Production by and isolation of exopolysaccharides from Streptococcus thermophilus grown in a milk medium and evidence for their growth-associated biosynthesis

Four Streptococcus thermophilus strains ( Strep. thermophilus BTC, Strep. thermophilus LY03, Strep. thermophilus 480 and Strep. thermophilus Sfi20) have been examined for their exopolysaccharide production capacity. All strains produced a polymer composed of the neutral sugars glucose and galactose, but in different ratios. It was clearly shown that the biosynthesis of exopolysaccharides from Strep. thermophilus LY03 is growth-associated and hence displays primary metabolite kinetics. The monomer ratio of the exopolysaccharide synthesized did not vary throughout the fermentation cycle. The production kinetics and exopolysaccharide yields were strongly dependent on the fermentation conditions. Physical factors such as temperature, pH and oxygen tension as well as chemical factors (medium composition, initial lactose concentration, carbon/nitrogen levels) were of utmost importance.     

UDP-N-acetylglucosamine 4-epimerase activity indicates the presence of N-acetylgalactosamine in exopolysaccharides of Streptococcus thermophilus strains

The monomer composition of the exopolysaccharides (EPS) produced by Streptococcus thermophilus LY03 and S. thermophilus Sfi20 were evaluated by high-pressure liquid chromatography with amperometric detection and nuclear magnetic resonance spectroscopy. Both strains produced the same EPS composed of galactose, glucose, and N-acetylgalactosamine. Further, it was demonstrated that the activity of the precursor-producing enzyme UDP-N-acetylglucosamine 4-epimerase, converting UDP-N-acetylglucosamine into UDP-N-acetylgalactosamine, is responsible for the presence of N-acetylgalactosamine in the EPS repeating units of both strains. The activity of UDP-N-acetylglucosamine 4-epimerase was higher in both S. thermophilus strains than in a non-EPS-producing control strain. However, the level of this activity was not correlated with EPS yields, a result independent of the carbohydrate source applied in the fermentation process. On the other hand, both the amounts of EPS and the carbohydrate consumption rates were influenced by the type of carbohydrate source used during S. thermophilus Sfi20 fermentations. A correlation between activities of the enzymes alpha-phosphoglucomutase, UDP-glucose pyrophosphorylase, and UDP-galactose 4-epimerase and EPS yields was seen. These experiments confirm earlier observed results for S. thermophilus LY03, although S. thermophilus Sfi20 preferentially consumed glucose for EPS production instead of lactose in contrast to the former strain.     

Requirement for phosphoglucomutase in exopolysaccharide biosynthesis in glucose- and lactose-utilizing Streptococcus thermophilus

To study the influence of phosphoglucomutase (PGM) activity on exopolysaccharide (EPS) synthesis in glucose- and lactose-growing Streptococcus thermophilus, a knockout PGM mutant and a strain with elevated PGM activity were constructed. The pgmA gene, encoding PGM in S. thermophilus LY03, was identified and cloned. The gene was functional in Escherichia coli and was shown to be expressed from its own promoter. The pgmA-deficient mutant was unable to grow on glucose, while the mutation did not affect growth on lactose. Overexpression of pgmA had no significant effect on EPS production in glucose-growing cells. Neither deletion nor overexpression of pgmA changed the growth or EPS production on lactose. Thus, the EPS precursors in lactose-utilizing S. thermophilus are most probably formed from the galactose moiety of lactose via the Leloir pathway, which circumvents the need for a functional PGM.     

Correlation of activities of the enzymes alpha-phosphoglucomutase, UDP-galactose 4-epimerase, and UDP-glucose pyrophosphorylase with exopolysaccharide biosynthesis by Streptococcus thermophilus LY03

The effects of different carbohydrates or mixtures of carbohydrates as substrates on bacterial growth and exopolysaccharide (EPS) production were studied for the yoghurt starter culture Streptococcus thermophilus LY03. This strain produces two heteropolysaccharides with the same monomeric composition (galactose and glucose in the ratio 4:1) but with different molecular masses. Lactose and glucose were fermented by S. thermophilus LY03 only when they were used as sole energy and carbohydrate sources. Fructose was also fermented when it was applied in combination with lactose or glucose. Both the amount of EPS produced and the carbohydrate source consumption rates were clearly influenced by the type of energy and carbohydrate source used, while the EPS monomeric composition remained constant (galactose-glucose, 4:1) under all circumstances. A combination of lactose and glucose resulted in the largest amounts of EPS. Measurements of the activities of enzymes involved in EPS biosynthesis, and of those involved in sugar nucleotide biosynthesis and the Embden-Meyerhof-Parnas pathway, demonstrated that the levels of activity of alpha-phosphoglucomutase, UDP-galactose 4-epimerase, and UDP-glucose pyrophosphorylase are highly correlated with the amount of EPS produced. Furthermore, a weaker relationship or no relationship between the amounts of EPS and the enzymes involved in either the rhamnose nucleotide synthetic branch of the EPS biosynthesis or the pathway leading to glycolysis was observed for S. thermophilus LY03.     

郑州部分市售酸奶中乳酸菌的分离鉴定与益生特性的研究

目的调查郑州部分市售的酸奶中嗜热乳酸链球菌与保加利亚乳酸杆菌存在情况与益生特性。方法无菌采取样品进行乳酸菌分离,并对分离菌进行形态学、生化特性、产酸性、耐酸性、耐胆汁性、高温耐受性和抑菌性等生物学特性研究。结果从8种品牌中分离到4株保加利亚乳酸杆菌、7株嗜热链球菌,且分离菌株均能产酸、对高温具有一定的耐受性,在pH 3.0以上均能生长良好,而且分离菌的抑菌物质具有一定的热稳定性,保加利亚乳酸杆菌经70、100℃热处理抑菌活性基本不变。嗜热链球菌经70℃热处理后保留90%以上的抑菌活性,但经100℃高温处理后抑菌活性有所降低。分离菌产生的抑菌物质对胰蛋白酶敏感,保加利亚乳酸杆菌在偏中性条件下抑菌活性较高,而嗜热链球菌在偏中性条件下丧失抑菌活性。结论本研究为河南市售酸奶质量以及为消费者提供有利信息奠定基础。     

Presence of additional peptidases in Streptococcus thermophilus CNRZ 302 compared to Lactococcus lactis

Streptococcus thermophilus is widely used in the dairy industry but little is known about its peptidase system. The aim of this study was to determine the biochemical and genetic characteristics of this system, and to compare it to the well known system of Lactococcus lactis . We separated the intracellular proteins of Strep. thermophilus CNRZ 302 and L. lactis NCDO 763 by ion-exchange chromatography and we detected the activity of the different types of peptidases. In both L. lactis and Strep. thermophilus strains, we showed 13 different peptidase activities with biochemical homologies between both species. Streptococcus thermophilus also possessed two peptidases which we did not find in L. lactis : an aminopeptidase and an oligopeptidase. We performed Southern blot experiments and among the eight peptidase genes tested, only the genes encoding the general aminopeptidases, pepC and pepN , were homologous between the L. lactis and Strep. thermophilus strains. Besides biochemical and genetic similarities, the peptidase systems of Strep. thermophilus and L. lactis thus differed by the presence of additional peptidases in Strep. thermophilus .     

Characterization of a bacteriocin, Thermophilin 1277, produced by Streptococcus thermophilus SBT1277

AIMS: To assess the inhibitory activity and the influence of culture condition on the growth and bacteriocin, Thermophilin 1277, production by Streptococcus thermophilus SBT1277. METHODS AND RESULTS: Thermophilin 1277, which was produced by S. thermophilus SBT1277, showed an antimicrobial activity against several lactic acid bacteria and food spoilage bacteria including Clostridium butylicum, C. sprogenes and Bacillus cereus. Thermophilin 1277 was inactivated by proteinase K. Heating treatment did not affect the antimicrobial activity. The partially purified Thermophilin 1277 had an apparent molecular mass of 3.7 kDa. N-terminal sequence analysis revealed 15 amino acid residues that correspond with amino acid sequence of the lantibiotics bovicin HJ50 produced by Streptococcus bovis HJ50. The effects of culture condition for the bacteriocin production by S. thermophilus SBT1277 were studied. During the batch fermentation, Thermophilin 1277 was produced in M17 broth, but no bacteriocin production occurred in the sucrose-tryptone (ST) broth. Bacteriocin production was detected in pH controlled ST broth at pH values of 5.5-6.5. CONCLUSIONS: Thermophilin 1277 production from S. thermophilus strain depended on the culture conditions. Some characters and N-terminal amino acid sequence of Thermophilin 1277 differed from bacteriocins produced by S. thermophilus reported previously. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptococcus thermophilus SBT1277 or its bacteriocin which has a wide inhibitory spectrum has a potential use as a biopreservative in dairy products.     

Plasmid transfer via transduction from Streptococcus thermophilus to Lactococcus lactis

Using Streptococcus thermophilus phages, plasmid transduction in Lactococcus lactis was demonstrated. The transduction frequencies were 4 orders of magnitude lower in L. lactis than in S. thermophilus. These results are the first evidence that there is phage-mediated direct transfer of DNA from S. thermophilus to L. lactis. The implications of these results for phage evolution are discussed.     

Isolation and Characterization of a Streptococcus thermophilus Plasmid Closely Related to the pMV158 Family

Twenty-two Streptococcus thermophilus strains used for milk fermentations were analyzed for their plasmid content and 13 of them (59%) were found to contain one or two plasmids. Fifteen S. thermophilus plasmids were divided into four groups using DNA homology. Ten plasmids were classified within group A and they shared homologies with all the previously sequenced S. thermophilus plasmids. Three plasmids (group B) hybridized with each other and two plasmids only hybridized with themselves (groups C and D). Single-stranded DNA was detected within strains containing plasmids of groups A, C, and D, indicating that they replicate via a rolling-circle mode. The only plasmid of group C, named pSMQ172, was further characterized. This 4230-bp plasmid replicates in Escherichia coli, Lactococcus lactis, and Streptococcus salivarius and does not confer phage resistance. Comparisons with databases showed that pSMQ172 was related to pMV158 of Streptococcus agalactiae and to pSSU1 of Streptococcus suis. These results suggest that genetic exchanges may have occurred between pathogenic and nonpathogenic streptococci.     

Molecular genetics of Streptococcus thermophilus

Abstract The metabolism and genetics of Streptococcus thermophilus (presently Streptococcus salivarius ssp. thermophilus ) have only been investigated recently despite its widespread use in milk fermentation processes. The development of recombinant DNA technology has allowed impressive progress to be made in the knowledge of thermophilic dairy streptococci. In particular, it has permitted a careful analysis of phenotypically altered variants which were derived from a mother strain by plasmid or chromosomal DNA reorganization. While natural phage defense mechanisms of S. thermophilus remain poorly documented, information on the bacteriophages responsible for fermentation failures has accumulated. The lysogenic state of two S. thermophilus strains has also been demonstrated for the first time. Gene transfer techniques for this species have been established and improved to the point that targeted manipulation of their chromosomal determinants is now feasible. Cloning and expression vectors have been constructed, and a few heterologous genes were successfully expressed in S. thermophilus . The first homologous genes, involved in carbohydrate utilization, have been cloned and sequenced, shedding some light on the molecular organization of key metabolic steps.     

Complete genome sequence of the pigmented Streptococcus thermophilus strain JIM8232

Streptococcus thermophilus is a dairy species commonly used in the manufacture of cheese and yogurt. Here, we report the complete sequence of S. thermophilus strain JIM8232, isolated from milk and which produces a yellow pigment, an atypical trait for this bacterium.     

Regulation of branched-chain amino acid biosynthesis by alpha-acetolactate decarboxylase in Streptococcus thermophilus

AIMS: To demonstrate the presence of an active alpha-acetolactate decarboxylase in Streptococcus thermophilus and to investigate its physiological function. METHODS AND RESULTS: Streptococcus thermophilus CNRZ385 contains a gene encoding an alpha-acetolactate decarboxylase. Comparison of the production of alpha-acetolactate and its decarboxylation products, by the parent strain and an alpha-acetolactate decarboxylase-deficient mutant, demonstrated the presence of a control of the pool of alpha-acetolactate by valine, leucine and isoleucine. This control occurs via an allosteric activation of the alpha-acetolactate decarboxylase. Cell-free extracts of S. thermophilus were not able to decarboxylate the isoleucine precursor alpha-acetohydroxybutyrate. CONCLUSIONS: These results strongly suggest that one of the physiological functions of the alpha-acetolactate decarboxylase in S. thermophilus is to regulate leucine and valine biosynthesis by diverting the flux of alpha-acetolactate towards acetoin when the branched-chain amino acids are present at a high concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: Regulation of branched-chain amino acid biosynthesis by alpha-acetolactate decarboxylase may occur in several other micro-organisms and explain some of their growth properties.     

Fermentation of milk by Streptococcus salivarius subspecies salivarius and Streptococcus salivarius subspecies thermophilus and their use to the yoghurt manufacturer

Unlike Streptococcus salivarius subspecies thermophilus, Streptococcus salivarius subspecies salivarius fails to grow symbiotically in milk in the presence of Lacto-bacillus bulgaricus , does not produce large quantities of the flavour volatiles, acetal-dehyde or diacetyl and is unable to stimulate growth of Lact. bulgaricus by producing formate. Although Strep, salivarius subspecies salivarius and thermophilus have similar DNA base composition and belong in the same DNA homology group, the former is unsuitable for milk fermentations such as yoghurt because fermentation of milk using this organism results in products with poor flavour, aroma and texture.     

嗜热链球菌胞外多糖EPS333对酸乳品质的影响

本研究以酸乳为基质,通过测试酸乳酸化速率曲线、脱水收缩敏感性、持水力、水分迁移、质构及流变特性等参数,研究了嗜热链球菌胞外多糖与市场常规应用多糖对酸乳品质的影响。研究结果表明:嗜热链球菌胞外多糖EPS333在酸乳凝胶过程中较其他几种多糖能明显减缓酸化趋势,后熟及储藏阶段减少"自由水"含量,降低流动性,改善其流变特性。     

Preventive effect of Streptococcus thermophilus YIT 2001 on dextran sulfate sodium-induced colitis in mice

We investigated the preventive effect of Streptococcus thermophilus YIT 2001, a lactic acid bacterum having high antioxidative activity, on acute colitis induced by 2.5% dextran sulfate sodium in mice, and compared the effect with that of S. thermophilus YIT 2084 which has lower antioxidative activity. Feeding S. thermophilus YIT 2001 decreased the disease activity index and level of lipid peroxide (the thiobarbituric acid reactive substance content) in the colonic mucosa. The hematocrit and hemoglobin concentrations in the blood of S. thermophilus YIT 2001-fed mice were higher than those of the control mice. S. thermophilus YIT 2084 had no significant effect on these parameters. The results suggest that the antioxidative activity of S. thermophilus YIT 2001 was involved in the improving effect on colitis.     

Metabolically improved exopolysaccharide production by Streptococcus thermophilus and its influence on the rheological properties of fermented milk

Altered levels of enzymes in the central carbon metabolism in Streptococcus thermophilus increased the exopolysaccharide (EPS) production 3.3 times over that of the parent strain. The influence of enhanced EPS production on the rheological properties of fermented milk is described for engineered strains of S. thermophilus which produce different levels of EPSs.     

Characterization of pER371-based Streptococcus thermophilus-Escherichia coli shuttle vectors

Native plasmid of Streptococcus thermophilus ST137, pER371 (2.7 kb) linearized at various unique restriction sites was individually subcloned into Escherichia coli plasmid pUC19 to generate the pUER-series recombinants. A selection cassette consisting of chloramphenicol- and erythromycin-resistance genes was spliced into each construct to generate the pMEU shuttle vectors. Electrotransformation of Streptococcus thermophilus with these vectors showed that a ca. 1.7 kb BstEII/ BanII fragment is essential for plasmid replication. A shuttle vector, pMEU149-1 (5.3 kb), was constructed using the minimally required fragment for replication. A chloramphenicol acetyltransferase ( cat) gene was successfully expressed in the ultimate S. thermophilus host by using pMEU149-1. Cloning vectors derived from pER371 should provide valuable alternative gene delivery vehicles for the genetic engineering of lactic acid bacteria.