Saccharomyces cerevisiae cx51 and application thereof in red wine brewing

Abstract

This invention relates to the field of microbial technology. Specifically, it relates to a highly colonizing and aroma-producing brewing yeast, CX51, and its application in red wine production. This brewing yeast, CX51, is deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 19, 2025, with accession number CGMCC No. 39063. In multi-strain mixed fermentation, CX51 effectively suppresses the proliferation of native brewing yeasts and maintains a significant competitive advantage over commercial brewing yeasts, exhibiting outstanding colonization ability. Fermentation of 'Marselle' wine solely with CX51 significantly increases the content of ethanol esters and acetate esters in the wine, enhancing floral and fruity aromas and optimizing the aroma composition and sensory quality. Brewing yeast CX51 has broad prospects for industrial application in the production of specialty red wines.

Description

Technical Field

[0001] This invention relates to the field of microbial technology. Specifically, it relates to a strong colonization ability of aroma-producing brewing yeast CX51 and its application in red wine production. Background Technology

[0002] Alcoholic fermentation in wine is a complex biological process dominated by yeast. Saccharomyces cerevisiae plays a central role in converting sugars in grape juice into alcohol, while also participating in the formation of aroma compounds through secondary metabolism. Whether the metabolic potential of Saccharomyces cerevisiae translates into flavor contributions in the final wine depends on its competitive ability within the fermentation system. The fermentation system is not a sterile environment, but a dynamic and competitive environment where various microorganisms coexist. Target strains must compete with native yeasts, non-Saccharomyces cerevisiae, and bacteria to acquire nutrient resources, maintain population dominance, and secure a dominant position in fermentation in order to complete alcoholic fermentation and contribute to the wine's style. This ability to establish a population, maintain activity, and achieve dominance in a complex environment is called colonization ability. Colonization ability is a core indicator for evaluating the application value of Saccharomyces cerevisiae, directly affecting whether it can achieve population dominance in a complex environment and contribute flavor compounds to the final wine.

[0003] The Helan Mountain East Foothills region of Ningxia, as an important emerging wine-producing area in China, has made significant progress in recent years in the exploration, isolation, identification, screening, and application of native wine yeast resources, providing core microbial support for the development of distinctive wines from the region. In the isolation and screening of native wine yeasts from the Helan Mountain East Foothills region of Ningxia, colonization ability is a core indicator. The value of studying native strains lies in their ability to better adapt to the local physicochemical conditions of grape juice and the natural microbial ecological environment. This adaptability must be determined through the ability to survive continuously in competition and establish population dominance. Only when the strain has effectively colonized the fermentation system can its unique metabolic characteristics and aroma potential truly be reflected in the perceived flavor quality of the final wine. However, existing research still has significant shortcomings. There is a lack of systematic and in-depth research on the colonization ability of these superior strains in real wine fermentation, making it difficult to fully realize the application value of native yeasts. Therefore, this invention addresses the above-mentioned technical needs by providing a native wine yeast strain with excellent colonization performance, superior brewing characteristics, and outstanding aroma production ability. Summary of the Invention

[0004] The technical objective of this invention is to overcome the existing challenges in yeast application and provide a brewing yeast strain, CX51, with outstanding colonization ability and excellent aroma-producing performance, and its application in red wine production. The brewing yeast CX51 can be used in the production of red wines such as 'Marselan', stably growing and multiplying during alcoholic fermentation, and significantly increasing the content of ester flavor compounds in the wine.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A strain of brewing yeast CX51, selected from naturally fermented Cabernet Sauvignon wine from the Helan Mountain East Foothills wine region in Ningxia, has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 19, 2025, with accession number CGMCC No. 39063.

[0007] After being mixed with 6 native Saccharomyces cerevisiae strains and 1 commercial yeast for fermentation, the Saccharomyces cerevisiae strain CX51 could coexist with the commercial yeast in the multi-strain mixed inoculation fermentation experiment, and the proportion reached 22.73% after fermentation.

[0008] The brewing yeast CX51 exhibited strong competition and colonization capabilities when fermented with commercial yeasts EC118, D254, QA23, and RX60 and native yeasts CX57, CX59, and M41. At the end of fermentation, the percentages of CX51 strains were as follows: EC118 fermentation group 41.67%, QA23 fermentation group 25%, D254 fermentation group 54.17%, RX60 fermentation group 100%, CX57 fermentation group 82.61%, and M41 fermentation group 54.17%.

[0009] The brewing yeast with accession number CGMCC No.39063 provided by this invention was preserved in YPD matrix at -80℃.

[0010] A highly colonizing aroma-producing wine yeast, CX51, and its application in red wine production.

[0011] CX51 was used for individual inoculation and fermentation.

[0012] The application can alter the body and aroma composition of wine, increasing its floral and fruity aromas.

[0013] The brewing yeast CX51 can increase the content of ethyl acetate, isoamyl acetate, ethyl isobutyrate, ethyl hexanoate, ethyl octanoate, ethyl laurate, and ethyl palmitate in 'Marselan' wine.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] The brewing yeast CX51 provided by this invention has a strong colonization ability and can improve the final aroma of wine to a certain extent, enhancing the floral and fruity aroma of wine.

[0016] Biological Preservation Instructions

[0017] CX51, *Saccharomyces cerevisiae*, was deposited on November 19, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 39063. Attached Figure Description

[0018] Figure 1 Interdelta fingerprints of 10 different genotypes of Saccharomyces cerevisiae;

[0019] Figure 2 The colonization of 10 different genotypes of brewer's yeast during mixed fermentation;

[0020] Figure 3 The colonization of CX51 and other strains through mixed fermentation in pairs;

[0021] Figure 4 The maximum growth of CX51 and other strains at high N concentrations;

[0022] Figure 5 The maximum growth of CX51 and other strains at low N concentrations;

[0023] Figure 6 The colonization status of CX51 and other strains at different stages in small-scale experiments;

[0024] Figure 7 The colonization status of CX51 and other strains at different stages in pilot-scale experiments;

[0025] Figure 8 The morphological characteristics of CX51 on the WLN plate are shown. Detailed Implementation

[0026] This invention provides a highly colonizing aroma-producing wine yeast, CX51, and its application in winemaking. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately alter and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0027] The composition of the culture medium involved in the examples is as follows:

[0028] YPD yeast nutrient medium: yeast extract, 10 g / L; peptone, 20 g / L; glucose, 20 g / L; agar, 20 g / L.

[0029] WLN nutrient agar medium: tryptone, 5 g / L; yeast extract, 4 g / L; glucose, 50 g / L; potassium dihydrogen phosphate, 0.55 g / L; potassium chlorate, 0.425 g / L; calcium chloride, 0.125 g / L; magnesium chloride, 0.125 g / L; ferric chloride, 0.0025 g / L; manganese sulfate, 0.0025 g / L; bromocresol green, 0.022 g / L; agar, 20 g / L.

[0030] Synthetic culture medium (simulated grape juice): Sugars and acids (g / L): Glucose (100), Fructose (100), Malic acid (5), Citric acid (5), Tartaric acid (3); Inorganic salts (mg / L): Potassium dihydrogen phosphate (750), Magnesium sulfate heptahydrate (250), Potassium sulfate (500), Sodium chloride (200), Calcium chloride dihydrate (155), Magnesium sulfate monohydrate (4), Zinc sulfate (4), Sodium molybdate dihydrate (1), Cobalt chloride hexahydrate (0.4), Copper sulfate pentahydrate (1), Boric acid (1), Potassium iodide (1); Vitamins (mg / L): Inositol (20), Nicotinic acid (2), Pyridoxine hydrochloride (0.25), Calcium pantothenate (1.5), Thiamine hydrochloride (0.25), Biotin (0.003); Nitrogen source (m g / L: Ammonium chloride (460), L-glutamine (505.3), L-threonine (75.9), L-leucine (48.4), L-arginine (374.4), L-tryptophan (179.3), L-alanine (145.3), L-glutamic acid (120.4), L-serine (78.5), L-aspartic acid (44.5), L-valine (44.5), L-phenylalanine (37.9), L-isoleucine (32.7), L-histidine (32.7), L-methionine (31.4), L-tyrosine (18.3), L-glycine (18.3), L-lysine (17.0), L-cysteine ​​(13.1), and L-proline (612.6).

[0031] The Saccharomyces cerevisiae CX51 of this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39063 and deposit date of November 19, 2025.

[0032] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0033] Example 1: Analysis of the colonization ability of mixed fermentation of 10 different genotype Saccharomyces cerevisiae strains

[0034] Four different genotypes of commercial Saccharomyces cerevisiae EC118, QA23, D254, and RX60 were activated and then inoculated in equal proportions into 100 mL shake flasks containing 50 mL of synthetic culture medium. The total inoculum size was 1 × 10⁻⁶. 6 The concentration of CFU / mL was increased, and 10 mL of sterile water was injected into the fermentation plug after inoculation and sealed. The fermentation process was measured using the carbon dioxide loss method. When the weight change was less than 0.2 g / 100 mL for two consecutive days, it was considered that no further weight loss had occurred, and the fermentation was considered to have ended.

[0035] After fermentation, 1 mL of wine sample was serially diluted. The appropriate dilutions were then plated onto WLN nutrient agar plates, with two replicates per dilution. The plates were incubated upside down at 28°C for 2-3 days. Plates with 30-100 single colonies were selected for colony morphology observation, and approximately 48 single colonies were randomly selected for Interdelta fingerprint analysis.

[0036] PCR amplification was performed using primers delta12: 5'-TCAACAATGGAATCCCAAC-3' (SEQ ID NO:1) and delta21: 5'-CATCTTAACACCGTATATGA-3' (SEQ ID NO:2) to obtain different genotypes within the *Saccharomyces cerevisiae* species. Amplification system: 1 μL template DNA, 1 μL each of primers delta12 and delta21, 12.5 μL 2 × Phanta® FlashMaster Mix (Dye Plus), and double-distilled water (dd H2O) to a final volume of 25 μL. PCR amplification conditions: pre-denaturation 98℃, 30 s; denaturation 98℃, 10 s; annealing 50℃, 5 s; extension 72℃, 30 s; 30 cycles. 6 μL of the amplification product was electrophoresed on a 1.5% agarose gel for 1.0 h at a constant voltage of 100 V. The results were displayed and images were acquired using the automated gel imaging and analysis system ChampGel6000.

[0037] Figure 1 Interdelta fingerprints of 10 *Saccharomyces cerevisiae* strains were obtained. After fermentation, 24 single colonies were randomly selected for DNA extraction and electrophoresis analysis. The population percentage of each strain is shown in the figure. Figure 2 Studies have confirmed that the colonization ability of CX51 in the mixed fermentation of 7 strains is affected by commercial yeasts. When mixed with D254 and RX60, the colonization ability is stronger and it can survive stably (Table 1). Figure 5 The colony morphology of CX51 on WLN plates.

[0038] Table 1. Colonization of CX51 in fermentation with a mixed inoculation of 7 strains.

[0039]

[0040] Example 2: Analysis of the colonization ability of Saccharomyces cerevisiae CX51 mixed with 6 different genotypes during fermentation.

[0041] Example 1 demonstrates that CX51 has a strong colonization ability after multi-strain mixed fermentation. To further prove its colonization, CX51 was mixed with commercial yeasts EC118, QA23, D254, RX60 and native yeasts CX59, M41 for inoculation in pairs. The colonization was investigated by the proportion of CX51 present after fermentation.

[0042] EC118, QA23, D254, RX60, CX59, M41, and CX51 were activated in YPD liquid medium. Then, CX51 and the other yeasts were inoculated at a 1:1 ratio into 100 mL shake flasks containing 50 mL of synthetic medium. After inoculation, 10 mL of sterile water was injected into the fermentation plug to seal the flask. The fermentation process was measured using the carbon dioxide loss method. Fermentation was considered complete when the weight change was less than 0.2 g / 100 mL for two consecutive days, indicating that no further weight loss had occurred.

[0043] After fermentation, 1 mL of wine sample was serially diluted. The appropriate dilutions were then plated onto WLN nutrient agar plates, with two replicates per dilution. The plates were incubated upside down at 28°C for 2-3 days. Plates with 30-100 single colonies were selected for colony morphology observation, and approximately 48 single colonies were randomly selected for Interdelta fingerprint analysis.

[0044] PCR amplification was performed using primers delta12: 5'-TCAACAATGGAATCCCAAC-3' (SEQ ID NO:1) and delta21: 5'-CATCTTAACACCGTATATGA-3' (SEQ ID NO:2) to obtain different genotypes within the *Saccharomyces cerevisiae* species. Amplification system: 1 μL template DNA, 1 μL each of primers delta12 and delta21, 12.5 μL 2 × Phanta® FlashMaster Mix (Dye Plus), and double-distilled water (dd H2O) to a final volume of 25 μL. PCR amplification conditions: pre-denaturation 98℃, 30 s; denaturation 98℃, 10 s; annealing 50℃, 5 s; extension 72℃, 30 s; 30 cycles. 6 μL of the amplification product was electrophoresed on a 1.5% agarose gel for 1.0 h at a constant voltage of 100 V. The results were displayed and images were acquired using the automated gel imaging and analysis system ChampGel6000.

[0045] After the fermentation of CX51 and yeast in a dual-strain mixture, the population proportions of each strain are detailed in Table 2. When CX51 was fermented with commercial yeast RX60, the strain proportion reached 100%, while when fermented with native yeast CX57, the proportion was 82.61%. These dual-strain fermentation results fully demonstrate that CX51 possesses excellent colonization ability and can effectively suppress the growth and reproduction of other reference strains.

[0046] Table 2. Colonization of CX51 in mixed fermentation of two strains

[0047]

[0048] Example 3: Growth of Saccharomyces cerevisiae CX51 and other strains under different nitrogen sources

[0049] In wine fermentation systems, nitrogen is a crucial nutrient affecting the growth and reproduction of brewer's yeast, the initiation of sugar metabolism, and the fermentation process. Nitrogen concentrations are generally categorized into three types: low nitrogen, normal nitrogen, and high nitrogen. Low nitrogen typically refers to a concentration of around 140–150 mg N / L, which can easily lead to slow yeast growth, delayed fermentation, or even stagnation. Normal nitrogen usually refers to a suitable nitrogen level that meets the basic growth needs of yeast and ensures smooth alcoholic fermentation, such as around 250–300 mg N / L. High nitrogen refers to an environment with sufficient or excessive nitrogen supply, such as 400 mg N / L and above. This can promote biomass accumulation and fermentation initiation in some strains, but excessive nitrogen can also lead to metabolic imbalance or affect cell survival in later stages. Therefore, in the screening process for strongly colonizing brewer's yeast, the maximum growth rate of strains under different nitrogen concentrations helps to determine the colonization ability of the strains.

[0050] The selected strongly colonizing strain CX51 and other strains were subjected to a 1×10⁻⁶ PCR. 6 CFU / mL were inoculated into high-nitrogen and low-nitrogen synthetic media, respectively, and incubated statically at 28°C. OD was measured every 12 h. 600nm Values, plotting maximum biomass (OD) max The comparison charts were used to analyze the growth of the strains under different nitrogen source concentrations and their correlation with colonization ability.

[0051] The results are as follows Figure 4 and Figure 5 As shown, strain CX51 exhibited excellent growth capacity under different nitrogen source concentrations. At high nitrogen concentrations, its biomass accumulation efficiency was significantly higher than all tested strains, indicating a stronger ability to absorb and utilize amino acids and ammonium nitrogen. It can rapidly proliferate and accumulate biomass under sufficient nitrogen conditions. This physiological characteristic allows it to maintain a high population proportion when competing with various commercial yeast strains, confirming that efficient biomass accumulation in high-nitrogen environments is essential for stable coexistence and maintaining a competitive advantage. At low nitrogen concentrations, CX51 still grew normally with a high maximum biomass, demonstrating good low-nitrogen tolerance, further confirming its strong growth capacity and providing important physiological support for its excellent colonization ability.

[0052] Example 4: Comparative Study of Colonization and Aroma Production Capacity of Saccharomyces cerevisiae CX51 and Other Strains in a Small-Scale Experiment

[0053] To investigate the colonization performance and aroma characteristics of the highly colonizing strain CX51 in actual winemaking, a fermentation experiment was conducted using 'Marselan' grape juice as the substrate. CX51 and other highly colonizing strains obtained through screening were inoculated into 100 mL of 'Marselan' grape juice, while commercial wine yeasts QA23, D254, and RX60 served as control groups; the inoculation amount was 1×10⁻⁶. 6 The fermentation process was monitored every 24 hours by weighing CFU / mL.

[0054] Samples were taken at four fermentation stages: early stage (relative density 1.060–1.070), middle stage (relative density 1.020–1.030), late stage (relative density 0.995–1.000), and end stage. Interdelta fingerprinting technology was used to dynamically track the population succession of each strain, and their colonization performance was evaluated based on the competitive ability among strains. At the same time, the volatile aroma components of the wine samples after fermentation were detected, and their composition and content were analyzed to comprehensively evaluate the brewing application performance of strains with high colonization ability.

[0055] The determination of aroma compounds in wine was performed using a headspace-solid phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS): 5 mL of wine sample was accurately added to a 20 mL sample vial, along with 1.5 g of sodium chloride and 10 μL of 4-methyl-2-pentanol internal standard (1.0009 g / L). The vial was sealed with a cap containing a PTFE septum and placed on a multi-functional autosampler. Extraction was performed using a polydimethylsilane / carbon sieve / divinylbenzene extraction head (DVB / CAR / PDMS 50 / 30 µm). Extraction conditions: adsorption at 40℃ for 30 min, followed by extraction with shaking at 500 r / min for 30 min. The capillary column used was an HP-INNOWAX 60m × 0.25 mm × 0.25 μm (Folsom, CA, USA), with a flow rate of 1 mL / min. The carrier gas was 99.999% pure helium, and the injection mode was 5:1 split injection. The column oven temperature program was as follows: initial column temperature 50℃, held for 1 min, then increased to 220℃ at 3℃ / min, held for 3 min. Finally, increased to 240℃ at 10℃ / min, held for 5 min, for a total run of 63 min. The mass spectrometry interface temperature was 280℃, and the ion source temperature was 230℃. Electron impact ionization (EI) was used as the ionization method, with an ion source energy of 70 eV and a mass scan range of 30–350 m / z. Two technical parallels were set up for each sample.

[0056] Qualitative analysis: Peak identification was performed using an Automated Modeling Convolution and Identification System (AMDIS), and the corresponding retention indices (RIs) were calculated based on the retention times of aroma compounds. The qualitative analysis of aroma compounds was then completed by matching the retention indices of these compounds with those of aroma standards, mass spectrometry information, or the National Institute of Standards and Technology (NIST) 2014 mass spectrometry database.

[0057] Quantitative Analysis: 4 g / L glucose and 4 g / L tartaric acid were added to an ethanol-water solution (15% v / v), and the pH was adjusted to 3.4 with sodium hydroxide to serve as a simulated solution for the wine standard curve. Aroma substance standards were dissolved in chromatographic-grade ethanol to prepare a standard stock solution. This stock solution was added to 50 ml of the simulated solution, and the solution was continuously diluted 15 times to obtain simulated solutions of different concentrations covering the range of aroma substance content in the wine sample. After analyzing the 15 simulated solutions, standard curves for each aroma substance were obtained. The ratio of the peak area of ​​the corresponding aroma substance standard to the peak area of ​​the internal standard response at different concentrations was calculated, and the concentration of each substance was obtained by substituting this ratio into the standard curve. For aroma substances with available standards, quantification was performed using their standard curves; for aroma substances without available standards, semi-quantitative analysis was performed using standard curves for aroma substances with similar carbon number, chemical structure, and functional groups.

[0058] (1) Colonization capacity analysis during fermentation

[0059] Depend on Figure 6 The results showed that strain CX51 has outstanding colonization advantage. In the early stage of fermentation, the proportion of this strain was as high as 98%, indicating that it can quickly adapt to the fermentation environment by rapidly absorbing environmental nutrients, gain fermentation advantage and achieve efficient colonization. By the end of fermentation, the proportion of CX51 can reach 100%, ensuring the stable and efficient completion of alcohol fermentation, which fully demonstrates the excellent environmental adaptability and fermentation stability of this strain.

[0060] (2) Analysis of aroma production characteristics of small-scale fermentation

[0061] The CX51 strain exhibits significant aroma advantages during wine fermentation. Its high content of higher alcohols such as isoamyl alcohol and isobutanol imparts a banana sweetness and enhances the body of the wine. Simultaneously, the strain has a low total fatty acid content, effectively preventing off-flavors and ensuring flavor purity. Among the esters contributing to the core fruit aromas, phenethyl acetate and ethyl 2-hydroxy-4-methylvalerate are present in the highest amounts among all strains, contributing rich coconut cream and tropical fruit aromas, creating a differentiated flavor profile. The content of benzene compounds is significantly higher than that of commercial strains, with phenethyl alcohol, accounting for over 99%, imparting a rich rose and honey texture to the wine, significantly enhancing the complexity of the floral aromas. Furthermore, its C6 / C9 alcohol concentration is moderate, and the key substance hexanol has an OAV value below 0.5, resulting in a gentle herbal aroma. The synergistic effect of various aroma components ultimately forms a stable flavor profile characterized by rich fruit aromas, a full-bodied flavor, and elegant floral notes.

[0062] Table 3. Concentration of aroma substances after alcoholic fermentation in the small-scale experiment.

[0063]

[0064]

[0065]

[0066]

[0067] Example 5: Comparative Study on Colonization and Aroma Production Capacity of Saccharomyces cerevisiae CX51 and Other Strains in Pilot-Scale Experiment

[0068] The above-mentioned small-scale fermentation results confirm that the brewer's yeast CX51 possesses excellent colonization ability. To further evaluate its application potential in the industrial production of wine, especially its aroma-producing performance, this study conducted a pilot-scale fermentation experiment on 'Marselan' wine using a 3L fermentation tank. A natural fermentation group (hereinafter referred to as ZR), a commercial yeast D254 group, and a native yeast M41 group were set up as control groups. After the grape juice was allowed to settle and the supernatant was collected, the activated D254, CX51, and M41 strains were respectively used at a concentration of 1×10⁻⁶. 6 The inoculum was individually injected into 3L of 'Marselan' grape juice at a concentration of CFU / mL and allowed to ferment statically at 18°C. The fermentation endpoint was determined by the carbon dioxide loss method, i.e., the weight change was less than 0.2 g / 100 mL for two consecutive days, which was considered as the completion of fermentation. Subsequently, the final fermentation sample was taken for detection and analysis of major metabolites and volatile aroma substances.

[0069] The sample was filtered through a 0.22 μm aqueous phase filter membrane (PES), and the contents of organic acids, glucose, fructose, glycerol, and ethanol were determined. The instrument used was an HPLC 1200 (Agilent Technologies, Santa Clara, CA, USA), and the ion exchange column was an HPX-87 HAminex ion-exchange column (300 × 7.8 mm, Bio-Rad Laboratories, Hercules, CA, USA). Isocratic elution was performed using 5 mmol / L H₂SO₄ as the mobile phase at a flow rate of 0.6 mL / min. Organic acids were determined using a UV detector (Agilent, USA), with an injection volume of 20 μL, a column temperature of 60 °C, and a measurement time of 25 min. Glucose, fructose, glycerol, and ethanol were determined using a differential refractive index detector (Agilent, USA), with an injection volume of 20 μL, a column temperature of 45 °C, and a measurement time of 30 min.

[0070] (1) Analysis of the colonization of CX51

[0071] The CX51 strain exhibits excellent colonization ability during the fermentation of 'Marselan' wine, second only to the commercial yeast D254, and has good adaptability to the fermentation environment. After inoculation, this strain can quickly enter the proliferation phase, with a population ratio of about 98% in the early stage of fermentation. It can effectively suppress the growth of miscellaneous bacteria by virtue of its population advantage, and can reach 100% in the middle of fermentation, quickly establishing a dominant population position, which fully demonstrates its outstanding colonization ability.

[0072] (2) Analysis of main metabolite results

[0073] As shown in Table 4, all experimental groups successfully completed alcoholic fermentation, with reducing sugar utilization rates exceeding 99%. The CX51 group had a higher glycerol content than the other three groups, and its citric acid content was also relatively high, second only to the M41 group. Furthermore, the contents of key metabolic components such as ethanol, tartaric acid, lactic acid, and acetic acid showed no significant differences compared to other groups. In summary, CX51 not only ensures complete fermentation but also possesses excellent metabolic characteristics, including strong glycerol accumulation and a relatively balanced organic acid profile.

[0074] Table 4. Content of main metabolites in each experimental group after alcoholic fermentation of 'Marselan' grape juice

[0075]

[0076] (3) Determination of aroma substances in pilot-scale experiments

[0077] As shown in Table 5, a total of 97 volatile aroma compounds were identified in all experimental groups, namely 7 C6 / C9 compounds, 14 higher alcohols, 6 fatty acids, 9 acetates, 27 ethyl esters, 12 other esters, 12 terpenes and norisoprene compounds, 3 benzenes, and 7 other compounds.

[0078] The total content of aroma compounds in 'Marselan' wines varied significantly. The content of native strain CX51 (576450.42 μg / L) and naturally fermented ZR group (579769.51 μg / L) was significantly higher than that of commercial yeast D254 group (517575.88 μg / L). The total aroma content of CX51 group was 11.4% higher than that of D254 group, indicating a stronger overall aroma synthesis ability.

[0079] Among the four core aroma categories—higher alcohols, acetates, ethyl acetates, and terpenes and norisoprene—the CX51 group had the highest content, increasing by 10.6%, 16.9%, 39.2%, and 57.9% respectively compared to the D254 group. This enhanced the aroma intensity and complexity of the red wine. Commercial yeast D254 was dominated only by benzene compounds, with other key aroma components lower than those in CX51. While the ZR group had a higher total content, it had the highest content of C6 / C9 compounds, which could easily lead to a raw, green odor. The aroma composition of the CX51 group included caprylic acid, isobutyric acid, isovaleric acid, ethyl laurate (lipidic and fruity aromas), ethyl palmitate (waxy and creamy aromas), α-terpineol (clove and sweet aromas), and β-damascone (floral and cooked apple aromas). The total content of higher alcohols, acetates, ethyl acetates, and terpenes was the highest among the four groups. The content of key aroma substances such as isobutanol, 1-heptanol, isoamyl acetate, ethyl laurate, α-terpineol, and geraniol was also the highest, which can impart a rich aroma of fresh fruit, rose, and clove to the wine. At the same time, the content of 1-octen-3-ol was significantly lower than that of the ZR group, and the furfural content was the lowest among the four groups, effectively reducing the risk of undesirable flavors such as mushroom. In summary, the local strain CX51 has a significant advantage in the synthesis of core aroma substances. The total aroma content is 11.4% higher than that of D254, the key aroma OAV value is higher, the fruit, floral and fermentation aromas are rich and complex, and the control of undesirable odor substances is better. The aroma balance is significantly better than that of the ZR group.

[0080] Table 5. Concentration of aroma compounds in 'Marselan' wines after alcoholic fermentation

[0081]

[0082]

[0083]

[0084]

[0085] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The preservation number is CGMCC No.39063, which is Saccharomyces cerevisiae CX51.

2. The brewing yeast CX51 according to claim 1, characterized in that, Using simulated grape juice as the fermentation substrate, commercial Saccharomyces cerevisiae strains EC118, QA23, D254, and RX60 with different genotypes of native Saccharomyces cerevisiae strains CX51, CX57, CX59, M41, M43, and XL312 were mixed in equal proportions for inoculation and fermentation. The total inoculation amount was 1×10⁻⁶. 6 CFU / mL; In multi-strain mixed inoculation fermentation, the brewing yeast CX51 can coexist stably with commercial brewing yeast and fully participate in the entire alcohol fermentation process, with its strain ratio reaching 22.73% after fermentation.

3. The brewing yeast CX51 according to claim 2, characterized in that, Using simulated grape juice as the fermentation substrate, the wine yeast CX51 was inoculated with commercial wine yeasts EC118, QA23, D254, and RX60, and native yeasts CX57 and M41 at a 1:1 ratio, with a total inoculation amount of 1×10⁻⁶. 6 CFU / mL; The brewing yeast CX51 exhibited strong competitive and colonization abilities in the double-inoculation fermentation experiment. At the end of fermentation, the percentages of CX51 strains were as follows: EC118 fermentation group 41.67%, QA23 fermentation group 25%, D254 fermentation group 54.17%, RX60 fermentation group 100%, CX57 fermentation group 82.61%, and M41 fermentation group 54.17%.

4. The application of the brewing yeast CX51 according to claim 2 or 3 in red wine brewing.

5. The growth of *Saccharomyces cerevisiae* CX51 under different nitrogen source concentrations according to claim 4, characterized in that, CX51 can maintain normal growth in environments with varying nitrogen source concentrations, demonstrating excellent growth capabilities.

6. The application of the brewing yeast CX51 according to claim 5 in small-scale winemaking, characterized in that, The wine in question is 'Marseille' wine; Commercial brewing yeasts D254 and RX60, and native brewing yeasts CX51, M41 and M43 were inoculated and fermented separately. In the application described, CX51 demonstrated a significant colonization ability in real grape juice, rapidly establishing population dominance and stably completing alcoholic fermentation. The brewing yeast CX51 can significantly increase the content of floral and fruity aroma compounds in the wine sample.

7. The application of the brewing yeast CX51 according to claim 6 in pilot-scale winemaking, characterized in that, The wine in question is 'Marseille' wine; The brewing yeast CX51 can quickly absorb nutrients and dominate the fermentation process; The brewing yeast CX51 has the characteristics of high fructose conversion rate, high glycerol accumulation, and balanced organic acid content. The brewing yeast CX51 can significantly increase the content of key ester flavor compounds such as ethyl acetate, isoamyl acetate, and ethyl hexanoate in 'Marselan' wine.

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