Basic characterization and partial purification of β-glucosidase from cell-free extracts of Oenococcus oeni ST81

Aims: This study was designed to characterize a β‐glucosidase of Oenococcus oeni ST81, a strain isolated from a Spanish wine of the origin appellation Ribeira Sacra. Methods and Results: The β‐glucosidase of O. oeni ST81 seems to have a periplasmic localization into the cells. This activity was strongly inhibited by gluconic acid, partially inhibited by glucose and not inhibited by fructose, lactate, malate, mannitol or sorbitol. Ethanol increased the activity of this enzyme up to 147%. Among the several metal ions assayed, only Fe²⁺ (10 mmol l^?1) and Cu²⁺ (5 mmol l^?1) exhibited a partial inhibitory effect (40%). This enzyme was partially purified using a combination of ammonium sulfate precipitation and chromatographic methods. The single peak because of β‐glucosidase in all chromatographic columns indicates the presence of a single enzyme with an estimated molecular mass of 140 kDa. The calculated K_m and V_max values for 4‐nitrophenyl‐β‐d ‐glucopyranoside were 0·38 mmol l^?1 and 5·21 nmol min^?1, respectively. The enzyme was stable at pH 5·0 with a value of t_1/2 = 50 days for the crude extract. Conclusions: The β‐glucosidase of O. oeni ST81 is substantially different from those characterized from other wine‐related lactic acid bacteria (LAB), such as Lactobacillus plantarum and Lactobacillus brevis; however, it appears to be closely related to a β‐glucosidase from O. oeni ATCC BAA‐1163 cloned into Escherichia coli. The periplasmic localization of the enzyme together with its high tolerance to ethanol and fructose, the low inhibitory effect of some wine‐related compounds on the enzymatic activity and long‐term stability of the enzyme could be of interest for winemaking. Significance and Impact of the Study: Information regarding a β‐glucosidase from O. oeni ST81 is presented. Although the release of aroma compounds by LAB has been demonstrated, little information exists concerning the responsible enzymes. To our knowledge, this study contains the first characterization of a native β‐glucosidase purified from crude extracts of O. oeni ST81.     

A β-glucosidase from <Emphasis Type="Italic">Oenococcus oeni</Emphasis> ATCC BAA-1163 with potential for aroma release in wine: cloning and expression in <Emphasis Type="Italic">E. coli</Emphasis>

Lactic acid bacteria (LAB) are responsible for olfactory changes in wine during malolactic fermentation (MLF). A side characteristic of MLF is the release of grape derived aroma compounds from their glycosylated precursors by β-glycosidase activities of these bacteria. Apart from Oenococcus oeni, which is regarded as the most promising species for MLF, glycosidic activities have also been observed in wine related members of the genera Lactobacillus and Pediococcus. Nevertheless, information on the involved enzymes including their potential use in winemaking is limited. In this study we report that β-glucosidases with similar protein sequences can be identified in the genomes of Lactobacillus brevis, O. oeni and Leuconostoc mesenteroides. TTG serves as start codon for the glucosidase gene of O. oeni. The β-glucosidase of O. oeni ATCC BAA-1163 was expressed in E. coli and partially characterized. The enzyme displayed characteristics similar to β-glucosidases isolated from L. brevis and L. mesenteroides. A pH optimum between 5.0 and 5.5, and a K _m of 0.17 mmol L⁻¹ pNP-β-d-glucopyranoside were determined. A glycosyltransferase activity was observed in the presence of ethanol. The enzyme from O. oeni was capable to hydrolyze glycosides extracted from Muskat wine. This study also contains a report on glycosidase activities of several LAB species including Oenococcus kitaharae.     

Impact of acetaldehyde- and pyruvic acid-bound sulphur dioxide on wine lactic acid bacteria

Aims: To investigate the impact of acetaldehyde‐ and pyruvic acid‐bound sulphur dioxide on wine lactic acid bacteria (LAB). Methods and Results: Growth studies were performed where Oenococcus oeni, Pediococcus parvulus, Ped. damnosus and Lactobacillus hilgardii were inoculated into media containing various concentrations of acetaldehyde or pyruvic acid and an equimolar concentration of SO₂ at pH 3·50 and 3·70. Low concentrations of acetaldehyde‐ and pyruvic acid‐bound SO₂ were inhibitory to the growth of all bacteria although acetaldehyde‐bound SO₂ was generally more inhibitory than pyruvic acid‐bound SO₂. Inhibition was greater at pH 3·50 than 3·70, and Lact. hilgardii was the most sensitive to acetaldehyde‐bound SO₂, while O. oeni was the most sensitive to pyruvic acid‐bound SO₂. Degradation of SO₂‐bound acetaldehyde was observed for all LAB, and aside from O. oeni, there was also complete degradation of SO₂‐bound pyruvic acid at both pH values. O. oeni only degraded pyruvic acid at pH 3·70. Degradation of SO₂‐bound acetaldehyde or pyruvic acid did not correlate with bacterial growth as inhibition was always observed in media containing bound SO₂. Conclusions: Acetaldehyde‐ and pyruvic acid‐bound SO₂ were inhibitory to wine LAB growth at concentrations as low as 5 mg l^?1. Despite this inhibition, all wine LAB degraded SO₂‐bound acetaldehyde and pyruvic acid suggesting that bound SO₂ may have a bacteriostatic rather than bacteriocidal action. Significance and Impact of the Study: Sulphur dioxide bound to acetaldehyde or pyruvic acid is inhibitory to growth of wine LAB and must be considered when conducting the malolactic fermentation or controlling the growth of spoilage bacteria such as Pediococcus and Lactobacillus.     

Optimization of electrotransformation conditions for Leuconostoc mesenteroides subsp. mesenteroides ATCC8293

Aims: To establish an efficient genetic transformation protocol for Leuconostoc species, methods for competent‐cell preparation and electroporation conditions were optimized. Methods and Results: Leuconostoc mesenteroides subsp. mesenteroides ATCC8293 cells were sequentially treated with penicillin G and lysozyme, and the plasmid pLeuCM was subsequently transformed into the cells. Our results demonstrated that transformation efficiencies were significantly increased (100‐fold), and increased electric field strength also contributed to enhance transformation efficiency. Maximum transformation efficiency (1 × 10⁴ or more transformants per μg DNA) was achieved when cells were grown in De Man, Rogosa, Sharpe (MRS) media containing 0·25 mol l^?1 sucrose and 0·8 μg ml^?1 penicillin G, followed by treatment with 600 U ml^?1 lysozyme and electroporation at a field strength of 10 kV cm^?1. When this protocol was used to transform pLeuCM into Leuc. mesenteroides, Leuconostoc gelidum, Leuconostoc fallax and Leuconostoc argentinun, successful transformations were obtained in all cases. Furthermore, this procedure was applicable to species belonging to other genera, including Lactobacillus plantarum, Pediococcus pentosaceus and Weissella confusa. Conclusions: The results demonstrate that the transformation efficiency for Leuconostoc spp. could be increased via optimization of the entire electroporation procedures. Significance and Impact of the Study: These optimized conditions can be used for the extensive genetic study and the metabolic engineering of not only Leuconostoc spp. but also different species of lactic acid bacteria.     

In vitro anti‐biofilm activity of Boswellia spp. oleogum resin essential oils

Aims: To evaluate the anti‐biofilm activity of the commercially available essential oils from two Boswellia species. Methods and Results: The susceptibility of staphylococcal and Candida albicans biofilms was determined by methyltiazotetrazolium (MTT) staining. At concentrations ranging from 217·3 μg ml^?1 (25% v/v) to 6·8 μg ml^?1 (0·75% v/v), the essential oil of Boswellia papyrifera showed considerable activity against both Staphylococcus epidermidis DSM 3269 and Staphylococcus aureus ATCC 29213 biofilms. The anti‐microbial efficacy of this oil against S. epidermidis RP62A biofilms was also tested using live/dead staining in combination with fluorescence microscopy, and we observed that the essential oil of B. papyrifera showed an evident anti‐biofilm effect and a prevention of adhesion at sub‐MIC concentrations. Boswellia rivae essential oil was very active against preformed C. albicans ATCC 10231 biofilms and inhibited the formation of C. albicans biofilms at a sub‐MIC concentration. Conclusions: Essential oils of Boswellia spp. could effectively inhibit the growth of biofilms of medical relevance. Significance and Impact of the Study: Boswellia spp. essential oils represent an interesting source of anti‐microbial agents in the development of new strategies to prevent and treat biofilms.     

A partial proteome reference map of the wine lactic acid bacterium Oenococcus oeni ATCC BAA-1163

Oenococcus oeni is the main lactic acid bacterium that carries out the malolactic fermentation in virtually all red wines and in some white and sparkling wines. Oenococcus oeni possesses an array of metabolic activities that can modify the taste and aromatic properties of wine. There is, therefore, industrial interest in the proteins involved in these metabolic pathways and related transport systems of this bacterium. In this work, we report the characterization of the O. oeni ATCC BAA-1163 proteome. Total and membrane protein preparations from O. oeni were standardized and analysed by two-dimensional gel electrophoresis. Using tandem mass spectrometry, we identified 224 different spots corresponding to 152 unique proteins, which have been classified by their putative function and subjected to bioinformatics analysis.     

Oenococcus oeni preference for peptides: qualitative and quantitative analysis of nitrogen assimilation

Optimization of malolactic fermentation in wine depends mainly on better understanding of nitrogen nutritional requirements of Oenococcus oeni. Four widely used starter strains and the reference ATCC BAA-1163 strain were grown in media containing different N sources: free amino acids, oligopeptides (0.5–10 kDa) or polypeptides (> 10 kDa). Amino acid auxotrophies were determined by the single omission technique. The tested strains were indifferent to only two to four amino acids and two of the starter strains appeared to be particularly demanding. Nitrogen consumption was investigated and a significant level of nitrogen was consumed by O. oeni only in the free amino acid medium. In media containing complex nitrogen sources, a global balance above 5 mg N l⁻¹ was enough to ensure biomass formation of all tested strains. Moreover, for all strains, bacterial growth yield was higher in the presence of nitrogen from peptides than that from free amino acids. However, no direct relationship between the bacterial growth level and the amount of nitrogen metabolized could be established. These findings were discussed in relation to the physiology of wine malolactic bacteria.     

High-throughput screening and characterization of xylose-utilizing, ethanol-tolerant thermophilic bacteria for bioethanol production

Aims: To develop a high‐throughput assay for screening xylose‐utilizing and ethanol‐tolerant thermophilic bacteria owing to their abilities to be the promising ethanologens. Methods and Results: Based on alcohol oxidase and peroxidase‐coupled enzymatic reaction, an assay was developed by the formation of the coloured quinonimine to monitor the oxidation of ethanol in the reaction and calculate the concentration of ethanol. This assay was performed in 96‐well microtitre plate in a high‐throughput and had a well‐linear detection range of ethanol from 0 up to 2·5 g l^?1 with high accuracy. The assay was then verified by screening soil samples from hot spring for xylose‐utilizing and ethanol production at 60°C. Three isolates LM14‐1, LM14‐5 and LM18‐4 with 3–5% (v/v) ethanol tolerance and around 0·29–0·38 g g^?1 ethanol yield from xylose were obtained. Phylogenetic and phenotypic analysis showed that the isolates clustered with members of the genus Bacillus or Geobacillus subgroup. Conclusions: The developed double enzyme‐coupled, high‐throughput screening system is effective to screen and isolate xylose‐utilizing, ethanol‐producing thermophilic bacteria for bioethanol production at the elevated temperature. Significance and Impact of the Study: Our research presented a novel high‐throughput method to screen thermophilic bacteria for producing ethanol from xylose. This screening method is also very useful to screen all kinds of ethanologens either from natural habitats or from mutant libraries, to improve bioethanol production from lignocellulosic feedstocks.     

Enhanced solid‐state citric acid bio‐production using apple pomace waste through surface response methodology

Aims: To evaluate the potential of apple pomace (AP) supplemented with rice husk for hyper citric acid production through solid‐state fermentation by Aspergillus niger NRRL‐567. Optimization of two key parameters, such as moisture content and inducer (ethanol and methanol) concentration was carried out by response surface methodology. Methods and Results: In this study, the effect of two crucial process parameters for solid‐state citric acid fermentation by A. niger using AP waste supplemented with rice husk were thoroughly investigated in Erlenmeyer flasks through response surface methodology. Moisture and methanol had significant positive effect on citric acid production by A. niger grown on AP (P < 0·05). Higher values of citric acid on AP by A. niger (342·41 g kg^?1 and 248·42 g kg^?1 dry substrate) were obtained with 75% (v/w) moisture along with two inducers [3% (v/w) methanol and 3% (v/w) ethanol] with fermentation efficiency of 93·90% and 66·42%, respectively depending upon the total carbon utilized after 144 h of incubation period. With the same optimized parameters, conventional tray fermentation was conducted. The citric acid concentration of 187·96 g kg^?1 dry substrate with 3% (v/w) ethanol and 303·34 g kg^?1 dry substrate with 3% (v/w) methanol were achieved representing fermentation efficiency of 50·80% and 82·89% in tray fermentation depending upon carbon utilization after 120 h of incubation period. Conclusions: Apple pomace proved to be the promising substrate for the hyper production of citric acid through solid‐state tray fermentation, which is an economical technique and does not require any sophisticated instrumentation. Significance and Impact of the Study: The study established that the utilization of agro‐industrial wastes have positive repercussions on the economy and will help to meet the increasing demands of citric acid and moreover will help to alleviate the environmental problems resulting from the disposal of agro‐industrial wastes.     

Combined cold, acid, ethanol shocks in Oenococcus oeni: Effects on membrane fluidity and cell viability

The effects of combined cold, acid and ethanol on the membrane physical state and on the survival of Oenococcus oeni were investigated. Membrane fluidity was monitored on intact whole O. oeni cells subjected to single and combined cold, acid and ethanol shocks by using fluorescence anisotropy with 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe. Results showed that cold shocks (14 and 8 °C) strongly rigidified plasma membrane but did not affect cell survival. In contrast, ethanol shocks (10-14% v/v) induced instantaneous membrane fluidisation followed by rigidification and resulted in low viability. Acid shocks (pH 4.0 and pH 3.0) exerted a rigidifying effect on membrane without affecting cell viability. Whatever the shock orders, combined cold (14 °C) and ethanol (14% v/v) shocks resulted in strong membrane rigidification. Interestingly, O. oeni survived combined cold and ethanol shocks more efficiently than single ethanol shock. Membrane rigidification was induced by ethanol-and-acid (10% v/v - pH 3.5) shock and correlated with total cell death. In contrast, O. oeni recovered its viability when subjected to cold (8 °C)-then-ethanol-and-acid shock which strongly rigidified the membrane. Our results suggested a positive short-term effect of combined cold, acid and ethanol shocks on membrane fluidity and viability of O. oeni.     

Damage of staphylococcal cytoplasmic membrane by Quercus infectoria G. Olivier and its components

Aims: In traditional Thai medicine, nutgall of Quercus infectoria G. Olivier is well‐documented as an effective agent for wound and skin infections. The present study was aimed to establish modes of action of the ethanol extract of the plant as well as its main constituents to induce anti‐methicillin‐resistant Staphylococcus aureus (MRSA) activity. Methods and Results: The minimal inhibitory concentration (MIC)/minimal bactericidal concentration (MBC) values of ethyl acetate I, ethyl acetate II, 95% ethanol and 30% ethanol fractions against MRSA were 0·06/0·25, 0·13/0·25, 0·25/0·5 and 0·5/1·00 mg ml^?1, respectively. Ellagic acid, gallic acid, syringic acid and tannic acid as major components of Q. infectoria nutgall extract were included in this study. Among these, gallic acid and tannic acid demonstrated good MIC/MBC values at 0·06/0·06 and 0·13/0·25 mg ml^?1, respectively. A lysis experiment demonstrated that the ethanol extract, ethyl acetate fraction I and all of the main components failed to lyse MRSA cells. In contrast, both MRSA and Staph. aureus ATCC 25923 treated with the ethanol extract, ethyl acetate fraction I, gallic acid and tannic acid displayed significant loss of tolerance to low osmotic pressure and high salt concentration. Conclusions: The results documented the effect of different fractions of Q. infectoria and purified compounds on MRSA and Staph. aureus. In addition, the study demonstrated that treatment with Q. infectoria extract and the purified compounds results in hypersensitivity to low and high osmotic pressure. Significance and Impact of the Study: This study provides scientific information to support the traditional uses of the nutgall extract and suggesting its anti‐MRSA mechanisms.     

Ethanol fermentation with Kluyveromyces marxianus from Jerusalem artichoke grown in salina and irrigated with a mixture of seawater and freshwater

Aims: To study fuel ethanol fermentation with Kluyveromyces marxianus ATCC8554 from Jerusalem artichoke (Helianthus tuberosus) grown in salina and irrigated with a mixture of seawater and freshwater. Methods and Results: The growth and ethanol fermentation of K. marxianus ATCC8554 were studied using inulin as substrate. The activity of inulinase, which attributes to the hydrolysis of inulin, the main carbohydrate in Jerusalem artichoke, was monitored. The optimum temperatures were 38°C for growth and inulinase production, and 35°C for ethanol fermentation. Aeration was not necessary for ethanol fermentation with the K. marxianus from inulin. Then, the fresh Jerusalem artichoke tubers grown in salina and irrigated with 25% and 50% seawater were further examined for ethanol fermentation with the K. marxianus, and a higher ethanol yield was achieved for the Jerusalem artichoke tuber irrigated with 25% seawater. Furthermore, the dry meal of the Jerusalem artichoke tubers irrigated with 25% seawater was examined for ethanol fermentation at three solid concentrations of 200, 225 and 250 g l^?1, and the highest ethanol yield of 0·467, or 91·5% of the theoretical value of 0·511, was achieved for the slurry with a solid concentration of 200 g l^?1. Conclusions: Halophilic Jerusalem artichoke can be used for fuel ethanol production. Significance and Impact of the Study: Halophilic Jerusalem artichoke, not competing with grain crops for arable land, is a sustainable feedstock for fuel ethanol production.     

Isolation and characterization of Oenococcus oeni from Aglianico wines

A technological characterization of Oenococcus oeni strains isolated from Aglianico wines was performed to select starter cultures for malolactic fermentation (MLF). One hundred and fifty six O. oeni isolates were extracted from Aglianico wines, and identified by using species-specific PCR. Malolactic activity (MLA), sulphur dioxide (SO₂) resistance, acetaldehyde metabolism and other technological characteristics were tested. Differences in the technologically relevant characteristics were observed. All O. oeni strains were able to grow at low temperature and none in presence of 14% of ethanol. About 80% of O. oeni degraded more than 80% of acetaldehyde, producing ethanol and acetic acid as final products. Among nine O. oeni chosen, four isolates were sensitive to 60 mg of SO₂ l⁻¹, while the other five had high resistance. Considering their technological characteristics, five O. oeni strains could be selected starter cultures for MLF in Aglianico.     

Inactivation of Cronobacter spp. (Enterobacter sakazakii) in infant formula using lactic acid,copper sulfate and monolaurin

Aims: To investigate the effect of lactic acid (LA), copper (II), and monolaurin as natural antimicrobials against Cronobacter in infant formula. Methods and Results: The effect of LA (0·1, 0·2 and 0·3% v/v), copper (II) (10, 50 and 100 μg ml^?1) and monolaurin (1000, 2000, and 3000 μg ml^?1) suspended into tween‐80? or dissolved in ethanol against Cronobacter in infant formula was investigated. Reconstituted infant formula and powdered infant formula were inoculated with five strains of Cronobacter spp. at the levels of c. 1 × 10⁶ CFU ml^?1 and 1 × 10³ CFU g^?1, respectively. LA at 0·2% v/v had a bacteriostatic effect on Cronobacter growth, whereas 0·3% v/v LA resulted in c. 3 log₁₀ reduction. Copper (II) at the levels of 50 μg ml^?1 and 100 μg ml^?1 elicited c. 1 and 2 log₁₀ reductions, respectively. The combination of 0·2% LA and 50 μg ml^?1 copper (II) resulted in a complete elimination of the organism. Monolaurin exhibited a slight inhibitory activity against Cronobacter (c. 1·5 log₁₀ difference) compared to the control when ethanol was used to deliver monolaurin. Conclusions: A complete elimination of Cronobacter was obtained when a combination of sublethal concentrations of LA (0·2%) and copper (II) (50 μg ml^?1) was used. Significance and Impact of the Study: The use of the synergistic interactive combination of LA and copper (II) could be beneficial to control Cronobacter in the infant formula industry.     

Ethanol synthesis from glycerol by Escherichia coli redox mutants expressing adhE from Leuconostoc mesenteroides

Aims: Analysis of the physiology and metabolism of Escherichia coli arcA and creC mutants expressing a bifunctional alcohol‐acetaldehyde dehydrogenase from Leuconostoc mesenteroides growing on glycerol under oxygen‐restricted conditions. The effect of an ldhA mutation and different growth medium modifications was also assessed. Methods and Results: Expression of adhE in E. coli CT1061 [arcA creC(Con)] resulted in a 1·4‐fold enhancement in ethanol synthesis. Significant amounts of lactate were produced during micro‐oxic cultures and strain CT1061LE, in which fermentative lactate dehydrogenase was deleted, produced up to 6·5 ± 0·3 g l^?1 ethanol in 48 h. Escherichia coli CT1061LE derivatives resistant to >25 g l^?1 ethanol were obtained by metabolic evolution. Pyruvate and acetaldehyde addition significantly increased both biomass and ethanol concentrations, probably by overcoming acetyl‐coenzyme A (CoA) shortage. Yeast extract also promoted growth and ethanol synthesis, and this positive effect was mainly attributable to its vitamin content. Two‐stage bioreactor cultures were conducted in a minimal medium containing 100 μg l^?1 calcium d ‐pantothenate to evaluate oxic acetyl‐CoA synthesis followed by a switch into fermentative conditions. Ethanol reached 15·4 ± 0·9 g l^?1 with a volumetric productivity of 0·34 ± 0·02 g l^?1 h^?1. Conclusions: Escherichia coli responded to adhE over‐expression by funnelling carbon and reducing equivalents into a highly reduced metabolite, ethanol. Acetyl‐CoA played a key role in micro‐oxic ethanol synthesis and growth. Significance and Impact of the Study: Insight into the micro‐oxic metabolism of E. coli growing on glycerol is essential for the development of efficient industrial processes for reduced biochemicals production from this substrate, with special relevance to biofuels synthesis.     

Conversion of biomass hydrolysates and other substrates to ethanol and other chemicals by Lactobacillus buchneri*

Aims: A Lactobacillus buchneri strain NRRL B‐30929 can convert xylose and glucose into ethanol and chemicals. The aims of the study were to survey three strains (NRRL B‐30929, NRRL 1837 and DSM 5987) for fermenting 17 single substrates and to exam NRRL B‐30929 for fermenting mixed substrates from biomass hydrolysates. Methods and Results: Mixed acid fermentation was observed for all three L. buchneri strains using various carbohydrates; the only exception was uridine which yielded lactate, acetate and uracil. Only B‐30929 is capable of utilizing cellobiose, a desired trait in a potential biocatalyst for biomass conversion. Flask fermentation indicated that the B‐30929 strain can use all the sugars released from pretreated hydrolysates, and producing 1·98–2·35 g l^?1 ethanol from corn stover hydrolysates and 2·92–3·01 g l^?1 ethanol from wheat straw hydrolysates when supplemented with either 0·25× MRS plus 1% corn steep liquor or 0·5× MRS. Conclusions: The L. buchneri NRRL B‐30929 can utilize mixed sugars in corn stover and wheat straw hydrolysates for ethanol and other chemical production. Significance and Impact of the Study: These results are valuable for future research in engineering L. buchneri NRRL B‐30929 for fermentative production of ethanol and chemicals from biomass.     

Characterization of lactic acid bacteria from musts and wines of three consecutive vintages of Ribeira Sacra

Aims: This study was designed to isolate and characterize the lactic acid microbiota of the musts and wines of a young denomination of origin area, Ribeira Sacra in north‐west Spain. Methods and Results: Over three consecutive years (2007, 2008 and 2009), we examined musts and wines from four cellars in different zones of the region. Through biochemical and genetic tests, 459 isolates of lactic acid bacteria (LAB) were identified as the following species: Lactobacillus alvei (0·7%), Lactobacillus brevis (1·7%), Lactobacillus frumenti (0·9%), Lactobacillus kunkeei (12%), Lactobacillus plantarum (6·5%), Lactobacillus pentosus (0·9%), Lactococcus lactis ssp. lactis (3%), Leuconostoc citreum (0·7%), Leuconostoc fructosum (synon. Lactobacillus fructosum) (3·7%), Leuconostoc mesenteroides ssp. mesenteroides (2·8%), Leuconostoc pseudomesenteroides (0·2%), Oenococcus oeni (59%), Pediococcus parvulus (7%) and Weisella paramesenteroides (synon. Leuconostoc paramesenteroides) (0·9%). Of these species, O. oeni was the main one responsible for malolactic fermentation (MLF) in all cellars and years with the exception of Lact. plantarum, predominant in 2007, in one cellar, and Lact. brevis, Lact. frumenti and Ped. parvulus coexisting with O. oeni in one cellar in 2009. Different strains (84) of LAB species (14) were identified by biochemical techniques (API strips, the presence of plasmids, enzyme activities and MLF performance) and molecular techniques (PCR). All assays were carried out with every one of the 459 isolates. To select candidates for use as culture starters, we assessed malolactic, β‐glucosidase and tannase activities, the presence of genes involved in biogenic amine production and plasmid content. Conclusions: A high diversity of LAB is present in the grape musts of Ribeira Sacra but few species are responsible for MLF; however, different strains of such species are involved in the process. As far as we are aware, this is the first report of Lact. frumenti thriving in wine. Significance and Impact of the Study: Information on LAB populations in must and wine is presented. A large collection of well‐characterized strains of LAB are available as starter cultures to winemakers.     

Technological properties of Oenococcus oeni strains isolated from typical southern Italian wines

Aims: To isolate indigenous Oenococcus oeni strains suitable as starters for malolactic fermentation (MLF), using a reliable polyphasic approach. Methods and Results: Oenococcus oeni strains were isolated from Nero di Troia wines undergoing spontaneous MLF. Samples were taken at the end of alcoholic fermentation and during MLF. Wine samples were diluted in a sterile physiological solution and plated on MRS and on modified FT80. Identification of O. oeni strains was performed by a polymerase chain reaction (PCR) experiment using strain‐specific primers. Strains were further grouped using a multiplex RAPD‐PCR analysis. Then, six strains were inoculated in two wine‐like media with two different ethanol concentrations (11 and 13% vol/vol) with a view to evaluate their capacity to grow and to perform MLF. In addition, a quantitative PCR (qRT‐PCR) approach was adapted to monitor the physiological state of the strains selected. Conclusion: A positive correlation between the malolactic activity performance and the ability to develop and tolerate stress conditions was observed for two selected O. oeni strains. Significance and Impact of the Study: The results reported are useful for the selection of indigenous MLF starter cultures with desired oenological traits from typical regional wines. It should be the base for the improvement in organoleptic quality of typical red wine.     

Improved ethanol production of a newly isolated thermotolerant Saccharomyces cerevisiae strain after high-energy-pulse-electron beam

Aims: To isolate thermotolerant Saccharomyces cerevisiae with high‐energy‐pulse‐electron (HEPE) beam, to optimize the mutation strain fermentation conditions for ethanol production and to conduct a preliminary investigation into the thermotolerant mechanisms. Methods and Results: After HEPE beam radiation, the thermotolerant S. cerevisiae strain Y43 was obtained at 45°C. Moreover, the fermentation conditions of mutant Y43 were optimized by L3³ orthogonal experiment. The optimal glucose content and initial pH for fermentation were 20% g l^?1 and 4·5, respectively; peptone content was the most neglected important factor. Under this condition, ethanol production of Y43 was 83·1 g l^?1 after fermentation for 48 h at 43°C, and ethanol yield was 0·42 g g^?1, which was about 81·5% of the theoretical yield. The results also showed that the trehalose content and the expression of the genes MSN2, SSA3 and TPS1 in Y43 were higher than those in the original strain (YE0) under the same stress conditions. Conclusions: A genetically stable mutant strain with high ethanol yield under heat stress was obtained using HEPE. This mutant may be a suitable candidate for the industrial‐scale ethanol production. Significance and Impact of the Study: High‐energy‐pulse‐electron radiation is a new efficient technology in breeding micro‐organisms. The mutant obtained in this work has the advantages in industrial ethanol production under thermostress.     

Genetic manipulation tools for Dietzia spp.

Aim: To develop an applicable vector system and a transformation method for the manipulation of Dietzia spp. Methods and Results: The pNV18 Nocardia‐E. coli shuttle vector was tested and found to be a replicating plasmid in Dietzia sp. E1. With the use of pNV18, an electroporation method was optimized for the transformation of Dietzia sp. E1, and a transformation efficiency suitable for genetic manipulations was achieved (2·18 × 10⁴ transformants μg^?1 DNA). The method was also applied for the transformation of Dietzia cinnamea, D. maris, D. natronolimnaea and D. psychralcaliphila. Conclusions: The first applicable vectors and a simple electroporation protocol enabling the manipulation of several Dietzia spp. are presented. Significance and Impact of the Study: Dietzia spp. have clinical, industrial and great environmental importance; however, the analysis of the Dietzia genus is currently hampered by the lack of manipulation techniques. The presented basic tools allow the genetic analysis of several Dietzia species, including the human disease‐associated Dietzia maris.