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Article

Chemical Fingerprints of Honey Fermented by Conventional and Non-Conventional Yeasts

by
Dorota Kregiel
1,*,
Urszula Dziekonska-Kubczak
2,
Karolina Czarnecka-Chrebelska
3 and
Katarzyna Pielech-Przybylska
2
1
Department of Environmental Biotechnology, Lodz University of Technology, Wolczanska 171/173, 90-530 Lodz, Poland
2
Institute of Fermentation Technology and Microbiology, Lodz University of Technology, Wolczanska 171/173, 90-530 Lodz, Poland
3
Department of Biomedicine and Genetics, Medical University of Lodz, Mazowiecka 5, 92-215 Lodz, Poland
*
Author to whom correspondence should be addressed.
Molecules 2025, 30(11), 2319; https://doi.org/10.3390/molecules30112319
Submission received: 1 May 2025 / Revised: 23 May 2025 / Accepted: 24 May 2025 / Published: 26 May 2025
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Abstract

:
Previous studies have shown the positive effects of non-conventional Metschnikowia spp. yeasts in mixed cultures with Saccharomyces cerevisiae on the properties of fruit wines. In this study, we investigated the effects of using conventional S. cerevisiae and non-conventional Metschnikowia pulcherrima yeasts as starter cultures in controlled mixed fermentations of honey wort. Other non-conventional yeasts were also tested for comparison, including Wickerhamomyces anomalus, Dekkera/Bretannomyces bruxellensis, and Wickerhamomyces anomalus. We evaluated the tolerance of the tested yeasts to high sugar content and analyzed the metabolic profiles of both monocultures and mixed systems. The M. pulcherrima strain showed the highest tolerance to 30% w/v glucose. The chemical complexity of fermented honey was improved using M. pulcherrima in co-starters with S. cerevisiae. The fermented honey samples were characterized by lower ethanol content, higher glycerol level, and rich volatilomes containing higher levels of both esters (ethyl acetate, 3-methylbutyl acetate, 2-methylpropyl acetate) and aliphatic alcohols (2-methylpropan-1-ol, 3-methylbutan-1-ol, and 2-methylbutan-1-ol). Similar characteristics were obtained using mixed populations of four strains: S. cerevisiae, M. pulcherrima, D. bruxellensis, and W. anomalus.

1. Introduction

Non-conventional yeasts have until recently been regarded as undesirable microorganisms in winemaking, responsible for cloudiness, incomplete fermentation, low alcohol formation, and the production of undesired aromas in final products [1]. However, non-Saccharomyces yeasts, including Metschnikowia sp., are now being marketed as active yeast preparates [2,3,4]. Currently, there are more than 80 validated Metschnikowia species. The pigmented Metschnikowia strains are the most frequently used for winemaking [5], in particular, M. pulcherrima and M. fructicola, belonging to the M. pulcherrima clade [3]. Metschnikowia spp. are generally recommended in winemaking for their contribution to improving aromatic complexity. This is due to their enzymatic properties (e.g., β-D-glucosidase, cysteine, β-lyase, esterase), which lead to interesting wine flavors (esters, higher alcohols) [6,7,8]. Numerous studies have described the positive effects of Metschnikowia strains in mixed cultures with the classical wine yeast S. cerevisiae [9,10,11,12]. Lately, studies have also shown that the M. pulcherrima clade can be used to ferment apple and chokeberry wines with improved aromatic complexity, especially in the case of apple wines using the Metschnikowia spp. as co-starters with S. cerevisiae Tokay [13]. The use of the M. pulcherrima clade for the bioprotection of fruit musts as an alternative to using sulphites has also been investigated [14].
Various mechanisms have been proposed to explain the antimicrobial antagonism exhibited by the M. pulcherrima clade. One proposed mechanism is related to their ability to produce pulcherrimin. The cells secrete pulcherriminic acid into the environment, which forms an insoluble pulcherrimin chelate with ferric ions. The lack of iron caused by the bonding of ferric ions inhibits the growth of many undesirable microorganisms. As a result, the M. pulcherrima clade shows strong biocontrol activity against yeast genera detrimental in enology (e.g., Brettanomyces/Dekkera, Wickerhamomyces, Hanseniaspora, Pichia), as well as filamentous fungi (e.g., Penicillium, Aspergillus and Fusarium). However, it has no or only a slight negative impact on conventional wine yeasts [7,13,15].
The promising results of the use of Metschnikowia species in enology encouraged further research into their application in the production of mead. Mead is a traditional alcoholic beverage obtained by fermenting honey wort. References to mead have been found dating back 3000 years. The origins of the drink can be traced back to African countries. It was later produced throughout the Mediterranean basin and Europe, playing an essential role in early ancient civilizations [16,17]. Traditionally, honey can be diluted with different proportions of water or juice, such as 1:0.5, 1:1, 1:2, or 1:3. In traditional mead, small amounts of fruits, spices, and herbs may also be added, but their incorporation should not overpower the unique flavor and aroma of the mead [16]. Mead contains 8 to 18% v/v of ethanol. It has not only a long history but also an expanding global market [18].
The yeasts used in the production of mead are usually conventional S. cerevisiae, similar to those used in wine or champagne production. These yeasts are able to ferment sugars, such as glucose and fructose, resulting in the formation of ethanol and carbon dioxide. However, given the high sugar levels in honey and wine must and the low nitrogen concentrations in honey, it was hypothesized that these strains may not be the most suitable for mead production [19]. In previous studies conducted by our team, some M. pulcherrima strains showed high osmotic tolerance (30% w/v) [20], which suggested potential for honey fermentation and mead production.
In the present study, we evaluate the chemical properties of Polish honey fermented by yeast monocultures and co-cultures of conventional and non-conventional yeasts. Particular attention was paid to the compatibility of the strains S. cerevisiae and M. pulcherrima in the presence of other non-conventional yeasts during the first stages of fermentation. The quality of the fermented honey samples was evaluated on the basis of their chemical characteristics, particularly their aroma profiles.

2. Results and Discussion

2.1. Yeast Growth at Different Concentrations of Glucose

The results of the growth of yeast monocultures after 7-day incubation in YPD medium are presented in Table 1. The tested yeasts were able to grow in the media with the highest concentration of glucose (30% w/v), but their growth was very diverse.
With increasing glucose concentrations, M. pulcherrima showed higher osmotolerance, with a notable increase at 30% glucose concentration in comparison to other tested strains. The increase was statistically significant compared to D. bruxellensis at 20% and 30% glucose (p = 0.019; p = 0.013, respectively, K-W test).
The statistical analysis showed that as the glucose concentration increased, the growth of the tested yeasts decreased, and this decrease in multiplication was statistically significant for all tested strains at a glucose concentration of 30% compared to the initial analyzed glucose concentration of 1% w/v (Supplementary, Table S1, Figure S1).
It should be emphasized that the incubation conditions used were aerobic (shaking cultures); however, in S. cerevisiae, a yeast exhibiting a strong Crabtree effect, higher glucose concentrations induced fermentation processes [21]. In unconventional yeasts, due to their Crabtree-negative or petite-positive characteristics, glucose metabolism at higher sugar concentrations is directed towards multiplication processes, which was visible in the form of higher optical density values [22]. Moreover, M. pulcherrima yeasts known to be oleaginous microorganisms [23]. The ability to produce oil may contribute to a degree of osmotolerance in yeasts [24]. However, similar properties have been observed for the non-oleaginous yeast W. anomalus. Osmotolerance has often been described in this yeast [25,26,27]. It should therefore be assumed that the mechanisms of osmotolerance in yeasts may be very different. Cronwright et al. [28] studied the induction of glycerol production in yeast as a protective mechanism against an osmotic environment. It was noted that glycerol uptake and synthesis systems contribute to the osmotic tolerance of Kluyveromyces marxianus [29]. Dušková and co-workers similarly observed that glycerol uptake systems contribute to high osmotolerance in Zygosaccharomyces rouxii [30].
Numerous experiments have shown that yeast strains that are tolerant to high sugar concentrations effectively produce ethanol in media containing up to 25% w/v glucose [31]. Yeasts identified as S. cerevisiae, M. pulcherrima, and W. anomalus are often isolated from the nectar of flowering plants and sugar-rich food as spoiling microbiota [20,31,32,33]. M. pulcherrima is a good candidate for obtaining beverages with low ethanol content but interesting volatile profiles. W. anomalus strains can be used in oenology, due to their tolerance of up to 12.5% (v/v) ethanol [34]. However, Brettanomyces/Dekkera bruxellensis is a particularly troublesome wine spoilage yeast [35].

2.2. Fermentation Performance of Tested Yeast Strains

Figure 1 shows CO2 production during fermentation trials. The highest fermentation rates were noted in the samples fermented with S. cerevisiae. As monocultures, the non-conventional strains showed poor fermentation dynamics during the first 15 days. S. cerevisiae started gas production on the second day after inoculation.
Unconventional strains started producing CO2 after the sixth day. The fermentation process was slow in all tested variants, even after 15 days of incubation. Interestingly, the population consisting of S. cerevisiae and M. pulcherrima cultures showed better fermentation performance than the S. cerevisiae monoculture. Similar dynamics characterized the mixed populations of SC + MP + DB + WA and SC + DB + WA. A statistical analysis of the obtained results is presented in Table S2 (Supplementary).
The use of non-Saccharomyces species as co-starter cultures with S. cerevisiae is becoming a common practice in the oenological industry. According to the literature, the growth of non-Saccharomyces yeasts can affect alcoholic fermentation by S. cerevisiae [36]. Such “cooperation” has been reported between S. uvarum and M. pulcherrima by Contreras et al. [9]. Mencher et al. [37] studied the transcriptional responses of S. cerevisiae to short-term co-cultivation with M. pulcherrima and other non-conventional yeasts. The results showed over-expression of the gluco-fermentative pathway, which was much stronger than with the other yeast species. Moreover, a strong repression of the respiration pathway was observed in response to Metschnikowia sp. The authors suggested that a direct interaction stress response may occur between S. cerevisiae and the other yeasts, which, under excess sugar conditions, induces transcription of the hexose transporters, triggers glucose flow towards fermentation, and inhibits respiration, leading to an increase in both metabolic flow and population dynamics. Recently, the results of transcriptomic analyses have confirmed interspecific communication between S. cerevisiae and M. pulcherrima. Mejias-Ortiz and co-workers [38] reported the upregulation of yeast metabolism in response to competing species. This finding points to the presence of signals that yeast cells may perceive as cues, indicating the presence of competitors.

2.3. HPLC Analysis

The honey worts fermented with mono- and co-cultures of yeasts were characterized by diverse chemical profiles (Table 2 and Table 3). The S. cerevisiae monoculture showed the best glucose and fructose consumption during the fermentation of honey broth. The monocultures of non-conventional yeasts, especially W. anomalus and D. bruxelensis, demonstrated the weakest sugar utilization.
The monoculture of M. pulcherrima was characterized by fairly good glucose consumption in the fermentation trials. Fructose was either barely utilized or not utilized by the non-conventional yeast monocultures. This indicates the common phenomenon of glucose repression, since fructose will not be metabolized when glucose is available [39]. A statistical analysis of the results is presented in Figure S3 (Supplementary).
The fermented honey wort after 15-day fermentation by a monoculture of S. cerevisiae contained 45.75 g/L ethanol and 5.73 g/L glycerol, with a residual sugar content of 175.36 g/L. The fermented alcoholic beverage obtained using an M. pulcherrima monoculture contained a significantly lower content of ethanol (12.781 g/L) and higher contents of glycerol (7.91 g/L) and residual sugars (248.97 g/L) in comparison to the beverage from S. cerevisiae (p > 0.05). The beverages obtained using other monocultures had the lowest ethanol content (10.53–12.49 g/L) but similar residual sugar content (318–319 g/L) and glycerol concentrations (1.4–1.9 g/L). Better results were obtained with mixed populations. These fermented honey broths contained higher levels of ethanol (57.7–63.9 g/L) and glycerol (7.03–7.16 g/L). The best results were noted for the SC + MP population (Supplementary, Table S3; Figure S4).
In all samples after fermentation, small amounts of methanol were detected. Yeasts are able to produce methanol through hydrolysis of pectins; alternatively, this compound may be synthesized from glycine [40,41,42]. In this case, the second option seems more likely because there is practically no pectin in honey, but glycine may be present in this source as one of several amino acids [42].
M. pulcherrima strains are widely used as starter strains in wine fermentation, both to improve the properties and for biocontrol in grape wines [43,44,45]. They can also be used as co-cultures for fruit wines. M. pulcherrima with S. cerevisiae produced wines with lower ethanol content and higher glycerol levels [13]. Glycerol is a major by-product of ethanol fermentation. Under anaerobic growth conditions, yeast cells produce glycerol to help maintain a cytosolic redox state conducive to sustaining glycolytic catabolism. In addition, glycerol has an important physiological function, because it is accumulated intracellularly when cells are exposed to decreased extracellular water activity [27,28]. Glycerol in wine can contribute to flavor intensity and aroma volatility [46,47]. Therefore, the increased production of glycerol has a positive effect both on the physiology of yeast cells and the sensory characteristics of alcoholic beverages. Our results obtained after HPLC analysis confirmed that honey broth is a suitable raw material for alcoholic fermentation by mixed cultures, especially those containing M. pulcherrima cells.

2.4. GC Analysis

S. cerevisiae and non-conventional yeasts presented different volatile characteristics. Their diverse secondary metabolic pathways and enzymatic profiles may contribute to the increased diversity of flavor phenotypes [13,47]. Our analysis of the main volatile compounds using GC-MS identified the main components of the volatilomes, with concentrations above 0.0001 mg/L (Supplementary, Table S3). The main compounds in the volatile profiles of all the obtained samples were as follows: acetaldehyde, propan-1-ol, ethyl acetate, 3-methylbutan-1-ol, and 2-methylbutan-1-ol. The lowest number of the major volatile compounds was obtained from the samples fermented with D. bruxelensis (8 compounds). The highest number was obtained for M. pulcherrima monoculture (13 compounds) and co-cultures SC + MP + DB + WA and SC + DB + WA (15–16 compounds). The samples fermented by W. anomalus and S. cerevisiae strains contained 9 and 11 compounds, respectively. However, despite having a greater variety of volatiles, the honey broth fermented with the co-cultures showed lower overall concentrations of volatiles (188.516–206.859 g/L) compared to the monoculture of M. pulcherrima (261.389 g/L).
The main volatile present in the highest concentrations was acetaldehyde. This metabolite was produced both by monocultures (3.535–138.881 g/L) and by co-cultures (35.761–47.128 g/L). The highest levels of acetaldehyde were recorded for the M. pulcherrima monoculture (138.881 g/L) and the co-culture with S. cerevisiae (SC + MP) (47.128 g/L). It is worth noting that the levels of this compound were significantly lower for honey broth fermented by mixed cultures. According to the literature, at low levels acetaldehyde can play a positive role in some specific aromatic contexts, while at higher levels it has negative effects associated with the generic presence of other aldehydes [48].
Another compound formed in higher amounts was ethyl acetate. This metabolite was detected especially in samples with W. anomalus and M. pulcherrima strains, both as monocultures (83.121 g/L and 32.536 g/L) and in co-cultures with the following strains: SC + MP + DB + WA (72.141 g/L) and SC + DB + WA (48.485 g/L). Ethyl acetate is an important component of the volatile profile, significantly contributing to the wine or mead aroma [48,49].
The following aliphatic higher alcohols were also found in the fermented honey samples: 3-methylbutan-1-ol, 2-methylpropan-1-ol, and 2-methylbutan-1-ol. The first compound, 3-methylbutan-1-ol, was produced by both the monoculture of S. cerevisiae (52.439 g/L) and co-cultures with this conventional yeast: SC + MP (56.083 g/L), SC + MP + DB + WA (61.214 g/L), and SC + DB + WA (65.180 g/L). 2-Methylpropan-1-ol was detected at the highest level in the samples fermented by the M. pulcherrima monoculture (25.038 g/L) as well as by the co-culture SC + MP + DB + WA (18.768 g/L). 2-Methylbutan-1-ol was produced mainly by the monocultures of S. cerevisiae (14.448 g/L) and M. pulcherrima (12.350 g/L), but it was also detected in the mixed fermentations of SC + MP (16.228 g/L), SC + MP + DB + WA (16.210 g/L), and SC + DB + WA (17.324 g/L). These aliphatic alcohols are often present in wines and meads and contribute desirable complexity to aroma at moderate concentrations. It is worth noting that concentrations of higher alcohols in the range of 300–400 mg/L are acceptable, but concentrations below 300 mg/L give a desirable, pleasant character [50,51]. Therefore, all the tested samples were within the limits of organoleptic acceptability. Unlike S. cerevisiae and other monocultures, the M. pulcherrima strain formed pentanal (0.027 g/L) but was not able to produce propanal, in contrast to DB (0.039 g/L) and WA (0.041 g/L). However, these aliphatic aldehydes were present in very small amounts in the fermented honey samples.
Principal component analysis (PCA) was performed to assess the effect of the yeast strains on the profile of volatile aromatic compounds in the analyzed samples. PCA allowed for the extraction of principal components (PCs) that significantly contributed to explaining the total variance of all the variables studied. A double criterion was used in the selection of PCs: the Kaiser criterion (eigenvalues of components > 1) and the criterion of the degree of explained variability >80%. The preliminary analysis showed that in the case of all fermentation variants using different yeast strains, the eigenvalues >1 were for the first three factors. Therefore, the components PC1, PC2, and PC3 were finally adopted as the three dimensions determining the space of the considered parameters (Table 4).
The three principal components, PC1, PC2, and PC3, which are responsible in turn for the largest sources of variation in the data (81.1%), were used to prepare the plot space. Figure 2 shows a three-dimensional PCA biplot, incorporating both observations (samples) and variables (volatile compounds). The common visualization of variables and observations facilitates the inference of relationships between volatile compounds and the distribution of observations to each other, the identification of compounds that differentiate the tested samples to the greatest extent, and the identification of samples with a similar chemical profile.
The observations distributed in the three-dimensional plot demonstrated clear variation. The samples fermented by MP yeast and a mixed culture of all the strains employed in the study (SC + MP + DB + WA) exhibited a strong positive shift along PC1 and PC2, while the sample fermented by the three strains, i.e., SC + DB + WA, demonstrated a clear positive shift along PC1. These findings indicate that the significant influence of the variables highly correlated with these components and the unique aromatic profile of these samples concerning the other fermentation variants. Conversely, the sample fermented by the SC yeast strain exhibited a moderate shift toward positive PC1 and PC2 values, showing only partial similarity to the sample fermented by MP yeast. In contrast, the control sample, as well as the two samples fermented by the DB and WA yeast monocultures, are located in opposite areas of the plot, suggesting different volatile compound profiles.
The main component of PC1 strongly positively correlated with variables from the group of higher alcohols (propan-1-ol, 2-methylpropan-1-ol, 3-methylbutan-1-ol, and 2-methylbutan-1-ol) and esters (ethyl hexanoate, 3-methylbutyl acetate, ethyl 2-methylpropanoate, 2-methylpropyl acetate, ethyl hexanoate, ethyl octanoate, and ethyl butanoate). These are compounds with characteristic fruity notes. The samples fermented by MP monoculture and mixed cultures of SC + MP + DB + WA and SC + DB + WA yeasts correlated well with PC1, which indicates a rich profile of volatile compounds. The presence of ethyl esters and esters of higher alcohols should be considered particularly important, as they indicate the high technological or sensory potential of the yeast strains.
In turn, the PC2 showed a strong positive correlation with acetaldehyde and diethyl acetaldehyde acetal (1,1-diethoxyethane), explaining 92 and 86% of the variance of these two variables, respectively. The PC2 also positively correlated with ethyl propanoate, explaining 70% of the variance. An evaluation of the position of the observation representing the sample fermented by MP yeast revealed that, in comparison to other samples, it was distinguished by a higher concentration of these compounds.
A positive correlation was shown between PC3 and propanal, attributable to a four-times-higher concentration of this compound in samples fermented by a mixed culture of SC and MP yeasts compared to those fermented by DB, WA yeasts, and a mixed culture of SC + MP + DB + WA yeasts. On the other hand, in the case of ethyl acetate, the selected three components (PC1–PC3) explained a mere 41.6% of the variance in this variable, of which PC1 accounted for 22%. The correlation shown distinguishes, in terms of ethyl acetate concentration, fermentation variants that involve WA yeast, whether as a monoculture or in a mixed culture (SC + MP + DB + WA and SC + DB + WA).
In summary, PCA revealed differences between the yeast strains in terms of the profiles of volatile compounds. M. pulcherrima and mixed yeast cultures SC + DB + WA and SC + MP + DB + WA were characterized by high fermentation activity and the production of volatile compounds, including acetal (1,1-diethoxyethane), aldehydes (acetaldehyde and pentanal), higher alcohols (3-methylbutan-1-ol and 2-methylbutan-1-ol), and esters (ethyl propanoate, ethyl 2-methylpropanoate, 2-methylpropyl acetate, ethyl butanoate, 3-methylbutyl acetate, ethyl hexanoate, and ethyl octanoate).
Previous studies have shown that some non-conventional yeasts may also be used for mead production, with good results [50,52,53,54]. Non-Saccharomyces yeasts belonging to the Torulaspora genus used as pure cultures or mixed with S. cerevisiae increase the aromatic complexity of mead [53]. Mixed culture of different Torulaspora strains and S. cerevisiae show good fermentative performance in under 10 days. Recently, analysis of the chemical parameters of meads obtained by S. cerevisiae and other non-conventional strains of Hansenula uvarum in different combinations also showed differences between the samples in terms of residual sugars, acetic acid, glycerol, ethanol, and volatile organic compounds. Pleasant characteristics of sweetness, honeyness, and floralness were found in the mead fermented with co-cultures of S. cerevisiae and H. uvarum, while mead samples obtained using a monoculture of S. cerevisiae were dry, balanced, and free from foreign odors and tastes. These results show that the controlled use of conventional S. cerevisiae and non-conventional H. uvarum yeasts can be a promising approach to improving the quality of meads [54].
The potential of diverse non-Saccharomyces yeast strains applied in mead production is still unknown, which has greatly limited their use in practice [55,56]. Our research indicates that mixed fermentation using S. cerevisiae with M. pulcherrima and other non-conventional yeasts remarkably improves the sensory profiles of young meads. Metabolic analysis showed that M. pulcherrima had the greatest potential to improve the “balance” and “fullness” notes of young meads. These positive effects on the metabolic profiles of fermented young meads proved the important role of non-S. cerevisiae existing briefly in the early stages of fermentation.

3. Materials and Methods

3.1. Yeast Cultures

The conventional and non-conventional yeast strains used in this study are presented in Table 5. The yeasts strains were stored at −18 °C in a microbank storage system (Microbank®, Biomaxima, Lublin, Poland). A single bead was placed in wort broth (Merck Millipore, Darmstadt, Germany) to activate the strain. After 48 h of cultivation at 28 °C, one loop (10 μL) of yeast suspension was streaked onto potato dextrose agar (PDA) [potato extract 0.4% w/v; dextrose 2.0% w/v; agar 1.5% w/v] (Merck Millipore, Darmstadt, Germany) to ensure the purity of the yeast culture and incubated at 28 °C for two days.

3.2. Yeast Osmotolerance

To determine the growth profiles of the yeasts under varying glucose concentrations (0.5–30% w/v), YPD medium [yeast extract 2.5% w/v, peptone 5.0% w/v, glucose with variable concentrations] was used. The yeast inoculums were prepared in sterile Ringer solution. The optical density of the inoculum suspensions was 1.0 degrees according to the MacFarland scale (°McF), determined using a DEN-1 densitometer (Merck Millipore, Darmstadt, Germany). The intensity of yeast growth at 28 °C after 7 days in a rotary shaker (170 rpm) was measured using a DEN-1 densitometer and expressed on the McFarland scale.

3.3. Honey Wort Preparation

Multiflorous honey was obtained from a local beekeeper from the Malopolska region (Poland) and delivered by Datan Sp. z o.o. (Krakow, Poland) (50°03′41″ N, 19°56′18″ E). The honey was diluted with sterile distilled water in a volumetric proportion of 1:2 (honey:water). Sugar content was measured refractometrically (Anton Paar 6000 densimeter, Graz, Austria) and standardized to 36 °Bx. The mead wort was topped up with the addition of (NH4)2HPO4 (0.04% w/v), which is a popular supplement in mead-making [56]. The honey wort was gently heated (90 °C) for 30 min. The foam formed during heat treatment was systematically removed. After cooling, the specific gravity was controlled and set to 35 °Bx with sterile distilled water.

3.4. Fermentation Trials

The following yeast strains as monocultures and co-cultures were used in the experiments (Table 6).
Sterile Erlenmeyer flasks (volume 100 mL) were filled with 50 mL of the pasteurized honey wort. All samples were inoculated with 2.5 mL of standardized (6 °McF) yeast suspension (5% v/v). Mixed populations were prepared in volumetric proportions of 1:1 (two strains), 1:1:1 (three strains), or 1:1:1:1 (four strains). The flasks were closed with fermentation airlocks and silicone stoppers to allow CO2 to escape. The fermentation samples were incubated without agitation at 25 °C. The weight loss of the flasks due to the release of CO2 was monitored every day during the 2-week fermentation period. This fermentation time was chosen because non-Saccharomyces yeasts play a substantial role in the early stages of wine fermentation [57,58,59]. After fermentation, the samples were centrifuged (10 °C, 10,000× g, 10 min) using a centrifuge 5804R (Eppendorf, Wesseling-Berzdorf, Germany). The supernatant was collected and analyzed using chromatographic techniques. Prior to chromatography, clear liquid samples were prepared by filtration using 0.45 μm polyethersulfone membranes (Merck Millipore, Darmstadt, Germany).

3.5. HPLC Analysis

The profiles of the main saccharides, acetic acid, glycerol, methanol, and ethanol in the young meads were determined using an HPLC (Agilent 1260 Infinity, Agilent Technologies, Santa Clara, CA, USA) with a Hi-Plex H column (7.7 × 300 mm, 8 m, Agilent Technologies, Santa Clara, CA, USA) and a refractive index detector at 55 °C. The column temperature was maintained at 60 °C. As a mobile phase, a 5 mM solution of H2SO4 was used at a flow rate of 0.7 mL/min with a sample volume of 20 L [13,60]. The samples were analyzed as received and after 10 rounds of dilution with ultrapure water. The data were processed using OpenLab CDS Chemstation software Rev. C.01.06 (Agilent Technologies, Santa Clara, CA, USA).
Standard solutions of pure reagents in ultrapure water were prepared to quantify the concentration of the analyzed compounds in the range of 1.5–30.0 g/L for glucose, fructose, and ethanol; 0.04–9.2 g/L for glycerol; and 0.02–1.25 g/L for acetic acid. The linearity of the obtained calibration curves was satisfactory across the whole tested range, with an R2 value of at least 0.9997. The limit of detection (LOD) and limit of quantification (LOQ) were calculated according to the method proposed by Haubax and Vos [61].

3.6. GC-MS Analysis

To identify and quantify the volatiles in the obtained fermented honey samples, we used an Agilent 7890A GC (Agilent Technologies, Santa Clara, CA, USA) gas chromatograph equipped with an Agilent MSD 5975C quadrupole mass spectrometer and an Agilent 7697A headspace analyzer. The headspace sampler was connected to the gas chromatograph via a transfer line through the split–splitless injector. An Rxi-5 ms capillary column (60 m, 0.25 mm, 0.25 m; Restek, Bellefonte, PA, USA) was used to separate the compounds. The initial GC oven temperature was set to 30 °C and held for 6 min, then ramped up by 5 °C/min to 80 °C (held for 3 min), and ramped up again 10 °C/min to 230 °C. This final temperature was maintained for 6 min. The carrier gas was helium, with a flow rate of 1.2 mL/min. Before analysis, a 20 mL headspace vial was filled with a 7 mL sample of wine and closed tightly. The headspace conditions were as follows: the temperatures of the oven, loop, and transfer line were set at 50 °C, 60 °C, and 70 °C, respectively. The equilibration time and injection duration were 20 min and 0.7 min, respectively. During sample equilibration, the vial was shaken (136 shakes/min). The temperature of the injector was 250 °C. Injections were made in split mode (10:1). The temperatures of the MSD ion source, transfer line, and quadrupole were 230 °C, 250 °C, and 150 °C, respectively. The ionization energy was 70 eV.
Qualitative analysis was initially performed in full scan ion monitoring mode (SCAN). The volatile compounds in the young mead samples were identified by comparing their mass spectra with those of standard compounds and with the mass spectra of the NIST/EPA/NIH Mass Spectra Library (Version 2.0g). Next, quantitative analysis of the volatile compounds in the tested samples was performed using the external calibration method. Quantitative analysis was performed in selected ion monitoring mode (SIM). The calibration standards were prepared using a dilution series of an external analytical standard mixture. The linearity of the calibration curves was tested over the following concentration ranges: 2–100 mg/L for ethyl acetate and acetaldehyde, 1–200 mg/L for 3-methylbutan-1-ol and 2-methylbutan-1-ol, 0.5–100 mg/L for 2-methylpropan-1-ol, 0.25–10 mg/L for propan-1-ol, 0.005–0.25 mg/L for propanal and pentanal, 0.1–1 mg/L for furan-2-carbaldehyde, 0.1–5 mg/L for butane-2,3-dione and 3-methylfuran, 0.005–1 mg/L for 1,1-diethoxyethane, 0.01–5 mg/L for ethyl formate, 0.01–25 mg/L for methyl acetate, 2–50 g/L for pentan-2one, and 0.5–100 g/L for other esters (ethyl propanoate, ethyl-2-methylpropanoate, 2-methylpropyl acetate, ethyl butanoate, 2-methylbutyl acetate, 3-methylbutyl acetate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, ethyl 2-methylbutanoate, and ethyl-3-methylbutanoate). The correlation coefficients of the calibration curves of the external standards were 0.99 on average. The values of LOD and LOQ were calculated based on the standard deviation of the response and the slope of the calibration curve at levels approximating the LOD [61]. The obtained data were analyzed using Agilent MassHunter software B.07.00 (Agilent Technologies, Santa Clara, CA, USA).

3.7. Statistics

The results of the statistical analysis were presented as the mean ± SD of three separate experiments (each variant of wine was prepared in three replicates, with one technical sample). As the results did not follow a normal distribution (Shapiro–Wilk test), non-parametric tests were used for the statistical analysis of the following experiments: yeast strain growth profiles under varying glucose concentrations, weight loss during the fermentation process, and changes in the chemical composition of the wort. Differences in the analyzed parameters were determined using the Kruskal–Wallis test (KW test), followed by a multiple comparisons test (MCT) to indicate significant differences between the groups. A p-value < 0.05 was considered statistically significant. The KW, MCT, and independent sample t-Tests were performed using Statistica®13.1 (StatSoft, Tulsa, OK, USA). In addition, to investigate the ability of different yeast strains used as monocultures and mixed cultures to synthesize volatile compounds during fermentation, principal component analysis (PCA) was performed with Statistica®13.3 (TIBCO Software Inc., San Ramon, CA, USA).

4. Conclusions

In this study, we investigated the effects of using conventional S. cerevisiae and non-conventional yeasts as starter cultures in controlled mixed fermentations of honey wort. Various species of unconventional yeast were selected based on their physiology and fermentation characteristics. Particular attention was paid to the yeast M. pulcherrima, due to the growing interest in this yeast in winemaking. The obtained profiles showed that mixed cultures strongly altered the aromatic profiles of fermented honey samples in comparison to the corresponding monocultures. In general, the aromatic complexity of the fermented honey wort was improved by using non-conventional yeasts, especially the M. pulcherrima strain, as a co-starter. After co-inoculation with S. cerevisiae, the young mead showed a lower ethanol content, higher glycerol level, and higher concentration of volatile substances. Inoculation with other unconventional yeasts from the genera Dekkera and Wickerhamomyces did not change this beneficial effect. This preliminary study is a first step towards the preparation of honey worts using conventional and non-conventional yeasts, and evaluation of their chemical properties after fermentation. Future studies will investigate different yeast strains, inoculation methods, and honey broth supplements, which may require longer fermentation times. We also plan to explore a wider range of honey varieties and conduct larger-scale fermentations.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules30112319/s1, Figure S1: Box-and-whisker plots showing mean yeast growth values at 20% and 30% glucose concentrations; Figure S2: Box-and-whisker plots showing mean CO2 formation after 7- and 15-day fermentation; Figure S3: Box-and-whisker plots showing glucose and fructose content in monocultures and mixed populations; Figure S4: Box-and-whisker plots showing the compound content (glycerol, acetic acid, methanol, ethanol) in fermented honey beverages produced by tested yeast strains as monocultures and mixed populations; Table S1: The comparison of yeast growth depending on the glucose concentration in the culture medium; Table S2: Fermentation performance of tested yeast strains as monocultures and mixed populations; Table S3: Volatilomes of honey wort fermented by monocultures and mixed populations.

Author Contributions

Conceptualization, D.K.; methodology, D.K., U.D.-K. and K.P.-P.; formal analysis, K.C.-C.; investigation, D.K., U.D.-K. and K.P.-P.; resources, K.C.-C.; data curation, K.C.-C.; writing—original draft preparation, D.K., U.D.-K. and K.P.-P.; writing—review and editing, D.K.; visualization, K.C.-C. and K.P.-P.; supervision, D.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Molecules 30 02319 g001
Figure 1. Fermentation performance (CO2 formation) of the tested yeast strains as monocultures and mixed populations. SC—Saccharomyces cerevisiae, MP—Metschnikowia pulcherrima, WA—Wickerhamomyces anomalus, DB—Dekkera bruxellensis.
Figure 1. Fermentation performance (CO2 formation) of the tested yeast strains as monocultures and mixed populations. SC—Saccharomyces cerevisiae, MP—Metschnikowia pulcherrima, WA—Wickerhamomyces anomalus, DB—Dekkera bruxellensis.
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Molecules 30 02319 g002
Figure 2. 3D biplot of principal components PC1, PC2, and PC3; active observations—fermentation variants: SC, MP, DB, WA, SC + MP, SC + MP + DB + WA, SC + DB + WA, control sample. SC—S. cerevisiae, MP—M. pulcherrima, WA—W. anomalus, DB—D. bruxellensis.
Figure 2. 3D biplot of principal components PC1, PC2, and PC3; active observations—fermentation variants: SC, MP, DB, WA, SC + MP, SC + MP + DB + WA, SC + DB + WA, control sample. SC—S. cerevisiae, MP—M. pulcherrima, WA—W. anomalus, DB—D. bruxellensis.
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Table 1. Yeast growth [°McF] with different concentrations of glucose [% w/v] in the culture medium. The results are presented as the mean ± SD. Statistically significant differences in growth (KW test, followed by MCT) are indicated in bold, The p-value is given below the data for each strain. The letters indicate between which strains there is a statistically significant difference.
Table 1. Yeast growth [°McF] with different concentrations of glucose [% w/v] in the culture medium. The results are presented as the mean ± SD. Statistically significant differences in growth (KW test, followed by MCT) are indicated in bold, The p-value is given below the data for each strain. The letters indicate between which strains there is a statistically significant difference.
Yeast StrainGlucose Concentration in the Culture Medium [% w/v]
1102030
S. cerevisiae8.967 ± 0.2527.600 ± 0.4584.867 ± 0.3063.567 ± 0.321
M. pulcherrima8.367 ± 0.2087.933 ± 0.5516.667 ± 0.321 A5.900 ± 0.100 B
D. bruxellensis8.067 ± 0.4936.767 ± 0.3213.833 ± 0.153 A2.867 ± 0.153 B
W. anomalus8.967 ± 0.1157.867 ± 0.2086.233 ± 0.4164.167 ± 0.404
p valuep > 0.05p > 0.05A 0.019B 0.013
Table 2. Glucose and fructose content in honey worts fermented by monocultures and mixed populations. The control sample is the honey wort before fermentation. The results are presented as the mean ± SD. Statistically significant differences (KW test, followed by MCT) are indicated in bold. The letters indicate between which groups there is a statistically significant difference.
Table 2. Glucose and fructose content in honey worts fermented by monocultures and mixed populations. The control sample is the honey wort before fermentation. The results are presented as the mean ± SD. Statistically significant differences (KW test, followed by MCT) are indicated in bold. The letters indicate between which groups there is a statistically significant difference.
YeastsSampleGlucose [g/L]Fructose [g/L]
Control182.466 ± 6.513189.375 ± 8.407
MonoculturesSC *65.555 ± 1.092 A110.476 ± 5.472 B
MP89.211 ± 2.321159.559 ± 10.990
WA145.564 ± 4.127173.857 ± 5.550 B
DB152.324 ± 4.052 A166.734 ± 4.202
Mixed populations SC + MP70.123 ± 3.830128.480 ± 2.703
SC + MP + WA + DB79.390 ± 2.683130.322 ± 6.252
SC + WA + DB73.015 ± 2.804126.511 ± 2.984
p-valuep = 0.013p = 0.013
* SC—S. cerevisiae, MP—M. pulcherrima, WA—W. anomalus, DB—D. bruxellensis.
Table 3. The metabolic profiles in fermented honey wort obtained by monocultures and co-cultures of yeasts. The results are presented as the mean ± SD. The control sample is the honey wort before fermentation. Statistically significant differences (KW test, followed by MCT) in compound content are indicated in bold. The letters indicate between which groups there is a statistically significant difference.
Table 3. The metabolic profiles in fermented honey wort obtained by monocultures and co-cultures of yeasts. The results are presented as the mean ± SD. The control sample is the honey wort before fermentation. Statistically significant differences (KW test, followed by MCT) in compound content are indicated in bold. The letters indicate between which groups there is a statistically significant difference.
Yeast StrainsCompound [g/L]
GlycerolAcetic AcidMethanolEthanol
Control0.197 ± 0.0060.000 ± 0.0000.000 ± 0.0000.000 ± 0.000
MonoculturesSC *5.762 ± 0.3450.858 ± 0.0162.281 ± 0.103 C45.563 ± 0.903
MP7.928 ± 0.178 A0.562 ± 0.0111.247 ± 0.19312.781 ± 0.547
WA1.873 ± 0.0540.387 ± 0.0070.153 ± 0.01712.442 ± 0.586
DB1.362 ± 0.057 A0.227 ± 0.010 B0.126 ± 0.007 C10.495 ± 0.497 D
Mixed populationsSC + MP7.155 ± 0.0460.928 ± 0.0261.242 ± 0.12263.511 ± 1.948 D
SC + MP + WA + DB7.107 ± 0.0971.087 ± 0.028 B2.081 ± 0.08557.630 ± 1.377
SC + WA + DB7.019 ± 0.1860.944 ± 0.0431.241 ± 0.11959.549 ± 1.899
p-value0.0080.0080.0080.008
* SC—S. cerevisiae, MP—M. pulcherrima, WA—W. anomalus, DB—D. bruxellensis.
Table 4. Eigenvalues with % of the total variance explained by the principal components.
Table 4. Eigenvalues with % of the total variance explained by the principal components.
Principal ComponentEigenvalueVariability [%]Cumulative [%]
PC17.50544.14744.147
PC24.30125.29869.445
PC31.98211.65881.103
Table 5. Yeast strains used in the study.
Table 5. Yeast strains used in the study.
StrainOriginStrain AbbreviationReferences
Saccharomyces cerevisiae Tokay LOCK0203LOCK *SC[13,20]
Metschnikowia pulcherrima NCYC747NCYC **MP[13,20]
Dekkera bruxellensis NCYC D5300NCYCDB[13,20]
Wickerhamomyces anomalus NCYC D5299NCYCWA[13,20]
* Collection of Pure Cultures of Industrial Microorganisms, Lodz University of Technology, Poland, ** National Collection of Yeast Cultures; Norwich, United Kingdom.
Table 6. Yeast cultures used for fermentation trials.
Table 6. Yeast cultures used for fermentation trials.
MonoculturesMixed Populations
Saccharomyces cerevisiae
SC		S. cerevisiae SC + M. pulcherrima MP
Metschnikowia pulcherrima
MP		S. cerevisiae SC + M. pulcherrima MP +
W. anomalus WA + D. bruxellensis DB
Dekkera bruxellensis
DB
Wickerhamomyces anomalus
WA		S. cerevisiae SC + W. anomalus WA +
D. bruxellensis DB
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Kregiel, D.; Dziekonska-Kubczak, U.; Czarnecka-Chrebelska, K.; Pielech-Przybylska, K. Chemical Fingerprints of Honey Fermented by Conventional and Non-Conventional Yeasts. Molecules 2025, 30, 2319. https://doi.org/10.3390/molecules30112319

AMA Style

Kregiel D, Dziekonska-Kubczak U, Czarnecka-Chrebelska K, Pielech-Przybylska K. Chemical Fingerprints of Honey Fermented by Conventional and Non-Conventional Yeasts. Molecules. 2025; 30(11):2319. https://doi.org/10.3390/molecules30112319

Chicago/Turabian Style

Kregiel, Dorota, Urszula Dziekonska-Kubczak, Karolina Czarnecka-Chrebelska, and Katarzyna Pielech-Przybylska. 2025. "Chemical Fingerprints of Honey Fermented by Conventional and Non-Conventional Yeasts" Molecules 30, no. 11: 2319. https://doi.org/10.3390/molecules30112319

APA Style

Kregiel, D., Dziekonska-Kubczak, U., Czarnecka-Chrebelska, K., & Pielech-Przybylska, K. (2025). Chemical Fingerprints of Honey Fermented by Conventional and Non-Conventional Yeasts. Molecules, 30(11), 2319. https://doi.org/10.3390/molecules30112319

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