Honey-must fermentation: structural trends, hive-associated yeasts, and the shift to precision biotechnologyABSTRACT:
Honey-must fermentation is expanding within the functional beverage market. However, industrial production remains constrained by a reliance on generic wine yeasts, ill-suited for the honey’s hyper-osmotic and nitrogen-deficient environment. This systematic review consolidates the biotechnological advancements, distinguishing between traditional Saccharomyces applications and the emerging domestication of hive-associated microbiota. Following the PRISMA 2020 guidelines, 383 records were identified, and 42 studies were selected for qualitative synthesis. The statistical breaking-point analysis revealed a structural inflection in research output in 2019, correlating with a surge in scientific interest, with the rising global demand for natural, immune-boosting products. Assessment of process variables indicated that mitigating yeast stress requires precise control of temperature (22 °C -25 °C) and pH (3.6-4.0), along with organic nitrogen supplementation. Essentially, the review identified a paradigm shift toward wild yeasts isolated from honey and pollen, such as Zygosaccharomyces rouxii and Lachancea thermotolerans. These “hive-native” strains possess evolutionary adaptations to osmotic stress and offer superior aromatic complexity compared to the traditional commercial strains. The future of honey-based beverages lies in a “hive-to-bottle” approach that combines native biodiversity with precision nutrition, to overcome fermentation bottlenecks and create distinct, terroir-driven sensory profiles, thereby transitioning the field from empirical methods to controlled industrial innovation.
Key words:
yeast isolation; yeast screening; mead; fermentation parameters
RESUMO:
A fermentação de mosto de mel está em expansão no mercado de bebidas funcionais, embora a produção industrial ainda seja limitada pela dependência de leveduras, vinícolas genéricas inadequadas ao ambiente hiperosmótico e deficiente em nitrogênio do mel. Neste sentido, esta revisão sistemática consolidou avanços biotecnológicos, distinguindo entre as abordagens tradicionais com Saccharomyces e a emergente domesticação da microbiota associada à colmeia. Seguindo as diretrizes PRISMA 2020, foram identificados 383 registros, dos quais 42 estudos foram selecionados para síntese qualitativa. A análise estatística do ponto de ruptura revelou uma inflexão estrutural na produção científica em 2019, associando o aumento do interesse acadêmico à demanda global por produtos naturais e imunomoduladores. Além disso, a avaliação das variáveis de processo demonstrou que a mitigação do estresse das leveduras requer controle preciso da temperatura (22-25 °C) e do pH (3,6-4,0), além da suplementação com nitrogênio orgânico. Essencialmente, a revisão identificou uma mudança de paradigma no uso de leveduras selvagens isoladas do mel e do pólen, como Zygosaccharomyces rouxii e Lachancea thermotolerans. Essas linhagens “nativas da colmeia” possuem adaptações evolutivas ao estresse osmótico e apresentam complexidade aromática superior em comparação às estirpes comerciais genéricas. Portanto, o futuro das bebidas à base de mel reside em uma abordagem “da colmeia à garrafa”, combinando a biodiversidade de linhagens nativas com estratégias de nutrição de precisão para superar gargalos fermentativos e criar perfis sensoriais distintos, transicionando o campo de métodos artesanais empíricos para a inovação industrial controlada
Palavras-chave:
isolamento de leveduras; seleção de leveduras; hidromel; parâmetros fermentativos
INTRODUCTION
Honey has long been appreciated as a natural sweetener and is increasingly valued for its health-promoting properties, positioning it as a strategic ingredient in the functional beverage sector (BHAT et al., 2020; MEENAKSHI et al., 2025). Rising consumer interest in health-oriented products, intensified by the global disruption due to the COVID-19 pandemic, has spurred a “renaissance” of traditional fermented drinks such as mead driving the industry, which putsmore effort into developing more nutritious and functional alternatives to the standard alcoholic beverages (MEENAKSHI et al., 2025). Honey-based fermentation offers a unique pathway to craft beverages with distinctive flavor profiles, shaped by the botanical origin of the honey, the specific yeast strain employed, and the nutritional supplementation strategy (GARCÍA et al., 2024; YU et al., 2024). However, controlling honey-must fermentation remains a biotechnological challenge, as the substrate presents a unique, high-stress environment (characterized by extreme osmotic pressure and a critical lack of nitrogen) that differs significantly from grape-must or gra-in wort (FERLA et al., 2024).
Even as Saccharomyces cerevisiae remains the industrial workhorse due to its robustness and high ethanol yield, reliance on generic commercial strains often results in standardized sensory profiles that fail to capture the botanical complexity of the origin of honey (DOOLAM et al., 2024). Furthermore, the metabolic stress induced by the nutrient-poor matrix frequently leads to sluggish fermentation and production of off-flavors (HU et al., 2024). Consequently, optimizing process parameters have become an active area of research to improve both the quality and efficiency of honey-derived alcoholic beverages. A promising frontier in this field is exploring the “hive ecosystem,” as a source of specialized biological solutions. Honey itself serves as a reservoir of resilient yeasts adapted to low water activity (typically 0.500.65) and antimicrobial compounds. Harnessing this biodiversity offers a pathway to differentiate products through unique aromatic signatures (BHAT et al., 2020; DIMOPOULOU et al., 2023).
Recent efforts to isolate wild yeasts from honey and hive environments identified candidate strains, such as, Zygosaccharomyces rouxii, Lachancea thermotolerans, and osmotolerant S. cerevisiae variants, with the potential to improve fermentation resilience and complexity (CHAIJAK et al., 2024; ARAÚJO et al., 2021a). However, the successful implementation of these wild isolates requires a deep understanding of their nutritional needs and kinetic behaviors. Therefore, the shift from empirical artisanal methods to precision biotechnology inherently requires the biotechnological domestication of these wild hive-associated yeasts, transforming them from unpredictable environmental agents into reliable starter cultures. In this context, the present article conducted a systematic review to consolidate the current knowledge on honey-must fermentation, employing the rigorous Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) methodology to map the structural shift in research focus after 2019, and critically evaluatethe interaction between ‘native hive’ yeasts and precision nutrition.
DEVELOPMENT
This review followed PRISMA 2020 guidelines for transparency and reproducibility. Scopus was selected as the primary database after a preliminary comparison with Web of Science, PubMed, ScienceDirect, and SpringerLink, as it showed that it encompassed all relevant records, both overlapping and unique. The search, performed on November 27, 2025, used the string: TITLE-ABS-KEY (honey OR mead OR “honey wine” OR spirit OR “honey spirit” OR “honey-shine” OR “honeyshine” OR “honey shine”) AND (fermentation OR distillation OR “fermented honey” OR “honey fermentation” OR “distilled mead” OR “mead distillation”) AND (yeast OR “yeast isolation” OR “isolated yeast” OR screening) ).
The timeframe (2010-2026) was chosen due to the field’s exponential growth since 2010. The inclusion criteria targeted original English articles on honey-must fermentation, yeast nutrition, spirit production, and yeast isolation from hive environments. We excluded Reviews (except for mapping), conference articles, and studies without quantitative fermentation data.
As illustrated in the PRISMA flow diagram (Figure 1), the selection process identified 383 records, with 120 retained for scientometric mapping via VOSviewer (v.1.6.20). Methodological guidance followed ANDREO-MARTÍNEZ et al. (2020) and ZAKARIA et al. (2021). Thresholds included: journals (≥3 publications), countries (≥1 article and 100 citations), keywords (occurrence ≥10), and authors (≥4 articles). Fractional counting was applied to author networks, to balance the contributions, while full counting was used for other metrics. Total Link Strength (TLS) measured interconnectedness and Average Normalized Citations (AvNC) were applied to mitigate time-dependent citation bias.
PRISMA 2020 flow diagram illustrating the systematic selection process of studies. The diagram details the identification of records from the Scopus database (n = 383), the screening phase excluding unrelated topics (n = 263), and the final eligibility assessment. A total of 120 articles were retained for scientometric analysis, while 42 studies met the specific criteria for the qualitative synthesis of fermentation parameters.
For qualitative synthesis, 42 studies were selected after a full-text assessment of 120 articles, with 78 excluded for insufficient process data. The eligible articles were categorized into: (i) fermentation parameters (temperature, pH, sugar, nitrogen, and time) and (ii) yeast selection (Saccharomyces and non-Saccharomyces isolates). Data extraction focused on experimental conditions, must composition, and reported outcomes.
The temporal evolution of 120 articles (2010-2025) reflected a field transitioning from nascent to established research. A breaking point analysis (strucchange package, R) identified a significant structural shift in 2019 (Figure 2), with output growing from 20 to 50 documents annually by 2025. This surge correlated with the post-pandemic demand for functional, immune-boosting products, and a global honey market projected to reach USD 15.59 billion by 2032 (MEENAKSHI et al., 2025). Simultaneously, an expanding alcoholic beverage market and a rising interest in the per capita consumption drive for wild yeast isolation, to differentiate products through unique sensory profiles (HU et al., 2024; MOLINET & CUBILLOS, 2020).
Annual evolution of scientific publications on honey-must fermentation and yeast selection (2010-2025). The solid line represents the number of documents retrieved from the Scopus database per year. The vertical dashed line indicates the structural breaking point identified in 2019, marking a statistically significant shift in research growth trends.
Journal analysis (Table S1) shows that the Journal of the Institute of Brewing was the most prolific, while Food Chemistry held the highest impact (64.50 citations/article). Metrics such as Normalized Citations identified LWT, the Journal of the Institute of Brewing, and Food Microbiology were high-impact venues. The latter two, along with the International Journal of Food Microbiology, exhibited high Total Link Strength (TLS), indicating they are central to the academic network. Conversely, journals like Yeast appeared less integrated into the core of honey-fermentation citation network.
Geographically (Table S2), Brazil led in volume and citations, leveraging its biodiversity and stingless bee honey to diversify products (SILVA et al., 2022). China, Portugal, and Turkey focused on agricultural productivity and economic development (BHAT et al., 2020). In contrast, New Zealand prioritizes value over volume, focusing on Manuka honey’s bioactive traits, to justify premium functional beverages (CLARE et al., 2024).
Keyword co-occurrence analysis (Table S3) confirms “fermentation,” “yeast,” and “Saccharomyces” as the field’s conceptual pillars. While metabolic and characterization terms (ethanol, microbiology) dominated; emerging interests in “antioxidant”, “aroma profile”, and “nitrogen” showed high TLS, indicating they are well-integrated specializations.
Co-authorship clusters (VOSviewer, Table S4) revealed distinct collaborative hubs. Cluster 1 (Portugal) led in impact, focusing on S. cerevisiae optimization and microbiology. Cluster 2 (Italy) led in volume, but showed lower interdisciplinary exchange. Clusters 3 and 4 (Brazil) bridged biochemistry and food technology; although, they faced systemic barriers to collaboration, such as, funding and geographic distance (MARTINEZ & SÁ, 2020).
Yeast strains and their role in the honey-must fermentation
Applying the inclusion criteria reduced the dataset from 120 to 36 studies that specifically address honey-must fermentation for producing alcohol or alcoholic beverages, providing strong discussions of the results and process variables.
S. cerevisiae was the most frequently applied yeast in honey-must fermentation (Table S5). This yeast is widely used in both research and commercial applications for its ability to ferment a wide range of sugars, yielding high ethanol concentrations. S. cerevisiae is particularly valuable in honey fermentation, because honey is rich in sugars, which can create a challenging environment for other yeast species (DOURADINHO et al., 2023; FERLA et al., 2024). The resilience of S. cerevisiae enables it to manage the high sugar concentration typical of honey-must, as well as variations in pH and temperature, making it a dependable choice for consistent, high-quality ethanol production (DOOLAM et al., 2024). Additionally, this strain’s higher tolerance to stress factors - such as increased alcohol concentrations, temperature fluctuations, and pH variations - helps maintain a stable fermentation process in conditions that could inhibit other yeasts, such as sensitive wild strains from the Kloeckera and Metschnikowia genera (SALAS-NAVARRETE et al., 2022).
Other species within the Saccharomyces genus, including S. bayanus, S. pastorianus, and S. uvarum, are also cited for their contributions to the flavor complexity and sensory profile of fermented honey beverages. S. bayanus is recognized for its unique fermentation profile, which enhances the aromatic complexity of the final product, adding subtle fruity and floral notes to the flavor profile (ARAÚJO et al., 2020; DIMOPOULOU et al., 2023). Meanwhile, S. pastorianus and S. uvarum are valued for producing cleaner, more balanced flavors, characterized by restrained ester production and low levels of higher alcohols. However, these species generally exhibit moderate alcohol tolerance and ferment more efficiently at lower temperatures (8 °C -15 °C), thereby prolonging the fermentation process (DIMOPOULOU et al., 2023; MAICAS & MATEO, 2023). Despite this, their contributions to sensory balance and refinement make them valuable in making honey-based beverages with a more nuanced, controlled taste (TIMOUMA et al., 2021).
Alongside Saccharomyces strains, non-Saccharomyces yeasts also play a complementary role in honey-must fermentation, with species such as Candida, Hanseniaspora, Metschnikowia, and Pichia being frequently examined. These yeasts are often combined with S. cerevisiae to improve the beverage’s sensory qualities (MAICAS & MATEO, 2023). Non-Saccharomyces yeasts release a variety of volatile compounds, including esters, higher alcohols, and organic acids, which collectively contribute to the fruity, floral, and even spicy aromas in the final product (BORREN & TIAN, 2020). For example, Hanseniaspora uvarum is known for producing fruity esters that significantly enhance the aromatic complexity and create a desirable sensory experience in honey-based alcoholic beverages (SOLA et al., 2024).
Some non-Saccharomyces yeasts have demonstrated a potential in reducing sulfur dioxide (SO2) levels. SO2 is widely used in the fermentation industry for its antimicrobial and antioxidant properties, helping control unwanted microbial growth, and preventing oxidation in beverages (GIMÉNEZ et al., 2023). However, excessive levels of SO2 can adversely affect the sensory qualities and consumer perception of fermented products, imparting undesirable “struck match” or “chemical” flavors that mask the natural aromas and tasting notes (WANG et al., 2024). These yeasts thereby improve the quality of the fermentation environment and enhance microbial stability. Metschnikowia pulcherrima, for example, has been studied for its ability to lower SO2 levels in white grape-must, which is also relevant to honey fermentation (GIMÉNEZ et al., 2023; TORRES-DÍAZ et al., 2024).
This yeast consumes oxygen, helping to limit enzymatic browning reactions and reducing the need for sulfur-based preservatives, which are often undesirable in fermented products (GIMÉNEZ et al., 2023). Additionally, M. pulcherrima can produce antimicrobial compounds, providing a natural means of biological control and reducing sulfur compounds, thereby improving product quality and consumer acceptance (TORRES-DÍAZ et al., 2024). Similarly, certain Pichia species - such as. P. membranifaciens, P. guilliermondii, P. kudriavzevii, P. fermentans, and P. kluyveri - are known to produce killer toxins that inhibit unwanted microorganisms, including competing yeasts, fungi, and bacteria, thus enhancing microbial stability during fermentation (BELDA et al., 2017). Using non-Saccharomyces yeasts in combination with S. cerevisiae can therefore result in a synergistic effect, in which, each contributes to different aspects of fermentation quality. While S. cerevisiae provides high ethanol yields necessary for alcoholic honey beverages, non-Saccharomyces yeasts bring a broader spectrum of volatile compounds and beneficial characteristics, enhancing both sensory complexity and microbial stability.
Critical process parameters in honey-must fermentation
Controlling key variables (specifically temperature, soluble solids (°Brix), fermentation time, pH, and nutrient supplementation) is fundamental to optimizing yeast metabolism, ethanol yield, and the sensory profile of the final product (CUENCA et al., 2022; GARCÍA et al., 2024). Table S5 summarizes the effects of these variables across multiple studies, highlighting that deviations from optimal ranges often lead to yeast stress or organoleptic defects (GUZZON et al., 2020; DOURADINHO et al., 2023).
Temperature is widely recognized as a determining factor in fermentation kinetics. Although, the studies reviewed in Table S5 explored temperatures ranging from 18 °C to 30 °C, the optimal balance between kinetics and sensory quality was consistently reported at 22 °C -25 °C. The research indicated a clear operational trade-off between fermentation kinetics and sensory quality. Studies maximizing kinetic efficiency and high ethanol yields, such as those by ARAÚJO et al. (2020) and GARCÍA et al. (2024), successfully employed elevated temperatures of 30 °C and 33 °C, respectively, achieving ethanol concentrations as high as 15.5% v/v. However, when the focus shifted to aroma preservation, the literature strongly supported lowering the temperature, to prevent excessive accumulation of undesirable volatile metabolites. For instance, BÉNES et al. (2015) demonstrated that at 24 °C, the process achieved ethanol yields between 13.60% and 17.47%, v/v, while inducing elevated levels of acetaldehyde and 1-propanol. Consequently, to maintain low levels of fusel alcohols and achieve optimal aromatic profiles, studies often restricted temperatures to 20 °C -22 °C; within this range, stable ethanol production of 11.4%-14.4% v/v was reported (FERLA et al., 2024; PEREIRA et al., 2013). Even at extremely low temperatures like 12 °C, high ethanol yields of 13.1% v/v possibly remained (PASCOAL et al., 2019); although, this required significantly extended fermentation periods. Therefore, maintaining the process between 20 °C and 24 °C provides the ideal compromise between robust conversion rates and a refined, defect-free sensory profile (CUENCA et al., 2016; PEREIRA et al., 2013).
Regarding substrate concentration, soluble solids were adjusted between 15°Brix and 35°Brix across the reviewed studies. As honey is primarily composed of glucose and fructose, the initial sugar concentration dictates both the potential ethanol content and the osmotic pressure exerted on yeast cells. A range of 18-24°Brix emerged as the most effective for balancing sugar availability with cell viability, preventing sluggish fermentation caused by osmotic shock (DOURADINHO et al., 2023). Within this range, GARCÍA et al. (2024) demonstrated efficient sugar conversion rates (0.27 g/L·h at 22°Brix), and ARAÚJO et al. (2020) reported high ethanol yields using naturally sugar-rich dark honey.
The physicochemical environment was further defined by pH and process duration. Fermentation times varied significantly (7-30 days) depending on volume and inoculum density, with larger volumes (5-10 L) typically requiring up to 30 days for complete development of volatile compounds. To ensure microbial stability and optimal enzymatic activity, the initial pH was generally adjusted to a range of 3.2 to 4.5, using organic acids. Even as CASTILLEJA et al. (2017) suggested pH 4.5 as optimal for Saccharomyces growth, Table S5 highlighted successful outcomes at lower pH levels; for instance, high ethanol yielded (8%-12% v/v) and favorable aromatic profiles were achieved at pH 3.5-3.7. Conversely, pH values below 3.5 can hinder cell function, particularly as ethanol accumulates (TORRES-GUARDADO et al., 2022; CHEN et al., 2024).
Finally, nutrient supplementation is indispensable due to the honey’s naturally low nitrogen content. The addition of diammonium phosphate (DAP), pollen, or yeast extract is a standard practice to prevent stuck fermentations. KEMPKA et al. (2015) observed maximum cell growth with 30 g/L of pollen or yeast extract, while ROLDÁN et al. (2011) reported that a moderate addition of 10 g/L pollen was sufficient for sensory enhancement. Inorganic sources like DAP also proved effective, increasing ethanol yields at concentrations ranging from 267 mg/L to 1 g/L. Synthesizing the data from Table S5, the most effective fermentation conditions converge on a specific profile: A temperature of 22 °C -25 °C, sugar concentration of 22-24°Brix, initial pH of 3.6-4.0, and supplementation with organic nitrogen sources (e.g., 30 g/L pollen). Managing these core variables provided a solid foundation for consistent, high-quality honey-must fermentation.
Yeast nutrition for honey-must fermentation
Honey lacks some essential nutrients, especially nitrogen, which is crucial for yeast growth and metabolism (RIBEIRO et al., 2020). The levels of nutrients and minerals vary depending on the type of honey; however, the average yeast assimilable nitrogen (YAN) content in honey-must is typically be below 50 mg/L (ROLDÁN et al., 2011). Approximately, 150 to 200 mg/l of YAN is required to provide sufficient nitrogen for proper fermentation, otherwise, the yeast cells may become stressed and struggle to ferment effectively, leading to stuck or sluggish fermentations, reduced ethanol yields, impairment of the desirable flavor and aroma compounds, and the production of unwanted by-products, such as, hydrogen sulfide (H2S), among other issues (CUENCA et al., 2022).
Besides nitrogen, vitamins and minerals are also necessary. They play a vital role in enzymatic functions and metabolic pathways, whereas, minerals are important for cell structure, enzyme activation, and osmotic balance. Table S6 summarizes findings from multiple studies that compare the average levels of vitamins and minerals in honey and pollen, with the concentrations typically required in “must” for optimal fermentation (alcohol and alcoholic beverage production). According to the published data in Table S5, BARRY (2018) and ROMANO (2021) were the exceptions that did not incorporate a compound with nutritional value for the yeasts. These studies reported ethanol concentrations below 7.0% (v/v).
Diammonium phosphate (DAP) is the most frequently used nutrient source, cited in 12 studies (Table S5) for providing assimilable nitrogen and phosphorus, which are essential for yeast protein synthesis, energy metabolism, and fermentation activity. Studies using DAP, as reported in Table S5, showed ethanol concentrations ranging from 10% to 15% v/v, accompanied by improved sugar conversion and fermentation stability. However, as DAP alone does not provide vitamins, amino acids, and other growth cofactors, it is often combined with more complex organic nutrients to avoid sensory degradation or incomplete fermentations.
Yeast extract has been used in six studies (Table S5). It provides a rich mix of amino acids, B-vitamins, trace minerals, and growth factors, making it especially effective in supporting yeast viability, rapid fermentation, and flavor complexity. In ARAÚJO et al. (2020) study, 5 g/L yeast extract contributed to ethanol production above 13% (v/v). Conversely, CUENCA et al. (2022) observed an enhancement of ester profiles and a reduction in the formation of fusel alcohols. There are; however, some drawbacks to supplementing “musts” with yeast extracts. It is relatively expensive and may influence flavor, which may or may not be desirable, depending on the style of the mead being produced (ARAÚJO et al., 2020; RIBEIRO et al., 2020).
Pollen was also used in six studies (Table S5). It contains sugars, amino acids, vitamins (especially from the B complex), and minerals (Table S6). It also supplies 500-1000 mg/100 g of YAN (ROLDÁN et al., 2011) and has been shown to enhance the fermentation rate, cell growth, and sensory profiles. KEMPKA et al. (2015) found that 30 g/L pollen led to high cell growth. ROLDÁN et al. (2011) reported optimal flavor development with only 10 g/L. Despite its nutritional benefits, pollen composition is highly variable depending on the floral origin and harvest conditions, which may lead to inconsistencies in fermentation results.
Ammonium sulfate ((NH₄)₂SO₄) was reported in four studies (Table S5). This inorganic nitrogen source supplies ammonium ions, but lacks vitamins or amino acids, thus requiring supplementation from more complex nutrients. When combined with other nutrients, (NH4)2SO4 supported the production of 10%-15% (v/v) ethanol. Its low cost and ease of application make it suitable for industrial processes, although it is rarely used on its own due to its limited nutrient profile. Other less common supplements reported, included, ammonium chloride (NH4Cl) and potassium phosphate (K2HPO4). NH4Cl serves as a direct nitrogen source, but lacks micronutrients. K2HPO4 supports phosphorus metabolism and stabilizes pH, but does not supply nitrogen or growth cofactors. Both nutrients required additional supplementation for optimal fermentation performance and flavor development. Supplementing with 0.2 g/L NH4Cl results in an ethanol yield of 12% (v/v) at pH 3.7. When utilizing 0.1 g/L K2HPO4 at pH 3.5, the ethanol yield is 8% (v/v).
Conversely, the addition of individual vitamins and mineral salts to the must has also been reported (Table S5). Magnesium chloride (0.05-0.1 g/L) and vitamin B1 (0.5 mg/L) were added to enhance enzymatic activity and cofactor availability. Although, these nutrients were not primary nitrogen sources, they supported yeast metabolism and fermentation robustness, helping to stabilize the process. They are never used alone.
Commercial nutrient blends were also used (Table S5). They combined ammonium salts, vitamins, yeast hulls, and trace minerals into balanced formulations. They were designed to ensure a full spectrum of nutrients and reduce the risk of fermentation. Although, these products improved ethanol production and sensory balance in multiple studies, their multi-component nature made it challenging to isolate the impact of the individual ingredients.
In line with this, the use of plant extracts was also reported (Table S5). ARAÚJO et al. (2020) evaluated cowpea extract as a low-cost alternative to nutrients. The extract was rich in proteins and amino acids and was applied at concentrations of up to 30 g/L, improving cell growth and fermentation efficiency and yielding 13.6%-15.5% (v/v) ethanol. Cowpea extract was especially effective when combined with inorganic salts, demonstrating a potential for use in resource-constrained or alternative production systems (ARAÚJO et al., 2020).
In summary, adequate yeast nutrition is crucial for honey-must fermentation, as it enhances ethanol production efficiency and the sensory quality of the final beverage, with each nutrient offering unique advantages and limitations.
Ethanol yields and aroma production
The optimization of ethanol yields in honey-must fermentations was heavily influenced by the fermentation conditions mentioned above. All the data are summarized in Table S5. High ethanol concentrations (12%-15% v/v) were often achieved by fermenting musts with a high initial sugar content (typically 30°Brix), at elevated temperatures (around 30 °C), frequently with inorganic nitrogen supplementation (ALMEIDA et al., 2020; ARAÚJO et al., 2020). For example, adding 0.2 g/L NH4Cl to honey-must and fermenting at 30°Brix and 30 °C yielded approximately 12% ethanol (MENDES-FERREIRA et al., 2010). Similarly, 1.0 g/L DAP supported strong fermentation and ethanol yields in the 12%-15% range (ALMEIDA et al., 2020). Ethanol levels were also observed to range from 13.6% to 15.5% at 30 °C using DAP and (NH4)2SO4 (ARAÚJO et al., 2020). These results supported the idea that high sugar and nitrogen levels, combined with higher temperatures, encouraged vigorous yeast activity and efficient sugar conversion. It was reported consistently that sweet sorghum juice fermentation was most effective at 37 °C, reaching ethanol concentrations of 101.81 g/L (~12.9% v/v) (CHAIJAK et al., 2024). To achieve this result, a thermotolerant yeast strain was employed. Such findings indicated that higher temperatures could boost ethanol productivity when yeast strains had adapted to heat stress and were provided with sufficient nutrients.
However, higher fermentation temperatures are also associated with increased production of volatile compounds, such as, esters and higher alcohols. Numerous studies have shown that these conditions can lead to excessive levels of volatile metabolites, potentially compromising the sensory quality (ZHAO et al., 2024). For example, BÉNES et al. (2015) have reported high levels of acetaldehyde and 1-propanol in meads fermented at higher temperatures using S. bayanus. Similarly, CUENCA et al. (2022) have observed increased ester production and decreased fusel alcohols through temperature control and nutrient supplementation. Compounds such as ethyl acetate, which impart solvent-like notes and acetaldehyde, associated with green apple aromas can be accepted only at very low concentrations. To address this concern, more moderate fermentation temperatures between 25 °C -28 °C are recommended for honey-based fermentations, as this range seems to optimize metabolic efficiency and preserve sensory qualities. Studies, such as, those by GUZZON et al. (2020), GARCÍA et al., (2024), and ARAÚJO et al. (2020) reported that temperatures within this range enable stable ethanol production and a more refined aromatic profile.
Strategies for isolating and screening wild yeasts
Among the extensive literature reviewed, six studies have provided specific, actionable data on the isolation and screening of wild yeast strains directly from honey and hive-associated environments (e.g., pollen, beeswax) for alcoholic beverage production (Table S7). This selection highlighted a critical niche, as the beverage industry increasingly seeks to move beyond standard commercial starters, to craft distinctive, innovative products, even as, commercial strains offer a consistency, as wild strains harbored in apiary environments possess an untapped potential for enhanced metabolic variation and unique aromatic complexity (MOLINET & CUBILLOS, 2020).
The screening protocols identified in these studies were rigorous, designed to evaluate yeast robustness under stress conditions that mimic the harsh environment of honey-must fermentation. The key selection criteria included high osmotic tolerance (20%-30% w/w glucose), ethanol resistance (5%-15% v/v), pH adaptability (3.0-5.0), and thermal resilience (25 °C -35 °C). These parameters are essential to ensure that selected strains not only survive, but also thrive in high-sugar, acidic, and alcohol-accumulating conditions of mead production.
Ethanol tolerance emerged as a decisive trait. Notably, strains isolated by SILVA et al. (2020) and ARAÚJO et al. (2021b) demonstrated viability at ethanol concentrations of up to 15% v/v, the highest threshold recorded among the reviewed studies. Similarly, osmotic stress management was observed in robust S. cerevisiae and S. boulardii isolates (PEREIRA et al., 2009; CHAIJAK et al., 2024), as well as in Z. rouxii, which successfully fermented media with up to 30% glucose (RODRÍGUEZ MACHADO et al., 2024). Regarding the pH, the ability to function between 3.0 and 5.0 was common across isolates from various geographic origins (PEREIRA et al., 2009; CHAIJAK et al., 2024; RODRÍGUEZ MACHADO et al., 2024). It was a range that optimized nutrient uptake, while naturally inhibiting spoilage organisms, such as, Enterobacteriaceae and lactic acid bacteria.
Beyond S. cerevisiae, the screening processes detailed in Table S7 identified a diverse array of non-Saccharomyces species with specific biotechnological applications for honey fermentation. These included Schizosaccharomyces pombe and L. thermotolerans, isolated from honey and pollen by SILVA et al. (2020) and ARAÚJO et al. (2021b), as well as Z. rouxii, Rhodosporidiobolus ruineniae, Clavispora lusitaniae, and Metschnikowia chrysoperlae (RODRÍGUEZ MACHADO et al., 2024). Additionally, comparative screenings of Africanized and stingless bee honeys have highlighted the excellent fermentative potential of S. bayanus (DANTAS et al., 2023).
The functional characterization of these species provides a valuable toolbox for producers. For ensuring high ethanol yields, S. cerevisiae, S. boulardii, and C. lusitaniae are optimal choices (PEREIRA et al., 2009; CHAIJAK et al., 2024; RODRÍGUEZ MACHADO et al., 2024). When the goal is to enhance sensory complexity and aromatic profiles, L. thermotolerans, M. chrysoperlae, and S. bayanus are pivotal (SCHWARZ et al., 2020; RODRÍGUEZ MACHADO et al., 2024). Furthermore, specialized osmophilic strains like Z. rouxii and S. pombe are uniquely suited to overcome the antimicrobial hurdles of high-sugar musts (ARAÚJO et al., 2021a; RODRÍGUEZ MACHADO et al., 2024), while the isolation of R. ruineniae opens avenues for functional beverages enriched with bioactive compounds such as carotenoids (RODRÍGUEZ MACHADO et al., 2024).
The transition from identifying wild strains to their application in standardized fermentations defines the process of biotechnological domestication. This domestication is guided by the selective pressure imposed by the parameters detailed in Table S7, in which isolates must demonstrate resilience to the ‘hostile’ environment of honey-must. For instance, the domestication of strains such as S. cerevisiae and S. boulardii involve validating their ability to maintain high ethanol production (up to 10.83% v/v) and glucose tolerance (up to 30% w/w) under acidic conditions (pH 3.0-5.0) (PEREIRA et al., 2009). Similarly, the screening of the non-Saccharomyces species, such as, L. thermotolerans and S. pombe, for ethanol tolerance up to 15% v/v (SILVA et al., 2020; ARAÚJO et al., 2021a) exemplifies how wild microbiota are ‘tamed’ to meet the industrial requirements of precision biotechnology. By aligning evolutionary wild traits with rigorous screening criteria, researchers can effectively domesticate these yeasts, transforming them from unpredictable wild agents into specialized biotechnological tools, for high-quality mead production.
Strategic and biotechnological horizons: a roadmap for precision honey fermentation
The comprehensive analysis conducted in this review offers a dual roadmap, strategic and technical, for the advancement of honey-based beverages. Strategically, the application of a statistical breaking point analysis provided empirical validation of the field’s maturation, pinpointing 2019 as a structural inflection point. This shift correlated with the surge in biotechnological research, with the global post-pandemic demand for functional and immune-boosting foods (MEENAKSHI et al., 2025). For the scientific community, this confirmed that honey fermentation has evolved from a niche artisanal topic to a consistently growing research domain, thereby justifying the transition from empirical trials to precision biotechnology.
Furthermore, the geographical analysis revealed a critical strategic dichotomy: “Volume vs. Value”. Although, major producers have driven research through biodiversity exploration, the premiumization model observed in certain markets highlights the need to turn from commodity-based research to value-added functional products. To achieve this sophistication, the technical paradigm must shift from generic fermentation to precision ecosystem management. The current industrial practices largely rely on commercial yeast strains, which often lack the specific adaptations required for the hyper-osmotic and antimicrobial environment of honey. A critical analysis of the isolation studies reviewed has suggested that the “domestication” of the hive microbiome is the most promising avenue for innovation (MOLINET & CUBILLOS, 2020; ARAÚJO et al., 2021a).
Strains isolated directly from honey, pollen, and beeswax, such as, the specific Z. rouxii and the osmo-tolerant S. cerevisiae variants, appeared to possess an evolutionary competitive advantage. Unlike commercial strains adapted to grape must, these wild isolates from honey have likely evolved mechanisms to thrive in high-sugar, low-protein matrices (RODRÍGUEZ MACHADO et al., 2024). However, unlocking the potential of these “hive-native” strains requires overcoming the “Nitrogen Gap”. As evidenced in this review, the innovation opportunity lies not merely in adding generic supplements, but in tailoring the Yeast Assimilable Nitrogen (YAN) profile to the specific metabolic needs of wild strains to prevent off-flavors and ensure sensory complexity (KEMPKA et al., 2015; ROLDÁN et al., 2011).
Finally, the convergence of bibliometric trends toward a “sensory profile” and “aroma,” indicated that the market demands differentiation. This fact may drive the strategic deployment of non-Saccharomyces species, such as L. thermotolerans, for natural acidification, and Metschnikowia pulcherrima for enzymatic release of bound aromas, not as competitors to S. cerevisiae, but as bio-flavoring tools in sequential inoculations. Ultimately, by integrating the strategic insights of bibliometrics with the ecological “Hive-to-Bottle” approach, the industry can overcome traditional limitations and define the next generation of high-value, distinctly flavored, honey-based alcoholic beverages.
CONCLUSION
This systematic review has confirmed that honey-must fermentation has transitioned from a niche topic to a more dynamic research field, characterized by a pronounced structural expansion, since 2019, driven by a global demand for natural and functional products. The synthesis of technical data has revealed that while fundamental parameters, such as, temperature control (22 °C -25 °C) and pH management (3.6-4.0), remain essential, the true frontier for innovation lies in the “domestication” of hive-associated microbiota. The use of wild yeasts, isolated directly from honey and pollen, specifically adapted to high osmotic stress, represents a superior biotechnological strategy, as compared to generic wine strains. When coupled with precision nitrogen supplementation to bridge the nutritional gap of honey, these native strains offer a viable pathway to overcome the stuck fermentations and unlock unique sensory profiles. Ultimately, the adoption of this “hive-to-bottle” ecological approach serves as the cornerstone for the next generation of premium, terroir-driven, honey-based beverages.
SUPPLEMENTARY MATERIAL
All supplementary files for this manuscript are hosted in a permanent repository and are available for consultation at: <https://docs.google.com/document/d/1Ux42nQu8J6qA_2du37Qo4yPORO7_Xpmx/edit?usp=sharing&ouid=100087523703515496736&rtpof=true&sd=true>.
ACKNOWLEDGMENTS
The authors gratefully acknowledged the financial support provided by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and SAMARCO Mineração (process number 4500181394, SEI 23109.004168/2020-33). We also thank the Postgraduate Program in Environmental Engineering of the Universidade Federal de Ouro Preto (UFOP) for the institutional support. We also thank the APICRIM Research Group and the Laboratory of Cellular and Molecular Biotechnology at UFOP for the infrastructure and scientific support provided throughout this study. We extend our special thanks to Professor C. Lucas for kindly dedicating her time to help improve the cohesion and organization of this manuscript. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.
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