Yeast-Bacteria Coinoculation

2019

Abstract Yeasts and lactic acid bacteria (LAB) coinoculation is the simultaneous, or close in time inoculation, of both types of microorganisms in winemaking. Coinoculation has been used mainly to early accomplish malolactic fermentation (MLF), employing generally the couples Oenococcus oeni/Saccharomyces cerevisiae and Lactobacillus plantarum/S. cerevisiae. Early completion of MLF decreases the overall vinification time, reduces the microbial spoilage risk, and even prevents the biogenic amines synthesis. LAB/yeasts coinoculation could be also used to increase the acidity of wines when inoculated in grape must, using sugar to synthesize lactic acid and producing ethanol diminution. Coinocu…

A novel culture medium for Oenococcus oeni malolactic starter production

2015

Malolactic fermentation is essential in wine quality. One of the strategies used to control this fermentation involves the inoculation of selected lactic acid bacteria, mainly Oenococcus oeni. Laboratory media usually produce large amounts of biomass, but with little or no adaptability to wine. We propose a culture medium to grow and pre-adapt O. oeni cells, and the steps to scale-up production. To achieve this objective, 27 different media were tested. All contained grape must and wine, and nutritional supplements in order to benefit bacterial growth. Those media contained different ethanol levels, pH values, and grape must concentrations. The optimized culture medium named Oenococcus Prod…

Ability of Kocuria varians LTH 1540 To Degrade Putrescine: Identification and Characterization of a Novel Amine Oxidase.

2015

This work describes the identification and characterization of an amine oxidase from Kocuria varians LTH 1540 (syn. Micrococcus varians) primarily acting on putrescine. Data from MALDI-TOF MS/MS and the identification of Δ(1)-pyrroline as degradation product from putrescine indicate that the enzyme is a flavin-dependent putrescine oxidase (PuO). Properties of partially purified enzyme have been determined. The enzyme oxidizes diamines, putrescine and cadaverine, and, to a lesser extent, polyamines, such as spermidine, but not monoamines. The kinetic constants (Km and Vmax) for the two major substrates were 94 ± 10 μM and 2.3 ± 0.1 μmol/min·mg for putrescine and 75 ± 5 μM and 0.15 ± 0.02 μmo…

Factors affecting the production of putrescine from agmatine by Lactobacillus hilgardii X1B isolated from wine

2008

Aims: To elucidate and characterize the metabolic putrescine synthesis pathway from agmatine by Lactobacillus hilgardii X1B. Methods and Results: The putrescine formation from agmatine by resting cells (the normal physiological state in wine) of lactic acid bacteria isolated from wine has been determined for the first time. Agmatine deiminase and N-carbamoylputrescine hydrolase enzymes, determined by HPLC and LC-Ion Trap Mass Spectrometry, carried out the putrescine synthesis from agmatine. The influence of pH, temperature, organic acids, amino acids, sugars and ethanol on the putrescine formation in wine was determined. Conclusions: Resting cells of Lact. hilgardii X 1B produce putrescine …

Immobilisation of yeasts on oak chips or cellulose powder for use in bottle-fermented sparkling wine

2019

[EN] Sparkling wine production comprises two successive fermentations performed by Sacharomyces cerevisiae strains. This research aimed to: develop yeast immobilisation processes on two wine-compatible supports; study the effects of yeast type (IOC 18-2007 and 55A) and the immobilisation support type (oak chips and cellulose powder) on the fermentation kinetics, the deposition rate of lees and the volatile composition of the finished sparkling wine; compare the fermentation parameters of the wines inoculated with immobilised or non-immobilised cells. Proper immobilisation of yeast on oak chips and cellulose powder was demonstrated by electron microscopy. Total sugar consumption occurred in …

Influence of yeast strains on managing wine acidity using Lactobacillus plantarum

2018

Abstract Lactobacillus plantarum has been used for deacidifying wines through malolactic fermentation (MLF), and more recently for acidifying them. The species Oenococcus oeni is mainly responsible for MLF in wines at a pH below 3.5. However, the Lactobacillus and Pediococcus species can carry out this reaction in wines at a higher pH. MLF generally begins once yeasts have completed alcoholic fermentation, but nowadays some winemakers prefer carrying out MLF simultaneously to alcoholic fermentation. The advantages of this strategy are shorter times in which to complete wine vinification and better wine color stabilization. Lactobacillus plantarum is preferred to O. oeni for performing early…

Aurintricarboxylic acid as a nuclease inhibitor in fungal protoplasts

1986

Aurintricarboxylic acid (ATA), a potent nuclease inhibitor, has been shown to prevent foreign DNA degradation in transformation of Penicillium chrysogenum and Saccharomyces cerevisiae. ATA may be useful not only in vitro but also in vivo.

Biogenic amines in wines from three Spanish regions.

2005

One hundred and sixty-three wines from La Rioja, Utiel-Requena, and Tarragona were analyzed to determine if there were any differences in the concentrations of six biogenic amines that are found in these three regions. The influence of grape variety, type of vinification, wine pH, malolactic fermentation, and storage in bottle on biogenic amine concentrations was studied. Results show important differences in putrescine and histamine concentrations among regions, varieties of grape, and type of wine; differences were less appreciable for the remaining biogenic amines studied. Low pH prevented biogenic amine formation. Malolactic fermentation and short storage periods in bottle (3-6 months) …

Penicillium auxotrophic mutants can be detected by suing xanthene dyes

1987

Auxotrophic mutants ofPenicillium spp. have been directly isolated after mutagenic treatment from agar plates containing Xanthene dyes. They grow as characteristic small colored colonies. Some strains were tested and they showed a differential response depending on the Xanthene dye used.

The effects of freezing and freeze-drying ofOenococcus oeniupon induction of malolactic fermentation in red wine

2000

Summary The use of Oenococcus oeni starter cultures for the induction of malolactic fermentation (MLF) in wine permits control over the timing of the process and the quality of the wine. Successful inoculation of bacterial starter cultures into wine depends on the selection of suitable strains and on the preparation and conservation of those cultures. Medium for Leuconostoc oenos (MLO) is the best medium for easy and rapid growth of O. oeni cultures under laboratory controlled conditions for isolation and identification. However, this study showed that O. oeni cells inoculated in MLO failed to induce MLF in wine while cells grown in Medium of Preculture (MP) or wine, stored at −20 °C or fre…

Conjugative plasmid pIP501 undergoes specific deletions after transfer from Lactococcus lactis to Oenococcus oeni

2003

Conjugal transfer of plasmids pIP501 and its derivative pVA797 from Lactococcus lactis to Oenococcus oeni was assayed by filter mating. Plasmid pIP501 was transferred to a number of O. oeni strains whereas a single transconjugant of O. oeni M42 was recovered when pVA797 was used. Physical analysis of the transconjugant plasmids revealed that pIP501 and pVA797 underwent extensive deletions in O. oeni that affected the tra region (conjugal transfer) and SegB region (stability). All derivatives showed segregational instability in O. oeni, but were stably maintained in L. lactis. These differences correlated with the different plasmid copy numbers and the extent of deletions within the SegB reg…

Lactobacillus aquaticus sp. nov., isolated from a Korean freshwater pond.

2009

A Lactobacillus strain, IMCC1736T, was isolated recently from a Korean freshwater pond following an extensive study of the microbial community in this ecosystem. Its 16S rRNA gene was sequenced and phylogenetic analysis placed this strain within the Lactobacillus salivarius group, closely related to Lactobacillus satsumensis NRIC 0604T, with 97.9% sequence similarity. In the present work, the taxonomic status of strain IMCC1736T has been re-evaluated. It was characterized phylogenetically, genotypically and phenotypically and, based on DNA-DNA hybridization values, this strain represents a novel Lactobacillus species. Strain IMCC1736T can be differentiated genotypically from its closest rel…

NAD(P)H regeneration is the key for heterolactic fermentation of hexoses in Oenococcus oeni

2002

Oenococcus oeni (formerly Leuconostoc oenos) can perform malolactic fermentation, converting L-malate to L-lactate and carbon dioxide, in wines. The energy and redox potential required to support the growth of the micro-organism are supplied mainly by the consumption of carbohydrates via the heterolactic pathway. In the first steps of hexose metabolism two molecules of NAD(P)(+) are consumed, which must be regenerated in later reactions. The aim of this work was to test if aerobic growth of O. oeni promotes higher cell yields than anaerobic conditions, as has been shown for other lactic acid bacteria. O. oeni M42 was found to grow poorly under aerobic conditions with glucose as the only car…

Erwinia piriflorinigrans sp. nov., a novel pathogen that causes necrosis of pear blossoms

2010

Eight Erwinia strains, isolated from necrotic pear blossoms in València, Spain, were compared with reference strains of Erwinia amylovora and Erwinia pyrifoliae, both of which are pathogenic to species of pear tree, and to other species of the family Enterobacteriaceae using a polyphasic approach. Phenotypic analyses clustered the novel isolates into one phenon, distinct from other species of the genus Erwinia, showing that the novel isolates constituted a homogeneous phenotypic group. Rep-PCR profiles, PCR products obtained with different pairs of primers and plasmid contents determined by restriction analysis showed differences between the novel strains and reference strains of E. amylovo…

Effect of micro-oxygenation on the evolution of aromatic compounds in wines: Malolactic fermentation and ageing in wood

2009

Abstract The effect of micro-oxygenation on the aromas of two varietal wines (Tempranillo and Cabernet Sauvignon) when alcoholic fermentation is complete was examined. Several factors, such as whether malolactic fermentation (MLF) took place in barrels or stainless steel vats and ageing time in oak barrels, were taken into account. Major aroma components were studied using GC-FID and minor compounds were studied by GC–MS analysis before and after MLF and after 4 and 8 months of maturation in oak barrels. Sensorial analysis was performed to describe and quantify the different wine notes. Micro-oxygenation affects different compounds but the effect depends to a great extent on the grape varie…

Improved detection and enumeration of yeasts in wine by Cells-qPCR

2018

Abstract Quantitative PCR by directly sampling (Cells-qPCR) has been adapted to detect and quantify total yeasts, and B. bruxellensis, S. cerevisiae and Z. bailii species, in grape musts and wines. To increase assay sensitivity, the effects of a previous cell wall lysis, by both enzymatic and mechanical methods, were evaluated. Cell wall disruption by mechanical methods showed the best results to enhance assay sensitivity. Numerous standard curves were constructed by mechanically lysed cells in culture medium, and in white and red grape musts and wines. Good regression values (>0.99) and efficiencies (>0.99) were obtained, and it was possible to detect one single cell per reaction in all th…

A polyphasic approach in order to identify dominant lactic acid bacteria during pasta manufacturing

2010

Using a polyphasic approach, we have isolated and identified, lactic acid bacteria (LAB) in samples directly collected from an artisanal pasta-making manufactory located in Puglia (South Italy). Samples were collected during several steps of pasta processing and LAB were isolated on MRS and M17 plates. Furthermore, strains were grouped in a total of eight species by means of randomly amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) typing and 16S rDNA sequencing. The majority of strains were identified as belonging to Pediococcus pentosaceus and Enterococcus faecium species. The remaining strains were characterized and assigned to Weissella confusa, Pediococcus acidilactici,…

Biogenic amine determination in wine fermented in oak barrels: Factors affecting formation

2008

Changes in biogenic amines (histamine, putrescine, tyramine and cadaverine) were monitored during the industrial production of Tempranillo and Cabernet Sauvignon wines. The origin of these amines in relation to the presence of different lactic bacteria (indigenous or inoculated) during malolactic fermentation, while aging in oak barrel, and the correlations between amines and their corresponding amino acids were statistically evaluated. In this study we found that there was a greater increase in amines post malic acid depletion than during malolactic fermentation and that more amines were produced in wine of the Tempranillo variety. The total amino acid concentration was 1.6 times greater i…

Tyramine and phenylethylamine production among lactic acid bacteria isolated from wine.

2007

The ability of wine lactic acid bacteria to produce tyramine and phenylethylamine was investigated by biochemical and genetic methods. An easy and accurate plate medium was developed to detect tyramine-producer strains, and a specific PCR assay that detects the presence of tdc gene was employed. All strains possessing the tdc gene were shown to produce tyramine and phenylethylamine. Wines containing high quantities of tyramine and phenylethylamine were found to contain Lactobacillus brevis or Lactobacillus hilgardii. The main tyramine producer was L. brevis. The ability to produce tyramine was absent or infrequent in the rest of the analysed wine species.

The use of core-shell high-performance liquid chromatography column technology to improve biogenic amine quantification in wine

2015

BACKGROUND HPLC column technology has been improved, providing better resolution of closely eluting compounds, better analyte sensitivity, and shorter analysis times. The core-shell technology columns offer a faster analysis through the use of shorter columns without compromising resolution. The aim of this work was to improve the methods for determination of biogenic amines (BAs) in wine using the new HPLC PFP core-shell column technology. RESULTS Two different elution programs were designed to quantify BAs with the core-shell PFP column. Program I flow rate was 2 mL min−1. The total elution time was 10 min. In elution program II, the flow rate was 0.8 mL min−1 and the total elution time w…

Nucleotide sequence of plasmid p4028, a cryptic plasmid from Leuconostoc oenos.

1996

Abstract TheLeuconostoc oenosplasmid p4028 was cloned in pBlueScript (SK+), and its complete nucleotide sequence was determined. The analysis of the nucleotide sequence revealed five open reading frames, all of them located on the same strand and grouped in two clusters separated by a short noncoding stretch. A similarity search against the other sequences deposited in the EMBL and GenBank databases showed that p4028 has no significant similarity with any of the sequences checked. Nevertheless, a putative ATP-binding motif was found in ORF2. A more detailed analysis of this ORF suggests that it could encode for a DNA-dependent ATPase.

The potential of positively-charged cellulose sponge for malolactic fermentation of wine, using Oenococcus oeni

2001

Malolactic fermentation (MLF) is a secondary bioconversion developed in some wines involving malic acid decarboxylation. The induction of MLF in wine by cultures of free and immobilized Oenococcus oeni cells was investigated. This work reports on the effect of surface charges in the immobilization material, a recently described fibrous sponge, as well as the pH and the composition of the media where cells are suspended. A chemical treatment provided positive charge to the sponges (DE or DEAE) and gave the highest cell loadings and subsequent resistance to removal. Preculture media to grow the malolactic bacteria before the immobilization procedure were also evaluated. We have established fa…

16S-ARDRA, a tool for identification of lactic acid bacteria isolated from grape must and wine.

2003

Lactic acid bacteria (LAB) are found in a great variety of habitats, including grape must and wines. There is a close relationship between the species of LAB which develop during fermentation and the eventual quality of the wine. For these reasons analytical techniques allowing fast and reliable identification of wine LAB are needed. In this work a simple and accurate protocol for identifying species of LAB isolated from grape must and wine is presented. This protocol is based on the amplification, directly from colony, of 16S rDNA and later digestion with one of the following restriction enzymes BfaI, MseI and AluI. A sequential use of the three enzymes is proposed to simplify LAB wine ide…

Polyphasic study of wine Lactobacillus strains: taxonomic implications

2005

One hundred and seventy-eight lactobacilli isolated from wine were characterized by a polyphasic approach. Strains were phenotypically identified at genus and species level by classical tests including the analysis of cell morphology, homo/heterofermentative character, sugar fermentation patterns, growth at different temperatures and the optical nature of the isomer of lactic acid produced from glucose. Molecular techniques such as random amplification of polymorphic DNA (RAPD), amplified 16S rDNA restriction analysis (16S-ARDRA), PFGE-RFLP and ribotyping were used to characterize strains, and their potential for identification and/or typing was evaluated. The information obtained with thes…

Lactobacillus uvarum sp. nov. - A new lactic acid bacterium isolated from Spanish Bobal grape must

2008

Five strains isolated from grape musts in Spain in 1997, have been characterized by several molecular techniques, and three of them have been identified as pertaining to a new species. All strains are Gram-positive rods, aerotolerant and homofermentative bacteria that do not exhibit catalase activity. Phylogenetic analysis based on 16S rRNA gene sequences placed these strains within the genus Lactobacillus, closely related to Lactobacillus mali. DNA-DNA hybridization experiments confirmed that strain 71 belongs to the lately described species L. satsumensis, strain 88 belongs to L. mali and the other three isolates have an independent status at species level. Restriction analysis of the amp…

Lactobacillus oeni sp. nov., from wine.

2009

Ten Lactobacillus strains, previously isolated from different Bobal grape wines from the Utiel-Requena Origin Denomination of Spain, were characterized phylogenetically, genotypically and phenotypically. The 16S rRNA genes were sequenced and phylogenetic analysis showed that they form a tight phylogenetic clade that is closely related to reference strains Lactobacillus satsumensis NRIC 0604T, ‘Lactobacillus uvarum’ 8 and Lactobacillus mali DSM 20444T. DNA–DNA hybridization results confirmed the separation of the strains from other Lactobacillus species. Genotypically, the strains could be differentiated from their closest neighbours by 16S amplified rDNA restriction analysis and random ampl…

Acetobacter musti sp. nov., isolated from Bobal grape must

2016

An acetic acid bacterium (strain Bo7T), obtained during a study of the microbial diversity of spontaneous fermentations of Bobal grape must, was subjected to a taxonomic study using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences allocated strain Bo7T to the genus Acetobacter, and revealed Acetobacter aceti and Acetobacter oeni to be nearest neighbours (99.57 % 16S rRNA gene sequence similarity between strain Bo7T and A. oeni CECT 5830T, and 98.76 % between strain Bo7T and A. aceti CECT 298T). Cells of strain Bo7T are Gram-negative, motile rods, catalase-positive and oxidase-negative. The DNA G+C content of strain Bo7T was 58.0 mol%. DNA–DNA hybridizations demo…

Regulation of hdc expression and HDC activity by enological factors in lactic acid bacteria.

2008

Aims:  The aim of this work was to study the influence of enological factors on the histidine decarboxylase gene (hdc) expression and on histidine decarboxylase enzyme (HDC) activity in Lactobacillus hilgardii, Pediococcus parvulus and Oenococcus oeni. Methods and Results:  Cell extracts and whole cells were used. Glucose, fructose, malic acid and citric acid diminished the hdc expression. Ethanol did not increase hdc expression or activity in cells, but increased HDC activity. Temperature and pH had effect on the activity of HDC but not on hdc expression. Tartaric acid and l-lactic acid, and sulphur dioxide (SO2) had no effect on enzyme synthesis and activity. Bacterial species differ in t…

Use of starter cultures ofLactobacillusto induce malolactic fermentation in wine

2017

Background and Aims Oenococcus oeni is the lactic acid bacteria species that best adapts to harsh wine conditions. This species is currently the main component of the malolactic starter cultures used in winemaking. Other species of lactic acid bacteria, however, such as Lactobacillus, can also conduct the malolactic fermentation, especially in low acidity wines. This study aimed to identify suitable Lactobacillus strains and inoculation methods to undertake the malolactic fermentation in wines with pH > 3.5. Methods and Results Six Lactobacillus strains of species L. mali (E4634), L. paracasei (E4539, E4541), L. plantarum (E4538, E4608) and L. satsumensis (E4555) were selected for their goo…

Molecular characterization of <em>Vitis vinifera</em> L. local cultivars from volcanic areas (Canary Islands and Madeira) using SSR marke…

2019

Aim: This study characterises and identifies 79 grapevine accessions from the Canary Islands and 14 from Madeira using simple sequence repeat (SSR) analysis.Methods and Results: A kit of 20 microsatellites or simple sequence repeats (SSRs) were used to obtain the molecular profiles of the 93 accessions in this study. The results allowed us to identify four new cultivars (Bienmesabe tinto, Burra volcanica, Vallera, Verijadiego negro), two new colour mutations (Listan rosa, Mollar cano rosado) and two unknown molecular profiles from Madeira. Furthermore, we propose that eight names of varieties be included in the Vitis International Variety Catalogue (VIVC) as prime names, and 38 accession na…

Protoplasts fromPodospora anserina: Isolation, purification, and transformation

1985

Protoplasts fromPodospora anserina mycelium were produced using the commercially available enzyme Novozym 234. Different parameters involved in protoplast isolation were analyzed in order to establish optimal conditions, and protoplast production was notably increased. For the purification of protoplasts, several techniques based on both centrifugation and filtration were assayed, with filtration yielding the best results. Regeneration of protoplasts was studied on different media and osmostic stabilizers, and about 80% regeneration was obtained. The good physiological condition of the protoplasts produced with this method is demonstrated by the lack of cell wall and high regeneration rate …

Molecular cloning ofTrichophyton mentagrophytes DNA sequences with promoter activity inEscherichia coli

1992

A promoter probe library from the dermatophyte fungusTrichophyton mentagrophytes has been constructed in the pVB32 plasmid vector. Using this library, a set ofT. mentagrophytes DNA sequences with promoter activity inEscherichia coli has been cloned. The size and the resistance phenotype conferred by these DNA fragments varied. Southern blot analysis confirmed that they were derived fromT. mentagrophytes genomic DNA.

Which lactic acid bacteria are responsible for histamine production in wine?

2005

Aims: To quantify the ability of 136 lactic acid bacteria (LAB), isolated from wine, to produce histamine and to identify the bacteria responsible for histamine production in wine. Methods and Results: A qualitative method based on pH changes in a plate assay was used to detect wine strains capable of producing high levels of histamine. Two quantitative, highly sensitive methods were used, an enzymatic method and HPLC, to quantify the histamine produced by LAB. Finally, an improved PCR test was carried out to detect the presence of histidine decarboxylase gene in these bacteria. The species exhibiting the highest frequency of histamine production is Oenococcus oeni. However, the concentrati…

A new fear in wine: Isolation of Staphylococcus epidermidis histamine producer

2016

Abstract Biogenic amines (BAs) are undesirable compounds in all foods and beverages because they may induce food-borne intoxications when consumed at high concentrations. The aim of this study was to identify the organism responsible for the synthesis of histamine in a Tempranillo red wine from Ribera de Duero D.O. (Denomination of origin), Spain. Bacteria present in wines after malolactic fermentation were isolated, identified and typed. Four strains of the species Oenococcus oeni and two strains of Staphylococcus epidermidis were found. None of the O. oeni strains produced histamine, cadaverine or putrescine, but one of the S. epidermidis strains (Pa) was able to produce all three in synt…

Selection of Lactobacillus strains to induce biological acidification in low acidity wines

2016

Abstract Because of global warming, wines are obtained nowadays with high pH values and low acidity. This results in wines with disturbed flavor and increased susceptibility of microbial spoilage. The aim of this work was the selection of Lactobacillus strains with ability to induce biological acidification in low acidity grape musts to obtain more acidic wines. A screening of Lactobacillus strains was carried out using several selection criteria. Lactobacillus strains that grew in must, carried out the malolactic fermentation, acidified grape must, synthesized lactic acid from sugars, and showed high resistance to lysozyme and sulfur dioxide were selected. Selected strains were characteriz…

Transposons Tn916 and Tn925 can transfer from Enterococcus faecalis to Leuconostoc oenos

1996

The streptococcal transposons Tn916 and Tn925 were transferred to several strains of Leuconostoc (Ln.) oenos using the filter mating method. The insertion of both transposons into the chromosome occurred at different sites. Transconjugants of Ln. oenos carrying Tn916 could serve as donors in mating experiments with Lactococcus lactis LM2301. Further analysis of L. lactis LM2301 transconjugants showed that the insertion of the transposon Tn916 into the chromosome was site-specific. These studies establish a basis for the initiation of genetic studies in this Leuconostoc species since there are no efficient conjugal or transformation systems previously described for this microorganism.

Characterization of Lactobacillus isolates from fermented olives and their bacteriocin gene profiles

2011

Near one hundred isolates of Lactobacillus paraplantarum, Lactobacillus pentosus and Lactobacillus plantarum from table olives were studied. Strains were genotyped by rep-PCR. Although the technique failed to differentiate some isolates at the species level, it proved a robust and easy procedure that could be useful for distinguishing between related strains of L. paraplantarum, L. pentosus and L. plantarum from a large pool of unrelated strains of these species. A PCR-based screening revealed the presence of the plantaricin encoding genes plnA, plnB, plnC, plnD, plnE/F, plnF, plnI, plnJ, plnK, plnG and plnN in most isolates of the three species. Sequences of bacteriocin genes present in L.…

Transformation of Aspergillus parasiticus using autonomously replicating plasmids from Aspergillus nidulans.

1994

A genetic transformation system for the aflatoxin-producing fungus Aspergillus parasiticus using two autonomously replicating plasmids from A. nidulans (ARp1 and pDHG25) is reported. Transformation frequencies using the plasmid pDHG25 were from 5 x 10(2) to 2.5 x 10(4) transformants per 10(6) viable protoplasts and microgram DNA. The stability of the plasmids in the transformants was also studied. This transformation system offers a new opportunity to clone genes related to aflatoxin production using appropriate aflatoxin-defective mutants.

Malolactic fermentation in white wines

2022

Abstract Malolactic fermentation (MLF) is an important process in wine production and involves the bioconversion of malic acid into lactic acid and CO2. However, many other biochemical reactions occur simultaneously, and several other grape must and wine compounds are also modified besides malic acid. Some can improve quality, while others may be detrimental to sensory quality or health. Among them, there are the synthesis of carbonyl compounds, esters, monoterpenes, volatile phenols, acetamide or mousy odor, methanethiol, dimethyl disulfide, methionol, 3-(methylsulfanyl) propionic acid, citrulline, or biogenic amines. The role of MLF in winemaking is triple: reducing the acidity of the win…

Presence of nucleosomes inPenicillium chrysogenum

1987

We have studied the chromatin structure ofPenicillium chrysogenum. This fungus presents the typical nucleosomal repeat and the core DNA size characteristic of all the eukaryotes. The repeat length (about 180 base pairs) is in the range of those obtained for most fungi (160–180 base pairs) and shorter than in higher eukaryotes. Knowledge aboutP. chrysogenum chromatin structure opens the way to the study of the mechanisms of genetic regulation in this filamentous fungus.

Rapid Plasmid Isolation. A Laboratory Experiment for Intermediate and Advanced Students

1989

Nucleotide sequence of a Trichophyton mentagrophytes HindIII mitochondrial DNA fragment containing at RNA gene cluster

1993

A 0.85-kb HindIII mitochondrial DNA fragment of the dermatophytic fungus Trichophyton mentagrophytes has been sequenced. The fragment contains eight complete genes which corresponds to a tRNA gene cluster. From 5′ to 3′, the sequenced genes code for tRNAthr, tRNAglu, tRNAval, tRNAmet1, tRNAmet3, tRNAleu, tRNAala, and tRNAphe. This tRNA gene cluster is located downstream of the larger ribosomal RNA gene. The particularities ofthe sequenced genes and their comparison with other fungal tRNA mitochondrial genes are reported.

Biogenic amine production by lactic acid bacteria, acetic bacteria and yeast isolated from wine

2007

Abstract Biogenic amines are undesirable in all foods and beverages because if consumed at too high concentration, they may induce food-borne intoxications. The biogenic amine production by 155 strains of lactic acid bacteria, 40 strains of acetic bacteria and 36 strains of yeast isolated from wine were analysed in wine, grape must and synthetic media by HPLC. We did not observe biogenic amine production by acetic bacteria and yeast; however, we found production of histamine, tyramine, phenylethylamine and putrescine by LAB. A correlation of 100% was observed between biogenic amine production in synthetic medium and wine and between activity and presence of gene. With the results expose in …

Improvement of volatile composition of wines by controlled addition of malolactic bacteria

1999

The effect of malolactic fermentation (MLF) on the volatile composition of red wines was studied by inoculation with selected lactic acid bacteria. Four wines were inoculated with different Oenococcus oeni (syn. Leuconostoc oenos) strains, the major malolactic species found in wines, and one was inoculated with a Lactobacillus sp. strain. A non inoculated wine was also analyzed to act as a control. Malolactic fermentation and evolution of non volatile compounds were followed by HPLC and after the depletion of the malic acid present in wine the volatile compounds were extracted and analyzed by gas chromatography with flame ionization and mass spectrometry. Wines which had undergone the MLF s…

Direct and Rapid Detection and Quantification of Oenococcus oeni Cells in Wine by Cells-LAMP and Cells-qLAMP

2018

Fast detection and enumeration of Oenococcus oeni in winemaking are necessary to determine whether malolactic fermentation (MLF) is likely to be performed or not and to decide if the use of a commercial starter is needed. In other wines, however, performing MLF can be detrimental for wine and should be avoided. The traditional identification and quantification of this bacteria using culture-dependent techniques in wine-related matrices require up to 14 days to yield results, which can be a very long time to perform possible enological operations. Loop-mediated isothermal amplification (LAMP) is a novel culture-independent technique that amplifies nucleic acid sequences under isothermal cond…

Recombinant laccase from Pediococcus acidilactici CECT 5930 with ability to degrade tyramine

2017

Biogenic amines degradation by bacterial laccases is little known, so we have cloned and heterologously expressed, in E. coli, a new laccase from Pediococcus acidilactici CECT 5930 (Lpa5930), a lactic acid bacterium commonly found in foods able to degrade tyramine. The recombinant enzyme has been characterized by physical and biochemical assays. Here we report the optimization of expression and purification procedures of this laccase. DNA encoding sequence of laccase from P. acidilactici was amplified by PCR and cloned into the expression plasmid pET28a for induction by isopropyl-β-D-thiogalactoipyranoside. Protein expression was performed in E. coli BL21(DE3) harboring pGro7 plasmid expres…

Lactobacillus bobalius sp. nov., a lactic acid bacterium isolated from Spanish Bobal grape must

2008

A Lactobacillus strain, designated 203(T), previously isolated from Bobal grape must was characterized phylogenetically, genotypically and phenotypically in order to establish whether it represents a novel species. On the basis of the 16S rRNA gene sequence, strain 203(T) was shown to belong to the genus Lactobacillus, falling within the Lactobacillus alimentarius-Lactobacillus farciminis group and being closely related to the type strains of L. alimentarius, Lactobacillus kimchii and Lactobacillus paralimentarius. DNA-DNA hybridization results confirmed the separate status of strain 203(T) at the species level. To establish the similarities and differences between 203(T) and the three afor…

Histamine, histidine, and growth-phase mediated regulation of the histidine decarboxylase gene in lactic acid bacteria isolated from wine

2006

Fermented foods are frequently contaminated by histamine that is generated by microorganisms with histidine decarboxylase activity. The ingestion of large amounts of histamine can cause serious toxicological problems in humans. A study of the effects of histamine, histidine, and growth phase on histamine production by lactic acid bacteria isolated from wine is reported here. With northern blots and specific activity analysis, we observed that histidine induces the expression of the histidine decarboxylase gene (hdc) and that histamine causes a decrease in the expression of this gene. The expression of hdc is also mediated by the bacterial growth phase. Histidine and histamine do not affect …

Exploring the biodiversity of two groups of Oenococcus oeni isolated from grape musts and wines: Are they equally diverse?

2016

One hundred and four Oenococcus oeni isolates were characterised by the carbohydrate fermentation (CH) profile and DNA fingerprinting. Forty-four isolates came from grape must, and 60 from wines sampled at the end of alcoholic fermentation or during malolactic fermentation. The grape must isolates fermented more CH than the wine isolates. In genotypical terms, no clear boundary between grape must and wine isolates was found. Diversities were deduced by considering the isolates of grape must and of wine separately and jointly. By considering only CH fermentation abilities, the group of grape must isolates gave higher diversity index (DICH) values than those isolated from wine; i.e., these is…

Cells-qPCR as a direct quantitative PCR method to avoid microbial DNA extractions in grape musts and wines.

2017

A novel quantitative PCR assay called Cells-qPCR has been developed for the rapid detection and quantification of yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) directly from grape must and wine that does not require DNA extraction. The assay was tested on Brettanomyces bruxellensis, Saccharomyces cerevisiae, Lactobacillus plantarum, Oenococcus oeni, Acetobacter aceti and Gluconobacter oxydans in culture media, and in white and red grape musts and wines. Standard curves were constructed from DNA and cells for the six target species in all the matrices. Good efficiencies were obtained for both when comparing DNA and cells standard curves. No reaction inhibition was observe…

Comparative survey of putrescine production from agmatine deamination in different bacteria

2008

This article aims to study putrescine production in Lactobacillus hilgardii strain X1B, an agmatine degrader isolated from wine, and to compare it with three other different species, previously reported as putrescine producers from agmatine: Pseudomonas aeruginosa PAO1, Enterococcus faecalis ATCC11700 and Bacillus cereus CECT 148T. The effect of different biogenic amines, organic acids, cofactors, amino acids and sugars on putrescine production was evaluated. In some cases, a similar effect was found in all the strains studied but the magnitude differed. Arginine, glucose and fructose showed an inhibitory effect, whereas the presence of agmatine induced the production of putrescine in all m…

Lowering histamine formation in a red Ribera del Duero wine (Spain) by using an indigenous O. oeni strain as a malolactic starter

2016

This study demonstrates for the first time that a non-commercial selected autochthonous O. oeni strain has been used to conduct malolactic fermentation (MLF) while lowering histamine formation in the same winery. Lactic acid bacteria (LAB) were isolated from 13 vats before and after spontaneous MLF at the Pago de Carraovejas winery from the Ribera del Duero region (Spain). Only O. oeni were present, typed and characterized, and both histamine producer and non-producers existed. From the non-producers, one strain was selected to become a starter according to its genetic profile, prevalence in the different wines in the winery, resistance to alcoholic degree, resistance to high polyphenolic c…

Lactobacillus vini sp. nov., a wine lactic acid bacterium homofermentative for pentoses.

2006

Six strains with more than 99·5 % 16S rRNA gene sequence similarity, identical internal spacer region profiles and restriction analysis of the amplified 16S rRNA gene patterns were isolated from fermenting grape musts during independent studies carried out in France and Spain many years apart. Strains are Gram-positive, motile, facultatively anaerobic rods that do not exhibit catalase activity and have the ability to utilize pentose sugars (ribose and/or l-arabinose), although they are homofermentative bacteria. Strains ferment pentoses exclusively yielding lactic acid as the end product. A broad set of molecular techniques has been applied to characterize these strains and the results show…

Structural analysis and biochemical properties of laccase enzymes from two Pediococcus species

2021

Summary Prokaryotic laccases are emergent biocatalysts. However, they have not been broadly found and characterized in bacterial organisms, especially in lactic acid bacteria. Recently, a prokaryotic laccase from the lactic acid bacterium Pediococcus acidilactici 5930, which can degrade biogenic amines, was discovered. Thus, our study aimed to shed light on laccases from lactic acid bacteria focusing on two Pediococcus laccases, P. acidilactici 5930 and Pediococcus pentosaceus 4816, which have provided valuable information on their biochemical activities on redox mediators and biogenic amines. Both laccases are able to oxidize canonical substrates as ABTS, ferrocyanide and 2,6‐DMP, and non‐…

A selective medium for the isolation of malolactic mutants of Leuconostoc oenos

1994

We have developed a selective medium for the isolation of Leuconostoc oenos mutants defective in malolactic fermentation. Forty per cent of colonies isolated directly on selective plates after UV mutagenesis had lost their ability to degrade malate. None of the tested mutants showed any detectable malolactic activity and all lacked a protein band corresponding in size to that of the malolactic enzyme. The availability of such mutants provides a valuable tool both for physiological and genetic research on malolactic fermentation.

Development of specific fluorescent oligonucleotide probes for in situ identification of wine lactic acid bacteria.

2003

A rapid method for the identification of lactic acid bacteria (LAB) from wine has been developed. This method is based on fluorescence in situ hybridisation (FISH), using fluorescent oligonucleotide probes, homologous to 16S rDNA of those species of LAB commonly found in wines. The protocol for the specific detection of these bacteria was established through the hybridisation of 36 reference strains. The specificity of the probes was evaluated by using pure cultures. Probes were used to identify species in different wines, making it evident that direct identification and quantification from natural samples without culturing is also possible. The results show that FISH is a promising techniq…

Technological properties of Lactobacillus plantarum strains isolated from grape must fermentation

2016

Malolactic fermentation (MLF) is a secondary fermentation in wine that usually takes place during or at the end of alcoholic fermentation. Lactobacillus plantarum is able to conduct MLF (particularly under high pH conditions and in co-inoculation with yeasts) and some strains are commercially used as MLF starter cultures. Recent evidences suggest a further use of selected L. plantarum strains for the pre-alcoholic acidification of grape must. In this study, we have carried out an integrated (molecular, technological, and biotechnological) characterization of L. plantarum strains isolated from Apulian wines in order to combine the two protechnological features (MLF performances and must acid…

Saccharomyces cerevisiae and Oenococcus oeni immobilized in different layers of a cellulose/starch gel composite for simultaneous alcoholic and malol…

2013

Abstract The production of a two-layer composite biocatalyst for immobilization of two different microorganisms for simultaneous alcoholic and malolactic fermentation (MLF) of wine in the same bioreactor is reported. The biocatalyst consisted of a tubular delignified cellulosic material (DCM) with entrapped Oenococcus oeni cells, covered with starch gel containing the alcohol resistant and cryotolerant strain Saccharomyces cerevisiae AXAZ-1. The biocatalyst was found effective for simultaneous low temperature alcoholic fermentation resulting to conversion of malic acid to lactic acid in 5 days at 10 °C. Improvement of wine quality compared with wine fermented with S. cerevisiae AXAZ-1 immob…

Malic Enzyme and Malolactic Enzyme Pathways Are Functionally Linked but Independently Regulated in Lactobacillus casei BL23

2013

ABSTRACT Lactobacillus casei is the only lactic acid bacterium in which two pathways for l -malate degradation have been described: the malolactic enzyme (MLE) and the malic enzyme (ME) pathways. Whereas the ME pathway enables L. casei to grow on l -malate, MLE does not support growth. The mle gene cluster consists of three genes encoding MLE ( mleS ), the putative l -malate transporter MleT, and the putative regulator MleR. The mae gene cluster consists of four genes encoding ME ( maeE ), the putative transporter MaeP, and the two-component system MaeKR. Since both pathways compete for the same substrate, we sought to determine whether they are coordinately regulated and their role in l -m…

Influence of freezing temperatures prior to freeze-drying on viability of yeasts and lactic acid bacteria isolated from wine

2017

Aims To determine the effect of three different freezing temperatures on post-freeze-drying survival rates of wine yeasts and lactic acid bacteria (LAB). To know if a similar freeze-drying protocol can be used for both micro-organisms. Methods and Results Cells from liquid culture media were recovered and concentrated in appropriate lyoprotectants. Aliquots of each strain were frozen at −20, −80 and −196°C before vacuum drying. Viable cell counts were done before freezing and after freeze-drying. Survival rates were calculated. Freezing temperatures differently affected yeast and bacteria survival. The highest survival rates were obtained at −20 and −80°C for yeasts, but at −196°C for LAB. …