Applications of Metschnikowia pulcherrima in Wine Biotechnology

Abstract

Metschnikowia pulcherrima (Mp) is a ubiquitous yeast that frequently appears in spontaneous fermentations. The current interest in Mp is supported by the expression of many extracellular activities, some of which enhance the release of varietal aromatic compounds. The low fermentative power of Mp makes necessary the sequential or mixed use with Saccharomyces cerevisiae (Sc) to completely ferment grape musts. Mp has a respiratory metabolism that can help to lower ethanol content when used under aerobic conditions. Also, Mp shows good compatibility with Sc in producing a low-to-moderate global volatile acidity and, with suitable strains, a reduced level of H₂S. The excretion of pulcherrimin gives Mp some competitive advantages over other non-Saccharomyces yeasts as well as providing some antifungal properties.

1. Ecology and Physiology

Metschnikowia pulcherrima (Mp) is a globous/elliptical yeast that cannot be distinguished from Saccharomyces cerevisiae (Sc) by microscopy (Figure 1). Sometimes, it can be observed a single large, highly refractive oil droplet inside the cell. Mp is a teleomorph yeast belonging to an ascomycetous genus [1]. Its anamorph form is called Candida pulcherrima. Mp is a ubiquitous yeast that has been found in grapes, fruits (fresh and spoiled), flowers, nectars and tree sap fluxes. Several insects can work as vectors for this yeast. Mp strains can be identified through the use of selective and differential substrates; Mp strains showed both positive β-glucosidase enzyme activity and proteolytic activity [2]. Mp grows properly in either YPD or L-lysine media, and it can also can use arbutin as a carbon source in agar plates, indicating the expression of β-glucosidase activity (Figure 2) [3]. Recently, its nitrogen requirement was evaluated and slower consumption rates of ammonium were observed in Mp in comparison to other yeast genera [4]. This slow nitrogen uptake is indicative of its low fermentative ability [5].

The β-glucosidase activity related to Mp has been associated with different intracellular β-glucosidases, with the identification of three different bands observed when using fluorogenic substrates via an electrophoretic technique [6]. Of these three bands, the major band has similar physicochemical properties to those found in other studied yeasts, with high activity in ethanol and glucose concentrations often found in wines but low stability below pH 4. Mp is unable to develop in YPD at 37 °C and shows very weak or no growth in nitrate agar (Figure 2). It is able to use glucose, sucrose, fructose, galactose and maltose as carbon sources but shows weak or inexistent development in lactose [7]. It can grow properly under low temperature (15–20 °C) and pH conditions (3–6) [8]. Under environmental stress conditions such as a shortage of nitrogen, its recognition in optical microscopy is easy thanks to the appearance of a fat globule inside the cell at the beginning of the sporulation process [8]. In its sporulated form, the asci of Metschnikowia are long and clavate, containing one to two acicular to filiform spores [1].

The fermentative power of Mp is low, with many strains easily reaching 4% v/v in ethanol [3], although previous studies have observed the production of ethanol up to 6–7% v/v [9]. This feature, together with the fact that the presence of Mp in freshly pressed must is about 19–39% of the yeast ecology [9], makes it necessary to use Mp together with other yeast with a high fermentative power such as Sc or Schizosaccharomyces pombe to fully ferment grape sugars [10]. Its volatile acidity is also quite moderate, ranging from 0.3 to 0.4 g/L expressed as acetic acid [3]. Moreover, some strains are able to decrease the formation of H₂S during fermentation [11].

The fermentative performance of Mp is lower than that observed for other non-Saccharomyces species. The CO₂ production during fermentation yielded lower amounts for Mp than for Sc with 4.5 g per 100 mL vs. 12.9 g per 100 mL, respectively [12]. Mp has an intermediate acetoin production during alcoholic fermentation with respect to other species, such as S. cerevisiae and B. bruxellensis with low acetoin production and C. stellata and K. apiculata with the highest production of acetoin. The metabolic pathway for the production of this secondary metabolite from fermentation is shown in Figure 3. In addition, the amount of 2,3-butanediol produced by Mp is usually lower than that produced by Sc.

In mixed cultures with S. cerevisiae, viability was found to decrease rapidly after a few days of fermentation because of the low resistance to the ethanol produced by S. cerevisiae [14,15]. The use of emerging physical technologies that are able to strongly reduce the wild yeast content in grapes [16] can facilitate the prevalence of Mp during a longer period until the sequential inoculation of Sc, thus also increasing its effect on the sensory profile of the wines.

The sensibility of Mp to SO₂ is lower than that observed in Sc, Saccharomycodes ludwigii or S. pombe, but Mp shows a medium resistance compared with other non-Saccharomyces species [7]. A certain sensibility to some antimicrobials such as carvacrol and thymol has also been observed [17]. Regarding the use of dimethyl dicarbonate (DMDC), the growth of Mp strains during the fermentation of grape must is delayed, but not inhibited, after the addition of 400 mg/L DMDC [18]. The total inhibition of the microbial population can be achieved with 500 mg/L of DMDC. Sc can survive the addition of 200 mg/L DMDC, whereas the growth of other species of the genus Saccharomyces is inhibited with 150 mg/L DMDC.

2. Antimicrobial Bio-Tool

Mp can be used as a biological control agent thanks to its ability to produce natural antimicrobial compounds, namely pulcherrimin, an insoluble red pigment with antifungal activity. This peculiar antimicrobial activity is produced by the depletion of iron in the medium through the precipitation of iron(III) ions caused by the interaction with pulcherriminic acid, a precursor of pulcherrimin secreted by Mp. In this way, the environment becomes inhospitable to other microorganisms that require iron for their development. Pulcherrimin has shown effective inhibitory activity against several yeasts: Candida tropicalis and Candida albicans, as well as the Brettanomyces/Dekkera, Hanseniaspora and Pichia genera; and fungi: Botrytis cinerea, as well as Penicillium, Alternaria and Monilia spp. [19,20,21,22,23,24]. However, S. cerevisiae seems not to be affected by this antimicrobial activity [21,22]. Therefore, the use of Mp as a selected starter in sequential or mixed biotechnologies with Sc could be of great interest in modern enology.

Mp, as well as other yeast species such as Wickerhamomyces anomala (formerly Pichia anomala) and Torulaspora delbrueckii (Td), has a broad killer spectrum against some spoilage yeasts [25,26], of which C. glabrata had the highest sensitivity against the toxins from this species [27]. Mp has also been described as biofungicide capable of effectively reducing the incidence of Botrytis development in postharvest fruits [28]. Its antagonistic mechanism is mainly based on its competition for nutrients [29].

3. Aroma Compounds

The single use of Mp has led to excessive production of ethyl acetate with negative sensory repercussions [30]. However, the mixed use of Mp with Saccharomyces uvarum reduces the production of ethyl acetate, simultaneously favoring the formation of 2-phenyl ethanol and 2-phenylethyl acetate [30]. The use of co-inoculations of this type (mixed fermentations with Mp/Sc) has produced high contents of acetate esters and β-damascenone with lower levels of C₆ alcohols in ice wines made from the Vidal blanc grape variety [31]. An improvement in the aromatic complexity of the wines can be obtained by the use of Mp as a co-starter with Sc [3,32], mainly due to its high production of esters derived from its intense extracellular enzymatic activity [10,33]. Similarly, sequential fermentations with Mp showed a higher production of higher alcohols, with particularly high concentrations of isobutanol and phenylethanol [4].

4. Enzymatic Activities

Activities of the following enzymes have been described in Mp: pectinase, protease, glucanase, lichenase, β-glucosidase, cellulase, xylanase, amylase, sulphite reductase, lipase and β-lyase [11,33,34,35]. This is because Mp one of the non-Saccharomyces yeast species able to express more extracellular hydrolytic enzymes. Its high proteolytic activity makes it a very interesting fermentation partner for Sc, since the amino acids released (including those from autolysis) can serve as a source of nutrients for Sc [36]. In addition, its intense glucosidase activity [2], higher under aerobic conditions [37], promotes the release of varietal aromas from the grape by hydrolyzing bound monoterpenes. However, it is important to always remember that the intensity of the enzymatic activity depends not only on the species, but also on the strain [32].

Concerning aroma enhancement, the expression of β-D-glucosidase favors the release of free terpenes and this activity has been evaluated using the substrates 4-methylumbelliferyl-β-D-glucoside (MUG) and p-nitrophenyl-β-D-glucoside (pNPG), showing a good intensity with medium-to-low degradation of color by the effect on anthocyanin glucosides [38]. The commercial Mp L1781 (Flavia™ MP346, Lallemand) expresses α-arabinofuranosidase; this activity helps to release precursors of volatile terpenes [39,40] (Figure 4) and thiols [32,41], which help to enhance fruity smells in some varieties. This strain has shown an enzymatic specific activity of 0.22 U/mg when used as a dry yeast or fresh culture [41]. This has been measured by the hydrolysis of 11 μmol de p-nitrophenyl-α-L-arabinofuranosidase (pNPA) per minute [42].

Intracellular β-glucosidase of Mp has been purified by ion-exchange chromatography on amino agarose gel [6] and subsequently characterized. The optimum catalytic activity was observed at 50 °C and pH 4.5. The enzyme shows hydrolytic activity on β-(1→4) and β-(1→2) glycosidic bonds. The stability in alcoholic media (12% v/v) is good but it is affected by low pH.

5. Aerobic Metabolism/Alcohol Degree Reduction

The sequential use of Mp and Sc has proved to be somewhat effective in lowering the ethanol content of wine [11,43,44,45,46]. This is connected with the aerobic respiratory metabolisms of Mp that, in suitable aeration conditions, can aerobically metabolize more than 40% of sugars, thus significantly reducing the ethanol yield. An example of this application can be seen in the study developed by Contreras et al. (2014), where an average reduction in the alcoholic strength of 1.6% v/v was achieved when Mp was used in sequential fermentation with Sc (inoculated on the fourth day) in the production of red wine of the Syrah variety from a must with 240 g/L of sugars (potential alcoholic strength of 14% v/v). Therefore, the use of certain non-Saccharomyces yeast species, such as Mp, has been suggested as a biotechnological strategy aimed at producing wines with lower levels of ethanol [47]. In this last study, a kind of “collaboration” was seen between populations of Mp and S. uvarum, that is, a synergistic effect, achieving a lower ethanol production than in pure fermentations with each yeast. Recently, Mestre Furlani et al. (2017) evaluated the metabolic behavior of different non-Saccharomyces native yeasts to reduce the ethanol content during winemaking. They report that two out of the three strains of Mp isolated from grapes have a sugar to ethanol conversion ratio greater than >19 g/L/% v/v [48]. This confirms the usefulness of Mp to obtain wines with lower ethanol content.

6. Improvement of Wine Color Stability

Some non-Saccharomyces adsorb lower contents of anthocyanins during fermentation than Sc [49]. In Sc, the adsorption can range between 1 and 6% in total content of anthocyanins [50], but can reach up to 30% for some specific anthocyanins [51]. Adsorption is influenced by the composition and structure of the yeast cell wall. Mp shows a low adsorption of anthocyanins in cell walls when compared with other yeasts such as Sc, Td or Lachancea thermotolerans (Lt) in grape skin agar (Figure 5), according to the methodology described by Caridi et al. [52].

The effect of Mp in the formation of stable pigments (pyranoanthocyanins and polymers) during fermentation has been studied in sequential fermentations with Sc and S. pombe [10].

7. Conclusions

The versatility of Metschnikowia pulcherrima lies in its ability to ferment in combination with other yeast species as well as modulate the synthesis of secondary metabolites of fermentation to improve the sensory profile of the wine. It is characterized by a medium fermentation power and a high enzymatic capacity to release aromatic precursors from the grape. In addition, this yeast has potential as a biocontrol agent in order to limit competition with other yeasts in the fermentation medium.

The abovementioned applications and features of Metschnikowia pulcherrima may be of great interest in order to address one of the major concerns in today’s winemaking industry, such as excessive alcoholic strengths and the increasing prevalence in the market of flat wines from a sensory point of view. Mp could help solve these issues. The only important thing is to select the proper combination, as well as the right time and ratio of inoculation, between Mp and another yeast species capable of completing the alcoholic fermentation.