High-Performance Indigenous Lactiplantibacillus plantarum Strains for Enhanced Malolactic Fermentation and Wine Quality

1. Introduction

Malolactic fermentation (MLF) is a critical process in winemaking, contributing to acidity reduction, flavor enhancement, increased complexity, and improved microbial stability [1,2]. This transformation is crucial for the production of the majority of red wines and selected white wines[3].

During winemaking, the primary lactic acid bacteria (LAB) genera frequently encountered include Oenococcus spp., Lactobacillus spp., Pediococcus spp., and Leuconostoc spp. [4,5]. Among these, Oenococcus oeni (O. oeni) dominates due to its superior adaptability to the wine environment [6,7]. Lactiplantibacillus plantarum (L. plantarum) exhibits a certain ability to survive in the harsh wine environment [8,9]. Among its notable traits, the ability to produce bacteriocins stands as a key advantage [10]. This trait can aid in maintaining strain dominance during fermentation and reducing reliance on SO₂. [11]. Furthermore, L. plantarum exhibits a more diverse repertoire of wine-associated enzymes compared to O. oeni [12], a feature that enhances wine aroma and subsequently improves overall sensory quality [13]. However, L. plantarum remains underutilized as a wine fermentation starter, with only the ML Prime™ strain from LALLEMAND finding commercial application in fermentation agents. This limited adoption stems from its susceptibility to low pH and high ethanol levels. Rising alcohol levels in wines, driven by global warming, coupled with the cool climatic conditions prevalent in northern China, present a growing challenge to the survival and fermentation efficiency of malolactic bacteria (MLB) [16,17]. Additionally, the prevalent utilization of commercial starter cultures across global wine regions may diminish regional distinctiveness and increase product homogeneity. In recent years, major wine-producing countries such as Spain, Italy, Argentina, and Chile have increasingly prioritized the development and application of indigenous MLB strains [18-25]. Thus, identifying novel potential L. plantarum strains capable of adapting to winemaking stressors holds significant importance for China’s wine industry. The Jiaodong Peninsula—encompassing key regions such as Qingdao and Yantai, ranks among China’s premier grape-growing areas, boasting exceptionally favorable geographical and climatic conditions [14,15]. However, the indigenous MLB resources of this region have not yet been systematically explored.

Herein, we performed a comprehensive analysis and screening of 342 L. plantarum strains isolated from the Jiaodong Peninsula under stress conditions. Two superior strains, J43 and P101, were subjected to a thorough evaluation of their fermentation performance and their impacts on multiple aspects of Marselan wine. Our research contributes to the discovery of novel, elite indigenous LAB strains in China and enhances understanding of their MLF characteristics, thereby providing technical and experimental foundations for the innovation of wine LAB starter cultures and the improvement of wine region characteristics and quality.

2. Materials and Methods

2.1. Experimental Strains and Culture Conditions

A total of 342 L. plantarum strains were isolated from spontaneous MLF wine samples of different grape varieties collected from wineries in the Jiaodong Peninsula, specifically including: Chateau Nine Peaks (Chardonnay, 54 strains); Pula Valley (Chardonnay and Marselan, 52 strains); Taiyihu Winery (Chardonnay, Cabernet Sauvignon, and Marselan, 104 strains); Greatwall Longji Winery (Chardonnay and Marselan, 31 strains); and Aweihai Winery (Marselan, 91 strains). L. plantarum XJ25, preserved in the Microbiology Laboratory of the College of Enology at Northwest A&F University, was used as the type strain. [26]. All L. plantarum strains were cultured in MRS medium under optimal conditions: 37°C in a constant-temperature anaerobic incubator for 24h. The MRS liquid medium composition was as follows: peptone (10 g/L), beef extract (10 g/L), glucose (20 g/L), yeast extract (5 g/L), CH₂COONa (5 g/L), K₂HPO₄ (2 g/L), diammonium hydrogen citrate (2 g/L), MgSO₄·7H₂O (0.2 g/L), MnSO₄·H₂O (0.05 g/L), and Tween 80 (1 mL/L). The medium was sterilized at 115°C for 15min prior to use. For MRS solid medium, 15 g of agar was added to 1 L of the liquid medium.

2.2. Stress Tolerance Analysis of L. Plantarum Strains

Simulated wine contained 10 mL/L grape juice, 2 g/L glucose, 2 g/L D-fructose, 0.2 g/L NaCl, 1 g/L (NH₄)₂SO₄, 2 g/L K₂HPO₄, 0.05 g/L MnSO₄, 0.2 g/L MgSO₄, 4 g/L yeast, and 3 g/L L-malic acid. Absolute ethanol was added per formulation conditions; pH was adjusted with HCl/NaOH after constant volume. Sterile filtration (0.22 μm organic membrane) was performed in a clean bench for decontamination. Three simulated wines mimicked regional conditions:

• Simulated wine A: 12% (v/v) ethanol, pH 3.60
• Simulated wine B: 10% (v/v) ethanol, pH 3.30
• Simulated wine C: 14% (v/v) ethanol, pH 3.80

Activated L. plantarum was inoculated into wine A at 1.0% and incubated at 20°C for 48 h. Initial and 48h OD₆₀₀ were measured to calculate relative OD₆₀₀ (48h OD₆₀₀ - 0h OD₆₀₀). Top 30 strains were selected for survival tests in Simulated wine A, B, and C. Twice-activated L. plantarum was centrifuged (8000 r/min, 5 min), resuspended in saline, and inoculated into A, B, C at 10⁸ CFU/mL. After 6 h incubation at 20°C, survival rate was calculated as (6h viable count / 0 h viable count) × 100%. Viable counts were determined via dilution plating.

2.3. Analysis of L-Malic Acid Content and Viable Bacterial Count

During the process, samples were collected at 24h intervals to determine L-malic acid content and monitor the progress of MLF in simulated wine A. L-malic acid concentration was measured using a highly specific L-malic acid kit (Biosystems, Barcelona, Spain) following the manufacturer’s instructions. Measurements were conducted with the Enology Y15 automatic analyzer (Biosystems, Barcelona, Spain). Viable bacterial counts were determined by the dilution plate coating method at 24h intervals.

2.4. Analysis of Physicochemical Indices and Organic acid Contents

The concentrations of glucose, fructose, glycerol, ethanol, succinic acid, citric acid, lactic acid, and tartaric acid were determined via High Performance Liquid Chromatography (HPLC). Wine samples were diluted 4-fold with ultrapure water and filtered through a 0.22 μm organic filter membrane. The HPLC conditions were as follows: column, BIO-RAD 910-5025; mobile phase, 10 mmol/L H₂SO₄ solution; flow rate, 0.6 mL/min; column temperature, 60°C; injection volume, 20 μL. Organic acids were detected using channel Ch3 at a wavelength of 280 nm, while sugar alcohols were detected via channel B-Ch1. Additionally, pH, total phenol content, and total acidity were analyzed in accordance with Chinese national standard test methods.

2.5. Analysis of Anthocyanin Contents and CIELAB Color Parameters

The concentrations of anthocyanins in wine were analyzed using HPLC. 1 mL wine sample was filtered using a 0.22 μm organic filter membrane. The chromatographic conditions were mobile phase A: pure H₂O: acetonitrile: formic acid =800:100:25. Mobile phase B: pure H₂O: acetonitrile: formic acid =400:500:25. The column was Agilent EC-C18, the flow rate was 1 mL/min, the column temperature was 40°C, and the injection volume was 25 μL. The elution program was 0-4 min, 3% B; 4-14 min, 3%-18% B; 14-16 min, 80% B; 16 min, 80%-3% B; 16-18 min, 3% B. Anthocyanin content was quantified using dimethicalin-3-O-glucoside. The color intensity was quantitatively assessed using the W100 wine color analyzer (China Hanon Co., Ltd., Jinan, China) [48].

2.6. Analysis of Individual Phenolic Contents in Marselan Wine

The concentrations of gallic acid, catechin, vanillic acid, epicatechin, chlorogenic acid, caffeic acid, p-coumaric acid, trans-ferulic acid, quercetin, and kaempferol were determined by HPLC. To 1 mL of wine sample, a mixture of dispersing agent (acetonitrile, 0.5 mL) and extraction agent (ethyl acetate, 1 mL) was added. After agitation for 10s, the mixture was centrifuged at 8000 r/min for 15min, and the supernatant was transferred to a 10 mL centrifuge tube. This procedure was repeated twice. The combined supernatants were rotary evaporated and then brought to a constant volume of 1 mL with methanol, followed by filtration through a 0.22 μm organic filter membrane. Chromatographic conditions included a Synergi Hydro-RP C18 column, with mobile phase A consisting of ultrapure water: acetonitrile:glacial acetic acid (800:100:1, v/v/v) and mobile phase B as ultrapure water: acetonitrile:glacial acetic acid (400:500:1, v/v/v); the flow rate was 1 mL/min, injection volume 20 μL, and elution procedure was as follows: 0–45 min, 0%–35% B; 45–50 min, 35%–100% B; 50–55 min, 100% B; 55–56 min, 100%–0% B; 56–62 min, 0% B. Detection wavelengths were set as follows: 280 nm for gallic acid, catechin, vanillic acid, and epicatechin; 259 nm for protocatechuic acid; 320 nm for chlorogenic acid, caffeic acid, and trans-ferulic acid; and 360 nm for quercetin and kaempferol. Quantification was performed using reference standards.

2.7. Volatile Compound Analysis

Volatile compounds were quantified using headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS). For extraction, 5 mL wine sample and 1 g NaCl were added to a headspace vial, followed by 10 μL of 4-methyl-2-pentanol as the internal standard. The mixture was incubated at 40°C with stirring for 1h, then subjected to SPME extraction. GC-MS conditions were as follows: helium (He) was used as the carrier gas at a constant flow rate of 1 mL/min. The oven temperature program was set as: initial hold at 40°C for 3min, ramped to 160°C (ramp rate not specified), then increased to 230°C at 7°C/min and held for 8min. Mass spectrometry parameters included a scan range of 33–450 m/z, electron ionization (EI) in positive ion mode, and an ion source temperature of 230°C. Unknown compounds were identified by comparing their retention times with those of standard aroma components, and quantification was performed accordingly.

2.8. Statistical Analysis

In this experiment, three parallel tests were performed. All data were analyzed using SPSS (version 22.0; IBM, Armonk, NY, USA) for a oneway analysis of variance (ANOVA) and Duncan’s test (p < 0.05). Images were drawn using Origin 2024 (OriginLab Corporation, Northampton, MA, USA) and GraphPad Prism version8.0.2 (GraphPad Software, Boston, MA, USA).

3. Results and Discussion

3.1. Combined Stress Tolerance Screening of L. plantarum Strains

A total of 342 L. plantarum strains were isolated and identified from spontaneous MLF samples sourced from various winemakers across the Jiaodong Peninsula. These strains underwent initial stress screening in simulated wine A (12% ethanol, pH 3.60), a formulation mirroring the actual wine conditions of the Jiaodong Peninsula. Among the 342 isolates, 30 strains exhibited a relative OD₆₀₀ exceeding 0.04 in simulated wine A, earning them a spot in subsequent screening rounds. In simulated wine A, six strains—P5, LM18, LM6, J43, P49, and LM12a—stood out with significantly higher 6-hour survival rates than the reference strain XJ25 (129.07%) (Figure 1A). XJ25, a well-regarded indigenous Chinese L. plantarum, is widely utilized in wine MLF research [27,28]. Notably, LM12a, P49, and J43 achieved survival rates of 149.92%, 148.94%, and 148.51% respectively, surging past XJ25 and signaling robust resilience to the dual stress of 12% ethanol and pH 3.6. In stark contrast, 14 strains struggled with 6-hour survival rates below 100%, with P32 bottoming out at 47.52%. Under simulated wine B conditions (10% ethanol, pH 3.30), XJ25 maintained a 105.15% survival rate, while 10 strains—P5, LM66, YM157, YM167, PM121, P32, L28, LM12a, YC41, and J43—outperformed it (Figure 1B). As acidity rose and ethanol levels dipped, strains like LM66, YM157, YM167, PM121, P32, L28, LM12a, YC41, and J43 saw their survival rates climb, revealing a stronger aptitude for thriving in low-pH, low-ethanol environments. Among these, PM121, P32, L28, LM12a, YC41, and J43 displayed statistically significant advantages over XJ25, with J43—already a star performer in simulated wine A—leading the pack at 183.64%. Simulated wine C (14% ethanol, pH 3.80) brought another shift: XJ25 logged a 2005.63% survival rate, while five strains surpassed the 2000% mark (Figure 1C). P43, YM152a, J43, and P101 outshone XJ25 with survival rates of 2465.16%, 2575.00%, 3165.95%, and 3273.96% respectively. Their survival rates in this high-ethanol, low-acidity milieu far outpaced those in A and B, underscoring their impressive adaptability to such conditions. Conversely, LM90 limped in with a mere 14.22% survival rate, starkly illustrating the stifling impact of high ethanol on its growth. O. oeni is recognized as the dominant species in MLF due to its exceptional adaptability to wine environmental stresses [5,29]. However, certain L. plantarum strains have also demonstrated considerable adaptive capacity [30,31], a finding supported by our screening results. Strain J43 exhibited robust adaptability across all three stress-induced simulated wine conditions, with particularly notable performance under high-acid and high-ethanol stress. In contrast, LM12a showed greater suitability for high-acid environments but was significantly impacted by elevated ethanol concentrations. Collectively, strains J43, P49, LM12a, PM121, P32, L28, P43, YM152a, and P101 displayed superior adaptability in the stress screening across the three simulated wine formulations.

3.2. Viable Cell Counts and L-Malic Acid Consumption During MLF with Different L. plantarum Strains

Ten wild-type L. plantarum strains (J43, P49, LM12a, PM121, P32, L28, P43, YM152a, YC41, and P101) that exhibited superior stress tolerance in simulated wine assays were selected for MLF trials in simulated wine A (12% ethanol, pH 3.60), with strain XJ25 serving as the control. Temporal dynamics of L-malic acid concentration and viable cell counts were systematically monitored throughout fermentation. All selected strains displayed the most rapid decline in L-malic acid content within the first 24h (Figure 2A). This phenomenon may be attributed to the highest viable cell density and biological activity observed in the simulated wine during this initial period, which drove a significant reduction in malic acid levels. The control strain XJ25 consumed the largest amount of malic acid within the first 24h, with a decrease of 0.63 g. Strain J43 also induced a rapid reduction in malic acid content during this period. After 24h, the rate of L-malic acid consumption by all strains slowed significantly. Among the tested strains, J43 and P101 consumed the highest amounts of L-malic acid over the entire fermentation period in wine A, ultimately reducing the malic acid concentration to below 2 g/L. Figure 2B illustrates that the viable cell counts of P101 and J43 remained relatively stable within the first 24 h, after which they began to decline. By day 9, P101 maintained the highest viable cell count, remaining above 10⁶ CFU/mL. The viable count of J43 was second only to P101, staying above 10⁵ CFU/mL—significantly higher than that of the control strain XJ25. Notably, strains P101 and J43 not only sustained high viable cell counts and robust biological activity throughout MLF but also exhibited superior L-malic acid utilization capacity compared to XJ25 during simulated wine fermentation. These findings indicate that P101 and J43 possess excellent fermentation potential. Consequently, P101 and J43 were selected for Marselan wine fermentation trials.

3.3. Physicochemical Indices of Wines after MLF with Different L. plantarum Strains

The basic physicochemical indices of wine samples subjected to MLF with various L. plantarum strains were analyzed, with details presented in Table 1. Results revealed significant alterations in several key parameters (p<0.05) in wines inoculated with the screened L. plantarum strains for MLF compared to non-MLF controls. These parameters included pH, alcohol content, glucose, fructose, total acidity, glycerol, and total phenols. Post-MLF, a notable decrease in total acidity was observed, accompanied by a significant increase in pH levels—consistent with the decarboxylation of malate to lactate during MLF [5]. All wine samples exhibited reduced fructose content after fermentation, indicating fructose metabolism by these strains. Glycerol concentrations also differed significantly post-fermentation, suggesting glycerol metabolism by the three strains; J43 showed the lowest glycerol concentration at 4.34 g/L, representing a 0.51 g reduction. Glycerol undergoes aerobic metabolism via the glycerol kinase pathway, where it is phosphorylated to glycerol-3-phosphate and subsequently oxidized to dihydroxyacetone phosphate. Notably, 13 glycerol-metabolizing L. plantarum strains have been isolated from Australian wines [32]. Alcohol content decreased significantly in J43-inoculated wines, reaching 13.68% (v/v), while only slight reductions were observed in XJ25 and P101 samples. Additionally, total phenol content decreased post-MLF in all inoculated wines, with J43-inoculated samples showing the lowest total phenol content (3,047.19 mg/L)—a value significantly different from pre-fermentation levels.

3.4. Changes in Viable L. plantarum Counts and Organic acid Contents in Marselan Wine

During MLF, the viable cell counts of the three strains remained consistently above 10⁷ CFU/mL, with only a slight reduction, indicating good adaptability and viability in Marselan wine (Figure 3A). Acid stress significantly impacts industrial microbial processes [33], particularly in food fermentation, where it inhibits microbial growth, disrupts metabolic activity, and prolongs fermentation duration [34]. As shown in Figure 3B–D, the contents of citric acid, tartaric acid, and succinic acid in wines inoculated with XJ25, P101, and J43 remained essentially unchanged throughout MLF. Citric acid can serve as a carbohydrate source to provide energy and accelerate the growth of lactic acid bacteria; studies have shown that citrate supplementation can increase the abundance of Micrococcus and Lactobacillus [35]. However, in this experiment, the three L. plantarum strains did not metabolize citric acid, resulting in no significant change in citric acid content in the wine.

The initial concentration of L-malic acid in the wine was 3.07 g/L, with an initial lactic acid content of 0.53 g/L. P101 and J43 exhibited a rapid rate of L-malic acid consumption in the first 5 days, which slowed thereafter. By day 8, the L-malic acid concentrations were 0.41 g/L for P101 and 0.68 g/L for J43 (Figure 3E). The lactic acid concentrations reached 2.21 g/L for J43 and 2.35 g/L for P101 (Figure 3F), with no significant difference in the extent of lactate accumulation between the two strains. XJ25 showed the fastest malate consumption rate: by day 5, L-malic acid in the wine was completely depleted, and the lactate concentration remained essentially unchanged, marking the end of MLF. While J43 and P101 consumed L-malic acid at a slower pace than XJ25, they maintained robust viable cell counts throughout fermentation, achieved substantial lactate production, and preserved key organic acid profiles—collectively reflecting their excellent fermentation potential for winemaking.

3.5. Effects of L. plantarum on Wine Color and Anthocyanin Contents Before and After MLF

Table 2 reveals that MLF induced by L. plantarum in Marselan wine led to an increase in wine brightness, as reflected by a rise in L* value from the initial 16.86. Among the strains tested, P101 yielded the highest L* value (19.20), indicating significantly greater brightness and enhanced gloss. However, no significant differences were observed in other color parameters across treatments, including a*, b*, C*_ab (Chroma), and h*_ab (Hue angle). The total color differences(△E*_ab) between wines before and after MLF were below 3.0 for all strains, suggesting visually indiscernible color alterations. Collectively, these results indicate that MLF mediated by these strains exerts minimal impact on overall wine color.

Figure 4 illustrates that before MLF, malvidin-3-O-glucoside (Mv 3-O-Glu) was the most abundant anthocyanin in the wine at 566.54 mg/L, while peonidin-3-O-acetylglucoside (Pn 3-O-acetylglc) was the least abundant at 3.56 mg/L. After MLF, the content of all tested anthocyanins in wine samples had decreased, with no significant differences observed between fermentations using different strains. Compared to wines fermented with XJ25, those fermented with J43 and P101 showed higher levels of trans-malvidin-3-p-coumaroylglucoside (Mv 3-p-coumglc trans) and lower levels of cyanidin-3-O-glucoside (Cy 3-O-Glu), while the contents of the other seven anthocyanins were nearly identical. The detected anthocyanins content following MLF was lower than that observed prior to MLF. Critically, despite reduced monomeric anthocyanin content after MLF, combined with the observed color stability (ΔE*ab < 3.0; Table 2), this phenomenon likely stems from L. plantarum's ability to release acetaldehyde—a key mediator promoting anthocyanin polymerization and enhancing color stability [36].

Pearson correlation analysis was performed to explore intrinsic relationships between wine anthocyanin content and color parameters (Figure 5). Results showed positive correlations among L*, a*, b*, and C*_ab: higher brightness (L*) was associated with a more intense red hue (a*), more prominent yellow hue (b*), and greater saturation (C*_ab). The correlation between a and C*_ab was particularly strong (correlation coefficient = 0.99), indicating a high degree of linearity. All nine anthocyanins exhibited positive correlations with each other, with correlation values exceeding 0.8, reflecting an extremely strong linear relationship. In contrast, L*, a*, b*, and C*_ab were negatively correlated with the nine anthocyanins, suggesting that higher anthocyanin diversity and content were associated with lower wine brightness, less intense red and yellow hues, and reduced saturation. The hue angle (h*_ab) showed negative correlations with L* and b*, but positive correlations with a* and C*_ab. These results indicate that the reduction in anthocyanin content post-MLF contributed to brighter, more saturated, and more transparent wines, exerting a positive impact on overall wine color. Notable differences were observed in the correlations between anthocyanin profiles (types and contents) and color parameters when comparing wines undergoing MLF with O. oeni versus those fermented with L. plantarum [37].

3.6. Individual Phenolic Contents During MLF with Different L. plantarum Strains

Phenolic compounds are critical components of wine, influencing its color [38], taste, and structural properties, while also being associated with antioxidant activity [39] and potential health benefits [40]. Seven individual phenolics were detected via HPLC (Figure 6). Among these, kaempferol was the least abundant at 1.96 mg/L, whereas catechin was the most abundant at 354.92 mg/L. Gallic acid content decreased significantly post-MLF, with strain-specific variations: P101 induced the greatest reduction, with content dropping from an initial 90.02 mg/L to 60.55 mg/L. MLF mediated by the tested L. plantarum strains also reduced catechin content, with P101 causing the most substantial decline—from 354.9 mg/L to 313.6 mg/L. Syringic acid content remained relatively unchanged in J43-inoculated wines post-MLF but decreased in those fermented with XJ25 and P101. In contrast, chlorogenic acid content decreased specifically in wines fermented with J43.

3.7. Volatile Compound and PCA Analysis of Marselan Wines Fermented with Different L. plantarum Strains

L. plantarum strains exhibit a more diverse enzyme profile compared to O. oeni strains, particularly in terms of aroma-modifying enzymes such as β-glucosidase and phenolic acid decarboxylase [31]. Qualitative and quantitative analyses of volatile components in Marselan wine were conducted post-AF and post-MLF with three L. plantarum strains. As shown in Table. 2, a total of 62 aroma compounds were detected in Marselan wine after AF, comprising 20 esters, 13 alcohols, 14 terpenes, 5 Aldehydes and Ketones, 4 volatilephenols, 5 fatty acids, and 1 pyrazine. Alcohols accounted for the highest total content among all volatile compounds in the wine samples, with 4 substances exhibiting an odor activity value (OAV) exceeding 0.1. Post-MLF, the total alcohol content decreased: P101-fermented wines showed the highest total alcohol concentration (123,826.78 μg/L), while XJ25-fermented wines had the lowest (93,050.44 μg/L). Esters represented the most diverse class of volatile compounds, contributing fruity notes to the wine aroma. Among these, 5 substances had an OAV greater than 1. After MLF, the contents of ethyl acetate increased in all strains, with P101 showing significantly higher levels than XJ25. Notably, J43 fermentation resulted in a 3-fold increase in ethyl 2-methylbutyrate compared to pre-MLF levels, which is known to make a significant contribution to the fruity aroma characteristics of wine [43,44]. As illustrated in the heatmap (Figure 7A), MLF with L. plantarum strains increased ethyl lactate content, which may impart richer milk and butter notes, thereby enhancing the complexity and elegance of the wine aroma [45]. Additionally, MLF with L. plantarum strains led to increased contents of isobutanol, 1-heptanol, and benzyl alcohol in Marselan wine, which can contribute orange-like and fatty aromas, while other alcohols remained relatively unchanged.

Terpenes, as signature components of grape varieties, contribute floral, citrus, and tropical fruit aromas[46]. MLF with J43 and P101 strains increased the contents of linalool and nerol in wine samples, which are associated with floral and citrus aromas. MLF with all three L. plantarum strains increased the contents of fatty acids such as octanoic acid; at low concentrations, these can impart lactic acid notes, but at high concentrations, they may introduce fatty or other undesirable odors [47]. No significant differences in aldehyde and ketone contents were observed in wine samples before and after MLF.

To further characterize the differences in volatile compounds among wines fermented with different strains, principal component analysis (PCA) was performed on substances with OAV values exceeding 0.1 across all treatment groups. As shown in Figure 7B, the first principal component (PC1) accounted for 61.8% of the total variance, while the second principal component (PC2) explained 27.2%. Most esters, alcohols, and terpenes were concentrated in the first and second quadrants, with ethyl 2-methylbutyrate and nerol localized in the fourth quadrant and phenolic compounds in the third quadrant. Non-MLF wine samples clustered in the third quadrant, whereas wines fermented with J43 and XJ25 were positioned in the fourth quadrant, closely associated with ethyl 2-methylbutyrate and nerol—compounds known to contribute apple-like aroma and floral/grass characteristics to Marselan wine. Substances with positive contributions to wine aroma, including ethyl acetate, linalool, ethyl lactate, and ethyl isovalerate, were clustered in the first quadrant. Notably, P101-fermented wines also fell within this quadrant, exhibiting proximity to ethyl butanoate, ethyl acetate, and linalool, compounds associated with characteristic fruity aromas such as banana, strawberry, and pineapple. The clear separation observed in the PCA plot between Marselan wines subjected to L. plantarum-mediated MLF and non-MLF samples indicates that MLF with L. plantarum strains enhances the aroma complexity of Marselan wine.

Table 2. Concentrations, odor thresholds, and aroma descriptors of volatile compounds in the wines before and after MLF with different strains.

Compounds	Aroma Concentration (μg/L)						Thresholds	OAV	Description
	AF-END	J43	XJ25		P101
Esters (20)
Ethyl acetate	31,039.50±187.59d	33,513.88±331.35c	37,143.24±107.61b		39,475.58±2466.94a		7500	＞1	Banana, Strawberry
Ethyl butanoate	110.39±1.74b	101.86±1.36c	111.98±2.62b		122.11±5.25a		400	＞0.1	Strawberry, Banana, Pineapple
Ethyl 2-methylbutanoate	1.44±0.33b	6.71±0.07a	6.02±0.91a		0.00±0.00c			＞1	Apple
N-propyl propionate	0.00±0.00b	0.00±0.00b	0.00±0.00b		6.69±0.60a
Ethyl 3-methylbutanoate	0.70±0.02b	0.94±0.02a	0.93±0.03a		0.92±0.06a		3	＞0.1	Strawberry, Sweet Fruity
Isoamyl acetate	140.24±4.96a	131.82±2.54a	130.62±7.22a		141.67±0.60a		160	＞0.1	Banana, Fruity
Isobutyl isovalerate	86.09±1.00a	85.31±0.04a	87.45±4.63a		88.51±3.63a
Ethyl caproate	183.10±1.95a	126.95±1.86c	124.48±1.25c		145.04±5.67b		14	＞1	Green apple, Strawberry
Ethyl lactate	5,696.61±109.69c	8,452.46±72.65b	10,514.85±890.43a		10,125.87±617.30a		14000	＞0.1	Milk, Butter
Methyl caprylate	41.82±1.17a	0.21±0.04b	0.01±0.00b		0.44±0.06b
Ethyl caprylate	806.04±4.23a	483.09±10.60c	408.13±17.75d		549.00±18.75b		5	＞1	Pineapple, Pear, Floral
Ethyl 3-hydroxybutanoate	259.54±0.03b	282.64±3.83a	278.41±11.78ab		294.31±14.40a		200000	＜0.1
Ethyl pelargonate	12.95±0.15a	9.89±0.24b	7.31±0.62c		9.11±0.37b		200	＜0.1	Fruity
Ethyl caprate	551.25±10.49a	417.11±4.64b	339.74±17.62c		426.03±8.34b		200	＞1	Fruity
Diethyl succinate	101.82±1.08b	112.89±3.12a	105.02±0.91b		111.31±0.06a		6000	＜0.1	Fruity, Melon
Ethyl benzoate	3.99±0.00a	3.98±0.01a	3.95±0.01b		3.96±0.00b
Ethyl 9-decanoate	0.00±0.00c	1.09±0.12a	0.18±0.00bc		0.32±0.03b
Phenylethyl acetate	12.41±0.16a	12.21±0.39ab	10.97±0.34c		11.41±0.13bc		250	＜0.1	Rose, Sweet
Ethyl laurate	9.11±0.00ab	9.32±0.35a	8.43±0.31b		9.37±0.27a		1500	＜0.1	Sweet, Beeswax
Ethyl nonacosanoate	5.53±0.02a	5.66±0.07a	5.64±0.11a		5.65±0.06a
Alcohols (13)
1-Propanol	24,180.88±210.70ab	21,199.51±567.61b	26,965.99±2592.89a		26,482.49±1510.11a		306000	＜0.1	Mello, Mature fruity, Floral and Green
Isobutanol	20,490.93±572.41b	21,429.31±98.03a	21,817.10±117.27a		21,755.57±438.80a		40000	＞0.1	Chemical
1-Butanol	1,008.94±5.06b	1,007.35±0.65b	1,043.25±15.06a		1,043.41±3.64a		150000	＜0.1	Fruity, Green, Malt, Chemical, Alcohol
4-Methyl-2-pentanol	2,066.00±0.00a	2,066.00±0.00a	2,066.00±0.00a		2,066.00±0.00a
Isoamylol	59,476.92±258.64a	50,966.47±129.62c	26,789.52±816.49d		57,480.29±1242.33b		30000	＞1	Caramel, Lipid
4-Methyl-1-pentanol	20.08±0.04ab	18.84±0.31c	19.93±0.16b		20.47±0.09a		50000	＜0.1
3-Methyl-1-pentanol	16.08±0.13a	13.59±0.24b	15.62±0.23a		16.43±0.75a		500	＜0.1
Hexyl alcohol	837.77±1.03b	766.70±8.22c	817.96±7.20b		844.34±14.77a		8000	＜0.1
1-Heptanol	28.59±0.08a	27.23±0.15b	27.49±0.03b		27.74±0.22b		2500	＜0.1
Octanol	5.21±0.20a	3.14±0.01c	2.84±0.04c		3.50±0.21b		40	＞0.1	Floral
1-Decanol	28.41±2.12c	52.91±1.00a	46.23±1.94b		56.45±2.10a		400	＞0.1	Orange, Fatty
Benzyl alcohol	537.12±2.66c	589.82±11.65b	630.14±23.13a		618.40±20.07ab		200000	＜0.1	Roast, Fruity
Phenethyl alcohol	12,456.63±35.15a	12,143.24±1000.30a	11,895.77±395.40a		12,682.42±867.55a		400	＞1	Orange, Fatty
Terpenes (14)
Limonene	0.88±0.07a	0.30±0.00b	0.58±0.04ab		0.77±0.16ab		10	＞0.1	Sweet, Citrus, lemon
Linalool	21.66±0.01c	21.83±0.03a	21.74±0.01b		21.87±0.03a		25	＞0.1	Floral, Citrus
1-Octen-3-ol	15.11±0.10a	13.56±0.11c	13.30±0.23c		14.12±0.34b
(E)-3-Hexenol	38.01±1.45a	33.42±0.03b	35.02±0.37b		35.19±0.65b
(Z)-Rose oxide	3.35±0.00a	3.34±0.00c	3.35±0.00d		3.35±0.00b
(Z)-3-Hexenol	1.64±0.02d	20.21±0.10b			17.85±0.56c	24.47±1.86a
α-Terpineol	12.36±0.12b	19.96±6.09a			12.13±0.03b	12.70±0.11b	250	＜0.1	Lilac
Nerol	127.68±1.17c	145.60±10.33ab			148.60±2.52a	133.38±0.52b	400	＞0.1	Floral, Green
Ethyl geranyl ether	12.12±0.02a	12.02±0.03a			12.09±0.10a	12.16±0.11a
Terpinen-4-ol	9.88±0.02b	12.39±1.68a			10.38±0.05ab	10.38±0.03ab
(E)-Beta-farnesene	3,219.34±38.03a	2,256.72±40.30c			1,935.72±64.89d	2,417.84±61.92b
Citronellol	8.40±0.35a	6.87±0.69b			5.95±0.16b	6.50±0.09b	100	＜0.1	Green, Lilac, Rose
Nerolido	9.83±0.02a	9.95±0.21a			9.97±0.27a	9.83±0.02a	400	＜0.1	Green, Floral
Dodecatrienol	81.28±0.00b	83.38±1.62ab			85.22±0.95a	82.61±0.35b
Aldehydes and Ketones (5)
2-Hexenal	401.26±1.77a	244.66±11.87c			237.31±2.94c	291.43±16.09b
Nonanal	0.02±0.01a	0.00±0.00b			0.00±0.00b	0.01±0.01b	2.5	＜0.1	Citrus
α-Ionone	10.54±0.25a	9.96±0.19ab			8.74±0.10c	9.40±0.67bc
(E)-5-Butyldihydro-4-methyl-2(3H)-furanone	227.48±0.01c	227.84±0.15a			227.46±0.12c	227.69±0.09ab
Ionone	9.45±0.01a	9.47±0.01a			9.45±0.01a	9.45±0.00a
Volatile Phenols(4)
Phenol	16.16±0.01a	15.48±0.73ab			15.07±0.10b	15.27±0.01b	30	＞0.1
4-Ethylguaiacol	125.70±0.03b	125.56±0.02c			125.62±0.03c	125.79±0.04a
4-Ethyl-pheno	66.10±0.02a	66.16±0.09a			66.12±0.04a	66.21±0.03a
2,4-Di-t-butylphenol	177.36±0.10a	179.34±5.71a			185.39±8.94a	181.06±2.40a
Fatty acids (5)
Propanoic acid	8,232.04±120.08b	8,853.37±137.58a		8,869.48±261.78a		8,880.14±243.06a
Isovaleric acid	80.37±65.62c	196.47±16.12b		302.09±15.53a		202.13±16.33b	3000	＜0.1		Sour, Cheese
Octanoic acid	353.73±7.14b	498.00±65.80a		435.19±14.94ab		501.68±30.48a	500	＞1		Sour, Cheese, Fatty
Decanoic acid	188.28±2.28c	245.13±17.88ab		232.35±5.00b		255.88±5.69a	1000	＞0.1		Sour, Fatty
Benzoic acid	23,418.69±3.04a	23,416.63±5.84a		23,423.12±9.65a		23,433.27±9.87a
Other (1)
2-Methoxy-3-isobutyl pyrazine	0.94±0.00a	0.93±0.00b		0.93±0.00b		0.93±0.00b

The mean ± SD of measurements made in triplicates were used to reflect the characteristic values. Different lowercase letters in each of these columns indicate a significant difference (p < 0.05), and the same letter indicates that the difference is not significant (p > 0.05). AF-END indicates the wine before MLF.4. Conclusions.

Table 2. Concentrations, odor thresholds, and aroma descriptors of volatile compounds in the wines before and after MLF with different strains.

Compounds	Aroma Concentration (μg/L)						Thresholds	OAV	Description
	AF-END	J43	XJ25		P101
Esters (20)
Ethyl acetate	31,039.50±187.59d	33,513.88±331.35c	37,143.24±107.61b		39,475.58±2466.94a		7500	＞1	Banana, Strawberry
Ethyl butanoate	110.39±1.74b	101.86±1.36c	111.98±2.62b		122.11±5.25a		400	＞0.1	Strawberry, Banana, Pineapple
Ethyl 2-methylbutanoate	1.44±0.33b	6.71±0.07a	6.02±0.91a		0.00±0.00c			＞1	Apple
N-propyl propionate	0.00±0.00b	0.00±0.00b	0.00±0.00b		6.69±0.60a
Ethyl 3-methylbutanoate	0.70±0.02b	0.94±0.02a	0.93±0.03a		0.92±0.06a		3	＞0.1	Strawberry, Sweet Fruity
Isoamyl acetate	140.24±4.96a	131.82±2.54a	130.62±7.22a		141.67±0.60a		160	＞0.1	Banana, Fruity
Isobutyl isovalerate	86.09±1.00a	85.31±0.04a	87.45±4.63a		88.51±3.63a
Ethyl caproate	183.10±1.95a	126.95±1.86c	124.48±1.25c		145.04±5.67b		14	＞1	Green apple, Strawberry
Ethyl lactate	5,696.61±109.69c	8,452.46±72.65b	10,514.85±890.43a		10,125.87±617.30a		14000	＞0.1	Milk, Butter
Methyl caprylate	41.82±1.17a	0.21±0.04b	0.01±0.00b		0.44±0.06b
Ethyl caprylate	806.04±4.23a	483.09±10.60c	408.13±17.75d		549.00±18.75b		5	＞1	Pineapple, Pear, Floral
Ethyl 3-hydroxybutanoate	259.54±0.03b	282.64±3.83a	278.41±11.78ab		294.31±14.40a		200000	＜0.1
Ethyl pelargonate	12.95±0.15a	9.89±0.24b	7.31±0.62c		9.11±0.37b		200	＜0.1	Fruity
Ethyl caprate	551.25±10.49a	417.11±4.64b	339.74±17.62c		426.03±8.34b		200	＞1	Fruity
Diethyl succinate	101.82±1.08b	112.89±3.12a	105.02±0.91b		111.31±0.06a		6000	＜0.1	Fruity, Melon
Ethyl benzoate	3.99±0.00a	3.98±0.01a	3.95±0.01b		3.96±0.00b
Ethyl 9-decanoate	0.00±0.00c	1.09±0.12a	0.18±0.00bc		0.32±0.03b
Phenylethyl acetate	12.41±0.16a	12.21±0.39ab	10.97±0.34c		11.41±0.13bc		250	＜0.1	Rose, Sweet
Ethyl laurate	9.11±0.00ab	9.32±0.35a	8.43±0.31b		9.37±0.27a		1500	＜0.1	Sweet, Beeswax
Ethyl nonacosanoate	5.53±0.02a	5.66±0.07a	5.64±0.11a		5.65±0.06a
Alcohols (13)
1-Propanol	24,180.88±210.70ab	21,199.51±567.61b	26,965.99±2592.89a		26,482.49±1510.11a		306000	＜0.1	Mello, Mature fruity, Floral and Green
Isobutanol	20,490.93±572.41b	21,429.31±98.03a	21,817.10±117.27a		21,755.57±438.80a		40000	＞0.1	Chemical
1-Butanol	1,008.94±5.06b	1,007.35±0.65b	1,043.25±15.06a		1,043.41±3.64a		150000	＜0.1	Fruity, Green, Malt, Chemical, Alcohol
4-Methyl-2-pentanol	2,066.00±0.00a	2,066.00±0.00a	2,066.00±0.00a		2,066.00±0.00a
Isoamylol	59,476.92±258.64a	50,966.47±129.62c	26,789.52±816.49d		57,480.29±1242.33b		30000	＞1	Caramel, Lipid
4-Methyl-1-pentanol	20.08±0.04ab	18.84±0.31c	19.93±0.16b		20.47±0.09a		50000	＜0.1
3-Methyl-1-pentanol	16.08±0.13a	13.59±0.24b	15.62±0.23a		16.43±0.75a		500	＜0.1
Hexyl alcohol	837.77±1.03b	766.70±8.22c	817.96±7.20b		844.34±14.77a		8000	＜0.1
1-Heptanol	28.59±0.08a	27.23±0.15b	27.49±0.03b		27.74±0.22b		2500	＜0.1
Octanol	5.21±0.20a	3.14±0.01c	2.84±0.04c		3.50±0.21b		40	＞0.1	Floral
1-Decanol	28.41±2.12c	52.91±1.00a	46.23±1.94b		56.45±2.10a		400	＞0.1	Orange, Fatty
Benzyl alcohol	537.12±2.66c	589.82±11.65b	630.14±23.13a		618.40±20.07ab		200000	＜0.1	Roast, Fruity
Phenethyl alcohol	12,456.63±35.15a	12,143.24±1000.30a	11,895.77±395.40a		12,682.42±867.55a		400	＞1	Orange, Fatty
Terpenes (14)
Limonene	0.88±0.07a	0.30±0.00b	0.58±0.04ab		0.77±0.16ab		10	＞0.1	Sweet, Citrus, lemon
Linalool	21.66±0.01c	21.83±0.03a	21.74±0.01b		21.87±0.03a		25	＞0.1	Floral, Citrus
1-Octen-3-ol	15.11±0.10a	13.56±0.11c	13.30±0.23c		14.12±0.34b
(E)-3-Hexenol	38.01±1.45a	33.42±0.03b	35.02±0.37b		35.19±0.65b
(Z)-Rose oxide	3.35±0.00a	3.34±0.00c	3.35±0.00d		3.35±0.00b
(Z)-3-Hexenol	1.64±0.02d	20.21±0.10b			17.85±0.56c	24.47±1.86a
α-Terpineol	12.36±0.12b	19.96±6.09a			12.13±0.03b	12.70±0.11b	250	＜0.1	Lilac
Nerol	127.68±1.17c	145.60±10.33ab			148.60±2.52a	133.38±0.52b	400	＞0.1	Floral, Green
Ethyl geranyl ether	12.12±0.02a	12.02±0.03a			12.09±0.10a	12.16±0.11a
Terpinen-4-ol	9.88±0.02b	12.39±1.68a			10.38±0.05ab	10.38±0.03ab
(E)-Beta-farnesene	3,219.34±38.03a	2,256.72±40.30c			1,935.72±64.89d	2,417.84±61.92b
Citronellol	8.40±0.35a	6.87±0.69b			5.95±0.16b	6.50±0.09b	100	＜0.1	Green, Lilac, Rose
Nerolido	9.83±0.02a	9.95±0.21a			9.97±0.27a	9.83±0.02a	400	＜0.1	Green, Floral
Dodecatrienol	81.28±0.00b	83.38±1.62ab			85.22±0.95a	82.61±0.35b
Aldehydes and Ketones (5)
2-Hexenal	401.26±1.77a	244.66±11.87c			237.31±2.94c	291.43±16.09b
Nonanal	0.02±0.01a	0.00±0.00b			0.00±0.00b	0.01±0.01b	2.5	＜0.1	Citrus
α-Ionone	10.54±0.25a	9.96±0.19ab			8.74±0.10c	9.40±0.67bc
(E)-5-Butyldihydro-4-methyl-2(3H)-furanone	227.48±0.01c	227.84±0.15a			227.46±0.12c	227.69±0.09ab
Ionone	9.45±0.01a	9.47±0.01a			9.45±0.01a	9.45±0.00a
Volatile Phenols(4)
Phenol	16.16±0.01a	15.48±0.73ab			15.07±0.10b	15.27±0.01b	30	＞0.1
4-Ethylguaiacol	125.70±0.03b	125.56±0.02c			125.62±0.03c	125.79±0.04a
4-Ethyl-pheno	66.10±0.02a	66.16±0.09a			66.12±0.04a	66.21±0.03a
2,4-Di-t-butylphenol	177.36±0.10a	179.34±5.71a			185.39±8.94a	181.06±2.40a
Fatty acids (5)
Propanoic acid	8,232.04±120.08b	8,853.37±137.58a		8,869.48±261.78a		8,880.14±243.06a
Isovaleric acid	80.37±65.62c	196.47±16.12b		302.09±15.53a		202.13±16.33b	3000	＜0.1		Sour, Cheese
Octanoic acid	353.73±7.14b	498.00±65.80a		435.19±14.94ab		501.68±30.48a	500	＞1		Sour, Cheese, Fatty
Decanoic acid	188.28±2.28c	245.13±17.88ab		232.35±5.00b		255.88±5.69a	1000	＞0.1		Sour, Fatty
Benzoic acid	23,418.69±3.04a	23,416.63±5.84a		23,423.12±9.65a		23,433.27±9.87a
Other (1)
2-Methoxy-3-isobutyl pyrazine	0.94±0.00a	0.93±0.00b		0.93±0.00b		0.93±0.00b

The mean ± SD of measurements made in triplicates were used to reflect the characteristic values. Different lowercase letters in each of these columns indicate a significant difference (p < 0.05), and the same letter indicates that the difference is not significant (p > 0.05). AF-END indicates the wine before MLF.4. Conclusions.

In this study, 342 indigenous L. plantarum strains were characterized, among which 30 strains exhibited robust tolerance to harsh winemaking environments. Through comprehensive evaluation, the superior strains J43 and P101 were identified, showing excellent malic acid consumption capacity and adaptability to winemaking stress factors. During MLF, these strains efficiently consumed L-malic acid and produced lactic acid within 8 days. MLF mediated by J43 and P101 significantly influenced anthocyanin contents in wines, while altering organic acid contents. However, these strains had almost no effect on wine basic physicochemical parameters, color parameters, and individual phenolic contents. Comprehensive aroma profiling of Marselan wines undergoing MLF revealed that J43 and P101 enhanced the production of some key aroma compounds, Increased contents of ethyl acetate, ethyl lactate, ethyl 2-methylbutyrate, and nerol, and other such compounds contributing to more intense aromatic characteristics. The selection of these indigenous strains offers distinctive wine attributes while supporting oenological biodiversity. As L. plantarum has not been widely adopted as wine starter culture, the strains J43 and P101 present significant potential for application in wine production and as innovative fermentation agents, thereby creating new avenues for enhancing winemaking quality and diversity. This study also highlights the importance of regional microbial biodiversity in developing locally adapted winemaking solutions, which contribute to innovation and sustainability in the global wine industry.