Screening Of Acid-protease Strain And Enzymatic Hydrolysis Of Condensed Fish Soluble To Amino Acid Solution

Through the screening of acid-protease strains,combined with modern fermentation techniques,the present study attempted to develop new amino acid organic fertilizers.the main findings are as follows:An acid-protease strain named Zju-C was screened from the hydrolytic liquid of fish meal using the transparent hydrolysis circle method and the Folin-Phenol method.Morphological observation and 26S rRNA molecular identification were performed,The BLAST of 26S rRNA sequence in GenBank indicated that the strain belonged to Pichia pastoris and the phylogenic tree of the strain,which was generated by Vector NTI Suite 7 software based on 26S rRNA sequence,showed that it was most close to Pichia galeiformis strain with 100%of identity.In order to improve the ability of the target bacteria to produce acid protease,the research was carried out by using physical mutagenesis methods such as ultraviolet light and heat treatment.The results showed that the compound mutagenesis had a significant effect on the improvement of the enzyme production performance of the target bacteria,and finally,a relatively high-yield dominant strain Pichia galeiformis strain.Zju-C UV-R2 was selected.Compared with the original strain,the lag phase of the strain was 4 h earlier,and the enzyme activity could reach194.3±1.6 U/mL,it is 1.61 times the original enzyme activity.Three significant factors affecting the yield of rhamnolipid were screened by plackett-burman design and the single-factor experiment:Na2HPO4 concentration,MgSO4 concentration,and salinity.The steepest climbing experiment was used to determine the center point of the response surface analysis.Finally,the significant influence factors were modeled and analyzed by response surface analysis to determine the best.Medium formulation(g/L):Waste molasses 50;soybean meal 50;NaH2PO4 11.55;magnesium sulfate 1.14;salinity 22.76;pH 5.0 and the inoculation amount was 3%.The theoretical prediction of enzyme activity was 390.7 U/mL,and the actual enzyme activity was 391.6±2.3 U/mL.The two were very similar,indicating that the second-order regression model had good repeatability and reliability.On the basis of the previous shaking flask experiment,a fermentation test was conducted in a 7.5 L fermentor.Under the condition of 4 L/min and 400 r/min,the fermentation was transferred for 24 h with 3%inoculation,and the enzyme activity of the crude enzyme solution reached 419.4±4.5 U/mL.In order to obtain a better effect of the development of fluid fertilizer of amino acid,the preliminary study on the enzyme activity of crude enzyme solution showed that the enzyme activity of crude enzyme solution could be activated by adding 0.9 g/L manganese ion at pH 3.5 and 45?.It reached 542.9±2.7 U/mL,which was 182%higher than the enzyme activity before optimization.On this basis,preliminary research on amino acid solution was carried out,after 7 days of enzymatic hydrolysis and fermentation amplification of 7.5L fermenter.Finally,the amino acid content in the fermentation broth can be increased to 69.5±1.7 mg/mL,which is 39%more than before enzymatic hydrolysis.The raw material of enzymatic hydrolysis is condensed fish soluble,which is also called hydrolysis of fish protein.The preparation method is to partially or completely hydrolyze the aquatic protein protein by acid,alkali or enzyme to make it soluble protein.After component analysis,The amino acid solution obtained in this study can be used as fish-protein-organic-liquid fertilizer for production and application.And compared the standard of amino acid liquid fertilizer,Although there were some deficiencies in amino acid content,it was very close to the successful development of liquid fertilizer.Besides,through field experiments,it was found that amino acid solution had a significant effect on enhancing crop stress resistance.