Malolactic Starter Research Articles

Effects of Heterologous Expression of Genes Related L-Malic acid Metabolism in Saccharomyces uvarum on Flavor Substances Production in Wine.

During malolactic fermentation (MLF) of vinification, the harsh L-malic acid undergoes transformation into the milder L-lactic acid, and via decarboxylation reactions it is catalyzed by malolactic enzymes in LAB. The use of bacterial malolactic starter cultures, which usually present challenges in the industry as the suboptimal conditions after alcoholic fermentation (AF), including nutrient limitations, low temperatures, acidic pH levels, elevated alcohol, and sulfur dioxide concentrations after AF, lead to "stuck" or "sluggish" MLF and spoilage of wines. Saccharomyces uvarum has interesting oenological properties and provides a stronger aromatic intensity than Saccharomyces cerevisiae in AF. In the study, the biological pathways of deacidification were constructed in S. uvarum, which made the S. uvarum carry out the AF and MLF simultaneously, as different genes encoding malolactic enzyme (mleS or mleA), malic enzyme (MAE2), and malate permease (melP or MAE1) from Schizosaccharomyces pombe, Lactococcus lactis, Oenococcus oeni, and Lactobacillus plantarum were heterologously expressed. For further inquiry, the effect of L-malic acid metabolism on the flavor balance in wine, the related flavor substances, higher alcohols, and esters production, were detected. Of all the recombinants, the strains WYm1SN with coexpression of malate permease gene MAE1 from S. pombe and malolactic enzyme gene mleS from L. lactis and WYm1m2 with coexpression of gene MAE1 and malate permease gene MAE2 from S. pombe could reduce the L-malic acid contents to about 1 g/L, and in which the mutant WYm1SN exhibited the best effect on the flavor quality improvement.

Analysis of the Genome Architecture of Lacticaseibacillus paracasei UNQLpc 10, a Strain with Oenological Potential as a Malolactic Starter

The Lacticaseibacillus paracasei UNQLpc 10 strain was isolated from a Malbec wine produced in North Patagonia, Argentina, and identified by 16S rRNA gene sequencing. The aim of this work was to obtain the fully assembled genome of the UNQLpc 10 strain, analyze its structure, and evaluate the possible functions of the predicted genes with regard to its oenological potential as a malolactic starter. UNQLpc10 is the first whole assembled genome of an oenological strain of Lcb. paracasei reported in databases. This information is of great interest inexpanding the knowledge of diversity of oenological lactic acid bacteria and in searching for new candidate species/strains to design starter cultures. The in silico genome-wide analysis of UNQLpc 10 confirms the existence of genes encoding enzymes involved in the synthesis of several metabolites of oenological interest, and proteins related to stress responses. Furthermore, when UNQLpc 10 was incubated in synthetic wine, it exhibited a very good survival and L-malic acid consumption ability.

Molecular tools for the analysis of the microbiota involved in malolactic fermentation: from microbial diversity to selection of lactic acid bacteria of enological interest.

Winemaking is a complex process involving two successive fermentations: alcoholic fermentation, by yeasts, and malolactic fermentation (MLF), by lactic acid bacteria (LAB). During MLF, LAB can contribute positively to wine flavor through decarboxylation of malic acid with acidity reduction and other numerous enzymatic reactions. However, some microorganisms can have a negative impact on the quality of the wine through processes such as biogenic amine production. For these reasons, monitoring the bacterial community profiles during MLF can predict and control the quality of the final product. In addition, the selection of LAB from a wine-producing area is necessary for the formulation of native malolactic starter cultures well adapted to local winemaking practices and able to enhance the regional wine typicality. In this sense, molecular biology techniques are fundamental tools to decipher the native microbiome involved in MLF and to select bacterial strains with potential to function as starter cultures, given their enological and technological characteristics. In this context, this work reviews the different molecular tools (both culture-dependent and -independent) that can be applied to the study of MLF, either in bacterial isolates or in the microbial community of wine, and of its dynamics during the process.

Use of Apple Pomace as Substrate for Production of Lactiplantibacillus plantarum Malolactic Starter Cultures

The by-products of the food industry are an economic alternative as a source of nutrients to obtain biomass. At the same time, theiruse could solve the environmental problem related to their disposal, which is highly polluting due to their elevated biochemical oxygen demand. In this work, we seek to optimize the production of cellular biomass of two native Patagonian strains of Lactiplantibacillus plantarum (UNQLp 11 and UNQLp155), selected for its oenological and technological properties, using apple pomace (AP), a residue from the juice and cider industry. The supplementation of AP with yeast extract, salts, and Tween 80 (sAP), proved to maintain the growth of the Lpb. plantarum strains, similar to the commercial medium used to grow LAB (De Man, Rogosa, Sharpe, MRS). Cultures grown in sAP medium showed good tolerance to wine conditions (high ethanol content and low pH), demonstrated by its ability to consume L-malic acid. The subsequent inoculation of these cultures in sterile wines (Merlot and Pinot noir) was carried out at laboratory scale, evaluating cell viability and L-malic acid consumption for 21 days at 21 °C. Cultures grown in sAP media showed a similar performance to MRS media. Thus, sAP media proved to be a suitable substrate to grow oenological Lpb. plantarum strains where cultures (with high size inoculums) were able to drive malolactic fermentation, with an L-malic acid consumption higher than 90%.

Whey permeate as a substrate for the production of freeze-dried Lactiplantibacillus plantarum to be used as a malolactic starter culture.

The aim of this work was to obtain freeze-dried biomass of the native Patagonian Lactiplantibacillus plantarum strain UNQLp 11 from a whey permeate (WP)-based medium and compare it with the growth in commercial MRS broth medium. Survival and activity of the freeze-dried Lb. plantarum strain were investigated after inoculation in wine as a starter culture for malolactic fermentation (MLF). The effect of storage and rehydration condition of the dried bacteria and the nutrient supplementation of wine were also studied. The freeze-dried cultures from WP and those grown in MRS showed similar survival results. Rehydration in MRS broth for 24h and the addition of a rehydration medium to wine as nutrient supplementation improved the survival under wine harsh conditions and guaranteed the success of MLF. Storage at 4°C under vacuum was the best option, maintaining high cell viability for at least 56days, with malic acid consumption higher than 90% after 7days of inoculation in a wine-like medium. These results represent a significant advance for sustainable production of dried malolactic starter cultures in an environmentally friendly process, which is low cost and easy to apply in winemaking under harsh physicochemical conditions.

Effect of mixed fermentations with Starmerella bacillaris and Saccharomyces cerevisiae on management of malolactic fermentation

This work aims to improve the management of the malolactic fermentation (MLF) in red wines by elucidating the interactions between Starmerella bacillaris and Saccharomyces cerevisiae in mixed fermentations and malolactic bacteria. Two Starm. bacillaris strains were individually used in mixed fermentations with a commercial S. cerevisiae. MLF was performed using two autochthonous Lactobacillus plantarum and one commercial Oenococcus oeni inoculated following a simultaneous (together with S. cerevisiae) or sequential (at the end of alcoholic fermentation) approach. The impact of yeast inoculation on the progress of MLF was investigated by monitoring the viable microbial populations and the evolution of the main oenological parameters, as well as the volatile organic composition of the wines obtained in mixed and pure micro-scale winemaking trials. Our results indicated that MLF was stimulated, inhibited, or unaffected in mixed fermentations depending on the strains and on the regime of inoculation. O. oeni was able to perform MLF under all experimental conditions, and it showed a minimal impact on the volatile organic compounds of the wine. L. plantarum was unable to perform MLF in sequential inoculation assays, and strain-depending interactions with Starm. bacillaris were indicated as factor affecting the outcome of MLF. Moreover, uncompleted MLF were related to a lower aromatic complexity of the wines. Our evidences indicate that tailored studies are needed to define the appropriate management of non-Saccharomyces and malolactic starter cultures in order to optimize some technological parameters (i.e. reduction of vinification time) and to improve qualitative features (i.e. primary and secondary metabolites production) of red wines.

Effect of exogenous proline on the ethanolic tolerance and malolactic performance of Oenococcus oeni.

The use of malolactic starter cultures, often offer no guarantee of microbiological success due to the chemical and physical factors (pH, ethanol, SO2, nutrient availability) that occur during the winemaking process. This study was born with the aim of improving the performance of the lactic acid bacteria used as a starter culture in the de-acidification of wines. Two commercial strains of Oenococcus oeni, were used. Was evaluated the effect of exogenous l-proline added during the bacterial growth, on the improvement of their survival in the presence of different ethanol concentrations and their ability to degrade l-malic acid in synthetic wine with the presence of 12% (v/v) and 13% (v/v) of ethanol. The results showed that l-proline improve ethanol tolerance and so the malolactic performances of O. oeni. This work represents an important strategy to ensure good vitality and improve the performance of the malolactic starter.

Inoculum Strategies and Performances of Malolactic Starter Lactobacillus plantarum M10: Impact on Chemical and Sensorial Characteristics of Fiano Wine.

Malolactic fermentation (MLF) is a biological process that, in addition to deacidifying, also improves biological stability and changes the chemical and sensorial characteristics of wines. However, multiple biotic and abiotic factors, present in must and wine, make the onset and completion of MLF by indigenous malolactic bacteria or added commercial starters difficult. This work illustrates the metabolic and fermentative dynamics in winemaking Fiano wine, using a commercial starter of Saccharomyces cerevisiae and the selected strain Lactobacillus plantarum M10. In particular, an inoculum of malolactic starter was assessed at the beginning of alcoholic fermentation (early co-inoculum), at half alcoholic fermentation (late co-inoculum), and post alcoholic fermentation (sequential inoculum). The malolactic starter, before its use, was pre-adapted in sub-optimal growth conditions (pH 5.0). In sequential inoculum of the Lb. plantarum M10, even in a wine with high acidity, has confirmed its good technological and enzymatic characteristics, completing the MLF and enriching the wine with desirable volatile compounds.

Design of a low-cost culture medium based in whey permeate for biomass production of enological Lactobacillus plantarum strains.

The production of malolactic starter cultures requires the obtention of suitably large biomass at low-cost. In this work it was possible to obtain a good amount of biomass, at laboratory scale, of two enological strains of Lb. plantarum, by formulating a culture medium based on whey permeate (WP), a by-product of the cheese industry usually disposed as waste, when this was supplemented with yeast extract (Y), salts (S) and Tween 80 (T) (WPYST). Bacteria grown in WPYST medium exhibited good tolerance to stress conditions of synthetic wine (pH 3.5, ethanol 13% vol/vol). However, when WPYST was added with 8% vol/vol ethanol, cultures inoculated in synthetic wine, showed a lower viability and capacity to consume L-malic acid than when they were cultured in WPYST without ethanol. Subsequently, strains grown in WPYST were inoculated in sterile wine samples (final stage of alcoholic fermentation) of the red varietals Merlot and Pinot noir, and incubated at laboratory scale. Cultures from WPYST, inoculated in Pinot noir wine, showed a better performance than bacteria grown in MRS broth, and exhibited a consumption of L-malic acid higher than 90%. However, cultures from WPYST or from MRS broth, inoculated in sterile Merlot wine, showed a lower survival. This study allowed the formulation of a low-cost culture medium, based on a by-product of the food industry, which showed to be adequate for the growth of two enological strains of Lb. plantarum, suggesting their potentiality for application in the elaboration of malolactic starter cultures.

Lactobacillus plantarum as a malolactic starter culture in winemaking: A new (old) player?

Malolactic fermentation (MLF) is a process in winemaking responsible for the conversion of L-malic acid to L-lactic acid and CO2, which reduces the total acidity, improves the biological stability, and modifies the aroma profile of wine. MLF takes place during or after alcoholic fermentation and is carried out by one or more species of lactic acid bacteria (LAB), which are either present in grapes and cellars or inoculated with malolactic starters during the winemaking process. Although the main bacterium among LAB used in commercial starter cultures for MLF has traditionally been Oenococcus oeni, in the last decade, Lactobacillus plantarum has also been reported as a malolactic starter, and many works have shown that this species can survive and even grow under harsh conditions of wine (i.e., high ethanol content and low pH values). Furthermore, it has been proved that some strains of L. plantarum are able to conduct MLF just as efficiently as O. oeni. In addition, L. plantarum exhibits a more diverse enzymatic profile than O. oeni, which could play an important role in the modification of the wine aroma profile. This enzymatic diversity allows obtaining several starter cultures composed of different L. plantarum biotypes, which could result in distinctive wines. In this context, this review focuses on showing the relevance of L. plantarum as a MLF starter culture in winemaking.

Impact of malolactic fermentation on the volatile composition of Turkish Kalecik karası red wines

The effects of spontaneous malolactic fermentation (MLF) and a malolactic starter culture (Oenococcus oeni) on the volatile compounds of Kalecik karası red wines were investigated. The volatile compounds were extracted using liquid–liquid extraction and quantified by GC–MS–flame ionisation detection. Starter cultures of O. oeni had a positive effect in shortening MLF and producing lower volatile acidity. After MLF, the total volatile compounds increased in both the inoculated wines and the spontaneously fermented wine. Ethyl lactate, diethyl succinate and γ-butyrolactone increased upon inoculation with cultures of O. oeni strains PN4 and VP41. MLF increased the diacetyl concentration in all Kalecik karası wines. Strain dependent differences were found in the level of diacetyl, with O. oeni PN4 producing more diacetyl than O. oeni VP41. After MLF, the concentrations of acetoin, 4-ethylguaicol and 4-ethylphenol increased in spontaneously fermented wine compared with wines inoculated with O. oeni. Accordingly, the use O. oeni PN4 is recommended for a controlled MLF to produce better quality Kalecik karası wines. © 2018 The Institute of Brewing & Distilling

Diverse physiological and metabolic adaptations by Lactobacillus plantarum and Oenococcus oeni in response to the phenolic stress during wine fermentation

Understanding the mechanisms of combating phenolic stress in wine is important for optimization of starter culture for improved competitiveness. The cellular membrane adaptations, as well as metabolic transformations driven by malo-lactic starter cultures, i.e. Lactobacillus plantarum (Lp2565) and Oenococcus oeni (Oo2219) with response to wine phenolic compounds were studied. The morphological changes based on scanning electron microscopy indicated the higher tolerance of Oo2219 to phenolic stress than Lp2565. Further, the fatty acid profiling suggested that the membrane fluidization in Lp2565 was attributed to higher unsaturated fatty acids whereas the rigidification in Oo2219 was by incorporation of saturated fatty acid in the membrane. The metabolic transformation of phenolic compounds suggested that Lp2565 has more versatile phenolic detoxification enzyme systems compared to Oo2219. The metabolic conversions resulted in degradation of phenolic compounds into volatile phenols, aromatic alcohol, and phenyl propionic acids, thus indicating the possible involvement of oxidoreductases, decarboxylases, and demethylases.

Malic Acid in Wine: Origin, Function and Metabolism during Vinification

The production of quality wines requires a judicious balance between the sugar, acid and flavour components of wine. L-Malic and tartaric acids are the most prominent organic acids in wine and play a crucial role in the winemaking process, including the organoleptic quality and the physical, biochemical and microbial stability of wine. Deacidification of grape must and wine is often required for the production of well-balanced wines. Malolactic fermentation induced by the addition of malolactic starter cultures, regarded as the preferred method for naturally reducing wine acidity, efficiently decreases the acidic taste of wine, improves the microbial stability and modifies to some extent the organoleptic character of wine. However, the recurrent phenomenon of delayed or sluggish malolactic fermentation often causes interruption of cellar operations, while the malolactic fermentation is not always compatible with certain styles of wine. Commercial wine yeast strains of Saccharomyces are generally unable to degrade L-malic acid effectively in grape must during alcoholic fermentation, with relatively minor modifications in total acidity during vinification. Functional expression of the malolactic pathway genes, i.e. the malate transporter (mae1) of Schizosaccharomyces pombe and the malolactic enzyme (mleA) from Oenococcus oeni in wine yeasts, has paved the way for the construction of malate-degrading strains of Saccharomyces for commercial winemaking.

Phage-host interactions analysis of newly characterized Oenococcus oeni bacteriophages: Implications for malolactic fermentation in wine

Nowadays, only few phages infecting Oenococcus oeni, the principal lactic acid bacteria (LAB) species responsible for malolactic fermentation (MLF) in wine, have been characterized. In the present study, to better understanding the factors affecting the lytic activity of Oenococcus phages, fifteen O. oeni bacteriophages have been studied in detail, both with molecular and microbiological methods. No correlations were found between genome sizes, type of integrase genes, or morphology and the lytic activity of the 15 tested phages. Interestingly, though phage attack in a wine at the end of alcoholic fermentation seems not to be a problem, it can indeed represent a risk factor for MLF when the alcohol content is low, feature that may be a key point for choosing the appropriate time for malolactic starter inoculation. Additionally, it was observed that some phages genomes bear 2 or 3 types of integrase genes, which point to horizontal gene transfer between O. oeni bacteriophages.

Use of starter cultures ofLactobacillusto induce malolactic fermentation in wine

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 good growth performance and high malolactic activity in grape must, although these characteristics differed among them. Freeze-dried starter cultures were obtained for strains E4538, E4608, E4555 and E4634. These starter cultures showed a high rate of malic acid consumption in grape must. Inoculation of the Lactobacillus strains in grape must, prior to fermentative yeasts, was selected as the best inoculation strategy to promote the malolactic fermentation. Conclusions Inoculation of the starter cultures of the selected Lactobacillus strains before yeast inoculation in grape must effectively allows malolactic fermentation in wines. Significance of the Study Inoculation of Lactobacillus strains in grape must, prior to fermentative yeasts, is an effective alternative to O. oeni to undertake malolactic fermentation in wines of pH > 3.5.

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

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 content, inability to synthesise histamine, growth kinetics and malolactic activity. This starter was produced at semi-industrial levels to inoculate 20,000L of Tempranillo red wine. The inoculated vat showed 5-fold less histamine than the non-inoculated control vat. After 1year, the barrel-ageing histamine concentrations were 3-fold lower in the inoculated vat than in the non-inoculated vat.

Comparative vinification assays with selected Patagonian strains of Oenococcus oeni and Lactobacillus plantarum

The performance of Patagonian Lactobacillus plantarum and Oenococcus oeni strains as malolactic starter cultures was compared. Two autochthonous strains of each species were selected, based on the presence of aroma-related genes, and inoculated in sterile wine of high ethanol content. The effects of initial inoculum size and acclimation treatment on the efficiency of malolactic fermentation (MLF) were analyzed for each strain. O. oeni strains were able to successfully conduct the MLF only when the inoculum concentration was higher than 1.108 CFU/mL and cells were acclimated in sublethal ethanol concentrations. The increase of ethanol concentration in the acclimation medium also improved the kinetics of malic acid consumption. Successful MLF with L. plantarum strains required lower inocula and no acclimation treatment. In addition, these strains showed a better profile of aroma-related genes than O. oeni. L. plantarum strains appeared to be more efficient than O. oeni strains as candidates for malolactic starter cultures to be used in Patagonian red wines.

Selection and characterization of Oenococcus oeni strains for use as new malolactic fermentation starter cultures

Oenococcus oeni (O. oeni) strains are critically important for winemaking. They bring microbial stabilization, de-acidification and sensory improvement through malolactic fermentation (MLF) to almost all red wines and some white wines. In this study, 23 strains of O. oeni isolated from China were evaluated for use as MLF starter cultures. Strains were characterized and screened in wine-like media with increasing selective pressure, such as low pH and high ethanol concentration. Their abilities to conduct MLF were affected significantly by ethanol concentration and pH. Eight strains were selected and subsequently evaluated by detecting the enzyme-encoding genes involved in biogenic amines synthesis and wine aroma modification. Seven well-performing strains were further assessed in newly alcohol-fermented Cabernet Sauvignon wine. The results show that strains O. oeni CS-4b and O. oeni ME-5b exhibited better performance than the commercial starter O. oeni 31MBR in wine. Therefore, they were selected as potential MLF starters suitable for wine with high ethanol concentration and low pH.

Advances in wine analysis by PTR-ToF-MS: Optimization of the method and discrimination of wines from different geographical origins and fermented with different malolactic starters

PTR-ToF-MS has been previously used to analyse the headspace of wine, but it is not fully exploited in the field due to problems related to the high ethanol concentration. In the case of alcoholic fermentation during bread-making, we have recently proposed improvements to the method by introducing argon in the system in order to reduce fragmentation and formation of ethanol clusters. In this study, we optimize the experimental setup in the case of wine by (i) boosting the sampling protocol (sample headspace flushing and incubation); (ii) determining the optimal E/N value while using argon as carrier gas and (iii) proving that the optimized protocol reduces the effect of ethanol. The new protocol has been verified to discriminate eight French wines coming from three different regions (Gers, Gironde, Languedoc) and, in order to assess the applicability of the method in a relevant problem of oenological interest, we also tested it on a set of samples consisting of a red wine fermented with two different commercial preparations of Oenococcus oeni. Using principal component analysis of selected m/z signals, differentiation among wines from different geographical origin was achievable. Samples corresponding to the reference wine and to wines inoculated with two different commercial preparations were clearly separated. Intriguingly, our approach suggests the selective degradation of volatile organic compounds by O. oeni in wine as new possible feature of malolactic starter cultures in wine.

A novel culture medium for Oenococcus oeni malolactic starter production

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 Production Medium (OPM) contained diluted commercial 4X concentrate white must (1:6), ASv = 4%, and a pH of 3.8. The total time to obtain 80 L of an O. oeni liquid starter culture from the stock culture at the laboratory, with a final population of CFU = 1 × 109 mL−1, was 22 d. The starter culture was efficiently scaled-up, and preserved at 4 °C, −20 °C, or freeze-dried. This new culture medium also allowed adaptation of bacteria to the wine conditions, consuming all malic acid (3 g/L) in 7 d with an inoculum of CFU = 1 × 106 mL−1.