Isolation, Identification, Antibacterial Peptide Activities And Fermentation Performance Of Lactic Acid Bacteria

The quality of lactic acid bacteria (LAB) is directly related to the quality of yogurt. During preservation and passage of LAB, seed strains contamination, variation and/or degeneration are common problems. Therefore, regular identification, rejuvenation and selection of excellent strains are of great importance for high quality yogurt production. In this study, seven LAB strains were isolated and identified from the three bacterial seed stocks in the Experimental Dairy Farm of Yangzhou University from1990to2006. Their performaces in yogurt fermentation, antimicrobial peptide production and suitability for preparation of yogurt starters were studies with the following results.(1) According to their colony morphorlogy, staining property and biochemical reaction characteristics,7LAB strains were isolated by streaking3lactic acid bacterial stocks on MRS agar plates, which were named as strains ?-1, ?-2, ?-3, ZK, G, ST and393, respectively. To identify further the LAB strains,16S ribosomal DNA (rDNA) were amplified for sequence analysis. On the phylogenetic tree generated, the seven LAB strains belonged to Bacillus acidophilus (strain ?-1), Lactobacillus bulgaricus (strain ?-3), Lactococcus lactis (strain393) or Streptococcus thermophilus (strains ?-2, ZK, G and ST). Some isolated strains differed with the labelings of the bacterial seed stocks. These results suggest mis-labeling, impurity, contamination and/or degeration of the seed stocks during passage and/or storage, and thus regular rejuvenation, isolation, identification and selection of the bacterial stocks are needed for high quality yogurt production.(2) Yogurt fermentation tests were carried out with the representative strains of different lactic acid bacteria. The result showed that differences in the clotting time, acid production speed, viscosity, yogurt flavor and/or quality existed not only between different species of LABs, but also between different strains of the same species. Among them, Streptococcus thermophilus strain ?-2has the same acid production characteristics as Lactobacillus bulgaricus strain ?-3, but different from Streptococcus thermophilus strains G, ST and ZK. Among the seven LAB strains tested, Streptococcus thermophilus strain G and ST strains had a better performance in yogurt fermentation, including higher acid production speed, shorter solidify time, higher viscosity and sensory evaluation. Strain IV-3performed better in flavor, quality and comprehensive evaluation. The other strains had some unilateral advantages. These findings suggest that the phylogenetic tree based on the16S rDNA sequences can be used for accurate identification of LABs, but can't replace the conventional methodsd for selection of excellent yogurt fermentation strains.(3) Two representative strains with better performance in yogurt fermentation, Streptococcus thermophilus strain ST and Lactobacillus bulgaricus strain IV-3, were selected for further study on antibacterial peptide production. The result showed that the antibacterial peptides produced by the two strains had a brord inhibitory profile against Grams-positive and negative bacteria. By using Staphylococcus aureus as the indicator bacterium, the antibacterial peptides had the highest antibacterial activity at10h after incubation. The two strains had the highest peptide production levels after culturing for22-24h. The antibacterial peptide from strain IV-3was more active than that from strain ST. Both antibacterial peptides had a good thermostability without loss of antibacterial activity after boiling for20min. They were also stable under acidulous and neutral conditions, but can be easily hydrolyzed by proteases.(4) Streptococcus thermophilus strain ST and Lactobacillus bulgaricus strain IV-3were mixed in1:10,1:1,10:1or20:1as the fermentation starter agent, which are called zhl, zh2, zh3and zh4, respectively. Yogurt fermentation experiments showed that the mixed strain had a better complementary performance than the single strains as yogurt fermentation agent, including shorter milk-clotting time, quicker lactic acid production speed, higher viscosity, more live bacterial number and better flavor of the product. When storage at4?, the yogurt had a weaker postacidification, the viscosity was slightly increased with a relatively large number of viable lactic acid bacteria. After storage for24h, the contents of acetaldehyde and diacetyl reached to the highest levels but the ratio was within the proper concentration range. The fermented milk remained stable with a longer shelf life and thus the starter culture was suitable for yogurt production. The overall performance of the zh3starter agent was the best among the four starter agents tested.