A Study On The Fermentation And Preparation Of Active Peptides From Bacillus SCSIO06063

Purpose:To convert marine mollusc protein into functional peptide by enzymatic hydrolysis is an effective way to improve its utilization efficiency.However,due to the large and complex structure of marine mollusk proteins,most of them exist in the form of glycoproteins,and the hydrolysis sites are shielded by sugar-based side chains,and it is difficult to hydrolyze into functional domain-integrated functional peptides under mild conditions.In the earlier stage of the laboratory,the enzyme-producing marine microorganisms were screened and studied for this common problem.It was found that the extracellular protease produced by marine-derived Bacillus sp.SCSIO 06063 was used to enzymatically degrade scallop protein under mild conditions.The product had significant anti-oxidation properties and showed good potential for application.Based on this study,the enzyme-producing fermentation of SCSIO 06063 was optimized to explore the optimal conditions for the large-scale production of SCSIO 06063 protease,and to further investigate the product of its catalytic scallop protein,and to identify the antioxidant active ingredients,in order to form a complete new enzyme application technology.Methods:Methods:1.Optimization of fermentation conditions:Protease activity was used as a detection target to optimize the culture conditions for production of proteases by Bacillus subtilis SCSIO 06063.Using the single-factor method,the carbon source,nitrogen source,metal ion,and salinity were optimized based on the LB medium.The effects of temperature,initial pH,inoculum volume,and rotational speed on enzyme production by fermentation were investigated.The response surface method was used to optimize the main factors affecting the production of enzymes,and enzyme solutions and scallop enzymatic solutions were prepared under optimal conditions.2.Antioxidative peptides in the scallop SCSIO 06063 hydrolysate:The antioxidant activity was evaluated by the DPPH and OH radical scavenging rates.The ultrafiltration and reversed-phase high-performance liquid chromatography were used to separate the enzymatic hydrolysate repeatedly to purify the antioxidant Peptides and the MALDI-TOF-MS techniques were used to identify the amino acid sequences of these peptides.Result:1.The study found that the optimum nitrogen source for SCSIO 06063 production was urea and the most suitable carbon source was maltose.The three factors that had the greatest impact on the production of enzyme were maltose,urea and CaCl2.Three factors and three levels of response surface methodology were used.When the maltose concentration was 32.21 g/L,the urea concentration was 9.85 g/L,and the CaCl2 concentration was 2.39 g/L,the theoretical protease activity was 176.22 U/mL.For the accuracy of the model,three sets of parallel experiments were performed under the optimal combined concentration of the above theories.The average protease activity was 169.96U/mL,and the predicted value error was 3.55%.This model can be used to predict the production of protease by strain SCSIO06063.Finally,the optimal composition of fermentation medium was determined as follows:32.21g/L maltose,9.85g/L urea,2.39g/LCaC12,1g./MgCl2,and 20g/LNaCl were added on the basis of LB medium.Under optimal conditions,the enzyme activity in the 10L fermentation tank reached 456.72U/mL,which was 3.45 times higher than that before optimization.2.After the scallop hydrolysate was ultrafiltered and separated by repeated RP-HPLC,three peptides,SHE-?-1,3,and 5,were isolated and purified.The matrix-assisted laser desorption time-of-flight mass spectrometry(MALDI-TOF MS)was used to identify these peptides as His-Ala-Ile-Ile-Met-Met-Val(SHE-?-1),Leu-Asn-Pro-Lys-Leu-Ile-Met-Val(SHE-?-3),Met-Ile-Leu-Ile-Leu-Phe-Arg(SHE-?-5),respectively.The BLAST search in the NCBI database did not find the homologous sequence of SHE-?-1,indicating that this is a new structural peptide.The DPPH free radical scavenging assay was used to evaluate the antioxidant activity.The clearance rates of SHE-?-1,3,and 5 were 51.60%,55.42%and 53.44%,respectively.Conclusion:The protease produced by Bacillus subtilis SCSIO06063 has been greatly improved after optimization of the composition of the medium and the culture conditions.The enzymatic hydrolysis of the scallops by the use of the crude enzyme solution has resulted in the formation of novel structural peptides.This study provides useful information for the peptides preparation using protease efficiently and for the optimization of protease production processes.