Non-Saccharomyces yeasts, particularly
Candida oleophila and
Candida boidinii, have gained little attention in enological research, unlike other commercial strains including
Torulaspora delbrueckii,
Metschnikowia pulcherrima, and
Lachancea Thermotolerans [
16,
17,
18,
19,
20,
21,
22,
23,
24]. Actually, only one study reported on the fermentative potential of
C. oleophila and
C. boidinii and their capacity to reduce ethanol during Sauvignon Blanc wine production [
33]. However, its antimicrobial qualities have not yet been studied in wine fermentation, despite the potential demonstrated in other food production areas.
C. oleophila has strong antifungal action, particularly for
Penicillium spp. in post-harvest studies [
28,
29,
30,
31], while
C. boidinii has been studied for olive production processes, with proven effects against the spoilage microorganisms
Enterobacteriácea,
Coliform, and
Shigella [
32].
4.1. Candida spp. Yeasts Produce Antimicrobial Effects in Grape Must
The results indicated that the bio-protective capacity of a yeast against a spoilage microorganism depends on the yeast species used, the inoculum concentration, and the spoilage microorganism under study.
In the case of lactic acid bacteria,
C. oleophila yeast (
Table 2) showed bio-protective behavior greater than that generated by
C. boidinii and by CO/CB across fermentation, and a better antimicrobial capacity than sulfites during pre-fermentative stages (<72 h), followed by protection comparable with the control until the end of fermentation. These results align with Escribano-Viana [
16], where a mixed inoculation of
T. delbrueckii/L. thermotolerans on LAB production in red wine must had a slightly better controlling effect than sulfites, followed by equaling the effect of SO
2 at the end of fermentation. In turn, bio-protection rose after increasing the initial inoculum concentration from 10
6 cells/mL to 10
7 cells/mL, showing that lactic acid bacteria were negatively affected by greater initial concentrations of bio-protective
Candida oleophila.
Currently, no studies indicate a reduction of lactic acid bacteria when using
Candida spp. strains, although
Candida oleophila has been associated with controlling post-harvest rot diseases in fruits, mainly related to fungal control [
28,
29,
30,
31]. In fact, yeasts including
M. Pulcherrima and
L. thermotolerans; two strains that are highly studied and proven bio-controllers against LAB [
16,
18,
19,
20], are also used as antimicrobial agents for post-harvest diseases [
38], meaning that there could be a relation between both potentials. However, further study is needed to support this.
For acetic acid bacteria,
Candida boidinii yeast (
Table 1) presented greater antimicrobial behavior than that of
Candida oleophila across fermentation, as well as being better than the strategy with sulfites and CO/CB in pre-fermentative stages (<72 h). At the end of fermentation, AAB concentrations were similar and without significant differences from sulfites and CO/CB strategies, probably due to the anaerobic conditions arising after inoculation with
S. cerevisiae on the third day [
16], since AAB only developed in the presence of oxygen [
21]. In turn, both
Candida boidinii 1 × 10
6 and
Candida boidinii 1 × 10
7 strategies presented concentrations without significant differences, meaning that no direct relation was observed between the concentration of the initial inoculum and the bio-protective effect against AAB with
Candida boidinii. However, when increasing
C. oleophila concentrations from 1 × 10
6 to 1 × 10
7 cells/mL, there was a strong reduction in AAB concentration, from 6.33 × 10
4 to 3.33 × 10
4 CFU/mL in the sample at 72 h, with an observable dependence on the initial inoculum concentration. It would be interesting to use larger
C. oleophila concentrations in future studies, since although there was a strong drop in AAB concentration at the end of the fermentation, the results did not exceed the protective effect generated by either
C. boidinii or sulfites.
In turn,
Brettanomyces bruxellensis could be effectively controlled with the
Candida boidinii 1 × 10
7 and mixed inoculum strategies (
Table 3 and
Figure 4), showing greater efficacy than
Candida oleophila until the end of fermentation. When compared with the sulfites control, the
Candida boidinii 1 × 10
7 strategy showed better antimicrobial behavior than sulfites in pre-fermentative stages, while the mixed CO/CB strategy showed such behavior until the end of fermentation, despite initially presenting higher
Brettanomyces bruxellensis concentrations. In this case,
C. boidinii presented a bio-protective behavior dependent on the initial inoculum concentration, where higher concentrations correlated with greater antimicrobial effects against
B. bruxellensis.The cause of the controlling effect presented by
C. boidinii may be the rapid fermentative power of this yeast, which can finish fermentation more quickly than the other modalities whether with simple inoculation or in mixed inoculation CO/CB (
Figure 1b,c), leading to swiftly exhausting nutrients in the medium and producing ethanol. In fact, the
Candida boidinii 1 × 10
7 strategy presented an ethanol production of 12.78 ± 0.15 %
v/
v (
Table 4), a significantly higher concentration than that obtained by the sulfites control and the other strategies. This behavior was unexpected since the literature reports that
non-saccharomyces yeasts in general, and
Candida boidinii in particular, are yeasts that can reduce ethanol levels in wine [
33,
39]. However, this may be due to the aforementioned lack of control over LAB (
Table 2), given that the presence of hetero-fermentative LAB can increase CO
2 and ethanol concentrations in wine [
40]. The rapid fermentation caused by
C. boidinii yeast compared with
C. oleophila had already been reported in the literature [
33], where sequential fermentation with the yeasts
C. boidinii–
S. cerevisiae finished fermentation by day 8, while the
C. oleophila–
S. cerevisiae duo finished by day 15. The importance of this lies in the fact that ethanol is considered an active bio-controlling compound [
15] since most microorganisms are inhibited by high concentrations of this compound. Strategies that present a rapid fall in density (
C. boidinii 1 × 10
6,
C. boidinii 1 × 10
7, and mixed inoculum) (
Figure 1b,c), produce ethanol earlier, altering microorganisms susceptible to this metabolite. The ones affected in this case are AAB and
B. bruxellensis yeast.
In summary, the strategies with Candida oleophila were more effective at suppressing LAB than Candida boidinii strategies, whereas the latter was more effective against AAB and B. bruxellensis than Candida oleophila strategies.
4.2. Final Wine Quality Is Comparable to That Obtained with SO2
Quality wines were obtained with the experimental conditions used, without significant differences between the bio-protected strategies and sulfite in producing L-malic acid, L-lactic acid, glycerol, tartaric acid, and residual sugar (
Table 4). All strategies were characterized by low L-lactic acid concentrations and the same L-malic acid concentration, despite differences in the final population of lactic acid bacteria (
Table 2 and
Figure 3). Indeed, the antimicrobial-free strategy was expected to present a higher concentration of lactic acid, given the lack of control obtained over LAB (
Table 2). However, the reason for this could be explained by the inhibition of malolactic fermentation (MLF), a process responsible for transforming malic acid into lactic acid due to the presence of certain compounds such as medium-chain fatty acids, organic acids, and peptides [
41], which could have been present in the final wines, preventing the production of lactic acid. On the other hand, the desired temperature for MLF to occur should be around 25 °C; in fact, it is known that temperatures between 12 and 20 °C, such as those used in this research, inhibit the metabolic activity of LAB, preventing the production of their metabolites [
42]. Additionally, another inhibitory compound is alcohol, where concentrations between 12 and 15%
v/
v alter the bacterial cell membrane, disrupting their metabolism; indeed, the strategies with
C. boidinii and antimicrobial-free presented higher alcohol concentrations (
Table 4), potentially causing the inactivation of lactic bacteria that prevented differences in the final chemical profile [
42]. Lastly, the type of strain must be considered, given that
Oenococcus oeni is the main bacterial species responsible for carrying out this biochemical stage, due to its ability to tolerate the harsh physicochemical properties of the wine once fermentation is complete [
43]; therefore, the presence of other LAB species may have prevented malic acid from transforming into lactic acid. However, to test this hypothesis, metabolomic-level analyses such as quantitative PCR would be required.
All strategies also presented a low final concentration of residual sugars (glucose and fructose), meaning that the final wines were dry and fermentative yeast action was unaffected by bio-protectors. The bio-protection modality with
Candida oleophila showed significantly high acetic acid concentrations (0.267 g/L) compared with the sulfite strategy. These findings align with the microbiological analysis, where
C. oleophila did not achieve effective bio-control against AAB (
Table 1 and
Figure 2), the main producers of this metabolite. However, the maximum allowable level in wines was not exceeded (1.2 g/L) [
44], preventing negative quality impacts.
With regard to volatile composition, we obtained wines with desirable organoleptic properties mainly associated with floral and fruity character.
Candida oleophila presented high concentrations of phenethyl alcohol (2-phenylethanol) associated with rose scents, significantly differing from the other strategies that stood out due to the production of ethyl esters from linear fatty acids (
Table 5). This may be due to high concentrations of
Brettanomyces bruxellensis, a spoilage microorganisms present in musts protected with
C. oleophila (
Figure 4) and which can produce phenylethyl alcohol through the Ehrlich pathway [
45]. However, studies have only been conducted on the fermentation of carrot pomace, and not on wine. There is current research about the potential for
Candida albicans to produce phenylethyl alcohol [
46], so
Candida oleophila may also produce this compound; however, there are no studies in this regard. Another alternative could be related to lipids released by the early cellular death of
Candida oleophila (
Table A1), which repress the acetyl transferase enzymes (ATF), decreasing esters’ synthesis [
47].
Another relevant aspect is the volatile profile obtained using the
Candida boidinii strategy (
Table 5), which produced high concentrations of ethyl-9-decenoate (13.61% abundance), associated with fruity peach aromas and significantly differing from the other strategies. This may be due to the presence of LAB which are not effectively controlled by the bio-protector
Candida boidinii. Wang et al. [
48], in a study on the effect of initiating cultures on aromatic compounds and wine microbiota, showed that wines with a greater population of
Leuconostoc and
Lactobacillus bacteria were positively correlated with ethyl 9-decenoate. However, no direct relation has yet been established between lactic bacteria and the production pathways of this compound. The study was also conducted on Cabernet Sauvignon rather than Sauvignon Blanc, although it could aid future studies.
The initially spontaneous strategies (sulfites and antimicrobial-free) and mixed strategy were characterized by the presence of fatty acid esters, including ethyl hexanoate, ethyl octanoate, and ethyl decanoate. These results align with the literature reports on Sauvignon Blanc wines fermented with
Saccharomyces Cerevisiae [
49]. In fact, a study evaluating the effect of SO
2 on Sauvignon Blanc must showed that the ethyl esters ethyl hexanoate, ethyl octanoate, and ethyl decanoate were produced in greater quantities in wines after adding sulfites compared to those without sulfites [
49].
In general, the volatile profile was not negatively affected by bio-protectors. In fact, they added aromatic complexity to the wine, particularly Candida boidinii and Candida oleophila, which showed positive differences from the control. In future studies, it would be interesting to complement the results with genetic identification techniques, including a quantitative PCR and MALDI-TOF MS, which would help identify microorganism species and verify the proper implantation of the bio-protected strain.