Screening And Identification Of Probiotics In The Sea Cucumber Intestine

At present,sea cucumber culture has become one of the most important aquaculture species in the north of China.However,antibiotics frequently misuse with outbreaking diseases,which seriously hindered the enlargement of breeding scale.To find a safe and nontoxic replacer is imperative.Therefore,the research of feed additives made by probiotics gradually is paid more attention.Marine microorganisms have their own unique advantages because of the diversities and uniqueness of the marine environment.Thus,screening of gut probiotics of sea cucumber is a primary condition to research and develop the probiotic addtivies for sea cucumber.This study focused on screening of sea cucumber intestinal probiotics.Based on alkaline tolerance,acid tolerance,calcium-dissolving ring and bacteriotatic test,one lactobacillus was isolated,named as HS-R9;Based on phenolsulfte acid method,two exopolysaccharide-producing yeasts were isolated,named as HS-J4 and HS-J9.Based on the morphological and 16S rDNA sequence analysis,the bacterium HS-R4 was identified as Lactobacillus plantarum.Its retrieval number is KF668239 in GenBank.Based on the morphological and 26S rDNA sequence analysis,the yeasts HS-J4 and HS-J9 were identified as Rhodotorula mucilaginosa and Meyerozyma guilliermondii.Their retrieval number are KF668240 and KF668241 in GenBank.The yield of exopolysacchatide synthesized by yeasts was estimated by phenolsulfte acid method,and the fermentation conditions of exopolysaccharide production by strains HS-J4 and HS-J9 were optimized by response surface method(RSM)to maximize the yield of polysaccharide.The pH,inoculum volume and fermentation time were chosen as the main factors,and the yield of exopolysaccharide as response value based on one factor test.The extracellular polysaccharide production of those important factors were optimized by Central Composite Design(CCD)of Box-behnken methods.The response analysis was done by Design expert 8.0 and the final result was validated by actual experiment.The optimum fermentation conditions of yeast HS-J4 were obtained as follows:pH 6.5,inoculum volume 4%and fermentation time 39h.The experimental expolysaccharides yield could reach 0.560mg/mL under the optimized condition,which was increased by 18.10%compared to the original conditions(0.474mg/mL).The optimum fermentation conditions of yeast HS-J9 were obtained as follows:pH 6.6,inoculum volume 4%and fermentation time 41.3h.The experimental expolysaccharides yield could reach 0.762mg/mL under the optimized condition,which was increased by 23.67%compared to the original conditions(0.616 mg/mL).The study results laid the foundation for the application and development of probiotics in aquaculture.