Hansenula polymorpha and application thereof

Abstract

The invention discloses hansenula polymorpha var. Uvarum and application thereof. The preservation number of the hansenula polymorpha var. Uvarum CJ2 is CGMCC (China General Microbiological Culture Collection Center): 34391; the hansenula polymorpha CJ2 provided by the invention has good fermentation capacity, low H2S yield, good alcohol production capacity and ester production capacity, and can tolerate 35% glucose concentration, 250mg / L SO2 concentration, 2.9 pH value, 12% ethanol concentration and 10 DEG C temperature; good activity of producing glycosidase, protease and esterase is achieved; the content of malic acid and tartaric acid in the longan dry white wine fermented by hansenula polymorpha CJ2 is relatively low, and the acerbity and hardness taste of the wine is reduced.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology, specifically relating to a grape juice spore-bearing Hansenula yeast and its application. Background Technology

[0002] Wine is an alcoholic beverage made from fresh grapes or grape juice through partial or complete alcoholic fermentation. Yeast is a key microorganism in winemaking, and its metabolic activity directly influences and determines the fermentation process and the quality of the wine. Currently, in industrial wine production, winemakers typically use specific commercial yeasts for fermentation to ensure the controllability and stability of the fermentation process. However, the widespread use of commercial yeasts may reduce the flavor complexity and typicality of wines and lead to homogenization of wine products. With the development of the wine industry, consumers have placed higher demands on the quality of wines, and the production of high-quality wines urgently requires wines that possess regional characteristics and reflect local features and styles.

[0003] Non-sacchariculture yeasts were initially isolated from spoiled wines and were once considered harmful strains due to their association with the production of off-flavor substances such as acetic acid, acetaldehyde, and hydrogen sulfide. However, recent studies have shown that non-sacchariculture yeasts play a positive role in enhancing the regional characteristics and flavor complexity of wines. In-depth research indicates that in the early stages of fermentation, they can secrete enzymes such as glycosidases, proteases, and esterases, which interact with substrates in grape juice to generate aromatic compounds such as esters, terpenes, higher alcohols, and acids, thus improving the aroma and complexity of the wine and contributing positively to its flavor. However, the high sugar and low pH fermentation environment can severely affect yeast activity. Therefore, screening for yeasts with good environmental tolerance 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

[0004] To address the problems of poor environmental tolerance of existing non-brewing yeasts and the lack of distinct regional style characteristics in wine products, this invention provides a Hansenula polymorpha yeast for grape juice and its application.

[0005] To achieve the aforementioned objective, the present invention employs the following technical solution: This invention provides a grape juice spore-forming Hansenula polymorpha yeast, which is derived from grapes, and its 26S rDNA nucleotide sequence is shown in SEQ ID No. 1.

[0006] Preferably, the *Hansenula polymorpha* from grape juice is named *Hansenula polymorpha* from grape juice CJ2; its Latin name is: Hansenia sporauvarumIt was deposited on April 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC:34391, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The biological characteristics of Hansenula polymorpha CJ2 in grape juice provided by this invention are as follows: on WL medium, the colonies are round with neat borders, spherical protrusions, dark green in color, and smooth in surface.

[0008] The *Hansenula polymorpha* CJ2 strain in grape juice provided by this invention exhibits good environmental tolerance. The β-glucosidase activity of this strain is 61.54±1.01 mU / mL, the β-xylosidase activity is 54.51±1.02 mU / mL, the protease activity is 144.51±2.42 U / mL, the C2 esterase activity is 28.67±1.03 mU / mL, the C4 esterase activity is 13.20±1.24 mU / mL, the C6 esterase activity is 23.04±1.03 mU / mL, and the C8 esterase activity is 23.69±1.03 mU / mL.

[0009] Preferably, the grape juice contains Hansenula polymorpha CJ2, which is selected from the natural fermentation liquid of longan grapes in Huaizhuo grape production area of ​​Zhangjiakou City, Hebei Province.

[0010] Preferably, the present invention also provides a grape juice Hansenula polymorpha CJ2 inoculum, comprising the grape juice Hansenula polymorpha CJ2.

[0011] Preferably, the grape juice containing Hansenula polymorpha CJ2 inoculum is a liquid inoculum, a semi-liquid inoculum, or a solid inoculum.

[0012] Preferably, the grape juice containing *Hansenula polymorpha* CJ2 is inoculated into YPD liquid medium and fermented until the viable count is ≥10⁻⁶. 8 The concentration of cfu / mL was increased to obtain a liquid bacterial agent.

[0013] The liquid bacterial agent is concentrated, and the supernatant is removed to obtain a semi-liquid bacterial agent.

[0014] The concentration can be achieved using conventional concentration methods in the art, such as centrifugation and filtration, and the present invention does not impose any special requirements.

[0015] Preferably, the liquid bacterial agent is centrifuged and concentrated at 4°C and 8000 r / min to remove the supernatant and obtain a semi-liquid bacterial agent.

[0016] Add buffer solution and lyophilization protectant to the semi-liquid bacterial agent to adjust the viable count to 10⁻⁶. 10 cfu / mL ~10 12After pre-freezing the bacterial cells at -80℃ for 5 hours, the cells were rapidly transferred to a vacuum freeze-drying apparatus and freeze-dried at -50~-60℃ for 12 hours to obtain a solid bacterial agent.

[0017] The buffer solution can be a conventional buffer solution in the art, such as PBS buffer or physiological saline, and the lyophilization protectant can also be a commonly used lyophilization protectant in the art, such as at least one of skim milk powder, maltodextrin, dextran or glycerol.

[0018] Preferably, the viable concentration of *Hansenula polymorpha* CJ2 inoculum in grape juice is 10⁻⁶ after activation in an incubator at pH 3-4 and 28°C for 2 hours. 8 cfu / mL ~10 12 cfu / mL, inoculum size 1×10 6 cfu / mL ~1×10 8 cfu / mL.

[0019] The present invention also provides the application of the aforementioned grape juice containing Hansenula polysaccharide CJ2 or an inoculum containing the aforementioned grape juice containing Hansenula polysaccharide CJ2 in winemaking, particularly in the production of dry white wine.

[0020] The grape juice Hansenula polysaccharide CJ2 strain provided by this invention can be widely used in the winemaking of various grape varieties in China, especially suitable for winemaking in the Huaizhuo production area of ​​Zhangjiakou, Hebei Province. In terms of the typicality of Longyan dry white wine, it is significantly better than commercial strains, indicating that the selected grape juice Hansenula polysaccharide CJ2 strain can reflect the typicality of local wines and has practical application value.

[0021] The present invention also provides a method for producing wine, wherein grape raw materials are fermented using the aforementioned grape juice containing Hansenula polysaccharide CJ2 or grape juice containing Hansenula polysaccharide CJ2 inoculum to obtain wine.

[0022] Specifically, the method for producing wine involves the following steps: Select fresh, unspoiled longan grapes, destem, crush, and press them to extract the juice. Divide the grape juice into Erlenmeyer flasks, inoculate the grape juice with Hansenula polysaccharide CJ2 or a Hansenula polysaccharide CJ2 inoculum, vortex mix evenly at 3000 r / min, and ferment for 11-12 days at pH 3-4 and 12-13℃ to obtain dry white wine.

[0023] Preferably, the inoculum size of *Hansenula polymorpha* CJ2 in the grape juice is 1 × 10⁻⁶. 6 cfu / mL ~1×10 8 cfu / mL.

[0024] Fermentation of grape juice with Hansenula polysaccharide CJ2 produces a series of metabolites that can impart a pleasant and unique flavor to wine. The esters in the metabolites are directly affected by the proliferation density of Hansenula polysaccharide CJ2 cells in grape juice. The optimal inoculation amount can ensure that the content of flavor substances in the wine is appropriate, making the wine taste richer and more harmonious.

[0025] Preferably, the grape juice raw material is obtained by crushing, destemming, and pressing longan grapes from Huaizhuo production area in Zhangjiakou City, Hebei Province.

[0026] It should be noted that the grape juice Hansenula polymorpha CJ2 needs to be activated before fermentation. Conventional activation methods in the field can be used, such as inoculating the grape juice Hansenula polymorpha CJ2 into YPD liquid medium and culturing it overnight in a shaker at 28°C.

[0027] The *Hansenula polymorpha* CJ2 strain provided by this invention can tolerate a glucose concentration of 35%, an SO2 concentration of 250 mg / L, a pH of 2.9, a 12% ethanol concentration, and a temperature of 10°C. The β-glucosidase activity of this strain is 61.54 ± 1.01 mU / mL, the β-xylosidase activity is 54.51 ± 1.02 mU / mL, the protease activity is 144.51 ± 2.42 U / mL, the C2 esterase activity is 28.67 ± 1.03 mU / mL, the C4 esterase activity is 13.20 ± 1.24 mU / mL, the C6 esterase activity is 23.04 ± 1.03 mU / mL, and the C8 esterase activity is 23.69 ± 1.03 mU / mL.

[0028] The beneficial effects of this invention are as follows: The grape juice of this invention contains Hansenula polymorpha CJ2, which has strong environmental resistance and good activity in producing glycosidases, proteases, and esterases. When applied to winemaking, it can reduce the content of malic acid and tartaric acid in wine, significantly increase the content of esters in wine, help increase the flavor complexity of wine, and give wine a rich aroma of flowers, pineapple, apple, pear, and banana, as well as sweetness. Thus, the wine has regional characteristics, reflects local features and style, and meets people's higher demand for wine quality.

[0029] Secondly, compared with commercial non-brewing yeast NSD, the grape juice containing Hansenula polysaccharide CJ2 provided by this invention can result in lower levels of malic acid and tartaric acid in the brewed dry white wine, reducing the wine's acidity and harshness; it also results in higher ester content (3082.70±28.93μg / L), increasing its rich floral and fruity aromas, which are elegant and harmonious, exhibiting distinct regional aroma and flavor characteristics, and are of great significance to promoting the development of the wine industry. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 The colony morphology of *Hansenula polymorpha* CJ2 in grape juice on WL solid medium; Figure 2 This is a schematic diagram showing the organic acid content in the wine sample. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all 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.

[0033] In the following examples, *Hansenula polymorpha* CJ2 from grape juice was deposited on April 28, 2025, at the China General Microbiological Culture Collection Center, accession number: CGMCC:34391.

[0034] Culture medium used in the examples: YPD liquid culture medium (1L): 20g glucose, 20g peptone, 10g yeast extract, 1000mL water, natural pH, sterilized at 121℃ for 20min before use.

[0035] YPD solid medium (1L): 20g glucose, 20g peptone, 10g yeast extract, 20g agar, 100mg chloramphenicol (pre-dissolved in a small amount of ethanol), 1000mL water, natural pH, sterilized at 121℃ for 20min before use.

[0036] WL medium (1L): 5g yeast extract, 5g acid-hydrolyzed casein, 50g glucose, 0.55g potassium dihydrogen phosphate, 0.425g potassium chloride, 0.125g calcium chloride, 0.125g magnesium sulfate, 0.0025g ferric chloride, 0.0025g manganese sulfate, 0.022g bromocresol green, 17g agar. Sterilize at 121℃ for 20 minutes before use.

[0037] Fermentation medium (1L): Tween 80 10mL, yeast extract 10g, peptone 20g, glucose 20g, ammonium nitrate 3g, magnesium sulfate heptahydrate 0.5g / L, potassium dihydrogen phosphate 4g, sterilized at 121℃ for 20min and ready for use. Example

[0038] 1. Selection of starting strains 1.1 Isolation and screening of fruit surface yeasts Fresh, ripe longan grapes were manually destemmed and crushed, then placed in a fermentation container for skin maceration fermentation at 16–18°C. Under sterile conditions, 1 mL of the fermentation broth was added to 9 mL of sterilized 0.90% sodium chloride solution, shaken to mix, and the above steps were repeated, with serial dilutions of 10 mL each time. 2 ~10 8 The 100µL dilution was evenly spread on YPD solid medium containing 100mg / L chloramphenicol and incubated at 28℃ for 2-3 days. After colony growth, colonies with typical yeast morphology were selected for streaking purification and further purified by the four-zone streak method on WL differential medium. The color, size, moisture and morphology of the colonies were observed to make a preliminary identification of the isolated yeast. After three repeated purifications, single colonies were obtained. The purified single colonies were picked and inoculated into YPD liquid medium and cultured for 24h (28℃, 180r / min). The obtained bacterial suspension was mixed with 40% glycerol (1:1) to prepare glycerol tube preservation solution. Each sample was prepared in triplicate and stored in an ultra-low temperature freezer at -80℃.

[0039] 1.2 Determination of basic characteristics of the strain 1.2.1 Determination of fermentation power of strains Add 10 ml of LYPD liquid medium to a test tube and insert a Durham tube. After sterilization, add 2% activated bacterial solution to the test tube, gently shake to ensure uniform distribution of the bacterial solution, and check for air bubbles in the Durham tube. Incubate statically at 28°C and observe the air bubble production in the Durham tube every 12 hours to screen out yeast strains with strong gas production capacity.

[0040] The gas produced by strain CJ2 can completely fill the Durham tube within 24 hours, indicating that the strain starts fermenting rapidly and has a strong fermentation capacity.

[0041] 1.2.2 H2S Production Capacity Determination The strain was spread on BIGGY medium and incubated at 28°C for 3–5 days. The H2S production capacity of the strain was evaluated based on the color change of the colonies on the medium. The colony color from light to dark indicated that the H2S production capacity went from no production to high production.

[0042] The strain CJ2 was yellowish-brown on BIGGY medium, indicating that it produced low levels of H2S.

[0043] 1.2.3 Determination of alcohol production capacity After the strain was activated, it was inoculated into the lower layer of TTC medium and cultured at 28°C for 3 days. Then, the colonies were covered with 15mTTC upper layer medium and cultured at 28°C in the dark for 3-4 hours. The colony color changes were as follows: dark red indicates high ethanol production, rose red indicates medium ethanol production, and light pink indicates weak ethanol production.

[0044] The CJ2 strain is magenta, indicating that it produces alcohol.

[0045] 1.2.4 Determination of ester production characteristics The strain was inoculated onto an ester-producing solid medium and cultured at 28°C for 3 days. Milky white colonies indicated that the strain had a weak ester-producing ability; light yellow colonies corresponded to strains with moderate ester-producing ability; and dark yellow colonies indicated strains with high ester-producing ability.

[0046] The strain CJ2 is deep yellow, indicating that it has a high ester production capacity.

[0047] 1.3 Determination of strain tolerance The tolerance of the strains was determined using 10ml LYPD liquid medium and Durham tubes. For high glucose tolerance, glucose concentrations of 18%, 25%, 27%, 30%, and 35% were used; for SO2 tolerance, concentrations of 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, and 250 mg / L were used; for acidity tolerance, pH values ​​of 2.9, 3.2, 3.5, 3.8, and 4.1 were used; for ethanol tolerance, ethanol concentrations (v / v) of 6%, 8%, 10%, 12%, and 14% were used; and for temperature tolerance, temperature gradients of 10℃, 12℃, 14℃, 16℃, and 18℃ were used. Each strain was inoculated with 2% activated bacterial solution and incubated at 28℃. Gas volume in the Durham tubes was observed after 72 hours.

[0048] The results showed that strain CJ2 could tolerate 35% glucose concentration, 250 mg / L SO2 concentration, pH value of 2.9, 12% ethanol concentration, and temperature of 10℃.

[0049] 1.4 Determination of enzyme production characteristics 1.4.1 Glycosidase assay Yeast culture was inoculated at a ratio of 2% into YPD liquid medium and activated for 48 h (28℃, 180 rpm). The activated solution was then inoculated into fermentation medium at a ratio of 5% and cultured on a shaker for 72 h (28℃, 180 rpm). After the culture was completed, the fermentation broth was centrifuged at low temperature (8000 rpm, 10 min), and the supernatant was collected to obtain crude enzyme solution.

[0050] β-glucosidase and β-xylosidase activity assays: 0.75 mL of citrate-phosphate buffer (pH=5.0), 0.25 mL of 1 mmol / L reaction substrate (p-nitro-β-D-glucosinolate / p-nitro-β-D-xyloside), and 0.1 mL of frozen-centrifuged crude enzyme solution were added sequentially to centrifuge tubes. The reaction was carried out at 40℃ for 30 min. After the reaction, 1 mL of 0.5 mol / L sodium carbonate was added to terminate the reaction. The absorbance was measured at 400 nm. YPD liquid medium without bacterial inoculation was used as a blank control. Glucosidase activity refers to the amount of enzyme required to produce 1 μmol pNP per minute at 40℃ and pH 5.0.

[0051] The β-glucosidase activity of strain CJ2 was 61.54±1.01 mU / mL, and the β-xylosidase activity was 54.51±1.02 mU / mL.

[0052] 1.4.2 Protease Assay Centrifuge the activated yeast fermentation broth at low temperature for 10 minutes (8000 rpm), and take the supernatant as the required crude enzyme solution.

[0053] Protease activity assay: Add 2.5 mL of 0.2 mol / L Tris-HCl buffer (pH=8.0) containing 0.5% casein and 0.5 mL of crude enzyme solution to a test tube sequentially. Incubate at 60℃ for 5 min. After the reaction, add 2.5 mL of 110 mmol / L trichloroacetic acid to stop the reaction. Incubate at room temperature for 50 min, then centrifuge (8000 rpm, 4℃, 10 min). In a new centrifuge tube, add 2.5 mL of 0.5 mol / L sodium carbonate solution, 0.5 mL of 0.5 mol / L Folin-Ciocalteu solution, and 1 mL of centrifuged filtrate sequentially. Measure the absorbance at 660 nm. Protease activity is defined as the amount of enzyme required to produce 1 μmol of tyrosine per minute at 60℃ and pH 8.0.

[0054] The protease activity of strain CJ2 was 144.51 ± 2.42 U / mL.

[0055] 1.4.3 Esterase Assay Add 0.86 mL of citrate-phosphate buffer (pH=5.0), 0.04 mL of 25 mmol / L reaction substrate (C2, C4, C6, and C8 esterase substrates dissolved in ethanol), and 0.1 mL of refrigerated centrifuged bacterial culture to a centrifuge tube sequentially. Incubate at 37°C for 1 h. After the reaction, terminate the reaction by adding 0.1 mL of 0.5 mol / L sodium hydroxide solution. Measure the absorbance at 400 nm. Esterase activity is defined as the amount of enzyme required to produce 1 μmol of p-nitrophenol per minute at 37°C and pH 5.0.

[0056] The C2 esterase activity of strain CJ2 was 28.67±1.03 mU / mL, the C4 esterase activity was 13.20±1.24 mU / mL, the C6 esterase activity was 23.04±1.03 mU / mL, and the C8 esterase activity was 23.69±1.03 mU / mL. Example

[0057] Identification of enzyme-producing strains Molecular biological identification The bacterial culture to be identified was sent to Genewiz Biotechnology Co., Ltd. for DNA extraction and 26S rDNA D1 / D2 region sequencing. The sequencing results were compared using the NCBI Blast homology database, and the strain CJ2 was identified as *Hansenula polymorpha* CJ2 of grape juice. Hanseniaspora grapes Its 26S rDNA nucleotide sequence is shown in SEQ ID No. 1:

[0058] The grape juice contains Hansenula polymorpha CJ2 ( Hanseniaspora grapes The strain was deposited on April 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC:34391. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Example

[0059] Grape juice containing Hansenula polymorpha CJ2 is used in the production of dry white wine. 1. Fermentation experiment Sampling was conducted at a perennial high-quality vineyard in Huailai County, Zhangjiakou City, Hebei Province. Healthy, fresh longan grapes with intact skin were harvested and destemmed and crushed. 800 mL of grape juice was dispensed into 1000 mL Erlenmeyer flasks. 16 mL of activated Hansenula polysaccharide CJ2 suspension was inoculated into the grape juice. After 48 h, local wine yeast HS32 (C+S) was inoculated. Commercial active dry yeast NSD and local wine yeast HS32 (N+S) were used as controls. Static fermentation was carried out at 12-13℃. The fermentation process was monitored regularly, and the weight was measured every 12 h. Fermentation was stopped when the weight change was <0.5 g for two consecutive times. The fermentation liquid was centrifuged (8000 r / min, 10 min), and the supernatant was used as a wine sample for subsequent experimental analysis.

[0060] 2. Determination of organic acid content Wine samples were filtered using a 0.22 μm needle filter for high-performance liquid chromatography (HPLC) analysis. Standard solutions of six organic acids—malic acid, tartaric acid, lactic acid, citric acid, succinic acid, and oxalic acid—were prepared, and standard curves were plotted. The analytical conditions were as follows: a Diamonsil Plus C18 column; mobile phase consisting of phase A (0.1% phosphoric acid solution) and phase B (methanol); a flow rate of 0.8 mL / min; isocratic elution mode; a phase A to phase B volume ratio of 97:3; elution time of 20 min; column temperature of 40 °C; detector wavelength of 210 nm; and sample injection volume of 10 μL.

[0061] The organic acid content in the wine sample is as follows Figure 2As shown, malic acid and tartaric acid are the main organic acids in wine. Malic acid plays a central role in yeast metabolism and is a key component of the tricarboxylic acid cycle (TCA cycle). Excessive amounts can affect the taste of wine. Tartaric acid can balance the sweetness and fruitiness in wine, making it refreshing, but excessive amounts can make the wine acidic, astringent, and harsh. The levels of malic acid and tartaric acid in the C+S wine sample were lower than those in the N+S wine sample. Lactic acid is mainly produced by yeast during alcoholic fermentation. Its content is not only important for the quality of wine but also beneficial to health, such as aiding in lactose digestion, having anti-cancer effects, and maintaining cholesterol levels, and can enhance the roundness of the wine's taste. Citric acid has a refreshing citrus flavor and is an intermediate in the TCA cycle. The levels of lactic acid and citric acid in the two wine samples were similar. Succinic acid is a byproduct of yeast cell nitrogen metabolism during fermentation. The levels of succinic acid in the C+S wine sample were slightly higher than those in the N+S wine sample. Oxalic acid is a commonly used chelating agent. In the human body, it may combine with calcium ions to form calcium oxalate precipitate, which may lead to the formation of kidney stones. The oxalic acid content in both wine samples was very low.

[0062] 3. Detection of volatile compounds Extraction of volatile aroma components: The aroma was determined by a semi-quantitative internal standard method. 8 mL of wine was pipetted into a 20 mL headspace vial, and 10 µL of 3-octanol solution (300 mg / L) was added as an internal standard and 2 g of NaCl to aid aroma volatilization. The vial was then placed in a 40 °C water bath for 15 min for equilibration, 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.

[0063] 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 NIST14 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. Aromas with VIP > 1 were obtained using the orthogonal partial least squares-discriminant analysis (OPLS-DA) model, as shown in Table 1.

[0064] Table 1

[0065] During the alcoholic fermentation of wine, esters are a typical byproduct, mainly including acetates, ethyl fatty acid esters, and other ester compounds. These substances significantly contribute to the fruit and floral aromas of wine, enhancing the complexity and intensity of the aromas, thereby improving the overall quality of the wine. The ester content of the C+S wine sample (3082.70±28.93μg / L) was significantly higher than that of the N+S wine sample. The C+S wine sample contained significantly higher levels of esters such as ethyl hexanoate, ethyl octanoate, isoamyl acetate, hexyl acetate, phenylhexyl acetate, and ethyl 9-decenoate than the control N+S wine sample. Ethyl hexanoate has sweet fruit aromas of pineapple and banana; ethyl octanoate has aromas of brandy and banana. These substances work synergistically to form the core fruit aroma characteristics of the wine.

[0066] The formation of higher alcohols in wine is a complex biochemical process regulated by multiple factors, contributing to a sweeter and more mellow body. These alcohols primarily originate from the grapes themselves and the alcoholic fermentation process, specifically involving pathways such as glycoside hydrolysis, amino acid and fatty acid metabolism. Isoamyl alcohol imparts aromas of banana and cheese, while phenethyl alcohol carries a rose-like fragrance. The isoamyl alcohol content has a dual impact on the sensory quality of wine; within a reasonable concentration range, it contributes to a rich flavor, while excessive amounts can lead to a noticeable bitterness. Isoamyl alcohol levels were relatively high in N+S wine samples. 2,3-Butanediol imparts creamy and buttery notes to wine, and its content was relatively high in C+S wine samples.

[0067] Acids also have a significant impact on the taste and complexity of wine. These substances mainly originate from fermentation byproducts and the grape berries themselves. A suitable concentration of acid infuses the wine with richer flavor layers, enhancing its complexity and balance.

[0068] The results above show that the *Hansenula polymorpha* CJ2 strain of grape juice, with accession number CGMCC:34391, selected in this invention, exhibits excellent resistance to high sugar, SO2, acid, alcohol, and temperature. It also possesses the superior characteristic of high production of glycosidases, proteases, and esterases. Wines brewed using this strain possess local biochemical characteristics and produce more prominent and richer regional aroma components, which is of great significance for brewing distinctive and high-quality dry white wines. Finally, it should be noted that the terms "comprising," "including," or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus.

[0069] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0070] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A Saccharomyces uvarum, characterized in that, The grape juice Hanseniaspora is derived from grapes, and the nucleotide sequence of its 26S rDNA is shown as SEQ ID No.

1.

2. The Hansenula polymorpha strain of claim 1, wherein, The grape juice Hanseniaspora is named as grape juice Hanseniaspora CJ2, which has been preserved in the China General Microbiological Culture Collection Center on April 28, 2025, and the preservation number is CGMCC: 34391.

3. The Hansenula polymorpha strain of claim 2, wherein the strain is Hansenula polymorpha DGA1 -. The beta-glucosidase activity of the grape juice Hanseniaspora CJ2 is 61.54±1.01 mU / mL, the beta-xylosidase activity is 54.51±1.02 mU / mL, the protease activity is 144.51±2.42 U / mL, the C2 esterase activity is 28.67±1.03 mU / mL, the C4 esterase activity is 13.20±1.24 mU / mL, the C6 esterase activity is 23.04±1.03 mU / mL, and the C8 esterase activity is 23.69±1.03 mU / mL.

4. The Hansenula polymorpha of claim 3, characterized in that, The grape juice Hanseniaspora CJ2 has a yellow-brown color on the BIGGY culture medium, indicating that it produces low H2S.

5. A Saccharomyces Hensenii CJ2 inoculant characterized by, The grape juice Hanseniaspora CJ2 of claim 2 is used for fermentation, and the grape juice Hanseniaspora CJ2 is inoculated into YPD liquid medium to obtain a liquid inoculum.

6. The Hansenula polymorpha CJ2 inoculum of grape juice according to claim 5, characterized by, The liquid inoculum is concentrated, and the supernatant is removed to obtain a semi-liquid inoculum.

7. The Hansenula polymorpha CJ2 inoculum of grape juice according to claim 6, characterized by, To the semi-liquid inoculant, a buffer solution is added, a lyophilization protective agent is added, and the viable cell number is adjusted to 10 10 cfu / mL ~ 10 12 cfu / mL, and freeze-dried to obtain a solid inoculant.

8. The Hansenula polymorpha CJ2 inoculum of grape juice according to claim 7, characterized by that, The active bacteria concentration of the grape juice Hansenula polymorpha CJ2 in the grape juice Hansenula polymorpha CJ2 inoculant is 10 8 cfu / mL~10 12 cfu / mL after the grape juice Hansenula polymorpha CJ2 inoculant is activated at 28℃ for 2h in a culture box at pH 3~4 6 cfu / mL~1×10 8 cfu / mL.

9. The Hansenula polymorpha CJ2 inoculum of grape juice according to claim 7, characterized by that, The buffer is a PBS buffer or physiological saline, and the freeze-drying protective agent is at least one of skimmed milk powder, malt dextrin, dextran, or glycerol.

10. The grape juice Hanseniaspora CJ2 or grape juice Hanseniaspora CJ2 inoculum of any one of claims 1-9 is used for brewing grape wine.

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