 8.29%respectively;17 kinds of amino acids were detected with a total content of 55.74%and essential amino acids accounted for the total content of amino acids 41.68%,with the highest glutamic acid content?7.93%?and a high hydrophobic amino acid content?22.78%?.The main restricted amino acids are methionine and cystine,and the essential amino acid index EAAI is 85.23.The results show that squid processing by-products can be used as a high-quality source for the preparation of antihypertensive peptides.?2?Using angiotensin-converting enzyme-??ACE?inhibitory activity and peptide content as the main indicators,the squid processing by-product hydrolysis protease was screened,and the response surface center combination design was used to further optimize the enzymatic hydrolysis process to establish ACE inhibitory activity.A quadratic regression model of peptide content and enzymatic hydrolysis temperature,hydrolysis time,p H,and enzyme addition,through analysis of variance and verification experiments,it was concluded that the model can better reflect the ACE inhibitory activity and peptide content of the squid processing by-product hydrolysate The changing law.The results showed that the optimal hydrolysis process conditions were enzymolysis temperature 52.4?,enzymolysis time 5.7 h,p H 7.1,and the amount of enzyme added was 8151 U·g-1.Under this condition,its ACE inhibition rate and peptide content were up to 84.26%,229.09 mg·g-1.?3?Using Bacillus natto fermentation squid processing by-products,using ACE inhibition rate and peptide content as indicators,through a single factor test to study the fermentation time,fermentation temperature,inoculation amount and liquid-to-material ratio process parameters,And adopt Box-Behnken response surface analysis to optimize the process conditions,and establish a quadratic polynomial mathematical model.The results show that the regression model can better reflect the relationship between the level of each factor and the response value.The order of the influence of each factor on the ACE inhibition rate and peptide content is fermentation temperature>inoculation amount>liquid-to-material ratio>fermentation time.The best technological conditions for the preparation of ACE inhibitory peptides by the by-product of squid fermentation by Bacillus spp.were fermentation time 37.5h,fermentation temperature 42.1?,inoculation amount 6.0%,and liquid-to-material ratio 22.7.The ACE inhibition rate of the freeze-dried product prepared under the above fermentation conditions reached 82.22%,and the peptide content was as high as 208.18 mg·g-1.?4?Separation of the enzymatic?E?and fermentation?F?hydrolysate obtained under the optimal process conditions into five different molecular weight and size peptide components?<1 k D,1?3 k D,3?5 k D,5?10 k D and>10 k D?;Evaluate the ACE and renin inhibitory activity of hydrolysate and each polypeptide component,and explore the stability of the best active component through simulated digestion of the gastrointestinal tract.The results show that:components with<1 k D?components with<1 k D obtained by enzymatic hydrolysis are denoted as E-1 and components with fermentation products<1 k D are denoted as F-1?have the largest ACE inhibitory activity?E-1 inhibition rate was 87.48±1.76%,F-1 inhibition rate was86.50±1.84%?and renin inhibitory activity?E-1 inhibition rate was 69.72±1.16%,F-1inhibition rate was 81.09±1.23%?;The IC50 values of ACE and renin of component E-1 were1.34±0.12 mg·m L-1 and 1.47±0.06 mg·m L-1 respectively;the IC50 values of ACE and renin of component F-1 were 1.01±0.08 mg·m L-1,1.15±0.09 mg·m L-1;after the components were digested by artificial gastric juice 120 min+artificial intestinal fluid 120 min,the final ACE inhibition rate of E-1 component dropped to 35.15±1.31%,renin inhibition rate It decreased to43.17±1.42%,the final ACE inhibition rate of F-1 component decreased to 48.24±1.18%,and the renin inhibition rate decreased to 49.37±1.16%.Generally speaking,the anti-hypertensive activity and stability of the F-1 component are stronger.The results showed that the enzymatic hydrolysis,fermentation hydrolysate and membrane separation components of<1k D of squid processing by-product protein may be used as functional ingredients for the development of blood pressure-related functional foods and health products.The results of this study provide a theoretical basis for the high-value utilization of squid processing waste and the research and development of food-derived antihypertensive peptides. |
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