Aspartate biosynthesis is essential for the growth of Streptococcus thermophilus in milk, and aspartate availability modulates the level of urease activity

We investigated the carbon dioxide metabolism of Streptococcus thermophilus, evaluating the phenotype of a phosphoenolpyruvate carboxylase-negative mutant obtained by replacement of a functional ppc gene with a deleted and inactive version, Deltappc. The growth of the mutant was compared to that of the parent strain in a chemically defined medium and in milk, supplemented or not with L-aspartic acid, the final product of the metabolic pathway governed by phosphoenolpyruvate carboxylase. It was concluded that aspartate present in milk is not sufficient for the growth of S. thermophilus. As a consequence, phosphoenolpyruvate carboxylase activity was considered fundamental for the biosynthesis of L-aspartic acid in S. thermophilus metabolism. This enzymatic activity is therefore essential for growth of S. thermophilus in milk even if S. thermophilus was cultured in association with proteinase-positive Lactobacillus delbrueckii subsp. bulgaricus. It was furthermore observed that the supplementation of milk with aspartate significantly affected the level of urease activity. Further experiments, carried out with a p(ureI)-gusA recombinant strain, revealed that expression of the urease operon was sensitive to the aspartate concentration in milk and to the cell availability of glutamate, glutamine, and ammonium ions.     

Anaerobic fermentation of Lactobacillus bulgaricus and streptococcus thermophilus on 3% nonfat dry milk with pure and mixed culture

Two different strains of Lactobacillus bulgaricus (ATCC-11842 and Microlife) and two strains of Streptococcus thermophilus (ATCC-19258 and Microlife) were grown anaerobically on 3% nonfat dry milk in pure and mixed culture using batch followed by fed batch culture. Samples were collected every 30 min. Concentrations of lactose, galactose, lactic acid, and other products present were measured using high pressure liquid chromatography. A spectrophotometric method from the literature was modified and used to determine microbial biomass concentrations. Relative cell numbers of the two organisms were measured microscopically in mixed culture. The results are presented in tabular form. S. thermophilus (Microlife) showed different growth characteristics compared to the other cultures. This culture utilized most of the galactose that was formed and produced greater amounts of lactic acid and biomass.     

Production of natural folates by lactic acid bacteria starter cultures isolated from artisanal Argentinean yogurts

Folate is a B-group vitamin that cannot be synthesized by humans and must be obtained exogenously. Although some species of lactic acid bacteria (LAB) can produce folates, little is known about the production of this vitamin by yogurt starter cultures. Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains were isolated from artisanal Argentinean yogurts and were grown in folate-free culture medium (FACM) and nonfat milk after which intracellular and extracellular folate production were evaluated. From the initial 92 isolated LAB strains, 4 L. delbrueckii subsp. bulgaricus and 32 S. thermophilus were able to grow in the absence of folate. Lactobacillus delbrueckii subsp. bulgaricus CRL 863 and S.?thermophilus CRL 415 and CRL 803 produced the highest extracellular folate levels (from 22.3 to 135?μg/L) in FACM. In nonfat milk, these strains were able to increase the initial folate concentrations by almost 190%. This is the first report where native strains of L. delbrueckii subsp. bulgaricus were shown to produce natural folate. The LAB strains identified in this study could be used in developing novel fermented products bio-enriched in natural folates that could in turn be used as an alternative to fortification with the controversial synthetic chemical folic acid.     

Growth and activity of Bulgarian yogurt starter culture in iron-fortified milk

Bulgarian yogurts were manufactured and fortified with 8, 15 and 27 mg of iron kg(-1) of yogurt. The growth and acidifying activity of the starter culture bacteria Streptococcus thermophilus 13a and Lactobacillus delbrueckii subsp. bulgaricus 2-11 were monitored during milk fermentation and over 15 days of yogurt storage at 4 degrees C. Fortifying milk with iron did not affect significantly the growth of the starter culture during manufacture and storage of yogurt. Counts of yogurt bacteria at the end of fermentation of iron-fortified milks were between 2.1 x 10(10) and 4.6 x 10(10) CFU ml(-1), which were not significantly different from numbers in unfortified yogurts. In all batches of yogurt, the viable cell counts of S. thermophilus 13a were approximately three times higher than those of L. delbrueckii subsp. bulgaricus 2-11. Greater decrease in viable cell count over 15 days of storage was observed for S. thermophilus 13a compared to L. delbrueckii subsp. bulgaricus 2-11. Intensive accumulation of lactic acid was observed during incubation of milk and all batches reached pH 4.5 +/- 0.1 after 3.0 h. At the end of fermentation process, lactic acid concentrations in iron-fortified yogurts were between 6.9 +/- 0.4 and 7.3 +/- 0.5 g l(-1). The acidifying activity of starter culture bacteria in the control and iron-fortified milks was similar. There was no increase in oxidized, metallic and bitter off-flavors in iron-fortified yogurts compared to the control. Iron-fortified yogurts did not differ significantly in their sensorial, chemical and microbiological characteristics with unfortified yogurt, suggesting that yogurt is a suitable vehicle for iron fortification and that the ferrous lactate is an appropriate iron source for yogurt fortification.     

Analysis of exponential growth data for yoghurt cultures

Obtaining accurate estimates of maximum specific growth rate, growth yield, and product yield is important for many fermentation processes. A systematic procedure is presented to select the exponential growth region and estimate the maximum specific growth rate using the covariate adjustment method with all the available measured variables (i.e. biomass, substrate, and product). The procedure is applied to data collected during growth of pure and mixed cultures of Lactobacillus bulgaricus and Streptococcus thermophilus on 3% dry milk under anaerobic conditions. The estimation procedure gives good estimates with relatively narrow confidence intervals even though biomass concentration is measured by an indirect method. The estimated values of maximum specific growth rate range from 0.2805 h(-1) for S. thermophilus (ATCC-19258) to 0.4672 h(-1) for S. thermophilus (Microlife). Growth and product yields are estimated using regression analysis and the data for the exponential growth region. The growth yields are compared to their theoretical maximum values.     

Isolation and characterization of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus from plants in Bulgaria

One of the traditional ways of preparation of yogurt starter in Bulgaria is placing a branch of a particular plant species into boiled sheep's milk maintained at about 45°C, which is further incubated until a dense coagulum is obtained. To investigate the possible origin of the yogurt starter bacteria, Lactobacillus delbrueckii ssp. bulgaricus ( L. bulgaricus ) and Streptococcus thermophilus ( S. thermophilus ), the traditional way of yogurt-starter preparation was followed. Hundreds of plant samples were collected from four regions in Bulgaria and incubated in sterile skim milk. The two target bacteria at low frequencies from the plant samples collected were successfully isolated. Phenotypic and genotypic characteristics of these bacterial isolates revealed that they were identified as L. bulgaricus and S. thermophilus . Twenty isolates of L. bulgaricus and S. thermophilus , respectively, were selected from the isolated strains and further characterized with regard to their performance in yogurt production. Organoleptic and physical properties of yogurt prepared using the isolated strains from plants were not significantly different from those prepared using commercial yogurt-starter strains.
It was therefore suggested that L. bulgaricus and S. thermophilus strains widely used for commercial yogurt production could have originated from plants in Bulgaria. To our knowledge, this is the first report on the isolation and characterization of L. bulgaricus and S. thermophilus strains from plants.     

Selective enumeration of Lactobacillus casei from yogurts and fermented milk drinks

A selective medium (LC agar) was developed for enumeration of Lactobacillus casei populations from commercial yogurts and fermented milk drinks that may contain strains of yogurt bacteria (Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus), probiotic bacteria (Lactobacillus acidophilus and bifidobacteria) and L. casei. Appropriate dilutions were pour-plated in specially formulated LC agar acidified to pH 5.1 and the plates incubated at 27°C for 72 to 96 h under anaerobic conditions. Growth of S. thermophilus was prevented by adjusting pH to 5.1. L. delbrueckii ssp. bulgaricus did not ferment ribose as the carbon source, as a result the organisms did not form colonies. L. acidophilus formed colonies on MRS-ribose agar; however, this organism did not grow in the specially formulated LC agar containing ribose. Similarly, Bifidobacterium spp. did not form colonies in LC agar. L. casei formed colonies on LC agar. © Rapid Science Ltd. 1998     

Estimation of the true growth yield and maintenance coefficient for yoghurt cultures

Point and 95% confidence interval estimates of the true growth yield and maintenance coefficient are reported for four pure cultures andtwo mixed cultures of Lactobacillus bulgaricus and Streptococcus thermophilus grown anaerobically on 3% nonfat dry milk using batch followed by fed batch culture. An improved method of data analysis, which includes separate analysis of the exponential growth region and a new method of selecting smoothing spline functions, is utilized. The parameter estimation results are compared with the theoretical maximum yields for one and two moles of ATP produced per mole of glucose and to the growth yield estimated from the exponential growth region analysis.     

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。     

Detection of extracellular bound proteinase in EPS-producing lactic acid bacteria cultures on skim milk agar

AIMS: Skim milk agar was developed to investigate extracellular cell-bound proteinase in yogurt cultures, Streptococcus thermophilus and Lactobacillus bulgaricus. METHODS AND RESULTS: The Lact. bulgaricus cultures produced more extracellular cell-bound proteinase than did Strep. thermophilus cultures. Strong positive correlations between the size of the exopolysaccharide (EPS) layer and extracellular cell-bound proteinase were found for both Streptococcus and Lactobacillus cultures. CONCLUSION: Strong positive linear relationships existed between the EPS size and colony size and the diameter of clear zone and colony size for Streptococcus cultures, whereas weak positive linear relationships were observed for Lactobacillus cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: These data are useful to validate the relationship between extracellular proteinase and the EPS size of LAB. Also, a convenient medium to detect the presence of extracellular cell-bound proteinase of LAB is valuable for dairy industries.     

cse, a Chimeric and variable gene, encodes an extracellular protein involved in cellular segregation in Streptococcus thermophilus

The isolation of a Streptococcus thermophilus CNRZ368 mutant displaying a long-chain phenotype allowed us to identify the cse gene (for cellular segregation). The N terminus of Cse exhibits high similarity to Streptococcus agalactiae surface immunogenic protein (SIP), while its C terminus exhibits high similarity to S. thermophilus PcsB. In CNRZ368, deletion of the entire cse open reading frame leads to drastic lengthening of cell chains and altered colony morphology. Complementation of the Deltacse mutation with a wild-type allele restored both wild-type phenotypes. The central part of Cse is a repeat-rich region with low sequence complexity. Comparison of cse from CNRZ368 and LMG18311 strains reveals high variability of this repeat-rich region. To assess the impact of this central region variability, the central region of LMG18311 cse was exchanged with that of CNRZ368 cse. This replacement did not affect chain length, showing that divergence of the central part does not modify cell segregation activity of Cse. The structure of the cse locus suggests that the chimeric organization of cse results from insertion of a duplicated sequence deriving from the pcsB 3' end into an ancestral sip gene. Thus, the cse locus illustrates the module-shuffling mechanism of bacterial gene evolution.     

Utilization of UF-permeate for production of beta-galactosidase by lactic acid bacteria

Four lactobacilli strains (Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacilus casei and Lactobacillus reuteri) were grown in MRS broth and three lactococci strains (Streptococcus thermophilus, Lactococcus lactis subsp. Lactis and Lactococcus lactis subsp. lactis biovar. diacetilactis) were grown in M17 broth. L. reuteri and S. thermophilus were chosen on the basis of the best mean beta-galactosidase activity of 10.44 and 10.01 U/ml respectively, for further studies on permeate-based medium. The maximum production of beta-galactosidase by L. reuteri was achieved at lactose concentration of 6%, initial pH 5.0-7.5, ammonium phosphate as nitrogen source at a concentration of 0.66 g N/L and incubation temperature at 30 degrees C/24 hrs to give 6.31 U/ml. While in case of S. thermophilus, maximum beta-galactosidase production was achieved at 10% lactose concentration of permeate medium, supplemented with phosphate buffer ratio of 0.5:0.5 (KH2PO4:K2HPO4, g/L), at initial pH 6.0-6.5, ammonium phosphate (0.66g N/L) as nitrogen source and incubation temperature 35 degrees C for 24 hrs to give 7.85 U/ml.     

Exopolysaccharide-producing strains of thermophilic lactic acid bacteria cluster into groups according to their EPS structure

AIMS: To compare galactose-negative strains of Streptococcus thermophilus and Lactobacillus delbrueckii subspecies bulgaricus isolated from fermented milk products and known to produce exopolysaccharides (EPSs). METHODS AND RESULTS: The structures of the EPSs were determined using nuclear magnetic resonance (NMR) and their genetic relationships determined using restriction endonuclease analysis (REA) and random amplification of polymorphic DNA (RAPD). Similar groupings were apparent by REA and RAPD, and each group produced an EPS with a particular subunit structure. CONCLUSION: Although none of the strains assimilated galactose, all inserted a high proportion of galactose into their EPS when grown in skimmed milk, and fell into three distinct groups. Significance and Impact of the Study: This information should help in an understanding of genetic exchanges in lactic acid bacteria.     

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.