Study On Screening Of Kelp-Degrading Bacteria And Enzymolysis Optimization And Application Of Fermentation Broth In Plant Tissue Culture

With the development of marine science,seaweed and its derivative seaweed polysaccharides are increasingly concerned by the scientific community because of their strong biological activity,development value and application prospects.However,the application of seaweed polysaccharide is limited due to its easy coagulability.Its degradation product algal oligosaccharide has a small molecular weight and is easy to be used by passive plants.These characteristics make up for the defects in the application of algal polysaccharide.Alginate lyase,as a tool enzyme,is often used to degrade seaweed polysaccharides.The production of algin oligosaccharides by using enzymes has gradually become the main way,with mild reaction conditions,strong specificity and high yield.In order to efficiently degrade kelp and effectively utilize algal oligosaccharides,a strain that could decompose kelp and produce high alginate lyase was screened from kelp degrading flora,and the strains were identified by 16Sr DNA sequencing.The single factor test was used to optimize the fermentation conditions of the strain and the decomposition conditions of kelp by alginate lyase,and the products obtained by degradation of kelp were analyzed.The effect of kelp fermentation broth on plant growth was studied by adding kelp fermentation broth to plant tissue culture.The experimental results are as follows:（1）Biodiversity analysis,isolation and screening of kelp degrading bacteria.High-throughput sequencing technology was used to analyze three groups of highly efficient kelp degrading flora to study microbial diversity.The results showed that the dominant genera among the three flora were Paenibacillus,Jonesia,and Sphingopyxis.The dominant bacteria in the three bacterial groups were isolated and purified,and 9dominant bacteria were screened out and identified by 16Sr DNA sequencing.Furthermore,the selective culture medium with pectin,hydroxymethylcellulose sodium and alginate sodium as the only carbon source was used to analyze the types of enzyme production,and the enzyme activity was detected by DNS.A strain that can degrade kelp efficiently and produce alginate lyase was selected,which was Sphingopyxis sp.（2）Optimization of enzyme production conditions of Sphingopyxis sp.Single factor experiment was used to optimize the fermentation conditions of Sphingopyxis to produce alginate lyase,and the results were obtained by detecting the biomass and enzyme activity in the fermentation broth.The results were as follows:In the fermentation medium,the inoculated amount of seed liquid was 5%,the initial p H was 6,the optimal carbon source was kelp,the optimal concentration was 25%,the optimal nitrogen source was ammonium sulfate,the optimal concentration was 0.5%,and the optimal concentration of sodium chloride was 1%,and the culture was placed at 30℃,130rpm.Under the optimum culture conditions,the activity of alginate lyase increased by 2.1times.（3）Optimization of enzymolysis conditions and product analysis of kelp.The conditions for degradation of kelp by alginate lyase were optimized,and the optimal conditions for enzymolysis were obtained by detecting the relative enzyme activity and total sugar content in the reaction system.The optimum conditions for enzymolysis were obtained,the optimum substrate for enzymatic hydrolysis was 25%kelp mud,the optimal inoculation amount is 10%,and the optimal p H system is the buffer system of Na₂HPO₄-Na H₂PO₄with p H7,and the optimal sodium chloride concentration is 0.5%.The crude extracts were obtained by centrifugal filtration and ethanol precipitation of the enzymolysis solution and identified.The results of Molish,Philling and ninhydrin experiments showed that the enzymolysis product was non-reducing sugar polysaccharide and did not contain amino acids.By thin layer chromatography,it was found that the development position of the crude extract on the silica gel plate was between sucrose diosaccharide and threosaccharide tetrasaccharide,which suggested that the oligosaccharide in the crude extract was triosaccharide.（4）Effects of kelp fermentation broth on plants growth and accumulation of effective components in tissue culture.The kelp fermentation broth prepared by fermentation of Sphingopyxis was added to the plant medium,we found:under the same condition of dilution of kelp fermentation broth,the promotion effect of adding the fermentation broth filtered by filter membrane on the proliferation of adventitious buds of greenwort callus was higher than that of using high temperature sterilization fermentation broth;adding 250 times,500 times,1000 times,2000 times diluted kelp fermentation broth,and using the medium without the addition of kelp fermentation broth as the control,the dilution of 1000 times,2000 times can promote the proliferation of vermilion callus,and the content of phenol and protein increased by34%and 77.5%when treated with kelp fermentation liquid diluted by 2000 times,and polysaccharide content increased by 9.5%when diluted by kelp fermentation liquid diluted by 1000 times.