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PubMed1988

Enhancement of host resistance against Listeria infection by Lactobacillus casei: efficacy of cell wall preparation of Lactobacillus casei.

Cell wall, cytoplasm, polysaccharide, and peptidoglycan fractions prepared from Lactobacillus casei, L. plantarum, and L. acidophilus were examined for their efficacies to enhance resistance of host mice against Listeria monocytogenes infection. Intraperitoneal injections of those cellular fractions of L. casei led to elicitation of inflammatory cells in the peritoneal cavity and the efficacy was highest in the case of peptidoglycan. Macrophage ratio in the resultant peritoneal exudate cells was also highest in mice given peptidoglycan. Macrophages induced with cell wall fraction of L. casei showed the most potent phorbol myristate acetate (PMA)-triggered respiratory burst (chemiluminescence and O2- production determined on the basis of nitroblue tetrazolium reduction) followed by those elicited with peptidoglycan. All the macrophages induced with cell wall of L. casei (two strains) and L. acidophilus enhanced O2- production in response to PMA but L. plantarum did not enhance O2(-)-producing ability in such a manner. The L. casei-cell wall also enhanced in vitro listericidal activity of mouse peritoneal macrophages, but such an activity was not noted in the case of L. acidophilus-cell wall. When mice were intravenously given the cellular fractions 7 or 13 days before L. monocytogenes infection, cell wall fractions of L. casei caused the most potent protective activity. A weak protective activity was also found in peptidoglycan of L. casei. Therefore, the protective action of L. casei against L. monocytogenes infection in host mice may be attributed to cell wall compounds and partially to the peptidoglycan moiety.

Animals↗
PubMed1980

Role of bacterial interactions in the colonization of oral surfaces of Actinomyces viscosus.

The effects of several microbial residents of human oral cavities on the attachment of Actinomyces viscosus T14V to tooth surfaces were assessed by using the saliva-coated hydroxyapatite (sHA) in vitro system. Attachment to sHA by A. viscosus T14V was not inhibited in the presence of Streptococcus mutans GS5, either in the presence or absence of sucrose. Precoating sHA beads with S. mutans in the presence of sucrose also did not retard strain T14V attachment. However, the presence of Streptococcus sanguis M5 during strain T14V attachment markedly inhibited the interaction of A. viscosus T14V with sHA, whereas another S. sanguis strain, strain G9B, produced relatively weak inhibition. The inhibitory effect of S. sanguis M5 appeared to result from the direct interaction of this organism with A. viscosus T14V. Conversely, the presence of strain T14V markedly increased the attachment of S. sanguis M5 to sHA while partially inhibiting the attachment of S. sanguis G9B. Moreover, sHA beads precoated with A. viscosus T14V bound much higher levels of S. sanguis M5 relative to uncoated sHA, whereas S. sanguis G9B attachment was only weakly inhibited. It was also observed that sHA beads precoated with either S. sanguis M5 or G9B inhibited subsequent attachment by strain T14V. These results suggest the possibility that the attachment sites for both S. sanguis strains partially overlap those for A. viscosus T14V. Streptococcus mitis weakly inhibited attachment of strain T14V to sHA, and both Streptococcus salivarius and Lactobacillus casei had little effect on attachment. In addition, both a radioisotope attachment assay and fluorescent microscopy demonstrated no significant attachment of A. viscosus T14V to human epithelial cells.

Actinomyces↗
PubMed1981

Oral ecology and virulence of Lactobacillus casei and Streptococcus mutans in gnotobiotic rats.

Lactobacilli comprise a small percentage of the normal oral microbial flora of humans and are isolated commonly from saliva and frequently from an active caries lesion. We have compared the pathogenesis and colonization pattern of Lactobacillus casei with that of Streptococcus mutans strain 6715 in gnotobiotic rats. Of the two L. casei strains tested, L. casei strain ATCC 4646 caused slightly more caries than L. casei strain ATCC 11578. However, the level of caries induced by either L. casei strain was significantly lower (P less than 0.01) than that observed in similar-aged rats monoassociated with S. mutans strain 6715. When groups of rats were infected with mixtures of L. casei strain ATCC 4646 and S. mutans strain 6715, or with L. casei followed by S. mutans, higher numbers of L. casei than S. mutans were found associated with the tongue and in saliva; S. mutans always predominated in plaque. The level of caries observed in these groups of rats was similar to that seen with rats monoassociated with S. mutans except when L. casei comprised greater than 1% of the plaque microflora. In this latter situation, the level of caries was significantly lower (P less than or equal to 0.05) than that obtained in S. mutans-monoassociated rats. The results of this study suggest that L. casei colonizes sites in the oral cavity (including the tongue and saliva) other than the tooth surface in rats. The effect of L. casei in plaque toward reduction of S. mutans-induced dental caries in rats is discussed.

Animals↗
PubMed2002

The effect of Arg209 to Lys mutation in mouse thymidylate synthase.

Mouse thymidylate synthase R209K (a mutation corresponding to R218K in Lactobacillus casei), overexpressed in thymidylate synthase-deficient Escherichia coli strain, was poorly soluble and with only feeble enzyme activity. The mutated protein, incubated with FdUMP and N(5,10)-methylenetetrahydrofolate, did not form a complex stable under conditions of SDS/polyacrylamide gel electrophoresis. The reaction catalyzed by the R209K enzyme (studied in a crude extract), compared to that catalyzed by purified wild-type recombinant mouse thymidylate synthase, showed the K(m) value for dUMP 571-fold higher and V(max) value over 50-fold (assuming that the mutated enzyme constituted 20% of total crude extract protein) lower. Thus the ratios k(cat, R209K)/k(cat, 'wild') and (k(cat, R209K)/K(m, R209K)(dUMP))/( k(cat, 'wild')/K(m, 'wild')(dUMP)) were 0.019 and 0.000032, respectively, documenting that mouse thymidylate synthase R209, similar to the corresponding L. casei R218, is essential for both dUMP binding and enzyme reaction.

Amino Acid Substitution↗
PubMed1987

Proteolytic activity of crude cell-free extract of Lactobacillus casei and Lactobacillus plantarum.

Crude cell-free extracts of some strains of each L. casei and L. plantarum were assayed for their amino-, imino- and endopeptidase as well as the caseinolytic activity. L-alanine-, L-phenylalanine- and L-leucine-p-nitroanilide but not L-glutamic acid-p-nitroanilide, were hydrolyzed by all the strains indicating an amino-peptidase activity. The activity of proline iminopeptidase was very low compared to that of the aminopeptidase. L. casei could be distinguished from L. plantarum by its high endopeptidase activity against succinyl-phenylalanine- and glutaryl-phenylalanine-p-nitroanilide. The caseinolytic activity of cell-free extract of L. casei ATCC 393 was about one seventh the caseinolytic activity of intact cells, suggesting that the bulk of the cellular proteinase activity is located in the cell wall. It appears that a metallo aminopeptidase and a probable cysteine one are involved in the hydrolysis of amino acid-p-nitroanilide, whereas the endopeptidase activity appears to be related to a cysteine enzyme. Incubation of gels with L-leucine-p-nitroanilide after electrophoresis allowed the revealing of 2 zones of aminopeptidase activity in a strain of L. casei and only one in two others, but in L. plantarum it did not allow the revealing of any. The high proteolytic activities of L. casei compared to those of L. plantarum may explain its more frequent occurrence in cheese and its probable role in the ripening of many cheese varieties.

Dithiothreitol↗
PubMed1987

Antibody for detection and quantitation of membrane-associated folate-binding protein from Lactobacillus casei.

Lactobacillus casei cells contain a 25 kDa, membrane-associated, folate-binding protein (fbp), which is a component of the folate transport system. Polyclonal antibody to fbp (anti-fbp) has been prepared, and conditions have been established for detection and quantitation of the protein. Anti-fbp did not block [3H]folate transport or binding in L. casei cells. As judged by Western blots, the antibody reacted only with fbp on sodium dodecyl sulfate electrophoretograms of Triton X-100 extracts of L. casei membranes. Anti-fbp showed no cross-reactivity with L. casei dihydrofolate reductase, L. casei 5,10-methenyltetrahydrofolate synthetase, L1210 dihydrofolate reductase, rat liver dihydrofolate reductase, or L1210 folate-binding protein. Enzyme-linked immunosorbent assay measurements indicated the presence of an fbp in membranes of Lactobacillus salivarius and two transport-defective sublines of L. casei. Anti-fbp was used to demonstrate selective extraction, with n-butanol, of fbp from a mixture of Triton-solubilized L. casei membrane proteins; repression of fbp in membranes of L. casei cells grown on high levels of folate; and localization of fbp by electron microscopy, using anti-fbp in conjunction with goat anti-rabbit IgG gold conjugate, in L. casei membranes.

Animals↗
PubMed2005

In vitro evaluation of probiotics microorganisms adhesion to an artificial caries model.

OBJECTIVE: This in vitro study compared the adhesion of two probiotics microorganisms (Lactobacillus casei Shirota and Lactobacillus acidophilus) to an artificial caries model. METHOD: In total, 30 bovine teeth were longitudinally sectioned, excluding the lingual half surface. The specimens were covered with nail varnish, except for an area of 3 by 5 mm in dentin, suspended in an artificial caries solution promoted by Streptococcus mutans, and incubated at 37 degrees C. After 14 days, the specimens were separated equally into two groups and transferred to a brain heart infusion culture media containing L. acidophilus (group A) and L. casei Shirota (group S), at 37 degrees C. After 48 h, the exposed area of dentin was washed with 1 ml of distilled water and the caries dentin was removed and dispersed in 1 ml of saline solution. The samples of distilled water and caries dentin were diluted and inoculated in Rogosa selective Lactobacillus agar. The results in CFU/ml were analysed by the Mann-Whitney test. RESULTS: There was no significant difference between Groups A and S for the lactobacilli count in samples of distilled water (P = 0.237). CONCLUSIONS: The amount of L. acidophilus in the artificially caries dentin was significantly superior compared to L. casei Shirota (P = 0.047), suggesting an inferior adhesion potential for this microorganism.

Animals↗
PubMed1995

Optimizing conditions for the growth of Lactobacillus casei YIT 9018 in tryptone-yeast extract-glucose medium by using response surface methodology.

This study was undertaken to find optimum conditions of tryptone, yeast extract, glucose, Tween 80, and incubation temperature for the growth of Lactobacillus casei YIT 9018 and to assess the effects of these factors by use of response surface methodology. A central composite design was used as an experimental design for allocation of treatment combinations. A second-order polynomial regression model, which was used at first for analysis of the experiment, had a significant lack of fit. Therefore, cubic and quartic terms were incorporated into the regression model through variable selection procedures. Effects involving incubation temperature, yeast extract, glucose, and tryptone were significant, whereas the only significant effect involving Tween 80 was the interaction effect between temperature and Tween 80. It turned out that growth of L. casei YIT 9018 was most strongly affected by the incubation temperature. Estimated optimum conditions of the factors for growth of L. casei YIT 9018 are as follows: tryptone, 3.04%; yeast extract, 0.892%; glucose, 1.58%; Tween 80, 0%; incubation temperature, 35 degrees C.

Bacteriological Techniques↗
PubMed1997

Antagonistic activity exerted in vitro and in vivo by Lactobacillus casei (strain GG) against Salmonella typhimurium C5 infection.

The aim of this study was to compare the antagonistic properties of Lactobacillus casei GG exerted in vitro against Salmonella typhimurium C5 in a cellular model, cultured enterocyte-like Caco-2 cells, to those exerted in vivo in an animal model, C3H/He/Oujco mice. Our results show that a 1-h contact between the invading strain C5 and either the culture or the supernatant of L. casei GG impeded the invasion by the Salmonella strain in Caco-2 cells, without modifying the viability of the strain. After neutralization at pH 7, no inhibition of the invasion by C5 was observed. The antagonistic activity of L. casei GG was examined in C3H/He/Oujco mice orally infected with C5 as follows: (i) L. casei GG was given daily to conventional animals as a probiotic, and (ii) it was given once to germ-free animals in order to study the effect of the population of L. casei GG established in the different segments of the gut. In vivo experiments show that after a single challenge with C5, this strain survives and persists at a higher level in the feces of the untreated conventional mice than in those of the treated group. In L. casei GG germ-free mice, establishment of L. casei GG in the gut significantly delayed the occurrence of 100% mortality of the animals (15 days after C5 challenge versus 9 days in germ-free mice [P < 0.01]). Cecal colonization level and translocation rate of C5 to the mesenteric lymph nodes, spleen, and liver were significantly reduced during the first 2 days post-C5 challenge, although the L. casei GG population level in the gut dramatically decreased in these animals.

Animals↗
PubMed1976

Total folate binding capacity of normal human plasma, and variations in uremia, cirrhosis, and pregnancy.

The current study presents evidence that all human serum contains a class of high-affinity folate binders (KA=2.8 X10(10 liters/mole), which migrate as a single peak on gel filtration. Failure of previous studies to detect this characteristic in all but a minority of subjects is attributable to its variable, often total, saturation. Direct measurement of the total folate binding capacity (TFBC) has been made possible by dissociation of endogenous folate-binder complexes at acid pH, removal of free folate by coated charcoal, and radiofolate tagging. This procedure does not appear to significantly denature the binders, which release and rebind similar quantities of 3H-PGA. In 20 normal subjects, TFBC ranged from 100 to 325 pg/ml (mean+/-SE = 174+/-16), and was always at least 33% saturated. In three clinical conditions, all associated with elevated unsaturated folate binding capacity, three different patterns emerged when TFBC was also measured. Uremic subjects had significantly elevated mean TFBC with normal saturation. In cirrhotic subjects, mean TFBC approximated normal, but saturation was significantly decreased. In pregnancy, two groups were seen: one with increased TFBC and the other with a normal TFBC, some of whom had decreased saturation. Lactobacillus casei serum folate level was about 30 times greater than the TFBC; there was no correlation between the two measurements.

Adsorption↗
PubMed1988

Direct experimental evidence for competitive inhibition of dihydrofolate reductase by methotrexate.

Steady-state enzyme kinetic techniques at very low enzyme concentration (0.4 nM) were used successfully to measure the inhibition constant (53 pM) for the dissociation of methotrexate from the ternary complex of methotrexate, NADPH and dihydrofolate reductase from Lactobacillus casei; and to demonstrate unequivocally that the inhibition was, indeed, competitive with respect to dihydrofolate.

Binding, Competitive↗
PubMed2000

Integrative food-grade expression system based on the lactose regulon of Lactobacillus casei.

The lactose operon from Lactobacillus casei is regulated by very tight glucose repression and substrate induction mechanisms, which made it a tempting candidate system for the expression of foreign genes or metabolic engineering. An integrative vector was constructed, allowing stable gene insertion in the chromosomal lactose operon of L. casei. This vector was based on the nonreplicative plasmid pRV300 and contained two DNA fragments corresponding to the 3' end of lacG and the complete lacF gene. Four unique restriction sites were created, as well as a ribosome binding site that would allow the cloning and expression of new genes between these two fragments. Then, integration of the cloned genes into the lactose operon of L. casei could be achieved via homologous recombination in a process that involved two selection steps, which yielded highly stable food-grade mutants. This procedure has been successfully used for the expression of the E. coli gusA gene and the L. lactis ilvBN genes in L. casei. Following the same expression pattern as that for the lactose genes, beta-glucuronidase activity and diacetyl production were repressed by glucose and induced by lactose. This integrative vector represents a useful tool for strain improvement in L. casei that could be applied to engineering fermentation processes or used for expression of genes for clinical and veterinary uses.

Escherichia coli↗
PubMed2006

Essential roles of monocytes in stimulating human peripheral blood mononuclear cells with Lactobacillus casei to produce cytokines and augment natural killer cell activity.

We examined the effect of a probiotic strain, Lactobacillus casei strain Shirota, on cytokine production and natural killer (NK) cell activity in human peripheral blood mononuclear cells (PBMNC). The cellular mechanisms of immunoregulation by L. casei strain Shirota were also investigated. L. casei strain Shirota stimulated PBMNC to secrete interleukin-12 (IL-12), gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and IL-10. However, depletion of monocytes from PBMNC eliminated the induction of these cytokines. L. casei strain Shirota was phagocytosed by monocytes and directly stimulated them to secrete IL-12, TNF-alpha, and IL-10. IFN-gamma production was diminished by the addition of anti-IL-12 antibody to the PBMNC cultures. Purified T cells, but not NK cells, produced IFN-gamma effectively when stimulated with L. casei strain Shirota in the presence of monocytes, indicating that monocytes triggered by L. casei strain Shirota help T cells to produce IFN-gamma through secreting IL-12. In addition, NK cell activity and CD69 expression on NK cells increased after cultivation of PBMNC with L. casei strain Shirota. When monocytes were depleted from PBMNC, L. casei strain Shirota did not enhance NK cell activity. These results demonstrate that monocytes play critical roles in the induction of cytokines and following the augmentation of NK cell activity during the stimulation of human PBMNC with L. casei strain Shirota.

Adult↗
PubMed1982

Cloning and expression of the beta-D-phosphogalactoside galactohydrolase gene of Lactobacillus casei in Escherichia coli K-12.

Lactose metabolism in Lactobacillus casei 64H is associated with the presence of plasmid pLZ64. This plasmid determines both phosphoenolpyruvate-dependent phosphotransferase uptake of lactose and beta-D-phosphogalactoside galactohydrolase. A shotgun clone bank of chimeric plasmids containing restriction enzyme digest fragments of pLZ64 DNA was constructed in Escherichia coli K-12. One clone contained the gene coding for beta-D-phosphogalactoside galactohydrolase on a 7.9-kilobase PstI fragment cloned into the vector pBR322 in E. coli strain chi 1849. The beta-D-phosphogalactoside galactohydrolase enzyme isolated from E. coli showed no difference from that isolated from L. casei, and specific activity of beta-D-phosphogalactoside galactohydrolase was stimulated 1.8-fold in E. coli by growth in media containing beta-galactosides. A restriction map of the recombinant plasmid was compiled, and with that information, a series of subclones was constructed. From an analysis of the proteins produced by minicells prepared from transformant E. coli cells containing each of the recombinant subclone plasmids, it was found that the gene for the 56-kilodalton beta-D-phosphogalactoside galactohydrolase was transcribed from an L. casei-derived promoter. The gene for a second protein product (43 kilodaltons) was transcribed in the opposite direction, presumably under the control of a promoter in pBR322. The relationship of this second product to the lactose metabolism genes of L. casei is at present unknown.

Cloning, Molecular↗
PubMedDate not supplied

Carotenoid production by lactoso-negative yeasts co-cultivated with lactic acid bacteria in whey ultrafiltrate.

Two strains were selected--the lactoso-negative yeast Rhodotorula rubra GED2 and the homofermentative Lactobacillus casei subsp. casei Ha1 for co-cultivation in cheese whey ultrafiltrate (WU) and active synthesis of carotenoids. Under conditions of intensive aeration (1.0 l/l min, 220 rpm), a temperature of 30 degrees C, WU with 55.0 g lactose/l, initial pH = 5.5, the carotenoid content in the cells reached a maximum, when the growth of the cultures had come to an end, i.e. in the stationary phase of the yeast. The maxima for dry cell accumulation (27.0 g/l) and carotenoid formation (12.1 mg/l culture medium) did not coincide on the 5th and 6th day, respectively. A peculiarity of the carotenoid-synthesizing Rh. rubra GED2 strain, co-cultivated with L. casei Ha1, was the production of carotenoids with high beta-carotene content (46.6% of total carotenoids) and 10.7% and 36.9% for torulene and torularhodin, respectively.

Carotenoids↗
PubMedDate not supplied

The effect of supplementation with milk fermented by Lactobacillus casei (strain DN-114 001) on acute diarrhoea in children attending day care centres.

The objective of this study was to determine if supplementation with milk fermented by yogurt cultures and Lactobacillus casei (strain DN-114 001) could lessen acute diarrhoea in healthy children. The study was conducted over six months, with 287 children aged 18.9 (SD 6.0) months, comprising three periods of one month supplementation, each month being followed by one month without supplementation. Subjects were supplemented daily with either 125 g or 250 g (according to age) of one of three tested dairy products: standard yogurt, milk fermented by yogurt cultures and Lactobacillus casei (10(8) cfu/ml), or a jellied milk (control product). A daily record was kept of the number and type of stools. Although the incidence of diarrhoea was not shown to be different between the groups, the severity of diarrhoea over the six-month study was significantly decreased (4.3 days) with the supplementation of L. casei fermented milk compared with the jellied milk (8.0 days) (p = 0.009).

Acute Disease↗