Application Of Alkaline Protease BaApr1 In The Preparation Of Antioxidant Peptides From Grass Carp Scales

Grass carp is the most productive freshwater fish in the world today,and a large amount of fish scale waste has been produced during the industrialization process.Fish scale is a rich protein raw material and an ideal source of antioxidant peptides.After protease-assisted hy-drolysis and purification,antioxidant peptides with high economic value can be obtained and used in the market.In this paper,a microbial protease BaApr1 derived from Bacillus altitudi-nis W3 was used to hydrolyze grass carp fish scales.By optimizing the hydrolysis process,a hydrolysate with high antioxidant activity was obtained,and three antioxidant peptides were obtained through a series of separation and purification.Finally,their in vitro activity was evaluated,and their structure-activity relationship was analyzed.The main research results were as follows:（1）The expressed BaApr1 crude enzyme solution was purified by ammonium sulfate precipitation,nickel column affinity chromatography and desalting.The final purification fold was 30.06 times and the recovery rate was 30.44%.The optimum reaction temperature of BaApr1 was 50℃,and the optimum reaction p H was 9.5.It showed good activity stability after being placed under moderate and alkaline conditions for 9 h,respectively.The activation effect of Ca²⁺on BaApr1 was the most obvious,which can be increased to 1.80 times at 5mmol·L^-1.（2）The optimal decalcification conditions for fish scales were:a HCl concentration of0.4 mol·L^-1,a solid-liquid ratio of 1:15（m/v）and a decalcification time of 120 min.The de-calcified fish scales were hydrolyzed by eight proteases,among which the hydrolysate of BaApr1 showed the best antioxidant activity compared with other products.The single-factor optimization results for the hydrolysis of fish scales by BaApr1 were as follows:an enzyme dosage of 1250 U·g^-1,a hydrolysis time of 7 h,a temperature of 50℃and a p H of 9.5.The optimal conditions obtained by the orthogonal experimental design were:an enzyme dosage of 1250 U·g^-1,a hydrolysis time of 7 h,a temperature of 50℃,and a p H of 9.0.Grass carp scale hydrolysate（GCSH）was prepared under the optimal conditions.（3）The protein content of GCSH was 88.15%,the total hydrophobic amino acid content was 28.27%,the total essential amino acid content was 19.51%,and the total delicious amino acid content was 60.54%,which proved that GCSH has good nutritional value and application value.The solubility of GCSH can reach more than 90%in the range of p H 3.0-11.0,and the highest was 98.06%at p H 6.0.In addition,GCSH has a certain tolerance to the conditions of temperature 0-100°C,p H 2.0-12.0,gastrointestinal digestion,which can ensure the stability of antioxidant activity and meet the needs of food industrial applications.（4）GCSH was purified to extract high-efficiency antioxidant peptides,and the sca venging ability of DPPH·and ABTS⁺·of each fraction was used as an index.The fr action UF-3（<3 k Da）was obtained from GCSH after ultrafiltration,then the AEC-3(0.5 mol·L^-1 Na Cl elution)was obtained by anion exchange chromatography,and then the GFC-1 was obtained by gel filtration chromatography.Finally,the components SH P2,SHP4 and SHP5 were obtained by ultra-high performance liquid chromatography.Three peptides were identified by secondary mass spectrometry as:Tyr-Val-Gln-Ala-Gly-Ala-Ala-Gly-Ala-Ala-Ala-His（SHP2）,Val-Lys-Leu-Tyr-Val-Leu-Leu-Val-Pro（SHP4）,an d Val-Gln-Val-Leu-Ala-Gly-Pro-Val-Val-Lys-Leu-Tyr（SHP5）.（5）The activities of SHP2,SHP4 and SHP5 were evaluated,and their semi-elimination concentration（EC50）values for DPPH·scavenging activity were 4.08 mg·m L^-1,5.82mg·m L^-1 and 4.52 mg·m L^-1,respectively;ABTS⁺·scavenging activity were 0.23 mg·m L^-1、0.55 mg·m L^-1 and 0.37 mg·m L^-1,respectively;HO·scavenging activity were 2.78 mg·m L^-1、3.70 mg·m L^-1 and 3.16 mg·m L^-1,respectively.In addition,SHP2 also exhibited the strongest reducing power,reaching 0.64 at 5 mg·m L^-1.SHP5 showed the best lipid peroxidation inhibi-tion,and inhibiting 79.81%of linoleic acid lipid peroxidation within 7 d and 33.23%of beef lipid peroxidation within 15 d.The antioxidant activities of SHP2,SHP4 and SHP5 are relat-ed to their molecular weight range（1043-1285 Da）and amino acids such as hydrophobic amino acids（Ala,Val,Leu,Pro）,aromatic amino acids（Tyr）,Gly and His present in their primary structures.