[Show abstract][Hide abstract] ABSTRACT: Lysozyme and the reduction of metabisulfite addition to red wine were evaluated during a winemaking process and after malolactic fermentation (MLF). Treatment, with lysozyme, of the must from Tempranillo grapes and at the end of alcoholic fermentation (AF) caused the 100% implantation of the inoculated bacterial strain and shortened the duration of MLF by 7 days. At the end of the MLF, wines treated with lysozyme showed lower volatile acidity, color intensity, and biogenic amine content. The differences in color intensity disappeared during wine stabilization. The lysozyme addition after MLF led to lower histamine concentrations in wines. These phenomena occurred irrespective of the lactic acid bacteria (LAB) proliferation control and of the Oenococcus oeni dominant strain identified at this period. The results of this study show the significance of preventive use of lysozyme in vinification of red wine to maintain low histamine levels and ensure a successful implantation of inoculated O. oeni starters. Sulfur dioxide is commonly used to control spoilage bacteria in wines, but may cause some unwanted effects, such as negative sensory characteristics, and allergic reactions in sensitive people, or can be a source of hydrogen sulfite formation. The antimicrobial activity of SO 2 decreases as wine pH becomes higher, making it more difficult to microbiologically stabilize wines with low acidity. Other antimicrobials such as sorbic acid and dimethyl carbonate are active against yeasts but have limited activity against bacteria [7, 8]. Nisin is a bacteriocin that may be an alternative [3, 45], but at present its addition to wine is not approved. Lysozyme is an enzyme isolated from hen egg albumen  and its lytic activity against most lactic acid bacteria (LAB) associated with wine has recently been found to be useful in controlling bacterial activity in wines [8, 16, 38]. However, it does not substitute the antioxidant properties of sulfur dioxide , and although it is highly active against Gram-positive bacteria, it has little activity against Gram-negative bacteria and is inactive against the eukaryotic cell wall . Moreover, the bacteriolytic action of lysozyme in wines is affected by the polyphenolic components, which may bind to lysozyme . It has been observed that lysozyme is more active in white wines than in red ones, which is attributed to the difference in polyphenolic content . Lysozyme has several applications, including delaying malolactic fermentation (MLF) in red wines, allowing longer macerations and micro-oxygenation to improve color stabilization and polyphenolic extraction [20-22, 27, 40]. The lysozyme addition can also prevent the increase of volatile acidity during stuck or sluggish alcoholic fermentation (AF) and delay or inhibit MLF in white wines [16, 17, 19]. In recent years, wine industries have moved towards using pure starter cultures of selected LAB to promote a reliable and rapid malic acid bioconversion, and thus ensuring a better control and predictability of the reaction . Commercial strains directly inoculated into wine improved significantly the control of MLF , but in many cases, the implantation of the cultures did not always occur and the indigenous microbiota carried out MLF [4, 31, 33] with the risk of volatile acidity, biogenic amine production, and unnecessary costs. In this context, addition of lysozyme can decrease endogenous LAB populations and favor LAB inoculated implantation by avoiding LAB competition.
[Show abstract][Hide abstract] ABSTRACT: Genetic typification of 120 bacterial isolates of Lactobacillus plantarum and Oenococcus oeni from different Rioja musts and wines was performed by numerical analysis of pulsed-field gel electrophoresis (PFGE) patterns
with endonuclease SfiI, and 46 of them were also studied by randomly amplified polymorphic DNA (RAPD)-PCR. A comparative study of both typification
methods applied to L. plantarum and O. oeni oenological strains was performed. Bacterial species was determined both by biochemical identification methods and by specific
PCR analysis. A wide variety of restriction digest patterns were detected by PFGE among L. plantarum strains (36 unrelated patterns and one closely related pattern, out of 48 isolates), as well as among O. oeni strains (18 unrelated patterns out of 72 isolates). PFGE was shown to be a suitable method for strain differentiation and
to determine which strains are present in wine fermentations, with a discriminatory power to type L. plantarum and O. oeni strains higher than that of RAPD-PCR.
European Food Research and Technology 06/2008; 227(2):547-555. · 1.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: : Malolactic fermentations (MLF) of wines inoculated with selected lactic acid bacteria strains of the species Oenococcus oeni and Lactobacillus plantarum were studied and compared with spontaneous MLF. Bacterial populations were monitored along the whole process of MLF and bacteria identifications were carried out at species and strain level. Macrorestriction analysis with SfiI endonuclease and subsequent PFGE was carried out in order to identify O. oeni individual strains. L. plantarum active lyophila did not survive competing with the indigenous microbiota in a wine with 15.3 % (vol/vol) alcohol, whereas the selected O. oeni strains carried out wine MLF. The highest production of histamine took place during MLF in those wines that underwent spontaneous MLF with a mixed population of indigenous strains. The lowest levels of histamine were obtained with the selected commercial O. oeni strain that imposed 100% over the indigenous microbiota. Results indicate that development of MLF leaded by selected O. oeni active lyophila provides negligible histamine levels in red wines of quality and susceptible of subsequent ageing in wood.
Key words: Lactobacillus plantarum; Oenococcus oeni; Malolactic fermentation; Wine; PFGE
[Show abstract][Hide abstract] ABSTRACT: Two hundred and four bacterial isolates from Rioja red wines undergoing spontaneous malolactic fermentation (MLF) were studied.
Bacterial species was determined both by microbiological identification methods and by specific PCR analysis. Oenococcus oeni was shown to be the predominant species (98.5% of total isolates). Pulsed field gel electrophoresis (PFGE) of chromosomal
DNA digested with SfiI was used to differentiate individual strains of O. oeni. A wide variety of restriction digest patterns were detected, which indicated a rich biodiversity of indigenous strains.
Most fermentations (37 out of 41) showed from 2 to 6 clones growing in the same tank. Five O. oeni strains were the most frequently found, appearing in more than three of the 13 studied wineries, and most times in combination
with other less frequently found strains. PFGE was shown to be a suitable method for strain differentiation, for monitoring
individual strains and determining which strains actually survive and carry out MLF. A high genotypic heterogeneity of wild
O. oeni strains was demonstrated and 90% of the studied wines showed mixed populations of O. oeni strains during MLF.
European Food Research and Technology 01/2007; 226(1):215-223. · 1.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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°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.
[Show abstract][Hide abstract] ABSTRACT: Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria.
Applied and Environmental Microbiology 12/2003; 69(11):6801-7. · 3.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, we looked at the yeast population present in four spontaneous alcoholic fermentations in the Rioja appellation
(D.O.Ca. Rioja, Spain). The study was conducted through the identification of the yeasts via the PCR–RFLP technique of the
ITS region of rDNA. In a first harvest, the qualitative diversity of the species present in spontaneous alcoholic fermentation
was studied, and in a second harvest, their quantitative significance. In spontaneous fermentations, 17 different yeast species
were found, although only two of them, Candida stellata and Kloeckera apiculata, as well as Saccharomyces cerevisiae, appeared in major proportions during fermentation. The significance of the non-Saccharomyces yeasts during the spontaneous alcoholic fermentation was conditioned by the presence of Saccharomyces cerevisiae in the medium. Species not cited in literature in connection with winemaking and yeasts associated with wines spoilage have
been detected in all the alcoholic fermentations carried out in the qualitative study.
European Food Research and Technology 230(6):885-891. · 1.44 Impact Factor