Study On The Preparation Of Oligopeptides From By-products Of Grass Carp Processing By Fermentation Of Bacillus Subtilis

Grass carp will produce large amounts of by-products in the processing,such as fish skin,fish viscera,fish bone and so on.By-products of grass carp processing contain abundant protein and lipid resources.If they are discarded directly,they will not only lead to the waste of resources,but also cause environmental pollution.If the protein resources in them are used rationally to prepare oligopeptides,the high-value utilization of grass carp processing by-products can be realized.Using microorganism to degrade protein is a good method.Bacillus subtilis is widely present in nature and can produce proteases to degrade proteins.However,there has been no report on the production of oligopeptides by Bacillus subtilis fermentation of grass carp processing by-products,and this research needs to be strengthened.In this study,the raw materials of grass carp processing byproducts were taken as the object,and the high-yielding protease Bacillus subtilis isolated and screened from grass carp viscera was used as the starting strain.Bacillus subtilis fermentation method was used to degrade by-products of grass carp processing to produce oligopeptides.The production process was optimized,and the fermentation process was simulated.Oligopeptides can be directly produced from wet raw materials of grass carp processing by-products,reducing the energy consumption of subsequent oligopeptides separation,and providing theoretical basis and data support for industrial production of oligopeptides from by-products of grass carp processing based on Bacillus subtilis fermentation and endogenous enzyme catalytic coupling method.The main research results are as follows:（1）Isolate and screen protease-producing strain from grass carp viscera.The proteinase-producing strain was isolated and purified by enriched culture of beef extract peptone medium and selected culture of casein solid medium.The strain was identified from three levels:morphology,physiology,biochemistry and molecular（gene sequencing technology）.The results showed that strain was Bacillus subtilis,which could produce neutral protease.During the growth period of the strain,protease activity was positively correlated with bacterial growth.After the growth of the strain entered a stable period,protease continued to accumulate,and the enzyme activity reached the highest value of 175.43 U/m L after culturing 48h.（2）The fermentation of Bacillus subtilis was used to degrade by-products of grass carp processing to produce oligopeptides.The chemical composition of grass carp processing byproducts was analyzed,and the grass carp protein was degraded by the fermentation of Bacillus subtilis to prepare oligopeptides.The fermentation process of Bacillus subtilis(inoculation amount of strain（0%,5%,10%,15%,20%,25%and 30%）,initial p H of reaction（5.0,6.0,7.0,8.0 and 9.0）,fermentation time（0h,12 h,24 h,36 h and 48 h）,temperature（22℃,27℃,32℃,37℃and 42℃）,the physicochemical properties of oligopeptides were analyzed.The results showed that the protein content was 19%.The optimal fermentation process was as follows:Bacillus subtilis inoculum dosage of 15%,initial reaction p H 7.0,fermentation time24 h,temperature 37℃,oligopeptide content could be increased to 65 mg/g.The oligopeptides were rich in acidic amino acids,isoelectric point was 1.88,relative molecular mass were 317-774 Da.The solubility capacity of oligopeptides was 91%.The oil holding capacity of oligopeptides was 7%.The foaming ability of oligopeptides was 37%.The emulsifying activity of oligopeptides was 32%.The IC₅₀values of oligopeptide products for ABTS⁺·and O₂^-·scavenging and Fe²⁺chelating capacity were 0.28 mg/m L,7.81 mg/m L and 2.38 mg/m L,respectively.（3）The fermentation process of Bacillus subtilis fermentation method degradation of grass carp processing by-products to produce oligopeptides was simulated.According to the metabolic changes of viable bacteria number,oligopeptide content and protein content in the fermentation process,Logistic model,partial correlation model and Luedeking-piret model were used to construct the bacterial growth,substrate consumption and product generation kinetic models.The model was fitted with experimental data and verified experimentally.The results were as follows:the model could well reflect the process of preparing oligopeptides from by-products of grass carp processing by Bacillus subtilis fermentation.The established kinetic mathematical model was in good agreement with the experimental values.The correlation coefficients R² of three models were all above 0.9725,indicating that three kinetic models could well describe the bacterial growth,protein consumption and oligopeptide generation.It would have a guiding significance for optimization and control of fermentation process of fish preparation oligopeptides,and provide theoretical basis and data support for the development of automatic production process of fish preparation oligopeptides.