Adaptation of lactic acid bacteria to unfavorable growth conditions

The adaptation of lactic acid bacteria (LAB) to unfavorable growth conditions, e.g., depletion of nutrient sources, overthreshold cell density of a population, or antibiotic impact, was shown to include: (1) formation of cyst-like dormant cells (CDC) providing for survival and species preservation and (2) realization of intra-population phenotypic variability, which is demonstrated by development of non-dominant colonies on plates inoculated with CDC suspensions. In Lactobacillus plantarum, the dormant cells, which retained viability and heat resistance for a long time, were formed in 10- and 20-fold concentrated suspensions of the stationary phase cells. In 4-month cell suspensions, two types of cells were present, CDC and L-forms. The CDC of Lactococcus lactis were formed in (1) post-stationary cultures grown under glucose limitation and (2) in stationary phase cultures resuspended in starvation medium (without glucose). Populations of CDC stored for different periods of time varied in the ability for phase variation; as a result, both variants exhibited a shift of the population’s CDC spectrum to the transition of the dominant S-colony type to the R-type up to complete substitution (by day 25). In Lactobacillus acidophilus AT-41, CDC appeared in (1) post-stationary cultures grown on a nitrogen-limited medium; (2) autolyzing cultures treated with ampicillin or erythromycin; and (3) concentrated (10- and 20-fold) suspensions of stationary-phase cells. At plating of L. acidophilus CDC, the substitution of the S-type for the dominant R-type in variants (1) (day 30), (2) (100 μg/ml ampicillin, day 10), and (3) (day 25) was 68.6%, 30.1%, and 61.2%, respectively. The S-variant of L. acidophilus was used for development of a novel lactofermented product based on vegetable (beet) juice fermentation, which sustained high titer of viable cells (2 × 10⁶ cells/ml).     

Comparative and functional analysis of the rRNA-operons and their tRNA gene complement in different lactic acid bacteria

The complete genome sequences of the lactic acid bacteria (LAB), Lactobacillus plantarum, Lactococcus lactis, and Lactobacillus johnsonii were used to compare location, sequence, organisation, and regulation of the ribosomal RNA (rrn) operons. All rrn operons of the examined LAB diverge from the origin of replication, which is compatible with their efficient expression. All operons show a common organisation of 5'-16S-23S-5S-3' structure, but differ in the number, location and specificity of the tRNA genes. In the 16S-23S intergenic spacer region, two of the five rrn operons of Lb. plantarum and three of the six of Lb. johnsonii contain tRNA-ala and tRNA-ile genes, while L. lactis has a tRNA-ala gene in all six operons. The number of tRNA genes following the 5S rRNA gene ranges up to 14, 16, and 21 for L. lactis, Lb. johnsonii and Lb. plantarum, respectively. The tRNA gene complements are similar to each other and to those of other bacteria. Micro-heterogeneity was found within the rRNA structural genes and spacer regions of each strain. In the rrn operon promoter regions of Lb. plantarum and L. lactis marked differences were found, while the promoter regions of Lb. johnsonii showed a similar tandem promoter structure in all operons. The rrn promoters of L. lactis show either a single or a tandem promoter structure. All promoters of Lb. plantarum contain two or three -10 and -35 regions, of which either zero to two were followed by an UP-element. The Lb. plantarum rrnA, rrnB, and rrnC promoter regions display similarity to the rrn promoter structure of Esherichia coli. Differences in regulation between the five Lb. plantarum promoters were studied using a low copy promoter-probe plasmid. Taking copy number and growth rate into account, a differential expression over time was shown. Although all five Lb. plantarum rrn promoters are significantly different, this study shows that their activity was very similar under the circumstances tested. An active promoter was also identified within the Lb. plantarum rrnC operon preceding a cluster of 17 tRNA genes.     

Comparative studies of lactic acid dehydrogenases in lactic acid bacteria

The stability, pH-dependence and kinetic properties of the Mn²⁺ and FDP-activated NAD-dependent lactic acid dehydrogenases from Lactobacillus casei ssp. casei (ATCC 393) and L. curvatus (DSM) 20010) were studied after the enzymes were purified to homogeneity by affinity chromatography. Both enzymes are virtually unidirectional, catalysing efficiently only the reduction of pyruvate. They are similar with respect to the effector requirement and pH-optimum. They differ, however, in their electrophoretic mobility, heat stability, pH-dependence of the Mn²⁺ requirement and several kinetic properties. It is suggested that most of these differences are caused by differences of the negative charges in the vicinity of the FDP-binding site or the site responsible for the interaction of the subunits of the enzymatically active oligomeres.Abbreviations l-LDH l-Lactic acid dehydrogenase - FDP Fructose-1,6-bisphosphate - DTE Dithioerythrol AddendumIn the case of the L. casei-LDH the shape of the NADH saturation curve is not changed by omitting the effectors FDP and Mn 2⁺. The K _M under these conditions is 3 fold higher (10.10 ^–5 M).     

Identification and characterization of lactic acid bacteria in ragi tape

One hundred and eighteen lactic acid bacteria (LAB) were isolated from five different types of ragi tape, a traditional dry-starter of Balinese rice wine. The isolates could be classified into three groups based on the cell shape and capability to produce gas from glucose. Group I contained 66 homofermentative cocci, group II contained seven homofermentative rods, and group III contained 45 heterofermentative rods. Among these 118 isolates, 21 isolates representing these groups were selected and were first identified using phenotypic characters. The identification performed phenotypically was confirmed by sequencing of variable region 8 (V8) of the 16S rDNA. The comparative studies led to the identification of Pediococcus pentosaceus, Enterococcus faecium, Lactobacillus curvatus, Weissella confusa, and W. paramesenteroides from the ragi tape examined.     

Interaction of yeasts and some nitrogen fixing bacteria on nodulation of legumes

Summary Combined inoculation ofRhizobium trifolii withSaccharomyces cerevisiae and other yeasts generally enhanced the number of nodules, length of plants and dry weight of Egyptian clover (Trifolium alexandrinum) seedlings grown on agar slopes. Similar effects were observed when seedlings were inoculated withR. trifolii in the presence of dialyzed culture filtrate ofS. cerevisiae.     

Isolation and characterization of a novel lactic acid bacterium for the production of lactic acid

We isolated a novel lactic acid bacterium from a Korean traditional fermented food, soybean paste. The newly isolated strain, dubbed RKY2, grew well on glucose, sucrose, galactose, and fructose, but it could not utilize xylose, starch, or glycerol. When the partially amplified 16S rDNA sequence (772 bp) of the strain RKY2 was compared with 10 reference strains, it was found to be most similar toLactobacillus pentosus JCM 1588^T, with 99.74% similarity. Therefore, the strain RKY2 was renamedLactobacillus sp. RKY2, which has been deposited in the Korean Collection for Type Cultures as KCTC 10353BP.Lactobacillus sp. RKY2 was found to be a homofermentative lactic acid bacterium, because its end-product from glucose metabolism was found to be mainly lactic acid. It could produce more than 90 g/L of lactic acid from MRS medium supplemented with 100 g/L of glucose, with 5.2 g L⁻¹ h⁻¹ of productivity and 0.95 g/g of lactic acid yield.     

Molecular characterization of clinical Saccharomyces cerevisiae isolates and their association with non-clinical strains

We assessed the molecular characterization of 96 clinical isolates of S. cerevisiae from a Spanish medical institution and we compared them with 6 non-clinical strains isolated from wine, beer and bread and 1 S. boulardii strain collected from a commercial preparation. The strains were subjected to HinfI mtDNA restriction analysis and PCR amplification of delta sequences. Although both techniques are appropriate for routine clinical analysis, that based on PCR turned out to be the most discriminating. This study, apart from providing tools for clinical application, deals with the relationships between clinical and non-clinical strains. The two baker's yeasts analysed shared mtDNA and PCR patterns with a group of 31 clinical isolates. An exogenous entry of a strain was also reflected in the case of 19 clinical isolates and the therapeutic strain S. boulardii. Both baker's yeasts and S. boulardii were identified respectively among 32.3% and 19.8% of the clinical isolates and there seemed to be a connection between their ability to colonize humans and their ability to cause vaginal infection. The rest of food isolates were not grouped with clinical strains.     

Genetic identification of naturalSaccharomyces sensu stricto yeasts from Finland,Holland and Slovakia

Genetic and karyotypic studies of naturalSaccharomyces sensu stricto yeasts from Finland, Holland and Slovakia revealed three wild sibling-species:Saccharomyces cerevisiae, Saccaromyces bayanus andSaccharomyces paradoxus.     

大曲来源淀粉利用型乳酸菌的筛选及其淀粉利用特性

【背景】乳酸菌是面包、馒头等发酵食品中的重要功能微生物,对改善质地和风味均具有重要作用。淀粉利用能力高的乳酸菌,因其能够在生面粉中更好地定殖而具有重要的应用价值。【目的】筛选获得淀粉水解型乳酸菌并研究其淀粉利用特性。【方法】以浓香型白酒大曲为筛选源,采用淀粉基质碳源对大曲中乳酸菌进行定向富集,结合淀粉发酵能力筛选高淀粉利用能力菌株,并对筛选得到的优良菌株展开淀粉酶表达及其酶活力研究。【结果】以贮存3-6个月的大曲为优秀筛选源,以生面糊传代富集方法可较快筛选出具有良好淀粉利用能力的乳杆菌,主要物种为植物乳杆菌和类食品乳杆菌。对其中一株具有淀粉利用能力的类食品乳杆菌LBM12001的淀粉水解特征和淀粉酶活力展开研究,该菌株淀粉水解能力达10 g/L,并且其在面糊中具有良好的定殖能力;酶活力测定表明,其α-淀粉酶和麦芽糖淀粉酶为胞外酶;麦芽糖淀粉酶水解淀粉的最适pH值为3.5,比酶活为1 240 U/mg。【结论】建立起从我国传统白酒发酵大曲中高效筛选淀粉水解型乳酸菌的富集筛选方法,以及菌株的水解能力评价方法,获得的胞外麦芽糖淀粉酶分泌型乳杆菌在酸面团、馒头等需进行生面粉发酵食品的生产中具有重要应用前景。     

Effects of pressure on cell morphology and cell division of lactic acid bacteria

The effect of pressure and temperature on the growth of the mesophilic lactic acid bacteria Lactococcus lactis and Lactobacillus sanfranciscensis was studied. Both strains were piezosensitive. Lb. sanfranciscensis failed to grow at 50 MPa and the growth rate of Lc. lactis at 50 MPa was less than 30% of that at atmospheric pressure. An increase of growth temperature did not improve the piezotolerance of either organism. During growth under high-pressure conditions, the cell morphology was changed, and the cells were elongated as cell division was inhibited. At atmospheric pressure, temperatures above the optimal temperature for growth caused a similar effect on cell morphology and cell division in both bacteria as that observed under high-pressure conditions. The segregation and condensation of chromosomal DNA were observed by DAPI staining and occurred normally at high-pressure conditions independent of changes in cell morphology. Immunofluorescence microscopy of Lc. lactis cells demonstrated an inhibitory effect of high pressure on the formation of the FtsZ ring and this inhibition of the FtsZ ring formation is suggested to contribute to the altered cell morphology and growth inhibition induced by high pressure.Communicated by K. Horikoshi     

Molecular characterization of alanine racemase from Bifidobacterium bifidum

Bifidobacterium bifidum is a useful probiotic agent exhibiting health-promoting properties, and its peptidoglycans have the potential for applications in the fields of food science and medicine. We investigated the bifidobacterial alanine racemase, which is essential in the synthesis of -alanine as an essential component of the peptidoglycans. Alanine racemase was purified to homogeneity from a crude extract of B. bifidum NBRC 14252. It consisted of two identical subunits with a molecular mass of 50 kDa. The enzyme required pyridoxal 5′-phosphate (PLP) as a coenzyme. The activity was lost in the presence of a thiol-modifying agent. The enzyme almost exclusively catalyzed the alanine racemization; other amino acids tested, except for serine, were inactive as substrates. The kinetic parameters of the enzyme suggested that the B. bifidum alanine racemase possesses comparatively low affinities for both the coenzyme (9.1 μM for PLP) and substrates (44.3 mM for -alanine; 74.3 mM for -alanine). The alr gene encoding the alanine racemase was cloned and sequenced. The alr gene complemented the -alanine auxotrophy of Escherichia coli MB2795, and an abundant amount of the enzyme was produced in cells of the E. coli MB2795 clone. The enzymologic and kinetic properties of the purified recombinant enzyme were almost the same as those of the alanine racemase from B. bifidum NBRC 14252.     

降解尿酸乳酸菌复合菌系的筛选与菌株组合提升降解效果

【背景】高尿酸症由血液中尿酸含量明显升高而导致,利用乳酸菌对人体的益生作用缓解高尿酸血症越来越受到关注。【目的】获得具有降解尿酸能力的乳酸菌复合菌系与纯培养菌株。【方法】以泡菜为样品来源,以尿酸为底物,采用MRS培养基筛选降解尿酸的乳酸菌复合菌系,通过高效液相色谱法测定复合菌系对尿酸的降解能力。【结果】得到一组乳酸菌复合菌系,当培养温度为37 °C、pH值为6.20、静置培养72 h后复合菌系对尿酸的降解率为12.08%;通过优化培养条件,当该菌系在以牛肉膏为单一氮源、初始pH值为5.00、温度为35 °C的条件下培养72 h,尿酸降解率上升至17.19%,降解率比优化前提高了42.3%;从该菌系中分离出两株具有尿酸降解能力的菌株UA-1与UA-2,它们的尿酸降解率分别为10.85%和8.65%;通过形态学观察和16S rRNA基因序列分析,经鉴定两株菌均为布氏乳杆菌(Lactobacillus buchneri)。将两株单菌组合降解尿酸试验发现,UA-1与UA-2比例为2:1的尿酸降解率为20.2%,比原复合菌系的降解能力提高了67.22%。【结论】研究证明了乳酸菌复合菌系对尿酸的降解能力优于单个菌株,为后续利用乳酸菌复合菌系应用提供了数据支持。     

Fermentation of maltotriose by brewer's and baker's yeasts

Two brewer's yeasts and one baker's yeast grew with 95% (w/w) pure maltotriose as carbon source in the presence of antimycin A to block respiration. Biomass yields (0.15 and 0.24 g dry yeast g^–1 sugar, respectively, with and without antimycin A) were similar for growth on maltose and maltotriose, and yields of ethanol were 80% of stoichiometric. Yeasts harvested during growth on glucose and containing low maltose transport activity did not begin to use maltotriose in the presence of antimycin A until after a long lag phase (up to 50 h), but yeast harvested during growth on maltose, and containing high maltose transport activity, began to use maltotriose after about 25 h. Much shorter lags were observed before growth started in the absence of antimycin A.     

Molecular characterization of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> nasal isolates from Turkey

Methicillin-resistant Staphylococcus aureus (MRSA) colonize most frequently in the anterior nares of the nose and cause serious infections all over the world. The aim of this study was to determine the nasal carriage rate of S. aureus and MRSA strains in Turkish elementary school children. We also analyzed molecular characterizations of MRSA strains by using pulse field gel electrophoresis (PFGE), multi locus sequence typing (MLST), staphylococcal chromosomal cassette mec (SCCmec) typing, and detection of the Panton-valentine leucocidin (PVL) gene. The nasal swabs were obtained from 4,050 children during a 4 month period in Ankara. In vitro antimicrobial susceptibility testing to 1 μg oxacillin and 30 μg cefoxitin was determined by a disk diffusion method. We found that the 1,001 of 4,050 (24.7%) children were colonized with S. aureus. Three S. aureus strains were resistant to oxacillin and cefoxitin. The rate of MRSA among all children was 0.07%. The MRSA strains revealed three different PFGE pattern. All MRSA isolates by harbored the SCCmec type IV element, but not the PVL gene. The two MRSA isolate belonged to sequence type (ST) 30, whereas the other one was a unique type. The results of this study demonstrated that S. aureus nasal carriage rate was consistent with previous studies. However, MRSA carriage rate was low. This study also indicated that the ST30-type IV without PVL gene MRSA clone may be expected to spread in Turkish community.     

Molecular structure and genetic regulation of SFA, a gene responsible for resistance to formaldehyde in Saccharomyces cerevisiae, and characterization of its protein product

Summary A 3.7 kb DNA fragment of yeast chromosome IV has been sequenced that contains the SFA gene which, when present on a multi-copy plasmid in Saccharomyces cerevisiae, confers hyper-resistance to formaldehyde. The open reading frame of SFA is 1158 by in size and encodes a polypeptide of 386 amino acids. The predicted protein shows strong homologies to several mammalian alcohol dehydrogenases and contains a sequence characteristic of binding sites for NAD. Overexpression of the SFA gene leads to enhanced consumption of formaldehyde, which is most probably the reason for the observed hyper-resistance phenotype. In sfa:LEU2 disruption mutants, sensitivity to formaldehyde is correlated with reduced degradation of the chemical. The SFA gene shares an 868 by divergent promoter with UGX2 a gene of yet unknown function. Promoter deletion studies with a SFA promoter-lacZ gene fusion construct revealed negative interference on expression of SFA by upstream sequences. The upstream region between positons – 145 and – 172 is totally or partially responsible for control of inducibility of SFA by chemicals such as formaldehyde (FA), ethanol and methyl methanesulphonate. The 41 kDa SFA-encoded protein was purified from a hyper-resistant transformant; it oxidizes long-chain alcohols and, in the presence of glutathione, is able to oxidize FA. SFA is predicted to code for a long-chain alcohol dehydrogenase (glutathione-dependent formaldehyde dehydrogenase) of the yeast S. cerevisiae.     

Immobilization and characterization of Saccharomyces cerevisiae in crosslinked,prepolymerized polyacrylamide-hydrazide

Summary Baker's yeast (Saccharomyces cerevisiae) was immobilized in gels made of prepolymerized, linear, water soluble polyacrylamide, partially substituted with acylhydrazide groups. Gelation was effected by the addition of controlled amounts of dialdehydes (e.g. glyoxal). The immobilized yeasts retained full glycolytic activity. Moreover, the entrapped cells were able to grow inside the chemically corsslinked gel during continuous alcohol production. Glyoxal was found to be the most favourable crosslinking agent for this system. the system employed allowed for the free exchange of substrate and products. The gel surrounding the entrapped cells had no effect on temperature stability profile. On the other hand, substantial enhancement in survival of cells in presence of high ethanol concentrations was recorded for the entrapped yeast. The capability of the immobilized yeast to carry out continuous conversion of glucose to ethanol was demonstrated.     

Synergism among lactic acid, sulfite, pH and ethanol in alcoholic fermentation of Saccharomyces cerevisiae (PE-2 and M-26)

Summary The industrial production of ethanol is affected mainly by contamination by lactic acid bacteria besides others factors that act synergistically like increased sulfite content, extremely low pH, high acidity, high alcoholic content, high temperature and osmotic pressure. In this research two strains of Saccharomyces cerevisiae PE-2 and M-26 were tested regarding the alcoholic fermentation potential in highly stressed conditions. These strains were subjected to values up to 200 mg NaHSO₃ l⁻¹, 6 g lactic acid l⁻¹, 9.5% (w/v) ethanol and pH 3.6 during fermentative processes. The low pH (3.6) was the major stressing factor on yeasts during the fermentation. The M-26 strain produced higher acidity than the other, with higher production of succinic acid, an important inhibitor of lactic bacteria. Both strains of yeasts showed similar performance during the fermentation, with no significant difference in cell viability.