Malolactic Fermentation Starter Cultures Research Articles

Malolactic fermentation of lactic acid bacteria isolated from southern Brazilian red wine.

The objective was to isolate lactic acid bacteria (LAB) from southern Brazil's wines and investigate their potential as starter cultures for malolactic fermentation (MLF) in Merlot (ME) and Cabernet Sauvignon (CS) wines through the fermentative capacity. The LAB were isolated from CS, ME, and Pinot Noir (PN) wines in the 2016 and 2017 harvests and evaluated for morphological (color and shape of the colonies), genetic, fermentative (increase in pH, acidity reduction, preservation of anthocyanins, decarboxylation of L-malic acid, yield of L-lactic acid, and content of reduced sugars), and sensory characteristics. Four strains were identified as Oenococcus oeni [CS(16)3B1, ME(16)1A1, ME(17)26, and PN(17)65], one as Lactiplantibacillus plantarum [PN(17)75], and one as Paucilactobacillus suebicus [CS(17)5]. Isolates were evaluated in the MLF and compared to a commercial strain (O. oeni), as well as a control (without inoculation and spontaneous MLF), and standard (without MLF). CS(16)3B1 and ME(17)26 isolates finished the MLF for CS and ME wines, respectively, after 35 days, similar to the commercial strain, and CS(17)5 and ME(16)1A1 isolates ended the MLF in 45 days. In the sensory analysis, ME wines with isolated strains received better scores for flavor and overall quality than the control. Compared to the commercial strain, CS(16)3B1 isolate obtained the highest scores for buttery flavor and taste persistence. CS(17)5 isolate received the higher scores for a fruity flavor and overall quality and the lowest for a buttery flavor. The native LAB displayed MLF potential, regardless of the year and grape species from which they were isolated.

Isolation and Characterization of High-Ethanol-Tolerance Lactic Acid Bacteria from Australian Wine.

Lactic acid bacteria are very important in winemaking. In this study, 108 lactic acid bacteria isolates were obtained from high-ethanol-content (~17% (v/v)) Grenache wines during uninoculated malolactic fermentation (MLF). The 16S rRNA and species-specific PCR showed that 104 of these were Oenococcus oeni, three were Lactobacillus hilgardii, and one was Staphylococcus pasteuri. AFLP of HindIII and MseI digests of the genomic DNA of the O. oeni strains was developed for the first time to discriminate the strains. The results showed that the method was a suitable technique for discriminating the O. oeni strains. Based on the cluster analysis, nine O. oeni strains were chosen for inclusion in an ethanol tolerance assay involving monitoring of optical density (ABS600nm) and viable plating. Several O. oeni strains (G63, G46, G71, G39) survived and grew well in MRS-AJ with 17% (v/v) ethanol, while the commercial O. oeni reference strain did not. Strain G63 could also survive and grow for 168 h after inoculation in MRS-AJ medium with 19% (v/v) ethanol. These results suggest that O. oeni G63, G46, G71, and G39 could potentially be used as MLF starters for high-ethanol-content wines. All three L. hilgardii strains could survive and grow in MRS-AJ with 19% (v/v) ethanol, perhaps also indicating their suitability as next-generation MLF starter cultures.

Biogenic amines and the importance of starter cultures for malolactic fermentation

Biogenic amines are compounds produced mainly by microorganisms from the decarboxylation of some amino acids which may cause several health issues in susceptible people. In the winemaking process, one of the factors with the greatest impact on the final concentration of biogenic amines in wine is the malolactic fermentation, which mainly involves the action of lactic acid bacteria. This article gathers scientific literature on the importance of the use of inoculation in winemaking, especially for the potential formation of biogenic amines in spontaneous fermentations, with emphasis on malolactic fermentation. It establishes the set of attributes that influence the formation of biogenic amines in winemaking and aspects related to their toxicity, especially histamine and tyramine. With the growing interest of the market in products with less chemical appeal, the safety of wines, whether from non-conventional or traditional winemaking, must increasingly be observed. Therefore, the importance of using cultivated starter cultures is discussed, along with the safety of organic wines and the presentation of articles that seek to reconcile the production of organic wine with the preservation of the ‘microbiological terroir’ in a safe way, using selected wild yeasts and lactic acid bacteria.

Lactobacillus plantarum, a New Biological Tool to Control Malolactic Fermentation: A Review and an Outlook

Malolactic fermentation (MLF) in wine is an important step in the vinification of most red and some white wines, as stands for the biological conversion of l-malic acid into l-lactic acid and carbon dioxide, resulting in a decrease in wine acidity. MLF not only results in a biological deacidification, it can exert a significant impact on the organoleptic qualities of wine. This paper reviews the biodiversity of lactic acid bacteria (LAB) in wine, their origin, and the limiting conditions encountered in wine, which allow only the most adapted species and strains to survive and induce malolactic fermentation. Of all the species of wine LAB, Oenococcus oeni is probably the best adapted to overcome the harsh environmental wine conditions and therefore represents the majority of commercial MLF starter cultures. Wine pH is most challenging, but, as a result of global warming, Lactobacillus sp. is more often reported to predominate and be responsible for spontaneous malolactic fermentation. Some Lactobacillus plantarum strains can tolerate the high alcohol and SO2 levels normally encountered in wine. This paper shows the potential within this species for the application as a starter culture for induction of MLF in juice or wine. Due to its complex metabolism, a range of compositional changes can be induced, which may positively affect the quality of the final product. An example of a recent isolate has shown most interesting results, not only for its capacity to induce MLF after direct inoculation, but also for its positive contribution to the wine quality. Degrading hexose sugars by the homo-fermentative pathway, which poses no risk of acetic acid production from the sugars, is an interesting alternative to control MLF in high pH wines. Within this species, we can expect more strains with interesting enological properties.

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.

Selection and Characterisation of Oenococcus oeni and Lactobacillus plantarum South African Wine Isolates for Use as Malolactic Fermentation Starter Cultures

This study focused on characterising 23 Oenococcus oeni and 19 Lactobacillus plantarum strains isolated from the South African wine environment for the development of potential commercial malolactic fermentation (MLF) starter cultures. These strains were characterised with regards to oenological important characteristics, including the genetic screening for enzyme-encoding genes (enzymes that are involved/implicated in wine aroma modification, as well as enzymes pertaining to the wholesomeness of the final wine product), their fermentation capabilities, the ability to maintain viability during MLF, as well as the volatile acidity production. A total of three O. oeni and three L. plantarum strains were selected at the completion of this study. These six strains showed the most potential during the characterisation stages of the study and were able to successfully complete MLF in Pinotage wine. It was also found that L. plantarum strains displayed a more diverse enzyme profile than O. oeni strains, particularly with regards to the presence of the aroma-modifying enzymes β-glucosidase and phenolic acid decarboxylase (PAD), which implies the future use of this species in the modification of the wine aroma profile and use as commercial starter culture.

Protective role of glutathione addition against wine-related stress in Oenococcus oeni

Oenococcus oeni is the main species responsible for the malolactic fermentation (MLF) of wine due to its ability to survive in this environment. Some wine-related stress factors, such as ethanol and low pH, may alter the cell redox balance of O. oeni. For the first time, the ability to uptake glutathione (GSH), an almost universal tripeptide with antioxidant properties, has been associated to the improvement of stress response in O. oeni. Despite the inability of O. oeni to synthesize GSH, this bacterium can capture it from the media. The ability of 30 O. oeni strains to uptake GSH was assessed in this study. Although all of the strains tested were able to import GSH, substantial variability among them was detected. To assess the physiological function of GSH, three strains with different GSH-import capacities were selected. Significant changes in membrane fatty acids composition were observed due to GSH addition. The most relevant was the increase of cyclopropane fatty acids in cell membrane, in both the exponential and the stationary phases. Cells grown with GSH showed an improved survival against ethanol shock (14% v/v). GSH addition also increased biomass production during the adaptation to wine stress conditions (pH4, pH3.4 and 6% ethanol). The results suggest that GSH enrichment could improve the resistance to stress to O. oeni, which could be useful for the adaptation of MLF starter cultures.

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.

Technological properties of Lactobacillus plantarum strains isolated from grape must fermentation

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 acidification aptitudes). Several parameters such as sugar, pH and ethanol tolerance, resistance to lyophilisation and behaviour in grape must were evaluated. Moreover, the expression of stress gene markers was investigated and was linked to the ability of L. plantarum strains to grow and perform MLF. Co-inoculation of Saccharomyces cerevisiae and L. plantarum in grape must improves the bacterial adaptation to harsh conditions of wine and reduced total fermentation time. For the first time, we applied a polyphasic approach for the characterization of L. plantarum in reason of the MLF performances. The proposed procedure can be generalized as a standard method for the selection of bacterial resources for the design of MLF starter cultures tailored for high pH must.

Growth and consumption of L-malic acid in wine-like medium by acclimated and non-acclimated cultures of Patagonian Oenococcus oeni strains.

Five Oenococcus oeni strains, selected from spontaneous malolactic fermentation (MLF) of Patagonic Pinot noir wine, were assessed for their use as MLF starter cultures. After the individual evaluation of tolerance to some stress conditions, usually found in wine (pH, ethanol, SO2, and lysozyme), the behavior of the strains was analyzed in MLO broth with 14% ethanol and pH3.5 in order to test for the synergistic effect of high ethanol level and low pH and, finally, in a wine-like medium. Although the five strains were able to grow in MLO broth under low pH and/or high ethanol, they must be acclimated to grow in a wine-like medium. Additionally, glycosidase and tannase activities were evaluated, showing differences among the strains. The potential of the strains to ferment citrate was tested and two of the five strains showed the ability to metabolize this substrate. We did not detect the presence of genes encoding histidine, tyrosine descarboxylase, and putrescine carbamoyltransferase. All the strains tested exhibited good growth capacity and ability to consume L-malic acid in a wine-like medium after cell acclimation, and each of them showed a particular enzyme profile, which might confer different organoleptic properties to the wine.

Biodiversity of Lactobacillus plantarum from traditional Italian wines.

In this study, 23 samples of traditional wines produced in Southern Italy were subjected to microbiological analyses with the aim to identify and biotype the predominant species of lactic acid bacilli. For this purpose, a multiple approach, consisting in the application of both phenotypic (API 50CHL test) and biomolecular methods (polymerase chain reaction-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing) was used. The results showed that Lactobacillus plantarum was the predominant species, whereas Lb. brevis was detected in lower amount. In detail, out of 80 isolates 58 were ascribable to Lb. plantarum and 22 to Lb. brevis. Randomly amplified polymorphic DNA-polymerase chain reaction was used to highlight intraspecific variability among Lb. plantarum strains. Interestingly, the cluster analysis evidenced a relationship between different biotypes of Lb. plantarum and their origin, in terms of wine variety. Data acquired in this work show the possibility to obtain several malolactic fermentation starter cultures, composed by different Lb. plantarum biotypes, for their proper use in winemaking processes which are distinctive for each wine.

Patagonian red wines: selection of Lactobacillus plantarum isolates as potential starter cultures for malolactic fermentation

The aim of this study was to evaluate fifty-three Lactobacillus plantarum isolates obtained from a Patagonian red wine, molecularly identified and typified using RAPD analysis, in order to select starter cultures for malolactic fermentation (MLF). The results obtained suggest a considerable genetic diversity, taking into account that all L. plantarum isolates were obtained from one cellar and one vintage. Based on the capacity to tolerate a concentration of 14% ethanol in MRS broth for 2days, eight isolates were selected for the subsequent analysis. The incidence of various wine stress factors (ethanol, acid pH, lysozyme and sulfur dioxide) on isolates growth was studied. Besides, glucosidase and tannase activities were evaluated, and the presence of genes involved in the synthesis of biogenic amines was examined by PCR. A previously characterized indigenous Oenococcus oeni strain was included with comparative purposes. Differences in technologically relevant characteristics were observed among the eight L. plantarum selected isolates, revealing an isolate-dependent behavior. Detectable glucosidase and tannase activities were found in all isolates. The presence of genes encoding histidine and tyrosine descarboxylases and putrescine carbamoyltransferase was not detected. The ability of L. plantarum isolates to grow and consume L-malic acid in simulated laboratory-scale vinifications revealed that two of them could be considered as possible MLF starter cultures for Patagonian red wines. These isolates will be subjected to further analysis, for a final winery technological characterization.

Comparative metabolic profiling to investigate the contribution ofO. oeniMLF starter cultures to red wine composition

In this research work we investigated changes in volatile aroma composition associated with four commercial Oenococcus oeni malolactic fermentation (MLF) starter cultures in South African Shiraz and Pinotage red wines. A control wine in which MLF was suppressed was included. The MLF progress was monitored by use of infrared spectroscopy. Gas chromatographic analysis and capillary electrophoresis were used to evaluate the volatile aroma composition and organic acid profiles, respectively. Significant strain-specific variations were observed in the degradation of citric acid and production of lactic acid during MLF. Subsequently, compounds directly and indirectly resulting from citric acid metabolism, namely diacetyl, acetic acid, acetoin, and ethyl lactate, were also affected depending on the bacterial strain used for MLF. Bacterial metabolic activity increased concentrations of the higher alcohols, fatty acids, and total esters, with a larger increase in ethyl esters than in acetate esters. Ethyl lactate, diethyl succinate, ethyl octanoate, ethyl 2-methylpropanoate, and ethyl propionate concentrations were increased by MLF. In contrast, levels of hexyl acetate, isoamyl acetate, 2-phenylethyl acetate, and ethyl acetate were reduced or remained unchanged, depending on the strain and cultivar evaluated. Formation of ethyl butyrate, ethyl propionate, ethyl 2-methylbutryate, and ethyl isovalerate was related to specific bacterial strains used, indicating possible differences in esterase activity. A strain-specific tendency to reduce total aldehyde concentrations was found at the completion of MLF, although further investigation is needed in this regard. This study provided insight into metabolism in O. oeni starter cultures during MLF in red wine.

Investigation of the Volatile Composition of Pinotage Wines Fermented with Different Malolactic Starter Cultures Using Comprehensive Two-Dimensional Gas Chromatography Coupled to Time-of-Flight Mass Spectrometry (GC×GC-TOF-MS)

Headspace solid phase microextraction in combination with comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (HS-SPME-GC × GC-TOF-MS) was used for the detailed investigation of the impact of malolactic fermentation (MLF) using three commercial Oenococcus oeni strains on the volatile composition of Vitis vinifera cv. Pinotage wines. GC × GC allowed the identification of 115 volatile compounds, including both major constituents and trace-level compounds, in a single analysis. A number of compounds differing in mean concentration levels between the control wines and those fermented with different starter cultures were shown for the first time to be influenced by MLF and/or the bacterial strain. Principal component analysis (PCA) provided excellent separation between the wines fermented with different MLF starter cultures and the control wine. Significantly different levels for some volatile compounds in wines fermented with one of the LAB starter cultures could be indicative of metabolic differences of this strain.

Lactobacillus: the Next Generation of Malolactic Fermentation Starter Cultures—an Overview

Malolactic fermentation (MLF) is a secondary wine fermentation conducted by lactic acid bacteria (LAB). This fermentation is important in winemaking as it deacidifies the wine, it contributes to microbial stability and lastly it contributes to wine aroma through the production of metabolites. Oenococcus oeni is the main species used in commercially available starter culture currently, but research has indicated that different Lactobacillus species also partake in MLF and this has shifted the focus in the MLF field to evaluate the potential of lactobacilli as starter cultures for the future. There are 17 different species of Lactobacillus associated with winemaking either being associated with the grapes/beginning of alcoholic fermentation or the MLF and wine. Lactobacillus associated with wine is mainly facultative or obligatory heterofermentative and can withstand the harsh wine conditions such as high ethanol levels, low pH and temperatures and sulphur dioxide. Wine lactobacilli contain the malolactic enzyme encoding gene, but sequence homology shows that it clusters separate from O. oeni. Lactobacillus also possesses more enzyme encoding genes compared to O. oeni, important for the production of wine aroma compounds such as glycosidase, protease, esterase, phenolic acid decarboxylase and citrate lyase. Another characteristic associated with wine lactobacilli is the production of bacteriocins, especially plantaricins which would enable them to combat spoilage LAB. All these characteristics, together with their ability to conduct MLF just as efficiently as O. oeni, make them suitable for a new generation of MLF starter cultures.

Technological properties ofOenococcus oenistrains isolated from typical southern Italian wines

To isolate indigenous Oenococcus oeni strains suitable as starters for malolactic fermentation (MLF), using a reliable polyphasic approach. 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 winelike 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. 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. 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.

Genetic screening of wine-related enzymes inLactobacillusspecies isolated from South African wines

The objective of this study was to investigate the presence of genes coding for enzymes of oenological relevance in wine Lactobacillus strains isolated from South African grape and wine samples during the 2001 and 2002 harvest seasons. A total of 120 wine lactobacilli isolates belonging to Lactobacillus plantarum, Lactobacillus hilgardii, Lactobacillus brevis, Lactobacillus pentosus, Lactobacillus paracasei, Lactobacillus sakei and Lactobacillus paraplantarum were genetically screened for enzyme-encoding genes using PCR with primers specific for beta-glucosidase, protease, esterase, citrate lyase and phenolic acid decarboxylase. The results of PCR screening showed that the Lactobacillus strains possessed different combinations of enzymes and that some strains did not possess any of the enzymes tested. Confirmation analysis with gene sequencing also showed high similarity of genes with those available in GenBank database. In this study, we have demonstrated the existence of genes coding for wine-related enzymes in wine lactobacilli that could potentially hydrolyse wine precursors to positively influence wine aroma. An expansion of knowledge on the genetic diversity of wine-associated lactic acid bacteria will enable the selection of novel malolactic fermentation starter cultures with desired oenological traits for the improvement of the organoleptic quality of the wine, and hence wine aroma.

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.