3.2.1. Carbohydrate Utilization
Using carbohydrates, such as glucose and maltose, is essential when characterizing potential starter cultures for the food industry, for example, to produce alcoholic beverages like beer. The results of the yeast isolates’ metabolism are shown in
Figure 1 and
Table 5 and
Table 6, representing the behavior of the yeasts
Z. rouxii,
Candida sp.,
S. pombe,
R. ruineniae,
C. lusitaniae, and
M. chrysoperlae over a 72 h incubation period at a temperature of 27 °C and 150 rpm agitation. The growth threshold was defined at an optical density read at 600 nm (OD
600) of 0.4 in the diagrams [
35]; values higher than this indicate significant growth, while values below indicate that the yeasts have not experienced significant growth.
All six yeast strains can efficiently utilize both glucose and maltose sugars, as evidenced by the increase in OD600. In the case of growth in the defined glucose medium, the yeasts Z. rouxii with OD600 2.45 and C. lusitaniae with OD600 2.43 exhibit the highest cellular growth, compared to R. ruineniae with OD600 1.76, which shows the lowest growth.
In the case of the defined maltose medium, the yeasts
Z. rouxii,
Candida sp.,
S. pombe, and
C. lusitaniae exhibit very similar growth, reaching the highest values with OD
600 2.36, OD
600 2.28, OD
600 2.26, and OD
600 2.27, respectively. A different scenario was observed for
M. chrysoperlae, which showed the lowest growth, with OD
600 1.73. The ability of the isolated yeast to grow in sugar-rich environments might be related to their osmotolerant ability; having been isolated from honey, these yeasts have an adaptive response to extracellular osmotic pressure, which allows them to pump ions from the cell exterior to the interior, and synthesize and concentrate various solutes (such as sugars, polyalcohols, amino acids, and glycerol) which allow them to maintain the cell integrity [
36,
37].
As part of the fermentative metabolism, yeasts are able to produce certain metabolites, and
Table 5 shows the concentrations of glucose, ethanol, glycerol, lactic acid, and acetic acid at the end of the fermentation in the glucose-defined medium.
S. pombe produced the highest ethanol concentration (2.26 ± 0.04%
v/
v), lactic acid concentration (0.23 ± 0.01 g/L), acetic acid concentration (0.59 ± 0.20), and was the second-highest producer of glycerol (1.56 ± 0.06 g/L). It consumed the highest amount of sugars compared to the other yeasts, with values of sucrose reaching 0.53 ± 0.03 g/L at the end of fermentation and consumed all of the fructose and glucose.
In the case of the medium supplemented with maltose (60 g/L),
Table 6 shows the same behavior for the yeast
S. pombe. The highest concentrations of lactic acid (0.27 ± 0.01 g/L) and acetic acid (0.74 ± 0.3 g/L) were obtained, but a higher concentration of ethanol (1.62 ± 0.06%
v/
v) was produced. Regarding sugars, the yeast consumed all glucose at the end of fermentation.
Little information is available associated with the fermentative metabolism of yeast isolated from honey. Matraxia et al. [
35] reported the percentage of sugar consumption in various fermentation treatments using yeast isolated from fermented honey. Regarding glucose, the yeasts in that study demonstrated the ability to consume 99.40 ± 0.02%, while for fructose, they consumed 97.30 ± 0.05%. In the case of sucrose, the consumption range in that study varied from 37.90 ± 0.39% to 100 ± 0%. These results are consistent with ours, as the honey-isolated yeasts also consumed virtually all the glucose and, to a lesser extent, sucrose during the phenotypic characterization tests. Regarding the produced metabolites, the authors of the previously mentioned study indicate that the concentration of ethanol produced varied between 0.52 and 5.16% (
v/
v), while acetic acid ranged between 0.03 and 0.26 g/L. As for glycerol, the values fluctuated between 1.28 and 3.80 g/L. In our research, the ethanol ratio falls between 0.07 ± 0.008% and 2.26 ± 0.04%, with the maximum concentration being lower than reported by these authors. Regarding acetic acid, the yeasts studied in this work produced slightly higher concentrations than those reported in the previously mentioned work. In contrast, the concentrations of glycerol generated were lower than those mentioned by the previously cited authors. These differences might be attributed to the different species evaluated in our study; as they are non-
Saccharomyces yeasts, they show a different metabolism.
In another study, Prestianni et al. [
38] studied
Saccharomyces cerevisiae and
Hanseniaspora uvarum isolated from honey by-products to evaluate the influence of taste and olfactory attributes in mead. Regarding sugar consumption, the authors reported that, in various fermentation treatments, a residual glucose percentage ranging from 0.72 to 31.86 g/L was obtained, while for fructose, it was from 1.05 to 40.76 g/L. Compared to our results, the residual sugar concentration reported by these authors is higher than what we observed in our research, where these sugars were consumed almost entirely. Regarding the produced metabolites, the authors of the other work reported concentrations of acetic acid ranging from 0.29 ± 0.02 g/L to 0.71 ± 0.06 g/L, ethanol from 5.31 ± 0.81% to 12.37 ± 1.07%, and glycerol from 4.31 ± 0.14 g/L to 7.25 ± 0.66 g/L. When comparing these results with ours, we observe that, in the case of acetic acid, our concentrations range from 0.17 ± 0.03 g/L to 0.74 ± 0.30, obtaining similar results to those reported by the authors of the other study. The ethanol percentage obtained in our research is lower than that reported by these authors, possibly because they carried out sequential fermentation treatments. Regarding glycerol, we also obtained concentrations, in most cases, below those reported by these authors, possibly due to the use of different yeast strains with different metabolism.
3.2.2. Ethanol Tolerance
Ethanol tolerance was studied at two concentrations, 5% (
v/
v) and 8% (
v/
v), for the six yeast strains.
Figure 2 shows the behavior of these yeasts under both treatments. The minimum growth threshold was set at an OD
600 of 0.4.
In the case of the fermentation carried out at 5% (v/v), the yeasts Z. rouxii, Candida sp., S. pombe, and R. ruineniae exhibited good growth, easily surpassing the established growth threshold, with OD600 values of 1.52, 1.06, 1.84, and 1.18, respectively. However, the yeasts Candida sp. and M. chrysoperlae remained below the growth threshold, indicating that they are not tolerant to ethanol concentrations commonly reached in the fermentation process for beer making. Nevertheless, these yeasts could be considered for non-alcoholic fermentation or obtaining beverages with an ethanol concentration lower than 5% (v/v).
During fermentation at 8% (v/v), a lag phase of more than 20 h was observed for all the yeast isolates; after this time, increases in optical density were observed to finally reach values above DO600 of 2, which suggest that the yeast needed to become used to the ethanol-rich medium before increasing in cell concentration. However, only the yeasts Z. rouxii, S. pombe, and R. ruineniae exhibited optimal growth above the threshold, reaching OD600 values of 0.56, 1.69, and 0.90, respectively. Meanwhile, Candida sp., C. lusitaniae, and M. chrysoperlae were inhibited at this ethanol concentration.
In general, our results showed that the yeast
S. pombe exhibited the best performance compared to others in both treatments regarding its fermentative metabolism and ethanol tolerance. This suggests that it is an interesting candidate for the alcoholic beverage industry. The yeast species has been previously reported as a starter culture for the production of wine. Interestingly, the utilization of the yeast allowed a deacidification of the wines without the use of lactic acid bacteria, given the yeast’s capacity to utilize malic acid along with the alcoholic fermentation [
39,
40,
41]. García et al. [
42] analyzed the ethanol resistance of the yeast
S. pombe, isolated from the spontaneous fermentation of Malvar grapes. In this study, the authors assessed growth at 5% (
v/
v) ethanol, where the yeast exhibited growth exceeding a 100% yield, while in the study at 8% (
v/
v) ethanol, they achieved a growth yield of 80%. When studying stress resistance at a concentration of 13%
v/
v ethanol, the cellular growth performance was less than 20%.
Additionally, this yeast possesses other characteristics that contribute to its suitability for the beverage industry. It has been reported that
S. pombe can assimilate glucose, maltose, sucrose, and raffinose, as well as D-gluconate as a carbon source [
43]. It exhibits a remarkable ability to carry out malo-alcoholic fermentation and exceptionally deacidify malic acid [
44], with a unique deacidification ability, as most
S. pombe strains achieve the complete deacidification of malic acid, and have been reported to resist high levels of ethanol, even up to 16% [
45]. In addition, it is resistant to preservatives such as sorbic or benzoic acid, with documented tolerance up to 600 mg/L. These characteristics make the yeast a suitable starter for the production of alcoholic beverages, both wines and beers.
Another study conducted by Yao et al. [
46] investigated the resistance of
Z. rouxii to ethanol concentrations of 0%, 5%, 7%, 8%, 9%, and 10%
v/
v. These authors reported that the yeast only showed a low cell yield at concentrations of 8%, 9%, and 10%
v/
v of ethanol. This finding aligns with our study, as
Z. rouxii exhibited a meager cell growth rate at the concentration of 8%
v/
v ethanol. In the same study, they concluded that, when
Z. rouxii was cultured alone, survival rates ranged from 6% to 10%, while co-culturing with
T. halophilus improved the ethanol tolerance of
Z. rouxii. The tolerance improved with an increase in the co-culturing time.
A similar study was conducted by Fan et al. [
47], who evaluated the ethanol tolerance of
C. lusitaniae. In this study, the yeast maintained an excellent cell growth rate only up to an ethanol concentration of 6% (
v/
v). This result is similar to the findings in this work, as
C. lusitaniae exhibited good cell growth at 5% (
v/
v) ethanol but did not perform well fermentatively at 8% (
v/
v) ethanol. The authors also reported studies on tolerance to high temperatures of 50 °C, where the yeast could grow over a wide pH range (pH 1–11) and exhibited tolerance to osmotic pressure, growing in media containing 80% glucose. All these results are similar to those obtained in this work when conducting tests for resistance to pH 3.5, high temperatures of 37 °C, and ethanol concentrations of 5% (
v/
v) and 8% (
v/
v) for the yeast
C. lusitaniae.
In the study conducted by Boro and Narzary [
48], they reported the isolation of two strains of
R. ruineniae, which they classified as non-amylolytic, non-proteolytic, non-cellulolytic, and non-ethanol producers, with a low tolerance of only 2% (
v/
v) ethanol. Compared with this study,
R. ruineniae exhibited a high growth rate in ethanol stress analyses at concentrations of 5% (
v/
v) and 8% (
v/
v) ethanol, suggesting that the metabolism of this yeast is strain-dependent.
3.2.4. Cross-Tolerance between Ethanol and Hops
To better assess the potential of the isolated yeasts as starter cultures for beer making, a cross-tolerance test was conducted, in which the yeast was subjected to fermentation with both hop and ethanol at different concentrations.
Figure 4 shows the results obtained.
In the treatment with 50 IBU + 5% (v/v) ethanol, the yeasts that exhibited good cellular growth after 72 h were R. ruineniae with OD600 2.02, Z. rouxii with OD600 1.98, S. pombe with OD600 1.91, Candida sp. with OD600 1.26, and C. lusitaniae with OD600 0.54. M. chrysoperlae was the only yeast with a poor fermentative performance, falling below the established growth threshold.
In the case of the treatment with 50 IBU + 8% (v/v) ethanol, the behavior was different. S. pombe and R. ruineniae exhibited good fermentative performance with OD600 values of 1.35 and 1.56, respectively. Meanwhile, Z. rouxii showed poor performance with an OD600 of 0.47. Yeasts demonstrating suboptimal fermentative performances included Candida sp., C. lusitaniae, and M. chrysoperlae, falling below the critical growth threshold.
The treatment with 90 IBU + 5% (v/v) ethanol revealed that the yeasts showing poor fermentative performances were C. lusitaniae and M. chrysoperlae, with OD600 values of 0.17 and 0.25, respectively, falling below the established growth threshold. In contrast, the remaining yeasts Z. rouxii, Candida sp., S. pombe, and R. ruineniae demonstrated good fermentative performances, with OD600 values of 1.21, 1.16, 1.79, and 1.52, respectively.
In the treatment with 90 IBU + 8% (v/v) ethanol, the results are different, indicating that the factor affecting the growth of some of these yeasts is ethanol, depending on the concentration used. In this case, the only yeasts that demonstrated good fermentative behavior were S. pombe and R. ruineniae, with OD600 values of 1.69 and 1.29, respectively. Meanwhile, Z. rouxii, Candida sp., C. lusitaniae, and M. chrysoperlae exhibited poor fermentative performances, falling below the established growth threshold, with OD600 values of 0.36, 0.14, 0.12, and 0.23, respectively.
These results suggest that ethanol would be a determining factor when selecting any of these yeasts. For instance, the behavior of yeasts fermented at 5% (v/v) ethanol generally showed that Z. rouxii, Candida sp., S. pombe, and R. ruineniae demonstrated good fermentative performances, with only C. lusitaniae and M. chrysoperlae exhibiting poor fermentative behavior under these conditions. However, in the case of the treatment with 8% (v/v) ethanol, the results were different, and yeasts S. pombe and R. ruineniae showed high fermentative performances, Z. rouxii exhibited lower performance, while C. lusitaniae and M. chrysoperlae showed poor fermentative performances.
In similar studies conducted by Michel et al. [
49], in which they analyzed the ethanol–hops cross-tolerance of non-
Saccharomyces yeasts,
T. delbrueckii, isolated from different sources (cheese brine, Pils beer, and sorghum spirits), exhibited good fermentative performance at 50 IBU + 5% (
v/
v) ethanol. Similar results were obtained for strains of
T. delbrueckii isolated from other sources (wheat beer and Pils beer) in the treatment with 90 IBU + 5% (
v/
v) ethanol.
In the study conducted by Matraxia et al. [
35], a total of 404 yeasts were isolated from fermented honey by products, identified as
Saccharomyces cerevisiae,
Wickerhamomyces anomalus,
Zygosaccharomyces bailii,
Zygosaccharomyces rouxii, and
Hanseniaspora uvarum. This work focused on analyzing five strains of
H. uvarum to assess their beer production capability. Regarding cross-tolerance to ethanol and hops, it was observed that the YGA36 and YGA38 strains were able to grow in the presence of up to 5% ethanol and up to 90 IBU, exhibiting faster growth than the control strain
S. cerevisiae US-05. The researchers concluded that all strains showed low fermentative power, highlighting especially the ability of the YGA34 strain of
H. uvarum to overgrow under these stress conditions, making it the selected strain for beer production.
Different studies have assessed the potential of
Z. rouxii,
S. pombe, and
M. chrysoperlae for producing alcoholic beverages. According to the study conducted by Petruzzi et al. [
50],
Zygosaccharomyces rouxii was reported to lack the ability to ferment maltose, making it an ideal candidate for the industrial production of beers with low ethanol levels or non-alcoholic beers. This organism is employed to inhibit alcohol production through biological processes, allowing the creation of innovative, unique beers with intense aromatic profiles. In another study by Callejo et al. [
51], it was concluded that
Schizosaccharomyces pombe was capable of increasing the quantity of volatile compounds such as (1-propanol, isobutanol, 2-methyl-1-butanol, 3-methyl-1-butanol), enhancing the ethanol content, acetaldehyde, and, simultaneously, improving the consistency and persistence of foam, compared to yeasts
Torulaspora delbrueckii,
Saccharomycodes ludwigii, and
Lachancea thermotolerans.
In the study conducted by Liu et al. [
52], the behavior of different strains of non-
Saccharomyces yeasts combined with
Saccharomyces cerevisiae was analyzed to improve the analytical composition of wines. The authors concluded that three varieties of
Metschnikowia, one
M. chrysoperlae and two
M. fructicola, exhibited a quite similar production of volatile compounds, showing higher levels of isoamyl acetate and (Z)-3-hexenyl acetate than the wine inoculated solely with
S. cerevisiae. These three wines stood out for their floral and fruity characteristics, with the wine containing
M. chrysoperlae mainly associated with fruity, tropical, and elderflower notes.
Metschnikowia strains proved promising for producing Solaris wines with more pleasant flavor profiles.
In the research carried out by Li et al. [
53], the performance of sequential fermentation with
Zygosaccharomyces rouxii and
Saccharomyces cerevisiae was examined to enhance low-alcohol kiwi wine’s antioxidant activity and aroma. The results of this study indicated that sequential fermentations generated significant increases in total flavonoids, phenols, and antioxidant activity while remarkably improving the aromatic profile. Furthermore, a higher percentage of
Z. rouxii inoculation contributed significantly to the complexity of volatile compounds, enhancing the sweet aroma of the wines. On the other hand, an equal proportion of inoculation between
Z. rouxii and
S. cerevisiae resulted in a significant increase in volatile contents, intensifying the tropical flavor. These findings provide valuable insights into applying non-
Saccharomyces yeasts in producing innovative beverages. Considering these findings, along with ethanol tolerance, sugar utilization ability, hop resistance, and volatile character, there is a possibility that these yeasts are alternative choices for wine and beer production.
On the other hand, the other three yeasts isolated have limitations for their use in food products. The genus
Candida sp. Is considered one of the most common endogenous fungi for humans, with
Candida albicans being the most abundant. Studies have identified the intestinal population of
C. albicans as one of the main sources of infection [
54].
C. lusitaniae, also known as
Candida lusitaniae, is an opportunistic pathogen that infrequently causes invasive candidiasis [
55].
R. ruineniae, a fungus belonging to the
Basidiomycetes genus [
56], is an opportunistic microbe and an endophyte of various plant species [
48]. Therefore, these yeasts were excluded from the probiotic characterization.