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Research Report

Bacterial Diversity and Enological Properties of Fructophilic Lactiplantibacillus plantarum during Fermentation of Traminette Grape

Adrienne Goppold, Louis Conradie, Folarin A. Oguntoyinbo
Am J Enol Vitic.  2025  76: 0760026  ; DOI: 10.5344/ajev.2025.25006

Abstract

Background and goals This study investigated the bacterial diversity of Traminette, an important hybrid grape used for winemaking in the midwestern and eastern United States. The role of wild fructophilic lactic acid bacteria (FLAB) in the formation of lactic acid and mannitol during fructose metabolism, as well as its possible role in malolactic fermentation, was also determined.

Methods and key findings High-throughput DNA metabarcoding sequencing of DNA extracted from samples at various stages of wine production identified an abundance of the operational taxonomic unit that includes genera such as Faecalibacterium, Peptoclostridium, Lactiplantibacillus, Roseburia, Bacillus, and Veillonella. It also identified Clostridium, Listeria, and Enterococcus, genera which may be of safety concern in wine. Across the different wine production stages, beta diversity and differential abundance indicated that yeast nutrients play a role in bacteria dynamics. In addition, Venn analysis of the relative abundance indicated that 53 common unique genera were shared in the Traminette R fermentation tank, and 45 genera were shared in the Traminette L fermentation tank. Cultured FLAB strains identified using sequencing of the almost-complete 16S rRNA gene included Leuconostoc citreum, Lactococcus lactis subsp. hordniae, and Lactiplantibacillus plantarum. Although fructophilic Lp. plantarum F4 demonstrated fructose metabolism similar to that of the reference strains of Lp. plantarum and the Fructobacillus fructosus FB3a strains, randomly amplified polymorphic DNA gel fingerprints revealed differences in the genomes of the strains. Lp. plantarum F4 did not express the mannitol dehydrogenase gene but did express the mle gene which is responsible for the decarboxylation of malic acid in artificial wine medium. High-performance liquid chromatography indicated no residual fructose or l-malic acid in artificial wine medium at pH 3.8 and 12% ethanol when used for fermentation.

Conclusions and significance This study identified the abundance of Bacillota genera during Traminette grape fermentation and suggests the use of fructophilic Lp. plantarum F4 during secondary fermentation of wine to reduce residual fructose and convert l-malic acid to l-lactic acid. These results indicated the diversity of bacteria associated with Traminette grape and the possible ecological applications during wine production.

Introduction

Traminette is a hybrid grape variety produced by crossing Gewürztraminer with Joannes Seyve 23.416 (Reisch et al. 1996). With partial resistance to fungal disease and good cold hardiness, this variety shows strong productivity, particularly in areas where cold winter temperatures can be problematic (Reisch et al. 1997, Ji and Dami 2008). Grapes ripen in late midseason and produce wines that showcase aromatics similar to those of Gewürztraminer. Potential aromas include perfume, flowers (roses), citrus (orange, mandarin), and spice (coriander) (Ji and Dami 2008). Traminette has good flavor and has been found useful for producing excellent white and sparkling wine with aromatic qualities of fruit and wine that are superior to those of its parent (Ji and Dami 2008, Skinkis et al. 2010).

Wine production worldwide is affected by many parameters that determine terroir and quality. Production remains a natural fermentation system, although inoculation with Saccharomyces cerevisiae and malolactic fermentation (MLF) using Oenococcus oeni are both prevalent in the industry (Alexandre et al. 2004, Betteridge et al. 2015, Parapouli et al. 2020). The microorganisms that occur naturally in grapes, fermentation vessels, and the environment actively participate in wine production and help to define its metabolic composition and flavor development; thus, it is necessary to investigate wild microbes in various grape-producing regions (Mtshali et al. 2012, Berbegal et al. 2016). Many members of the phylum Bacillota have been identified in wines worldwide, and their genetic identities are well documented (Barata et al. 2012, Portillo and Mas 2016, Franquès et al. 2017). Recently, metagenomic approaches using 16S amplicons have revealed that previously underreported microbes associated with winegrapes and their survival during fermentation are relevant in profiling wine microbial communities and describing wine terroir (Bokulich et al. 2016, Morgan et al. 2017). Because grape varieties vary in composition and viticultural practices, little is known about the functions of many nondominant microbes in grapes used for winemaking. For example, glucose and fructose levels in grapes such as Traminette may differ across the variety, with resulting effects on safety and quality during and after fermentation.

One of the most daunting problems facing wine production is the residual sugar composition, which often results in defects and affects the sensory profile (Reboredo-Rodríguez et al. 2015). Residual sugar is known to result from the fact that Saccharomyces has a greater affinity for glucose than fructose. When alcohol concentration increases and nutrients are reduced, the capability of yeast to ferment fructose is very low, often resulting in stuck fermentations. Therefore, when grapes and hybrids such as Traminette with high total soluble solids (TSS)—up to 22 (±2) Brix (% sugar w/w)—and a high fructose content are used in wine production, residual fructose is often created, which can support the postfermentation growth of wild spoilage bacteria. As fructose and glucose comprise the main sugars available for fermentation, the fructose-to-glucose ratio in grapes is an important consideration in winemaking and microbes that can metabolize fructose should be properly studied (Reisch et al. 1997, Berthels et al. 2008).

Little is known about bacterial fructose metabolism during wine production using Traminette grapes. However, members of the phylum Bacillota, such as O. oeni, are the main bacteria used for industrial MLF fermentation (Lonvaud-Funel 1999). Because O. oeni rarely consumes sugar before it finishes consuming l-malic acid, its ability to reduce residual fructose during wine fermentation is limited. Other mesophilic Bacillota such as Lactiplantibacillus plantarum provide alternatives for MLF fermentation, although the mechanism for fructose utilization by Lp. plantarum and the pathway used to achieve it is still not well researched and understood. Genera that are fructophilic lactic acid bacteria (FLAB) occupy fructose-rich ecological niches with limited genetic capability for carbohydrate utilization; some are obligate fructose fermenters, whereas others are facultative fructophilic species (Endo et al. 2009, Endo 2012, Neveling et al. 2012). FLAB are also referred to as acid-producing LAB that utilize D-fructose. They prefer fructose over glucose and have been consistently isolated from fructose-rich ecological niches (Endo et al. 2009). Although winegrapes are very rich in fructose and FLAB have been repeatedly shown biochemically to prefer fructose to glucose, little is known about FLAB during wine fermentation (Endo 2012). Many FLAB strains isolated from assorted fruits, honeydew nectar, and sugar-rich foods such as raw cocoa have been characterized (Gustaw et al. 2018, Viesser et al. 2020). Endo et al. (2018) showed the uniqueness of fructose-fermenting microbes; they are heterofermentative and produce lactate from fructose with deleted alcohol/acetate dehydrogenase (adhE). Although Apilactobacillus kunkeei isolated from grape juice can ferment various sugars (including glucose and fructose), none of its strains have been studied for fructose metabolism under different wine-production conditions (Endo et al. 2012).

Some FLAB are heterofermentative LAB that produce mannitol in large amounts by using fructose as an electron acceptor; a defect in lactate dehydrogenase activity has been shown to be responsible for mannitol production in homofermentative LAB (Wisselink et al. 2002). Accumulation of mannitol in wine is a defect as well as an indication of wine spoilage (Bartowsky 2009). To effectively utilize FLAB during wine production, their production of mannitol from fructose should be investigated.

The presence of FLAB in grapes can be linked to a high percentage of fructose, and their biotechnological benefits can be determined and maximized for enological advantages. Here, we studied Bacillota diversity using high-throughput DNA metabarcoding sequencing, cultured FLAB isolated from Traminette, and hypothesized that fructophilic bacteria isolated from wine could reduce residual fructose and perform decarboxylation of l-malic acid to form lactic acid during grape fermentation. Research into MLF fermentation by fructophilic bacteria and their stress response under extreme conditions in wine could be a starting point for their use in the industry. Therefore, understanding the diversity and functional roles of the wild bacteria associated with the fermentation of this grape is a prerequisite to addressing problems related to residual sugars, l-malic acid metabolism, safety, and quality in wine. This study determines the diversity and relative abundance of genera of bacteria associated with Traminette grape fermentation, studies the roles of yeast nutrients in bacterial dynamics, and evaluates the fructose and malic acid fermentation potential of some isolated fructophilic bacteria.

Materials and Methods

Wine production and microbial strains

During the 2021 grape harvest season, two half-ton Traminette grape parcels (TSS = 20.5 Brix, pH = 3.24, titratable acidity = 7.65 g/L) donated by Dynamis Estate wine in Jonesville, NC were crushed into two separate 600-L open-top fermenters (denoted “L” and “R”) and left on skins for ~4 days with the lids on for spontaneous fermentation.

The two Traminette grape fermentation trials were then sampled to determine bacterial diversity. In fermentation tank “Traminette R”, 20 g/hL Fermaid O yeast nutrient (Lallemand Oenology) was added at 17.7 Brix, then 20 g/hL Fermaid O and 12.5 g/hL Fermaid K at 13.4 Brix were added to achieve fermentation security. In the other fermentation tank, “Traminette L”, 40 g/hL Stimula Sauvignon blanc O (Lallemand Oenology) was added at 17.1 Brix, and 20 g/hL Fermaid O was added at 13.4 Brix to enhance the varietal character of the wine. After 4 days, S. cerevisiae var. bayanus yeast (QA23, Lallemand) was added. This yeast was selected as a strong fermenter that enhances varietal character in wine. The following samples were taken: must (day 1), yeast added (day 4), fermented must (day 15), and finished wine samples (day 30). The samples were analyzed immediately for pH and microbial population, then stored at −20°C for later metagenomic analysis.

Lp. plantarum 3058, 65493, 4306, and B813 were obtained from the Agricultural Research Service Culture Collection of the National Center for Agricultural Utilization Research (Peoria, IL). Fructobacillus fructosus FB3a was obtained from the Collection of the Fermentation Sciences of Appalachian State University (Boone, NC). The strains were first precultured in DeMan–Rogosa–Sharpe (MRS) broth and incubated at 30°C for 24 hr before subculture in adapted medium at pH 4.6 and 6% ethanol, as described (Miller et al. 2011).

Preprocessing, DNA extraction, and DNA quantification

To extract DNA from grapes, fermented grape must, and finished wine samples (obtained from refrigeration at −20°C), the FastDNA Spin Kit for Soil (MP Biomedicals) was used in conjunction with a preprocessing step that separated the microbial cells from large solid particles in the sample, following an optimized method described previously (Díaz et al. 2019). First, 20 mL of the sample was mixed with 10 mL cold, ultrapure water by vigorous vortexing. The solid particles were removed by centrifugation at 800×g for 1 min at 4°C, and the supernatant was retained. A further 10-mL water was added, and the process was repeated three times total, resulting in a final supernatant volume of ~30 mL. Cells were harvested from the particle-free supernatants by centrifugation at 3000×g for 20 min at 4°C. The supernatant was discarded and the pellet was washed three times with a 1-mL phosphate-buffered saline. After centrifugation at 14,000×g for 2 min, the pellet was resuspended in 978-μL sodium phosphate buffer and 122-μL MT buffer, incubated for 1 hr at 4°C, and homogenized for 60 sec at a speed setting of 6.5 m/sec, using a FastPrep-24 instrument (MP Biomedicals). This process was repeated three times, and samples were kept on ice for 5 min between each homogenization step. Otherwise, DNA extraction was performed according to manufacturer instructions. The extracted DNA was resuspended in 50-μL elution buffer. Total DNA extracted from the fermented samples was quantified using a Quickdrop UV spectrophotometer (Molecular Device) and quality was verified at wavelengths of 230 and 280 nm.

Illumina high-throughput sequencing

The V4 hypervariable region of the 16S rRNA gene was amplified using specific primers 515F (5′-GTGYCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACNVGGGTWTCTAAT-3′) (Caporaso et al. 2010). All PCRs were carried out with Phusion High-Fidelity PCR Master Mix (New England Biolabs). Libraries generated with the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) were sequenced using paired-end Illumina sequencing (2 × 250 base pairs) on the novaseq6000 platform (Illumina).

Bioinformatics

The sequence data from the samples were generated as FASTQ files; mapping/metadata files, read QC, filtering, and trimming were done in QIIME 2. The demultiplexed paired-end reads were filtered of substitution and chimera errors and merged using DADA2 (Callahan et al. 2016). Bacterial taxonomic assignment was performed at 97% similarity using a Naive Bayes classifier trained on the Silva ver. 132 99% operational taxonomic unit (OTU) database (Quast et al. 2013).

Isolation and characterization of FLAB

A 10-mL sample of Traminette grape must was homogenized and mixed with 90-mL sterile minimal recovery diluent (Peptone Saline Diluent; Oxoid). The sample was further diluted in a 10-fold dilution series, and 0.1-mL samples of suitable dilutions were spread onto plates containing MRS agar medium (Merck) or MRS-glucose + calcium carbonate (CaCO3), as described (Endo et al. 2012). Clear zones around colonies on the MRS-glucose + CaCO3 serve as indications of acid production. The plates were incubated aerobically at 30°C for 24 hr. Representative dominant colonies were picked from the plates of the highest dilutions and purified by repeated streaking onto the same nutrient agar medium. Stock cultures were kept in MRS broth (VWR) containing 50% glycerol and stored at −80°C. Of the 90 LAB isolates, only the strains that were positive on modified MRS-fructose without glucose (mMRS) medium were selected for further characterization.

Bacterial genotypic characterization

Extraction of genomic DNA from isolated pure cultures was performed as previously described (Diaz et al. 2019). PCR was performed using the primer pair O8F and 1391R to amplify the complete 16S rDNA gene. The PCR solution contained 5-μL PCR buffer for Taq polymerase (Go Taq, Promega), 25-mM dNTP (Bioline), 20 mM of each primer, and 5 units of Taq polymerase. PCR was performed using a thermocycler BiometraT300 (Biometra) using the following program: 94°C for 2 min; 30 cycles of 94°C for 20 sec, 58°C for 20 sec, and 65°C for 1 min; and 65°C for 10 min final extension. PCR amplicons were purified using the SureClean kit (Bioline). Sequencing reactions were prepared using primers O8F and 1391R and the BigDye Terminator v3.1 Cycle Sequence Kit (Applied Biosystems), according to manufacturer guidelines. Reactions were analyzed with an ABI 3730×l Analyzer (Applied Biosystems). Forward and reverse reads were manually checked then assembled into a contiguous sequence using the BioEdit software. BLAST Search was used to compare the nearly full-length 16S rDNA gene sequences that were obtained against those sequences deposited in GenBank. Sequences that showed more than 97% similarity were considered to belong to the same OTU (Altschul et al. 1997). Consensus sequences were imported into Mega 11 software, in which a similarity matrix and dendrogram were created based on the neighbor-joining method, and the sequences were deposited to GenBank to obtain accession numbers PP250159 to PP250166.

Genomic differentiation between strains of Lp. plantarum and reference strains was performed using random amplified polymorphic DNA (RAPD)-PCR fingerprinting analyses. Each reaction mixture contained premixed Taq (1 unit Go Taq Polymerase and 1× Go Taq Polymerase buffer; Promega), 2.5 mM dNTP, and 25 pmol primer M13 (‘5-GAGGGTGGCGGTTCT-3′) (Andrighetto et al. 2001). The final volume of the reaction mixture was adjusted to 50 μL with ultrapure water. PCR was carried out in 35 cycles of 94°C for 1 min and 40°C for 20 sec, then ramped to 72°C at 0.6°C/sec for 2 min and held at 72°C for 2 min. The PCR products were separated on 1.6% agarose gels at 48 V for 17 hr. Band patterns were visualized after staining with ethidium bromide and photographed under ultraviolet illumination.

Fructophilic screening under wine conditions

All fructophilic bacteria were screened for catalase production using 10% hydrogen peroxide and tested for their ability to metabolize fructose on modified fructose-MRS (fMRS) agar, in which glucose was substituted for fructose (2% w/v); the medium was prepared as previously reported (Endo et al. 2012). Strains were further tested for their ability to produce acid from fructose and glucose metabolism in wine synthetic media as previously described (Ugliano et al. 2003). The artificial wine was adjusted to pH 3.4 and pH 3.8, as well as 12% and 15% ethanol, inoculated with 1 mL of washed, medium-free cells (~1 × 106 cells), and incubated at 30°C (the optimum growth temperature). The optical density at 600 nm was measured daily. The samples obtained were analyzed for glucose, fructose, lactic acid, and malic acid.

Analytical methods

Concentrations of glucose, fructose, sucrose, mannitol, sorbitol, and organic acids (malic and lactic acid) were determined using high-performance liquid chromatography with refractive index detector (HPLC-RI) (Thermo Fisher Scientific), following the procedure described by Vrancken et al. (2008) and Ortiz et al. (2017).

Reverse transcription PCR (RT-PCR)

RT-PCR was used to determine expression of the MLF enzyme gene (mle) (MleF 3′-ATGACAAAAACTGCAAGTGA-5′ and MleR 3′-CTATTTGCTGATGGCCCGGTA-5′) (Miller et al. 2011) and the mannitol dehydrogenase gene (mdh) (MdhF 3′-TCAFCTGTTGGTTACCCG-5′ and MdhR 3′-GCAGGGATCTTGTCACCGTT-5′) (Aarnikunnas et al. 2002). Total RNA was extracted using the Direct-zol RNA Mini Prep Plus isolation kit (Zymo Research) with ~1 × 108 Lp. plantarum cells originally grown in MRS broth at 35°C to the end of the logarithmic growth phase (~5 hr). Then, 1 mL of washed, medium-free cells (~1 × 106 cells) were inoculated into 50-mL artificial wine medium and incubated at 30°C for 10 days (optimum growth condition). Samples were taken daily for RNA extraction following manufacturer instructions. The concentration of RNA was determined using Quickdrop at 260 nm (Molecular Devices, LLC). Expression of the gene coding for mannitol dehydrogenase and malic dehydrogenase in Fructobacillus were determined during in vitro wine fermentation using cDNA generated from RNA extracted from the Lp. plantarum by reverse transcriptase (iScript Reverse Transcription Supermix, 25 × 20 μL reactions, 100 μL; Biorad) and amplified using primer forward (5′-ATGACAAAAACTGCAAGTGA-3′) and reverse (5′-CTATTTGCTGATGGCCCGGTA-3′) (Miller et al. 2011).

Statistical analysis

Significant differences in alpha diversity between groups were calculated using the alpha-group-significance script in QIIME 2, which performs the Kruskal-Wallis test. Beta diversity was determined using the Bray-Curtis dissimilarity, and the differential abundance was computed using Maaslin (Mallick et al. 2021) with default parameters. The shared and unique OTU were shown as a Venn diagram using InteractiVenn (Heberle et al. 2015). Significant differences in bacterial community structure among the groups were evaluated by Microbiome DB (https://microbiomedb.org/mbio/app). A p value ≤ 0.05 was considered statistically significant. Evolutionary relationships of taxa were determined using MEGA 11 and the bootstrap consensus tree inferred from 100 replicates (Tamura et al. 2021).

Results and Discussion

High-throughput DNA metabarcoding sequencing

Our first aim was to determine the diversity of bacteria associated with the fermentation of Traminette grapes. Next-generation sequencing has become an important tool for profiling the microbiota and microbiome of different fermentation microbial communities. DNA samples extracted at different stages of Traminette fermentation were sequenced and profiled for diversity and dynamics based on the amplicon of the V4 regions of the 16S rRNA gene. The sequencing generated a total of 2,254,328 quality sequences that were mapped to bacterial taxa and used to generate OTU at 97% nucleotide sequence similarity. The rarefaction curves of the OTU for observed features and the Shannon diversity index reached a plateau, showing that the sequencing coverage was sufficient to capture the majority of the bacterial diversity (Supplemental Figure 1A and 1B). Similar observations have been reported previously in studies of fermented foods and beverages using next-generation sequencing (Diaz et al. 2019, Rezende et al. 2024).

Bacterial diversity and differential abundance studies

To determine whether the addition of yeast nutrients during winemaking affects bacterial dynamics during fermentation, we sampled two fermentation tanks (R and L) with different nutrients at different fermentation stages. The alpha diversity of bacterial richness, as observed using the Shannon diversity index and estimated by the Chao1 statistical estimator, was higher in the Traminette R tank (Figure 1A and 1B). The analysis of the alpha diversity also shows that—among samples taken at fermentation stages ranging from must to wine—the highest species diversity was observed during the fermenting must stage, at 14 days (Figure 1A and 1B). The beta diversity (as measured by a Bray Curtis dissimilarity of p < 0.05) between the genera of bacteria in the Traminette R and Traminette L tanks revealed that the difference between the two treatments was influenced by different yeast nutrients (Figure 2A). However, the beta diversity results showed similarity in the OTU of the genera at day 15 (fermented must) and in the final wine in the Traminette L tank. The diversity distance between the two treatments was dissimilar in the OTU assigned to the final wine (Figure 2A). The ranked abundance (p < 0.05) between the two yeast nutrient treatments is shown (Figure 2B). The Traminette L tank was up by two genera (gram negative and Sarcina), whereas the Traminette R tank was up by nine genera (Aerococcaceae, Actinomyces, Roseisolibacter, Deinococcus, Fournierella, gram negative, Clostridium, and two unknown genera). In the ranked abundance comparison of the OTU of must with that of wine (p < 0.05), the results show 22 genera were up in the must, which increased to 40 in wine; this indicates that bacteria diversity changed and increased during the fermentation (Figure 2C).

Figure 1
Figure 1

Box plots of the Shannon diversity and Chao1 of the alpha diversity for the observed operational taxonomic units of bacterial species in the two separate 600-L open-top fermenter tanks (Traminette L and Traminette R) used for the study (nutrient treatments, upper plots), and in samples from different stages of wine processing (lower plots) (p < 0.05).

Figure 2
Figure 2

A) Multivariate differentiation of two fermentation treatments conducted in separate 600-L open-top fermenter tanks (Traminette R [blue dots] and Traminette L [red dots]), B) differential abundance between the Traminette L (green dots) and Traminette R (red dots) tanks, and C) differential abundance between must (green dots) and wine (red dots) processing stages.

Examination of phylum Bacillota

The relative abundance results from a heatmap analysis show the richness at the phylum level, with the highest OTU ranking rated as 5, which includes Pseudomonadota, Actinobacteriota, Bacillota, and Bacteroidota (Figure 3). Analysis with DADA2 to rank the abundance of the OTU had results similar to those of the heatmap profile generated by QIIME 2. Both fermentation treatments (R and L) showed very high richness of the phylum Bacillota (Figure 3). This encouraged us to analyze the Bacillota OTUs at the genera level, and we identified Faecalibacterium, Peptoclostridium, Lactiplantibacillus, Roseburia, Bacillus, Veillonella, Streptococcus, Turicibacter, Blautia, Staphylococcus, and Lysinibacillus as the dominant Bacillota genera at all fermentation stages (Figure 4).

Figure 3
Figure 3

Heatmap of the relative abundance at the phylum level for the two separate 600-L open-top fermenter tanks (Traminette L and Traminette R) used for the study. The values of the population of each genera were scored; the legend indicates key scores as blue = highest abundance; red = lowest abundance.

Figure 4
Figure 4

Relative abundance of the operational taxonomic units of genera of the phylum Bacillota in samples taken from must, when yeast was added, during fermentation, and from the final wine for the two separate 600-L open-top fermenter tanks (Traminette R and Traminette L) that were used for the study. Different nutrient treatments were used for each tank.

Faecalibacterium have been previously isolated from human feces (Zou et al. 2021), and its high abundance in samples ranging from grape must to wine indicates that grapes may not be totally free from bacteria originating from human or animal gut or fecal material. Other Bacillota genera were identified that could pose safety challenges, including Clostridum, Enterococcus, and Listeria (Figure 4). However, detection of the DNA corresponding to these bacteria does not necessarily translate to their viability in wine.

The genera Brevibacillus, Desulfosporosinus, Lachnospiraceae, Acidibacillus, Incertae, Thermoflavimicrobium, Sporacetigenium, and Cohnella were detected in low abundance in only the wine sample. This result confirmed that DNA of some previously unreported bacteria exists in wine samples and may survive wine’s low acidity and 10 to 12% alcohol level. A metagenomic analysis on wine reported a wide microbial consortium (Zambianchi et al. 2023), and other studies have indicated that metagenomic and other -omics techniques have the potential to increase understanding of wine microbiomes and corresponding metabolism (Sirén et al. 2019, Rezende et al. 2024). Yet issues such as production of biogenic amines and transfer of genetic elements during wine fermentation have been reported (Lonvaud-Funel 2001) and indicate that attention should be paid to the microbial quality and safety of wine.

The prevalence of the genus Lactiplantibacillus throughout fermentation agrees with the findings of previous studies that monitored microbial succession from grape to wine (Berbegal et al. 2016, Ding et al. 2023). The results of the 16S amplicon sequencing showed that Bacillota associated with the grape progressively increased from the beginning of the fermentation to the final wine (Figure 4). The general distribution and dynamics of the genus are shown (Figure 4). The Kruskal-Wallis rank sum test showed that the richness of the genus Lactobacillus was significantly different (p < 0.05) between the must, yeast added, fermented must, and finished wine samples. The data also indicated a high abundance of other LAB such as Enterococcus, Pediococcus, Lactococcus, Leuconostoc, and Fructobacillus in the two fermentation treatments. The fermentation period is a very important stage during wine production and often involves increases in alcohol production, acidity, and carbon dioxide. Enterococcus is known to be more tolerant of environmental stress—including temperature, pH, salinity, and even desiccation—than Lactiplantibacillus (Gaca and Lemos 2019). In the current study, the OTU of Enterococcus increased from must to wine in both treatments (Figure 4). The roles of Enterococcus in wine and its mechanism of survival should be further investigated. Although increased alcohol and acidity may be responsible for the abundance of some genera such as Listeria and Clostridium in the final wine, evidence from previous studies has shown wine to have a low food safety risk for consumers (Azevedo et at 2016). Overall, Lactobacillus was most abundant, increasing as the fermentation progressed from must to wine and then declining in the final wine.

We then compared the dynamics and shared bacteria at the different stages of fermentation between the two fermentation treatments. Figure 5 shows the Venn diagram of shared unique OTU. In Traminette R, 53 (44.3%) of the common genera were shared across the must, yeast added, fermented must, and finished wine samples, while in Traminette L, 45 genera (37.7%) were shared across the four samples. The results further confirm that the core genera belonging to the phylum Bacillota participated in the fermentation of both Traminette treatments. Species of Bacillota are well known for their various roles during wine production (Lonvaud-Funel 1999).

Figure 5
Figure 5

Venn diagram showing the unique and shared bacteria genera across the two separate 600-L open-top fermenter tanks (Traminette R [A] and Traminette L [B]) that were used for the study and the operational taxonomic units among four different sampling stages during wine production.

Metabolism of glucose and fructose by fructophilic bacteria

Fructose and glucose are major sugars in Traminette grape and this study aimed to confirm that fructophilic bacteria are found in Traminette grape samples, to determine whether they play a role during fermentation, and to address issues of residual sugars due to late metabolism of fruit by yeast. Bacterial enrichment and examination for fructose fermentation were performed; positive isolates on mMRS with fructose showed clear zones around the colonies (Supplemental Figure 2). The phylogenetics of fructophilic bacteria were determined using data obtained from the sequencing of the almost-complete 16S rRNA gene. Figure 6 shows the phylogenetic tree of the FLAB and how it compares to other closely related LAB, particularly Fructobacillus, which has been previously isolated from grapes. Fructophilic activity was widespread among rod-shaped facultative LAB, such as Lp. plantarum and Fructobacillus, and similar observations have been previously reported (Endo 2012). Furthermore, the Lp. plantarum F4 strain obtained in this study was compared with other Lp. plantarum strains (i.e., F4,3; NRRL 3058,4; NRRL 65493,5; NRRL 4306,6; and NRRL B813) using RAPD-PCR. Lp. plantarum F4 was shown to be a unique strain, with band patterns distinct from those of other Lp. plantarum strains that originated from different ecosystems (Supplemental Figure 3). It is well established that strains should be investigated for their unique characteristics to determine their functional advantages and potential biotechnological uses (Colagrande et al. 1994).

Figure 6
Figure 6

Phylogenetic tree based on aligned 16S rRNA gene sequences of fructophilic lactic acid bacteria isolated from Traminette must and fermenting wine.

Enological properties

S. cerevisiae is the main starter culture for many wine fermentations. Its enological properties show a preference for glucose and it ferments fructose much later during grape fermentation. If a fermentation is not managed properly and the yeast are stressed, sluggish or stuck fermentations can occur and cause undesirable sweetness in wines due to residual sugar, which also contributes to spoilage (Tronchoni et al. 2009). Other factors that might cause high fructose levels in grapes include heat waves or high temperatures during the growing season. Also, hybrid grape varieties like Traminette naturally tend to have higher levels of fructose than glucose.

Wine can also encounter problems during fermentation when winemakers promote wild fermentation and the natural microbiome is unable to complete the fermentation. To address this challenge, FLAB isolates were screened for their survival under wine fermentation conditions. Table 1 shows the FLAB isolates and their growth under different pH levels at 6% v/v alcohol. All FLAB tested grew at pH 5 and above. Only Lp. plantarum and F. fructosus FB3 grew at pH 4 and below with 6.0% v/v alcohol. These two positive isolates were further tested for growth in 12 and 15% alcohol medium to investigate their potential use of fructose when alcohol accumulates in the fermentation. In our results, sugars were generally poorly utilized in the 15% alcohol growth medium (Table 2). HPLC analysis showed that Lp. plantarum F4 can metabolize fructose better than F. fructosus. In growth medium containing 12% alcohol, Lp. plantarum F4 had 0.05 mg/mL residual fructose, whereas F. fructosus had 0.3 mg/mL residual fructose. A similar observation was observed for glucose (Table 2). Comparatively, the fermentation of fructose under stressful conditions of low pH, high acidity, and high alcohol percentage appears to be more successful with Lp. plantarum F4.

View this table:
Table 1

Growth of different strains of catalase-negative fructophilic lactic acid bacteria (FLAB) at different pH levels on plates containing DeMan–Rogosa–Sharpe agar medium with calcium carbonate. Strains were isolated from must and fermenting wine at 6% alcohol concentration. FLAB formation from fructose on calcium carbonate was present for each strain. A dash (-) indicates that acid formation was not present at a given pH level; different pH levels were generated by adjustment of pH with acid.

View this table:
Table 2

Glucose, fructose, malic acid, and lactic acid in growth medium of Fructobacillus fructosus FB3a and Lactiplantibacillus plantarum F4, grown at pH 3.8 and at different alcohol concentrations.

Lp. plantarum F4 was tested for conversion of l-malic acid to l-lactic acid, as the significance of MLF fermentation in wine production has been repeatedly reported (Miller et al. 2011). The l-malic acid assay showed that Lp. plantarum F4 metabolized l-malic acid from its original 12 mg/mL in artificial wine to 0% in a medium that contains 0% alcohol. The l-malic acid concentration was reduced to 1.45 mg/mL when the alcohol level in the medium was increased to 15%. Moreover, at pH 3.8, Lp. plantarum F4 produced l-lactic acid in medium containing 12% alcohol or less (Table 2). Although malic acid concentration in wine is higher than 12 mg/mL, the result is an indication that malic acid could be metabolized by Lp. plantarum F4. Further testing in a medium containing a higher concentration of malic acid is recommended.

Lp. plantarum undergoes MLF fermentation at 0% ethanol at pH 3.8. Lp. plantarum F4 did not favor MLF metabolism at pH 3.2 with ethanol (data not shown). This confirms that Lp. plantarum F4 can perform MLF fermentation at wine’s acidic pH 3.8 and 12 to 15% ethanol concentration, as demonstrated using a wine medium. The ability of Lp. plantarum F4 to express the mle gene under wine fermentation conditions was investigated in an RT-PCR experiment, and this gene coding for conversion of l-malic acid to lactic acid was successfully expressed (Supplemental Figure 4). Similar results have previously been observed among Lp. plantarum associated with grapes or isolated from must (Berbegal et al. 2016). However, we determined that the mannitol dehydrogenase gene was not expressed by the Lp. plantarum F4 strain. This shows that the bacterium can ferment fructose without accumulation of mannitol and also suggests an effective way to use a facultative homofermenter LAB for MLF fermentation without producing spoilage metabolites.

Conclusion

This study is the first to use high-throughput sequencing to examine the diversity of the genera belonging to the phylum Bacillota during the fermentation of Traminette grapes. The results confirmed the diverse genera belonging to Faecalibacterium, Peptoclostridium, Lactobacillus, Roseburia, Bacillus, and Veillonella as the dominant wild bacteria involved with the fermentation. Other identified genera that could be of safety concern include Clostridium, Listeria, and Enterococcus. Yeast nutrients could play a role in the bacterial diversity and dynamics during Traminette grape fermentation, as the shared bacterial genera were different between the two fermentation treatments.

Among the cultured FLAB identified using almost-complete 16S rRNA gene sequencing were Leuconostoc citreum, Lactococcus lactis subsp. hordniae, and Lp plantarum. Lp. plantarum F4 demonstrated strong fructophilic properties, and RT-PCR analysis indicated no expression of the mannitol dehydrogenase gene. The strain decarboxylated l-malic acid to lactic acid and actively expressed the mle gene in an artificial wine medium and model wine. HPLC analysis indicated no residual fructose in a fermented artificial wine with pH 3.8 and 12% alcohol inoculated with Lp. plantarum F4 and also detected lactic acid postfermentation. Lp. plantarum could thus be used to eliminate the problem of residual fructose during wine production and could be used for MLF fermentation during cofermentation or following yeast fermentation.

Supplemental Data

The following supplemental materials are available for this article in the Supplemental tab above:

Supplemental Figure 1 Observed features rarefaction curves of observed operational taxonomic units (OTU) and Shannon rarefaction curves of observed OTU.

Supplemental Figure 2 Screening for fructophilic lactic acid bacteria on DeMan–Rogosa–Sharpe agar supplemented with calcium carbonate. Clear zones around colonies indicate acid production.

Supplemental Figure 3 Random amplified polymorphic DNA genome strain differentiation of Lactiplantibacillus plantarum with fructophilic properties. Lane identification: 1, 1-kb ladder; 2, Lp. plantarum F4; 3, Lp. plantarum NRRL 3058; 4, Lp. plantarum NRRL 65493; 5, Lp. plantarum NRRL 4306; 6, Lp. plantarum NRRL B813; 7, 1-kb ladder.

Supplemental Figure 4 Reverse transcription expression of mle gene in synthetic wine medium. Lane identification: 1, 100-base pair ladder; 2, Fructobacillus fructosus FB3a at 0 hr; 3, F. fructosus at 12 hr; 4, Lactiplantibacillus plantarum F4 at 0 hr; 5, Lp. plantarum F4 at 12 hr.

Data Availability

All data underlying this study are included in the manuscript and its supplemental information.

Footnotes

  • This work was funded by an Appalachian State University Research Council (URC) grant.

  • Goppold A, Conradie L and Oguntoyinbo FA. 2025. Bacterial diversity and enological properties of fructophilic Lactiplantibacillus plantarum during fermentation of Traminette grape. Am J Enol Vitic 76:0760026. DOI: 10.5344/ajev.2025.25006.

  • By downloading and/or receiving this article, you agree to the Disclaimer of Warranties and Liability. If you do not agree to the Disclaimers, do not download and/or accept this article.

  • Received January 2025.
  • Accepted September 2025.
  • Published online November 2025

This is an open access article distributed under the CC BY 4.0 license.

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Bacterial Diversity and Enological Properties of Fructophilic Lactiplantibacillus plantarum during Fermentation of Traminette Grape
Adrienne Goppold, Louis Conradie, Folarin A. Oguntoyinbo
Am J Enol Vitic.  2025  76: 0760026  ; DOI: 10.5344/ajev.2025.25006
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