Characterization of lactobacilli by Southern-type hybridization with a Lactobacillus plantarum pyrDFE probe

Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum (M.-C. Curk, J.-C. Hubert, and F. Bringel, Int. J. Syst. Bacteriol. 46:595-598, 1996) can hardly be distinguished on the basis of their phenotypes. Unlike L. plantarum and L. paraplantarum, L. pentosus ferments glycerol and xylose but not melezitose. We identified two L. pentosus strains (CNRZ 1538 and CNRZ 1544) which ferment glycerol and melezitose but not xylose. alpha-Methyl-D-mannoside was fermented by 66% of the L. plantarum strains tested but not by L. paraplantarum strains. In this paper we describe a simple method to identify L. plantarum, L. pentosus, and L. paraplantarum. This method is based on nonradioactive Southern-type hybridization between BglI DNA digests of the lactobacilli tested and a DNA probe (L. plantarum pyrDFE genes from strain CCM 1904). A total of 68 lactobacilli were classified into five groups on the basis of the bands detected. Two groups contained L. plantarum strains; one of these groups contained 31 strains, including the type strain, and was characterized by bands at 7, 4, and 1 kb, and the other group contained strain LP 85-2 and was characterized by bands at 5 and 1.1 kb. Only one band (a band at around 7 kb) was detected in the strains belonging to the L. pentosus group, and two bands (at 4 and 1 kb) were found in the strains belonging to the L. paraplantarum group. No hybridization was detected in the last group, which contained Lactobacillus casei, Lactobacillus coryniformis, Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus delbrueckii, and Lactobacillus leichmannii strains.     

Transthyretin amyloidosis: a new mutation associated with dementia

NaCl-tolerant lactic acid bacteria (LAB) strains LC-10 (Lactobacillus casei) and LP-15 (Lact. plantarum) and NaCl were used as additives to sorghun (Sorghum bicolor). Numbers of LAB were significantly (P < 0.05) higher in all the additive-treated silages than in the control silage at an early stage of ensiling. During the fermentation process, addition of NaCl or LAB effectively inhibited the growth of aerobic bacteria and clostridia, but not yeasts. All the additive-treated silages had significantly (P < 0.05) lower pH, ammonia nitrogen content, dry matter loss and gas production but significantly (P < 0.05) higher lactic acid content and residual water soluble carbohydrates compared with the control silage. The improvement in silage quality was in the order: LAB > NaCl > control. Yeast counts were high in all additive-based silages and they increased during the exposure of the silages to air. As a result, these silages suffered aerobic deterioration, whereas the control silage was stable. The results confirmed that the NaCl or LAB improved fermentation quality but did not prevent aerobic deterioration of the silage.     

Characterization and frequency distribution of species of lactic acid bacteria involved in the processing of mawè, a fermented maize dough from Benin

Lactic acid bacteria involved in the natural fermentation of both home-produced and commercial mawè were investigated during a 72 h fermentation period. Lactobacillus spp. constitute the majority (94%) of the strains of the lactic acid bacteria isolated, among which 89% represent the Betabacterium group. They include L. fermentum (biotype cellobiosus) (41%), L. fermentum or L. reuteri (19%), L. brevis (26%), L. confusus (less than 2%), L. curvatus (less than 1%) and L. buchneri (less than 1%). Other isolated lactic acid bacteria were L. salivarius, Lactococcus lactis, Pediococcus pentosaceus, Pediococcus acidilactici and Leuconostoc mesenteroides. Several species were detected at the early stage of fermentation, but the final stage was dominated by L. fermentum (biotype cellobiosus) and L. fermentum or L. reuteri totalling 90% of the isolated strains. The trend was the same for both home-produced and commercial mawè. No strains of L. plantarum, generally reported as dominating lactic acid bacteria at the final stage of fermentation of most plant foods, were isolated.     

Taxonomy and physiology of probiotic lactic acid bacteria

The current taxonomy of probiotic lactic acid bacteria is reviewed with special focus on the genera Lactobacillus, Bifidobacterium and Enterococcus. The physiology and taxonomic position of species and strains of these genera were investigated by phenotypic and genomic methods. In total, 176 strains, including the type strains, have been included. Phenotypic methods applied were based on biochemical, enzymatical and physiological characteristics, including growth temperatures, cell wall analysis and analysis of the total soluble cytoplasmatic proteins. Genomic methods used were pulsed field gel electrophoresis (PFGE), randomly amplified polymorphic DNA-PCR (RAPD-PCR) and DNA-DNA hybridization for bifidobacteria. In the genus Lactobacillus the following species of importance as probiotics were investigated: L. acidophilus group, L. casei group and L. reuteri/L. fermentum group. Most strains referred to as L. acidophilus in probiotic products could be identified either as L. gasseri or as L. johnsonii, both members of the L. acidophilus group. A similar situation could be shown in the L. casei group, where most of the strains named L. casei belonged to L. paracasei subspp. A recent proposal to reject the species L. paracasei and to include this species in the restored species L. casei with a neotype strain was supported by protein analysis. Bifidobacterium spp. strains have been reported to be used for production of fermented dairy and recently of probiotic products. According to phenotypic features and confirmed by DNA-DNA hybridization most of the bifidobacteria strains from dairy origin belonged to B. animalis, although they were often declared as B. longum by the manufacturer. From the genus Enterococcus, probiotic Ec. faecium strains were investigated with regard to the vanA-mediated resistance against glycopeptides. These unwanted resistances could be ruled out by analysis of the 39 kDa resistance protein. In conclusion, the taxonomy and physiology of probiotic lactic acid bacteria can only be understood by using polyphasic taxonomy combining morphological, biochemical and physiological characteristics with molecular-based phenotypic and genomic techniques.     

A Darwinian theory for the origin of cellular differentiation

The use of starter cultures to control and run the fermentative process is a usual way of manufacturing sausages in meat industries. The first stage in the starter culture designing process is to characterize the lactic acid bacteria isolated from these meat products, in order to select the best strains. The strains used for this study were isolated from different dry fermented sausages, obtained during the manufacturing process. The main tests used to identify the isolated bacteria were: microscopic-morphologic characteristics, catalase activity, production of gas, growth at 8, 15 and 45 degrees C, fermentation of carbohydrates and production of lactic acid isomers. A total of 194 strains were identified. Lactobacillus sake and Lactobacillus plantarum were the most frequent species. Other microbiological tests were also performed, and three strains of Lactobacillus sake were found which did not produce dextran from sucrose.     

Evidence for frequent lysogeny in lactobacilli: temperate bacteriophages within the subgenus Streptobacterium

A total of 17 of 21 Lactobacillus strains of the subgenus Streptobacterium were lysogenic. Two different temperate phages isolated from such lysogens are very similar to Lactobacillus casei phage PL-1. The narrow host range of bacteriophage PL-1 appears to be caused by homoimmunity.     

Acid tolerance response and survival by oral bacteria

Using 21 species of oral bacteria, representing six acidogenic genera, we undertook to determine whether the pH-limiting exponential growth is related to the ability of the organisms to generate an acid-tolerance response that results in enhanced survival at low pH. The lower pH limit of exponential growth varied by more than two units with that of Neisseria A182 at pH 6.34; growth of Lactobacillus casei RB1014 stopped at pH 3.81, with species of Actinomyces, Enterococcus, Prevotella and Streptococcus falling between these limits. The working hypothesis was that the organisms with the higher pH limits for growth are unable to respond to acidic environments in order to survive, whereas the more aciduric organisms would possess or acquire acid tolerance. Adaptation to acid tolerance was tested by determining whether the prior exposure of exponential-phase cells to a low, sub-lethal pH would trigger the induction of a mechanism that would enhance survival at a pH killing pH 7.5 control cells. The killing pH varied from pH 4.5 for Prevotella intermedia ATCC 25611 to pH 2.3 for the three Lactobacillus casei strains in the study, with the three Streptococcus mutans strains killed at pH 3.0 for 3 h. The adaptation experiments revealed three groups of organisms: non-acid-responders, generally representing strains with the highest terminal pH values; weak acid-responders in the middle of the pH list, generating low numbers of survivors at one or two pH values, and the aciduric, strong responders generating a high number of survivors at pH values in the range 6.0 to 3.5, but not at pH 7.5. Predominant among the latter group were the S. mutans and Lactobacilli casei strains, with the most significant adaptive response exhibited by S. mutans LT11 and S. mutans Ingbritt, involving a process that required protein synthesis. Time course experiments with the latter organisms indicated that 90-120 min was required after exposure to the triggering pH before the acid response was fully functional. These results indicate that the sudden exposure of strains of oral streptococci and lactobacilli, as well as Enterococcus faecalis, to pH values between 6.0 and 3.5 results in the induction of an acid tolerance response that enhances the survival of these strains at or below pH 3.5.     

Characterisation and selection of probiotic lactobacilli for a preliminary minipig feeding trial and their effect on serum cholesterol levels, faeces pH and faeces moisture content

Three out of 297 Lactobacillus strains isolated from pig faeces were selected for a feeding trial on account of their high bile-salt hydrolase (BSH) activity, bile-salt resistance, low pH tolerance and the production of antimicrobial substances. Two strains were identified as Lactobacillus johnsonii and one as Lactobacillus reuteri by DNA-DNA hybridisation. L. johnsoniii BFE 1061 produced a bacteriocin active against a range of lactic acid bacteria (LAB) and nonrelated bacteria including Clostridium perfringens. Six minipigs were maintained on a high-fat, high-cholesterol ('Western Style') diet for 17 weeks after which the diet was supplemented with the 'probiotic mixture' containing the above mentioned three Lactobacillus strains at 2 x 10(12) CFU per pig per day for five weeks. The mixture was given as a resuspended lyophilisate. During a two week follow-up period the minipigs received only the 'Western-style' diet without probiotic supplementation. A lowering effect on serum cholesterol levels was indicated after three weeks probiotic feeding, concomitant with an increase in the moisture content of the faeces and Lactobacillus cell numbers. Triglycerides, pH and number of lactic acid bacteria in faeces were not significantly influenced by probiotic supplementation.     

16S-23S and 23S-5S intergenic spacer regions of lactobacilli: nucleotide sequence, secondary structure and comparative analysis

Lactobacilli have been used as industrial starters for a long time, but in several cases their identification was, and still is, neither easy nor reliable. The aim of the present work was to examine whether the intergenic spacer regions could be of value in the identification of Lactobacillus species. For that purpose, the polymerase chain reaction (PCR) was used to amplify 16S-23S and 23S-5S spacer regions of Lactobacillus (L.) acidophilus, L. delbrueckii subsp. bulgaricus, L. casei, L. helveticus and L. curvatus. The PCR products were directly sequenced, and two forms of ribosomal RNA (rrn) operons were identified in each species studied: one with tandem tRNA(Ile)/tRNA(Ala) genes and the other one without tRNA genes. Our study revealed that the rrn operons of Lactobacillus species studied comprise the genes of 16S, 23S and 5S rRNA, in that order. Only the tRNA genes and the rRNA processing stems are highly conserved in spacer regions of lactobacilli. The divergence between the lactobacilli spacer region sequences arises from insertions and deletions of short sequences. These sequences could be interesting candidates for the development of species-specific probes. Theoretical RNA/RNA secondary structure models of the interaction between the two spacer region sequences were constructed. In conclusion, the two spacer region sequences may prove to be a useful alternative to 16S and 23S rDNA sequencing for designing species-specific probes and for establishing phylogenetic relationships between closely related species such as L. curvatus and L. casei or L. acidophilus and L. helveticus.     

The effect of lactic acid bacteria on intestinal metabolism and metabolic profile in gnotobiotic pigs

The effect of inoculation of Lactobacillus casei on selected parameters of metabolic profile and intestinal metabolism of gnotobiotic piglets was investigated during the first three weeks of their life. The experiment was carried out on 8 germ-free piglets. The experimental group was inoculated once a day with the Lactobacillus casei subsp. casei strain. The inoculum contained 1 x 10(8) microorganisms in 1 ml. The control group of piglets received no inoculum. Lactobacillus casei colonized jejunum and ileum in the numbers from 5.63 to 6.06 log 10 cm-2 and their numbers in the jejunal and ileal contents were in the range 8.38-9.87 log 10.ml-1. The daily consumption of milk by the inoculated animals was significantly higher (p < 0.001). The average weight of inoculated piglets at the end of the period investigated was higher by 29.7%. Lactobacillus casei affected several parameters investigated. Piglets inoculated with lactobacilli showed significantly lower (p < 0.05-0.01) values of pH of the jejunal content, numbers of erythrocytes, values of haematocrit, urea, glucose, total lipids, cholesterol and calcium in the serum and significantly higher values (p < 0.05-0.01) of lactic acid in the jejunal content. The values of phagocytic activity and the index of phagocytic activity in the piglets of the experimental group were two to three-fold higher in comparison with those detected in the control group. The application of Lactobacillus casei affected positively the growth of gnotobiotic piglets, their intestinal metabolism, the level of cholesterol in the serum and phagocytic activity.     

The effect of consumption of milk fermented by Lactobacillus casei strain Shirota on the intestinal microflora and immune parameters in humans

OBJECTIVE: To determine the effect of consumption of milk fermented by Lactobacillus casei strain Shirota (L. casei Shirota) on the composition and metabolic activities of the intestinal microflora, and immune parameters in humans. SUBJECTS: Twenty healthy male subjects aged 40-65 years were selected. DESIGN: A placebo-controlled trial was performed in which 10 subjects were randomly assigned to a control and 10 to a treatment group. During the first and last two weeks of the 8-week study the subjects received a strictly controlled diet without fermented products. The same controlled diet was given during the intermediate 4-week test period but then the treatment group received three times daily 100 ml of fermented milk containing 10(9) CFU L. casei Shirota/ml, whereas the same amount of unfermented milk was given to the subjects in the control group. RESULTS: In comparison to the control group, the consumption of L. casei Shirota-fermented milk resulted in an increase of the Lactobacillus count in the faeces in which the administered L. casei Shirota was predominant at the level of 10(7) CFU/g wet faeces. This was associated with a significant increase in Bifidobacterium counts (P < 0.05). Some shifts in the other bacterial species were found, such as a decreased number of Clostridium; however the differences were not statistically different between the treatment and the control groups. The beta-glucuronidase and beta-glucosidase activities per 10(10) bacteria decreased significantly (P < 0.05) at the second week of the 4-week test period with the consumption of L. casei Shirota-fermented milk. Furthermore, the consumption of the fermented milk product resulted in a slight but significant increase in the moisture content of the faecal samples (P < 0.05). No treatment effects were observed for any of the immune parameters measured (including natural killer (NK) cell activity, phagocytosis and cytokine production). CONCLUSIONS: The results suggest that consumption of L. casei Shirota-fermented milk is able to modulate the composition and metabolic activity of the intestinal flora and indicate that L. casei Shirota-fermented milk does not influence the immune system of healthy immunocompetent males.     

Cell surface characteristics of Lactobacillus casei subsp. casei, Lactobacillus paracasei subsp. paracasei, and Lactobacillus rhamnosus strains

Hydrophilic and electrostatic cell surface properties of eight Lactobacillus strains were characterized by using the microbial adhesion to solvents method and microelectrophoresis, respectively. All strains appeared relatively hydrophilic. The strong microbial adhesion to chloroform, an acidic solvent, in comparison with microbial adhesion to hexadecane, an apolar n-alkane, demonstrated the particularity of lactobacilli to have an important electron donor and basic character and consequently their potential ability to generate Lewis acid-base interactions with a support. Regardless of their electrophoretic mobility (EM), strains were in general slightly negatively charged at alkaline pH. A pH-dependent behavior concerning cell surface charges was observed. The EM decreased progressively with more acidic pHs for the L. casei subsp. casei and L. paracasei subsp. paracasei strains until the isoelectric point (IEP), i.e., the pH value for which the EM is zero. On the other hand, the EM for the L. rhamnosus strains was stable from pH 8 to pH 3 to 4, at which point there was a shift near the IEP. Both L. casei subsp. casei and L. paracasei subsp. paracasei strains were characterized by an IEP of around 4, whereas L. rhamnosus strains possessed a markedly lower IEP of 2. The present study showed that the cell surface physicochemical properties of lactobacilli seem to be, at least in part and under certain experimental conditions, particular to the bacterial species. Such differences detected between species are likely to be accompanied by some particular changes in cell wall chemical composition.     

Partial characterisation of pediocin PO2 and comparison with nisin for biopreservation of meat products

A plasmid associated bacteriocin (pediocin PO2) was isolated by ammonium sulphate precipitation from cell-free growth media and subsequent studies showed that the partially purified pediocin PO2 was most likely identical (molecular mass approximately 3200 daltons in size by SDS-PAGE, stable to low pH and heat at 121 degrees C for 15 min, inactivated by various proteolytic enzymes and resistant to treatment with a range of solvents, except 10% formaldehyde) to other pediocins (PA-1 and AcH) previously reported. The antagonistic spectrum of activity of pediocin PO2 was compared with nisin and showed a narrower host-range, but a much greater activity against Listeria species including strains of Listeria monocytogences, than did nisin. A rapid method of reflectance colorimetry was used to quantitate growth and acid production (as determined by the colour change in bromcresol purple) of Lactobacillus curvatus, added to a meat product model system. The combined effects of refrigeration temperature, microbial load and bacteriocin concentration were determined in the model over 15 days storage. Both nisin and pediocin demonstrated inhibitory activity against Lactobacillus curvatus in the model system. However, when bacteriocins were incorporated into a manufactured cooked meat product only low nisin activity and no pediocin activity was detected, after challenge of vacuum packaged slices of product with Lactobacillus curvatus, over a 21 day storage trial under refrigeration temperatures.     

Effect of acute ethanol administration on serum magnesium concentration in the rabbit

The size of the Lactobacillus plantarum CCM 1904 chromosome was determined by pulse-field gel electrophoresis. It was found to be 3.3-3.4 Mb using SfiI or AscI restriction endonucleases, compared to 3-4 Mb found for the other L. plantarum strains tested. L. plantarum CCM 1904 5S rDNA was clonedl by polymerase chain reaction, sequenced, and used as a probe to characterize strains. At least five rrn loci were found. The pulsed-field gel electrophoresis macrorestriction patterns were strain-specific, while the rDNA restriction hybridization patterns were species-specific.     

Cocoa fermentations conducted with a defined microbial cocktail inoculum

Cocoa fermentations were performed in wooden boxes under the following four experimental regimens: beans naturally fermented with wild microflora; aseptically prepared beans with no inoculum; and beans inoculated with a defined cocktail containing microorganisms at a suitable concentration either at zero time or by using phased additions at appropriate times. The cocktail used consisted of a yeast, Saccharomyces cerevisiae var. chevalieri, two lactic acid bacterial species, Lactobacillus lactis and Lactobacillus plantarum, and two acetic acid bacterial species, Acetobacter aceti and Gluconobacter oxydans subsp. suboxydans. The parameters measured were cell counts (for yeasts, filamentous fungi, lactic acid bacteria, acetic acid bacteria, and spore formers, including reisolation and identification of all residual cell types), sugar, ethanol, acetic acid, and lactic acid contents (and contents of other organic acids), pH, and temperature. A cut test for bean quality and a sensorial analysis of chocolate made from the beans were also performed. The natural fermentation mimicked exactly the conditions in 800-kg boxes on farms. The aseptic box remained largely free of microflora throughout the study, and no significant biochemical changes occurred. With the zero-time inoculum the fermentation was almost identical to the natural fermentation. The fermentation with the phased-addition inoculum was similar, but many changes in parameters were slower and less pronounced, which led to a slightly poorer end product. The data show that the nearly 50 common species of microorganisms found in natural fermentations can be replaced by a judicious selection and concentration of members of each physiological group. This is the first report of successful use of a defined, mixed starter culture in such a complex fermentation, and it should lead to chocolate of more reliable and better quality.     

Effects of lactobacilli on yeast-catalyzed ethanol fermentations

Normal-gravity (22 to 24 degrees Plato) wheat mashes were inoculated with five industrially important strains of lactobacilli at approximately 10(5), approximately 10(6), approximately 10(7), approximately 10(8), and approximately 10(9) CFU/ml in order to study the effects of the lactobacilli on yeast growth and ethanol productivity. Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus #3, Lactobacillus rhamnosus, and Lactobacillus fermentum were used. Controls with yeast cells but no bacterial inoculation and additional treatments with bacteria alone inoculated at approximately 10(7) CFU/ml of mash were included. Decreased ethanol yields were due to the diversion of carbohydrates for bacterial growth and the production of lactic acid. As higher numbers of the bacteria were produced (depending on the strain), 1 to 1.5% (wt/vol) lactic acid resulted in the case of homofermentative organisms. L. fermentum, a heterofermentative organism, produced only 0.5% (wt/vol) lactic acid. When L. plantarum, L. rhamnosus, and L. fermentum were inoculated at approximately 10(6) CFU/ml, an approximately 2% decrease in the final ethanol concentration was observed. Smaller initial numbers (only 10(5) CFU/ml) of L. paracasei or Lactobacillus #3 were sufficient to cause more than 2% decreases in the final ethanol concentrations measured compared to the control. Such effects after an inoculation of only 10(5) CFU/ml may have been due to the higher tolerance to ethanol of the latter two bacteria, to the more rapid adaptation (shorter lag phase) of these two industrial organisms to fermentation conditions, and/or to their more rapid growth and metabolism. When up to 10(9) CFU of bacteria/ml was present in mash, approximately 3.8 to 7.6% reductions in ethanol concentration occurred depending on the strain. Production of lactic acid and a suspected competition with yeast cells for essential growth factors in the fermenting medium were the major reasons for reductions in yeast growth and final ethanol yield when lactic acid bacteria were present.