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PubMed2007

Characterization and molecular cloning of a heterodimeric beta-galactosidase from the probiotic strain Lactobacillus acidophilus R22.

Beta-galactosidase from the probiotic strain Lactobacillus acidophilus R22 was purified to apparent homogeneity by ammonium sulphate fractionation, hydrophobic interaction, and affinity chromatography. The enzyme is a heterodimer consisting of two subunits of 35 and 72 kDa, as determined by gel electrophoresis. The optimum temperature of beta-galactosidase activity was 55 degrees C (10-min assay) and the range of pH 6.5-8, respectively, for both o-nitrophenyl-beta-D-galactopyranoside (oNPG) and lactose hydrolysis. The Km and Vmax values for lactose and oNPG were 4.04+/-0.26 mM, 28.8+/-0.2 micromol D-glucose released per min per mg protein, and 0.73+/-0.07 mM, 361+/-12 micromol o-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of oNPG with Ki,s=31.7+/-3.5 mM. The enzyme showed no specific requirements for metal ions, with the exception of Mg2+, which enhanced both activity and stability. The genes encoding this heterodimeric enzyme, lacL and lacM, were cloned, and compared with other beta-galactosidases from lactobacilli. Beta-galactosidase from L. acidophilus was used for the synthesis of prebiotic galacto-oligosaccharides (GOS) from lactose, with the maximum GOS yield of 38.5% of total sugars at about 75% lactose conversion.

Bacterial Proteins↗

PubMed1995

Cloning and nucleotide sequence of the gene for acidocin 8912, a bacteriocin from Lactobacillus acidophilus TK8912.

Acidocin 8912 is a bacteriocin produced by Lactobacillus acidophilus TK8912. The acidocin 8912 structural gene, acdT, was cloned and determined. It was located on the 14-kb plasmid pL103 and encoded a 46 amino acid precursor including a 20 amino acid N-terminal extension. The precursor sequence of the acdT gene shows a conservation of the general structural characteristics of the bacteriocin precursors from some lactic acid bacteria.

Amino Acid Sequence↗

PubMed1975

N-6-(delta-2-isopentenyl) adenosine: hydrolysis by a mucleosidase isolated from Lactobacillus acidophilus cells.

A nucleosidase activity has been isolated from Lactobacillus acidophilus which rapidly hydrolyses N-6 (delta-2-isopentenyl) adenosine to its corresponding base, N-6(delta-2-isopentenyl) adenine. The activity can be distinguished from the spleen exzyme (EC. 2.4.2.1), a purine nucleoside transferase, on the basis of its substrate specificity, electrophoretic behavior, and nondependence on phosphate. The bacterial enzyme hydrolyzes both inosine and isopentenyl adenosine, giving Km values of 63.3muM and 177 muM respectively. The presence of this enzyme in bacteria counts for the rapid conversion of the parent nucleoside to isopentenyl adenine, which has been observed in these cells. The enzyme thus assumes importance as one of the catabolic activities available to the cell for metabolizing the cytokinin, N-6-(delta-2-isopentenyl) adenosine.

Adenosine↗

PubMed2001

[Isolation and identification of the broad spectrum antimicrobial peptide AP311 produced by Lactobacillus acidophilus].

The heat stable antimicrobial peptide (AP311) produced by Lactobacillus acidophilus was isolated and identified. The AP311 has broad spectrum of inhibition including many Gram-positive and Gram-negative bacteria. The inhibition activity of AP311 was lost upon treatment with trypsin, subtilisin, proteanase K, chymotrypsin and pepsin. Inhibition activity of AP311 was decreased with pH increasing. The AP311 is very stable at acidic condition (pH2-4) even if heating at 100 degrees C for 30 min. But it was inactivated at basic pH (pH12) and the activity was restored completely upon reversion to acidic. The AP311 was not precipitated and inactivated by various organic solvents, except n-butanol. When the precipitate was redissolved in 0.02 mol/L HC1, its activity was restored. Based on its proteinaceous nature, broad spectrum of inhibition, we propose that AP311 should be considered the broad spectrum antimicrobial peptide.

Anti-Bacterial Agents↗

PubMed1973

Sites of cellular autolysis in Lactobacillus acidophilus.

Ultrastructural changes which occur during cellular autolysis of Lactobacillus acidophilus strain 63AM Gasser in 0.05 M citrate buffer, pH 5.0, were examined. Early in the process, randomly distributed electron-dense patches were seen on the wall surface, along with an accompanying eversion of mesosomes. Later, after a loss of about 20% of the initial cellular turbidity, dissolution from the outside of nascent cross walls was seen. This observation was related to the normal process of cell separation. After this stage, short lengths of the cylindrical portion of the wall appeared to be completely removed in a random manner over the entire surface. This dissolution produced gaps in the wall which allowed the extrusion of membrane and cytoplasm. Although membrane was usually extruded through one major, polar, subpolar, or septal site, other secondary points of membrane extrusion were also frequently seen in the same cell section.

Autolysis↗

PubMed2005

Optimization of cholesterol removal, growth and fermentation patterns of Lactobacillus acidophilus ATCC 4962 in the presence of mannitol, fructo-oligosaccharide and inulin: a response surface methodology approach.

AIMS: To optimize cholesterol removal by Lactobacillus acidophilus ATCC 4962 in the presence of prebiotics, and study the growth and fermentation patterns of the prebiotics. METHODS AND RESULTS: Lactobacillus acidophilus ATCC 4962 was screened in the presence of six prebiotics, namely sorbitol, mannitol, maltodextrin, hi-amylose maize, fructo-oligosaccharide (FOS) and inulin in order to determine the best combination for highest level of cholesterol removal. The first-order model showed that the combination of inoculum size, mannitol, FOS and inulin was best for removal of cholesterol. The second-order polynomial regression model estimated the optimum condition of the factors for cholesterol removal by L. acidophilus ATCC 4962 to be 2.64% w/v inoculum size, 4.13% w/v mannitol, 3.29% w/v FOS and 5.81% w/v inulin. Analyses of growth, mean doubling time and short-chain fatty acid (SCFA) production using quadratic models indicated that cholesterol removal and the production of SCFA were growth associated. CONCLUSIONS: Optimum cholesterol removal was obtained from the fermentation of L. acidophilus ATCC 4962 in the presence of mannitol, FOS and inulin. Cholesterol removal and the production of SCFA appeared to be growth associated and highly influenced by the prebiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: Response surface methodology proved reliable in developing the model, optimizing factors and analysing interaction effects. The results provide better understanding on the interactions between probiotic and prebiotics for the removal of cholesterol.

Cholesterol↗

PubMed2000

Survival and therapeutic potential of probiotic organisms with reference to Lactobacillus acidophilus and Bifidobacterium spp.

The present paper provides an overview on the use of probiotic organisms as live supplements, with particular emphasis on Lactobacillus acidophilus and Bifidobacterium spp. The therapeutic potential of these bacteria in fermented dairy products is dependent on their survival during manufacture and storage. Probiotic bacteria are increasingly used in food and pharmaceutical applications to balance disturbed intestinal microflora and related dysfunction of the human gastrointestinal tract. Lactobacillus acidophilus and Bifidobacterium spp. have been reported to be beneficial probiotic organisms that provide excellent therapeutic benefits. The biological activity of probiotic bacteria is due in part to their ability to attach to enterocytes. This inhibits the binding of enteric pathogens by a process of competitive exclusion. Attachment of probiotic bacteria to cell surface receptors of enterocytes also initiates signalling events that result in the synthesis of cytokines. Probiotic bacteria also exert an influence on commensal micro-organisms by the production of lactic acid and bacteriocins. These substances inhibit growth of pathogens and also alter the ecological balance of enteric commensals. Production of butyric acid by some probiotic bacteria affects the turnover of enterocytes and neutralizes the activity of dietary carcinogens, such as nitrosamines, that are generated by the metabolic activity of commensal bacteria in subjects consuming a high-protein diet. Therefore, inclusion of probiotic bacteria in fermented dairy products enhances their value as better therapeutic functional foods. However, insufficient viability and survival of these bacteria remain a problem in commercial food products. By selecting better functional probiotic strains and adopting improved methods to enhance survival, including the use of appropriate prebiotics and the optimal combination of probiotics and prebiotics (synbiotics), an increased delivery of viable bacteria in fermented products to the consumers can be achieved.

Adjuvants, Immunologic↗

PubMed1990

Utilization of sugars by Lactobacillus acidophilus strains.

Utilization of various carbohydrates viz., glucose, fructose, sucrose, lactose and galactose by Lactobacillus acidophilus strains was investigated in Lactobacillus Selection Broth. Maximum viable counts, acid production and sugar utilization by different test strains were in the order: glucose greater than or equal to fructose greater than sucrose greater than or equal to lactose greater than galactose. The generation time of the tested strains was shorter in glucose medium as compared to sucrose or lactose medium.

Animals↗

PubMed1998

Use of small ruminants' milk supplemented with available nitrogen as growth media for Bifidobacterium lactis and Lactobacillus acidophilus.

Growth of, and acid production by Bifidobacterium lactis and Lactobacillus acidophilus using ovine and caprine milk as media were evaluated for their potential use in cheese-making. A protein hydrolysate (MHP, obtained from incubation of bovine milk with protease) or a mixture of free amino acids (FAA, similar to the amino acid fraction of MHP) was added as a nitrogen enrichment source. Bifidobacterium lactis and Lact. acidophilus were inoculated at 50 ml l-1 and incubated at 37 degrees C with growth supplements added at ratios in the range 25-50 ml l-1. The maximum viable counts of Bif. lactis were lower in plain ovine and caprine milk than in nitrogen-enriched milk, and MHP was a better growth promoter than FAA. A similar trend was observed with the acidity values developed, and attempts to correlate growth with acidity were successfully performed. The highest uptake rates of amino acids in ovine milk were observed for lysine, isoleucine, leucine and proline, but only isoleucine was taken up at a similar rate in caprine milk. Final bacterial viable counts of Lact. acidophilus in the plain and enriched forms of ovine milk did not differ greatly from each other, although FAA was statistically a better growth promoter than MHP. Unlike results in ovine milk, cultures of Lact. acidophilus in caprine milk exhibited drops of 1-1.5 log cycles in viable cell counts by 24 h of fermentation, irrespective of the nature of the nitrogen source. Parallel studies indicated that the excess of fatty acid residues in caprine milk could be responsible for the poor growth of Lact. acidophilus.

Amino Acids↗

PubMed1982

Commensalistic Interaction Between Lactobacillus acidophilus and Propionibacterium shermanii.

Propionibacterium shermanii and Lactobacillus acidophilus were grown in batch mixed culture in a 5-liter fermenter under controlled conditions of pH 5.8 and 35 degrees C on a semisynthetic medium with glucose as an energy source. Cellular efficiencies and fermentation balances were developed for this pair and compared with P. shermanii grown in pure culture on glucose, lactate, and a mixture of these substrates and with L. acidophilus grown on glucose. P. shermanii had ATP yield coefficient values of 17 for each substrate alone but had an average value of 30 for substrate mixtures. Growth rates were similar for P. shermanii on glucose or lactate but higher cell yields were observed for glucose. P. shermanii used both lactate and glucose in mixed substrate until lactate was exhausted, and growth rates slowed thereafter. L. acidophilus had a similar ATP yield coefficient of 15 but produced lower cell yields than did P. shermanii on glucose. Mixed culture of both microorganisms on glucose resulted in much faster and nearly equal growth rates for both and no lactate accumulation in the medium. Acetic acid production rates per generation were lower in mixed culture, suggesting use by the growing culture. The cause of the synergistic effect was not determined but may be due to the rapid production and removal of lactate or CO(2) enhancement in mixed culture.

Journal Article↗

PubMed1988

Influence of Storage at Freezing and Subsequent Refrigeration Temperatures on beta-Galactosidase Activity of Lactobacillus acidophilus.

The ability of three strains of Lactobacillus acidophilus to survive and retain beta-galactosidase activity during storage in liquid nitrogen at -196 degrees C and during subsequent storage in milk at 5 degrees C was tested. The level of beta-galactosidase activity varied among the three strains (0.048 to 0.177 U/10 organisms). Freezing and storage at -196 degrees C had much less adverse influence on viability and activity of the enzyme than did storage in milk at 5 degrees C. The strains varied in the extent of the losses of viability and beta-galactosidase activity during both types of storage. There was not a significant interaction between storage at -196 degrees C and subsequent storage at 5 degrees C. The strains that exhibited the greatest losses of beta-galactosidase activity during storage in milk at 5 degrees C also exhibited the greatest losses in viability at 5 degrees C. However, the losses in viability were of much greater magnitude than were the losses of enzymatic activity. This indicates that some cells of L. acidophilus which failed to form colonies on the enumeration medium still possessed beta-galactosidase activity. Cultures of L. acidophilus to be used as dietary adjuncts to improve lactose utilization in humans should be carefully selected to ensure that adequate beta-galactosidase activity is provided.

Journal Article↗

PubMed1999

Inhibition of lipid peroxidation by Lactobacillus acidophilus and Bifidobacterium longum.

The inhibition of lipid peroxidation by Lactobacillus acidophilus and Bifidobacterium longum was investigated using two lipid model systems. All eight strains, including six strains of L. acidophilus and two strains of B. longum, demonstrated an inhibitory effect on linoleic acid peroxidation. The inhibitory rates on linoleic acid peroxidation ranged from 33 to 46% when 1 mL of intracellular cell-free extract was tested. In the second model system, the cell membrane of osteoblast was used as the source for biological lipid. The results indicated that all strains were able to protect biological lipids from oxidation. The inhibition rates on cell membrane lipid peroxidation ranged from 22 to 37%. The effect of L. acidophilus and B. longum on inhibition of fluorescent tissue pigment accumulation was also obtained for osteoblastic cells. The inhibition rates on fluorescent tissue pigment accumulation ranged from 20 to 39%. The antioxidative effect of each milliliter of intracellular cell-free extract of L. acidophilus and B. longum was equivalent to 104-172 ppm of butylated hydroxytoluene (BHT). These results indicated that all strains demonstrated high antioxidative activity. The scavenging ability of lipid peroxidation products, tert-butyl hydroperoxide and malondialdehyde, was also evaluated. The results showed that L. acidophilus and B. longum were not able to scavenge the tert-butyl hydroperoxide. Nevertheless, malondialdehyde was scavenged well by these strains.

Animals↗

PubMed2000

Changes in the surface potential of Lactobacillus acidophilus under freeze-thawing stress.

The zeta potential of Lactobacillus acidophilus CRL 640, a measure of the net distribution of electrical charges on the bacterial surface, is a function of the glucose concentration in the growing media. With 2% glucose, cells in the stationary phase showed a zeta potential of -45 +/- 2 mV. With these cells, the zeta potential after freezing and thawing decreased to -32 +/- 2 mV and there was a decrease in viability. The changes in the surface potential correlated with damage to the cell surface as shown by electron microscopy. Freeze-thawed cells incubated in a rich medium recovered a zeta potential of -38 +/- 2 mV without cell growth. L. acidophilus CRL 640 showed the same value of surface potential as control cells when they were frozen and thawed in 2 M glycerol.

Cell Membrane↗

PubMed1996

The presence of two S-layer-protein-encoding genes is conserved among species related to Lactobacillus acidophilus.

Previously we have shown that the type strain of Lactobacillus acidophilus possesses two S-protein-encoding genes, one of which is silent, on a chromosomal segment of 6 kb. The S-protein-encoding gene in the expression site can be exchanged for the silent S-protein-encoding gene by inversion of this slp segment. In this study the presence of S-protein and corresponding S-protein-encoding genes of strains belonging to species that are closely related to L. acidophilus was determined. All strains investigated were identified by numerical comparison of highly standardized one-dimensional SDS-PAGE whole-cellular-protein patterns. Western blot and Southern blot methods were used to identify the presence of, and homology between, S-proteins and S-protein-encoding genes. From these analyses we conclude that strains of L. acidophilus, L. crispatus, L. amylovorus and L. gallinarum possess an S-layer and contain two slp genes. Strains of L. helveticus possess an S-layer but have only one intact slp gene. Strains of L. gasseri, L. johnsonii and L. delbrueckii subsp. bulgaricus have neither an S-layer nor S-protein-encoding genes hybridizing with probes derived from the L. acidophilus slpA or slpB region. The presence of a highly conserved 5' region in the slp genes of strains of L. acidophilus, L. crispatus, L. amylovorus and L. gallinarum suggests that S-layer variation is a common feature for strains of these species.

Bacterial Proteins↗

PubMed1966

Inhibitory effect of saliva on glutamic acid accumulation by Lactobacillus acidophilus and the role of the lactoperoxidase-thiocyanate system.

Clem, W. H. (University of Washington, Seattle), and S. J. Klebanoff. Inhibitory effect of saliva on glutamic acid accumulation by Lactobacillus acidophilus and the role of the lactoperoxidase-thiocyanate system. J. Bacteriol. 91:1848-1853. 1966.-Saliva contains an antimicrobial system which inhibits the growth of Lactobacillus acidophilus, as well as a number of other organisms, in complete growth medium. This antimicrobial system consists of the salivary peroxidase (lactoperoxidase) and thiocyanate ions, and requires the presence of H(2)O(2). Saliva inhibits the accumulation of glutamic acid and certain other amino acids by resting cells. This effect of saliva is decreased by dialysis, and thiocyanate ions restore the inhibitory effect of dialyzed saliva. The inhibitory effect of saliva is decreased by heat (100 C, 10 min), and lactoperoxidase restores the inhibitory effect of heated saliva. Thus, the inhibition of glutamic acid accumulation by saliva appears to be due in part to the lactoperoxidase-thiocyanate antimicrobial system. H(2)O(2) increases the inhibitory effect of both saliva and the lactoperoxidase-thiocyanate system on glutamic acid accumulation. The inhibition of glutamic acid accumulation is not preceded by a loss in microbial viability. The glutamic acid accumulated by L. acidophilus under the conditions employed remains largely (over 90%) as free glutamic acid. This suggests that saliva and the lactoperoxidase-thiocyanate-H(2)O(2) system inhibit the net transport of glutamic acid into the cell.

Carbon Isotopes↗

PubMed1995

Survival during frozen and subsequent refrigerated storage of Lactobacillus acidophilus cells as influenced by the growth phase.

Cells of Lactobacillus acidophilus propagated in peptonized milk nutrient broth maintained at pH 5 were harvested during the late logarithmic (log) phase and at 6 h into the stationary phase of growth. Concentrated cultures were prepared from each age of cells, frozen, and stored at -196 degrees C. The concentrated cultures were assayed for numbers of total and bile-tolerant lactobacilli, beta-galactosidase activity, and ability to assimilate cholesterol before and after storage at -196 degrees C. Nonfermented acidophilus milk, prepared following 28 d of storage at -196 degrees C from each concentrated culture, was tested before and after 0, 7, 14, 21, and 28 d of storage at 7 degrees C. No decrease in numbers of total or bile-tolerant lactobacilli, beta-galactosidase activity, or amount of cholesterol assimilated was observed for any of three strains studied during storage at -196 degrees C. There were no significant differences between the two harvest times during storage at this temperature for any of the three strains. However, when the cells were suspended in milk and stored at 7 degrees C, the numbers of total and bile-tolerant lactobacilli declined over time as did the beta-galactosidase activity and the ability to assimilate cholesterol. The cells from L. acidophilus 223, harvested 6 h into the stationary phase, exhibited significantly less decrease in total numbers during extended storage at 7 degrees C than did the cells harvested in the late logarithmic phase. There was little or no difference in the decline in numbers of total lactobacilli between the two harvest times for strains 606 and 107. All strains decreased significantly in beta-galactosidase activity and the ability to assimilate cholesterol during storage in milk at 7 degrees C with no significant differences between the two harvest times.

Animals↗

PubMed1970

Effect of actinomycin D on the transfer of ribonucleic acid from nucleus to cytoplasm in Lactobacillus acidophilus.

After starvation for deoxyribosides, the deoxyribonucleic acid (DNA) of Lactobacillus acidophilus is restricted to a localized region of the cell. (3)H-uracil is first incorporated into such a restricted region but subsequently is found throughout the cell. This spread occurs despite the absence of protein synthesis and a major reduction in the rate of ribonucleic acid (RNA) synthesis. However, blocking RNA synthesis with actinomycin D restricts incorporation to a localized region of the cell. It is concluded that uracil is first incorporated into RNA in the bacterial nucleus from which it subsequently spreads through the cell. Actinomycin D could prevent this spread by preventing the completion of RNA molecules, which therefore do not dissociate from the DNA template.

Autoradiography↗

PubMed1997

Antibacterial effect of the adhering human Lactobacillus acidophilus strain LB.

The spent culture supernatant of the human Lactobacillus acidophilus strain LB produces an antibacterial activity against a wide range of gram-negative and gram-positive pathogens. It decreased the in vitro viability of Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Shigella flexneri, Escherichia coli, Klebsiella pneumoniae, Bacillus cereus, Pseudomonas aeruginosa, and Enterobacter spp. In contrast, it did not inhibit lactobacilli and bifidobacteria. The activity was heat stable and relatively sensitive to enzymatic treatments and developed under acidic conditions. The antimicrobial activity was independent of lactic acid production. Activity against S. typhimurium SL1344 infecting human cultured intestinal Caco-2 cells was observed as it was in the conventional C3H/He/oujco mouse model with S. typhimurium C5 infection and oral treatment with the LB spent culture supernatant.

Animals↗