The Screen Of Agarase-Producing Microbes From Sea Shellfish Gut And Optimization Of The Responsible Enzyme's Fermentation Conditions

With the deep of research and utilization to the marine biological resources,researchers found that the oligosaccharides and new oligosaccharides from enzymatic degradating agar had many physiological function,such as inducing cell apoptosis,antioxidation,antitumor,anti-inflammatory,decay resistance,promoting Bifidabacterirm sp growth,preventing diabetes and so on.It could be widely used in food industry,chemical industry,feedstuff industry,cosmetics industry,pharmaceutical industry and other fields.But,the few source,low activity and the expensive price of agarase limited market applications of agar oligosaccharide.This study was designed to provide basic screening ways of agar-degrading microorganism from marine shellfish gut,and provide a theoretical basis and technical support for the production of algae oligosaccharides.Agarose was used as the only carbon source,forty-four strains of agar-degrading bacteria were isolated from marine shelfish gut.By Lugosi iodine staining and DNS method,the strain A-001 presented bigger transparent circle around colony than the other,and it's maximum enzyme activity was 13.01 U·mL-1.The colony morphologies of the Strain A-001 was ivory,gram-negative and short rod.Through electron microscopy observation,the strain A-001 had a flagellum.Based on physiological and biochemical experiments and 16SrDNA sequencing,.the strain A-001 was identified as Vibrio alginolyticus.Through single factor experiment and response surface method,and using the agarase activity as an index,the medium of producing agarase was optimized.The optimal medium composition was as follows:agar powder 40 g·L-1,Nacl 30 g·L-1,peptone 5 g·L-1,yeast extract 10 g·L-1,FeSo3·7H2O 0.1 g·L-1,water 1L.When the strain A-001 was cultivated for 32h in optimal fermentation medium,the enzymatic activity of the supernatant of fermentation medium was up to 15.645 U·mL-1,which was more than 1.2 times of the enzymatic activity(13.01 U·ML-1)in unoptimal medium.The fermentation conditions for the strain A-001 was optimized in optimal medium by single factor experiment.The results showed that the optimal fermentation conditions were as follow:Initial pH7.0,inoculum 11%,liquid volume 30mL,culture temperature 36 rotation speed 150 r·min-1,fermentation time 42h.Under optimal fermentation conditions,the enzymatic activity of the supernatant of fermentation medium was up to 18.09 U·mL-1,more than 1.39 times of the enzymatic activity(13.01 U·mL-1)in unoptimal fermentation conditions.