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Addition of tomato juice to skimmed milk stimulated the growth of Lactobacillus acidophilus. It resulted in higher viable counts, shorter generation time and improved sugar utilization with more acid produced and lower pH for the strains tested. Varying results were obtained for the five strains examined.
Chromosomal DNA of nine strains of the Lactobacillus acidophilus (Hansen and Mocquot) group in which heterogeneity had previously been revealed by biochemical characteristics and DNA-DNA hybridization studies were further investigated by restriction analysis, Southern hybridization, conventional and pulsed-field gel electrophoresis (PFGE) analyses. Industrial strains were characterized and identified as Lact. acidophilus. For this species, the number of rRNA gene clusters was estimated to be at least four from analyses of rRNA gene restriction patterns. The chromosome size of type-strains of Lact. acidophilus and Lact. gasseri was estimated from PFGE analysis to be 1.85 and 2.02 Mb respectively and Lact. gasseri strains were found to contain large-sized linear plasmids.
The effect of temperature and growth phase on the autolysis of Lactobacillus acidophilus CRL 640 was studied. The maximal rate of autolytic activity (ca 48% cell lysis) was found at 45 degrees C. At this temperature, two peaks were detected: the first one at the early exponential phase of growth and the second lysis peak during the transition stage from the exponential to the stationary phase. The release of intracellular compounds absorbing at 260 and 280 nm was also detected at 45 degrees C. The microscopic observations revealed morphological changes and the presence of ghost cells. At 37 degrees C, the low autolytic activity obtained would be related to the normal cell cycle of growth.
In order to correlate the lipid composition of the membrane of Lactobacillus acidophilus CRL 640 with the freeze-thaw behaviour of the cultures grown at different temperatures, fatty acid methyl esters (FAMEs) from extracts grown at 25, 30, 37 and 40 degrees C were obtained and compared. Cultures grown at 25 degrees C (M25) exhibited more resistance to the freeze-thaw process probably because of an increase in C18:2 and C16:0 fatty acids. This culture also exhibited a lesser amount of phospholipids as shown by the sugar: phosphorus ratio. In all cases, the presence of the uncommon 10-hydroxyoctadecanoic acid was determined. From the extracts of the M25 and M37 cultures, diacylphosphatidylglycerol, cardiolipin, diglycosyldiglycerides, triglycosyldiglycerides and neutral lipids were isolated and identified. The structural elucidation was carried out by FAMEs and sugar analysis and by mass spectrometry using fast atom bombardment ionization. The changes in lipid composition due to different growth temperatures could be indicative of the resistance of the bacteria to freeze-thaw processes.
The cells of 28 strains of the Lactobacillus acidophilus group were evaluated for hemagglutination (HA) activity. The activity was found in the surface layer (SL) protein fraction extracted by 2 M guanidine hydrochloride. The most SL proteins from the A group strains (L. acidophilus (A1), L. crispatus (A2), L. amylovorus (A3), and L. gallinarum (A4)) showed HA activity, but the proteins from the B group strains (L. gasseri (B1) and L. johnsonii (B2)) showed no activity. The SL proteins from the A group strains were composed in common of a main component having molecular mass of about 40-45 kDa on SDS-PAGE. The SL proteins from JCM 1034 strain that showed the highest HA activity was fractionated by CM-Toyopearl ion-exchange chromatography. The highest HA activity was detected in the major protein of 41 kDa. This protein was purified and shown to be composed of about 50% of hydrophobic amino acids. The HA activity of the protein (1034 lectin) was specifically inhibited by fetuin and bovine lactoferrin at the concentrations of 80 and 160 micrograms/ml, respectively. The removal of N-acetylneuraminic acid from fetuin significantly decreased the inhibitory activity.
Salmonella typhimurium and enteropathogenic Escherichia coli (EPEC) were found to adhere to the brush border of differentiated human intestinal epithelial Caco-2 cells in culture, whereas Yersinia pseudotuberculosis and Listeria monocytogenes adhered to the periphery of undifferentiated Caco-2 cells. All these enterovirulent strains invaded the Caco-2 cells. Using a heat-killed human Lactobacillus acidophilus (strain LB) which strongly adheres both to undifferentiated and differentiated Caco-2 cells, we have studied inhibition of cell association with and invasion within Caco-2 cells by enterovirulent bacteria. Living and heat-killed Lactobacillus acidophilus strain LB inhibited both cell association and invasion of Caco-2 cells by enterovirulent bacteria in a concentration-dependent manner. The mechanism of inhibition of both adhesion and invasion appears to be due to steric hindrance of human enterocytic pathogen receptors by whole-cell lactobacilli rather than to a specific blockade of receptors.
The complete nucleotide sequence of plasmid pLA106 (2862 bp) from Lactobacillus acidophilus TK8912 was determined. Based on this sequence, the location of the genes for mobilization-plasmid recombination protein (mob), replication origin (ori), transcriptional repressor protein (repA) and replication initiation protein (repB) were predicted. Deletion analysis showed that the 1.4-kb PstI-EcoRV fragment carrying the ori, repA and repB genes is able to replicate in Lactobacillus and Escherichia coli cells. The plasmid pLA106 appears to have most features of the pLS1/pE194 plasmid family.
Bacteriocins are natural antimicrobial agents produced by food fermentative bacteria. Lactobacillus acidophilus DSM 20079 produces a small bacteriocin, with a molecular mass of 6.6 kDa, designated acidocin D20079. This antimicrobial peptide was extremely heat-stable (30 min at 121 degrees C) and was active over a wide pH range. It was found to be sensitive to proteolytic enzymes (trypsin, ficin, pepsin, papain, and proteinase K). Acidocin D20079 has a narrow inhibitory spectrum restricted to the genus Lactobacillus which includes L. sakei NCDO 2714, an organism known to cause anaerobic spoilage of vacuum-packaged meat. Maximum production of acidocin D20079 in MRS broth was detected at pH 6.0, and the peptide was purified by ammonium sulphate precipitation followed by sequential cation exchange and hydrophobic interaction chromatography. Purified acidocin D20079 spontaneously formed spherulite crystals during dialysis. As the N-terminus was found to be blocked for sequencing, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used to determine a partial sequence, and the molecular mass of the bacteriocin in the formed crystals (6.6 kDa). Estimates of the molecular weight of the partially purified peptide, using tricine-SDS-PAGE, in which bacteriocin activity was confirmed by overlayer techniques were in accordance with this value.
The influence of Lactobacillus acidophilus and zinc bacitracin alone, or in combination, on the growth of broiler chickens was monitored over a period of 8 weeks. 2. The maximum improvement in body weight over the controls was 10.8% with both additives in the diet but the use of bacitracin alone induced a 9.1% improvement. 3. Food conversion was reduced by zinc bacitracin alone but was improved by the use of L. acidophilus and bacitracin in combination. 4. The combination treatment increased abdominal fat deposition in the female chickens by 31%.
Bile salt hydrolase activity of Lactobacillus acidophilus was measured based on the disappearance of sodium glycocholate and sodium taurocholate from the reaction mixture using HPLC. The amount of sodium glycocholate and sodium taurocholate that disappeared was proportional to the amount of sodium cholate that appeared in the mixture as detected by HPLC. Sodium glycocholate did not precipitate at the enzyme reaction conditions (37 degrees C and pH 5.4) for determining bile salt hydrolase activity. The bile salt hydrolase assay was insensitive to low oxidation-reduction potential when measuring bile salt hydrolase from L. acidophilus, an intestinal microorganism. However, EDTA and freezing temperatures were necessary to maintain stability of the partially purified enzyme during storage.
The aim of the study was to investigate the influence of administration of probiotic bacteria on morphology of the gastrointestinal tract, liver and pancreas. The experiment was performed on 15 piglets at the age from 3 to 35 days, intragastrically administered with Bifidobacterium breve, B. animalis, and Lactobacillus acidophilus bacteria. In all piglets examined, the development of the gastrointestinal tract, liver and pancreas was observed to progress normally. After microflora administration to the piglets, an increase in the number of fibrocytes and fibroblasts was observed in the mucosa lamina propria of stomach and intestines. An increase was also reported in the number and activation of endocrine cells in the stomach and small intestines. The activity of alkaline and acid phosphatases, as well as succinic (SDH) and lactic (LDH) dehydrogenases, was found to be higher after the administration of probiotics. The administration of bacteria, especially of Lactobacillus acidophilus, caused an increase in the number of lymphocytes and lymphoid cells in lamina propria and intraepithelial lymphocytes in the small intestine. Enhanced proliferation of crypt cells was observed in the crypts of intestinal glands; however, there were no statistically significant differences in the PCNA index between the control and probiotic-administered groups. The performed study showed that the administration of probiotic bacteria has no negative impact on the morphology of the gastrointestinal tract, liver and pancreas and is found beneficial to its functioning and immune processes.
Four DNA probes were derived that hybridized specifically to DNA from Lactobacillus acidophilus O. The probes were constructed by randomly cloning lactobacillus DNA in plasmid vector pBR322. Two of the probes (pSR1 and pSR2) were composed of vector and plasmid DNA inserts (3.6 and 1.6 kb, respectively); the others (pSR3 and pSR4) were composed of vector and chromosomally derived inserts (6.9 and 1.4 kb, respectively). The probes were used to enumerate, by colony hybridization, strain O in digestive tract samples collected from piglets inoculated 24 hours previously with a culture of the strain. The probes did not hybridize to DNA from lactobacilli inhabiting the digestive tract of uninoculated piglets. Strain O made up about 10% of the total lactobacillus population of the pars esophagea and about 20% of the population in other digestive tract samples.
We have characterized three of at least five plasmids borne by Lactobacillus acidophilus 168 S. Restriction mapping indicates extensive sequence homology between at least two of them (p1 and p3). We have cloned them in Escherichia coli, and for the smallest (p1) we present the sequence of a region with two divergently arranged promoters which probably share a symmetrical (TTTAAA)-35 box and function efficiently in E. coli cells; an open reading frame contiguous to the promoter, which codes for a 120 amino acid protein of unknown function, and is transcribed in E. coli; and a transcription termination sequence next to this open reading frame. The promoter region contains an AT cluster which is similar to that of the ori2 region of the E. coli F plasmid, and is probably involved in the control of the replication of p1.
We reported previously that, when exposed to high osmotic pressure, Lactobacillus acidophilus IFO 3532 cells accumulated N,N,N-trimethylglycine (glycine betaine), which serves as a compatible intracellular solute. When grown in medium with high osmotic pressure, these cells also accumulated one amino acid, proline. The uptake of [3H]proline by resting, glucose-energized cells was stimulated by increasing the osmotic pressure of the assay medium with 0.5 to 1.0 M KCl, 1.0 M NaCl, or 0.5 M sucrose. The accumulated [3H]proline was not metabolized further. In contrast, there was no osmotic stimulation of [3H]leucine uptake. The uptake of proline was activated rather than induced by exposure of the cells to high osmotic pressure. Only one proline transport system could be discerned from kinetics plots. The affinity of the carrier for proline remained constant over a range of osmotic pressures from 650 to 1,910 mosM (Kt, 7.8 to 15.5 mM). The Vmax, however, increased from 15 nmol/min/mg of dry weight in 0.5 M sucrose to 27 and 40 nmol/min/mg of dry weight in 0.5 M KCl and in 1.0 M KCl or NaCl, respectively. The efflux of proline from preloaded cells occurred rapidly when the osmotic pressure of the suspending buffer was lowered.
Exponentially growing cultures of Lactobacillus acidophilus strain 60AM Gasser were previously shown to lose about one-third of their cell wall peptidoglycan per generation via turnover (Boothby, D., Daneo-Moore, L., Higgins, M. L., Coyette, J., and Shockman, G. D. (1973) J. Biol. Chem. 248, 2161-2169). We now show that 20 to 30% of the [3H]lysine initially present in insoluble peptidoglycan fractions was retained after 4 or more generations of continued exponential growth of cultures in the absence of label. Treatment of peptidoglycan fractions, before and after 6 or 8 generations of chase with lysozyme (EC 3.2.1.17), released soluble products containing [3H]lysine which had electrophoretic mobilities identical with the disaccharide-peptide derivatives obtained from the wall peptidoglycan of this species. Because protein is known to contaminate peptidoglycan residues, the double labeled technique was used to show that one-half or less of the label lysine present after 6 or 8 generations of chase could be attributed to protein contamination. This then left a minimum fraction of 10 to 20% of the peptidoglycan that was immune to turnover. The absence of turnover of peptidoglycan labeled during short pulses has now been quantitated to show that pulses shorter than 12% of a generation (6 to 7 min) did not turn over. This turnover-immune fraction is in reasonably good agreement with the immune fraction of 10 to 20% observed after long periods of chase of extensively labeled peptidoglycan.
Our previous report showed that supernatants of Lactobacillus acidophilus (LS) cultures possessed chemotactic and angiogenic properties. Specifically, LS stimulated gene expression and the secretion of tumor necrosis factor-alpha (TNF-alpha), the proliferation of immune cells in vitro, and blood vessel formation. Chemotaxis and proliferation of inflammatory cells in vivo were also stimulated by LS. In the current study, we hypothesized that LS stimulates the growth and development of other rapidly dividing cells, including embryonic cells. The stimulatory effects of LS on a neuroblastoma cell line (Neuro-2a), chicken embryos, and bovine embryos were examined. The addition of LS to Neuro-2a cultures caused a proliferation of cells in a concentration-dependent manner. Pretreatment of LS at 56 degrees C for 30 mins did not affect its stimulatory activity. The administration of LS to the chorioallantoic membrane (CAM) of chicken-embryonated eggs for 1-2 days resulted in extensive thickening of the membrane. The thickening was due to the influx and proliferation of fibroblasts and inflammatory cells, the accumulation of loose connective tissue composed primarily of mucopolysaccharides, and/or the formation of blood vessels. Stimulatory effects of LS on bovine embryos were also observed. The treatment with LS significantly promoted the development of zygotes to the four-cell stage and from the four-cell stage to blastocysts. These results have confirmed our hypothesis that LS exerts a stimulatory effect on the cells of embryonic stages including neuroblastoma cells, the CAM of chicken embryos, and bovine embryos from zygotes to blastocysts.
OBJECTIVE: To evaluate the biopotency of the viable probiotic Lactobacillus acidophilus (La1) in yoghurt matrix consumed by Egyptian children on their plasma vitamin B12 and folate levels, and their metabolic markers methylmalonic acid (MMA) and total homocysteine (t-Hcy). METHODS: A randomized nutritional supplementation trial (42 days duration) was performed in free-living children of both sexes (11 years old). The La1 in yoghurt matrix was administered to provide 1012 colony-forming units/subject/day. Blood sampling for the analysis of plasma vitamin B12, folate and t-Hcy was performed by standardized methods. Five-hour urine collection was used for the analysis of MMA and t-Hcy. RESULTS: Initially 33.3% of the children presented with biochemical vitamin B12 deficiency (<208 pg/ml), while one-fifth (21%) were biochemically deficient in folate (<3 ng/ml folate/ml plasma or 0.68 nmol/l). Fifty percent of the children presented with high plasma t-Hcy (>15.0 micromol/l). The daily consumption of the probiotic La1 yoghurt for 42 days significantly improved the mean levels of plasma vitamin B12 (P<0.05) and folate (P<0.01) among the studied children compared with the respective baseline data. On the other hand, the average levels of plasma t-Hcy and urinary MMA decreased significantly (P<0.05) at the termination of the 42-day nutritional supplementation, compared with the respective initial mean levels. The consumption of the probiotic yoghurt was also associated with a significant (chi2=8.0; P<0.01) reduction in the percentage prevalence of anemia (hemoglobin <120 g/l). CONCLUSION: The long-term ingestion of viable probiotic La1 potentially promoted the overall nutritional status of the studied children.
BACKGROUND: Specific strains of Lactobacillus acidophilus are known to inhibit intestinal cell adhesion and invasion by enterovirulent bacteria. As L. acidophilus can survive transiently in the human stomach, it may downregulate Helicobacter pylori infection. METHODS: The ability of L. acidophilus (johnsonii) La1 supernatant to interfere with H. pylori bacterial growth, urease activity, and adhesion to epithelial cells was tested in vitro. Its effect on H. pylori infection in volunteers was monitored in a randomized, double-blind, controlled clinical trial, using a drinkable, whey-based, La1 culture supernatant. H. pylori infected volunteers were treated 14 days with 50 ml of La1 supernatant four times a day combined with either omeprazole 20 mg four times a day or with placebo. Infection was assessed by breath test, endoscopy, and biopsy sampling, performed at inclusion, immediately at the end of the treatment (breath test only), and 4 weeks after the end of the treatment. RESULTS: La1 supernatant inhibited H. pylori growth in vitro, regardless of previous binding of H. pylori to epithelial cells. In 20 subjects (8 females, 12 males, mean age 33.1 years) a marked decrease in breath test values was observed immediately after treatment with La1 supernatant, both in the omeprazole and in the placebo group (median 12.3 vs. 28.8 and 9.4 vs. 20.4, respectively; p < 0.03). In both treatment groups, breath test values remained low 6 weeks after treatment (omeprazole treated 19.2, placebo treated 8. 3; p < 0.03 vs. pretreatment), but the persistence of H. pylori infection was confirmed in gastric biopsies. CONCLUSION: La1 culture supernatant shown to be effective in vitro has a partial, acid-independent long-term suppressive effect on H. pylori in humans.