Oenococcus oeni SDM1 and application thereof

Abstract

The invention discloses Oenococcus oeni SDM1 and application thereof, the Oenococcus oeni SDM1 is preserved in the China General Microbiological Culture Collection Center, the preservation name is Oenococcus oeni SDM1, the preservation date is April 28, 2025, the preservation number is CGMCC No.34393, and the preservation number is CGMCC No.34393; the oenococcus oeni SDM1 provided by the invention can produce beta-glucosidase (2.62 + / -0.08 mU / mL) at high yield, and has excellent tolerance (the oenococcus oeni SDM1 can survive under the conditions of 16% ethanol, pH 3.0 and 60 mg / L sulfur dioxide); compared with commercial oenococcus oeni OENO1, the oenococcus oeni SDM1 provided by the invention can enable the brewed dry red wine to have higher ester content (11658.21 + / -39.39 [mu] g / L), and the contents of isoamyl acetate, ethyl acetate, ethyl caprylate, ethyl caprate and ethyl hexanoate are obviously higher than those of the commercial oenococcus oeni OENO1, so that the brewed wine has rich flower fragrance and fruit fragrance, and the wine has the advantages of low cost, high yield and high quality. The tea has elegant and harmonious fragrance, mellow and full taste, and typical characteristics and production place characteristics.

Description

Technical Field

[0001] This invention relates to the field of industrial microbiology, and in particular to a type of chrysococcus faecium SDM1 and its applications. Background Technology

[0002] Wine is the world's second most consumed alcoholic beverage after beer, holding a significant position in global regional economies and international trade. In industrial wine production, selected commercial lactic acid bacteria are typically used for fermentation to ensure controllability and consistency. However, the widespread use of commercial lactic acid bacteria has led to product homogenization. Furthermore, domestic wine producers currently rely heavily on imported lactic acid bacteria agents. With the development of the wine industry, consumers are demanding higher quality wines, creating an urgent need for premium wines that reflect the characteristics of their respective regions and embody local features and styles.

[0003] In recent years, domestic and international studies have shown that *C. chrysogenum* can adapt to the harsh environmental conditions of wine, such as high ethanol content, low pH, high sulfur dioxide content, and low temperature. *C. chrysogenum* plays a positive role in enhancing the regional characteristics and flavor complexity of wine. The aroma substances in wine mainly include esters and alcohols, which impart fruity and floral aromas to wine, giving people a comfortable and pleasant feeling. For example, ethyl caprylate gives wine the flavors of apricot, pear, and banana; ethyl hexanoate has the flavor of green apple; and phenylethyl alcohol gives wine the flavors of rose and peach.

[0004] Furthermore, the β-glucosidase secreted by *C. sacchariformis* can hydrolyze bound aromatic compounds, releasing glycosides and monoterpenes that increase the aroma and chemical complexity of wine, raise the content of volatile compounds, and ultimately produce wines with distinctive regional flavors. Therefore, screening for *C. sacchariformis* strains with good environmental tolerance and high β-glucosidase production and applying them to wine production is of great significance for improving the flavor and quality of wines from specific regions. Summary of the Invention

[0005] The purpose of this invention is to provide a *Coccus vinifera* SDM1 and its application, in order to solve the problems mentioned above, such as poor environmental tolerance and low β-glucosidase activity of existing *Coccus vinifera* strains, as well as the lack of distinct regional style characteristics in wine products.

[0006] Oenococcus oeni SDM1, deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 28, 2025, with accession number CGMCC No. 34393.

[0007] The biological characteristics of the *S. chrysogenum* SDM1 provided by this invention are as follows: on ATB medium, the colonies are round, milky white, smooth, opaque, and less than 1 mm in diameter.

[0008] Oenococcus oeni SDM1 was selected from the natural fermentation liquid of Marselan grapes in Penglai, Shandong Province.

[0009] The *Sacchariformis* SDM1 strain provided by this invention can produce high levels of β-glucosidase and has strong environmental resistance. When applied to winemaking, it can significantly increase the total amount of aroma compounds and ester content in wine, helping to increase the flavor complexity of wine and giving it rich floral, pineapple, apple, pear, and banana aromas and sweetness. This allows the wine to have regional characteristics, reflect local features and style, and meet people's higher demands for wine quality.

[0010] Preferably, the 16S rDNA gene sequence of the *S. tumefaciens* SDM1 is shown in SEQ ID NO.1.

[0011] The β-glucosidase activity of *S. chrysogenum* SDM1 provided by this invention is (2.62±0.08 mU / mL); *S. chrysogenum* SDM1 can survive under conditions of 16% ethanol, pH 3.0 and 60 mg / L sulfur dioxide.

[0012] The application of *Saccharomyces cerevisiae* SDM1 in the preparation of a fermentation inoculum, comprising a fermentation inoculum containing the aforementioned *Saccharomyces cerevisiae* SDM1.

[0013] Preferably, the fermentation agent is a liquid agent, a semi-liquid agent, or a solid agent.

[0014] Chlorella vulgaris SDM1 was inoculated into ATB medium and fermented until the viable count reached 10⁻⁶. 9 A liquid bacterial agent was obtained with a concentration of cfu / mL or higher.

[0015] The above liquid bacterial agent was concentrated to obtain a semi-liquid bacterial agent.

[0016] The above concentration can be achieved using conventional concentration methods in the field, such as centrifugation and filtration, and the present invention does not impose any special requirements.

[0017] Add buffer solution and lyophilization protectant to the above semi-liquid bacterial agent to adjust the viable count to 10⁻⁶. 9 cfu / mL ~10 12 cfu / mL, freeze-dried to obtain solid bacterial agent.

[0018] The buffer solution can be a conventional buffer solution in the art, such as PBS buffer or physiological saline; the lyophilization protectant can also be a commonly used lyophilization protectant in the art, such as at least one of skim milk powder, trehalose, monosodium glutamate or glycerol.

[0019] Preferably, the viable concentration of *Saccharomyces cerevisiae* SDM1 in the fermentation agent is 10. 8 cfu / mL ~10 12 cfu / mL.

[0020] The application of *Sacchariformis SDM1* in winemaking, including the application of *Sacchariformis SDM1* or the aforementioned fermentation agent in winemaking; particularly in the fermentation of Cabernet Sauvignon wine.

[0021] The *S. sacchariformis* SDM1 strain provided by this invention can be widely used in the winemaking of various grape varieties in Chinese production areas, especially suitable for winemaking in Huailai County, Zhangjiakou City, Hebei Province. In terms of wine typicality, it is significantly superior to commercial lactic acid bacteria strains, indicating that the selected strain can reflect the typical characteristics of wines from its origin and has high application value.

[0022] The application of SDM1, a type of *Sacchariformis*, in a wine production method involves fermenting grape raw materials using either the aforementioned SDM1 or the aforementioned fermentation agent to obtain dry red wine.

[0023] Preferably, the specific steps of the wine production method include: adding potassium metabisulfite and pectinase to grape juice, mixing them evenly, inoculating the grape juice with the above-mentioned SDM1 of chrysophanolactam or the above-mentioned fermentation agent, mixing them evenly again, and fermenting to obtain dry red wine.

[0024] More specific steps are as follows: Select healthy Cabernet Sauvignon grapes, destem and crush them to obtain grape must. Using grape must as raw material, add potassium metabisulfite and pectinase to it. Then, inoculate the grape must with commercial wine yeast XPURE and the above-mentioned wine cocci SDM1 or the fermentation agent mentioned above. Select mixed fermentation and sequential fermentation as the fermentation method. Mix evenly and ferment at pH 3-4 and 20℃ to obtain dry red wine.

[0025] Preferably, the inoculum size of *Chlorella vulgaris* SDM1 is 1 × 10⁻⁶. 7 cfu / mL~1×10 8 cfu / mL.

[0026] Fermentation by *S. tumefaciens* SDM1 produces a series of metabolites that impart a unique and pleasant flavor to wine. The ester flavor compounds in these metabolites are directly influenced by the cell density of *S. tumefaciens* SDM1 and the fermentation method. Appropriate inoculum size and fermentation method can increase the content of flavor compounds in wine, resulting in a richer and more harmonious taste.

[0027] Preferably, the grape juice is obtained by destemming, crushing, and pressing Cabernet Sauvignon grapes from Huailai County, Zhangjiakou City, Hebei Province.

[0028] It should be noted that *C. tumefaciens* SDM1 needs to be activated before fermentation. Conventional activation methods in this field can be used, such as inoculating *C. tumefaciens* SDM1 into ATB medium and culturing it on a shaker at 28°C for 3 days.

[0029] Compared with the prior art, the present invention provides a *Chlorella vulgaris* SDM1 and its application, which has the following beneficial effects:

[0030] The *S. sacchariformis* SDM1 strain provided by this invention can produce high levels of β-glucosidase (2.62 ± 0.08 mU / mL) and exhibits excellent tolerance (it can survive under conditions of 16% ethanol, pH 3.0, and 60 mg / L sulfur dioxide).

[0031] Compared to the commercially available OENO1 sera hominis, the SDM1 sera hominis provided by this invention can produce dry red wines with a higher ester content (11658.21±39.39μg / L). Among them, the contents of isoamyl acetate, ethyl acetate, ethyl octanoate, ethyl decanoate, and ethyl hexanoate are significantly higher than those of the commercially available OENO1 sera hominis, giving the wines a rich floral and fruity aroma, an elegant and harmonious fragrance, a smooth taste, and typical characteristics and regional features. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 This is a schematic diagram of the colony morphology of *S. stomatologica* SDM1 on ATB solid medium according to the present invention.

[0034] Figure 2 This is a schematic diagram of the cell morphology of *S. chrysogenum* SDM1 in ATB medium according to the present invention. Detailed Implementation

[0035] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0036] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0037] Culture medium used in the examples:

[0038] ATB medium (1L): peptone 10g / L, glucose 10g / L, yeast extract 5g / L, magnesium sulfate heptahydrate 2g / L, manganese sulfate tetrahydrate 0.05g / L, tomato extract 25g / L, water 1000mL, pH 4.8±0.2, sterilized at 121℃ for 15min before use.

[0039] ATB isolation medium (1L): peptone 10g / L, glucose 10g / L, yeast extract 5g / L, magnesium sulfate heptahydrate 2g / L, manganese sulfate tetrahydrate 0.05g / L, tomato extract 25g / L, agar 20g, water 1000mL, pH 4.8±0.2, sterilized at 121℃ for 15min, then at approximately 60℃, 0.05g / L of actinomycin and 0.05g / L of vancomycin are prepared for use.

[0040] ATB solid medium (1L): peptone 10g / L, glucose 10g / L, yeast extract 5g / L, magnesium sulfate heptahydrate 2g / L, manganese sulfate tetrahydrate 0.05g / L, tomato extract 25g / L, agar 20g, water 1000mL, pH 4.8±0.2, sterilized at 121℃ for 15min before use.

[0041] ATB modified medium (1L): malic acid 3g / L, peptone 10g / L, beef extract 5g / L, Tween 80 1mL / L, dipotassium hydrogen phosphate 2g / L, yeast extract 4g / L, magnesium sulfate 0.2g / L, triammonium citrate 2g / L, glucose 20g / L, manganese sulfate 0.05g / L, sodium acetate 5g / L, water 1000mL, pH 4.8±0.2, sterilized at 121℃ for 15min before use.

[0042] MDA medium (1L): Magnesium sulfate heptahydrate 0.2g / L, manganese sulfate tetrahydrate 0.05g / L, ferrous sulfate heptahydrate 0.04g / L, calcium carbonate 0.1g / L, tryptone 5g / L, beef extract 8g / L, yeast extract 4g / L, Tween 80 0.5g / L, agar 20g / L, bromocresol purple 0.06g / L, amino acids (histidine, tyrosine, ornithine, lysine, phenylalanine, tryptophan) 5g / L each, pH 5.3±0.02, water 1000mL. Sterilize at 121℃ for 15min before use.

[0043] Example 1:

[0044] 1. Selection of starting strains.

[0045] 1.1 Isolation and screening of lactic acid bacteria;

[0046] For natural fermentation of wine, select fresh, ripe grapes. The grapes are not washed; instead, the skins are manually crushed and the stems removed. The processed grape must is placed in a 500mL Erlenmeyer flask, filling it to 70% capacity, leaving space for gas production. Fermentation is carried out naturally at 25℃ in an incubator. During the alcoholic fermentation stage, gas is continuously produced, forming a skin and pomace cap. Continuous stirring ensures full contact between the grape skins and juice. When constant weight is reached, alcoholic fermentation is considered complete. The skins and pomace are separated, the flask is sealed, and natural malolactic fermentation is initiated at 20℃. Under suitable conditions, the lactic acid bacteria present in the wine will begin to function. Whether natural malolactic fermentation has begun can be qualitatively determined using paper chromatography. For wine that has already started natural malolactic fermentation, 1mL of the solution is added to 9mL of sterilized 0.85% sodium chloride solution, gently shaken, and the above operation is repeated, with serial dilutions ranging from 10² to 10⁸ times. Using a micropipette, 0.1 mL of bacterial suspension at different concentration gradients was evenly spread onto ATB isolation medium. The ATB isolation medium was placed in an incubator and incubated upside down at 28°C, with three replicates for each concentration gradient. Single, round, milky-white, smooth, opaque colonies with a diameter less than 1 mm were picked from the ATB isolation medium and purified using the four-zone streak method. The pure cultures were numbered and recorded, mixed with 40% glycerol solution in an equal proportion, shaken thoroughly, and stored at -80°C.

[0047] 1.2 Screening of lactic acid strains that do not produce bioamines;

[0048] The selected lactic acid bacteria strains were inoculated into ATB medium and activated by shaking culture at 28°C for 72 h (180 r / min). The activated bacterial solution was picked up with an inoculation loop and spot-inoculated onto MDA medium and cultured at 28°C for 9 days. Three parallel experiments were set up. Strains that can produce phytoamines using amino acid precursors will show brown-red or purple areas around the colony or the tyrosine precipitate around the colony will disappear. If no color change is observed, the strain will not produce phytoamines.

[0049] Tests showed that *S. tumefaciens* SDM1 does not produce bioamines.

[0050] 1.3 Determination of β-glucosidase activity;

[0051] After activation, the strain was inoculated onto modified ATB medium at a rate of 1% and cultured at 28°C with shaking for 72 hours (180 r / min). The cultured bacterial solution was then placed in a refrigerated high-speed centrifuge and centrifuged at 8000 r / min for 10 minutes at 4°C. The supernatant, i.e., the crude enzyme solution, was collected and used for further analysis and detection.

[0052] Accurately pipette 1 mL of crude enzyme solution into a 15 mL centrifuge tube using a micropipette. Then add 1 mL of pNPG (10 mmol / L) substrate buffer solution (0.1 M citrate - 0.2 M disodium hydrogen phosphate) (pH 5.0), gently vortex to mix, and react in a water bath at 40 °C for 60 min. Add 8 mL of 1 mol / L sodium carbonate solution to terminate the reaction. After waiting 5 min for color development and stabilization, measure the absorbance under UV light at a wavelength of 401 nm. The blank is uninoculated ATB modified medium under the same treatment.

[0053] Enzyme activity unit (U) is defined as the amount of p-NP produced by 1 mL of bacterial culture in 1 min under the reaction conditions of p-NPG as substrate, pH 5.0, and 40℃.

[0054] The enzyme activity of SDM1 in *Chlorella vulgaris* was 2.62±0.08 mU / mL, which was significantly higher than that of OEN01 in commercial wine (1.94±0.08 mU / mL).

[0055] Example 2:

[0056] 2. Identification of strains.

[0057] 2.1 Ecological observation;

[0058] Strain SDM1 was inoculated onto ATB solid medium and incubated in a 28°C incubator for about 9 days. The morphology, color, and transparency of the colonies on the medium were observed, and the morphology of the cells and the Gram staining results were observed under an oil immersion microscope.

[0059] On ATB solid medium, strain SDM1 forms round, milky-white colonies with a smooth, opaque surface and a diameter of less than 1 mm. Figure 1 As shown.

[0060] Under an oil immersion microscope, strain SDM1 appears as oval or spherical cells, existing as single cells, in pairs, or in chains, and is Gram-positive. Figure 2 As shown.

[0061] 2.2 Molecular biological identification;

[0062] PCR amplification: 16S rDNA was amplified by PCR using universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-TACGGCTACCTTGTTACGACTT-3').

[0063] PCR amplification was performed using a 30 μL reaction system, which included: 15 μL of 2X EasyTaq SuperMix, 2 μL of template DNA, 1.0 μL of PCR forward primer (10 μM), 1.0 μL of PCR reverse primer (10 μM), and 11 μL of ddH2O.

[0064] PCR reaction conditions: First, pre-denaturation at 94℃ for 5 min; then denaturation at 94℃ for 30 s, annealing at 57℃ for 30 s, extension at 72℃ for 1.5 min, for a total of 30 cycles, and finally extension at 72℃ for 7 min.

[0065] PCR amplification product detection and gel extraction: 5 μL of PCR product was taken and the target band (approximately 1500 bp) was detected by 1.5% agarose gel electrophoresis (100V, 20-30 min). The target band was excised and purified using a magnetic bead gel extraction kit (Shanghai Somi). PCR amplification and sequencing were performed by Ruiboxing Biotechnology Co., Ltd., using the Sanger sequencing method. The sequencing result is SEQ ID NO.1:

[0066]

[0067] The 16S rDNA sequence of Oenococcus oeni strain SDM1 was compared with sequences in the GenBank database, confirming that strain SDM1 is Oenococcus oeni.

[0068] The Oenococcus oeni SDM1 strain was deposited on April 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 34393. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0069] Example 3:

[0070] 3. Evaluation of the stress resistance of lactic acid bacteria strains to ethanol, acid, and sulfur dioxide.

[0071] 3.1 Ethanol resistance evaluation;

[0072] Prepare ATB-modified medium with different ethanol concentrations (10%, 12%, 14%, 16%), and inoculate the activated *Chlorella vulgaris* SDM1 into each of the ATB-modified medium with different ethanol concentrations, with an inoculation volume of 10 μL. 7 The cfu / mL culture medium was shaken and cultured at 28°C for 6 days in an incubator. The OD 600nm was measured using conventional ATB modified medium with added bacterial solution as a control, and the relative OD 600nm (%) was calculated.

[0073] 3.2 pH;

[0074] The pH values ​​of the modified ATB medium were adjusted to 3.0, 3.3, 3.6, and 3.9, respectively. The activated *C. taurum* SDM1 was then inoculated into the modified ATB medium at each pH value, with an inoculation volume of 10 μL. 7 The cfu / mL culture medium was shaken and cultured at 28°C for 6 days in an incubator. The OD 600nm was measured using conventional ATB modified medium with added bacterial solution as a control, and the relative OD 600nm (%) was calculated.

[0075] 3.3 Sulfur dioxide;

[0076] Potassium metabisulfite was added to ATB modified medium to adjust the SO2 concentration to 30 mg / L, 40 mg / L, 50 mg / L, and 60 mg / L. The activated *C. tumefaciens* SDM1 was then inoculated into ATB modified medium with different SO2 concentrations at an inoculation volume of 10 μL. 7The cfu / mL culture medium was shaken and cultured at 28°C for 6 days in an incubator. The OD 600nm was measured using conventional ATB modified medium with added bacterial solution as a control, and the relative OD 600nm (%) was calculated.

[0077] Table 1

[0078]

[0079] As shown in the table, ethanol strongly inhibits the growth of *C. tumefaciens* SDM1 and commercial *C. tumefaciens* OENO1. This is mainly because ethanol disrupts the ordered structure of the cell membrane, reducing its fluidity. The SDM1 strain exhibits better ethanol resistance than the commercial strain OENO1. At 10% ethanol content, the growth of the strain is inhibited, with a relative OD600nm of 37.04%. Even at 16% ethanol content, the SDM1 strain maintains some tolerance, with a relative OD600nm of 8.78%, indicating that it is well-suited to the high-alcohol fermentation environment of wine.

[0080] At pH 3, the relative OD600nm of strain SDM1 decreased to 7.26%. As the pH of the culture medium increased, OD600nm increased to varying degrees. When the pH was between 3.3 and 3.9, the relative OD600nm of strain SDM1 was higher than that of the commercial strain OENO1. The pH of commercially available wines is generally between 3.3 and 3.8. Within this range, strain SDM1 showed better resistance, indicating that it is more suitable for the acid-base environment of wine fermentation.

[0081] The resistance of both strains decreased significantly with increasing sulfur dioxide concentration. SDM1 strain showed significantly higher resistance than OENO1 at concentrations of 30-60 mg / L, covering the commonly used sulfur dioxide addition range of 30-50 mg / L in winemaking. At a sulfur dioxide concentration of 60 mg / L, the relative OD600nm of SDM1 and OENO1 strains were 6.70% and 5.56%, respectively, both showing significant inhibition. This is because sulfur dioxide reacts with various cellular components (such as proteins), disrupting the integrity of membrane structures.

[0082] Example 4:

[0083] 4. SDM1 of *Cytosporum tobira* is used in the production of dry red wine.

[0084] 4.1 Fermentation experiment;

[0085] Sampling was conducted at a perennial high-quality vineyard in Huailai County, Zhangjiakou City. Healthy, fresh Cabernet Sauvignon grapes with intact skins were harvested. The destemmed and crushed Cabernet Sauvignon must was used as raw material. 3.5L of the must was placed in a clean 5L glass bottle, and 60mg / L potassium metabisulfite was added. After 2 hours, 60mg / L pectinase was added, and the mixture was cold-macerated at 4℃ for 3 days. Commercial brewing yeast XPURE was added at a dosage of 150mg / L of fermentation broth, and the fermentation temperature was maintained at 20℃. *Sacchariformis* SDM1 was selected, with the commercial strain OENO1 as a control. Two fermentation methods were used: simultaneous fermentation (lactobiotics were inoculated 24 hours after yeast inoculation) and sequential fermentation (lactobiotics were inoculated after complete alcoholic fermentation, i.e., when reducing sugar was less than 4g / L). The inoculation amount was 10... 7 If the malic acid content is less than 0.2 g / L and the CFU / mL, the malic acid-lactic acid fermentation is considered to be complete. The experiment is repeated 3 times.

[0086] 4.2 Detection of volatile compounds;

[0087] Extraction of volatile aroma components: 8 mL of wine was transferred to a 20 mL glass headspace vial, and 10 μL of 3-octanol aqueous solution (300 mg / L) was added as an internal standard, along with 2 g of NaCl to facilitate aroma volatilization. The aroma was determined using a semi-quantitative internal standard method. The vial was then equilibrated in a 40 °C water bath for 15 min, followed by SPME fiber headspace extraction at constant temperature for 40 min. Finally, the vial was manually injected into the GC inlet for 8 min for analysis.

[0088] Qualitative analysis was performed using full scan mode (SCAN), and quantitative analysis was performed using ion scan mode (SIM). Mass spectra were compared with the NIST 14 library, and only components with a matching degree of 80% or higher were analyzed. A semi-quantitative analysis of volatile compounds was performed using the internal standard method. The results are shown in Table 2.

[0089] Table 2

[0090]

[0091]

[0092] ND: indicates not detected.

[0093] In Cabernet Sauvignon dry red wine, the total aroma content of wines fermented simultaneously was significantly higher than that of wines fermented sequentially. In particular, the aroma content of wines treated with strain SDM1 reached (22935.90±133.56) μg / L, which was significantly higher than that of wines treated with commercial strain OENO1 (21460.05±64.09) μg / L, thus enhancing the aroma intensity of the wine.

[0094] Esters are the most abundant volatile aroma compounds in wine. The ester content in wine treated with strain SDM1 was significantly higher than that in wine treated with the commercial strain OENO1. Furthermore, the wine fermented simultaneously with SDM1 had the highest ester content (11658.21±39.39) μg / L. The levels of ethyl octanoate, ethyl decanoate, and isoamyl acetate in the wine fermented simultaneously with SDM1 were significantly higher than in other treatment groups, indicating that simultaneous fermentation with SDM1 enhanced the floral, pineapple, apple, pear, and banana aromas and sweetness of Cabernet Sauvignon wine.

[0095] Literature reports that when the concentration of higher alcohols is below 300 mg / L, it can effectively enhance the aroma layers and flavor complexity of wine. Therefore, alcohols in all treatment groups of wine samples had a positive impact on the aroma of the wine. The phenylethanol content in the simultaneously fermented wine was significantly higher than that in the other wines, for example, the SDM1 simultaneously fermented wine (2747.09±112.91) μg / L was higher than that in the SDM1 sequentially fermented wine (2277.27±72.09) μg / L, which imparted a rose aroma to the wine.

[0096] Volatile acids are the cause of fatty, rancid, and buttery flavors in wine. However, the concentrations of acids in the wine samples from each treatment group were low and the threshold was high, so they did not negatively affect the aroma of Cabernet Sauvignon wines. Simultaneously fermented wines had significantly higher acid content than sequentially fermented wines; for example, the simultaneous fermentation level of SDM1 wine (741.83±27.49) μg / L was higher than that of sequentially fermented SDM1 wine (440.75±20.81) μg / L.

[0097] 4.3 Determination of malic acid content;

[0098] Wine samples were filtered using a 0.22 μm needle filter for high-performance liquid chromatography (HPLC) analysis. A malic acid standard solution was prepared, and a standard curve was plotted. The analytical conditions were as follows: DiamonsilPlus C18 column; mobile phase consisting of phase A (0.1% phosphoric acid solution) and phase B (methanol); flow rate maintained at 0.8 mL / min; isocratic elution mode; phase A to phase B volume ratio set at 97:3; elution time 20 min; column temperature 40℃; detector wavelength 210 nm; sample injection volume 10 μL. The changes in malic acid content during wine fermentation are shown in Table 3.

[0099] Table 3

[0100]

[0101]

[0102] Changes in malic acid content are directly related to the malolactic fermentation process in wine. Both commercial strains OENO1 and SDM1 can completely degrade malic acid (<0.2 g / L), and their fermentation effectively shortens the total fermentation time of Cabernet Sauvignon dry red wine to only 11 days, while sequential fermentation requires 16 days to complete the entire fermentation process. Under the same fermentation conditions, the commercial strain OENO1 and the lactic acid bacteria SDM1 of this invention require the same amount of time to complete fermentation.

[0103] The results above show that the *S. sacchariformis* SDM1 strain with accession number CGMCC No. 34393 selected in this invention has good tolerance to alcohol, acid and sulfur dioxide, and has the excellent characteristic of high β-glucosidase production. Wines made from it have local biochemical characteristics and can produce more prominent and richer regional aroma components, which is of great significance for brewing distinctive and high-quality dry red and dry white wines.

[0104] In the description of this invention, it should be understood that the terms "longitudinal", "lateral", "up", "down", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this invention, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention.

[0105] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A type of *Chlorella vulgaris* SDM1, characterized in that, The preservation name of this Oenococcus oeni is: Oenococcus oeni SDM1; depositary institution: China General Microbiological Culture Collection Center (CGMCC); deposit date: April 28, 2025; accession number: CGMCC No. 34393.

2. The *S. sacchariformis* SDM1 according to claim 1, characterized in that, The 16S rDNA gene sequence of the *Chlorella vulgaris* SDM1 is shown in SEQ ID NO.

1.

3. The application of *Saccharomyces cerevisiae* SDM1 in the preparation of fermentation inoculants, characterized in that... A fermentation agent containing *Saccharomyces cerevisiae* SDM1 as described in claim 1.

4. The application of *Saccharomyces cerevisiae* SDM1 according to claim 3 in the preparation of fermentation inoculants, characterized in that, The fermentation agent is a liquid agent, a semi-liquid agent, or a solid agent.

5. The application of *Saccharomyces cerevisiae* SDM1 according to claim 3 or 4 in the preparation of fermentation inoculants, characterized in that, The viable concentration of *Coccus faecium* SDM1 in the fermentation agent is 10. 9 cfu / mL ~10 12 cfu / mL.

6. The application of *Saccharomyces cerevisiae* SDM1 in winemaking, characterized in that... The use of *S. tumefaciens* SDM1 as described in claim 1 or any one of the fermentation agents described in claims 3-5 in the production of wine.

7. The application of *Saccharomyces cerevisiae* SDM1 in a wine production method, characterized in that... Dry red wine is obtained by fermenting grape raw materials using the SDM1 of Chlorella vulgaris as described in claim 1 or any one of the fermentation agents of claims 3-5.

8. The application of *S. sacchariformis* SDM1 in a wine production method according to claim 7, characterized in that... The specific steps of the wine production method include: adding potassium metabisulfite and pectinase to grape juice, mixing them evenly, inoculating the grape juice with the SDM1 of the chrysophanolactam as described in claim 1 or the fermentation agent as described in any one of claims 3-5, mixing them evenly again, and then fermenting to obtain dry red wine.

9. The application of *S. sacchariformis* SDM1 in a wine production method according to claim 8, characterized in that: The inoculation amount of *Chlorella vulgaris* SDM1 was 1×10⁻⁶. 7 cfu / mL~1×10 8 cfu / mL.

10. The application of *S. sacchariformis* SDM1 in a wine production method according to claim 8 or 9, characterized in that: The grape juice was obtained by destemming, crushing, and pressing Cabernet Sauvignon grapes from Huailai County, Zhangjiakou City, Hebei Province.

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