Malolactic fermentation (MLF) is a desirable step in the vinification process of most red wines. It is conducted by certain specific species of lactic acid bacteria (LAB), by far the most important of which is Oenococcus oeni.
The result of a successful MLF is an increased microbial stability of wine through the consumption of key carbon sources and other nutrients, which might otherwise be used by spoilage microorganisms, and through the production of antimicrobial compounds by LAB [1
]. The organoleptic properties of wine can also be altered during MLF by the consumption and liberation of various metabolites [3
]. Residual sugars can be metabolized into lactic acid or lactic acid, carbon dioxide, and ethanol or acetic acid via the fermentative pathways of LAB. Acetic acid, and hence the oenological parameter volatile acidity, can also increase in wine from the degradation of citric acid during MLF [5
]. This MLF can be spontaneous, due to the microflora present in the winery or from the grape material, or it can be induced by inoculation with one of a number of selected starters, chosen according to their technological or quality attributes. As a consequence of their adaptation to the wine environment, in particular, their tolerance to wine’s acidity and alcohol concentration, strains of O. oeni
are the predominant LAB responsible for the MLF. Thus, starter cultures for the MLF are also predominantly selected from this species. The strain-level diversity of O. oeni
populations in wine ecosystems is very high, and it can be region- and winery-specific [6
], often contributing to recognized differences in wines. O. oeni
has been shown to genetically adapt according the type of wine (white or red), driven by the pH and the phenolic compounds present [8
Taxonomically, the species is ordered into three groups, with A and B being the two major phylogenetic groups, and C a putative group composed of a unique strain isolated from cider. Group A exclusively contains strains found in wine. All strains from cider, except that attributed to group C, are located in group B, while strains from Champagne and Burgundy are only from group A. It appears that most of the strains isolated from malolactic ferments derive from the domestication of ancestral O. oeni
strains during the process of the industrialization of wine and cider, rather than responding to geographical constraints [9
Several molecular techniques have been applied to determine the diversity of LAB in red wines. Polymerase chain reaction (PCR) -based techniques including 16S metabarcoding sequencing [12
], PCR-DGGE (denaturing gradient gel electrophoresis) [13
] and species-specific multiplex PCR [14
] are used on samples taken directly from wines, but these are as yet limited to differentiation to the species level at most. To reach an intra-species discrimination level, prior isolation stage is still required. Randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis of rare restriction enzyme digests (REA-PFGE) are molecular methods frequently employed to investigate the strain diversity of several species of wine LAB [15
]. PFGE proved to be a quick tool to study the O. oeni
community, and their fluctuation in wine and the level of discrimination of PFGE depends on the restriction enzyme used [7
]. To achieve a finer differentiation of isolated strains, other methods that have been applied to this area include multilocus sequence typing (MLST) [6
], multiple locus variable number of tandem repeat analysis (VNTR) [16
], differential display PCR [7
], and single nucleotide polymorphisms (SNPs) [6
In the context of wine, phenolic compounds are naturally occurring molecules that are derived from grape material. They are important components of wine, contributing many sensorially and technologically relevant traits to the finished product. Phenolics constitute a highly diverse group of compounds, having in common the possession of at least one phenolic ring in their structure, but varying greatly in structure and size, and consequently their impact on wine quality.
Many practices employed in the winemaking process influence the phenolics composition and concentration in pre-malolactic wines. These begin with viticulture (grape variety and clone, light exposure, degree of ripeness) and continue in the winery (destemming, crushing, pre-fermentation maceration, alcoholic fermentation, pressing) options and parameters, amongst others. Must freezing, cryogenic maceration, extended maceration, enzyme regime, and alcoholic fermentation temperature have been reported to increase phenolics concentrations in wines [17
]. On the other hand, according to Olejar et al., mechanical harvesting could contribute to decreases in phenolics through reactions with oxidative radicals [20
]. In the study of Caridi et al., one S. cerevisiae
strain used as a starter for alcoholic fermentation was shown to enhance red wine content in phenolic compounds, especially in trans
-caffeic acid, quercetin and (−)-epicatechin [21
Phenolic compounds found in wines are normally classified in two groups; the flavonoids, which include anthocyanins, flavan-3-ols and flavonols (and other flavone derivatives), and the non-flavonoids, which comprise hydroxycinnamic acids and stilbenes among others [22
]. Wine flavan-3-ols, mainly (+)-catechin and (−)-epicatechin, are primarily synthesized in seeds and stems, and are the precursors of procyanidins and condensed tannins, which contribute to the astringency and bitterness of wines. Flavonols, mainly represented by quercetin, myricetin, and kaempferol, are responsible for the yellow color of white wines. Hydroxycinnamic acids (HCA) are mostly found in grapes in their bound (tartaric ester) forms, located in the vacuoles of the skin and pulp cells. In their free forms (trans
-coumaric acid, trans
-ferulic acid, and trans
-caffeic acid), HCA are important compounds in the oxidation processes of wine, and they act as color stabilizers and flavor precursors [23
-resveratrol is the most relevant stilbene in wine, known for its antioxidant activity [26
] and mostly coming from grape skins, being biosynthesized via the phenylalanine pathway as a defense response to biotic and abiotic stresses [27
Some phenolic compounds have been shown to have a species and strain-dependent impact on the activity of bacteria, which are relevant to wine quality. Phenolics can activate or inhibit microbial growth and metabolism, depending on their structures and concentrations [28
]. Hydroxycinnamic acids, and especially trans
-coumaric acid, have been shown to exhibit a strong inhibitory effect on the growth and survival of malolactic starters and wine-spoilage strains [30
]. In addition to their antimicrobial effects, the presence of HCA has been observed to increase the cell membrane permeability of some wine LAB, to delay their metabolism of glucose and citric acid, and to increase the yield of lactic and acetic acid production from glucose [29
]. According to Devi et al. [35
], the microbial response to exposure to HCA is, in part, manifested as changes in membrane and enzyme compositions. Quercetin and (+)-catechin can stimulate cell growth and metabolism, or they have an antimicrobial effect, depending on their concentrations and the microorganisms targeted. Vaquero et al. [36
] investigated the negative impacts of both quercetin and (+)-catechin against pathogenic bacteria. Some other studies mention that flavonols possess antimicrobial activities linked to their antioxidant properties, and to their potential cytotoxicity [37
]. At high concentrations, (+)-catechin has been observed to have an inhibitory effect on bacterial development [39
]. However, according to another study, quercetin and (+)-catechin could stimulate MLF by O. oeni
under certain conditions [40
]. Moreover, previously published research suggest that some levels of (+)-catechin can activate the cell growth of some Pediococus pentosaceus
and Lactobacillus plantarum
]. Devi et al. [35
] noted that among all the phenolic compounds tested, (+)-catechin exercised the least stress on the LAB tested. As for trans
-resveratrol, this stilbene was described as a strong inhibitor against some contaminant yeasts and acetic acid bacteria [42
]. Furthermore, as reported by García-Ruiz et al. [43
], both stilbenes and flavonols may have a negative impact on the growth of the O. oeni, L. hilgardii
and P. pentosaceus
strains isolated from wine. In addition, flavonols, especially kaempferol, have found to strongly inactivate some LAB by damaging their membranes [44
]. Of course, the concentration of the compounds tested is critical to any effect, and most of the studies cited on this specific subject have been performed in culture media, with concentrations of phenolic compounds being far higher than those found in wines, and certainly not under real wine conditions.
In this present paper, the development of the malolactic microbiota was studied throughout, and immediately following MLF conducted with and without O. oeni starter inoculation. A post-alcoholic fermentation red wine, supplemented with varying concentrations of flavan-3-ols, HCA, flavonols, and trans-resveratrol was the matrix used in this experiment. The development of specific O. oeni strains was followed in the samples treated with flavonols and trans-resveratrol.
This study evaluated the impact of the addition of specific groups of phenolic compounds on the behavior of wine LAB, and more specifically, on the diversity of MLF starter O. oeni strains. The novelty of the work lies in the fact that it was performed directly in post-alcoholic fermentation wine supplemented with concentrations of phenolics that are within the range that is encountered in real wine situations.
In this present study, biochemical, microbiological, and molecular tools were used to study the development of lactic acid bacteria, and the diversity of Oenococcus oeni strains isolated from red wines treated with small, grape-derived, phenolic compounds. In order to analyze the effect of an increase of particular classes of phenolics on MLF, the concentrations of flavan-3-ols, hydroxycinnamic acids, flavonols, and trans-resveratrol were doubled and tripled in inoculated and non-inoculated, post-alcoholic fermentation wines.
Unlike the results obtained by Hernandez et al. [45
], the concentrations of all the phenolics tested, except trans
-coumaric acid, decreased during MLF for both inoculated and non-inoculated wines (Table S1
). This observation might be explained by the complexation or precipitation of these compounds, or it may indeed be linked to the specific microbial population of the wine and some interaction with it.
The results reported here show that the effect of the phenolics studied on the growth and metabolism of LAB during MLF, as well as on the intraspecific diversity of O. oeni, was influenced by the type of phenolic and their concentrations, the stage of the fermentation and whether the wines were inoculated.
The addition of all the compounds tested, except for the flavan-3-ols, caused a delay in the lactic acid production for the non-inoculated wines. The reduction of the lactic acid production rate by the microbiota of non-inoculated samples treated with flavonols and HCA is concomitant with the inhibitory nature of these compounds at these concentrations (Figure 1
The effect of flavan-3-ols on the LAB growth during fermentation was found to be dependent on the microbial population of the samples. No inhibitory effect was observed on the non-inoculated samples, but depending of the concentrations added, these compounds can negatively impact the LAB growth or delay the lactic acid production in the wine inoculated with the starter OenosTM
. These observations are in agreement with previous studies showing the variable effect of (+)-catechin on different LAB [35
The acetic acid yield was apparently repressed in the non-inoculated samples by HCA and trans
-resveratrol at the end of MLF (14 days), although it increased after 28 days. A possible shift in the metabolic pathway of glucose consumption in OenosTM
towards acetic acid production, caused by hydroxycinnamic acids by was previously observed by Campos et al. [34
] in experiments performed in growth medium. In our study, this observation is also noted for trans
-resveratrol’s effect on some indigenous LAB.
All of the compounds tested apparently caused a delay in citric acid degradation by the LAB in inoculated wines. A similar observation on citrate metabolism was shown by Campos et al. [34
], using the same O oeni
stain that was inoculated in the growth medium and supplemented with HCA and other benzoic acids.
As reported by García-Ruiz et al. [43
-resveratrol had a similarly negative impact on the growth and metabolism of LAB in non-inoculated samples as flavonols and HCA. Nevertheless, in this study, 28 days after initiation of incubation, the bacterial concentrations were higher in the wines treated with trans
Polyphenol extracts have been tested on the O. oeni
intra-diversity in wine [8
] but the effect of the addition of the individual families of phenolic compounds so far has not been explored. The peculiar activity of flavonols and trans
-resveratrol against the O. oeni
distribution in the wines was investigated in a preliminary way in this current study. PFGE-REA has been used successfully for strain typing O. oeni
in red wines [6
]. In this work, a PFGE-REA technique was employed on the 80 representative isolates from the non-treated wines, and the wines treated with flavonols and trans
-resveratrol and identified as O. oeni
by microscopic observation. Not
I was used as a restriction enzyme to study the diversity and evolution of the wine O. oeni
population. The isolates showed 22 different genetic profiles, indicating a considerable intra-specific diversity in the wines. One of the colonies that were randomly selected for subculture from the wines with no prior addition of phenolic compounds presented the same pattern as Oenos™, the starter that was employed in other parts of the experiment. The post-alcoholic fermentation wines used in this study were characterized by LAB concentrations of ~104
CFU/mL. Together with a high diversity of O. oeni
strains, these factors could explain why Oenos™, in inoculated experiments appears not to dominate the indigenous microbiota in this particular wine. The diversity of O. oeni
was more influenced by the fermentation time than the type of MLF (inoculated or not) and the concentration of the phenolic compounds under study. Indeed, as observed in Figure 2
, two clear clusters, corresponding to the two strain isolation time points, grouped all the patterns.
The effect of phenolics on O. oeni
seems also to be strain-dependent. Some authors [43
] have observed a strong inhibitory effect for quercetin against four different O. oeni
strains, with an IC50
of 0.148 to 0.454 g/L, while others [40
] have shown that the addition of 5–25mg/L of quercetin-activated malic acid degradation by another O. oeni
strain. In our study, the profile L, grouping the highest number of strains, was less represented in the phenolic-treated samples. Moreover, the profiles X, Z, and M were found only in the wines, with no prior addition of phenolic compounds. A possible interpretation is that these strains are less tolerant to flavonols and stilbenes. The same goes for the strains from profiles I and Q, which are possibly enhanced by trans
-resveratrol, whilst strains from profiles G and H might be activated by flavonols. On the other hand, strains from profiles J and O could be negatively impacted by trans
-resveratrol and those from profile Y, by flavonols. These results seem to indicate that flavonols and trans
-resveratrol could affect O. oeni
positively or negatively, depending on the specific strain.
In summary, the effects of the addition of specific phenolic compounds on LAB behavior during MLF in wine have been described in this paper, highlighting the importance, in this respect, of the pre-malolactic winemaking techniques that influence the phenolic composition of the wine. More particularly, the increase of flavonols and trans-resveratrol concentrations at this stage could influence the malolactic bacterial population at the strain level and therefore possibly also the metabolic activities occurring during this period. More work should be focused on the specific effect of these compounds on each PFGE profile. Current work is being concentrated upon the effects of small grape phenolics on the metabolite characterization of wines following inoculated and spontaneously conducted MLF.