| Probiotics are a class of bacteria which have beneficial effects on the host. However,probiotics are easy to be inactivated in the process of production, transportation, and storage.The number of functional living probiotic during shelf-life can not meet the productrequirements. Therefore, to choose a probiotic which has stronger resistance to externalenvironmental pressures, and to find a way to extend the shelf-life of probiotics is necessary.It means a lot to improving the application effect of probiotic products.A strain of potential probiotic B2-2which was preserved in the laboratory was studied.Its biological characteristics, including colony characteristics, cell morphology, growth curve,16S rDNA sequecing were performed. To reduce the cost of fermentation, a cheaper culturemedium was chosen from nutrient broth, PYE, molasses and brown sugar, and theconcentration was optimized. To optimize the final concentration of B2-2fermentation,different organic nitrogen and inorganic nitrogen were added to the culture medium tocompare their effects on growth of B2-2and to determine the best nitrogen sources andconcentration. Finally, in order to extend the shelf-life of B2-2preparation, the effects ofdifferent inorganic salt solutions and protective agents on prolonging preparation’s shelf-lifewere evaluated by accelerated test using the survival rate of viable bacterium10%as theshelf-life indicator. First, a best balanced salts solution was chosen by single-factorexperiments. Then,8different protective agents were added to the preparation respectively toscreen the4best by single-factor experiments. Taking these four protective agents as factors,the best composite protective ingredients were determined by L9(34) orthogonal experiment.To ensure the reliability of accelerated test, plate count method for measuring the shelf-lifewas used to verify the results of accelerated test.The Gram stain showed that probiotic B2-2is a long rod shaped Gram-positivebacterium. After48h of culturing, it can form round, moist, smooth and orange colonies withregular edge, shiny surface. Probiotic B2-2was preliminary identified as Bacillusvietnamensis by16S rDNA gene sequencing.According to the comprehensive comparison of B2-2growth under optimal concentration of each substrate,5%molasses, as a cheap and abundant culture medium, canachieve the same B2-2growth as nutrient broth and PYE. To adapt to large-scale industrialproduction and reduce cost,5%molasses was used as the best culture medium for subsequenthigh-density culturing. Through single-factor experiment, the optimum nitrogen source andconcentrations are as follows:1%yeast extract powder,0.5%ammonium sulfate. Insubsequent experiments, the selected medium components are: molasses,5%; yeast extractpowder,1%; ammonium sulfate,0.5%. Final concentration of B2-2can reach1012CFU/mL,two orders of magnitude higher than that of nutrition broth which is normally used in thelaboratory. The results of accelerated experiments showed that the optimal combination ofprotective agents under4℃is: trehalose,3%; sodium glutamate,7%; raffinose,5%; inositol,7%. In this case, shelf-life of B2-2preparation is1year10months. The optimal combinationof protective agents under25℃is: trehalose,3%; inositol,3%; sodium glutamate,7%;raffinose,5%. In this case, shelf-life of B2-2preparation is17days. Confirmatory test showedthat the composite protective agents can indeed play a significant role in prolonging theshelf-life of B2-2preparation, indicating that the results of shelf-life obtained by acceleratedtest are reliable. |
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