Characterization of a highly hop-resistant Lactobacillus brevis strain lacking hop transport

Resistance to hops is a prerequisite for lactic acid bacteria to spoil beer. In this study we analyzed mechanisms of hop resistance of Lactobacillus brevis at the metabolism, membrane physiology, and cell wall composition levels. The beer-spoiling organism L. brevis TMW 1.465 was adapted to high concentrations of hop compounds and compared to a nonadapted strain. Upon adaptation to hops the metabolism changed to minimize ethanol stress. Fructose was used predominantly as a carbon source by the nonadapted strain but served as an electron acceptor upon adaptation to hops, with concomitant formation of acetate instead of ethanol. Furthermore, hop adaptation resulted in higher levels of lipoteichoic acids (LTA) incorporated into the cell wall and altered composition and fluidity of the cytoplasmic membrane. The putative transport protein HitA and enzymes of the arginine deiminase pathway were overexpressed upon hop adaptation. HorA was not expressed, and the transport of hop compounds from the membrane to the extracellular space did not account for increased resistance to hops upon adaptation. Accordingly, hop resistance is a multifactorial dynamic property, which can develop during adaptation. During hop adaptation, arginine catabolism contributes to energy and generation of the proton motive force until a small fraction of the population has established structural improvements. This acquired hop resistance is energy independent and involves an altered cell wall composition. LTA shields the organism from accompanying stresses and provides a reservoir of divalent cations, which are otherwise scarce as a result of their complexation by hop acids. Some of the mechanisms involved in hop resistance overlap with mechanisms of pH resistance and ethanol tolerance and as a result enable beer spoilage by L. brevis.     

Purification and Characterization of Spirosomes in Lactobacillus brevis

Spirosomes, very fine spiral particles, were isolated from a protoplastlysate of Lactobacillus brevis ATCC 8287 by differential centrifugation and purified further by potassium tartrate density gradient centrifugation. The purified spirosome preparation showed a maximum peak around 275 nm on the ultraviolet absorption spectrum and it consisted of about 94.5% protein. The buoyant density in CsCl of the spirosomes was 1.320 g/cm³. The spirosomes were composed mainly of a single protein (spirosin) with an apparent molecular weight of about 95,000 as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The protein of the spirosomes was found to be composed predominantly of neutral amino acids accompanied by approximately equal amounts of acidic and basic amino acids. The spirosomes showed one antigenic determinant in the immunodiffusion test. The spirosomes were readily degraded by the action of proteolytic enzymes and lost their antigenicity, but they were not affected by treatment with either deoxyribonuclease or ribonuclease. The spiral structure of the spirosome was also found to be disintegrated by treatment with 1 m guanidine hydrochloride, 4 m urea or 0.1% SDS, but not by the action of deoxycholate, non-ionic detergents or mercaptoethanol, as observed in the electron microscope.     

Localization and Characterization of alpha-Glucosidase Activity in Lactobacillus brevis

Lactobacillus brevis is found together with the yeast Brettanomyces lambicus during the overattenuation process in spontaneously fermented lambic beer. An isolated L. brevis strain has been shown to produce an alpha-glucosidase with many similarities to the glucosidase earlier found in B. lambicus. The enzyme was purified by ammonium sulfate precipitation, gel (Sephadex G-150 and Ultrogel AcA-44) filtration, and ion-exchange chromatography (DEAE-Sephadex A-50). The molecular weights of the enzyme, as determined by gel chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were about 50,000 and 60,000, respectively. Optimum catalytic activity was obtained at 40 degrees C and pH 6.0. The enzyme showed a decrease of hydrolysis with an increase in the degree of polymerization of the substrate. The K(m) values for p-nitrophenyl-alpha-d-glucopyranoside, maltose, and maltotriose were 0.51, 3.0, and 5.2 mM, respectively. There was lack of inhibition by 0.15 mM acarbose and 0.5 M turanose, but the enzyme was inhibited by Tris (K(i) value of 25 mM). The alpha-glucosidase of L. brevis together with the enzyme of B. lambicus seems to be a key factor in the overattenuation of lambic beer, although the involvement of other lactic acid bacteria (pediococci) cannot be excluded.     

Bacteriocin properties of Lactobacillus fermenti, Lactobacillus brevis and Lactobacillus buchneri

Four bacteriocins of L. fermenti, 3 bacteriocins of L. brevis and 1 bacteriocin of L. buchneri were studied with respect to morphology of the inhibition growth zones of the indicator strains, capacity for diffusion through cellophane, sensitivity to high temperature, bacterial proteases, trypsin, chymotrypsin, pepsin, papain, nucleases and lysozyme. According to the differences in their properties the bacteriocins were classified as belonging to 8 types, including 4 types of L. fermenti bacteriocins and 3 types of L. brevis bacteriocins.     

Proteomic Approach for Characterization of Hop-Inducible Proteins in Lactobacillus brevis

Resistance to hops is a prerequisite for the capability of lactic acid bacteria to grow in beer and thus cause beer spoilage. Bactericidal hop compounds, mainly iso-α-acids, are described as ionophores which exchange H⁺ for cellular divalent cations, e.g., Mn²⁺, and thus dissipate ion gradients across the cytoplasmic membrane. The acid stress response of Lactobacillus brevis TMW 1.465 and hop adaptation in its variant L. brevis TMW 1.465A caused changes at the level of metabolism, membrane physiology, and cell wall composition. To identify the basis for these changes, a proteomic approach was taken. The experimental design allowed the discrimination of acid stress and hop stress. A strategy for improved protein identification enabled the identification of 84% of the proteins investigated despite the lack of genome sequence data for this strain. Hop resistance in L. brevis TMW 1.465A implies mechanisms to cope with intracellular acidification, mechanisms for energy generation and economy, genetic information fidelity, and enzyme functionality. Interestingly, the majority of hop-regulated enzymes are described as manganese or divalent cation dependent. Regulation of the manganese level allows fine-tuning of the metabolism, which enables a rapid response to environmental (stress) conditions. The hop stress response indicates adaptations shifting the metabolism into an energy-saving mode by effective substrate conversion and prevention of exhaustive protein de novo synthesis. The findings further demonstrate that hop stress in bacteria not only is associated with proton motive force depletion but obviously implies divalent cation limitation.     

Characterization of a Highly Hop-Resistant Lactobacillus brevis Strain Lacking Hop Transport

Resistance to hops is a prerequisite for lactic acid bacteria to spoil beer. In this study we analyzed mechanisms of hop resistance of Lactobacillus brevis at the metabolism, membrane physiology, and cell wall composition levels. The beer-spoiling organism L. brevis TMW 1.465 was adapted to high concentrations of hop compounds and compared to a nonadapted strain. Upon adaptation to hops the metabolism changed to minimize ethanol stress. Fructose was used predominantly as a carbon source by the nonadapted strain but served as an electron acceptor upon adaptation to hops, with concomitant formation of acetate instead of ethanol. Furthermore, hop adaptation resulted in higher levels of lipoteichoic acids (LTA) incorporated into the cell wall and altered composition and fluidity of the cytoplasmic membrane. The putative transport protein HitA and enzymes of the arginine deiminase pathway were overexpressed upon hop adaptation. HorA was not expressed, and the transport of hop compounds from the membrane to the extracellular space did not account for increased resistance to hops upon adaptation. Accordingly, hop resistance is a multifactorial dynamic property, which can develop during adaptation. During hop adaptation, arginine catabolism contributes to energy and generation of the proton motive force until a small fraction of the population has established structural improvements. This acquired hop resistance is energy independent and involves an altered cell wall composition. LTA shields the organism from accompanying stresses and provides a reservoir of divalent cations, which are otherwise scarce as a result of their complexation by hop acids. Some of the mechanisms involved in hop resistance overlap with mechanisms of pH resistance and ethanol tolerance and as a result enable beer spoilage by L. brevis.     

Comparative characterization of spirosomes isolated from Lactobacillus brevis, Lactobacillus fermentum, and Lactobacillus buchneri

Spirosomes, cytoplasmic fine spirals, were isolated and purified from Lactobacillus brevis ATCC 8287, L. fermentum F-1, and L. buchneri ATCC 4005, and their morphological, biochemical, and immunological properties were investigated. The spirosomes of these lactobacilli were morphologically indistinguishable from one another, and they had the same buoyant density of 1.320 g/cm3 in CsCl. All of the spirosomes were composed of a single protein, spirosin, with an apparent molecular weight of about 95,000 for L. brevis and L. fermentum and of about 96,000 for L. buchneri as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The spirosins from the three lactobacilli were compared by peptide mapping on SDS-PAGE after cleavage with N-chlorosuccinimide and limited proteolysis with Staphylococcus aureus V8 protease. The peptide map of the L. brevis spirosin was identical with that of the L. fermentum spirosin, whereas it was markedly different from the L. buchneri spirosin. The amino acid composition of the L. brevis spirosin was almost similar to that of the L. fermentum spirosin, while it differed appreciably from the L. buchneri spirosin. Using antiserum against the L. brevis spirosin, immunodiffusion test revealed that the antigenicity of the spirosomes from L. brevis was identical with that from L. fermentum, whereas it was partially different from that from L. buchneri.     

Lactobacillus brevis CD2: Fermentation Strategies and Extracellular Metabolites Characterization

Functional foods and nutraceuticals frequently contain viable probiotic strains that, at certain titers, are considered to be responsible of beneficial effects on health. Recently, it was observed that secreted metabolites might play a key role in this respect, especially in immunomodulation. Exopolysaccharides produced by probiotics, for example, are used in the food, pharmaceutical, and biomedical fields, due to their unique properties. Lactobacillus brevis CD2 demonstrated the ability to inhibit oral pathogens causing mucositis and periodontal inflammation and to reduce Helycobacter pylori infections. Due to the lack of literature, for this strain, on the development of fermentation processes that can increase the titer of viable cells and associated metabolites to industrially attractive levels, different batch and fed-batch strategies were investigated in the present study. In particular, aeration was shown to improve the growth rate and the yields of lactic acid and biomass in batch cultures. The use of an exponential feeding profile in fed-batch experiments allowed to produce 9.3 ± 0.45 × 10⁹ CFU/mL in 42 h of growth, corresponding to a 20-fold increase of viable cells compared with that obtained in aerated batch processes; moreover, also increased titers of exopolysaccharides and lactic acid (260 and 150%, respectively) were observed. A purification process based on ultrafiltration, charcoal treatment, and solvent precipitation was applied to partially purify secreted metabolites and separate them into two molecular weight fractions (above and below 10 kDa). Both fractions inhibited growth of the known gut pathogen, Salmonella typhimurium, demonstrating that lactic acid plays a major role in pathogen growth inhibition, which is however further enhanced by the presence of Lact. brevis CD2 exopolysaccharides. Finally, the EPS produced from Lact. brevis CD2 was characterized by NMR for the first time up to date.

     

Maltose-fructose co-fermentation by Lactobacillus brevis subsp. lindneri CB1 fructose-negative strain

The Lactobacillus brevis subsp. lindneri CB1 fructose-negative strain utilized fructose in co-fermentation with maltose or glucose. Compared to the maltose (17 g/l) fermentation, the simultaneous fermentation of maltose (10 g/l) and fructose (7 g/l) increased cell yield (A ₆₂₀from 2.6 to 3.3) and the concentrations of lactic acid and especially of acetic acid (from 2.45 g/l to 3.90 g/l), produced mannitol (1.95 g/l) and caused a decrease in the amount of ethanol (from 0.46 g/l to 0.08 g/l). The utilization of fructose depended on the continuous presence of maltose in the growth medium and the two carbohydrates were consumed in a molar ratio of about 2:1. The presence of tagatose (a fructose stereoisomer) partially inhibited fructose consumption and consequently caused a decrease of the end products of the co-metabolism. Since maltose was naturally present during sourdough fermentation, the addition of only 6 g fructose/kg wheat dough enabled the co-fermentation of maltose and fructose by L. brevis subsp. lindneri CB1. A higher titratable acidity and acetic acid concentration, and a reduced quotient of fermentation (2.7) were obtained by co-fermentation compared with normal sourdough fermentation. Some interpretations of the maltose-fructose co-fermentation are given.     

Lipase activity of Lactobacillus brevis

Summary The intracellular lipase from a strain of Lactobacillus brevis was partially purified and properties of the enzyme studied. Of the simple triglycerides, tripropionin was hydrolysed most easily by the enzyme as compared to others such as tributyrin, tricaproin and tricaprylin. Of the natural triglycerides such as butter oil and coconut oil, the former was degraded more readily than the latter. Among unsaturated triglycerides, the enzyme preferentially hydrolysed triolein as compared to olive oil. Highest enzymatic activity was observed at 30° C after 3.5 h incubation at pH 6.5. Salts of manganese, magnesium, sodium and calcium stimulated lipase activity while silver, mercury and Zinc were inhibitory. The enzyme was completely inactivated at 62.8° C after 30 min and at 71.7° C after 16 sec.     

Tyrosine decarboxylase activity of Lactobacillus brevis IOEB 9809 isolated from wine and L. brevis ATCC 367

Tyramine, a frequent amine in wines, is produced from tyrosine by the tyrosine decarboxylase (TDC) activity of bacteria. The tyramine-producing strain Lactobacillus brevis IOEB 9809 isolated from wine and the reference strain L. brevis ATCC 367 were studied. At the optimum pH, 5.0, K(m) values of IOEB 9809 and ATCC 367 crude extracts for L-tyrosine were 0.58 mM and 0.67 mM, and V(max) was higher for the wine strain (115 U) than the ATCC 367 (66 U). TDC exhibited a preference for L-tyrosine over L-DOPA as substrate. Enzyme activity was pyridoxal-5'-phosphate (PLP)-dependent and it was stabilized by the substrate and coenzyme. In contrast, glycerol and beta-mercaptoethanol strongly inhibited TDC. Tyramine competitively inhibited TDC for both strains. Citric acid, lactic acid and ethanol had an inhibitory effect on cells and crude extracts, but none could inhibit TDC at the usual concentrations in wines.     

Membrane-bound ATPase contributes to hop resistance of Lactobacillus brevis

The activity of the membrane-bound H+-ATPase of the beer spoilage bacterium Lactobacillus brevis ABBC45 increased upon adaptation to bacteriostatic hop compounds. The ATPase activity was optimal around pH 5.6 and increased up to fourfold when L. brevis was exposed to 666 microM hop compounds. The extent of activation depended on the concentration of hop compounds and was maximal at the highest concentration tested. The ATPase activity was strongly inhibited by N,N'-dicyclohexylcarbodiimide, a known inhibitor of FoF1-ATPase. Western blots of membrane proteins of L. brevis with antisera raised against the alpha- and beta-subunits of FoF1-ATPase from Enterococcus hirae showed that there was increased expression of the ATPase after hop adaptation. The expression levels, as well as the ATPase activity, decreased to the initial nonadapted levels when the hop-adapted cells were cultured further without hop compounds. These observations strongly indicate that proton pumping by the membrane-bound ATPase contributes considerably to the resistance of L. brevis to hop compounds.     

Membrane-Bound ATPase Contributes to Hop Resistance of Lactobacillus brevis

The activity of the membrane-bound H⁺-ATPase of the beer spoilage bacterium Lactobacillus brevis ABBC45 increased upon adaptation to bacteriostatic hop compounds. The ATPase activity was optimal around pH 5.6 and increased up to fourfold when L. brevis was exposed to 666 μM hop compounds. The extent of activation depended on the concentration of hop compounds and was maximal at the highest concentration tested. The ATPase activity was strongly inhibited by N,N′-dicyclohexylcarbodiimide, a known inhibitor of F_oF₁-ATPase. Western blots of membrane proteins of L. brevis with antisera raised against the α- and β-subunits of F_oF₁-ATPase from Enterococcus hirae showed that there was increased expression of the ATPase after hop adaptation. The expression levels, as well as the ATPase activity, decreased to the initial nonadapted levels when the hop-adapted cells were cultured further without hop compounds. These observations strongly indicate that proton pumping by the membrane-bound ATPase contributes considerably to the resistance of L. brevis to hop compounds.     

Enumeration of Carnobacterium divergens V41, Carnobacterium piscicola V1 and Lactobacillus brevis LB62 by in situ hybridizationflow cytometry

The specific detection and enumeration of Lactobacillus brevis LB62, Carnobacterium divergens V14 and Carnobacterium piscicola VI were studied by in situ hybridizationflow cytometry. The method was performed on the exponential growth phase with three probes targeting 16S rRNA labelled with fluorescein isothicyanate (FITC) : EUB338 probe universal for Eubacteria, Lb probe specific for Lact. brevis and Cb probe specific for the genus Carnobacterium . EUB338 was used to determine the permeabilization and hybridization conditions for the cells. The Lb probe gave no hybridization signal whereas the Cb probe allowed the detection and quantification by flow cytometry at 520 nm of the two Carnobacterium strains in pure culture or in mixtures with Listeria innocua F.     

Growth Response of Lactobacillus brevis to Aeration and Organic Catalysts

Under stationary and anaerobic conditions, greater cell yields of Lactobacillus brevis were obtained from autoclaved than from filter-sterilized glucose media. Fructose, tentatively identified as a product generated by the heating process, served as an excellent catalyst for inducing growth. The addition of micromolar quantities of pentoses or potential pentose precursors to the filter-sterilized medium was equally effective in stimulating growth. These organic catalysts were not essential for growth under aerobic conditions. Upon agitation, similar cell yields were obtained from the autoclaved and filter-sterilized media. The micromolar quantities of lactic acid produced per micromole of carbohydrate fermented appeared to be similar under aerobic and static conditions of incubation. The final concentration of acetic acid increased as the result of agitation. This increase in volatile acidity was accompanied by a significant decrease in ethyl alcohol production. The cell yield was increased nearly 50% under aerobic conditions.     

Analysis of S-layer proteins of Lactobacillus brevis

Abstract The presence of S-layer proteins in Lactobacillus brevis was examined by SDS-PAGE analysis. Thirty six out of a total of 41 L. brevis strains possessed S-layer proteins of molecular masses ranging from 38 to 55 kDa. Western blot analysis using antisera raised against whole cells of S-layer protein-carrying strains demonstrated the heterogeneity of L. brevis S-layer proteins. No clear relationship was observed between the presence of S-layer proteins or their immunological characteristics and the physiological activity of L. brevis as a beer spoilage organism.     

Random amplified polymorphic DNA-PCR based cloning of markers to identify the beer-spoilage strains of Lactobacillus brevis, Pediococcus damnosus, Lactobacillus collinoides and Lactobacillus coryniformis

AIMS: Beer-spoilage ability of lactic acid bacteria such as Lactobacillus brevis is a strain-dependent phenomenon in which the mechanism has not yet been completely clarified. In order to systematically identify genes that contribute to beer-spoilage, large-scale random amplified polymorphic DNA (RAPD)-based cloning methods was carried out. METHODS AND RESULTS: A systematic RAPD polymerase chain reaction (PCR) analysis using 600 primers was performed on beer-spoilage and on nonspoilage strains of L. brevis. Among 600 primers, three were found to amplify a single locus highly specific to beer-spoilage strains. DNA sequencing of this locus revealed a three-part operon encoding a putative glycosyl transferase, membrane protein and teichoic acid glycosylation protein. PCR analysis of typical beer-spoilage lactic acid bacteria suggested that this locus is highly specific to beer-spoilage strains. CONCLUSION: The cloned markers are highly specific to identify the beer-spoilage strains not only in L. brevis but also in Pediococcus damnosus, Lactobacillus collinoides and Lactobacillus coryniformis. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper proves that RAPD-PCR is an efficient method for cloning the strain-specific genes from bacteria. The markers described here is one of the most useful tools to identify the beer-spoilage strains of lactic acid bacteria.