Isolation, Screening, Application Of Fine Lactic Acid Bacteria From Yoghurt Self-made By Herdsman In Gannan Pastoral Area

Recently, as consumer demands for yogurt had increased quickly,factory also demands starter with high quality.Traditional starter is replacing by concentrated lactic acid bacteria starter which has more count of lively bacterial and more advantageous for it has less count of lively bacterial. It,s significant for promoting the industrial process of lacfic acid bacteria starter,in proving development of fermented dairy product; In this study,lactic acid bacteria were isolated from the samples of Yak Yoghourt collected from gannan region of Gansu. By first screening, further screening and combined fermentation experiments,the fine strains,combination of strans and the optimal fermantion conditions were determined.The technical conditions of its enrichment cultures, concentrated isolation and freeze-drying were studied; the enrichment mediums and were optimum in medium composition and environment factors. Finally, high-activity concentrated Yoghourt freeze-dry starter culture were obtained.Isolation of LAB. In this study, 96 strains of lactic acid bacteria(LAB) are Gram-positive, catalase-negative,producting-acid, were isolated from 22 Yak Yoghourt samples collected from Tianzhu region of Gansu.72 strains are Lactococcus,24 strans are Lactobacillus.Screening of LAB. First selecting:Genetic stability, coagulation time, titratable acidity, water-retention rate and sensory evaluation, 12 strains of genetically stable lactic acid bacteria were selected. Second selecting: souring rate, capability of postacidification, capability of acetaldehyde-producing, capability of diacetyle-producing, proteolytic activity. 8 strains with high fermenting performance were selected.The selecting of Functional characteristics: living bacterium number, acid-resistant capability, bile salts-resistant capability, NaCl-resistant capability. Finally, 4 strains lactobacillus and 2 strains lactococci may be screened as the starter culture of yogurt. The isolates were identified by biochemical characteristics, The identifiable results are: Q2 is Lactococcus lactis ssp. Lactis,Q5 is Streptoccus salivarius ssp.thermophilus,G1 is Lactobacillus curvatus, G3 is Lactobacillus delbrueckii ssp. Bulgaricus,G4 is Lactobacillus helveticus, G6 is Lactobacillus delbrueckii ssp.delbrueckii.Combined fermentation experiments.Finally,with the screen index: Aeidity, viscosity, values of sensory evaluation,the optimum compound strains breed fermentation of yogurt:Q2 and G4. Single factor experiment indicated that the fermentation condition is leaven temperature 43℃,seed volume 4%, Q2:G4 is 1:3, adding suger 6%.Based on single factor analysis, Orthogonal experiments showed that the optimum fermentation condition is leaven temPerature 43℃, seed volume 4%, Q2:G4 is 1:3, adding suger 6%. The sensory evaluation of fermented yogurt maximum value was obioned by the optimum conditions of fermentation,and was 96.63 points. By analysising the 16S rDNA sequences of Q2 and G4.The result showd Q2 was Streptoccus thermophilus,its accession number in Gene bank was GU338023;G4 was Lactobacillus delbrueckii ssp.bulgaricus,its accession number in Gene bank was GU338024.Study on the growth promoting culture of optimum environment conditions. In this study, the enrichment cultures, nutrition enhancement and freeze-drying protectant was studied in order to culture in high cell density. The optimum environment conditions of Q2: MRS culture medium, tomato juice 8 %, 4 % carrot juice, 4 % potato juice, buffer 3, 1.5% buffer 3 of adding time was 10 and 12h, the number of viable cells got to 1.17×1011CFU/mL. The optimum environment conditions of G4: skimmed milk culture medium, tomato juice 8 %, 4 % carrot juice, 6 % potato juice, buffer 3, 1.5% buffer 3 of adding time was 10 and 12h, the number of viable cells got to 1.28×1011CFU/mL.Study on conditions of microorganism collection. The collecting time knowed was 14 hour after culture, the optimum conditions of centrifuging were 8000 rpmin and 10 min, and the harvest rate was 87.45%.Screening of cryoprotectant for freeze-dried starter culture. The optimum cryoprotectant was determined as glycerol, skimmed milk, vitaminC and sucrose using 2-level factorial experiment. The final optimum cryoprotectant was determined as 8 mL/L glycerol, 15 % skimmed milk, 5 g/L vitaminC and 15% sucrose by orthogonal experiment. The survival rate of LAB was 88.15%.The study provided a scientific foundation of inudsrtialization application of activity concentrated yoghourt starter culture in our country.