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PubMed2005

Construction of a new reporter system to study the NaCl-dependent dnaK promoter activity of Lactobacillus sanfranciscensis.

A reporter system was developed to study gene expression in Lactobacillus sanfranciscensis. It was based on the Escherichia coli/Lactobacillus shuttle vector pLP3537 and the melA gene encoding alpha-galactosidase originating from Lactobacillus plantarum. melA was functionally expressed in E. coli and L. sanfranciscensis, and activity was easily monitored in vivo as well as in vitro by applying an optimized enzyme assay. The reporter system was validated by demonstrating the induction of the dnaK operon of L. sanfranciscensis by NaCl stress. The complete operon, which was composed of hrcA, grpE, dnaK, and dnaJ, was sequenced. A 299-bp sequence upstream of this operon, including a putative sigmaA-type promoter and a single conserved Controlling Inverted Repeat of Chaperone Expression element, was amplified. This amplicon was cloned directly upstream of melA. Both reporter enzyme activity and Northern hybridization analyses of dnaK and melA revealed a transcriptional induction, reaching its maximum when the culture was exposed to 0.75 M NaCl.

Base Sequence↗
PubMed2006

Improvement of protein production in lactic acid bacteria using 5'-untranslated leader sequence of slpA from Lactobacillus acidophilus. Improvement in protein production using UTLS.

The 5'-untranslated leader sequence (UTLS) of the slpA gene from Lactobacillus acidophilus contributes to mRNA stabilization by producing a 5' stem and loop structure, and a high-level expression system for the lactic acid bacteria was developed using the UTLS in this study. A plasmid, which expresses alpha-amylase under the control of the ldh promoter, was constructed by integrating the core promoter sequence with the UTLS. The role of the UTLS in increasing the copies of the alpha-amylase mRNA was proved by measuring alpha-amylase activity in the culture supernatant and the relative expression of alpha-amylase mRNA was determined by the quantitative real-time PCR analysis. Moreover, several expression systems were constructed by combining the core promoter sequence with the UTLS or with the partially deleted UTLS and the expression level was evaluated. The use of the UTLS led to the success in improving alpha-amylase expression in the two strains of Lactobacillus casei and Lactococcus lactis. The current study showed that the improvement in protein production using the UTLS could be applied to the expression system in the lactic acid bacteria.

5' Untranslated Regions↗
PubMed2006

Effective cellulose production by a coculture of Gluconacetobacter xylinus and Lactobacillus mali.

A microbial colony that contained a marked amount of cellulose was isolated from vineyard soil. The colony was formed by the associated growth of two bacterial strains: a cellulose-producing acetic acid bacterium (st-60-12) and a lactic acid bacterium (st-20). The 16S rDNA-based taxonomy indicated that st-60-12 belonged to Gluconacetobacter xylinus and st-20 was closely related to Lactobacillus mali. Cocultivation of the two organisms in corn steep liquor/sucrose liquid medium resulted in a threefold higher cellulose yield when compared to the st-60-12 monoculture. A similar enhancement was observed in a coculture with various L. mali strains but not with other Lactobacillus spp. The enhancement of cellulose production was most remarkable when sucrose was supplied as the substrate. L. mali mutants for exocellular polysaccharide (EPS) production were defective in promoting cellulose production, but the addition of EPS to the monoculture of st-60-12 did not affect cellulose productivity. Scanning electron microscopic observation of the coculture revealed frequent association between the st-60-12 and L. mali cells. These results indicate that cell-cell interaction assisted by the EPS-producing L. mali promotes cellulose production in st-60-12.

Cellulose↗
PubMed2000

Temperature-dependent variation in API 50 CH fermentation profiles of Lactobacillus species.

API 50 CH fermentation profiles of 45 Lactobacillus, one Atopobium, and three Weissella strains incubated at 30 degrees C and 37 degrees C were evaluated. Atopobium uli and ten species of Lactobacillus showed stable patterns despite the change in temperature. The rest of the type strains showed discrepancy between the two incubation temperatures: 18 strains lost, 12 additionally fermented another sugar, and 7 others fermented a different one in lieu. The variation was maximum in L. delbrueckii subsp. delbrueckii. L. malefermentans failed to ferment any of the substrates at 37 degrees C. Majority of the food and plant-associated strains (mainly heterofermenters) retained distinctive traits at 30 degrees C, while most of the animal-associated strains (mostly homofermenters) did so at 37 degrees C. No general trend was observed; 30 degrees C appeared to promote heterofermentation, while 37 degrees C favored homofermenters. Use of API 50 CH profiles for taxonomic purpose in most lactobacilli appears reproducible if a specific temperature for a species is strictly followed.

Fermentation↗
PubMed1997

Purification and partial amino acid sequence of brevicin 27, a bacteriocin produced by Lactobacillus brevis SB27.

Brevicin 27, a bacteriocin produced by Lacto bacillus brevis SB27, is inhibitory mainly against closely related Lactobacillus brevis and Lactobacillus büchneri strains. It was purified from the culture supernatant by a four-step purification procedure including ammonium sulfate precipitation, cation exchange, hydrophobic interaction, and reverse-phase, high performance liquid chromatographies. The purified bacteriocin was subjected to mass spectrometry, amino acid composition analysis, and sequencing by Edman degradation. It was shown to be an about 5200-Da basic protein containing a high proportion of lysine and of hydrophobic amino acids. The partial N-terminal amino acid sequence (25 residues) was unique when compared with the Protein Data Bank (PDB), Swiss Prot, and Protein Information Resource(PIR) data banks and to the translated Gen Bank.

Amino Acid Sequence↗
PubMed2000

Purification and characterization of invertase from Lactobacillus reuteri CRL 1100.

The invertase of Lactobacillus reuteri CRL 1100 is a glycoprotein composed by a single subunit with a molecular weight of 58 kDa. The enzyme was stable below 45 degrees C over a wide pH range (4.5-7.0) with maximum activity at pH 6.0 and 37 degrees C. The invertase activity was significantly inhibited by bivalent metal ions (Ca(++), Cu(++), Cd(++), and Hg(++)), beta-mercaptoethanol, and dithiothreitol and partially improved by ethylenediaminetetraacetic acid. The enzyme was purified 32 times over the crude extract by gel filtration and ion-exchange chromatography with a recovery of 17%. The K(m) and V(max) values for sucrose were 6.66 mM and 0.028 micromol/min, respectively. An invertase is purified and characterized for the first time in Lactobacillus, and it proved to be a beta-fructofuranosidase.

Chromatography, Ion Exchange↗
PubMed2004

The effect of Lactobacillus helveticus fermented milk on acute changes in calcium metabolism in postmenopausal women.

BACKGROUND: Milk fermented with Lactobacillus helveticus ( L. helveticus) has been shown to lower blood pressure and to increase bone mineral content in spontaneously hypertensive rats. The effect of L.helveticus may be due to better calcium availability. AIM OF THE STUDY: In the present study the effect of milk fermented with L. helveticus on acute changes in calcium metabolism and bone resorption in postmenopausal women was studied. METHODS: The study was performed as a randomised double-blind crossover study of 20 postmenopausal women (mean age 65, range 50-78). The study was carried out in two parts. Firstly, L. helveticus fermented milk was compared to a control milk. Secondly, juice containing peptides formed with L. helveticus bacteria was compared to a control juice. The acute effect on calcium metabolism was measured during the study day by serum ionised calcium (iCa), parathyroid hormone (PTH), calcium (Ca), phosphate (P), and urinary calcium. A direct marker of bone turnover, carboxyterminal telopeptide of type I collagen (ICTP), was measured from the serum. RESULTS: L. helveticus fermented milk reduced serum PTH (405.3 +/- 37 ng/l vs. 454.9 +/- 37, p = 0.012) and increased serum calcium (19.1 +/- 0.2 mmol/l vs. 18.8 +/- 0.2, p = 0.031) compared to the control milk. L. helveticus derived peptides had no significant acute effect on calcium metabolism, in fact, ionised calcium was lower and PTH higher after the juice containing peptides compared to the control juice. CONCLUSIONS: Fermentation of milk with Lactobacillus helveticus had a positive acute effect on calcium metabolism. This effect was not explained by the small peptides formed by L. helveticus.

Aged↗
PubMed2004

Fermentation of a milk-soymilk and Lycium chinense Miller mixture using a new isolate of Lactobacillus paracasei subsp. paracasei NTU101 and Bifidobacterium longum.

A milk-soymilk mixture was fermented using Lactobacillus paracasei subsp. paracasei NTU101 and Bifidobacterium longum BCRC11847 at different inoculum ratios (1:1, 1:2, 1:5, 2:1, and 5:1). When the inoculum ratio was 1:2, the cell numbers of both strains were balanced after 12 h of cultivation. The pH and titratable acidity were very similar at the various inoculum ratios of cultivation. The milk-soymilk mixture was supplemented with 5, 10, 15, and 20% Lycium chinense Miller juice and fermented with Lactobacillus paracasei subsp. paracasei NTU101 and B. longum BCRC11847. Sensory evaluation results showed that supplementation with 5% Lycium chinense Miller juice improved the acceptability of the fermented milk-soymilk. The fermented beverage was stored at 4 degrees C for 14 days; variations in pH and titratable acidity were slight. The cell numbers of L. paracasei subsp. paracasei NTU101 and B. longum BCRC11847 in the fermented beverage were maintained at 1.2x10(9) CFU/ml and 6.3x10(8) CFU/ml, respectively, after 14 days of storage.

Animals↗
PubMed2006

Metabolism associated with raised metabolic flux to sugar nucleotide precursors of exopolysaccharides in Lactobacillus delbrueckii subsp. bulgaricus.

Exopolysaccharide (EPS) metabolism was studied in a galactose-negative strain of Lactobacillus delbrueckii subsp. bulgaricus, using two different approaches. Firstly, using both the parent strain and a chemically induced mutant with higher yield and specific productivity of EPS than the parent, comparative information was obtained relating to enzyme activities and metabolite levels associated with EPS formation when grown on lactose. Under continuous culture conditions (D = 0.10 h(-1)), the higher metabolic flux towards EPS formation in the mutant strain relative to the parent appeared to be mediated by raised levels of UDP-glucose pyrophosphorylase (UGP). Marginally raised UDP-galactose 4-epimerase (UGE) activity in the mutant strain suggested that this enzyme could also play a role in EPS overproduction. The second approach involved investigating the effect of growth rate on sugar nucleotide metabolism in the parent, as it is known that EPS production is growth-associated in this strain. UGE activity in the parent strain appeared to increase when the growth rate was elevated from 0.05 to 0.10 h(-1), and further to 0.35 h(-1), conditions that can be associated with higher levels of metabolic flux to EPS formation. Concurrent with these increments, intracellular ATP levels in the cell were raised. In both investigations glucose-6-phosphate accumulated pointing to a constriction at this branch-point, and a limitation in the flow of carbon towards fructose-6-phosphate or glucose-1-phosphate. The changes in metabolism associated with enhanced flux to EPS provide guidance as to how the yield of Lactobacillus delbrueckii subsp. bulgaricus EPS can be improved.

Amino Sugars↗
PubMed2004

Delay of the first onset of pouchitis by oral intake of the probiotic strain Lactobacillus rhamnosus GG.

PURPOSE: Proctocolectomy with ileal pouch-anal anastomosis is the operation of choice for patients with refractory or fulminant ulcerative colitis. The most common long-term complication in these patients is pouchitis. This study was designed to investigate the efficacy of probiotic Lactobacillus rhamnosus GG in long-term delaying the first onset of pouchitis. METHODS: Between 1989 and 2001, a consecutive series of 127 patients presenting with ulcerative colitis underwent an ileal pouch-anal anastomosis at the Erasmus Medical Center in Rotterdam. Histopathologic investigation of the resected specimens revealed unsuspected Crohn's disease in five patients. Postoperative complications resulted in pouch excision in five patients. The remaining 117 patients were included in this study. All episodes of pouchitis occurring in this group were analyzed. Pouchitis was diagnosed on the basis of clinical symptoms and endoscopic and histologic features. The 39 patients, who underwent an ileal pouch-anal anastomosis between 1996 and 2001, started immediately after the operation with the daily intake of L. rhamnosus GG in a fermented product. The 78 patients, in whom an ileal pouch-anal anastomosis was performed between 1989 and 1996, received no L. rhamnosus GG. RESULTS: Except for the duration of follow-up, the patient characteristics, indications for proctocolectomy, number of postoperative complications, and functional outcome were similar in both groups. First episodes of pouchitis were observed less frequently in patients with a daily intake of L. rhamnosus GG (cumulative risk at 3 years: 7 vs. 29 percent; P = 0.011). CONCLUSIONS: Daily intake of fermented products containing L. rhamnosus GG provides significant clinical benefit, without side effects. Based on the results of this study, we recommend a daily intake of Lactobacillus rhamnosus GG (dose 1-2 x 10(10) bacteria) to delay the first onset of pouchitis.

Administration, Oral↗
PubMed1976

Control of and synthesis in Lactobacillus acidophilus Role of RNA and free amino acids.

In Lactobacillus acidophilus, the DNA synthesis in cells incubated in the absence of essential amino acids reaches levels corresponding to the initiation of further replication cycles than just to completing the cycles already running. This "relaxation of DNA synthesis" is stimulated by the presence of inhibitors of protein synthesis and by the presence of deoxyadenylate. These enhancements of DNA replication are cancelled by actinomycin D. In the presence of inhibitors of protein synthesis, the relaxtion of DNA synthesis is further stimulated by free amino acids. The effect of free amino acids, unlike in the former cases, is not inhibited by actinomycin D. It proposed that the chromosome replication in Lactobacillus acidophilus is regulated similarly as in some plasmids, i.e. independently of the synthesis of protein, depending on the synthesis of RNA and, in addition, on the presence of free amino acids.

Amino Acids↗
PubMed1992

Mobilization and location of the genetic determinant of chloramphenicol resistance from Lactobacillus plantarum caTC2R.

The mobilization of a nonconjugative plasmid (pCaT) that mediates chloramphenicol resistance in Lactobacillus plantarum caTC2R was achieved by comobilization with the conjugative plasmid pAM beta 1. The conjugation studies confirmed that the 8.5-kb pCaT in L. plantarum caTC2R contains the gene responsible for chloramphenicol resistance and that the plasmid has several unique restriction sites which make it useful for genetic studies in Carnobacterium spp. Cloning studies showed that the gene responsible for chloramphenicol resistance is located in the 2.6-kb EcoRV-SalI region of pCaT. This was confirmed by probing the 3.0-kb BglII fragment of pCaT with a biotin-labeled 1.6-kb BstEII-HpaII fragment from the streptococcal-derived plasmid pVA797(Cmr). Expression of chloramphenicol resistance in Carnobacterium as well as in other Lactobacillus species was achieved by electrotransformation using donor DNA from pCaT.

Bacterial Proteins↗
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↗
PubMed1991

Antibacterial activity of Lactobacillus sake isolated from dry fermented sausages.

Lactic acid bacteria isolated from Spanish dry fermented sausages were screened for antagonistic activities under conditions that eliminated the effects of low pH and hydrogen peroxide. From 720 isolates tested 119 were inhibitory to Lactobacillus fermentum CECT285. The isolates showing the largest inhibitory activity exhibited an antagonistic effect against several other lactobacilli and the selected foodborne pathogens Staphylococcus aureus and Listeria monocytogenes. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. The isolates were tentatively characterized as Lactobacillus sake. One of the isolates, L. sake 148 was chosen for further study. The compound excreted by L. sake 148 was active against various lactobacilli and several Gram-positive foodborne bacteria, but not against the Gram-negative bacteria tested. The antagonistic effects were almost eliminated by treatment with proteases, whereas they were heat resistant and bacteriostatic rather than bacteriocidal.

Animals↗
PubMed1995

Application of a mathematical model to describe the behaviour of the Lactobacillus spp. during the ripening of a Spanish dry fermented sausage (Chorizo).

The evolution of Lactobacillus spp. during the curing process of dry fermented sausage (Chorizo), has been studied under natural climatic conditions and in a controlled drying chamber, with regulated temperature and relative humidity. In order to fully understand the development of the microorganisms in each of these two cases, it applied a mathematical model based on a modified Gompertz equation. The proposed mathematical function is easy to use and statistically appropriate. Furthermore, it allows the distinction to be made between the four phases which characterise the behaviour of these microorganisms during the curing of the sausage (a) latency from an initial threshold level; (b) exponential growth; (c) stationary state; (d) depletion to a residual level. The mathematical analysis of this function has highlighted certain differences in the behaviour of the microbial flora depending on the technological conditions to which the drying of the sausages is subjected. Under the climatically controlled conditions of the drying process, a better development of the lactobacillus spp. is observed, but conversely the final stage of the exponential growth is brought forward, the length of the stationary phase is shortened and is reached before the end of the drying period, at which point the marginal decline of the microorganisms becomes ever more reduced.

Fermentation↗
PubMed1995

Degradation of sinigrin by Lactobacillus agilis strain R16.

Forty-two lactobacilli were screened for their potential to degrade glucosinolate sinigrin. One of them, strain R16, demonstrated a high level of sinigrin degradation; it was identified as Lactobacillus agilis. The sinigrin degrading activity of L. agilis R16 could only be demonstrated when intact cells were used. The products of sinigrin degradation are allyl-isothiocyanate (AITC) and glucose (which is further fermented to DL-lactic acid), suggesting that myrosinase activity is involved. The activity was induced by the presence of sinigrin. Glucose inhibited the myrosinase activity, even in induced cells. Lactobacillus agilis R16 was able to grow on an extract of brown mustard seed and caused glucosinolate degradation.

Biodegradation, Environmental↗
PubMed1996

Antibacterial activity of Lactobacillus plantarum UG1 isolated from dry sausage: characterization, production and bactericidal action of plantaricin UG1.

Lactobacillus plantarum UG1 isolated from dry sausage produced an antimicrobial substance that inhibited other strains of the genera Lactobacillus and Lactococcus, and some foodborne pathogens including Listeria monocytogenes, Bacillus cereus, Clostridium perfringens and Clostridium sporogenes. This antibacterial substance was inactivated by proteolytic enzymes and showed a bactericidal mode of action. Consequently, it was characterized as a bacteriocin, and was designated plantaricin UG1. This bacteriocin was stable in the pH range 4.5 to 7.0, partially inactivated by amylolytic enzymes and relatively thermostable. It was not affected by organic or lipolytic enzymes. Production of plantaricin UG1 was pH- and temperature-dependant and maximum yields were obtained in MRS broth cultures maintained at initial pH 6.5, and incubated at 25 degrees C to 30 degrees C, in the exponential to the early stationary growth phase of the producer organism. Ultrafiltration studies indicated that plantaricin UG1 has a molecular weight between 3 and 10 KDa. Curing experiments with L. plantarum UG1 resulted in the appearance of variants that lost bacteriocin production ability but were still immune to the bacteriocin. Plantaricin UG1 production appeared to be chromosomal encoded. Sensitive and insensitive Gram-positive bacteria adsorbed plantaricin UG1 irrespective of their susceptibility to it. In contrast, Gram-negative bacteria did not adsorb plantaricin UG1. The bactericidal action of plantaricin UG1 did not depend on the physiological state of the indicator culture and did not cause cell lysis. The resistance of two indicator strains to plantaricin UG1 has been studied.

Adsorption↗