Next Article in Journal
Exploring the Functionality of Microbes in Fermented Foods: Technological Advancements and Future Directions
Previous Article in Journal
Enhancing Steamed Bread Quality Through Co-Fermentation of Sourdough with Kazachstania humilis and Lactobacillus plantarum
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Fermentation
Volume 11
Issue 6
10.3390/fermentation11060299
fermentation-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Genomic and Fermentation Characterization of Kluyveromyces marxianus and Saccharomyces cerevisiae in Root Extract-Based Low-Alcohol Beverage

by
Eun-Ju Lee
†,
Seung-Hyun Choi
†,
Min-Ju Seo
†,
A-Reum Lee
,
Chan-Song Jang
,
Woong-Kwon Kwak
,
Jung-Ki Kwak
,
Jae-Ho Lee
,
Won-Joo Yoon
and
Seok-Min Yoon
*
LOTTE R&D Center, Seoul 07594, Republic of Korea
*
Author to whom correspondence should be addressed.
†
These three authors contributed equally to this work.
Fermentation 2025, 11(6), 299; https://doi.org/10.3390/fermentation11060299
Submission received: 25 April 2025 / Revised: 20 May 2025 / Accepted: 21 May 2025 / Published: 23 May 2025
(This article belongs to the Section Fermentation for Food and Beverages)
Browse Figures
Versions Notes

Abstract

Fermentation is widely recognized for enhancing the sensory attributes and nutritional value in foods, with recent research focusing on non-alcoholic and root-based functional beverages. In this study, the genomic and fermentation characteristics of Kluyveromyces marxianus LRCC8279 (KM8279) and Saccharomyces cerevisiae LRCC8293 (SC8293) were analyzed, specifically for their application in root extract-based low-alcohol fermentations. Whole-genome sequencing revealed that both strains harbored key genes involved in glucose, fructose, and sucrose metabolism and genes implicated in ethanol production. Although SC8293 harbored maltose-metabolizing genes, including MAL13 and MAL31, these genes were absent in KM8279. This genetic difference was evident in the fermentation performance, manifesting as distinct variations in alcohol production depending on the carbohydrate source. A further investigation of fermentation conditions demonstrated that both strains maintained low alcohol levels and exhibited a consistent growth at 15–20 °C within 72 h. Fermentation using extracts from Pueraria lobata, Arctium lappa (AL), Zingiber officinale (ZO), and Platycodon grandifloras revealed that KM8279 markedly increased the production of volatile compounds, contributing to floral and fruity sensory attributes in ZO and AL, whereas SC8293 contributed to a more complex flavor profile in AL. Notably, KM8279-ZO and KM8279-AL fermentations maintained alcohol contents below 1%, indicating their potential application in non-alcoholic beverages. Future studies are needed to investigate the relationship between the key volatile compound production and associated genetic characteristics, along with sensory evaluations, to develop optimized flavor modulation strategies.

1. Introduction

Fermentation is a biological process in which microorganisms metabolize organic substrates to generate diverse metabolic compounds. Traditionally, fermentation has been used to enhance preservation, improve sensory attributes, and increase the nutritional value of food [1,2]. Various microorganisms, including lactic acid bacteria, yeasts, and fungi, contribute to these processes and facilitate the production of bioactive compounds. This has led to the development of globally consumed fermented foods, including yogurt, cheese, kimchi, tempeh, and kombucha [2,3]. Fermented foods are widely studied for their unique sensory properties and potential health benefits, including gut health improvement and immune modulation, which have driven extensive research in this field [3,4]. Recent consumer trends favoring functional and minimally processed foods have accelerated the growth of the fermented food market [4]. Consequently, there is a growing demand for innovative fermented beverages that combine convenience with an enhanced sensory appeal and expand beyond traditional fermented food products [2,5].
Fermented beverages primarily utilize lactic acid bacteria (LAB) and yeast, each of which has distinct metabolic functions. LAB produce organic acids, including lactic acid, which lower the pH, enhance the microbiological stability, and improve sweetness and textures [6]. In contrast, yeasts metabolize sugars to generate volatile flavor compounds and carbonates, which play a crucial role in shaping the unique sensory attributes of fermented beverages. Notably, yeasts produce various terpenes, esters, and phenolic compounds that contribute to fruity and floral aromas; however, ethanol is inevitably generated as a byproduct of this process [7,8]. While low levels of ethanol (0.5–3%) can enhance flavor complexity [7,8,9], the growing consumer interest in health-conscious choices and the “Healthy-Pleasure” trend has driven the demand for non-alcoholic or low-alcohol fermented beverages [6,10]. Consequently, the development of yeast strains and fermentation processes that attenuate ethanol production while preserving the desirable flavor attributes is a key research focus.
Expanding fermentation substrates is another critical aspect in the development of fermented beverages. Consumers are increasingly prioritizing flavor enhancement with the incorporation of bioactive compounds with functional health benefits. In particular, root-based plant materials, which are recognized for their antioxidant, anti-inflammatory, and gut-health-promoting properties, have attracted considerable interest for their application in functional fermented beverages [11,12]. Traditionally, root extract-based fermented beverages have been primarily developed using LAB. For example, Kanji from northern India and Shalgam from Turkey are well-known beverages produced by fermenting root vegetables, including black carrots or beetroots, with LAB, particularly Pediococcus acidilactici. Additionally, the fermentation of root vegetable extracts, including red beet and carrot, using Lactiplantibacillus plantarum BR9, P35, and Lactobacillus acidophilus IBB801 has been reported to enhance antioxidant activity and exert inhibitory effects against pathogenic bacteria [13]. Furthermore, in Kombucha produced using Tartary buckwheat and burdock, fermentation is facilitated by a symbiotic culture of bacteria and yeast (SCOBY, primarily composed of Acetobacter, Gluconobacter, and Saccharomyces species), leading to the production of organic acids that enhance antioxidant activity and confer health benefits [14]. Moreover, ginger-based products, including ginger beer [15], and various beverages fermented with Saccharomyces cerevisiae and other yeast strains, have been extensively commercialized, demonstrating the growing interest in the development of root extract-based fermented beverages. However, these raw materials contain saponins, tannins, and volatile compounds that can impart bitterness and strong odors, potentially limiting consumer acceptance [16]. Fermentation has been explored as a strategy to address these challenges by facilitating the conversion or degradation of undesirable compounds, accompanied by the formation of new flavor precursors. Thus, screening for appropriate microbial strains and optimizing fermentation conditions are key factors in product development [1,3]. In previous research, as part of unpublished studies conducted by the authors, Kluyveromyces marxianus 8279 was isolated from yeotgireum (a traditional Korean malt), while Saccharomyces cerevisiae 8293 was isolated from nuruk (a traditional Korean koji). Especially, yeotgireum is a traditional Korean fermentation ingredient with a long history, produced by germinating and drying grains, followed by a slow maturation process. This natural process promotes the formation of diverse microorganisms and beneficial enzymes, which play a crucial role in enhancing saccharification during fermentation and imparting unique flavors and sensory characteristics to fermented products. These strains were subsequently registered in the LRCC (LOTTE R&D Culture Collections) strain banking database maintained by the LOTTE R&D Center. Based on our research, KM8279 produces a distinctive baking aroma in bakery and dessert fermentations. During screening, it exhibited superior aromatic properties compared to a commercial bakery yeast (Product ‘B’, L-Company, Marcq-en-Barœul, France). Accordingly, it has been applied to the large-scale production of baked foods. Similarly, SC8293 produces a unique whiskey-like aroma during distilled alcoholic beverage fermentation. In screening tests, it demonstrated a more pronounced whiskey-like aroma than a commercial whiskey yeast (Product ‘M’, P-Company, St. Louis, MO, USA). This strain has been filed for patent protection (Korean Patent Application No. 10-2023-0155180) due to its exceptional aroma production and robust fermentation capacity, making it ideal for premium alcoholic beverage applications. Although these strains have not been previously applied in root extract fermentation, previous research suggests that they are capable of producing desirable aroma profiles under appropriate conditions.
The development of appropriate strains cannot be reliably predicted using experimental tests alone; therefore, strain selection and process optimization require significant time and resources. For enhanced efficiency, various high-speed screening methods have been explored to assess the metabolic properties of fermentation strains [17]. In particular, whole-genome sequencing (WGS) has been used to evaluate genetic stability and identify key metabolic pathways involved in carbohydrate utilization and volatile compound production [18,19]. These genome-based analyses allow more accurate predictions of the fermentation performance under specific conditions, making them valuable tools for strain selection and process optimization.
This study focused on the genomic sequencing and fermentation characteristic analysis of two yeast strains, KM8279 and SC8293, with potential applications in root extract-based low-alcohol beverage production. These strains were pre-selected based on the authors’ previous research, where they were isolated, characterized, and shown to exhibit superior aromatic properties in non-beverage fermentation applications, such as bakery and distilled alcoholic products. Based on their established aroma profiles and industrial applicability, these strains were subjected to genomic sequencing to identify key metabolic pathways and were further evaluated for their fermentation characteristics under root extract-based conditions. This approach emphasizes the importance of leveraging well-characterized strains to optimize the flavor and quality in fermented beverages.

2. Materials and Methods

2.1. Bacterial Strains and Preparation

Kluyveromyces marxianus LRCC 8279 (KM8279) and Saccharomyces cerevisiae LRCC 8293 (SC8293), isolated by the Lotte R&D Center (Gangseo-gu, Seoul, Republic of Korea), were used in this study. These strains were selected at the Lotte R&D Center for their high ethanol production capabilities in distilled liquor production and are currently under a patent application. The strains were stored in glycerol stocks at −80 °C. Before use, they were streaked onto YPD agar plates (1% yeast extract, 2% peptone, and 2% glucose) and incubated at 30 °C for 24 h. A single colony from each plate was inoculated into 50 mL of YPD broth and cultured at 30 °C for 24 h. This culture was used as the inoculum for subsequent fermentation experiments.

2.2. DNA Extraction, WGS Analysis, and Gene Annotation

Genomic DNA was extracted from KM8279 and SC8293 cells using a Genomic DNA Extraction Kit (Intron Biotechnology, Seongnam, Republic of Korea), following the manufacturer’s protocol. The quality and purity of the extracted DNA were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), ensuring an absorbance ratio (A260/A280) of 1.8–2.0, indicating that high-quality DNA was suitable for WGS analysis [20]. Genomic libraries of KM8279 and SC8293 were prepared using a 20 kb SMRTbell Libraries Prep Kit (PacBio, Menlo Park, CA, USA) following the manufacturer’s instructions. The prepared libraries were sequenced using the PacBio RSII platform (PacBio, Menlo Park, CA, USA), which provides long-read sequences, enabling high-quality de novo genome assembly. Raw sequencing data were processed using PacBio SMRT Analysis software (version 2.3.0), and genome assembly was conducted using the Hierarchical Genome Assembly Process (HGAP, version 3.0). The assembled genomes of KM8279 and SC8293 were annotated using multiple bioinformatics tools for comprehensive functional characterization. Genome annotation was initially conducted using Rapid Annotations with Subsystems Technology (RAST) [21], summarizing subsystems related to metabolism, stress response, and cellular functions. The annotations were further validated and refined using the National Center for Biotechnology Information Prokaryotic Genomes Annotation Pipeline (version 4.1) and the PathoSystems Resource Integration Center (PATRIC, version 3.6.12) to ensure consistency across databases. To enhance functional resolution and identify orthologous genes, eggNOG-mapper was used [22].

2.3. Effects of Different Carbon and Nitrogen Sources on Alcohol Production

The fermentation characteristics of KM8279 and SC8293 were assessed using various carbon and nitrogen sources. Both strains were pre-cultured in YPD medium at 30 °C for 24 h, harvested by centrifugation (8000× g, 10 min), and washed with sterile saline solution (0.85% NaCl). The prepared inocula were diluted with a sterile saline solution to a final concentration of 106 colony-forming units (CFU)/mL for subsequent experiments. The tests were conducted in a basal medium composed of 20 g/L bactopeptone (Difco 211677, Becton, Dickinson and Company Co., Ltd., Franklin Lakes, NJ, USA) and 10 g/L yeast extract (Difco 212750, Becton, Dickinson and Company Co., Ltd., Franklin Lakes, NJ, USA), supplemented with glucose, fructose, sucrose, or maltose at a final concentration of 2%, with additional conditions using glucose and maltose at concentrations ranging from 0.5% to 2.0%. Additional experiments were conducted with glucose and maltose in a 1:1 ratio at final concentrations of 1.0, 1.6, 2.0, 3.0, and 4.0%. For nitrogen source comparisons, 2% (w/v) glucose was used as the sole carbon source, and the nitrogen sources used were yeast extract, soy peptone, peptides, and casein, each supplemented at 20 g/L. All fermentation experiments were conducted in triplicate (n = 3) using 250 mL bottles (DURAN® Original GL45, DWK Life Sciences GmbH, Wertheim, Germany) containing 200 mL of the medium, maintained at 30 °C for 72 h. Samples were collected 0, 24, 48, and 72 h after fermentation.

2.4. Effects of Different Temperatures and Cultivation Times on Alcohol Production

The impact of temperature on fermentation performance was examined at 10 °C, 15 °C, 20 °C, 25 °C, and 30 °C. Both strains were pre-cultured in YPD medium at 30 °C for 24 h, harvested by centrifugation (8000× g, 10 min), and washed with sterile saline solution (0.85% NaCl). All fermentations were conducted in triplicate (n = 3) using 250 mL bottles containing 200 mL of YPD medium for 72 h. The optimal cultivation time was further evaluated using glucose–maltose mixtures (1.0% and 1.5%, w/v). Samples were collected at 0, 24, 48, 72, 96, and 120 h for subsequent analysis.

2.5. Fermentation of Root Extracts

The root extracts of Pueraria lobata (PL), Arctium lappa (AL), Zingiber officinale (ZO), and Platycodon grandiflorus (PG) were obtained from a commercial food ingredient supplier (MSC Co., Ltd., Yangsan, Republic of Korea). Detailed nutritional compositions of the four root extracts are provided in Supplementary Table S1, with initial Brix values of 60.7 ± 0.3 for PL, 50.2 ± 0.4 for AL, 24.6 ± 1.1 for ZO, and 41.3 ± 0.8 for PG. Corresponding total sugar contents were 33.1 ± 1.8 g/100 mL, 29.7 ± 2.3 g/100 mL, 14.3 ± 1.6 g/100 mL, and 24.3 ± 2.3 g/100 mL, respectively. The raw materials were stored at 4 °C immediately after procurement and equilibrated at 25 °C for 1 h before processing. For fermentation, the soluble solid content of each root extract was measured using a digital refractometer (PAL-1; Atago Co., Ltd., Tokyo, Japan) and adjusted to 5 °Bx by diluting with sterile distilled water. The adjusted extracts were heat-treated at 90 °C for 30 min. Pre-cultured cells of each strain were inoculated into 150 mL of the prepared extracts. Based on preliminary tests (Supplementary Figure S1) indicating that fermentation at 15 °C maintained alcohol levels below 1% while ensuring active fermentation, all root extract fermentations were conducted in triplicate (n = 3) at 15 °C for 72 h under static conditions.

2.6. Determination of Alcohol Production and Culture Growth

Following fermentation, the culture broth was centrifuged at 8000× g for 10 min, and the supernatant was collected after filtration through a filter paper (Whatman No. 2, Cytiva, Marlborough, MA, USA). Subsequently, 100 mL of the supernatant was transferred to a volumetric flask and distilled using the VAPODEST system (Gerhardt, Königswinter, Germany) [23]. Alcohol content was determined using a density meter (DMA 5000M, Anton Paar, Graz, Austria) [24]. The cell growth was assessed using serial dilutions and agar plates. Fermented samples were serially diluted (10−1–10−7) with sterile saline solution and spread on YPD agar plates (1% yeast extract, 2% peptone, 2% glucose, and 1.5% agar). The plates were incubated at 30 °C for 48 h, a condition selected to ensure consistent and reliable evaluation of the two yeast strains, Kluyveromyces marxianus and Saccharomyces cerevisiae [25,26].

2.7. Analysis of Volatile Compounds in Fermented Beverages

Volatile compounds were analyzed using headspace solid-phase microextraction (HS-SPME), followed by gas chromatography with flame ionization detection (GC-FID; 7890A, Agilent Technologies Inc., Santa Clara, CA, USA), as described by Lee et al. [27]. For volatile compound extraction, 5 mL of the supernatant was transferred into a 20 mL headspace vial (PTFE/silicon septum, magnetic cap) containing 25% (w/v) NaCl to enhance ionic strength and improve extraction efficiency. The vial was hermetically sealed and subjected to magnetic stirring at 35 °C for 20 min to facilitate equilibrium between the liquid and headspace phases. Following equilibration, a 50/30 μm DVB/CAR/PDMS fiber (Supelco, Bellefonte, PA, USA) was exposed to the headspace for 40 min for analyte adsorption [28]. Separation of volatile compounds was performed using a DB-WAX capillary column (60 m × 0.25 mm × 0.25 μm, Agilent Technologies Inc., Santa Clara, CA, USA). The oven temperature was initially held at 40 °C for 2 min, increased at a rate of 2 °C/min to 220 °C, followed by a ramp of 20 °C/min to 240 °C, and maintained at 240 °C for 5 min. The injector temperature was set at 240 °C. Helium was used as the carrier gas at a constant flow rate. Compound identification was achieved by matching mass spectra with entries in the Wiley9Nist0.8 spectral library (version 5.0), according to the method described by Choi et al. [28].

2.8. Statically Analysis

All experiments were conducted in triplicate, and statistical analyses were performed using GraphPad Prism (version 9, GraphPad Software, Boston, MA, USA). Data were analyzed using a one-way analysis of variance, followed by Tukey’s honest significant difference test for multiple comparisons. Statistical significance was set at p < 0.05, and a post hoc analysis was conducted to identify significant differences between the treatment groups.

3. Results

3.1. Genomic Characterization

The WGS analysis provided insights into the genomic features of KM8279 and SC8293, and the key results are summarized in Table 1. The genome size of KM8279 was 11,008,819 bp, with 40.1% guanine and cytosine bases (GC content), whereas SC8293 exhibited a larger genome comprising 14,689,445 bp and a 38.2% GC content. SC8293 encoded a higher number of genes, including coding sequences and rRNA gene copies, while both strains exhibited similar tRNA gene counts.

3.2. Gene Annotation

The annotation results for the genes associated with carbohydrate metabolism and ethanol production are presented in Table 2. Both strains encoded key glycolytic enzymes, including hxk, pfk1, pfk2, and pyk, which mediate the conversion of glucose into pyruvate. Genes involved in sucrose metabolism, including suc, msc, glk, and hxk2, were identified in both strains. In addition, genes related to fructose metabolism, including frk1, fba1, and fbp1, were identified in both genomes. SC8293 harbored maltose-utilizing genes, including mal13, mal31 and mal61, mal63, and ypr196w; however, these genes were not detected in KM8279. However, the maa gene, encoding maltose O-acetyltransferase, was identified in both strains. Both genomes contain pdc1 and multiple alcohol dehydrogenase genes, including adh1, adh2, and adh6, which together constitute the core pathway for ethanol biosynthesis. Additional genes, such as gpd, nde, and ndi, were identified in both strains.

3.3. Effects of Carbon and Nitrogen Sources on Alcohol Production

The effects of various carbon and nitrogen sources on alcohol production are shown in Figure 1. Both KM8279 and SC8293 exhibited similar alcohol production levels with glucose, fructose, and sucrose as carbon sources, without significant differences between the two strains (Figure 1A). With 2% maltose as the carbon source, KM8279 produced 0.29 ± 0.06% alcohol, which was significantly lower than the 1.53 ± 0.06% produced by SC8293 (p = 0.0001). The effects of increasing glucose concentrations (0.5–2.0%) on the alcohol production are shown in Figure 1B. Both KM8279 and SC8293 demonstrated a concentration-dependent increase in alcohol production. Notably, SC8293 consistently produced slightly higher alcohol levels than KM8279, with significant differences observed at 1.5% glucose (p = 0.0506) and 2.0% glucose (p = 0.0028). SC8293 exhibited significantly higher ethanol production than KM8279 at all tested maltose concentrations except for 0.5%, with the most notable differences at 1.0% (SC8293: 0.91 ± 0.07% vs. KM8279: 0.32 ± 0.08%, p < 0.05) and 2.0% (SC8293: 1.65 ± 0.07% vs. KM8279: 0.29 ± 0.15%, p < 0.001).
In glucose–maltose mixed fermentations, SC8293 consistently produced higher alcohol concentrations than KM8279, with significant differences observed at sugar concentrations above 2.0%. Specifically, at 3.0% sugar SC8293 produced 2.73 ± 0.32% alcohol, compared to 1.74 ± 0.31% for KM8279 (p < 0.05). To evaluate the effects of nitrogen sources, yeast extract, soy peptone, peptide, and casein were added at a concentration of 20 g/L. The alcohol production exceeded 1.5% under all conditions, with no statistically significant differences observed between the two strains (Figure 1D).

3.4. Effects of Cultivation Temperature and Time on Alcohol Production

The alcohol production by KM8279 and SC8293 at various fermentation temperatures is shown in Figure 1E. Both strains exhibited an increased alcohol production as the fermentation temperature increased. SC8293 consistently produced higher alcohol levels than KM8279, with significant differences observed at temperatures of 15 °C or above. Specifically, at 15 °C SC8293 produced 1.47 ± 0.14% alcohol, which is significantly higher than KM8279 (0.71 ± 0.05%, p = 0.0007). Significant differences were also observed at 20 °C (p = 0.0001) and 30 °C (p = 0.0001), while no significant differences were detected at 10 °C and 25 °C. The effects of the optimal cultivation time using glucose–maltose mixtures (1.0% and 1.5%, w/v) are shown in Figure 1F. At 1.0% and 1.5% glucose with maltose, alcohol concentrations in KM8279 were maintained below 1% up to 48 h, and subsequently increased to 1.07–1.13 ± 0.08% at 72 h. In SC8293, a concentration of 2.12 ± 0.15% was measured at 48 h under the 1.5% condition, with no statistically significant change observed at later time points. At 1.5% glucose with maltose, SC8293 maintained significantly higher alcohol concentrations than KM8279 from 48 h to 96 h, demonstrating a consistent difference in fermentation performance.

3.5. Fermentation Characteristics of Beverages Using Root Extracts

The alcohol production and cell viability of KM8279 and SC8293 were evaluated using PL, AL, ZO, and PG extracts as substrates after fermentation (15 °C, 72 h) (Figure 2). The ethanol levels were significantly higher in SC8293 than in KM8279 during PL and AL fermentation. Notably, SC8293 produced 1.10 ± 0.19% alcohol in AL fermentation, which was 2.75-fold higher than that of KM8279 (0.40 ± 0.14%). In contrast, both strains produced low alcohol levels in the ZO and PG fermentation, with no significant difference observed in PG. The cell viability, expressed as Log CFU, was 6.5–6.9 for both strains for PL, AL, and PG fermentations. However, SC8293 cells exhibited a greater viability than KM8279 cells during the ZO fermentation.

3.6. Volatile Compound Profiles of Fermented Beverages

The volatile compound profiles of the PL, AL, ZO, and PG extracts are presented in Figure 3, demonstrating varied patterns that were dependent on both the fermentation substrate and microbial strain. Specific volatile compounds associated with ethereal, floral, fruity, fermented, and sour sensory attributes, which are characteristic of fermented beverages, were selected and summarized in Table 3. Ethyl acetate levels were significantly higher in the KM8279 fermentation using AL, ZO, and PG, whereas SC8293 exhibited increased levels only in PL. Volatile compounds contributing to floral attributes, including ethylhexanol, methyl salicylate, and phenethyl acetate, were predominantly detected in AL and ZO, with the highest concentrations observed during the KM8279 fermentation. SC8293 cells exhibited the highest levels of floral volatiles in AL. Fruity-associated volatiles, including butyl butanoate and isoamyl acetate, were the most abundant in the KM8279-ZO fermentation, followed by SC8293 fermentations using AL, ZO, and PG. Isoamyl and isobutyl alcohols were generally higher during the SC8293 fermentation than during the KM8279 fermentation. In contrast, both strains showed similar levels of acetic acid and isobutyric acid with no significant differences.

4. Discussion

Fermentation, which is traditionally employed for food preservation and flavor enhancement, has recently emerged as a global trend driven by changing consumer preferences. With the increasing demand for health- and wellness-oriented products, fermented beverages have been diversified, incorporating functional ingredients along with a sensory appeal. This study aimed to determine the optimal fermentation conditions for the development of a root extract-based beverage with a low alcohol content, while preserving desirable flavor characteristics and potential health benefits.
WGS-based genome characterization is a powerful approach for analyzing microbial genomes, allowing the identification and functional annotation of genes involved in key metabolic pathways [18,19]. It has been widely applied to predict the metabolic characteristics of fermentation strains and to facilitate the development of novel strains. In the present study, the WGS analysis of KM8279 and SC8293 was conducted to annotate the genes involved in carbohydrate metabolism and alcohol production. Both strains harbored essential genes involved in glucose, fructose, and sucrose metabolism. They also contain ethanol-associated genes, including pdc1, adh1, adh2, and adh6, indicating their functional capacity for glycolysis and alcohol fermentation. These findings are consistent with those of previous reports on Kluyveromyces marxianus and Saccharomyces cerevisiae, which have been shown to efficiently metabolize various carbon sources [29]. Among these, pdc1 encodes an enzyme that catalyzes the conversion of pyruvate to acetaldehyde, whereas the adh gene family is responsible for the reduction of acetaldehyde to alcohol, directly influencing the efficiency of alcohol fermentation [30]. However, notable differences in maltose metabolism were observed. SC8293 harbored mal13, mal31, mal61, mal63, and ypr196w, which are essential for maltose utilization; however, these genes were not detected in KM8279. An efficient maltose metabolism plays a key role in fermentation, particularly in brewing and other traditional alcoholic fermentation processes. The mal31 and mal61 genes encode maltose permeases responsible for transporting maltose into the cell, whereas mal13 and mal63 function as transcriptional regulators of maltose metabolism [31]. The presence of these genes indicated that SC8293 possesses an enhanced genetic capacity for maltose utilization. In contrast, the absence of these genes in KM8279 suggests a metabolic preference for monosaccharides, such as glucose and fructose [25]. These genetic differences are consistent with the observed differences in the fermentation performance across carbohydrate sources. Previous studies have demonstrated that S. cerevisiae efficiently metabolizes maltose and other disaccharides, whereas K. marxianus preferentially utilizes glucose and fructose as the major carbon sources [25,30]. Although these observations represent species-level metabolic characteristics, the fermentation performance can vary depending on strain-specific genetic compositions and regulatory mechanisms. Even within the same species, including K. marxianus and S. cerevisiae, variations in carbon source utilization, alcohol production, and fermentation kinetics have been reported and are largely determined by strain-specific genomic features and regulatory mechanisms. The findings of this study suggest that KM8279 exhibits a lower growth and alcohol production level than SC8293 when fermenting substrates with a high maltose content. In contrast, SC8293 cells demonstrated a consistent alcohol production across different sugar substrates. A precise understanding of strain-level fermentation traits is essential for optimizing beverage fermentation; however, this requires considerable time and effort. To minimize experimental efforts, WGS has emerged as a valuable approach for predicting the fermentation potential through the prior identification of key metabolic genes. The WGS-based genomic analysis enables the preliminary evaluation of the strain-specific fermentation potential before large-scale experimentation. The gene annotation conducted in this study highlights the applicability of genome-based predictions to guide strain selection [19]. The systematic identification of metabolic genes and their association with fermentation phenotypes may provide a reliable basis for the development of high-performance fermentation strains for future biotechnological research. In contrast, the WGS analysis of KM8279 and SC8293 did not reveal significant metabolic differences compared to commonly reported strains of Kluyveromyces marxianus and Saccharomyces cerevisiae. However, the WGS data generated in this study can serve as a foundational reference for optimizing strain selection and fermentation processes in various product applications. By establishing a WGS database of diverse yeast strains, it is possible to strategically align strain-specific metabolic profiles with available carbon sources in fermentation substrates. This approach can streamline the strain selection for specific product types, such as low-alcohol, high-alcohol, or minimally processed fermented beverages. Moreover, the WGS data can facilitate the precise formulation of fermentation media by identifying essential nutrients in substrates, allowing for an optimized nutrient supplementation or targeted carbon source utilization. However, this study has certain limitations. Specifically, the annotation focused primarily on carbon metabolism genes, while nitrogen metabolism genes and volatile organic compound synthesis pathways were not fully explored. Although nitrogen metabolism can also influence alcohol production, the focus of this study was primarily on carbon sources, which directly impact alcohol fermentation. Therefore, future studies should broaden the gene annotation to include a wider range of volatile compounds’ biosynthesis pathways, providing a more thorough understanding of strain-specific aroma characteristics. These comprehensive approaches could allow for a detailed investigation of the genetic factors that influence the flavor development in fermented beverages.
Temperature and cultivation time are critical factors that influence yeast metabolism. The higher ethanol production of SC8293 at 15–20 °C compared with that of KM8279 aligns with reports of previous studies, indicating that S. cerevisiae exhibits a robust fermentation activity, even at lower temperatures [32]. In contrast, KM8279 maintained consistent levels of alcohol production across the tested temperatures, which is consistent with the mesophilic fermentation profile of K. marxianus [33]. With respect to the alcohol production over the cultivation period, both the strains exhibited a decreased fermentation rate after 48 h, whereas SC8293 maintained a higher final alcohol concentration. This variation can be explained by the presence of maltose-metabolizing genes in SC8293, which enables efficient sugar utilization, whereas their absence in KM8279 limits its ability to ferment maltose. Although both strains exhibited a decreased alcohol production after a specific cultivation period, the difference in the final ethanol concentration indicated that the ability to utilize maltose was a critical determinant of fermentation efficiency.
In this study, KM8279 and SC8293 were used to ferment four different root extracts, and their suitability for non-alcoholic beverage (NAB) applications was assessed based on alcohol production and volatile compound profiles. NABs are generally defined as beverages containing less than 1% alcohol [10], underscoring the importance of understanding the fermentation behavior and flavor development in relation to both the strain selection and substrate composition. The fermentation of the 5 °Bx ZO extract resulted in a low alcohol production by both strains, with KM8279 and SC8293 yielding 0.1% and 0.4%, respectively. In contrast, the AL fermentation produced higher levels, with KM8279 generating 0.5 ± 0.1% and SC8293 producing 0.9 ± 0.2%, both exceeding the values observed in the ZO fermentation. Compared with conventional alcoholic fermentation, the alcohol production levels in this study were markedly lower. For instance, Lee et al. reported an 8% alcohol production from 32 °Bx ginger extract fermented with S. cerevisiae, underscoring the limited fermentability of the ZO extract [34]. ZO contains sucrose at 50–70 mg/g, glucose at 10–25 mg/g, and fructose at 8–20 mg/g [35]. In contrast, AL contains fructan at 9.0 ± 2.2 g per 100 g and inulin at 7.3 ± 0.1 g per 100 g, while the levels of simple sugars are considerably lower, with sucrose at 0.5 ± 0.1 g, glucose at 0.7 ± 0.1 g, and fructose at 0.8 ± 0.2 g per 100 g [36]. While KM8279 and SC8293 possess the metabolic capacity to utilize the primary carbohydrates present in ZO and AL, the low alcohol levels suggest that factors beyond carbohydrate composition may have contributed to the decreased fermentation efficiency. Ginger contains antimicrobial compounds, including gingerols and shogaols [37], which may inhibit yeast growth and metabolic activity.
Although there were no significant differences in alcohol production, significant differences in volatile compound profiles were observed. In particular, the AL and ZO fermentation with KM8279 have significantly increased levels of phenethyl alcohol, phenethyl butyrate, and methyl salicylate, along with elevated levels of butyl butyrate, butyl butanoate, and isoamyl acetate. A similar trend was observed in the AL fermentation by SC8293, which exhibited a more balanced composition of volatile compounds than KM8279. According to Rojas et al. [38], 2-phenylethyl acetate, hexyl ethanoate, and phenethyl alcohol are key contributors to the floral and fruity features of fermented beverages, thereby enhancing the aromatic profile. Braga et al. [39] reported that 2-phenylethyl acetate and ethyl hexanoate play crucial roles in the aroma profile of apple-based fermented beverages. Additionally, Swiegers et al. [7] identified ethyl acetate, ethyl hexanoate, 3-methylbutyl ethanoate, and isoamyl alcohol as the major contributors to the fruity aroma. Notably, butyl butyrate is recognized as an ester used to enhance the aroma of alcoholic beverages, exhibiting a fruity odor similar to that of ethyl butyrate, which is known for its sweet pineapple-like aroma and is present in various fruits, including apples, apricots, bananas, plums, and citrus [40]. Volatile compounds associated with floral and fruity characteristics positively influence consumer preferences [41]. Holt et al. highlighted the growing demand for floral and fruity aromas in the beverage industry, driving extensive research efforts to enhance these attributes [42]. The results of this study also showed that the fermentation using KM8279 and SC8293 resulted in an increased production of floral and fruity volatile compounds, with strain–substrate combinations, such as KM8279-ZO, KM8279-AL, and SC8293-AL, exhibiting the most significant effects. In particular, the KM8279-ZO and KM8279-AL combinations maintained ethanol levels below 1%, demonstrating their potential for consumer-preferred root extract-based fermented NAB applications. Although the PL and PG fermentation maintained ethanol levels below 1%, their volatile compound profiles did not exhibit differentiated sensory attributes.
In summary, this study characterized the genomic features of KM8279 and SC8293 and identified key genetic differences related to the carbohydrate metabolism and alcohol production. The fermentation characteristics, including nutritional sources, concentrations, cultivation times, and temperatures, were evaluated for both strains. Fermentation using four different root extracts demonstrated that KM8279 and SC8293 contributed to the production of appealing volatile compounds while maintaining low alcohol concentrations. These findings suggest that specific strain–substrate combinations can be used to develop low-alcohol fermented beverages with enhanced sensory properties and functional ingredients. Despite these promising results, this study had two key limitations. First, while metabolic genes related to carbohydrate metabolism and alcohol production were analyzed, a further investigation of the genes involved in volatile compound biosynthesis could improve the predictive accuracy of postfermentation aroma profiles. Future research should incorporate WGS-based analyses to identify genes associated with the biosynthesis of both volatile and taste compounds, in addition to metabolic pathways. Second, the experiments were conducted only at a scale below the pilot level. Large-scale fermentation experiments are required to evaluate whether alcohol production and volatile compound profiles can be consistently achieved on a commercial scale. These advancements will support the development of high-quality fermented beverages by integrating strain selection, fermentation conditions, and consumer preferences while also providing valuable tools for the efficient development of fermented products.

5. Conclusions

In this study, we investigated the genomic and fermentation characteristics of KM8279 and SC8293 to evaluate their potential for the development of low-alcohol fermented beverages using root extracts. The WGS analysis identified essential genes associated with carbohydrate metabolism and alcohol production, which were validated through fermentation experiments. Fermentation characteristics, including the nutritional source, concentration, cultivation time, and temperature, were also evaluated. ZO and Al root extract fermentations demonstrated that KM8279 enhanced the production of volatile compounds associated with floral and fruity sensory attributes while maintaining alcohol levels below 1%. In contrast, strain SC8293 produced a more balanced aroma profile during the AL fermentation. These findings provide a strain–substrate pairing strategy for developing NABs. This study highlights the potential of integrating strain selection, fermentation conditions, and consumer preferences for developing premium-quality fermented beverages. Future studies should extend the genomic analysis to include genes involved in the biosynthesis of volatile and taste compounds and assess the fermentation performance at both laboratory and industrial scales.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/fermentation11060299/s1, Figure S1: Effects of fermentation temperature (10 °C–30 °C) on alcohol production in roots-extract fermentation using KM8279 and SC8293; Table S1: Nutritional composition of four root-extracts used in fermentation tests.

Author Contributions

Conceptualization: E.-J.L. and M.-J.S.; Methodology: S.-H.C. and M.-J.S.; Validation: C.-S.J.; Investigation: E.-J.L., C.-S.J., J.-K.K. and J.-H.L.; Resources: W.-K.K. and W.-J.Y.; Writing—original draft preparation, E.-J.L. and M.-J.S.; Writing—review and editing: W.-K.K. and S.-M.Y.; Visualization, S.-H.C. and A.-R.L.; Supervision: J.-K.K. and J.-H.L.; Project administration: W.-J.Y. and S.-M.Y. 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 data presented in this study are available on request from the corresponding author.

Acknowledgments

The authors gratefully acknowledge the support provided by LOTTE Chilsung Beverage Co., Ltd., which enabled the use of facilities and equipment at the LOTTE R&D Center as part of its institutional infrastructure. We also thank the Savory & Sensory Team at the LOTTE R&D Center for their technical expertise and analytical support in conducting the comprehensive aroma analysis.

Conflicts of Interest

The authors declare no conflicts of interest. The research was conducted with general institutional support from LOTTE Chilsung Beverage Co., Ltd.; however, the supporting organization had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Abbreviations

The following abbreviations are used in this manuscript:
KMKluyveromyces marxianus
SCSaccharomyces cerevisiae
LABlactic acid bacteria
WGSwhole-genome sequencing
PLPueraria lobata
ALArctium lappa
ZOZingiber officinale
PGPlatycodon grandiflorus
CFUcolony-forming unit
GCguanine–cytosine
NABnon-alcoholic beverage

References

  1. Marco, M.L.; Heeney, D.; Binda, S.; Cifelli, C.J.; Cotter, P.D.; Foligné, B.; Hutkins, R. Health benefits of fermented foods: Microbiota and beyond. Curr. Opin. Biotechnol. 2017, 44, 94–102. [Google Scholar] [CrossRef] [PubMed]
  2. Tamang, J.P.; Cotter, P.D.; Endo, A.; Han, N.S.; Kort, R.; Liu, S.Q.; Mayo, B.; Westerik, N.; Hutkins, R. Fermented foods in a global age: East meets West. Compr. Rev. Food Sci. Food Saf. 2020, 19, 184–217. [Google Scholar] [CrossRef] [PubMed]
  3. Rezac, S.; Kok, C.R.; Heermann, M.; Hutkins, R. Fermented foods as a dietary source of live organisms. Front. Microbiol. 2018, 9, 1785. [Google Scholar] [CrossRef]
  4. Dimidi, E.; Cox, S.R.; Rossi, M.; Whelan, K. Fermented foods: Definitions and characteristics, impact on the gut microbiota and effects on gastrointestinal health and disease. Nutrients 2019, 11, 1806. [Google Scholar] [CrossRef]
  5. Aschemann-Witzel, J.; Gantriis, R.F.; Fraga, P.; Perez-Cueto, F.J.A. Plant-based food and protein trend from a business perspective: Markets, consumers, and the challenges and opportunities in the future. Crit. Rev. Food Sci. Nutr. 2021, 61, 3119–3128. [Google Scholar] [CrossRef]
  6. Arena, M.P.; Capozzi, V.; Spano, G.; Fiocco, D. The potential of lactic acid bacteria to colonize biotic and abiotic surfaces and the investigation of their interactions and mechanisms. Appl. Microbiol. Biotechnol. 2017, 101, 2641–2657. [Google Scholar] [CrossRef] [PubMed]
  7. Swiegers, J.H.; Bartowsky, E.J.; Henschke, P.A.; Pretorius, I.S. Yeast and bacterial modulation of wine aroma and flavour. Aust. J. Grape Wine Res. 2005, 11, 139–173. [Google Scholar] [CrossRef]
  8. Styger, G.; Prior, B.; Bauer, F.F. Wine flavor and aroma. J. Ind. Microbiol. Biotechnol. 2011, 38, 1145–1159. [Google Scholar] [CrossRef]
  9. Lilly, M.; Lambrechts, M.G.; Pretorius, I.S. Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates. Appl. Environ. Microbiol. 2000, 66, 744–753. [Google Scholar] [CrossRef]
  10. Okaru, A.O.; Lachenmeier, D.W. Defining No and Low (NoLo) alcohol products. Nutrients 2022, 14, 3873. [Google Scholar] [CrossRef]
  11. Martins, N.; Petropoulos, S.; Ferreira, I.C.F.R. Chemical composition and bioactive compounds of commonly consumed roots and tubers. Curr. Opin. Food Sci. 2016, 8, 36–41. [Google Scholar]
  12. Xiang, H.; Sun-Waterhouse, D.; Waterhouse, G.I.N.; Cui, C.; Ruan, Z. Fermentation-enabled wellness foods: A fresh perspective. Food Sci. Hum. Wellness 2019, 8, 203–243. [Google Scholar] [CrossRef]
  13. Zamfir, M.; Angelescu, I.-R.; Voaides, C.; Cornea, C.-P.; Boiu-Sicuia, O.; Grosu-Tudor, S.-S. Non-dairy fermented beverages produced with functional lactic acid bacteria. Microorganisms 2022, 10, 2314. [Google Scholar] [CrossRef]
  14. Lee, Y.J.; Kang, H.J.; Yi, S.H.; Jung, Y.H. Antioxidant properties of kombucha made with tartary buckwheat tea and burdock tea. Prev. Nutr. Food Sci. 2023, 28, 347–352. [Google Scholar] [CrossRef] [PubMed]
  15. Oliveira, L.I.G.; Costa, W.K.A.; Oliveira, F.C.; Bezerril, F.F.; Eireli, L.P.A.M.; Lima, M.S.; Noronha, M.F.; Cabral, L.; Wagner, R.; Pimentel, T.C.; et al. Ginger beer derived from back-slopping: Volatile compounds, microbial communities on activation and fermentation, metabolites, and sensory characteristics. Food Chem. 2024, 435, 137640. [Google Scholar] [CrossRef]
  16. Drewnowski, A.; Gomez-Carneros, C. Bitter taste, phytonutrients, and the consumer: A review. Am. J. Clin. Nutr. 2000, 72, 1424–1435. [Google Scholar] [CrossRef]
  17. Hittinger, C.T.; Steele, J.L.; Ryder, D.S. Diverse yeasts for diverse fermented beverages and foods. Curr. Opin. Biotechnol. 2018, 49, 199–206. [Google Scholar] [CrossRef] [PubMed]
  18. Fan, T.; Qu, J.; Wang, L.; Zhang, J. Genome sequencing, assembly, and characterization of Pichia fermentans Z9Y-3 as a non-Saccharomyces yeast with aroma enhancing potential. Food Biosci. 2023, 53, 102701. [Google Scholar] [CrossRef]
  19. Lu, Z.; Guo, L.; Chen, X.; Lu, Q.; Wu, Y.; Chen, D.; Wu, R.; Chen, Y. Omics sequencing of Saccharomyces cerevisiae strain with improved capacity for ethanol production. Fermentation 2023, 9, 483. [Google Scholar] [CrossRef]
  20. Lucchini, S.; Thompson, A.; Hinton, J.C. Microarrays for microbiologists. Microbiology 2011, 147, 1403–1414. [Google Scholar] [CrossRef]
  21. Aziz, R.K.; Bartels, D.; Best, A.A.; DeJongh, M.; Disz, T.; Edwards, R.A.; Formsma, K.; Gerdes, S.; Glass, E.M.; Kubal, M.; et al. The RAST Server: Rapid annotations using subsystems technology. BMC Genom. 2008, 9, 75. [Google Scholar] [CrossRef] [PubMed]
  22. Huerta-Cepas, J.; Forslund, K.; Coelho, L.P.; Szklarczyk, D.; Jensen, L.J.; von Mering, C.; Bork, P. Fast genome-wide functional annotation through orthology assignment by eggNOG-mapper. Mol. Biol. Evol. 2017, 34, 2115–2122. [Google Scholar] [CrossRef] [PubMed]
  23. Cloninger, L. Alcohol determination of malt-based beverages by rapid distillation. J. Am. Soc. Brew. Chem. 2018, 76, 21–23. [Google Scholar] [CrossRef]
  24. Bruner, J.; Marcus, A.; Fox, G. Dry-hop creep potential of various Saccharomyces yeast species and strains. Fermentation 2021, 7, 66. [Google Scholar] [CrossRef]
  25. Fonseca, G.G.; de Carvalho, N.M.B.; Gombert, A.K. Growth of the yeast Kluyveromyces marxianus CBS 6556 on different sugar combinations as sole carbon and energy source. Appl. Microbiol. Biotechnol. 2013, 97, 5055–5067. [Google Scholar] [CrossRef]
  26. Lo, S.-C.; Yang, C.-Y.; Mathew, D.C.; Huang, C.-C. Growth and autolysis of the kefir yeast Kluyveromyces marxianus in lactate culture. Sci. Rep. 2021, 11, 14552. [Google Scholar] [CrossRef] [PubMed]
  27. Lee, S.B.; Kim, D.H.; Park, H.D. Effects of protectant and rehydration conditions on the survival rate and malolactic fermentation efficiency of freeze-dried Lactobacillus plantarum JH287. Appl. Microbiol. Biotechnol. 2016, 100, 7853–7863. [Google Scholar] [CrossRef]
  28. Choi, K.T.; Park, C.W.; Lee, S.H.; Lee, Y.N.; Oh, J.Y.; Choi, J.S.; Choe, D.; Lee, S.B. Effects of co-fermentation by Saccharomyces cerevisiae and Hanseniaspora uvarum yeasts on the volatile aromatic compound and sensory quality of distilled soju. J. Korean Soc. Food Sci. Nutr. 2024, 53, 639–647. [Google Scholar] [CrossRef]
  29. Heux, S.; Cachon, R.; Dequin, S. Physiology of the yeast Kluyveromyces marxianus during batch and chemostat cultures with glucose as the sole carbon source. FEMS Yeast Res. 2006, 6, 469–476. [Google Scholar]
  30. Wang, D.; Wang, L.; Hou, L.; Deng, X.; Gao, Q.; Gao, N. Metabolic engineering of Saccharomyces cerevisiae for accumulating pyruvic acid. Ann. Microbiol. 2015, 65, 2323–2331. [Google Scholar] [CrossRef]
  31. Wang, X.; Bali, M.; Medintz, I.; Michels, C.A. Intracellular maltose is sufficient to induce MAL gene expression in Saccharomyces cerevisiae. Eukaryot. Cell 2002, 1, 696–703. [Google Scholar] [CrossRef] [PubMed]
  32. Hellborg, L. Yeast diversity in the brewing industry. In Beer in Health and Disease Prevention; Preedy, V.R., Ed.; Academic Press: Cambridge, MA, USA, 2009; pp. 77–88. [Google Scholar]
  33. Sene, L.; Tavares, B.; de Almeida Felipe, M.G.; dos Santos, J.C.; Pereira, F.M.; Tominc, G.C.; da Cunha, M.A.A. Ethanol production by Kluyveromyces marxianus ATCC 36907: Fermentation features and mathematical modeling. Biocatal. Agric. Biotechnol. 2023, 51, 102789. [Google Scholar] [CrossRef]
  34. Lee, J.H.; Son, H.S.; Jeong, J.H.; Noh, J.M.; Kim, J.M.; Choi, H.S.; Lee, Y.H. Quality characteristics of Takju, Yakju, and Spirit made from Phellinus linteus and ginger. Culin. Sci. Hosp. Res. 2015, 21, 103–119. [Google Scholar]
  35. Ghasemzadeh, A.; Jaafar, H.Z.E.; Karimi, E.; Ashkani, S. Changes in nutritional metabolites of young ginger (Zingiber officinale Roscoe) in response to elevated carbon dioxide. Molecules 2014, 19, 16693–16706. [Google Scholar] [CrossRef] [PubMed]
  36. Petkova, N.; Hambarlyiska, I.; Tumbarski, Y.; Vrancheva, R.; Raeva, M.; Ivanov, I. Phytochemical composition and antimicrobial properties of burdock (Arctium lappa L.) roots extracts. Biointerface Res. Appl. Chem. 2022, 12, 2826–2842. [Google Scholar]
  37. Semwal, R.B.; Semwal, D.K.; Combrinck, S.; Viljoen, A. Gingerols and shogaols: Important nutraceutical principles from ginger. Phytochemistry 2015, 117, 554–568. [Google Scholar] [CrossRef]
  38. Rojas, I.B.; Smith, P.A.; Bartowsky, E.J. Influence of choice of yeasts on volatile fermentation-derived compounds, colour and phenolics composition in Cabernet Sauvignon wine. World J. Microbiol. Biotechnol. 2012, 28, 3311–3321. [Google Scholar] [CrossRef]
  39. Braga, C.M.; Zielinski, A.A.F.; da Silva, K.M.; de Souza, F.K.F.; Pietrowski, G.A.M.; Couto, M.; Granato, D.; Wosiacki, G.; Nogueira, A. Classification of juices and fermented beverages made from unripe, ripe and senescent apples based on the aromatic profile using chemometrics. Food Chem. 2013, 141, 967–974. [Google Scholar] [CrossRef]
  40. Steingass, C.B.; Glock, M.P.; Lieb, V.M.; Carle, R. Light-induced alterations of pineapple (Ananas comosus [L.] Merr.) juice volatiles during accelerated ageing and mass spectrometric studies into their precursors. Food Res. Int. 2017, 100, 366–374. [Google Scholar] [CrossRef]
  41. George, J.; Pramanik, I.; Sanewski, G.; Nguyen, T.; Pun, S.; Edwards, D.; Currie, M.; Møller, S.; Hardner, C.; Lyons, P.; et al. Relationship between key aroma compounds and sensory attributes of Australian grown commercial pineapple cultivars. J. Agric. Food Chem. 2025, 73, 5839–5849. [Google Scholar] [CrossRef]
  42. Holt, S.; Miks, M.H.; de Carvalho, B.T.; Foulquié-Moreno, M.R.; Thevelein, J.M. The molecular biology of fruity and floral aromas in beer and other alcoholic beverages. FEMS Microbiol. Rev. 2019, 43, 193–222. [Google Scholar] [CrossRef] [PubMed]
Fermentation 11 00299 g001
Figure 1. Effects of nutritional sources, cultivation time, and temperature on alcohol production by Kluyveromyces marxianus LRCC8279 and Saccharomyces cerevisiae LRCC8293. (A) Alcohol production with varying carbon sources; (B) alcohol production with different concentration of carbon sources; (C) alcohol production with different concentration of mixed carbon sources; (D) alcohol production with varying nitrogen sources; (E) alcohol production across different cultivation times; and (F) alcohol production across different cultivation temperatures. 8279: Kluyveromyces marxianus LRCC8279; 8293: Saccharomyces cerevisiae LRCC8293. Significant differences between groups are indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; # Significantly different from the no-inoculation group or lowest condition in each subfigure (p < 0.05). No significant differences are not explicitly marked.
Figure 1. Effects of nutritional sources, cultivation time, and temperature on alcohol production by Kluyveromyces marxianus LRCC8279 and Saccharomyces cerevisiae LRCC8293. (A) Alcohol production with varying carbon sources; (B) alcohol production with different concentration of carbon sources; (C) alcohol production with different concentration of mixed carbon sources; (D) alcohol production with varying nitrogen sources; (E) alcohol production across different cultivation times; and (F) alcohol production across different cultivation temperatures. 8279: Kluyveromyces marxianus LRCC8279; 8293: Saccharomyces cerevisiae LRCC8293. Significant differences between groups are indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; # Significantly different from the no-inoculation group or lowest condition in each subfigure (p < 0.05). No significant differences are not explicitly marked.
Fermentation 11 00299 g001
Fermentation 11 00299 g002
Figure 2. Fermentation characteristics of root extract using Kluyveromyces marxianus LRCC8279 and Saccharomyces cerevisiae LRCC8293. Alc, alcohol concentration; LogCFU, cell viability expressed as the logarithmic value of colony-forming units; PL: Pueraria lobate; AL: Arctium lappa; ZO: Zingiber officinale; PG: Platycodon grandifloras; 8279: Kluyveromyces marxianus LRCC8279; and 8293: Saccharomyces cerevisiae LRCC8293. Significant differences between groups are indicated as * p < 0.05, and **** p < 0.0001. No significant differences are not explicitly marked.
Figure 2. Fermentation characteristics of root extract using Kluyveromyces marxianus LRCC8279 and Saccharomyces cerevisiae LRCC8293. Alc, alcohol concentration; LogCFU, cell viability expressed as the logarithmic value of colony-forming units; PL: Pueraria lobate; AL: Arctium lappa; ZO: Zingiber officinale; PG: Platycodon grandifloras; 8279: Kluyveromyces marxianus LRCC8279; and 8293: Saccharomyces cerevisiae LRCC8293. Significant differences between groups are indicated as * p < 0.05, and **** p < 0.0001. No significant differences are not explicitly marked.
Fermentation 11 00299 g002
Fermentation 11 00299 g003
Figure 3. Volatile compound profiles of the fermented root extract using Kluyveromyces marxianus LRCC8279 and Saccharomyces cerevisiae LRCC8293. 8279: Kluyveromyces marxianus LRCC8279; 8293: Saccharomyces cerevisiae LRCC8293. All data were expressed as the proportion of the peak area of each compound relative to the total volatile peak area.
Figure 3. Volatile compound profiles of the fermented root extract using Kluyveromyces marxianus LRCC8279 and Saccharomyces cerevisiae LRCC8293. 8279: Kluyveromyces marxianus LRCC8279; 8293: Saccharomyces cerevisiae LRCC8293. All data were expressed as the proportion of the peak area of each compound relative to the total volatile peak area.
Fermentation 11 00299 g003
Table 1. WGS-based genomic characteristics of Kluyveromyces marxianus LRCC8279 and Saccharomyces cerevisiae LRCC8293.
Table 1. WGS-based genomic characteristics of Kluyveromyces marxianus LRCC8279 and Saccharomyces cerevisiae LRCC8293.
ContentsLRCC8279LRCC8293
SpeciesKluyveromyces marxianusSaccharomyces cerevisiae
Genome size (bp)11,008,81914,689,445
GC content (%)40.138.2
Number of genes53177414
Number of CDSs51227059
Number of rRNAs186349
Number of tRNAs97
LRCC: LOTTE R&D Center; genome size (bp): total length of the genome in base pairs. GC content: percentage of guanine (G) and cytosine (C) bases in the genome. CDS, number of coding sequences; rRNAs, ribosomal RNA genes; and tRNAs, transfer RNA genes.
Table 2. Gene annotation of Kluyveromyces marxianus LRCC8279 and Saccharomyces cerevisiae LRCC8293 related to carbohydrate metabolism and alcohol production.
Table 2. Gene annotation of Kluyveromyces marxianus LRCC8279 and Saccharomyces cerevisiae LRCC8293 related to carbohydrate metabolism and alcohol production.
GenesEC No.Product82798293
GlucoseHxk2.7.1.1Hexokinase++
Pgi5.3.1.9glucose-6-phosphate isomerase−+
pkf1,22.7.1.11ATP-dependent 6-phosphofructokinase++
Pyk2.7.1.40pyruvate kinase++
MaltoseMaa2.3.1.79maltose O-acetyltransferase++
mal13−maltose fermentation regulatory−+
mal31−maltose permease−+
mal61−maltose permease−+
mal63−maltose fermentation regulatory−+
ypr196w−maltose fermentation regulatory−+
SucroseSuc3.2.1.26Invertase++
Msc−monosaccharide transporter++
Glk2.7.1.2Glucokinase++
hxk22.7.1.1hexokinase-2++
Fructosefrk12.7.1.4fructokinase 1++
fba14.1.2.13fructose-bisphosphate aldolase++
fbp13.1.3.11fructose-1,6-bisphosphatase++
Alcohol
production		pdc14.1.1.1pyruvate decarboxylase++
adh11.1.1.1alcohol dehydrogenase 1++
adh21.1.1.1alcohol dehydrogenase 2++
adh61.1.1.1aldehyde dehydrogenase 6++
Gpd1.1.1.8glycerol-3-phosphate dehydrogenase++
Nde1.6.5.3NADH dehydrogenase++
Ndi1.6.5.3NADH dehydrogenase++
+: Gene detected in the genome; −: Gene not detected in the genome; EC No.: enzyme commission number assigned to the gene product; Product: functional product or enzyme encoded by the gene; 8279: Kluyveromyces marxianus LRCC8279; and 8293: Saccharomyces cerevisiae LRCC8293.
Table 3. Identified volatile compounds associated with sensory attributes in fermented root extracts using Kluyveromyces marxianus LRCC8279 and Saccharomyces cerevisiae LRCC8293.
Table 3. Identified volatile compounds associated with sensory attributes in fermented root extracts using Kluyveromyces marxianus LRCC8279 and Saccharomyces cerevisiae LRCC8293.
Sensory
Characteristics		Volatile Compounds Identified (GC-MS Peak Area, ×106)
PL-82791PL-82931AL-82791AL-82931ZO-82791ZO-82931PG-82791PG-82931
Ethereal1.89 ± 0.56 c49.66 ± 2.25 b78.37 ± 1.74 a2.54 ± 0.54 c82.27 ± 11.99 a2.55 ± 0.45 c85.48 ± 5.25 a2.23 ± 0.14 c
Floral81.35 ± 2.56 c48.45 ± 11.03 d378.11 ± 15.43 a178.67 ± 9.83 b394.98 ± 8.62 a88.29 ± 0.69 c123.86 ± 11.23 c96.34 ± 8.24 c
Fruity42.66 ± 3.65 c78.78 ± 3.62 b57.40 ± 9.64 b129.50 ± 11.33 a150.89 ± 10.26 a101.84 ± 13.84 ab53.02 ± 0.53 b91.88 ± 5.44 ab
Fermented25.86 ± 2.14 c94.38 ± 7.22 b72.11 ± 8.54 b140.99 ± 2.52 a90.14 ± 4.27 b69.59 ± 6.29 b62.04 ± 2.65 bc81.15 ± 3.22 b
Sour0.00 ± 0.00 c0.00 ± 0.00 c6.17 ± 1.56 b0.00 ± 0.00 c14.46 ± 0.50 a0.00 ± 0.00 c3.83 ± 0.84 bc0.00 ± 0.00 c
Ethereal: the sum of volatile compounds associated with sweetness and fragrance (ethyl acetate). Floral: the sum of the volatile compounds related to a flower-like sweet fragrance (ethylhexanol, L-menthol, methyl salicylate, nonanol, phenethyl acetate, phenethyl butyrate, phenethyl alcohol, phenethyl propionate, phenyl acetaldehyde, and undecenol). Fruity: the sum of volatile compounds associated with fruit-like berries (benzaldehyde, butyl butanoate, butyl butyrate, ethyl hexanoate, and isoamyl acetate); fermented: the sum of the volatile compounds related to typical fermentation, musty berries (isoamyl alcohol and isobutyl alcohol). Sour: the sum of volatile compounds associated with acidity and tartness (acetic acid, isobutyric acid, and methylbutyric acid); PL: Pueraria lobate; AL: Arctium lappa; ZO: Zingiber officinale; PG: Platycodon grandifloras; 8279: Kluyveromyces marxianus LRCC8279; and 8293: Saccharomyces cerevisiae LRCC8293. Results are expressed as mean ± stand errors of means. abcd Means in the same columns with different lowercase superscript letters are significantly different at p < 0.05.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Lee, E.-J.; Choi, S.-H.; Seo, M.-J.; Lee, A.-R.; Jang, C.-S.; Kwak, W.-K.; Kwak, J.-K.; Lee, J.-H.; Yoon, W.-J.; Yoon, S.-M. Genomic and Fermentation Characterization of Kluyveromyces marxianus and Saccharomyces cerevisiae in Root Extract-Based Low-Alcohol Beverage. Fermentation 2025, 11, 299. https://doi.org/10.3390/fermentation11060299

AMA Style

Lee E-J, Choi S-H, Seo M-J, Lee A-R, Jang C-S, Kwak W-K, Kwak J-K, Lee J-H, Yoon W-J, Yoon S-M. Genomic and Fermentation Characterization of Kluyveromyces marxianus and Saccharomyces cerevisiae in Root Extract-Based Low-Alcohol Beverage. Fermentation. 2025; 11(6):299. https://doi.org/10.3390/fermentation11060299

Chicago/Turabian Style

Lee, Eun-Ju, Seung-Hyun Choi, Min-Ju Seo, A-Reum Lee, Chan-Song Jang, Woong-Kwon Kwak, Jung-Ki Kwak, Jae-Ho Lee, Won-Joo Yoon, and Seok-Min Yoon. 2025. "Genomic and Fermentation Characterization of Kluyveromyces marxianus and Saccharomyces cerevisiae in Root Extract-Based Low-Alcohol Beverage" Fermentation 11, no. 6: 299. https://doi.org/10.3390/fermentation11060299

APA Style

Lee, E.-J., Choi, S.-H., Seo, M.-J., Lee, A.-R., Jang, C.-S., Kwak, W.-K., Kwak, J.-K., Lee, J.-H., Yoon, W.-J., & Yoon, S.-M. (2025). Genomic and Fermentation Characterization of Kluyveromyces marxianus and Saccharomyces cerevisiae in Root Extract-Based Low-Alcohol Beverage. Fermentation, 11(6), 299. https://doi.org/10.3390/fermentation11060299

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop