An Initial Study Of DUAN NAI An Probiotics For Its Stability And Scaling-up Process

This study mainly focused on stability and scaling-up process of DUAN NAI AN probiotics which used for the prevention and treatment of piglets’ weaning diarrhea.1. Probiotics stability tests. Using traditional biochemical methods and molecular biological methods of 26 S r DNA for identification of bacteria before and after induction, and then measured growth curve in certain period of time, p H variation and the coherency by a method of direct counting with blood count board, the results showed that there was no mutation for the strain and as well as their performance included morphological, physiological and biochemical characteristics and molecular biological characteristics. And strain showed steadily increasing trend in 72 h, p H value gradually decreased from the initial 6 to 3.5 because of maltose fermentation, while cohesion value F1 equaled 0.243, which indicated that many cells suspending, difficult precipitation and clarification in the end of the fermentation, and proved that the probiotics could be better entering the gastrointestinal tract and affecting its prebiotic functionality.2. Tolerance test for DUAN NAI AN. Placed it in simulated gastric juice that p H value was 2 to 4 for 3 h, its number of viable cells rate in different p H environments were more than 100%; DUAN NAI AN strains growed well at a concentration of 0.1% to 1% of the artificial simulation intestinal fluid, also at a concentration of 0.3%, 0.6%, 1% of the bile salt environment, its survival rates were 100%, 90%, 77% respectively; And then cultured DUAN NAI AN in the Na Cl gradient concentration of 10% to 24% for 48 hours, it growed well in concentration of 10% to 16% and could survive in the high osmotic pressure environment of the Na Cl concentration of 22%; DUAN NAI AN temperature test, the results showed that its tolerance was at 58℃ for 10 min, 64℃ for 5min. Tolerance test results showed that in the range of tolerance for DUAN NAI AN probiotics, its higher survival rate and content of which could meet the best possible probiotic functional needs, indicated that the preparation has the basic conditions for the prevention of animal diseases, and also provided theoretical basis for the clinical application and a large-scale production in the future.3. Qualitative detection of DUAN NAI AN probiotics secreted proteins. Combination the methods of 7cm SDS-PAGE, capillary liquid chromatography, with Q-exactive mass spectrumanalysis, then rearched data in Mascot Knowledgebase in the Uni Prot database(Swiss-Prot/ Tr EMBL, www. expasy.org) and Gene Ontology(GO) Database(http: // www. geneontology.org/), after that, using Blast 2GO software for bioinformatics analysis. Protein spectrum identification results:the control group were identified 68 peptides, and 47 proteins groups, the DUAN NAI AN group were identified 241 peptides, and 121 proteins groups, which had a total of 109 annotated protein, thenthe first group of specific protein was 11, unique protein of second groups 63, common proteins of second group was 35. Finally, analyzed by bioinformatics with GO and KEGG pathway annotations indicated that DUAN NAI AN group not only were rich protein, also metabolic pathways. Meanwhile, bioinformatics analysis found that carbohydrate metabolism and energy metabolism in the DUAN NAI AN fermentation broth even more, also affected a small number of biological pathways, and DUAN NAI AN group had the unique protein annotation of electron carrier activity and transporter activity, which probably exercised the biological regulation, stimulus-response functions and positioning process.4. To optimize the DUAN NAI AN probiotics culture conditions.Using the methods of orthogonal design of L9(34) to optimize the shake flask culture conditions of DUAN NAI AN and fermentor culture conditions. Through shake flask cultivation, determined its culture medium formula and the basic culture conditions: inoculum of 1%, the temperature was at 25℃, p H value was of 6, shaker speed was 110 r/min; At the same time, research in 10 L fermentor, fluid volume, inoculum and speed had effects on the yeast, and also considered fermentation efficiency and cost, controlled fermentation conditions of p H was 6, the temperature was at 25℃, the DO value was less than 5% under the same conditions, and optimized yeast growth conditions of fermentor: speed was 200 r/min, inoculum of 5%, and with liquid quantity reached 4.5 L, then the content of the yeast could reach 2.80×108 cfu/ml. According to optimize the culture conditions step by step from seed liquid, shake flask to fermentor of which indicated that the probiotics had the potential to be a stable industrial mass production.