Shelf Life and Sensory Evaluation of a Potentially Probiotic Mead Produced by the Mixed Fermentation of Saccharomyces boulardii and Kombucha

Abstract

Mead is a fermented alcoholic beverage obtained through a diluted honey solution and the action of yeasts. Although a potentially probiotic mead obtained by mixed fermentation of Saccharomyces cerevisiae var. boulardii with kombucha has already been proposed in the scientific literature, aspects regarding the shelf life and sensory properties of this product must be evaluated in order to provide further knowledge for its potential market introduction. The present study aimed to evaluate the shelf life and sensory profile of potentially probiotic mead produced by mixed fermentation of S. boulardii and kombucha. The main results showed that the microorganisms in the mead exhibited fermentative metabolic activity, albeit reduced, under refrigerated storage conditions, with a decrease in soluble solids and an increase in alcohol content observed during storage. Mead with S. boulardii and kombucha maintained microbial viability above 6 log CFU/mL for both yeasts and lactic acid bacteria up to 60 days of storage, meeting the minimum recommended count for probiotic foods. For the sensory analysis, mead with S. boulardii and kombucha showed higher acceptance and purchase intention, being characterized by sensory attributes such as carbonated, effervescent, flavorful, honey taste, sweeter, refreshing, and less alcoholic. In conclusion, potentially probiotic mead produced with S. boulardii and kombucha presents a shelf life of 60 days and high sensory acceptability.

1. Introduction

Mead is a fermented alcoholic beverage obtained from the dilution of honey in water followed by fermentation by yeasts [1,2,3], and is considered one of the oldest alcoholic beverages known to humanity, with historical records dating back thousands of years. Its composition is characterized by the presence of simple carbohydrates, phenolic compounds, proteins, minerals, vitamins, and organic acids, which influence both its sensory properties and its antioxidant and functional potential [1,4,5]. Traditionally, mead production employs yeasts from the Saccharomyces cerevisiae genus, widely used in the alcoholic beverage industry due to their robustness and high fermentative capacity for alcohol production. However, recent advancements and research have encouraged the pursuit of alternative fermentation processes capable of incorporating probiotic and functional attributes into alcoholic beverages, such as the use of the probiotic yeast Saccharomyces cerevisiae var. boulardii for the development of beer, wine, and mead [1,3,5,6,7,8,9,10].

The application of the probiotic yeast Saccharomyces cerevisiae var. boulardii has proven to be a promising strategy for mead production [1,2,3,5,11,12]. This yeast is capable of surviving the gastrointestinal tract and exhibits health-promoting attributes such as modulation of the gut microbiota, resistance to bile and gastric pH, as well as antimicrobial and immunomodulatory properties [13,14]. Thus, with the aim of adding potential health benefits, studies in the literature have presented S. boulardii as a probiotic yeast suitable for the production of mead, in which it has shown resistance and survival to the alcoholic stress conditions of the drink [1,2,5,11].

A recent line of research that has been gaining prominence in scientific literature is the production of mead through mixed fermentation of S. boulardii with other microbial sources such as water kefir and kombucha, in such a way that the lactic acid bacteria present in these sources also aid and provide functionality to the products developed, since they are also sources of probiotics [1,2]. In this sense, Souza et al. [1] proposed the development of a new mead by mixed fermentation of S. boulardii and water kefir, in which these authors showed that the combination of 10 g/L of water kefir grains and 0.75 g/L of S. boulardii, fermented for 9 days at a temperature of 25 °C, produced a probiotic mead with viable cells exceeding 8 Log₁₀ CFU/mL of S. boulardii and also for lactic acid bacteria (LAB), respectively. It is also noteworthy that S. boulardii and LAB showed high survival under simulated in vitro gastrointestinal conditions, with counts exceeding 6 Log₁₀ CFU/mL for both microorganisms, since the mead developed also presented itself as a source of total phenolic compounds and antioxidants.

Recently, in another study, Souza et al. [2] proposed the development of a potentially probiotic mead using the probiotic yeast S. boulardii in mixed fermentation with kombucha, representing an innovative alternative for beverage development. In this study, the mead developed had a high count of S. boulardii and LAB, since these microorganisms were resistant to simulated gastrointestinal conditions in vitro, and the product was also a source of phenolic compounds and antioxidants. However, the shelf life and sensory characteristics of the mead developed by Souza et al. [2] should be investigated in order to provide valuable information for its potential introduction to the market.

In this context, the present study aimed to evaluate the shelf life and sensory profile of potentially probiotic mead obtained by mixed fermentation of S. boulardii and kombucha, thereby contributing essential information for its introduction into the market as a distinctive functional beverage.

2. Materials and Methods

For the development of this study, meads and/or batches of beverages produced by Souza et al. [2] were used, who, at the end of the production process, carried out the bottling and proper storage of the product. Thus, it should be noted that the values obtained for the parameters pH, acidity, soluble solids, alcohol content, phenolic compounds, and antioxidants at the initial storage time (zero days) in the shelf life analysis of this study are similar to those obtained by Souza et al. [2] after bottling the final product developed. The methods used for the shelf life and sensory analysis of mead are described in detail in this study.

2.1. Mead Production

Saccharomyces cerevisiae var. boulardii CCT 4308 (UFPEDA 1176) was obtained from the microbial culture collection of Fundação André Tosello (FAT, Research and Technology, Campinas, Brazil). Kombucha and mead were produced following the methodology described by Souza et al. [2]. Briefly, pure organic green tea (Camellia sinensis, Gunpowder variety, herbal flavor), imported from Hunan Province, China, was used for kombucha preparation. The infusion was prepared using potable water heated to 80 °C, with 6 g/L of green tea buds and leaves, and steeped for 10 min. After infusion, the liquid was filtered, and 50 g/L of commercial granulated sugar was added. The sweetened tea was cooled to 25 °C, followed by the addition of 10% (m/v) SCOBY and 10% (v/v) previously fermented kombucha as inoculum. The SCOBY and kombucha from the previous fermentation were acquired from the laboratory’s own culture collection, which are cultivated periodically for future studies, as they had the following characteristics: 4.8 °Brix of soluble solids, pH of 2.3, total acidity of 0.97% (expressed as lactic acid), and lactic acid bacteria count of 3.5 log CFU/mL. Fermentation was carried out in 300 mL glass containers, which were loosely covered to allow CO₂ release, and incubated at 25 °C for 20 days. Under these conditions, the resulting kombucha exhibited 4.63 °Brix of soluble solids, pH 2.73, total acidity of 0.75% (expressed as lactic acid), and a lactic acid bacteria (LAB) count of 3.4 log₁₀ CFU/mL, as reported by Souza et al. [2].

Accordingly, 3.0 L of mead were produced in 5 L polypropylene fermenters. Briefly, the required amounts of honey and potable water were calculated, and the water was heated to 65 °C. Honey was then dissolved in the heated water, maintaining the temperature at 65 °C, and the soluble solids content of the must was adjusted to 25 °Brix. The resulting mixture was subsequently pasteurized at a temperature of 65 °C for 30 min, and then cooled to 25 °C before fermentation. Then, approximately 0.75 g/L of S. boulardii CCT 4308 (UFPEDA 1176) and 25 mL/L of kombucha were added to the wort. Subsequently, a standard mead was prepared with the commercial yeast Saccharomyces cerevisiae Mangrove Jack’s M05, using the same microbial concentrations. The fermentation buckets were sealed with lids containing an airlock device to maintain anaerobic conditions inside. Mead fermentation was carried out for 9 days at a controlled temperature of 25 °C in a BOD-type incubator (model 347 CD, São Paulo, Brazil, MERSE). Three replicates were performed for each treatment, i.e., three fermentation buckets for treatment T1 (T1 = mead by mixed fermentation of S. boulardii and kombucha) and treatment T2 (T2 = mead by mixed fermentation of commercial S. cerevisiae and kombucha). During the production of mead, there was no filtration or clarification before bottling, as it is a probiotic product in which the presence of viable microorganisms is acceptable. Finally, the obtained mead was bottled in transparent glass bottles (previously cleaned and sanitized), with a volume of 275 mL, and stored under refrigeration at 7 ± 1 °C.

2.2. Shelf Life of the Prepared Product

2.2.1. Physicochemical Analyses

Physicochemical analyses were performed to determine pH using a benchtop pH meter (model PG 1800, FARMA, São Paulo, Brazil), total acidity according to AOAC methodology [15] and expressed as a percentage, soluble solids (°Brix) were obtained directly using a portable refractometer (model RSG-100ATC, Rainsun, Shanghai, China) containing a scale graduated in °Brix, and alcohol content (% ABV) using an ebulliometer (Kit-0700, CIENLAB, Campinas, Brazil). In addition to these analyses, total phenolic compounds were quantified according to Everette et al. [16], with absorbance measured at 700 nm using a JENWAY spectrophotometer (model 7305, Bibby Scientific Ltd., Stone, UK). A standard curve of gallic acid (0.01–0.05 mg/mL) was used, and the results were expressed in mg gallic acid equivalent (GAE)/100 mL. Antioxidant potential was measured using the Ferric Reducing Antioxidant Power (FRAP) method described by Benzie & Strain [17], with absorbance readings at 593 nm. A Trolox standard curve (2.5–15 μmol/L) was used, and results were expressed as μmol TE/100 mL. Furthermore, antioxidant capacity was also determined using the 2.2-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) method according to Re et al. [18], with absorbance measured at 734 nm. A Trolox standard curve (5.0–25 μmol/L) was used, and results were expressed as μmol TE/100 mL. All analyses were performed in triplicate.

2.2.2. Microbiological Analyses

Yeast colony counts were assessed according to Souza et al. [2], with adaptations. Approximately 1 mL of mead was mixed with 9 mL of sterile 0.1% peptone water (KASVI, K25-1402, Liofilchem, Roseto degli Abruzzi, Spain) and subjected to serial dilutions. Then, 100 µL aliquots were spread on plates containing rose bengal agar base (KASVI, K25-610237, Merck Life Science S.L.U., Madrid, Spain) and incubated at 35 °C for 48 h. Viable cells were counted directly on the plates, and results were expressed as Log CFU/mL. For total viable lactic acid bacteria counts, serial dilutions were performed using sterile 0.1% peptone water as described by Souza et al. [2]. Approximately 1 mL of diluted sample was pour-plated with a double layer of De Man, Rogosa and Sharpe (MRS) medium (KASVI, K25-1043, Liofilchem, Roseto degli Abruzzi, Spain). Plates were incubated inverted at 37 °C for 48 h. After incubation, colonies were counted directly on the plates, and results were expressed as Log CFU/mL. In addition, analyses were performed for contaminating microorganisms, counting total coliforms and Escherichia coli using compactDry™ EC microbiological kits (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan). In this regard, it should be noted that E. coli forms blue colonies, while coliforms acquire a pink-purple color on the plates. The total coliform count is considered to be the sum of the purple-pink and blue colonies. For Salmonella analyses, CompactDry™ SL microbiological kits (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) were used, based on pre-enrichment of the sample in lactose broth. The Salmonella colony count was performed based on color characteristics such as individual or merged black/green colonies and/or normal blue/purple to yellow color. All analyses were performed in triplicate.

2.3. Sensory Evaluation of the Prepared Product

To carry out the sensory analysis of the mead, approximately 120 untrained voluntary panelists of both sexes, legally permitted to consume alcoholic beverages (i.e., over 18 years of age), were invited and recruited. Sensory tests were conducted with members of the University of São Paulo community at the Fernando Costa Campus, Pirassununga/SP, including students, staff, and visitors. All participants were informed about the study’s objectives and ethical procedures. Those who agreed to participate signed an informed consent form, granting them the right to withdraw at any time without penalty. Mead samples were prepared following proper hygienic and sanitary procedures for food, and microbiological analyses confirmed negative results for coliforms at 35 °C and 45 °C, as well as absence of Escherichia coli and Salmonella spp. Sensory analysis was conducted in a sensory laboratory, in individual booths with white lighting and controlled temperature (26 ± 1 °C). Approximately 20 mL of chilled mead (7 ± 1 °C) were served per sample, which were coded with randomized three-digit numbers. Mineral water and unsalted crackers were provided to avoid interference between samples. Three mead samples were evaluated: treatment T1 (mead by mixed fermentation of S. boulardii and kombucha), treatment T2 (mead by mixed fermentation of commercial S. cerevisiae and kombucha), and treatment T3 (commercial mead available on the national market, produced with eucalyptus flower honey). The study was previously approved by the Research Ethics Committee involving Human Subjects of School of Animal Science and Food Engineering (FZEA), of University of São Paulo (USP), registered on Plataforma Brasil under number CAAE 68971123.9.0000.5422, with the date of approval being 11 July 2023.

2.3.1. Acceptance and Purchase Intention Test

Hedonic acceptance tests were applied using a 9-point structured verbal scale, ranging from “extremely disliked” (number 1) to “extremely liked” (number 9), with the midpoint “neither liked nor disliked” (number 5). The evaluated attributes were overall impression, color, aroma, flavor, and alcohol content. Purchase intention was assessed using a 7-point verbal attitude scale, ranging from “would never buy” (number 1) to “would always buy” (number 7), with the midpoint “might or might not buy” (number 4).

2.3.2. Check-All-That-Apply (CATA) Test

For the CATA test, panelists were asked to indicate which words appropriately described their experience with the evaluated sample. These words or terms referred to the sensory characteristics of mead and were as follows: Yellowish color-Brownish color-Clear-Cloudy-Honey flavor-More alcoholic-Less alcoholic-Refreshing-Sweeter-Less sweet-Dry-Acidic-Vinegary-Flavorful-Effervescent-Carbonated-Yeasty taste-Shiny-Bitter-Sparkling. Based on these predefined terms, panelists selected the words they found appropriate to describe the evaluated mead. It is important to note that the terms were based on sensory descriptions and characteristics of mead reported in previous scientific literature [2,11,19,20].

2.4. Statistical Analysis

To analyze the shelf life data, a completely randomized design (CRD) with a two-factor factorial scheme was used, with two fixed factors: type of treatment and storage time (days). Initially, assumptions of normality and homogeneity of variance of the residuals were verified. Then, factorial analysis of variance (ANOVA) was applied to assess the main effects of each factor and their interaction on the response variables. The null hypotheses tested considered the absence of main effects and interactions on the response variables. All shelf life analyses were performed using RStudio software (version 4.3.2).

In the hedonic test, the evaluated attributes included overall acceptance, color, aroma, flavor, alcohol content, and purchase intention. The data were analyzed by one-way ANOVA to verify whether significant differences existed among treatments for each sensory attribute. When a significant effect was detected, Tukey’s multiple comparison test was applied to determine which samples differed. Hedonic test analyses were conducted using Microsoft Excel^® 365, adopting a significance level of 5% (p ≤ 0.05).

For the CATA (Check-All-That-Apply) test, the selections made by panelists were statistically analyzed using the chi-square test of independence, to identify statistically significant associations between sensory attributes and the different evaluated samples. As in the hedonic test, this analysis was conducted using Microsoft Excel^® 365, adopting a significance level of 5% (p ≤ 0.05) in all interpretations.

3. Results and Discussion

3.1. Shelf Life of Mead

Figure 1 presents the physicochemical characteristics for pH, total acidity, soluble solids, and alcohol content of the mead during refrigerated storage at 7 ± 1 °C over 70 days. As shown in Figure 1A, the pH of the mead produced with the probiotic yeast S. boulardii (T1) remained lower, tending toward acidic pH, when compared with the mead produced with commercial S. cerevisiae (T2), with significant differences (p < 0.05) between treatments observed on days 10, 30, 40, 50, 60, and 70 of storage. Supporting the pH results, it was observed that T1 exhibited higher total acidity compared to T2, with significant differences (p < 0.05) throughout storage (Figure 1B). The lower pH values in T1 may be associated with a greater conversion of fermentable sugars into organic acids due to the association between kombucha and S. boulardii, thus contributing to increased acidity (Figure 1B), to the detriment of the high fermentative activity of the association between commercial S. cerevisiae and kombucha in the conversion of fermentable sugars into alcohol (Figure 1D) under refrigerated storage conditions.

Regarding soluble solids, a reduction of 2.46 °Brix for T1 and 0.96 °Brix for T2 was observed between the first and last day (70 days) of mead storage (Figure 1C). In parallel, it was found that as soluble solids decreased, alcohol content increased during refrigerated storage (Figure 1D). Specifically, the alcohol content in T1 increased by 2.4%, from 4.77% on day 0 to 7.17% on day 40, followed by a decrease to 6.7% at 70 days of storage. In T2, alcohol content increased by 1.5%, from 6.77% on day 0 to 8.27% on day 20, followed by a sharp decline to 7.3% at 70 days of storage. These behaviors observed for the reduction of soluble solids (Figure 1C) and increase in alcohol content (Figure 1D) in both T1 and T2 suggest that S. boulardii and commercial S. cerevisiae yeasts in association with kombucha exhibit fermentative metabolic activity, albeit reduced, under refrigerated storage conditions of the mead. A similar behavior was also observed by Terhaag et al. [10], who, while studying the development of a probiotic lychee beverage with Saccharomyces boulardii, noted an increase in ethanol content in LB12 (lychee beverage supplemented with sucrose at 12 °Brix) during storage, possibly due to continued yeast metabolism even under refrigeration. These authors confirm that refrigerated storage does not prevent ethanol synthesis in such products.

In Figure 1, although the sugar content progressively decreases (Figure 1C), for treatments T1, the alcohol content progressively increases until day 40, followed by a decrease while in the thesis for T2 (Figure 1D), alcohol content increased until day 20, and then decreased in the following days. This trend is unexpected, as a reduction in sugars would normally be associated with a proportional increase in ethanol during fermentation, but under reduced refrigerated storage conditions, microorganisms should have lower metabolic activity. However, according to the scientific literature, it is known that the microbial spectrum of the kombucha consortium is formed by various microorganisms, including lactic acid bacteria, acetic acid bacteria, and yeasts [2,21,22,23]. According to Villarreal-Soto et al. [21], the interaction of kombucha microorganisms can inhibit ethanol production, since some acetic acid bacteria (Acetobacter xylinoides, Acetobacter aceti, Acetobacter pasteurianus, and others) act by transforming ethanol into acetic acid and other acids such as gluconic and glucuronic acids, while other microorganisms can act on sugars such as glucose and synthesize compounds such as microbial cellulose (forming the biofilm that remains on the surface of the liquid), acids, and other secondary metabolites. These facts could be associated with the observed behavior (Figure 1D). However, we emphasize that further analysis would likely corroborate and provide a better explanation, such as volatile acidity, residual sugars, and specific acid analyses, although this is still a limitation of this study.

With regard to contaminating microorganisms in mead, the results for total coliforms, E. coli, and Salmonella counts were negative, and no counts of these microorganisms were observed during product storage. Regarding the count of viable microorganisms in the mead during refrigerated storage, the results are presented in Figure 2. Although the initial viable microorganism count in the study by Souza et al. [2] was slightly different from that in the present study, the results are comparable, considering that Souza et al. [2] performed the initial microbial count for simulated in vitro digestion analysis, whereas in the present study, the microbial count was performed for shelf life assessment. It was observed that during storage there was a pronounced reduction in lactic acid bacteria (LAB) counts in T1 and T2 at 70 days of storage (Figure 2A). Coincidentally, there was a reduction of 1.45 Log CFU/mL in both treatments T1 and T2 when comparing the initial and final storage times. Moreover, at the end of storage, LAB counts in T2 were significantly lower (p < 0.05) compared to T1, which may be related to the higher alcohol content in T2 (Figure 1D), potentially causing stress to the microorganisms in the more alcoholic environment and consequently leading to greater reduction and/or death of LAB.

Regarding the viable yeast cell count, the results are presented in Figure 2B. It can be observed that during refrigerated storage of the mead, there was a marked reduction in yeast cells, which may be related to the increase in alcohol content during storage (Figure 1D). Specifically, a reduction of 2.67 Log CFU/mL was observed for S. boulardii in T1 and 2.73 Log CFU/mL for commercial S. cerevisiae in T2 at the end of 70 days of storage (Figure 2B). However, it is noteworthy that up to 60 days of storage, the meads still presented yeast counts above 6 Log CFU/mL, whereas at 70 days, the yeast counts dropped to 5 Log CFU/mL. In this context, considering that 6 Log CFU/mL is regarded as the minimum therapeutic dose for probiotic products to confer beneficial effects on the host [1,2,3,5,24,25], it is evident that 60 days of refrigerated storage at 7 ± 1 °C represents the most appropriate shelf life for potentially probiotic mead obtained by mixed fermentation of S. boulardii and kombucha (T1) (Figure 2B).

Taking into account that 60 days of refrigerated storage at 7 ± 1 °C is established as the most suitable shelf life for the mead, the analysis of bioactive compounds was performed using storage times of zero and 60 days as reference points, with the results shown in Figure 3. According to Figure 3A, the total phenolic content in T1 increased by 3.62 mg GAE/100 mL at 60 days of storage, being significantly higher (p < 0.05) than at the initial storage time. The significant increase in total phenolics in T1 may be associated with the release of metabolites and interaction between the mixed culture microorganisms and the polyphenols present in honey. Lazo-Vélez et al. [26] reported that S. boulardii may contribute to the release of phenolic compounds through extracellular fractions, since the extracellular fraction of S. boulardii is rich in polyphenolic metabolites, such as vanillic acid, cinnamic acid, and vitamin B6. In contrast, T2 showed a significant reduction (p < 0.05) over the storage period and exhibited lower values than T1 at 60 days, suggesting lower bioconversion potential.

Regarding antioxidants measured by the ABTS method, a significant increase (p < 0.05) of 21.07 μmol TE/100 mL for T1 and 16.14 μmol TE/100 mL for T2 was observed at 60 days of storage, as shown in Figure 3B. It should be noted that, although there is a reduction in phenolic compounds in T2 (Figure 3A) during storage, there is an increase in antioxidants by ABTS in T2 (Figure 3B), showing that, in general, even though a higher amount of phenolic compounds is related to greater antioxidant power, the reduction in phenolics did not cause a decrease in antioxidant capacity by the ABTS method, suggesting that antioxidants that react with the ABTS cationic radical are the most prevalent. For the FRAP method, a significant reduction (p < 0.05) of 0.89 μmol TE/100 mL for T1 and 0.80 μmol TE/100 mL for T2 was observed at 60 days of storage, as shown in Figure 3C. These results reinforce that during the shelf life, mead retains antioxidant compounds, with particular emphasis on the increase in antioxidants measured by ABTS, despite the reduction observed by FRAP. Previous studies have reported that mead is a source of bioactive compounds, such as phenolic compounds and antioxidants [1,2,5,27].

3.2. Sensory Analysis

A total of 120 panelists participated in the sensory analysis, with ages ranging from 18 to 59 years; 31.67% were male (38 panelists) and 68.33% were female (82 panelists).

Table 1. Sensory profile for acceptance and purchase intention of mead.

Attributes	T1	T2	T3
Overall impression	7.75 ± 1.30 a	6.64 ± 1.63 b	6.89 ± 1.79 b
Color	7.30 ± 1.71 a	7.17 ± 1.63 a	6.89 ± 1.74 a
Aroma	7.19 ± 1.52 a	6.89 ± 1.61 a	7.31 ± 1.53 a
Flavor	7.84 ± 1.46 a	6.23 ± 1.96 b	6.68 ± 1.94 b
Alcohol content	7.56 ± 1.41 a	6.56 ± 1.77 b	6.64 ± 1.90 b
Purchase intention	4.98 ± 1.30 a	4.10 ± 1.54 b	4.00 ± 1.72 b

T1 = mead with kombucha and S. boulardii; T2 = mead with kombucha and commercial S. cerevisiae; T3 = commercial mead available in the national market, produced with eucalyptus flower honey. Different lowercase letters in the same row indicate statistically significant differences (p < 0.05) between treatments according to Tukey’s test. Identical letters denote no significant difference.

presents the results for mead acceptance and purchase intention. It is evident that the mead with S. boulardii and kombucha (T1) showed higher acceptance scores for the attributes of overall impression, flavor, and alcohol content, with significant differences (p < 0.05) compared to T2 (mead produced by mixed fermentation of commercial S. cerevisiae and kombucha) and T3 (commercial mead available in the national market). Regarding color and aroma attributes, all treatments were statistically similar (p > 0.05). It is worth noting that T1 presented mean scores above 7 points for all acceptance attributes, with emphasis on overall impression, which reached a score of 7.75 points. These results are in agreement with the study by Souza et al. [11], which also reported good acceptance for meads with S. boulardii, where the mead formulation with initial soluble solids content of 30 °Brix (formulation F2) was considered more acceptable, with an overall impression score of 7.63 ± 1.42 points. Furthermore, other studies in the literature have also demonstrated that meads possess aromatic complexity, pleasant organoleptic characteristics, and good sensory acceptance [11,28,29,30,31], corroborating the findings of this study.

For purchase intention (Table 1), it was found that the mead with S. boulardii and kombucha (T1) showed the highest purchase acceptance, with a significant difference (p < 0.05) compared to T2 and T3, which were statistically similar (p < 0.05). These results observed for purchase intention in T1 corroborate the higher acceptance of this mead, since, according to the literature, a favorable attitude can lead to a higher probability of use and/or purchase of a given product, whereas an unfavorable attitude can result in a lower likelihood of acquisition or purchase [11,32].

Regarding the frequency of descriptive terms in the CATA questionnaire, the results are shown in Figure 4. The CATA results revealed that the mead with S. boulardii and kombucha (T1) was mainly associated with the terms carbonated, effervescent, flavorful, honey taste, sweeter, refreshing, and less alcoholic (Figure 4), which is consistent with the lower alcohol content observed for T1, as presented in Figure 1D. Indeed, these sensory characteristics frequently mentioned by the panelists contributed to the higher acceptance and purchase intention for T1, as shown in Table 1.

On the other hand, the mead with commercial S. cerevisiae and kombucha (T2) was characterized by attributes frequently described by the terms yellowish, less sweet, and more alcoholic (Figure 4), which is consistent with the higher alcohol content observed for T2, as presented in Figure 1D. For the commercial mead available in the national market (T3), the terms most frequently selected by the panelists were dry, shiny, and clear (Figure 4). These characteristics described for T3 confirm that, despite rising production costs, some production steps such as clarification, filtration, and centrifugation to remove yeast cells are included in the process and are considered important for industrial products destined for market commercialization [31,33,34]. Interestingly, it is noteworthy that although T3 is a commercial mead with physical and chemical characteristics of 12.9 °Brix soluble solids, pH of 3.42, acidity of 0.34% (percentage of lactic acid), and alcohol content of 13%, i.e., it had a lower soluble solids content among the products and a higher alcohol content than T1 and T2, the panel members perceived T2 as the most alcoholic product, possibly due to the complexity and interaction of the sensory attributes described for this mead, which may have increased the perception of a higher alcohol content.

With regard to sensory analysis, it is important to note that the evaluations carried out in this study used samples referring to the initial storage time. Although these analyses indicated high acceptability and high purchase intention for mead fermented with S. boulardii and kombucha, it was observed that, during storage, the samples developed characteristics that were significantly different from those present at the beginning. These findings highlight the complexity inherent in potentially probiotic products containing viable microorganisms, especially with regard to maintaining stable sensory profiles during shelf life. In this study, it was found that the reduction in the metabolic activity of microorganisms during refrigerated storage resulted in measurable physicochemical changes—such as variations in soluble solids and alcohol content—when comparing the initial and final times. Such changes can directly influence the sensory perception of the product. Thus, conducting careful sensory analyses both during development and throughout the storage period is essential to understand and ensure that sensory attributes remain within acceptable limits until the end of shelf life.

Regarding the market potential for the proposed samples, some considerations are essential, especially because residual and continuous fermentation during storage results in a final product that is sensory and physicochemically distinct from that obtained immediately after production. This behavior can pose a challenge both for commercial insertion and for meeting regulatory requirements. Thus, one of the main limitations of the developed product is the alteration of its physicochemical parameters throughout its shelf life, a direct consequence of the presence of viable microbial cells capable of maintaining reduced but persistent metabolic activity. Additionally, there is a reinforced need for systematic sensory analyses—at the beginning, during, and at the end of storage—in order to ensure sensory consistency, detect possible deviations, and evaluate the overall stability of the product. From this perspective, the potentially probiotic mead produced may have commercial viability, especially in specific niches, such as the craft beverage and functional products market. However, further studies are needed to improve understanding of the factors that influence product quality and stability. Recommended investigations include: sensory characterization throughout storage; identification and quantification of specific bioactive compounds; detailed evaluation of the profile of organic acids and alcohols produced during fermentation and storage; as well as the development or selection of appropriate packaging to minimize undesirable changes and preserve quality until the end of shelf life.

4. Conclusions

It can be concluded that the potentially probiotic mead produced by the mixed fermentation of S. boulardii and kombucha has a shelf life of 60 days, maintaining viable yeast and lactic acid bacteria counts above 6 Log CFU/mL, which is the minimum recommended value for probiotic products. It can also be concluded that the microorganisms present in mead exhibit metabolic activity during refrigerated storage, even if reduced, contributing to an increase in the alcohol content of the product. In addition, mead with mixed fermentation between the probiotic yeast S. boulardii and kombucha showed greater acceptance and purchase intention by panel participants and was described by terms such as carbonated, effervescent, flavorful, honey taste, sweeter, refreshing, and less alcoholic. The good acceptability and positive purchase intention of mead may contribute to its future market entry and/or commercialization, demonstrating the possibility of use and acquisition. It should be noted that the application of S. boulardii in mixed fermentation with kombucha has shown to be a promising alternative for the development of mead that can stand out as an innovative, probiotic, and functional product. Thus, this study provides evidence supporting the application of probiotic strains in alcoholic matrices, expanding the possibilities for the development of products with health appeal. However, studies on the effects of mead on metabolism and modulation of the intestinal microbiota in in vivo animal and human models should be encouraged in order to investigate possible health-related aspects.