Factors affecting exocellular polysaccharide production by Lactobacillus delbrueckii subsp. bulgaricus grown in a chemically defined medium

We developed a chemically defined medium (CDM) containing lactose or glucose as the carbon source that supports growth and exopolysaccharide (EPS) production of two strains of Lactobacillus delbrueckii subsp. bulgaricus. The factors found to affect EPS production in this medium were oxygen, pH, temperature, and medium constituents, such as orotic acid and the carbon source. EPS production was greatest during the stationary phase. Composition analysis of EPS isolated at different growth phases and produced under different fermentation conditions (varying carbon source or pH) revealed that the component sugars were the same. The EPS from strain L. delbrueckii subsp. bulgaricus CNRZ 1187 contained galactose and glucose, and that of strain L. delbrueckii subsp. bulgaricus CNRZ 416 contained galactose, glucose, and rhamnose. However, the relative proportions of the individual monosaccharides differed, suggesting that repeating unit structures can vary according to specific medium alterations. Under pH-controlled fermentation conditions, L. delbrueckii subsp. bulgaricus strains produced as much EPS in the CDM as in milk. Furthermore, the relative proportions of individual monosaccharides of EPS produced in pH-controlled CDM or in milk were very similar. The CDM we developed may be a useful model and an alternative to milk in studies of EPS production.     

保加利亚乳杆菌H+-ATPase缺陷型菌株的筛选

【目的】从传统乳制品中筛选具有新霉素抗性的H+-ATPase缺陷的德氏乳杆菌保加利亚亚种自发突变株,为最终开发弱后酸化的酸奶发酵剂奠定基础。【方法】利用API 50 CH细菌鉴定系统和16s rRNA基因序列分析对菌株进行鉴定。新霉素作为筛选压力,筛选具有新霉素抗性自发突变菌株,比较亲本和突变菌株的H+-ATPase活力及其代谢情况。【结果】从内蒙古地区的传统发酵酸奶中分离鉴定出一株德氏乳杆菌保加利亚亚种（Lactobacillus delbrueckii subsp. bulgaricus）,并命名为KLDS 1.9201。以此为出发菌株,筛选出两株H+-ATPase缺陷的自发突变株,分别命名为KLDS 1.9201-1、KLDS 1.9201-4,它们的H+-ATPase活力分别比亲本KLDS 1.9201降低了46%和60%。在MRS培养基中生长24 h后,KLDS 1.9201、KLDS 1.9201-1和KLDS 1.9201-4对初始葡萄糖的代谢率分别为65%、41%和31%,终产物中乳酸的浓度分别为26g/L、18g/L和15g/L,突变菌株的生物量均低于亲本。【结论】H+-ATPase活力降低的德氏乳杆菌保加利亚亚种的自发突变株具有较低的生长速率和弱产酸能力,它们可被用于制作弱后酸化的酸奶发酵剂。     

Use of PCR-based methods for rapid differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis.

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412(T), which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains.     

Differentiation of Lactobacillus delbrueckii subspecies by ribotyping and amplified ribosomal DNA restriction analysis (ARDRA)

AIMS: To differentiate the subspecies of Lactobacillus delbrueckii, subsp. delbrueckii, subsp. lactis and subsp. bulgaricus. METHODS AND RESULTS: Amplified ribosomal DNA restriction analysis (ARDRA) and ribotyping were applied to over 30 strains. Both methods analyse the ribosomal genes which carry useful information about the evolutionary and taxonomic relationship among bacteria. The methods proved to be reliable and highly reproducible. ARDRA was applied to 16S rDNA, 23S rDNA and the IGS region, thus covering the whole rrn operon with eight restriction enzymes. Only EcoRI differentiated Lact. delbrueckii subsp. bulgaricus from Lact. delbrueckii subsp. delbrueckii/Lact. delbrueckii subsp. lactis, which confirmed the finding of other authors. Ribotyping with different enzymes under precisely optimized conditions revealed a high level of strain polymorphism. Only ribotyping with EcoRI allowed differentiation of the three subspecies on the basis of typical hybridization patterns. CONCLUSION: The successful differentiation of the three subspecies of Lact. delbrueckii by EcoRI ribotyping offers a new possibility for precise identification and differentiation of strains and new isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Both methods could be used for differentiation of Lact. delbrueckii subspecies.     

Strain dependency of the immunopotentiating activity of Lactobacillus delbrueckii subsp. bulgaricus

To obtain strains of Lactobacillus delbrueckii subsp. bulgaricus with high immunopotentiating activity, we screened 90 strains of this bacterial species for the proliferative response of murine spleen and beta-lactoglobulin-primed lymph node cells. In this screening, certain strains showed strong immunopotentiating activity. Among them, strain 1023 had the strongest mitogenic activity for murine Peyer's patch (PP) cells. Furthermore, strain 1023 induced IgA antibody production by PP cells as strongly as Bifidobacterium longum 6001, which had adjuvant activity when orally administered. Also in the assays using immune cells from human-flora-associated mice a few strains including 1023 showed strong immunopotentiating activity comparable to B. longum 6001. These results suggest that L. delbrueckii subsp. bulgaricus strains such as 1023 may be useful for the production of fermented milk with a more beneficial effect on the systemic and mucosal immune system.     

Deoxyribonuclease activities in Lactobacillus delbrueckii

DNase activity was examined in the extracellular and subcellular fractions of six non-transformable strains belonging to Lactobacillus delbrueckii subsp. lactis (L. lactis) and Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) and compared with the activity present in Lactobacillus johnsonii NCK 65, a transformable strain of Lactobacillus. In the extracellular fraction of the L. delbrueckii strains, a common protein band of 36 kDa was detected, while a band of 29 kDa was found in the same fraction of L. johnsonii. No nuclease activity was detected in the cytoplasmic fraction of this strain, indicating that the localization of the DNase activity could be a key factor in the uptake of foreign DNA.     

Lacticin, a bacteriocin produced by Lactobacillus delbrueckii subsp. lactis

Twenty-one strains of Lactobacillus delbrueckii and L. helveticus were tested for bacteriocin production against each other. Lactobacillus delbrueckii subsp. lactis JCM 1106 and 1107 produced an inhibitory agent active against L. delbrueckii subsp. bulgaricus JCM 1002 and NIAI yB-62, L. delbrueckii subsp. lactis JCM 1248 and L. delbrueckii subsp. delbrueckii JCM 1012. Lactobacillus delbrueckii subsp. lactis JCM 1248 inhibited only the growth of L. delbrueckii subsp. bulgaricus NIAI yB-62. These agents were sensitive to proteolytic enzymes and heating (at 60°C for 10min). These agents were considered to be bacteriocins and designated lacticin A and B.     

Exopolysaccharides produced by lactic acid bacteria of kefir grains

A Lactobacillus delbrueckii subsp. bulgaricus HP1 strain with high exopolysaccharide activity was selected from among 40 strains of lactic acid bacteria, isolated from kefir grains. By associating the Lactobacillus delbrueckii subsp. bulgaricus HP1 strain with Streptococcus thermophilus T15, Lactococcus lactis subsp. lactis C15, Lactobacillus helveticus MP12, and Sacharomyces cerevisiae A13, a kefir starter was formed. The associated cultivation of the lactobacteria and yeast had a positive effect on the exopolysaccharide activity of Lactobacillus delbrueckii subsp. bulgaricus HP1. The maximum exopolysaccharide concentration of the starter culture exceeded the one by the Lactobacillus delbrueckii subsp. bulgaricus HP1 monoculture by approximately 1.7 times, and the time needed to reach the maximum concentration (824.3 mg exopolysacharides/l) was shortened by 6 h. The monomer composition of the exopolysaccharides from the kefir starter culture was represented by glucose and galactose in a 1.0:0.94 ratio, which proves that the polymer synthesized is kefiran.     

Increased production of hydrogen peroxide by Lactobacillus delbrueckii subsp. bulgaricus upon aeration: involvement of an NADH oxidase in oxidative stress

The growth of Lactobacillus delbrueckii subsp. bulgaricus (L. delbrueckii subsp. bulgaricus) on lactose was altered upon aerating the cultures by agitation. Aeration caused the bacteria to enter early into stationary phase, thus reducing markedly the biomass production but without modifying the maximum growth rate. The early entry into stationary phase of aerated cultures was probably related to the accumulation of hydrogen peroxide in the medium. Indeed, the concentration of hydrogen peroxide in aerated cultures was two to three times higher than in unaerated ones. Also, a similar shift from exponential to stationary phase could be induced in unaerated cultures by adding increasing concentrations of hydrogen peroxide. A significant fraction of the hydrogen peroxide produced by L. delbrueckii subsp. bulgaricus originated from the reduction of molecular oxygen by NADH catalyzed by an NADH:H(2)O(2) oxidase. The specific activity of this NADH oxidase was the same in aerated and unaerated cultures, suggesting that the amount of this enzyme was not directly regulated by oxygen. Aeration did not change the homolactic character of lactose fermentation by L. delbrueckii subsp. bulgaricus and most of the NADH was reoxidized by lactate dehydrogenase with pyruvate. This indicated that NADH oxidase had no (or a very small) energetic role and could be involved in eliminating oxygen.     

Complete genome sequence of Lactobacillus delbrueckii subsp. bulgaricus strain ND02

Lactobacillus delbrueckii subsp. bulgaricus strain ND02 is a Chinese commercial dairy starter used for the manufacture of yoghurt. It was isolated from naturally fermented yak milk in Qinghai, China. Here, we report the main genome features of ND02 and several differences with two other published genomes of Lactobacillus delbrueckii subsp. bulgaricus strains.     

蒙古戈壁地区自然发酵乳中乳酸菌的分离鉴定

从采集自蒙古国戈壁地区的6份自然发酵乳中分离到14株乳酸菌,经过形态特征,生理生化特性,糖发酵试验和乳酸旋光性的测定,鉴定结果:乳酸球菌5株,包括Lactococcus lactissubsp.cremoris 1株,Pedio-coccus.(后缩写为Ped.).urinaeequi3株,Pediococcus.pentosaceus1株;乳杆菌9株,包括Lactobacillus.(后缩写为L.)helveticus8株,Lactobacillus.delbrueckii.subsp.bulgaricus1株。蒙古国戈壁地区自然发酵乳中的优势菌为Lactobacillus.helveticus,其次为Pediococcus.urinaeequi。     

Evolution of the bacterial species Lactobacillus delbrueckii: a partial genomic study with reflections on prokaryotic species concept

The species Lactobacillus delbrueckii consists at present of three subspecies, delbrueckii, lactis and bulgaricus, showing a high level of DNA-DNA hybridization similarity but presenting markedly different traits related to distinct ecological adaptation. The internal genetic heterogeneity of the bacterial species L. delbrueckii was analyzed. Phenotypic and several genetic traits were investigated for 61 strains belonging to this species. These included 16S rDNA sequence mutations, expression of beta-galactosidase and of the cell wall-anchored protease, the characterization of the lactose operon locus and of the sequence of lacR gene, galactose metabolism, and the distribution of insertion sequences. The high genetic heterogeneity of taxa was confirmed by every trait investigated: the lac operon was completely deleted in the subsp. delbrueckii, different mutation events in the repressor gene of the operon led to a constitutive expression of lacZ in the subsp. bulgaricus. Structural differences in the same genetic locus were probably due to the presence of different IS elements in the flanking regions. The different expression of the cell wall-anchored protease, constitutive in the subsp. bulgaricus, inducible in the subsp. lactis, and absent in the subsp. delbrueckii was also a consequence of mutations at the gene level. The galT gene for galactose metabolism was found only in the subsp. lactis, while no specific amplification product was detected in the other two subspecies. All these data, together with the absence of a specific IS element, ISL6, from the major number of strains belonging to the subsp. bulgaricus, confirmed a deep internal heterogeneity among the three subspecies. Moreover, this evidence and the directional mutations found in the 16S rDNA sequences suggested that, of the three subspecies, L. delbrueckii subsp. lactis is the taxon closer to the ancestor. Limitations of the current prokaryotic species definition were also discussed, based on presented evidences. Our results indicate the need for an accurate investigation of internal heterogeneity of bacterial species. This study has consequences on the prokaryotic species concept, since genomic flexibility of prokaryotes collides with a stable classification, necessary from a scientific and applied point of view.     

Production and monomer composition of exopolysaccharides by yogurt starter cultures

As components of starter cultures for Bulgarian yogurt, Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus revealed extensive exopolysaccharide (EPS) production activity when cultivated in whole cow's milk. The polymer-forming activity of thermophilic streptococci was lower (230-270 mg EPS/L) than that of the lactobacilli (400-540 mg EPS/L). Mixed cultures stimulated EPS production in yogurt manufacture, and a maximum concentration of 720-860 mg EPS/L was recorded after full coagulation of milk. The monomer structure of the exopolysaccharides formed by the yogurt starter cultures principally consists of galactose and glucose (1:1), with small amounts of xylose, arabinose, and/or mannose.     

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.     

Survival of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in the terminal ileum of fistulated Göttingen minipigs

The ability of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus administered in yogurt to survive the passage through the upper gastrointestinal tract was investigated with G?ttingen minipigs that were fitted with ileum T-cannulas. After ingestion of yogurt containing viable microorganisms, ileostomy samples were collected nearly every hour beginning 3 h after food uptake. Living L. delbrueckii subsp. bulgaricus and S. thermophilus were detected in the magnitude of 10(6) to 10(7) per gram of intestinal contents (wet weight) in all animals under investigation. A calculation of the minimum amount of surviving bacteria that had been administered is presented. Total DNA extracted from ileostomy samples was subjected to PCR, which was species specific for L. delbrueckii and S. thermophilus and subspecies specific for L. delbrueckii subsp. bulgaricus. All three bacterial groups could be detected by PCR after yogurt uptake but not after uptake of a semisynthetic diet. One pig apparently had developed an endogenous L. delbrueckii flora. When heat-treated yogurt was administered, L. delbrueckii was detected in all animals. S. thermophilus or L. delbrueckii subsp. bulgaricus was not detected, indicating that heat-inactivated cells and their DNAs had already been digested and their own L. delbrueckii flora had been stimulated for growth.     

Lactic acid bacteria isolated from dairy products inhibit genotoxic effect of 4-nitroquinoline-1-oxide in SOS-chromotest

Antigenotoxic activity against 4-nitroquinoline-1-oxide (4-NQO) of lactic acid bacteria isolated from commercial dairy products was studied using SOS-Chromotest. The supernatants from bacteria-genotoxin co-incubations in general exhibited a strong suppression on SOS-induction produced by 4-NQO on the tester organism Escherichia coli PQ37 (sfiA::lacZ). High genotoxicity inhibition (>75%) was found for 31/67 of the examined bacteria and the maximum values of some strains within the species were as follows: Lactobacillus casei, 99.1%; L. plantarum, 93.3%; L. rhamnosus, 93.4%; L. acidophilus, 90.9%; L. delbrueckii subsp. bulgaricus, 85.7% and Bifidobacterium bifidum, 89.6%; Strains with low antigenotoxicity (5-60%) were evidenced in both L. acidophilus and L. delbrueckii subsp. bulgaricus, whereas some inactive strains were found only in L. casei and L. delbrueckii subsp. bulgaricus. Cell exposure to 100 degrees C for 15 min prevented antigenotoxicity and no effect was evidenced for cell-free spent media. The active strains survived at 0.1 mM 4-NQO exposure and generally presented some relevant functional properties, such as tolerance to bile (0.5%) or acid environment (pH 2.0) and adherence to Caco-2 enterocytes. Antigenotoxicity was always associated with modification of the 4-NQO absorbance profile.     

Organization and nucleotide sequence of the glutamine synthetase (glnA) gene from Lactobacillus delbrueckii subsp. bulgaricus.

A 3.3-kb BamHI fragment of Lactobacillus delbrueckii subsp. bulgaricus DNA was cloned and sequenced. It complements an Escherichia coli glnA deletion strain and hybridizes strongly to a DNA containing the Bacillus subtilis glnA gene. DNA sequence analysis of the L. delbrueckii subsp. bulgaricus DNA showed it to contain the glnA gene encoding class I glutamine synthetase, as judged by extensive homology with other prokaryotic glnA genes. The sequence suggests that the enzyme encoded in this gene is not controlled by adenylylation. Based on a comparison of glutamine synthetase sequences, L. delbrueckii subsp. bulgaricus is much closer to gram-positive eubacteria, especially Clostridium acetobutylicum, than to gram-negative eubacteria and archaebacteria. The fragment contains another open reading frame encoding a protein of unknown function consisting of 306 amino acids (ORF306), which is also present upstream of glnA of Bacillus cereus. In B. cereus, a repressor gene, glnR, is found between the open reading frame and glnA. Two proteins encoded by the L. delbrueckii subsp. bulgaricus gene were identified by the maxicell method; the sizes of these proteins are consistent with those of the open reading frames of ORF306 and glnA. The lack of a glnR gene in the L. delbrueckii subsp. bulgaricus DNA in this position may indicate a gene rearrangement or a different mechanism of glnA gene expression.     

Regulation and Adaptive Evolution of Lactose Operon Expression in Lactobacillus delbrueckii

Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis are both used in the dairy industry as homofermentative lactic acid bacteria in the production of fermented milk products. After selective pressure for the fast fermentation of milk in the manufacture of yogurts, L. delbrueckii subsp. bulgaricus loses its ability to regulate lac operon expression. A series of mutations led to the constitutive expression of the lac genes. A complex of insertion sequence (IS) elements (ISL4 inside ISL5), inserted at the border of the lac promoter, induced the loss of the palindromic structure of one of the operators likely involved in the binding of regulatory factors. A lac repressor gene was discovered downstream of the beta-galactosidase gene of L. delbrueckii subsp. lactis and was shown to be inactivated by several mutations in L. delbrueckii subsp. bulgaricus. Regulatory mechanisms of the lac gene expression of L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis were compared by heterologous expression in Lactococcus lactis of the two lac promoters in front of a reporter gene (beta-glucuronidase) in the presence or absence of the lac repressor gene. Insertion of the complex of IS elements in the lac promoter of L. delbrueckii subsp. bulgaricus increased the promoter's activity but did not prevent repressor binding; rather, it increased the affinity of the repressor for the promoter. Inactivation of the lac repressor by mutations was then necessary to induce the constitutive expression of the lac genes in L. delbrueckii subsp. bulgaricus.     

Peptidase activity in various species of dairy thermophilic lactobacilli

AIMS: The aim of the present work was to evaluate the enzymatic potential manifested by aminopeptidase activity of different thermophilic Lactobacillus biotypes and to measure the influence of cell growth phase on enzyme expression. METHODS AND RESULTS: The activities were evaluated by the hydrolysis of beta-naphthylamide substrates for both whole and mechanically disrupted cells of L. helveticus, L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis strains, collected from both the exponential and the stationary growth phase. In general, activities were higher for cells in the exponential rather than in the stationary phase and the disrupted cells showed higher activities than the whole cells. The highest activity expressed by all strains corresponded to X-prolyl-dipeptidyl aminopeptidase while a moderate activity was observed towards Arg-betaNa, Lys-betaNa and Leu-betaNa. The lowest activity was observed for Pro-betaNa. CONCLUSIONS: It may be inferred that the cell structure and the cell physiology are crucial to define the level of efficiency of expression for aminopeptidase activity. The two species may be characterized by a different enzymatic system that hydrolyses N-terminal leucine. SIGNIFICANCE AND IMPACT OF THE STUDY: The differences of peptidase activities in L. helveticus and L. delbrueckii species acquires an importance to comprehend their role in the biochemical events occurring in cheese ripening.     

Complete sequencing and pan-genomic analysis of Lactobacillus delbrueckii subsp. bulgaricus reveal its genetic basis for industrial yogurt production

Lactobacillus delbrueckii subsp. bulgaricus (Lb. bulgaricus) is an important species of Lactic Acid Bacteria (LAB) used for cheese and yogurt fermentation. The genome of Lb. bulgaricus 2038, an industrial strain mainly used for yogurt production, was completely sequenced and compared against the other two ATCC collection strains of the same subspecies. Specific physiological properties of strain 2038, such as lysine biosynthesis, formate production, aspartate-related carbon-skeleton intermediate metabolism, unique EPS synthesis and efficient DNA restriction/modification systems, are all different from those of the collection strains that might benefit the industrial production of yogurt. Other common features shared by Lb. bulgaricus strains, such as efficient protocooperation with Streptococcus thermophilus and lactate production as well as well-equipped stress tolerance mechanisms may account for it being selected originally for yogurt fermentation industry. Multiple lines of evidence suggested that Lb. bulgaricus 2038 was genetically closer to the common ancestor of the subspecies than the other two sequenced collection strains, probably due to a strict industrial maintenance process for strain 2038 that might have halted its genome decay and sustained a gene network suitable for large scale yogurt production.