Screening Of Acid Protease Producing Strains And Application Of Fermentation Broth In Seed Germination

Acidic protease is widely used in the applications of food processing,leather production and feed industry.Since merely a small number of species of fungi（such as Aspergillus,Rhizopus,etc.）have been applied as the production strain in industry,the selections are limited.Moreover,the capacity for the production of protease in China is relatively lower compared with foreign countries.Therefore,some researches fouce on the development of novel microbe derived protease and on the screening of fungal stains with high-yield of protease.The processing of fish products will produce a large number of leftovers,which is low-cost and has a large value-added space.Except for water,the protein with the highest content is the main source of high-quality bioactive peptides（such as antioxidant peptides,antibacterial peptides,etc.）with good amino acid composition.Making full use of this part of protein resources is the key to improve the utilization rate of waste.Among many methods of waste material treatment,microbial fermentation is considered to be the most economical and applicable method for waste material treatment.The strain producing acid protease is directly added to the fish waste for fermentation to prepare acid liquid fertilizer.The use of the liquid fertilizer can alleviate the salinization of the land to a certain extent,which is of great significance to improve the production capacity of crops.In this study,a strain that can produce acidic protease was isolated from the soil in mangrove,which was identified as a member from the genus of Serratia according to the result of 16S r RNA sequencing.We further optimized the conditions for enzyme production,and also investigated the enzymatic properties,so as to improve the yield and activity of the protease.Our results laid a foundation for the application of the identified protease producing strain.This study has made the following achievements:1.A strain producing acidic protease is isolated from the soil in mangrove.After identification,it is found that this strain is genetically most related to Serratia rubidaea.The single factor experiment was utilized in combination with response surface methodology to optimize the conditions for enzyme production,with folin as the reagent for detecting the protease activity.The results showed that the optimal conditions for enzyme production for the strain were as follows:under the conditions of the inoculation amount of 0.01 OD₆₀₀and the liquid volume of 50 m L/500,the optimum culture conditions includes the temperature of 31℃,the use of 5 g/L glucose and 40 g/L peptone as the carbon and nitrogen sources respectively,t potassium dihydrogen phosphate at 10.12 g/L,the salinity at 4.94 g/L,and the optimum p H of5.5.After the optimization,the enzyme activity of this strain increased by 2.09 fold,from 158.53 U/m L to 331.34 U/m L.2.The extracts of protease was obtained using different concentrations of ammonium sulfate,and it was found that the highest enzyme activity was achiveved in the precipitaton of 50%ammonium sulfate.Anion exchange chromatography was applied to further purify the extracted protease,and the enzymatic properties were further investigated.The results showed that this enzyme exhibited the highest activity under the temperature of 40℃and p H of 6.This result proves that the enzyme beleongs to the type of acidic protease and it has stable activity at the temperature of10-50℃.3.Fermentation of golden pomfret residues was performed in the presence of this strain,and the composition of amino acids was determined by LC-MS before and after the fermentation.The result showed that the contents of glutamate,valine and proline increased significantly after the fermentation by this strain.And a certain concentration of fermentation liquid can promote the germination of cabbage seeds and the growth of pepper seedlings.