Leuconostoc mesenteroides and application thereof in wine brewing

Abstract

The invention relates to the technical field of microorganisms, in particular to leuconostoc mesenteroides and application of the leuconostoc mesenteroides in wine brewing, the leuconostoc mesenteroides is specifically leuconostoc mesenteroides NWAFU 4027, the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.29308, and the preservation date is December 13, 2023. The invention further discloses a preparation method of the leuconostoc mesenteroides. The low-malic acid dry red wine is prepared by using the leuconostoc mesenteroides leavening agent in combination with a commercial saccharomyces cerevisiae leavening agent on the basis of a mixed bacteria reverse sequence fermentation process, and the malic acid level in grape mash can be reduced to 0.1 g / L or below within 5 days; the method shortens the malic acid-lactic acid fermentation time in the wine brewing process, and has no adverse effect on alcoholic fermentation of saccharomyces cerevisiae and production of flavor compounds.

Description

A strain of Leuconostoc mesenteroides and its application in winemaking Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a strain of Leuconostoc mesenteroides and its application in winemaking. Background Technology

[0002] Malolactic fermentation is a crucial step in producing high-quality wine. Catalyzed by malate-lactase in lactic acid bacteria, malic acid in grape juice is converted into lactic acid. This process transforms the sharp taste of malic acid into the smooth taste of lactic acid, reducing the wine's acidity, promoting its microbial stability, and enhancing the complexity of its flavor and aroma, thereby improving the wine's sensory qualities.

[0003] To date, the most widely used malolactic fermentation strategy in the winemaking industry is sequential fermentation, which involves inoculating lactic acid bacteria after yeast alcoholic fermentation to carry out malolactic fermentation. However, the disadvantage of this process is that after alcoholic fermentation, the high ethanol content and low acidity in the wine are unfavorable to the survival of lactic acid bacteria, easily leading to slow or failed malolactic fermentation.

[0004] Mixed-culture reverse fermentation, which involves inoculating lactic acid bacteria before yeast inoculation for malolactic fermentation, can effectively shorten the malolactic fermentation time. This is mainly because grape juice is richer in nutrients than yeast fermentation broth and does not contain alcohol or other potential yeast-derived inhibitors, which is conducive to the growth and reproduction of lactic acid bacteria.

[0005] However, mixed-culture reverse fermentation processes place high demands on lactic acid bacteria strains. Conventional lactic acid bacteria strains are difficult to adapt to this special fermentation mode, and this problem is even more pronounced when considering the current application of *Leuconostoc mesenteroides* in wine fermentation. *Leuconostoc mesenteroides*, a typical strain of the *Leuconostoc* genus, is one of the most widely used lactic acid bacteria in malolactic fermentation of wine. With its strong malic acid degradation ability and positive flavor contribution characteristics, it has been proven to effectively improve the taste and enhance the complexity of wine aromas. It has already been applied to some extent in conventional sequential fermentation processes. Some studies and patents have also confirmed that it can enhance the aroma layers of wine by producing flavor substances such as diacetyl through metabolism, while simultaneously improving the microbial stability of the wine and preventing flavor degradation caused by subsequent contamination by other microorganisms. However, most of the Leuconostoc mesenteroides strains currently used in the industry are selected to be adapted to traditional sequential fermentation processes. Their characteristics do not match the special requirements of mixed reverse sequential fermentation. Furthermore, Leuconostoc mesenteroides strains are highly specific, with significant differences in metabolic capacity and environmental tolerance among different strains. Some strains may even produce excessive volatile acids and other undesirable metabolites, affecting the quality of the wine.

[0006] Specifically, the requirements for *Leuconostoc mesenteroides* in the mixed-culture reverse-sequential fermentation process are mainly reflected in the following aspects: 1. Tolerance to the native environment of grape must: It needs to tolerate the high levels of free sulfur dioxide, natural acidity, polyphenols, and antibacterial substances in fresh grape must to ensure rapid initiation of growth and metabolism after inoculation, without inhibition or delay. This differs from the low-acid, high-ethanol environment after alcoholic fermentation in traditional sequential fermentation, and is also a common shortcoming of conventional *Leuconostoc mesenteroides* strains. 2. Highly efficient degradation capacity for malic acid: It should possess a more efficient malic acid-lactic acid conversion capacity, enabling rapid and complete degradation of malic acid before the start of alcoholic fermentation, ensuring sufficient initiation and thorough conversion of malolactic fermentation. This is the core advantage of *Leuconostoc mesenteroides* as a malolactic fermentation strain. However, to adapt to reverse-sequential fermentation, its degradation efficiency needs to be further enhanced to meet the process requirements of shortening the fermentation cycle, aligning with its proven high-efficiency acid-reducing potential in fruit wine acid reduction. 3. Strong early growth and reproduction capacity, adaptable to low inoculum sizes: It requires rapid proliferation under grape must nutrient conditions to achieve sufficient cell density for fermentation efficiency, meeting the needs of industrial production with lower inoculum sizes and reducing production costs. This contrasts with the current application of *Leuconostoc mesenteroides* in other fruit wine fermentations, which require higher inoculum sizes to achieve ideal results. This is also one of the key areas for improvement in adapting it to reverse fermentation. 4. Compatibility with subsequent brewing yeasts: The strain's metabolism does not produce byproducts that inhibit yeast growth (such as excessive acetic acid or undesirable flavor compounds), and it does not interfere with normal alcoholic fermentation after yeast inoculation, ensuring a smooth transition between the two fermentation stages and avoiding fermentation failure due to strain imbalance. This is a key issue that urgently needs to be addressed in the application of *Leuconostoc mesenteroides* in mixed-culture fermentation, as most existing strains have not been screened and optimized for yeast compatibility. 5. Positive contribution to fermentation safety and flavor: It does not produce excessive volatile acids or other harmful substances, while improving the acidity of the wine, enhancing the complexity of the aroma and the smoothness of the taste, satisfying the sensory requirements of high-quality wines. However, when adapting to reverse fermentation, its flavor regulation ability needs to be further strengthened to avoid the generation of undesirable flavors caused by its strain specificity. Summary of the Invention

[0007] This invention provides a strain of Leuconostoc mesenteroides and its application in winemaking. A pure culture of Leuconostoc mesenteroides subsp. mesenteroides NWAFU 4027 is used in combination with a commercial brewing yeast starter to prepare low-malic acid dry red wine based on a mixed-culture reverse-sequential fermentation process. This can reduce the malic acid content in grape must to below 0.1 g / L within 5 days, shortening the malolactic fermentation cycle by about 10 days.

[0008] On the one hand, the present invention provides a Leuconostoc mesenteroides subsp. mesenteroides NWAFU 4027, which was deposited at the China General Microbiological Culture Collection Center on December 13, 2023, with accession number CGMCC No. 29308. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0009] The Enterobacter mesenchymalus NWAFU 4027 has a 16S rDNA sequence as shown in SEQ ID NO:1.

[0010] The Leuconostoc mesenteroides NWAFU 4027 was isolated from grape must during the mid-to-late stage of alcoholic fermentation at a Ningxia winery in October 2021. It has the following physiological characteristics: (1) It has good tolerance to acid: After being cultured at 25°C for 96 h in MRS liquid medium at pH 3.6, 3.4 and 3.2, the OD600 values ​​were 1.61±0.02, 1.29±0.03 and 0.22±0.01, respectively, and it can survive and proliferate slowly in a low pH environment.

[0011] (2) It has high tolerance to SO2. After being cultured at 25°C for 24 h in MRS liquid medium with SO2 concentrations of 50 mg / L, 75 mg / L and 100 mg / L, the OD600 values ​​were 2.20±0.01, 2.09±0.01 and 2.05±0.01, respectively.

[0012] (3) It has a high malic acid conversion capacity. After being cultured in Cabernet Sauvignon grape juice at a constant temperature of 25℃ for 24 h, the malic acid conversion rate reached 45.92±1.83%, which was significantly higher than that of other test strains and reference strains.

[0013] (4) It is tolerant to alcohol and can survive normally in an environment with an alcohol concentration of 12% (v / v).

[0014] On the other hand, the present invention provides a pure culture fermentation agent of Leuconostoc mesenteroides, the preparation method of which includes the following steps: (1) inoculating Leuconostoc mesenteroides NWAFU 4027 strain into lactic acid bacteria culture medium and continuously activating and culturing it at 30℃~40℃ for 2~4 generations; (2) isolating the bacterial cells from the activated bacterial culture and washing it 1-3 times with sterile buffer or sterile water; (3) resuspending the washed bacterial cells in sterile medium and correcting the viable count to 10. 8 ~10 11 The pure fermentation agent is obtained by measuring CFU / mL.

[0015] In another aspect, the present invention also provides an application of Leuconostoc mesenteroides in winemaking, specifically referring to the use of the above-mentioned Leuconostoc mesenteroides or pure culture starter culture in combination with commercial wine yeast starter culture, and the use of mixed culture reverse sequential fermentation process to prepare low malic acid dry red wine.

[0016] Furthermore, the application includes the following steps: after pretreatment by sequentially adding SO2 and pectinase to fresh grape must, first inoculating with Leuconostoc mesenteroides fermentation agent to start malolactic fermentation, and then inoculating with commercial wine yeast for alcoholic fermentation to obtain low malic acid dry red wine.

[0017] Furthermore, the amount of SO2 added is 45 mg / L to 55 mg / L, the amount of pectinase added is 15 mg / L to 25 mg / L, the inoculum size of Leuconostoc mesenteroides is 1% to 2%, and the inoculum size of commercial brewing yeast is 10 mg / L. 7 CFU / mL.

[0018] Furthermore, the amount of SO2 added is 50 mg / L; the amount of pectinase added is 20 mg / L; the inoculum amount of Leuconostoc mesenteroides is 1%; the temperature of the malic acid-lactic acid fermentation is 24℃~25℃, and the fermentation time is 4 d~5 d; the temperature of the alcoholic fermentation is 25℃, and the fermentation time is 15 d.

[0019] Furthermore, the low-malic acid dry red wine has a malic acid content of less than 0.1 g / L, a reducing sugar content of less than 3.0 g / L, and an alcohol content of approximately 12%.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The Leuconostoc mesenteroides NWAFU 4027 of the present invention has high acid tolerance, high SO2 tolerance and high malic acid conversion ability, and can tolerate 12% (v / v) alcohol environment, which solves the technical pain point that conventional Leuconostoc mesenteroides is difficult to adapt to the special environment of reverse fermentation, and fills the gap of special strains for this process.

[0021] (2) The pure culture fermentation agent of Leuconostoc mesenteroides of the present invention can reduce the malic acid content in grape must to below 0.1 g / L within 4-5 days, shortening the malic acid-lactic acid fermentation cycle by about 10 days compared with the traditional sequential fermentation, significantly improving fermentation efficiency and reducing time cost and energy consumption in industrial production.

[0022] (3) The Leuconostoc mesenteroides NWAFU 4027 of the present invention has good compatibility with commercial brewing yeast. The metabolic process does not produce adverse byproducts that inhibit yeast growth, ensuring a smooth transition between malic acid-lactic acid fermentation and alcoholic fermentation, avoiding the risk of fermentation stagnation or failure, and improving the stability and controllability of the process.

[0023] (4) The fermentation product of Leuconostoc mesenteroides NWAFU 4027 of the present invention has high safety, does not produce excessive volatile acids and other harmful substances, and can effectively improve the acidity of the wine, enhance the complexity of the aroma and the smoothness of the taste, preserve the regional flavor characteristics of the wine, solve the product homogenization problem caused by commercial strains, and significantly improve the sensory quality of the wine. Attached Figure Description

[0024] Figure 1 shows the colony morphology of Leuconostoc mesenteroides NWAFU 4027 on MRS agar plates; Figure 2 shows the variation of malic acid concentration in grape must with fermentation time during the reverse fermentation process of Leuconostoc mesenteroides NWAFU 4027 starter culture and Saccharomyces cerevisiae FX10 starter culture, with the inoculation time of lactic acid bacteria recorded as day 0; Figure 3 shows the variation of malic acid concentration in grape must with fermentation time during the reverse fermentation process of Leuconostoc mesenteroides NWAFU 4027 starter culture and Saccharomyces cerevisiae D254 starter culture, with the inoculation time of lactic acid bacteria recorded as day 0. Detailed Implementation

[0025] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0026] Example 1: Isolation and Identification of Leuconostoc mesenteroides strain NWAFU 4027: In October 2021, the applicant collected grape must samples from a wine fermentation tank (alcohol content approximately 6%, not yet inoculated with lactic acid bacteria starter) undergoing alcoholic fermentation at a winery in Ningxia Hui Autonomous Region. These samples were then transported to the College of Food Science and Engineering, Northwest A&F University. The researchers isolated the target strain using the streak plate method on MRS agar plates, initially designating it as NWAFU 4027. The researchers extracted total genomic DNA from this strain using a bacterial genomic DNA extraction kit. Subsequently, PCR was used to amplify its 16S rDNA sequence using universal bacterial primers 27F and 1492R. The Sanger assay was then used to determine its nucleic acid sequence, which is shown in SEQ ID NO:1. Comparison of this nucleic acid sequence with existing sequences in the GenBank database confirmed that the strain was Leuconostoc mesenteroides. Researchers further tested the carbohydrate fermentation capacity of this strain using the API 50CH kit from bioMérieux, France, and ultimately confirmed that it was Leuconostoc mesenteroides subsp. mesenteroides.

[0027] Example 21: Using MRS broth medium supplemented with 0.5 g / L L-cysteine ​​hydrochloride, *Leuconostoc mesenteriae* strain NWAFU 4027 was continuously activated for three generations at 37°C. The third generation was cultured for 18 h until the stationary phase (OD200). 600 Bacterial suspensions with a concentration of approximately 1.0 μg / mL were used for subsequent physiological characteristic assays. All assays included four replicates and one blank control group (no bacterial strain inoculated, only the corresponding culture medium / grape juice added). Results are expressed as mean ± standard deviation. Data were initially processed using Excel, and statistical analysis was performed using Minitab 18.0 software. One-dimensional analysis of variance (ANOVA) was used to compare differences between groups, and Tukey's method was used for multiple comparisons. A p < 0.05 was considered statistically significant.

[0028] 2. Acid tolerance test: Prepare MRS liquid culture medium, adjust the pH to 3.2, 3.4, and 3.6 with hydrochloric acid, sterilize and cool, and measure the initial bacterial density (OD). 600 =0.02 mg / L activated bacterial solution, incubated at 25°C for 96 h, and the OD of each group of bacterial solutions was measured. 600 value.

[0029] The test results showed that *Leuconostoc mesenteroides* strain NWAFU 4027 could survive in the pH range of 3.2–3.6, and the OD values ​​under different pH conditions were [not specified]. 600The values ​​were as follows: 0.22±0.01 at pH 3.2 (survivable, slow growth), 1.29±0.03 at pH 3.4 (good growth, significant proliferation), and 1.61±0.02 at pH 3.6 (vigorous growth, rapid proliferation). Statistical analysis showed that the OD values ​​of the strains in the pH 3.6 group were significantly higher than those in the pH 3.4 and pH 3.2 groups. 600 The values ​​showed a significant difference (p < 0.05), indicating that the strain has good acid resistance.

[0030] 3. SO2 tolerance test: MRS liquid culture medium was prepared, sterilized, and cooled. Potassium metabisulfite solution was aseptically added to prepare mediums with SO2 concentrations of 50 mg / L, 75 mg / L, and 100 mg / L. The initial bacterial density (OD) was used as the criterion for determination. 600 =0.02 mg / L activated bacterial solution, incubated at 25°C for 24 h, and the OD of each group of bacterial solutions was measured. 600 value.

[0031] The results showed that *Leuconostoc mesenteroides* strain NWAFU 4027 could grow normally within a SO2 concentration range of 50–100 mg / L. OD values ​​varied under different SO2 concentrations. 600 The values ​​were: 2.20±0.01 at 50 mg / L (vigorous growth, rapid proliferation), 2.09±0.01 at 75 mg / L (good growth, normal proliferation), and 2.05±0.01 at 100 mg / L (normal growth, no significant inhibition); statistical analysis showed that the OD values ​​of the strains varied among the different SO2 concentration groups. 600 The values ​​showed no significant difference (p>0.05), indicating that the strain has high SO2 tolerance.

[0032] 4. Malic acid conversion capacity determination: Fresh Cabernet Sauvignon grape juice was filtered and sterilized, and the initial bacterial density (OD) was used as the metric. 600 =0.02% inoculated with activated bacterial solution and incubated at 25℃ for 24 h. After incubation, the sample was serially diluted, filtered through a filter membrane, and the malic acid content was detected by LC-MS-MS method. The malic acid conversion rate was calculated, and Lactobacillus plantarum ATCC 14917 was set as the reference strain.

[0033] LC-MS detection conditions: The chromatographic column was a Thermo Accucore aQ column (150 mm). The column was 2.1 mm and 2.6 μm thick, with an injection volume of 4 μL, a column temperature of 40 °C, and a mobile phase of 0.1% formic acid aqueous solution (phase A) and acetonitrile (phase B). The elution program was 0 min (phase A 100%, phase B 0%) → 4 min (hold) → 4.1 min (phase A 20%, phase B 80%) → 8 min (hold) → 8.1 min (phase A 100%, phase B 0%) → 12 min (end), with a flow rate of 0.2 mL / min. The ion source was ESI negative ion mode, with an auxiliary gas temperature of 320 °C, an ion transfer tube temperature of 350 °C, a spray voltage of 3.0 kV, and a PRM monitoring method. The malic acid precursor ion m / z was 133.01425, and the daughter ion m / z was 115.00243.

[0034] The results showed that the malic acid content in the grape juice of the blank control group was 2.38±0.05 g / L; after inoculation with the *Leuconostoc mesenteroides* strain NWAFU 4027 of this invention, the malic acid content in the grape juice was 1.28±0.08 g / L, and the malic acid conversion rate was 45.92±1.83%; the malic acid conversion rate of the reference strain ATCC 14917 was 28.65±1.52%; statistical analysis showed that there was a significant difference in the conversion rate between the *Leuconostoc mesenteroides* strain NWAFU 4027 of this invention and the reference strain (p<0.05), indicating that this strain has a high malic acid conversion capacity.

[0035] 5. Alcohol tolerance test: Prepare MRS liquid culture medium, sterilize and cool, adjust the alcohol concentration to 12% (v / v), and use the initial bacterial density OD... 600 =0.02 mg / L activated bacterial solution, incubated at 25°C for 24 h, and the OD of the bacterial solution was measured. 600 The value was set up, and a control group without alcohol was also set up.

[0036] The test results showed that at a 12% (v / v) alcohol concentration, the OD of the strain was... 600 The value was 1.89±0.04, indicating normal survival and good growth; the OD value of the alcohol-free control group strain was... 600 The value was 2.15±0.02; statistical analysis showed that the OD values ​​of the two groups were... 600 The values ​​showed no significant difference (p>0.05), indicating that the strain has good alcohol tolerance.

[0037] Leuconostoc mesenteroides strain NWAFU 4027 exhibits good acid resistance (surviving and proliferating at pH 3.2-3.6), high SO2 resistance (growing normally at 50-100 mg / L SO2), high malic acid conversion rate (45.92±1.83% conversion rate in 24 h), and alcohol resistance (surviving normally at 12% (v / v) alcohol). Its physiological characteristics are stable and suitable for the requirements of mixed-culture reverse-sequential fermentation process in winemaking, making it suitable for the production of low-malic acid dry red wine.

[0038] Example 3: Preparation of pure culture fermentation agent of Leuconostoc mesenteroides NWAFU 4027 strain. A 1% culture of Leuconostoc mesenteroides NWAFU 4027 strain was inoculated into MRS broth and cultured continuously at 36℃~37℃ for three generations. The bacterial cells were then separated by centrifugation, washed twice with sterile water, and resuspended in the same sterile water. The viable count was corrected to 10⁻⁶. 10 CFU / mL was used to obtain pure culture fermentation agent of Leuconostoc mesenteroides NWAFU 4027.

[0039] Example 4: 1. This example sets up 4 fermentation groups, with 3 replicates in each group. The specific grouping is as follows: Control group 1 (FX10 single-strain group): only inoculated with commercial brewing yeast starter FX10 (trade name ZYMAFLORE) TM FX10 (purchased from LAFFORT, France); Experimental group 1 (NWAFU 4027 + FX10 mixed culture group): first inoculated with Leuconostoc mesenteroides NWAFU 4027 pure culture starter, then inoculated with commercial Saccharomyces cerevisiae starter FX10; Control group 2 (D254 single culture group): only inoculated with commercial Saccharomyces cerevisiae starter D254 (trade name LALVIN / ICV) starter. ® D254 ® The samples were purchased from DANSTAR FERMENT AG (LALLEMAND); Experimental group 2 (NWAFU 4027+D254 mixed culture group): Leuconostoc mesenteroides NWAFU 4027 pure culture starter culture was first inoculated, followed by commercial brewing yeast starter culture D254.

[0040] 2. Operation steps of mixed culture reverse sequence fermentation process (1) Grape must pretreatment: Take fresh grape must (initial glucose concentration 104.6 g / L, fructose concentration 107.4 g / L, reducing sugar concentration 216.8 g / L, pH 3.9, lactic acid concentration 0.1 g / L, acetic acid concentration 0.0 g / L, malic acid concentration 2.6 g / L, tartaric acid concentration 3.5 g / L, citric acid concentration 0.4 g / L, total acid concentration 6.6 g / L), add 50 mg / L SO2, shake and mix well, and let stand at room temperature for 30 min; then add 20 mg / L pectinase, mix well again, and let stand at 36℃~37℃ for 10 h.

[0041] (2) Inoculation and malic acid-lactic acid fermentation of experimental groups: 1% pure culture of Leuconostoc mesenteroides NWAFU 4027 (live count 10) was inoculated into the pretreated grape must of experimental groups 1 and 2. 10 Malic acid-lactic acid fermentation was completed by constant temperature fermentation at 24℃~25℃ for 5 days (CFU / mL).

[0042] (3) Inoculation of alcoholic fermentation in control and experimental groups: Experimental group 1 and experimental group 2: After the completion of malic acid-lactic acid fermentation, inoculate at 10 7 Inoculation with commercial brewing yeast FX10 and D254 at CFU / mL rates, respectively; Control groups 1 and 2: after grape must pretreatment, directly inoculated with 10 7 Inoculation with CFU / mL of commercial brewing yeast starter FX10 and D254 was carried out respectively; all groups were fermented at a constant temperature of 25℃ for 10 days to complete alcoholic fermentation, and the time of lactic acid bacteria inoculation was recorded as day 0.

[0043] 3. Methods and results of determination of physicochemical indicators On the 5th, 10th and 15th day of fermentation (the end point of alcoholic fermentation), samples were taken from each group of grape must and the following physicochemical indicators were determined: (1) Concentration of sugar compounds The grape must was mixed with an equal volume of acetone, then centrifuged, and the supernatant was taken. The sugar compounds in the sample were quantitatively analyzed by the OrbitrapQ Extractive Focus high-resolution liquid chromatography-mass spectrometry system of Thermo Fisher Scientific. The target compounds were separated on a C18 column, and then the level of the target compounds in the sample was quantified based on the negative ion ionization mode, the parallel reaction monitoring data acquisition mode and the peak area external standard method quantification mode. The reducing sugar level in the sample was quantitatively analyzed by the reducing sugar micro-reduction reagent kit. The changes of these compounds during fermentation are shown in Table 1.

[0044] Table 1. Concentration of carbohydrate compounds in grape must fermented with different starter cultures at different fermentation times.

[0045] Note: Data are expressed as mean ± standard deviation (n=3); there are significant differences between means that do not share the same letter in the same row (p<0.05); the inoculation time of lactic acid bacteria is recorded as day 0; the concentrations of glucose, fructose and reducing sugar in unfermented grape must are 104.6, 107.4 and 216.8 g / L, respectively.

[0046] During fermentation, glucose, fructose, and reducing sugars in the grape must are gradually consumed. Compared to single-strain fermentation, mixed-strain fermentation typically results in grape must with a higher reducing sugar content, but this did not significantly negatively impact the alcoholic fermentation process. On day 15 of fermentation, the reducing sugar content in the mixed-strain fermented grape must did not exceed 3 g / L, meeting the national standard GB / T 15037-2006 "Wine" regarding the limit of total sugar in dry red wine (≤4 g / L).

[0047] (2) The ethanol concentration of grape must sample was mixed with an equal volume of acetone, then centrifuged, and the supernatant was collected. The sugar compounds in the sample were quantitatively analyzed using a Shimadzu 2014C gas chromatograph. After separation by a highly polar DB-WAX quartz capillary column, the target analytes were detected by an FID detector, and quantified using the external standard method based on peak area. The results are shown in Table 2.

[0048] Table 2. Concentration of ethanol in grape must fermented with different starter cultures at different fermentation times.

[0049] Note: Data are expressed as mean ± standard deviation (n=3); there are significant differences between means that do not share the same letter within the same column (p<0.05); the time of lactic acid bacteria inoculation is recorded as day 0.

[0050] On day 10 of fermentation, the ethanol concentration of grape must in all groups was approximately 10%, but the alcoholic fermentation process was not yet complete. On day 15 of fermentation, the ethanol concentration of grape must in all groups increased to approximately 12% (12.0%–12.3%), and there was no significant difference in ethanol concentration among the groups (p<0.05). This indicates that *Leuconostoc mesenteroides* strain NWAFU 4027 had no significant inhibitory effect on the alcoholic fermentation process of grape must. Furthermore, when the fermentation period of grape must in all groups was extended to 20 days, no significant difference in ethanol concentration was found compared with that on day 15. This indicates that the alcoholic fermentation process reached its endpoint on day 15.

[0051] (3) pH and organic acid levels were determined using a pH meter. The total acid level of the sample was determined by titration according to the national standard GB / T 15038-2006 "General Analytical Methods for Wine and Fruit Wine". The grape must was mixed with an equal volume of acetone, centrifuged, and the supernatant was collected. The organic acids in the sample were quantitatively analyzed using an Orbitrap Q Extractive Focus high-resolution liquid chromatography-mass spectrometry system. The target analytes were separated on a C18 column, and then quantified based on negative ionization mode, parallel reaction monitoring data acquisition mode, and peak area external standard method. The results are shown in Table 3.

[0052] Table 3. pH and organic acid levels of grape must fermented with different starter cultures at different fermentation times.

[0053] Note: Data are expressed as mean ± standard deviation (n=3); ND, not detected; significant differences exist between means that do not share the same letter within the same row (p<0.05); lactic acid bacteria inoculation time is recorded as 0 d; the pH of unfermented grape must is 3.9, and its concentrations of lactic acid, acetic acid, malic acid, succinic acid, tartaric acid, citric acid and total acid are 0.1, 0.0, 2.6, 0.0, 3.5, 0.4 and 6.6 g / L, respectively.

[0054] After inoculating grape must with Leuconostoc mesenteroides NWAFU 4027 starter culture, the malic acid level in the grape must decreased with prolonged fermentation time. By day 5 post-inoculation, the malic acid level had decreased from an initial 2.6 g / L to below 0.1 g / L, while the lactic acid level increased from an initial 0.1 g / L to 2.6 g / L. After inoculation with yeast starter culture, the malic acid level in the grape must did not decrease further with prolonged fermentation time, but remained at approximately 0.1 g / L. By day 15 post-lactate inoculation, there was no significant difference in total acid levels among the groups (p>0.05), maintaining a level of approximately 6.0 g / L.

[0055] (4) Determination of Polyphenolic Compound Content Polyphenolic compounds are important flavor substances in wine. Key polyphenolic compounds in grape must were quantitatively analyzed using a Q Extractive Focus Orbitrap high-resolution liquid chromatography-mass spectrometry (LC-MS) system. The target compounds were separated on a C18 column, and then quantified using negative ionization mode, parallel reaction monitoring data acquisition mode, and external standard peak area method. The results are shown in Table 4.

[0056] Table 4. Concentrations of polyphenolic compounds in grape must fermented with different starter cultures on day 15 of fermentation.

[0057] Note: Data are expressed as mean ± standard deviation (n=3); there are significant differences between means that do not share the same letter within the same row (p<0.05); the time of lactic acid bacteria inoculation is recorded as day 0.

[0058] Fifteen days after lactic acid bacteria inoculation (reaching the fermentation endpoint), the concentrations of key polyphenolic compounds in the grape must were analyzed in each group. For grape must fermented with *Saccharomyces cerevisiae* FX10, there was no significant difference in the levels of each measured polyphenolic compound in the mixed strain fermentation group compared to the single yeast strain fermentation group (p>0.05). For grape must fermented with *Saccharomyces cerevisiae* D254, there was no significant difference in the levels of all polyphenolic compounds except gallic acid in the mixed strain fermentation group compared to the single yeast strain fermentation group (p>0.05), while the gallic acid level was significantly reduced by 7.5%. Overall, the *Leuconostoc mesenteriae* NWAFU 4027 starter culture had no significant negative impact on the levels of polyphenolic compounds in red wine.

[0059] (5) The content of aroma compounds was determined using a Shimadzu GCMS-QP2010 Ultra gas chromatograph-mass spectrometer (GC-MS) to quantitatively analyze the key volatile compounds in the samples. Grape must was mixed with an equal volume of acetone, centrifuged, and the supernatant was collected. The target compounds were separated on a DB-WAX quartz capillary column, and data were acquired using selected ion mode. The target compounds were quantified using the external standard method based on peak area. The results are shown in Table 5.

[0060] Table 5. Concentrations of volatile flavor compounds in grape must fermented with different starter cultures at day 15 of fermentation.

[0061] Note: Data are expressed as mean ± standard deviation (n=3); there are significant differences between means that do not share the same letter within the same row (p<0.05); the time of lactic acid bacteria inoculation is recorded as day 0.

[0062] Volatile compounds determine the main aroma characteristics of wine. After 15 days of lactic acid bacteria inoculation (reaching the fermentation endpoint), absolute quantitative analysis of key volatile flavor compounds in each group of grape must was performed. For grape must fermented with *Saccharomyces cerevisiae* FX10, compared with the single-strain fermentation group, the content of isoamyl alcohol and β-phenylethanol in the mixed strain fermentation group was significantly reduced (p<0.05), by 8.2% and 8.3%, respectively, while other compounds showed no significant changes (p>0.05). For grape must fermented with *Saccharomyces cerevisiae* D254, compared with the single-strain fermentation group, the content of ethyl acetate in the mixed strain fermentation group was significantly increased (p<0.05), by 16.7%, while other compounds showed no significant changes (p>0.05). β-Phenylene alcohol is a compound with a sweet, rose-like floral aroma, and its threshold in red wine is extremely low (1.8 mg / L). Isoamyl alcohol has a relatively high threshold in red wine (400 mg / L), and the isoamyl alcohol content in the grape must of all groups in this study was below this threshold. Therefore, the slight decrease in the concentrations of β-phenylethanol and isoamyl alcohol caused by the lactic acid bacteria inoculation mentioned above will not have a significant impact on the typical flavor characteristics of red wine. In conclusion, the Leuconostoc mesentery inoculum NWAFU 4027 fermentation inoculum has no significant negative impact on the levels of volatile flavor compounds in red wine.

[0063] The above provides a detailed description of the *Leuconostoc mesenteroides* strain provided by this invention and its application in the preparation of low-malic acid dry red wine. The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A strain of Leuconostoc mesenteroides, characterized in that, The Leuconostoc mesenteroides mentioned is specifically Leuconostoc mesenteroides subsp. mesenteroides NWAFU 4027, with accession number CGMCCNo.29308.

2. The Leuconostoc mesenteroides according to claim 1, characterized in that, The Enterobacter mesenchyme has a 16S rDNA sequence as shown in SEQ ID NO:

1.

3. A pure culture fermentation agent of Leuconostoc mesenteroides, characterized in that, The pure culture fermentation agent of Leuconostoc mesenteroides contains Leuconostoc mesenteroides NWAFU 4027 as described in claim 1.

4. The application of Leuconostoc mesenteroides in winemaking, characterized in that, The application specifically refers to using the Leuconostoc mesenteroides of claim 1 or 2 or the pure culture fermentation agent of claim 2 to brew and prepare low malic acid dry red wine.

5. The application according to claim 4, characterized in that, The application specifically refers to the use of Leuconostoc mesenteroides as described in claim 1 or the pure culture fermentation agent as described in claim 2 in combination with commercial brewing yeast fermentation agent, and the use of mixed culture reverse sequential fermentation process to prepare low malic acid dry red wine.

6. The application according to claim 5, characterized in that, Includes the following steps: After pretreatment by adding SO2 and pectinase to fresh grape must, malic acid-lactic acid fermentation is initiated by inoculating with Leuconostoc mesenteroides fermentation agent, followed by alcoholic fermentation with commercial wine yeast to obtain low malic acid dry red wine.

7. The application according to claim 6, characterized in that, The amount of SO2 added is 45 mg / L to 55 mg / L, the amount of pectinase added is 15 mg / L to 25 mg / L, the inoculum size of Leuconostoc mesenteroides is 1% to 2%, and the inoculum size of commercial brewing yeast is 10 mg / L. 7 CFU / mL.

8. The application according to claim 6, characterized in that, The temperature for malic acid-lactic acid fermentation is 24℃~25℃, and the fermentation time is 4 days~5 days; the temperature for alcoholic fermentation is 24℃~25℃, and the fermentation time is 10 days~15 days.

9. The application according to any one of claims 5 to 6, characterized in that, The low-malic acid dry red wine has a malic acid content of less than 0.1 g / L, a reducing sugar content of less than 3.0 g / L, and an alcohol content of 12%~13%.

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