Study On The Preparation,Purification And Antioxidant Activity Of Peptides From Cuttlefish (Sepia Pharaonis) Gonads

In order to realize the high value utilization of cuttlefish by-products,cuttlefish?Sepia pharaonis?gonads were used to prepare antioxidant peptides by liquid fermentation of Bacillus subtilis.The antioxidant activity in vitro and stability of cuttlefish gonad peptides were studied.And several isolation and purification technology were applied to peptides'sequential separation.The main research contents and conclusions are as follows:?1?The cuttlefish gonads were high protein?62.71 g/100g?and low fat?0.52 g/100g?,its amino acid is rich in variety and reasonable in composition and can be used as a high-quality protein source for the development of antioxidant peptides.The cuttlefish gonads were used as the sole fermentation substrate to study the dynamic changes of related indexes in the fermentation process of Bacillus subtilis.The results showed that Bacillus subtilis grew well in the fermentation medium and had a certain protease-producing ability to degrade the gonad proteins into peptides and amino acids,which in turn caused dynamic changes in amino nitrogen content,hydroxyl radical scavenging rate and reducing power in the fermentation broth.The protease activity and non-protein nitrogen content of the fermentation broth were used as evaluation indexes to study the effects of substrate concentration,initial pH value of the culture medium,fermentation temperature and fermentation time on the fermentation process.On the basis of the single factor experiment,the fermentation process was further optimized by orthogonal test.The optimum conditions were:substrate concentration of 30 mg/mL,initial pH6.0,fermentation temperature 34?and fermentation time 60 h.Under these conditions,the protease activity of the fermentation broth was 66.66 u/mL,and the non-protein nitrogen content was 1.10 mg/mL.?2?The crude protein content of cuttlefish gonad peptides was 52.37 g/100g and the protein utilization rate was 34.64%.Among its amino acid composition,lysine and aspartic acid were the highest,hydrophobic amino acid and antioxidant amino acid accounted for 39.1%and 63.3%of the total amino acid respectively,which had potential antioxidant activity.The cuttlefish gonad peptides had certain antioxidant activity in vitro,and its IC₅₀ value of DPPH radical scavenging rate,hydroxyl radical scavenging rate,superoxide anion radical scavenging rate and ferrous ion chelation rate were 14.99,21.22,7.95 and 1.75 mg/mL,respectively.But its reducing power was relatively weak,and the AC_0.5 value was 41.32 mg/mL.The stability of the antioxidant activity of the cuttlefish gonad peptides was studied.Results showed that temperature,food ingredients?including NaCl,sucrose and citric acid?,preservatives?including sodium benzoate and potassium sorbate?and K⁺,Mn²⁺and Ca²⁺concentration had little effect on the activity retention rate of peptides,while the strong acid environment and Zn²⁺concentration had a great influence on its activity retention rate.?3?With the DPPH radical scavenging rate as the main evaluation index,the cuttlefish gonad peptides were separated and purified by ultrafiltration,Sephadex G-25 gel filtration chromatography and RP-HPLC.The component SPGPs-2D-2 had the highest DPPH radical scavenging rate?70.61%,1 mg/mL?,which was increased by at least 30-fold compared with peptides before separation?67.26%,30 mg/mL?,and the cuttlefish gonad peptides were effectively enriched.