| Brown algae is the main aquatic economic algae in China.The cell wall of brown algae contains a large amount of brown algae polysaccharide,alginate,which has been used in many fields because of its natural physical and chemical properties.Alginate oligosaccharide is an oligosaccharide product with polymerization degree below 20 DP produced after the hydrolysis of alginate.It has biological activities such as antibacterial,anti-inflammatory,anti-aging and growth promotion,and has played an important role in the fields of food,medicine and daily chemicals.The alginate is mainly degraded by chemical,physical and biological methods.Compared with the former two methods,the biodegradation method has the advantages of controllable degradation reaction process,high activity of natural products and environmental friendlyness.However,there is no unified fermentation engineering bacteria and alginate lyase that can degrade alginate on the market at present.The main reason was that most of the strains that could ferment and degrade alginate had problems such as low enzyme production,unstable enzyme activity and easy inactivation.Failing to meet industrial production requirements.Therefore,the development of algina-degrading strains with high enzyme activity has been a key research direction,which is of great significance for the high-value utilization of Marine resources.The main work of this paper is as follows:(1)Samples were collected from the intestines of Apostichopus japonicus,Oratosquilla oratoria,Chlamys farreri,Haliotis diversicolor and shrimp paste,The culture medium was prepared with sodium alginate as the sole carbon source.The first screening was carried out by transparent ring method and the DNS method was used to screen.A high enzyme active alginate degrading strain B12 was screened from marine organisms.The strain was identified as Vibrio sp.by 16 S r DNA sequence analysis,physiological and biochemical experiments and electron microscopy.The five factors affecting the growth and enzyme production conditions of the strain were optimized by single factor test and response surface optimization test: initial p H value,fermentation temperature,Na Cl concentration,inoculation amount and liquid loading.The optimum conditions for the production of enzyme were p H6.52,28.20?,Na Cl concentration2.01%,inoculation amount 2.10%,and liquid loading 59.50 m L.Under the optimum fermentation conditions,the enzyme activity of B12 strain was 91.68 U/m L,which was38.50% higher than that before optimization.The time of enzyme production was 6 hours ahead of time,and the activity stability of the enzyme was better kept at 4? cold storage.(2)The intestinal flora of Apostichopus japonicus was cultured in laboratory and induced.Sodium alginate was used as the only carbon source to prepare the medium.The first screening was carried out by transparent ring method,and then screened by DNS and UV method.Four strains S1,S2,S10 and S11 were screened from the intestinal tract of the cultivated Rhododendron,and then The results of 16 S r DNA sequence analysis and electron microscopy showed that the four strains were Bacillus sp.,Microbacteria sp.,Vibrio sp.and Exiguobacterum sp.The four strains were mixed culture,and the best ratio of S2 and S11 was obtained.The dynamic changes of the abundance of the mixed strains were analyzed by high-throughput microbial diversity test.The four factors influencing the enzyme production conditions of the mixed strains were optimized by single factor test and response surface optimization test: initial p H value,Na Cl mass concentration,liquid loading and fermentation temperature It's not easy.The optimum conditions for producing enzyme of the mixed strains were as follows: the initial p H of the medium was 8.02,the concentration of Na Cl was 40.71 g/L,the content of the solution was 79.73 m L,and the culture temperature was 27.96?.Under the optimum fermentation conditions,the enzyme activity of the mixed strain was 94.78 U/m L,which was 43.90% higher than that before optimization. |
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