| Sargassum horneri are a type of large brown algae that are widely distributed in the ocean.Sargassum horneri cells are rich in polysaccharides such as alginate and fucoidan.And due to its unique molecular structure,alginate is widely used in food,medicine,and ecological protection.Alginate oligosaccharides,the degradation product of alginate,has various biological activities such as anti-oxidation,anti-cancer,anti-inflammatory,lowering blood pressure,lowering blood sugar,and promoting plant growth.Alginate and alginate oligosaccharides have the characteristics of high safety,and they have been studied in the fields of medicine and food.Therefore,Sargassum horneri has potential economic value.The use of alginate lyase to degrade alginate to produce alginate oligosaccharides is an efficient and reliable method.However,there are many types of alginate lyase and the enzymes show different activities.So many scholars chose to isolate alginate lyase from marine microorganisms.In this paper,the strains were isolated and screened from the digestive tract of Haliotis discus hannai,which can produce algin lyase efficiently.And the culture conditions of the strain producing enzymes were optimized.Finally,the fermentation conditions of the Sargassum horneri were studied using the screened strains.The specific research content and results are as follows:1.Using a selective medium with sodium alginate as the sole carbon source,16 strains with the ability to utilize sodium alginate were screened and named ALG1～16.The enzyme activity of the screened strains was determined,and the 8 strains with higher enzyme activity were identified.After 16 S r RNA gene amplification,sequencing,and comparison,it is determined that there are 4 species relative to the 8 strains.In order,ALG1,3,5 and 13 correspond to Vibrio algivours strain,ALG7 and ALG12 correspond to Vibrio rumoiensis strain,ALG8 correspond to Psychromonas aquatilis strain and ALG14 correspond to Vibrio aphrogenes strain.In addition,the similarity between ALG8 and Psychromonas aquatilis strain is 97.27%.After some physiological and biochemical identification experiments,it is considered that it may be a new strain.2.The conditions for the production of alginate lyase by the strain ALG7 were optimized.The optimal conditions for enzyme production were determined through single factor experiments.The results were 1.0% sodium alginate,0.6% peptone,3.0%Na Cl,initial p H 7.5,culture temperature 30℃,culture time 24 h.Then,orthogonal experiments were carried out on the four conditions of temperature,p H,time and Na Cl addition.The result of orthogonal optimization was 25℃,24 h,p H 7.5,and Na Cl 3.0%.Verification of the orthogonal results,the measured enzyme activity of 117.63 U/m L is lower than the result of the single factor optimal combination of 127.87 U/m L,so the final culture temperature is determined to be 30℃.3.The culture conditions of the alginate lyase-producing strain ALG8 were optimized.The single factor results were sodium alginate 1.2%,peptone 0.8%,Na Cl2.0%,initial p H 8.5,culture temperature 25℃,culture time 24 h.The same orthogonal experiment on temperature,time,p H and Na Cl addition was carried out,and the optimal culture condition for alginate lyase production was 2.5% Na Cl addition,p H 8.5,temperature 25℃,culture for 24 h.The enzyme activity is 112.32 U/m L after optimization.4.The strain ALG7 was cultured in the optimal enzyme-producing medium,and the cultured seed liquid was added to the copper algae solution.The bacterial fermentation of copper algae was studied.The concentration of total sugar and the concentration of reducing sugar in the fermentation broth were measured.The changes of sugar in fermentation broth were studied,and they were cultured in 60 m L conical flask,300 m L conical flask,and 1500 m L fermentation reactor.The results showed that the total sugar concentration in the fermentation broth showed a trend of first decreasing,then increasing and then decreasing,and the reducing sugar concentration first increasing and then decreasing.The smaller the system volume,the faster the growth of bacteria,and the faster the reduction of reducing sugar and total sugar concentration.Through experiments on the material-liquid ratio and the inoculum volume,it can be seen that the sugar concentration in the high material-liquid ratio group is higher,but the proportion of reducing sugars in the total sugar concentration is lower.In addition,reducing sugars decreased faster than total sugars in the high inoculation group.In this study,two bacterial strains that can efficiently produce alginate lyase were selected,named Vibrio rumoiensis strain ALG7 and Psychromonas.sp.strain ALG8.And ALG8 is tentatively scheduled as a new strain that has never been reported.The culture conditions for the production of alginate lyase from the two strains were optimized.The optimized enzyme activity was 127.87 U/m L and 112.32 U/m L,both strains showed good enzyme activity.Finally,Vibrio rumoiensis ALG7 was selected to conduct a preliminary study on the fermentation of Sargassum horneri,which laid the foundation for the production of alginate oligosaccharides by bacterial fermentation. |
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