Study On The Preparation Of Amino Acids Through The Fermentation Of Eel Visceral By Bacillus Amyloliquefaciens

China is a big country in eel farming and processing.A large number of byproducts visceral are produced during eel processing.Eel viscera is rich in protein and other nutrients.However,at present,the by-products visceral produced during eel processing has not been fully utilized in China.This not only causes the waste of resources,but also pollutes the environment.The protein degradation produces amino acids mainly through enzymatic method and microbial fermentation method.Compared with the enzymatic method,the microbial fermentation method avoids the high energy consumption of enzyme separation and the risk of inactivation of enzyme production and transportation during the production of enzyme preparation,and greatly reduces the cost of amino acid preparation.In this study,a Bacillus amyloliquefaciens with high-yield protease was obtained by isolation,screening,and identification from eel visceral,the process of enzyme production by Bacillus amyloliquefaciens fermentation was optimized,and the immobilization technology of Bacillus amyloliquefaciens was established.Furthermore,a method for fermenting eel viscera with immobilized Bacillus amyloliquefaciens to prepare amino acids was established,an efficient and low-cost amino acid production method was achieved.The main research content and conclusions are as follows:(1)A protease-producing strain G-1 was isolated and screened from eel visceral by enrichment culture,plate preliminary screening and shaker re-screening,and its protease activity was 42.39 U/m L.The strain was identified as Bacillus amyloliquefaciens from the morphological,physiological biochemical and molecular levels(gene sequencing),it was named Bacillus amyloliquefaciens G-1.(2)The process of enzyme production by Bacillus amyloliquefaciens G-1fermentation was optimized.The factors and levels were as follows: nitrogen source(yeast powder,peptone,beef paste,soybean meal and ammonium sulfate),soybean meal concentration(0.5%,1.0%,1.5%,2.0% and 2.5%),carbon source(sucrose,glucose,fructose,lactose and soluble starch),glucose concentration(0.5%,1.0%,1.5%,2.0% and 2.5%),initial p H(5.0,6.0,7.0,8.0 and 9.0),temperature(20,25,30,35 and40 °C),shaker speed(120,140,160,180 and 200 r/min),inoculation amount(1%,2%,3%,4% and 5%).The results showed that the optimal fermentation process of protease production by Bacillus amyloliquefaciens G-1 was as follows: 1.5% of soybean meal as nitrogen source,1.0% of glucose as carbon source,7.0 of initial p H,35℃ of temperature,180 r/min of shaker speed,and 3% of inoculation amount.After optimization,the protease activity of Bacillus amyloliquefaciens G-1 was 147.43 U/m L.It was about 3.5 times higher than before optimization.(3)The effect of immobilized Bacillus amyloliquefaciens G-1 by three methods(sodium alginate,sodium alginate-polyvinyl alcohol and sodium alginate-chitosan method)were investigated respectively,and the immobilization process of the most effective method was optimized.The results showed that sodium alginate-polyvinyl alcohol method was the most effective immobilization method,its optimal immobilization process was as follows: 3.0% of sodium alginate,8.0% of polyvinyl alcohol and 0.6% of calcium chloride,its corresponding protease activity of Bacillus amylolyticus G-1 was 76.77 U/m L.(4)The effect of immobilized Bacillus amyloliquefaciens G-1 fermented eel viscera to prepare amino acids was investigated,the fermenting eel visceral by the free Bacillus amyloliquefaciens G-1 and the crude enzyme liquid of Bacillus amyloliquefaciens G-1 were used as controls,respectively.On this basis,the process of fermenting eel viscera with immobilized Bacillus amyloliquefaciens G-1 to prepare amino acids was optimized.The factors and levels were as follows: material-liquid ratio(1:1,1:3,1:5,1:7 and 1:9),initial p H(5.0,6.0,7.0,8.0 and 9.0),inoculation amount(2%,4%,6%,8% and 10%),temperature(26,29,32,35 and 38°C),shaker speed(120,140,160,180 and 200 r/min).The results showed that the amino acid nitrogen content by the method of fermenting eel viscera with immobilized Bacillus amyloliquefaciens G-1 to prepare amino acids was about 1.2 times and 1.3 times higher than the controls,respectively.Its optimal fermentation process was as follows: 1:5 of material-liquid ratio,7.0 of initial p H,6% of immobilized Bacillus amyloliquefaciens G-1 inoculation amount,35℃ of temperature,180 r/min of shaker speed,its amino acid nitrogen content was 966 mg/L,its corresponding amino acid content was 5850mg/L.The fermentation broth is rich in aspartic acid,glycine,alanine and other flavored amino acids,which can be used as pure natural compound amino acid seasoning,etc.