Characteristics Of Acid And Flavor-Producing Streptococcu Thermophilus And Lactobacillus Bulgaricus,as Well As Their Functional Gene Typing And Expression

Streptococcus (S.) thermophilus and Lactobacillus (L.) bulgaricus, the most important dairy starter culture bacteria for fermentation, have important economic values and application potential. Rapid acidification of the milk is normally caused by the symbiotic interaction between the yogurt fermentation bacteria, and the interaction of these bacteria provides special flavor and desirable texture to the final yogurt product. In this thesis, S. thermophilus and L. bulgaricus isolated from different regions and different milk products including those currently preserved in the Lactic Acid Bacteria Culture Collection (LABCC) in Inner Mongolia Agricultural University were studied.43strains of S. thermophilus and39strains of L. bulgaricus were used to produce yogurts using traditional laboratory fermentation methods. The properties of the acid production and variation of flavor substances of the yogurt fermented by the two kinds lactic acid bacteria were recorded during the fermentation and storage period. Meanwhile multilocus sequence typing (MLST) method based on functional genes for acid and flavor formation of S. thermophilus and L. bulgaricus was used to study the phylogenetic and genetic evolution of these yogurt-producing strains. Several representative strains with different acid-producing and flavoring characteristics were selected for q-PCR analysis targeting their functional genes. The functional gene expression and the changes in phenotype indicators were correlated by q-PCR at different storage time points. The main results are listed as follows:First, the characteristics of pH, titratable acidity and viable counts of2kinds of LAB in fermented milk were monitored every two hours during the fermentation process. At the end of fermentation, the pH, titratable acidity, viable count, lactic acid content, acetaldehyde content, diacetyl content and other indicators of yogurt were measured after storage for0,8,12,24,36,48hours, and7,14, and21days. The acid-producing property and the variation pattern of production of flavor substances of selected strains of S. thermophilus and Lactobacillus bulgaricus were studied during the yogurt fermentation process and storage period. The results showed that the fermentation time of43S. thermophilus ranged between4.5-24.5h, among which the fermentation time of19strains was less than10h. The acid-producing rate of10strains was more than10°T/h. The fermentation time of39L. bulgaricus varied from8.5to24.5h, among which 23strains were less than15h. There are14strains with an acid-producing rate of more than3°T/h. From the results of acid production and fermentation characteristics, these bacteria hold good potential to be developed as starter cultures for industrial application.Secondly, a multilocus sequence typing (MLST) analysis based on functional genes associated with the acid and flavor formation characteristics of S. thermophilus and L. bulgaricus was performed to study the phylogenetic and genetic evolution of these strains. The results showed that the43strains of S. thermophilus formed19STs based on gene sequence analysis, while these strains were divided into four groups based on genotype of functional genes and phenotypic data such as fermentation time, acid and flavor substance formation. It was also found that the grouping based on functional genotype corresponded well with that of derived from the phenotyping characteristics. In particular, strains producing high, medium and low levels of acetaldehyde were clustered separately into different branches, which were also corresponding to their genotypes. The39L. bulgaricus strains could be divided into19STs types according to the functional gene sequence analysis. Moreover, these strains were grouped into four different clusters based on analysis combining functional gene sequence and phenotypic (including fermentation time, lactic acid content, and acetaldehyde content etc.) data. There was a good correlation between the genotype and phenotypic characteristics, specifically the fermentation time and lactic acid content. Strains with short (less than15h), moderate (15-20h) and long (more than20h) fermentation time were clustered into separate groups. Similar clustering trend was observed with the capacity of lactic acid production of these strains. The clustering pattern based on fermentation time or lactic acid production capacity was in agreement with the genotype grouping. The results showed that after a long term yogurt bacterial selection pressure and environmental adaptability, the genetic and genomic sequences of these bacteria have evolved to a high functionality in milk fermentation and yoghurt flavor formation in the environment. These results suggest that yogurt bacteria retained the functional gene sequences relating to milk fermentation and flavor formation under selection pressure and domestication of environmental adaptability in yogurt environment over a long period. Thus, based on these results, we grouped the strains that possessed desirable phenotypic properties with their functional gene sequences. This thesis has laid a foundation in rapid screening for strains with specific fermentation characteristics, and such traits are elucidated at the molecular biology and genetic levels.Thereafter, the representative strains with different fermentation characteristics and different flavor compounds were selected for q-PCR analysis that target to the related functional genes. Specifically, the correlation between the content of flavor substances (including diacetyl and acetaldehyde) and key functional gene expresrion was studied. The results showed that the main flavor substances of acetaldehyde content was significantly and positively correlated with the gene expression of lld, pdc, lld, etc. in S. thermophilus. There was significant positive correlation between the expression of the pfl gene and that of lld, pdc, and lld, but not other analyzed genes. The acetaldehyde content of L. bulgaricus was also significantly and positively correlated with the expression of the functional genes, pdc, lld, ald etc. The expression of ald gene was significantly and positively correlated with the expression of the lld gene, but not with the pdc gene expression. By monitoring two kinds of flavor chemicals, acetaldehyde and diacetyl, during yogurt storage, it was revealed that24to36hours of storage was the critical time for flavor production, and the flavor content decreased afterwards and along with the storage time.