Deoxyribonucleic acid hybridization among strains of lactobacilli

Hybridization of deoxyribonucleic acid (DNA) from Lactobacillus bulgaricus (ATCC 11842) with DNA of L. lactis (ATCC 12315), L. helveticus (ATCC 15009), and L. jugurt (ATCC 521) showed 86.0% reassociation with L. lactis, 4.8% with L. helveticus, and none with L. jugurt.     

Effect of medium composition on the ultrastructure of Lactobacillus strains

The growth of some locally isolated Lactobacillus strains forming D(-) or L(+) lactic acid, Lactobacillus helveticus ATCC 15009 and Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 was examined in different media. L. helveticus and Lactobacillus LBL strains formed atypical protoplast-like cells in LAPT medium, sensitive to SDS and proteinase. Specific morphological changes in the cell wall structure of these variants were revealed by transmission and scanning electron microscopy. The effect of glucose and various salts on their appearance was investigated. The prevalent role of metal cations, especially of Mg²⁺, was established.     

Lactobacillus helveticus MIMLh5-Specific Antibodies for Detection of S-Layer Protein in Grana Padano Protected-Designation-of-Origin Cheese

Single-chain variable-fragment antibodies (scFvs) have considerable potential in immunological detection and localization of bacterial surface structures. In this study, synthetic phage-displayed antibody libraries were used to select scFvs against immunologically active S-layer protein of Lactobacillus helveticus MIMLh5. After three rounds of panning, five relevant phage clones were obtained, of which four were specific for the S-layer protein of L. helveticus MIMLh5 and one was also capable of binding to the S-layer protein of L. helveticus ATCC 15009. All five anti-S-layer scFvs were expressed in Escherichia coli XL1-Blue, and their specificity profiles were characterized by Western blotting. The anti-S-layer scFv PolyH4, with the highest specificity for the S-layer protein of L. helveticus MIMLh5, was used to detect the S-layer protein in Grana Padano protected-designation-of-origin (PDO) cheese extracts by Western blotting. These results showed promising applications of this monoclonal antibody for the detection of immunomodulatory S-layer protein in dairy (and dairy-based) foods.     

Plasmid dna transformation of Lactobacillus strains by electropermeabilization

Summary Two thermophilic strains of Lactobacillus were transformed by electroporation; L.fermentum with a maximum of frequency of 1×10⁵/ug of plasmid vector pPSC₂₀DNA and 1.4×10³/ug pSA₃DNA. L.helveticus showed a very low frequency of transformation, from 9 to 26 transformants/ug DNA in all the experiments carried out with both the vectors. While L.fermentum transformants were very stable, in L.helveticus the acquired plasmid was lost after 30–50 generations.     

Nutritional Requirements and Nitrogen-Dependent Regulation of Proteinase Activity of Lactobacillus helveticus CRL 1062

The nutritional requirements of Lactobacillus helveticus CRL 1062 were determined with a simplified chemically defined medium (SCDM) and compared with those of L. helveticus CRL 974 (ATCC 15009). Both strains were found to be prototrophic for alanine, glycine, asparagine, glutamine, and cysteine. In addition, CRL 1062 also showed prototrophy for lysine and serine. The microorganisms also required riboflavin, calcium pantothenate, pyridoxal, nicotinic acid, and uracil for growth in liquid SCDM. The growth rate and the synthesis of their cell membrane-bound serine proteinases, but not of their intracellular leucyl-aminopeptidases, were influenced by the peptide content of the medium. The highest proteinase levels were found during cell growth in basal SCDM, while the synthesis of this enzyme was inhibited in SCDM supplemented with Casitone, Casamino Acids, or β-casein. Low-molecular-mass peptides (<3,000 Da), extracted from Casitone, and the dipeptide leucylproline (final concentration, 5 mM) play important roles in the medium-dependent regulation of proteinase activity. The addition of the dipeptide leucylproline (5 mM) to SCDM reduced proteinase activity by 25%.     

Slow milk coagulating variants ofLactobacillus helveticus

Curing experiments were performed onLactobacillus helveticus strain ATCC 15009 in order to find a correlation between the presence of three extrachromosomal elements and specific phenotypic traits. Mitomycin C treatment of the strain was found to result in an appearance of slow-coagulation variants unable to coagulate milk in 24 h at 42 °C. The effect of mitomycin C on phenotypic and genotypic characteristics ofL. helveticus was therefore further examined. The presence of mitomycin C appeared to act on the proteolytic system of the strain: slow variants exhibited a poor casein breakdown (50 % less) compared with the parental strain. Aminopeptidase activity and lactose utilization were unaffected. The phenotypic variation is possibly due to a point mutation of genetic patrimony. No loss of plasmid DNA was detected after mitomycin C treatment and the restriction pattern of plasmid and chromosomal DNA of the variants, after digestion with several restriction endonucleases, was identical to that obtained for the parental strain.     

Characterization of a K+-ATPase from Lactobacillus helveticus ATCC 15009

Lactobacillus helveticus ATCC 15009 (wild-type) membrane preparations hydrolyzed Mg²⁺-ATP as a function of K⁺ concentration (2–200 mM). Mg²⁺-ATP hydrolysis by L. helveticus membranes was strongly inhibited in the absence of exogenous K⁺, while it amounted to 6 nmol ATP hydrolyzed min^–1 (mg membrane protein)^–1 at 50 mM KCl (saturating conditions) and pH 7.2. The K⁺-dependent ATPase of L. helveticus displayed a relatively high affinity for potassium ions (K _m = 800 μM) and was not affected by pretreatment of membranes with N,N’-dicyclohexylcarbodiimide. Membrane preparations were subjected to hypotonic shock to obtain a maximum yield of open profiles. The formation of a maximum level of enzyme-phosphate complex with a molecular mass of approximately 82 kDa was induced upon treatment of L. helveticus membrane preparations with low concentrations of [γ-³²P]ATP in the presence of K⁺ and La³⁺ ions and was visualized by acidic SDS-PAGE. It was concluded that L. helveticus membranes contain an inwardly directed K⁺ pump whose presence is discussed in terms of its putative role in cytoplasmic pH regulation. Received: 16 December 1996 / Accepted: 14 May 1997     

Classification of Lactobacillus helveticus Strains by Immunological Differences in Extracellular Proteinases

Polyclonal antibodies against an extracellular proteinase of Lactobacillus helveticus CP53 were raised. The antibodies reacted with a 170-kDa enzyme with activity and a 53-kDa protein that seemed to be a degradation product of the 170-kDa proteinase from results of immunoblotting. The antibodies reacted also with a 45-kDa extracellular proteinase of L. helveticus CP790. However, monoclonal antibodies to the CP790 proteinase did not react with the proteinase of L. helveticus CP53. Seventeen strains of L. helveticus were tested for immunological reactivity with the two kinds of antibodies. The strains all had the same reactivity as either strain CP53 or strain CP790. Eleven strains with the 45-kDa proteinase were identified as L. helveticus biovar jugurti because they did not ferment maltose, four other strains with the 170- and 53-kDa proteins were identified as L. helveticus biovar helveticus because they fermented maltose. The remaining two strains dit not fit this pattern; they had both the 170- and 53-kDa proteins, but classification by their sugar utilization showed them to be L. helveticus biovar jugurti.     

Adaptation of Candida shehatae and Pichia stipitis to wood hydrolysates for increased ethanol production

Summary Candida shehatae ATCC 22984 and Pichia stipitis CBS 5776 were tested for ethanol production from xylose, glucose-xylose mixtures, and aspen wood total hydrolysates. Adaptation of these yeasts to wood hydrolysate solutions by recycling resulted in improved substrate utilization and ethanol production. Compared to the non-adapted cultures, recycled C. shehatae and P. stipitis in aspen hydrolysate increased g ethanol/g sugar consumed from 0.39 and 0.41 to 0.45 and 0.47; while ethanol production from a 70:30 glucose-xylose solution (total sugars 140 g/L) was 45 g/L in 24 h and 60 g/L in 72 h with the adapted yeasts compared to 15 g/L and 28 g/L in the same times with the parent strains.     

Citrate catabolism and production of acetate and succinate by Lactobacillus helveticus ATCC 15807

The citrate metabolism of Lactobacillus helveticus ATCC 15807 was studied under controlled-pH fermentations at pH 4.5 and pH 6.2. The micro-organism was able to co-metabolize citrate and lactose at both pH from the beginning of growth, which enhanced the rate of lactose consumption and lactic acid production, compared with cultures without citrate. The effect of citrate on cell growth was dependent on the balance between the ratio of dissociated to non-dissociated forms of the acetic acid produced and the extra ATP gained by the cells, both facts related to the citrate metabolism. The citrate catabolism determined a change in the fermentation pattern of L. helveticus ATCC 15807 from homolactic to a mixed-acid profile, regardless of the external pH. Within this new fermentation pattern, acetate was the major product formed (13–20 mM), followed by succinate (2.4–3.7 mM), while acetoine, dyacetile or butanediol were not detected. The mixed-acid profile displayed by L. helveticus ATCC 15807 was linked to NADH₂ oxidase activity rather than the acetate kinase enzyme.     

Effectiveness of Chemometric Techniques in Discrimination of Lactobacillus helveticus Biotypes from Natural Dairy Starter Cultures on the Basis of Phenotypic Characteristics

Lactobacillus helveticus is the dominant organism in natural starter cultures used for the production of typical Italian cheeses. In this study, 74 L. helveticus strains, isolated from grana and provolone cheese natural whey starters, were distinguished with respect to their origin by using both cell wall protein profiles and chemometric evaluation of some phenotypic parameters, such as the ability to acidify cultures and the presence of nonspecific proteolytic and peptidase activities. Cell wall protein patterns allowed L. helveticus strains to be distinguished with respect to their source of isolation. Among the different phenotypes studied, no single specific parameter permitted the two groups of strains to be separated. A good discrimination between the two groups of L. helveticus species was obtained by multivariate statistical techniques, which permitted the extraction of all of the discriminating information retained in the phenotypic activities. Associations between strain phenotype expression and dairy environmental ecosystem source are discussed.     

Instability of lactose and citrate metabolism of Leuconostoc strains

Summary The instability of Lac⁺ and Cit⁺ phenotypes was investigated inLeuconostoc mesenteroides subsp.cremoris ATCC 19245 and in four strains ofLeuconostoc mesenteroides subsp.dextranicum. The two phenotypes were linked respectively to a 14 Mdal and a 34 Mdal plasmid in Leuconostoc mesenteroides subsp.cremoris ATCC 19245. InLeuconostoc mesenteroides subsp.dextranicum the character Lac⁺ was linked to a 28 Mdal plasmid, while the Cit⁺ phenotype was stable.     

A Lactobacillus helveticus-Specific DNA Probe Detects Restriction Fragment Length Polymorphisms in This Species

A cloned 2-kb EcoRI fragment (fragment f) from a 34-kb plasmid of Lactobacillus helveticus CNRZ 1094 was shown by dot blot to specifically hybridize to total DNAs of 75 L. helveticus strains. No hybridization was found with L. acidophilus, L. crispatus, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. gasseri, or L. intestinalis strains. When Southern blots of EcoRI digests of L. helveticus strains were probed with fragment f, these strains displayed restriction fragment length polymorphisms on the basis of which they could be grouped into several clusters.     

Growth requirements and the establishment of a chemically defined minimal medium inZymomonas mobilis

Summary A chemically defined minimal medium which fulfils the growth requirements of differentZymomonas mobilis strains has been established. The kinetics of ethanol production of the strains ATCC 10988, CU1, CP4 and 11163 grown on the minimal medium at different glucose concentrations were measured. All strains produced ethanol at rates similar to those on complete medium. The minimal medium described is suitable to study spontaneous metabolic deficiciencies and regulation of enzyme activities inZ.mobilis.     

Assimilation of cholesterol by some cultures of lactic acid bacteria and bifidobacteria

Summary Cultures ofStr. thermophilus assimilated less cholesterol than those ofLactobacillus delbrueckii subsp.bulgaricus. A significant difference was found between strains ofL. delbrueckii subsp.bulgaricus — LB1 and LB2 and LB3 (p<0.001).Bif. bifidum actively assimilated cholesterol, but no significant difference was observed between their two strains (p>0.05). Cultures ofL. asidophilus assimilated significantly more cholesterol than those ofStr. thermophilus and a commercial yoghurt culture.     

Antibiotic resistance of potential probiotic bacteria of the genus <Emphasis Type="Italic">Lactobacillus</Emphasis> from human gastrointestinal microbiome

Thirteen Lactobacillus strains isolated from the gastrointestinal microbiome of people from the territory of the former Soviet Union have been studied for resistance to 15 antibiotics of different nature, namely, penicillins, aminoglycosides, macrolides, lincosamides, tetracyclines, chloramphenicol, and rifampicin. The strains included four strains of L. plantarum, four of L. helveticus, three of L. casei/paracasei, one of L. rhamnosus, and one of L. fermentum. All strains showed relative sensitivity to ampicillin, chloramphenicol, rifampicin, roxithromycin, erythromycin, and azithromycin, while none of them were sensitive to all tested antibiotics. L. plantarum strains had the broadest resistance spectra: one strain was resistant to tetracycline and three aminoglycosides and three strains were resistant to tetracycline and five aminoglycosides; one strain demonstrated high resistance to clindamycin and two strains to lincomycin. At the same time, two L. plantarum strains demonstrated resistance to benzylpenicillin coupled with sensitivity to ampicillin, another β-lactam antibiotic. Such resistance was clearly not related to the β-lactamase activity and could be explained by a specific mutation in one of the penicillin-binding proteins of the cell wall. Strains of L. helveticus, L. casei/paracasei, L. rhamnosus, and L. fermentum exhibited cross resistance to two to five different aminoglycosides. A PCR test of the resistance determinants for the widely clinically used antibiotics, tetracycline, chloramphenicol, and erythromycin revealed the presence of the tetM gene of conjugative transposon in L. casei/paracasei and two L. helveticus strains. Nucleotide sequence analysis of the amplified tetM fragments demonstrated their high homology with the tetM genes of Enterococcus faecalis and Streptococcus pneumoniae. The strains carrying tetM were tested for the genes of replication and conjugative transfer of plasmids in lactic acid bacteria. The results indicated that these strains contain genes identical or highly homologous to the rep and trsK genes of the plca36 plasmid and rep gene of the pLH1 and pLJ1 plasmids of lactic acid bacteria. The tetM gene is probably not expressed in strains sensitive to the corresponding antibiotic. However, the investigated lactobacilli cannot be directly used as probiotics, as they may serve as a source of genes for antibiotic resistance in the human microbiome.     

Inhibition of psychrotrophic bacteria in raw milk by immobilized lactic acid bacteria

Summary Cells ofLactococcus lactis orLactobacillus helveticus were immobilized in calcium-alginate beads, added to raw milk, and incubated 48 h at 7°C. The addition of 2.7×10⁷ immobilizedLc.lactis or 13×10⁷ immobilizedLb. helveticus cells per mL reduced the development of the psychrotrophic bacteria of raw milk by approximately 50%. The pH of the raw milk dropped 0.10 to 0.22 units under these conditions. Periodic agitation of the seeded raw milk increased the inhibitory activity of the immobilized lactic acid bacteria (LAB). Free LAB cells in the system were only of 0.5% of total LAB. The use of immobilized LAB to inhibit psychrotrophic bacteria might be extended to raw milks destined to the manufacture of non-fermented dairy products.     

Ethanolic fermentation of cane molasses by a highly flocculent yeast

Summary A study of the comparative kinetics of standardS.uvarum ATCC 26602 withS.cerevisiae Y-10 (an isolate) and a highly flocculent strain ofS.uvarum in batch mode has shown that both the isolate and the highly flocculentS. uvarum strain have more desirable characteristics than the standard strains for ethanol production from cane molasses.     

Functionality of the S-layer protein from the probiotic strain <Emphasis Type="Italic">Lactobacillus helveticus</Emphasis> M92

The objective of this study was the characterisation of the S-layer protein (SlpA) and its functional role in the probiotic activity of Lactobacillus helveticus M92. SlpA was isolated and identified by SDS-PAGE LC-MS/MS analysis. The slpA gene encoding the SlpA from L. helveticus M92 was sequenced and compared with other well characterised slpA genes. Sequence similarity searches revealed high homology with the SlpA of Lactobacillus strains. Purified SlpA showed significantly better immunomodulatory effects in orally immunised mice than L. helveticus M92 cells after SlpA removal. SlpA is involved in the autoaggregation of L. helveticus M92 cells and coaggregation of L. helveticus M92 with S. Typhimurium FP1 as these processes were negatively affected after SlpA removal from the cell surface. Therefore, the influence of oral treatment with L. helveticus M92 on an oral infection of mice by S. Typhimurium FP1 was investigated. Following the oral immunization of mice, with viable L. helveticus M92 and S. Typhimurium FP1 cells, the concentration in the luminal contents of total S-IgA and specific anti-Salmonella S-IgA antibodies, from all immunized mice was significantly higher compared to the control group or a group of mice infected only with S. Typhimurium FP1. These results demonstrate that the observed reduced infection by S. Typhimurium FP1 in mice with L. helveticus M92 is associated with competitive exclusion in the intestinal tract and enhanced immune protection conferred by the L. helveticus M92 and its SlpA.     

Doxycycline-Dependent Inducible and Reversible RNA Interference Mediated by a Single Lentivirus Vector

Most fermented milk prepared by strains of Lactobacillus helveticus showed significant antihypertensive effect in spontaneously hypertensive rats (SHR) by oral administration. However, milk fermented by other species of lactic acid bacteria did not show significant antihypertensive effects. Most of the whey fractions of the milk fermented by L. helveticus or Lactobacillus delbrueckii subsp. bulgaricus showed higher angiotensin I-converting enzyme (ACE) inhibitory activity than the activity of milk fermented by other species. Proteolytic activity in cell wall and peptide content of the fermented milk were higher in L. helveticus strains than other species.