Research And Development On Bacillus Natto Probiotics For Prevention And Control Of Vibrio Parahaemolyticus

Bacillus natto isolated from a functional food–natto is one of the12microbialstrains that can be directly used for animal feeding approved by China’s Ministry ofAgriculture. In this paper, Bacillus natto strains were isolated from three kinds ofJapanese natto, then they were purified and identified by physiological andbiochemical test. The inhibitory effects of these strains’ fermentation broth werecompared through their inhibition to the indicator bacteria Vibrio parahaemolyticusby the agar diffusion method. Their capacity of amylase and protease producing wascompared by starch plate and the casein plate method. Finally, a predominant strainwas filtered out.Then it was co-cultured with Vibrio parahaemolyticus to researchits antagonistic mechanism. Its temperature tolerance was researched. The fermentcondition of Bacillus natto cultured in soybean whey was optimized. On this basis,Bacillus natto was expanding cultured in a20L fermentation tank. Then，10%maltodextrin,5%soluble starch and5%nonfat dry milk were added into20hoursfermentation broth to protect live bacterial form high-temperature injury. The outlettemperature of spray drying was optimized by contrast the amount of live bacteriaand the water content of sprayed powder. Results:1. Isolation, purification and identification of Bacillus natto. Nine strains wereisolated according to the bacteria colony morphology. These strains were identifiedby physiological and biochemical test and the natto trial experiment. Finally, six ofthese strains were identified as Bacillus natto.2. Production strains screening. The strain NO.5was selected as productionstrain for its best performance on enzyme production inhibitory effect. Co-cultureexperiments showed that the Bacillus natto can effectively inhibit the proliferation ofVibrio parahaemolyticus as long as initial concentration is dominant. Temperaturetolerance tsts showed that Bacillus natto still remain viable under70℃for1h, but itwill inactivate rapidly when temperature is higher than80°C.3. Optimization of fermentation conditions. Single factor experiments showedthat temperature, initial pH, liquid volume and shake speed are the main factors.Fermentation conditions were optimized by orthogonal test. The optimal conditionsas follows: Liquid volumes20ml/100ml, temperature30℃, initial pH7.0, shakespeed200r/min. The confirmatory test showed that under these conditions theconcentration of live bacteria can reach up to1.83×109cfu/ml for20hoursfermentation.4. Selection of outlet air temperature. The feed rate of fermentation broth to bespray-dried is set to1.5L/h. Results showed that when outlet air temperature is70°C the powder has the best composite index.