| Decapterus Maruadsi is a kind of low-value South China Sea fish rich in high-quality protein and unsaturated fatty acids.Its main products are mainly processed fresh fish,processed into fishmeal or snack foods,and its comprehensive utilization value is low.In recent years,in-depth studies on the extraction of bioactive peptides from marine low-value fishes are of great significance for the development and utilization of low-value fishes and for their added value.Therefore,in this thesis,we used defatted Decapterus Maruadsi as a raw material to select the optimal protease and optimize the enzymolysis process to prepare a biologically active peptide with high antioxidant activity,and to separate and purify the enzymatic hydrolysate,and evaluate the Caenorhabditis elegans.Antioxidation and anti-aging activities in vivo provide a theoretical basis for the further development of functional foods such as antioxidant peptides.The main research contents and results are as follows:(1)Basic component analysis and structure determination of apoprotein from Amaranthus.Firstly,the basic components and amino acid composition of apocynchus olivum extracted from fish oil by low-temperature continuous phase-change extraction were studied.Fourier transform infrared spectroscopy(FTIR)and circular dichroism(CD)were used to determine the content of apolipoproteins.The secondary structure of the protein was determined and its amino acid composition was analyzed.Studies have shown that after extraction of fish oil by low temperature continuous phase-change extraction technology,the water content of A.lutescens apolipoprotein is 5.38%,ash is divided into 4.29%,crude fat is 0.23%,and crude protein is as high as 83.78%.Fitting the amide I band by FTIR combined with deconvolution method found that the content of ?-sheet in the debrided blister was reduced by 0.52%,the random curl was reduced by 0.22%,and the ?-helix was increased by 0.55%.The ?-turn angle increased by 0.09%.After verified by CD,it was found that after the fish oil was extracted by low temperature continuous phase transformation,the complete secondary structure was maintained inside the protein.The number of amino acids with antioxidant activity increased significantly.Among them,histidine increased by 0.51%,valine increased by 0.71%,leucine increased by 1.13%,alanine increased by 1.05%,and threonine increased.0.74%;Proline increased by 0.83%.(2)Screening of Enzymes in the Process of Deproteinized Proteolysis of.The alkaline protease,trypsin,pepsin,flavour protease and papain were used for enzymolysis of the apoprotein,and the optimal enzyme was screened by the in vitro antioxidant activity and degree of hydrolysis in the hydrolysate.The results showed that when alkaline protease was used for enzymolysis,the antioxidant index,DPPH radical scavenging rate,ORAC value,and ABTS radical scavenging rate in the hydrolysates reached their maximum at 6 h.The maximum values were 1.65,88.78%,3.45 ?mol/L,and 89.73%,respectively.At this time,the degree of hydrolysis in the hydrolysate was 30.67%.Therefore,the use of alkaline protease as the best enzyme,and optimize the process optimization.(3)Optimization of enzymolysis process of alkaline protease in Amorphophallus delavayi.With the degree of hydrolysis,DPPH free radical scavenging rate,ABTS free radical clarity,and ORAC value as indicators,the effects of different enzymatic hydrolysis temperatures,pH,feed-liquid ratio,and enzyme dosage on the enzymatic hydrolysis process were investigated and orthogonalized.The optimum process conditions for the enzymolysis were as follows: the ratio of solid to liquid was 1:30,the temperature of enzymolysis was 55°C,and the amount of enzyme added was 8000 U/g protein,pH 9.5.Under this condition,the DPPH radical scavenging rate of the hydrolyzate was 89.99%.(4)Separation and Purification of Apolipoprotein Peptide from Cyprinus.High molecular weight gel filtration chromatography was used to determine the molecular weight distribution in the hydrolysate and the hydrolysate was separated and purified by ultrafiltration,dextran gel column and preparative liquid phase method,respectively.The results showed that the anti-oxidation activity of the <3 kDa sample was higher in the ultrafiltered fractions.DPPH radical clearing rate,ABTS radical clearing ratio,reducing power,and ORAC value were 31.26%?46.34 %? 0.285?75.65 ?mol/L at 1mg/mL,respectively..Four fractions were obtained by Sephadex-15 column chromatography,and the activity of fraction 2 was better.DPPH radical scavenging rate,ABTS radical clearing rate,reducing power value,and ORAC value were 80.25%,77.49%,and 62.56 ?mol/L,0.35 respectively.And this component was separated by using a preparative liquid phase to obtain 8 components,wherein the antioxidant activity of component 5 is higher than that of other components.The DPPH radical scavenging rate,ABTS radical clearing rate,reducing power value,and ORAC value were 48.35%,39.79%,64.31 ?mol/L,and 0.43,respectively.The purity was analyzed by high performance liquid chromatography.The purity of the peak was approximately 90% based on the peak area normalization method,indicating that this component contained a peptide.After its structure was identified,its structure was ILGATIDNSK.(5)Caenorhabditis elegans model to evaluate the in vivo activity of antioxidant peptides.The obtained peptides were fed with Caenorhabditis elegans,and the antioxidative peptides were evaluated for their antioxidative and anti-aging activities by measuring the lifespan of the nematode,physiological indicators,in vivo biochemical indicators,and biochemical indicators after PQ treatment.The results showed that the purified samples significantly improved the lifespan of the nematode(p<0.05),and the head swing frequency,fecundity,swallowing frequency,moving force,and sinusoidal motor ability in the nematode after feeding the sample were similar to those of the control group.There has been a significant increase in the ratio.Under heat stress conditions,the purified sample group can significantly increase the lifespan of the nematode.In the determination of biochemical indicators in vivo,the content of ROS in the nematode after purification was significantly lower than that in the control group(p<0.05),and the SOD and CAT enzyme activities were also significantly increased.After the PQ-treated nematodes,the survival rate of the purified sample group was significantly higher than that of the control group(p<0.05).The SOD and CAT enzyme activities of the nematode in the sample group were higher than those of the control group.Therefore,the resistance was improved.Oxidized peptides have anti-oxidative and anti-aging effects on Caenorhabditis elegans. |
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