High tolerance of wild Lactobacillus plantarum and Oenococcus oeni strains to lyophilisation and stress environmental conditions of acid pH and ethanol

A total of 76 Lactobacillus plantarum and Oenococcus oeni wild strains were recovered from traditionally elaborated Spanish red wines and were investigated with respect to their response to acid pH, lyophilisation, temperature and ethanol concentrations which are normally lethal to lactic acid bacteria. Both L. plantarum and O. oeni strains were able to grow at pH 3.2, were highly resistant to lyophilisation treatment and proliferated in the presence of up to 13% ethanol at 18 degrees C. Therefore, it is shown that both species are highly tolerant to stress conditions and that similarly to O. oeni strains, L. plantarum strains are of interest in beverage biotechnology.     

High-level gene expression in Lactobacillus plantarum using a pheromone-regulated bacteriocin promoter

AIMS: To use promoters and regulatory genes involved in the production of the bacteriocin sakacin P to obtain high-level regulated gene expression in Lactobacillus plantarum. METHODS AND RESULTS: In a plasmid containing all three operons naturally involved in sakacin P production, the genes encoding sakacin P and its immunity protein were replaced by the aminopeptidase N gene from Lactococcus lactis (pepN) or the beta-glucuronidase gene from Escherichia coli (gusA). The new genes were precisely fused to the start codon of the sakacin P gene and the stop codon of the immunity gene. This set-up permitted regulated (external pheromone controlled) overexpression of both reporter genes in L. plantarum NC8. For PepN, production levels amounted to as much as 40% of total cellular protein. CONCLUSIONS: Promoters and regulatory genes involved in production of sakacin P are suitable for establishing inducible high-level gene expression in L. plantarum. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes a system for controllable gene expression in lactobacilli, giving some of the highest expression levels reported so far in this genus.     

Characterization of wine Lactobacillus plantarum by PCR-DGGE and RAPD-PCR analysis and identification of Lactobacillus plantarum strains able to degrade arginine

Summary Screening of strains isolated from red wine undergoing malolactic fermentation allowed the identification of lactic acid bacteria able to degrade arginine. A denaturing gradient gel electrophoresis approach, using the rpoB gene as the molecular target, was developed in order to characterize the isolated strains. Several strains were identified as Lactobacillus plantarum and were typed by RAPD-PCR with several randomly designed primers. Almost all of the␣L. plantarum strains identified were able to produce citrulline and ammonia, suggesting that the ability of␣L.␣plantarum to degrade arginine is a common feature in wine. During the characterization of the newly identified L.␣plantarum strains, the presence of genes coding for the arginine deiminase (ADI) pathway was observed in the strains able to produce citrulline, while the lack of this genes was observed in strain unable to produce citrulline. These results suggest that the degradation of arginine in L. plantarum is probably strain-dependent.     

Expression analysis of putative arcA, arcB and arcC genes partially cloned from Lactobacillus plantarum isolated from wine

AIMS: The aim of this paper was to study if homofermentative strains (Lacobacillus plantarum) capable of malolactic fermentation in wine can degrade arginine via the ADI pathway. METHODS AND RESULTS: Homofermentative lactic acid bacteria (LAB) isolated from a typical red wine were investigated for their ability to produce citrulline. Citrulline was formed suggesting that the arginine metabolism takes place via the arginine deiminase (ADI) pathway and not via the arginase/urease pathway. Ammonia was also detected with Nessler's reagent, and all the strains examined were able to produce ammonia. Identification of homofermentative LAB was performed using 16S ribosomal sequence analysis. The strains were further classified as belonging to L. plantarum species. Furthermore, the genes encoding for the three pathway enzymes (ADI, ornithine transcarbamylase, carbamate kinase) were partially cloned and gene expression was performed at two different pH values (3.6 and 4.5). CONCLUSIONS: The results suggest that citrulline production in wine, could be performed by homofermentative LAB. SIGNIFICANCE AND IMPACT OF THE STUDY: Homofermentative malolactic bacteria (L. plantarum) may degrade arginine through the ADI pathway.     

Optimization of a rapid method for studying the cellular location of beta-glucosidase activity in wine yeasts

AIMS: To improve a method for determining beta-glucosidase activity and to apply it in yeasts isolated from wine ecosystems from "La Mancha" region and to know its cellular location. METHODS AND RESULTS: A total of 82 wine yeasts were identified (PCR/RFLP) and evaluated for their beta-glucosidase activity. First, they were qualitatively evaluated by growth on YNB cellobiose, the activity was quantified using different culture media, under aerobic and anaerobic conditions and cells after 24-72 h of growth. To study the location activity, five fractions were obtained (supernatant, whole cell, cell wall, cytosol and cell membrane). The enzymatic assays were optimized, being: growth in YP cellobiose for 72 h in aeration conditions and, after cell removing, enzyme analysis with 128 g l(-1) of cellobiose as substrate, for 30 min at 30 degrees C. The genus that displayed the greatest activity were Pichia, Hanseniaspora and Rhodotorula, and the activity was intracellular. CONCLUSIONS: The study showed that beta-glucosidase activity was induced by the carbon source and was aerobic dependent. The non-Saccharomyces species displayed the greatest activity, which was intracellular and strain-dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: This study developed a reliable method for screening beta-glucosidase activity in yeasts isolated from wine ecosystems. This activity is very important in the release of monoterpenols from glycoside precursors for the enhancement of wine aromas.     

一个植物乳杆菌新质粒的序列分析与归类

[目的]分离鉴定植物乳杆菌PC518的质粒并分析滚环复制p C194家族复制起点特征。[方法]从植物乳杆菌PC518中提取质粒,HindⅢ单酶切后克隆测序,然后用反向PCR方法验证质粒序列的完整性。使用DNAMAN V6. 0软件和MEGA X软件对43个p C194家族质粒的复制起点序列和复制蛋白进行比对分析。[结果]分离得到一个3 325 bp的新质粒p LP325。43个p C194家族质粒复制起点中:24个在nick上、下游均有反向重复序列,12个只在nick上游有反向重复序列,4个只在下游有反向重复序列。复制蛋白的聚类与复制起点中反向重复序列的位置是对应的。[结论]p LP325的复制方式推定为滚环复制,属于p C194家族。p C194家族复制起点的bind以反向重复序列为特征,位于nick上游或下游。     

Isolation and basic characterization of a β‐glucosidase from a strain of Lactobacillus brevis isolated from a malolactic starter culture

Aims: To study glycosidase activities of a Lactobacillus brevis strain and to isolate an intracellular β‐glucosidase from this strain. Methods and Results: Lactic acid bacteria (LAB) isolated from a commercially available starter culture preparation for malolactic fermentation were tested for β‐glycosidase activities. A strain of Lact. brevis showing high intracellular β‐d ‐glucosidase, β‐d ‐xylosidase and α‐l ‐arabinosidase activities was selected for purification and characterization of its β‐glucosidase. The pure glucosidase from Lact. brevis has also side activities of xylosidase, arabinosidase and cellobiosidase. It is a homotetramer of 330 kDa and has an isoelectric point at pH 3·5. The K_m for p‐nitrophenyl‐β‐d ‐glucopyranoside and p‐nitrophenyl‐β‐d ‐xylopyranoside is 0·22 and 1·14 mmol l^?1, respectively. The β‐glucosidase activity was strongly inhibited by gluconic acid δ‐lactone, partially by glucose and gluconate, but not by fructose. Ethanol and methanol were found to increase the activity up to twofold. The free enzyme was stable at pH 7·0 (t_1/2 = 50 day) but not at pH 4·0 (t_1/2 = 4 days). Conclusions: The β‐glucosidase from Lact. brevis is widely different to that characterized from Lactobacillus casei ( Coulon et al. 1998 ) and Lactobacillus plantarum ( Sestelo et al. 2004 ). The high tolerance to fructose and ethanol, the low inhibitory effect of glucose on the enzyme activity and the good long‐term stability could be of great interest for the release of aroma compounds during winemaking. Significance and Impact of the study: Although the release of aroma compounds by LAB has been demonstrated by several authors, little information exists on the responsible enzymes. This study contains the first characterization of an intracellular β‐glucosidase isolated from a wine‐related strain of Lact. brevis.     

Study of beta-glucosidase production by wine-related yeasts during alcoholic fermentation. A new rapid fluorimetric method to determine enzymatic activity

AIMS: The beta-glucosidase activity is involved in the hydrolysis of several important compounds for the development of varietal wine flavour. The aim of the present study was to investigate the production of beta-glucosidase in a number of wine-related yeast strains and to measure and identify this activity over the course of grape juice fermentation. METHODS AND RESULTS: beta-glucosidase activity was measured as the amount of 4-methylumbelliferone released from 4-methylumbelliferyl-beta-d-glucopyranoside substrate. Intact cells of some grape and wine-spoilage yeasts showed beta-glucosidase activity much higher than those observed in wine yeasts "sensu stricto". During fermentation, three Saccharomyces cerevisiae strains, one Hanseniaspora valbyensis strain and one Brettanomyces anomalus strain showed beta-glucosidase activity both intra- and extracellularly. CONCLUSIONS: In the studied strains, beta-glucosidase activity was at its maximum when the cells were in the active growth phase. However, a lowering of medium pH to values around 3 during fermentation led to total loss of activity. SIGNIFICANCE AND IMPACT OF THE STUDY: During the course of this study, a new, rapid and reproducible method to assay beta-glucosidase activity was developed. The fact that Saccharomyces and non-Saccharomyces yeast strains are able to express beta-glucosidase activity during the alcoholic fermentation sheds new light on the contribution of these yeasts in the aroma expression of wines.     

Evidence for exopolysaccharide production by Oenococcus oeni strains isolated from non‐ropy wines

Aims: The aim of this study was to assess the exopolysaccharide (EPS) production capacities of various strains of Oenococcus oeni, including malolactic starters and strains recently isolated from wine . Methods and Results: Fourteen O. oeni strains displaying or not (PCR check on genomic DNA) the gtf gene generally associated with β‐glucan formation and ropiness were grown on grape juice medium, dialysed MRS‐derived medium or synthetic medium. The soluble polysaccharides (PS) remaining in the culture supernatant were alcohol precipitated, and their concentration was quantified by the phenol‐sulfuric method. Most of the O. oeni strains studied produced significant amounts of EPS, independently of their genotype (gtf+ or gtf?). The EPS production was not directly connected with growth and could be stimulated by changing the growth medium composition. The molecular weight distribution analysis and attempts to determine the PS chemical structure suggested that most strains produce a mixture of EPS. Conclusion: Oenococcus oeni strains recently isolated from wine or cultivated for many generations as a malolactic starter are able to produce EPS other than β‐glucan. Significance and Impact of the Study: These EPS may enhance the bacteria survival in wine (advantage for malolactic starters) and may contribute to the wine colloidal equilibrium.     

Evidence of different fermentation behaviours of two indigenous strains of Saccharomyces cerevisiae and Saccharomyces uvarum isolated from Amarone wine

Aims:  To explain the role of Saccharomyces cerevisiae and Saccharomyces uvarum strains (formerly Saccharomyces bayanus var. uvarum ) in wine fermentation.
Methods and Results:  Indigenous Saccharomyces spp. yeasts were isolated from Amarone wine (Italy) and analysed. Genotypes were correlated to phenotypes: Melibiose⁻ and Melibiose⁺ strains displayed a karyotype characterized by three and two bands between 225 and 365 kb, respectively. Two strains were identified by karyotype analysis (one as S. cerevisiae and the other as S. uvarum ). The technological characterization of these two strains was conducted by microvinifications of Amarone wine. Wines differed by the contents of ethanol, residual sugars, acetic acid, glycerol, total polysaccharides, ethyl acetate, 2-phenylethanol and anthocyanins. Esterase and β-glucosidase activities were assayed on whole cells during fermentation at 13° and 20°C. Saccharomyces uvarum displayed higher esterase activity at 13°C, while S. cerevisiae displayed higher β-glucosidase activity at both temperatures.
Conclusions:  The strains differed by important technological and qualitative traits affecting the fermentation kinetics and important aroma components of the wine.
Significance and Impact of the Study:  The contribution of indigenous strains of S. cerevisiae and S. uvarum to wine fermentation was ascertained under specific winemaking conditions. The use of these strains as starters in a winemaking process could differently modulate the wine sensory characteristics.     

Utilization of amino acids and dipeptides by Lactobacillus plantarum from orange in nutritionally stressed conditions

Aims:  To investigate amino acid and dipeptide utilization by Lactobacillus plantarum N4 isolated from orange peel, in a nutritionally depleted medium based on MRS (Mann, Rogosa, Sharpe).
Methods and Results:  In MRS with 0·1 g l⁻¹ of meat extract and without peptone and yeast extract, growth increased when essential and stimulatory amino acids and nonessential amino acid were added to the medium. Replacement of the essential amino acid, leucine, and the nonessential amino acid, glycine, by leucyl-leucine (Leu-Leu) and/or glycyl-glycine (Gly-Gly) significantly enhanced growth. Essential amino acids were mainly consumed and the dipeptides were almost completely used at the end of growth. Leucine and glycine accumulated internally from the peptides were higher than from the free amino acids. Glucose utilization increased in the media containing dipeptides compared with the medium containing free amino acids.
Conclusions:  In a N-depleted medium, Leu-Leu and/or Gly-Gly were more effective than the respective amino acids in supporting growth of the micro-organism. The more efficient internal accumulation of glycine and especially leucine from dipeptides confirmed the ability of the strain to assimilate mainly complex nitrogen molecules rather than simple ones.
Significance and Impact of the Study:  The ability of Lact. plantarum N4 to efficiently use dipeptides could contribute to spoilage development in the natural medium of the organism, orange juice.     

Beta-glucosidase activity from the thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose

An inducible mycelial beta-glucosidase from Scytalidum thermophilum was characterized. The enzyme exhibited a pI of 6.5, a carbohydrate content of 15%, and an apparent molecular mass of about 40 kDa. Optima of temperature and pH were 60 degrees C and 6.5, respectively. The enzyme was stable up to 1 h at 50 degrees C and exhibited a half-life of 20 min at 55 degrees C. The enzyme hydrolyzed p-nitrophenyl-beta-d-glucopyranoside, p-nitrophenyl-beta-d-xylopyranoside, o-nitrophenyl-beta-d-galactopyranoside, p-nitrophenyl-alpha-arabinopyranoside, cellobiose, laminaribiose and lactose. Kinetic studies indicated that the same enzyme hydrolyzed these substrates. Beta-Glucosidase was activated by glucose or xylose at concentration varying from 50 to 200 mM. The apparent affinity constants (K0.5) for glucose and xylose were 36.69 and 43.24 mM, respectively. The stimulatory effect of glucose and xylose on the S. thermophilum beta-glucosidase is a novel characteristic which distinguish this enzyme from all other beta-glucosidases so far described.     

Improvement of the fermentation performance of Lactobacillus plantarum by the flavanol catechin is uncoupled from its degradation

Aims: To determine the influence of the flavanol catechin on key metabolic traits for the fermentation performance of Lactobacillus plantarum strain RM71 in different media and to evaluate the ability of this strain to catabolize catechin. Methods and Results: Growth monitoring and time course of sugar consumption data tracking in chemically defined medium (CDM), revealed that growth of Lact. plantarum strain RM71 upon catechin was characterized by a noticeable shorter lag period, outcome of earlier sugar consumption and lactic acid production courses. Catechin gave rise to higher cell densities compared to controls because of an increased extension of sugar utilization. Fermentation of media relevant for practical fermentation processes with Lact. plantarum strain RM71 showed that catechin sped up malic acid decarboxylation, which besides quicker and extended consumption of several sugars, resulted in faster and higher lactic acid production and growth. Spectrophotometric evaluation of catechin by HPLC‐DAD and the lack of catechin concentration‐dependent effects showed that the observed stimulations were uncoupled from catechin catabolism by Lact. plantarum. Conclusions: The flavanol catechin stimulated the growth of Lact. plantarum strain RM71 by promoting quicker sugar consumption, increasing the extension of sugar utilization and stimulating malic acid decarboxylation. These stimulations are uncoupled from catechin catabolism as Lact. plantarum did not catabolize it during fermentation. Significance and Impact of the Study: This study, for the first time, examined the influence of the flavanol catechin on the fermentation performance of a Lact. plantarum strain in several media under different fermentation conditions. The information could be relevant to control the production and obtain high‐quality food products fermented by this micro‐organism.     

Lactobacillus plantarum LRCC 5273 isolated from Kimchi ameliorates diet-induced hypercholesterolemia in C57BL/6 mice

ABSTRACT

This study was designed to select potent cholesterol-lowering probiotic strains on HepG2 cell and investigate the effect of selected strain, Lactobacillus plantarum LRCC 5273 and LRCC 5279 in hypercholesterolemic mice. In the results, LP5273 group showed significantly reduced total and LDL cholesterol compared to HCD group. In addition to significantly up-regulated hepatic mRNA expression of LXR-α and CYP7A1, intestinal LXR-α and ABCG5 were significantly up-regulated in LP5273 group. With activation of hepatic and intestinal LXR-α and its target genes, fecal cholesterol and bile acid excretion were increased in LP5273 fed mice. These results suggest that LP5273 ameliorates hypercholesterolemia in mice through the activation of hepatic and intestinal LXR-α, resulting in enhancement of fecal cholesterol and bile acids excretion in the small intestine. The results of present study suggest mechanistic evidences for hypocholesterolemic effects of L. plantarum spp., and may contribute to future researches for prevention of hypercholesterolemia and cardiovascular disease.     

Effect of phenolic compounds on the co-metabolism of citric acid and sugars by Oenococcus oeni from wine

AIMS: The goal of this study was to examine the growth of Oenococcus oeni in the presence of phenolic compounds under wine conditions and to see how these compounds affect bacterial metabolism. METHODS AND RESULTS: Phenolic compounds have been added to a basal medium that simulates the composition of wine. Fifty milligrams per litre or more of phenolic compounds stimulated bacterial growth. Oenococcus oeni seemed to use citric acid and trehalose, if they were present, before glucose and fructose. Citrate was completely exhausted in three days and the yield of acetate was higher when phenolic compounds were present. CONCLUSIONS: Phenolic compounds reduced the rate of sugar consumption and enhanced citric acid consumption, increasing the yield of acetic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: This study allows a better knowledge of co-metabolism of citric acid and sugars by O. oeni in the presence of phenolic compounds of wine.