Pichia kluyveri ZFM-0524 and application thereof in waxberry wine fermentation

Abstract

The invention belongs to the technical field of biology, and particularly relates to pichia kluyveri ZFM-0524 and application of the pichia kluyveri ZFM-0524 in fermentation of waxberry fruit wine. The preservation number of the pichia kluyveri ZFM-0524 provided by the invention is CCTCC (China Center for Type Culture Collection) NO: M 2025281, and the preservation number of the pichia kluyveri ZFM-0524 provided by the invention is CCTCC NO: M 2025281. When the pichia kluyveri ZFM-0524 is used for fermenting the waxberry fruit wine, the content of volatile aroma substances in the waxberry fruit wine can be increased; the special ester substances capable of giving fragrance to flowers and fruits can also be generated; therefore, the flavor quality of the waxberry wine is improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a Pichia kluyveri ZFM-0524 and application of the yeast in the fermentation of bayberry wine. Background Art

[0002] Bayberry (Myrica rubra) is an evergreen tree native to China, belonging to the genus Myrica in the family Myricaceae. Its fruit is highly nutritious, rich in active ingredients such as anthocyanins, vitamins, and minerals. It has benefits such as antitussive, anti-vomiting, digestive, and dysentery treatment. However, due to its lack of a rind and its peak ripening period during hot and rainy seasons, bayberry is highly susceptible to microbial contamination and spoilage, resulting in annual postharvest losses of up to 20%-30%. To reduce postharvest losses, in addition to adopting preservation technologies such as cold chain transportation and modified atmosphere packaging, deep processing has become a key approach to efficiently utilize bayberry resources. Products include preserves, juices, vinegars, and wines. Fermented bayberry wine, which converts sugars into ethanol and flavor compounds through yeast metabolism, not only effectively reduces the risk of methanol production compared to traditional soaking processes, but also offers a moderate alcohol content, rich fruity aroma, and a higher retention of active ingredients, making it more suitable for modern health consumption. Its market share has increased significantly in recent years.

[0003] Yeasts play a crucial role in the production of fruit wine. Traditional bayberry wine brewing often relies on natural fermentation or the addition of commercial Saccharomyces cerevisiae. However, these methods often struggle to precisely control the fermentation process, resulting in a less rich aroma profile and a relatively monotonous flavor. Therefore, exploring new yeast resources and their application in fruit wine brewing has become an important approach to improving wine quality. Research has shown that non-Saccharomyces yeasts offer unique advantages in fruit wine brewing. Their metabolites, including volatile compounds such as esters, alcohols, and aldehydes, significantly enrich the aroma and flavor profile of fruit wine. Furthermore, non-Saccharomyces yeasts produce organic acids and polyols, which are crucial for regulating the acidity and body structure of fruit wine, resulting in a fresher, more rounded, harmonious, and balanced taste, significantly enhancing the sensory quality of fruit wine. Non-Saccharomyces yeasts are diverse and widely distributed across diverse regions. Each region exhibits unique metabolic properties and flavor contributions, influenced by factors such as geographic environment and climatic conditions. Therefore, by carefully screening and cultivating non-brewery yeast strains with excellent brewing performance, new fruit wine varieties with unique flavor and excellent quality can be developed to meet the growing diversified needs of consumers in the market.

[0004] CN118389305A, "A Pichia kluyveri PK-21 Strain and Its Application, Winemaking Agent, and Winemaking Method," provides a Pichia kluyveri PK-21 strain, with a deposit number of CCTCC NO: M2022229. Pichia kluyveri PK-21 is used to increase the content of higher alcohol acetate in wine. The higher alcohol acetate includes one or more of isobutyl acetate, isoamyl acetate, phenylethyl acetate, and ethyl acetate.

[0005] CN117229933A, "A Pichia kluyveri Strain High in Acetate Production and Its Application," provides a Pichia kluyveri PKL-A strain capable of producing acetate compounds, with a deposit number of CGMCC No. 27659. Volatile aroma substances produced by the fermentation process include isoamyl acetate, phenylethyl acetate, phenylethyl alcohol, and isoamyl alcohol.

[0006] The invention of CN112725204A, "A Pichia kluyveri strain HSP11 producing aroma substances and its use," screened and obtained a Pichia kluyveri strain HSP11 that can ferment grape juice to high yield aroma substances such as 1-octen-3-ol, rose oxide, and octyl acetate. The aroma substances are selected from the group consisting of isopentanol, n-octanol, n-heptanol, ethyl octanoate, 1-octen-3-ol, rose oxide, octyl acetate, phenylacetaldehyde, isovaleric acid, isobutyric acid, linalool, 3-methylthiopropanol acetate, geranyl acetate, epoxy-α-terpenyl acetate, phylloxetate, amyl acetate, isobutyl acetate, butyl acetate, ethyl isobutyrate, ethyl heptanoate, and ethyl propionate. The aroma substance is selected from the group consisting of 1-octen-3-ol, rose oxide, octyl acetate, phenylacetaldehyde, isovaleric acid, isobutyric acid, linalool, 3-methylthiopropanol acetate, geranyl acetate, epoxy-α-terpenyl acetate, phylloxetate, amyl acetate, isobutyl acetate, butyl acetate, ethyl isobutyrate, ethyl heptanoate, and ethyl propionate. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide Pichia kluyveri ZFM-0524 and its application in the fermentation of bayberry wine. The bayberry wine fermented by the yeast has a unique aroma and can improve the flavor quality of the bayberry wine.

[0008] To solve the above technical problems, the present invention provides a Pichia kluyveri ZFM-0524 strain, whose preservation number is CCTCC NO: M 2025281.

[0009] The present invention also provides an application of Pichia kluyveri ZFM-0524: the Pichia kluyveri ZFM-0524 is used for the fermentation of bayberry wine.

[0010] As an improvement of the application of the present invention, the yeast Pichia kluyveri ZFM-0524 can not only increase the content of volatile aroma substances in bayberry wine, but also produce unique ester substances that can impart floral and fruity aroma;

[0011] The volatile aroma substances with increased content are: ethyl acetate, isoamyl acetate, phenylethyl acetate, hexyl acetate, propyl acetate, isobutyl acetate, (Z)-3-hexenyl acetate, ethyl nonanoate, methyl benzoate, ethyl benzoate, 3-(methylthio)propyl acetate, 3-phenylpropyl acetate and γ-nonalactone;

[0012] The unique ester substances capable of imparting floral and fruity aroma are: butyl acetate, heptyl acetate, benzyl acetate, neryl acetate, and phenylethyl propionate.

[0013] Note: The above-mentioned "unique ester substances that can impart floral and fruity aroma" refers to the unique metabolites produced by the strain of the present invention during the fermentation process of bayberry juice - ester substances that can impart floral and fruity aroma, which are unique and irreplaceable.

[0014] The present invention also provides a method for producing bayberry wine, which comprises fermenting bayberry juice with Pichia kluyveri ZFM-0524 to obtain bayberry wine.

[0015] That is, the present invention provides a native non-brewery yeast with excellent fermentation performance and certain alcohol production capacity, and the use of the yeast in the fermentation of bayberry wine.

[0016] The Pichia kluyveri ZFM-0524 of the present invention is a wild non-brewer's yeast strain separated from Dongkui waxberry fruits sold in Xianju County, Taizhou.

[0017] The preservation information is as follows:

[0018] Deposit name: Pichia kluyveri ZFM-0524 Pichia kluyveri ZFM-0524, deposit unit: China Center for Type Culture Collection, deposit address: Wuhan University, Wuhan, China, deposit number: CCTCC NO: M 2025281, deposit date: February 24, 2025.

[0019] The Pichia kluyveri ZFM-0524 of the present invention has good fermentation performance and aroma production ability during the fermentation process of bayberry wine.

[0020] The present invention has the following beneficial effects: By studying the morphological and physiological characteristics of yeast, the present invention has screened a non-Saccharomyces yeast strain, Pichia kluyveri ZFM-0524, which exhibits strong fermentation performance and produces an ester aroma. The bayberry wine fermented with this yeast has an alcohol content of approximately 4-6%, a pH of approximately 3.5, a rich aroma, a rich ester flavor, a pure color, and a typical bayberry flavor. This is of great significance for improving the taste and quality of bayberry wine.

[0021] In summary, Pichia kluyveri ZFM-0524 not only possesses excellent fermentation properties but also enables the production of a wider variety of aroma components during the fermentation process, significantly enriching the aroma layering and complexity of bayberry wine. Bayberry wine fermented with this yeast is rich in esters, imparting a pure, bright color and rich, fragrant aroma. This provides a novel and effective approach to optimizing the bayberry wine production process. This invention not only promotes the diversified development of the bayberry industry but also provides consumers with a high-quality beverage option that is both delicious and healthy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The specific embodiments of the present invention are further described in detail below with reference to the accompanying drawings.

[0023] Figure 1 These are the plate morphology and microscope morphology of a single colony of Pichia kluyveri ZFM-0524.

[0024] Figure 2 The changes in sugar content (°Brix) and weight loss during the fermentation of bayberry wine.

[0025] Figure 3 It is a stacking diagram of the content and percentage of volatile aroma components in each experimental group in the specific embodiment. DETAILED DESCRIPTION

[0026] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0027] Example 1: Screening and separation of Pichia kluyveri ZFM-0524, comprising the following steps:

[0028] S1. Take bayberries sold in Xianju County, Taizhou, and remove dirt and other impurities. Use sterilized tools to remove the core of the bayberry fruit and then crush it to obtain the bayberry pulp.

[0029] Accurately weigh 100 g of bayberry pulp and 100 g of sterile water into a 500 mL glass bottle with a lid. The bottle cap was half-tightened and placed in a biochemical incubator at 16°C for incubation. Samples were taken every 3 days for a total of 3 samplings. The fermentation broth was diluted with 0.9% sterile saline to obtain a dilution.

[0030] illustrate:

[0031] Select bayberries that are uniform in size, intact on the surface, and of moderate hardness. In addition, the surface of the bayberry should be dry, bright red or dark red in color, and ripe and juicy. The fruit pitting and crushing process should be carried out in an ultra-clean workbench to avoid contamination of the experimental results by miscellaneous bacteria.

[0032] The mass ratio of bayberry pulp to sterile water is 1:1, thus ensuring that part of the fruit surface is exposed to the air.

[0033] S2. The dilution is evenly spread on YPD solid medium and grown in a 30°C constant temperature incubator for 1-2 days. Single colonies with typical yeast colony characteristics (homogeneous round colonies, uniform white or cream color, diameters between 1.5-3.0 mm, no obvious contamination by foreign bacteria and abnormal pigment deposition) are selected and inoculated into YPD liquid medium. The culture is incubated in a shaker at 30°C and 180 rpm for 24 hours. The bacterial solution is diluted according to its concentration and streaked onto YPD solid medium to further purify the yeast (the strain is purified using the plate streak method, and purification is performed 2-3 times according to the separation and purification effect to ensure that pure bacteria are obtained as much as possible). The growth status of the yeast is observed;

[0034] Note: The dilution gradient is generally 10 -5 -10 -7 , ensure that the yeast does not grow too densely in the solid culture medium, avoid interference during the growth process and affect observation.

[0035] Steps S1 and S2 are for preliminary screening of yeast, which is to screen out single colonies with different appearances in the culture medium, and to perform fermentation performance analysis on the strains in the single colonies to screen out yeast with the best performance.

[0036] The fermentation performance test is as follows: the strain is 6 CFU / mL was inoculated into bayberry juice, and the weight of the fermentation bottle was recorded every 24 h for monitoring.

[0037] The single bacteria screened by the present invention have different morphologies. Among them, the colonies of strain ZFM-0524 on YPD solid medium are round, opaque white, rough with protrusions, irregular edges, and easy to pick up. At the same time, pure single colonies on the YPD solid plate were picked and placed under a microscope to observe the cell morphology. (As shown in the figure, the colonies are round, opaque white, rough with protrusions, irregular edges, and easy to pick up. Figure 1shown).

[0038] Example 2: Identification of Pichia kluyveri ZFM-0524

[0039] A pure strain of ZFM-0524 was selected and inoculated into YPD liquid medium. Cultured at 30°C for 24 hours to activate the strain. An appropriate amount of activated bacterial culture was collected by centrifugation. Genomic DNA from the pure culture of the target strain was extracted using a kit. The conserved region of the 26S rDNA gene of the strain was amplified using primers NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL4 (5'-GGTCCGTGTTTCAAGACGG-3'). The amplified product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing analysis.

[0040] PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds; a 10-minute hold at 72°C, followed by cooling to 4°C. Strain sequencing results were compared using BLAST homology analysis using bioinformatics software, and the yeast standard strain sequence with the highest homology was selected.

[0041] The strains obtained by screening were deposited, and the deposit information is as follows:

[0042] Deposit name: Pichia kluyveri ZFM-0524, deposit unit: China Center for Type Culture Collection, deposit address: Wuhan University, Wuhan, China, deposit number: CCTCC NO: M 2025281, deposit date: February 24, 2025.

[0043] The 26S rDNA gene sequence is shown in SEQ ID NO: 1 in the sequence listing.

[0044] Example 3: Application of Pichia kluyveri ZFM-0524 in Bayberry Wine

[0045] The specific fermentation method includes the following steps:

[0046] S1. Raw Material Selection: Select bayberries that are uniform in size, intact, and of moderate firmness. Also, choose bayberries that are dry and bright or dark red in color.

[0047] Bayberry was purchased from Dongkui Bayberry in Xianju County, Taizhou City, Zhejiang Province.

[0048] S2. Raw material processing: Wash the bayberry fruits, dry them, and remove the cores using a sterilized knife.

[0049] S3. Juice extraction: Using gauze wrapping method, the bayberry pulp is placed in gauze, and then the gauze is gently squeezed with a heavy object to allow the juice to flow out, and then filtered with a filter (40 mesh filter) or fine gauze, and the resulting filtrate is bayberry juice;

[0050] S4. Juice clarification: The bayberry juice was allowed to stand at 4°C for 12 h to obtain preliminary clarification. The clarified bayberry juice was then centrifuged at 1000 rpm for 20 min at 4°C. The supernatant obtained by centrifugation was designated as clarified bayberry juice and stored at -20°C.

[0051] S5 juice adjustment: Determination of the initial pH and Brix values ​​of the clarified bayberry juice obtained in step S4, the initial Brix was 9-10 ° Brix, pH of about 3.5;

[0052] S6. Juice sterilization: The clarified bayberry juice obtained in step S4 was bottled and then sterilized using high-temperature pasteurization (95°C, 15 min) to obtain sterile bayberry juice, which was then naturally cooled for standby use; the sterile bayberry juice was cooled;

[0053] Strain activation: Use YPD medium to activate non-Saccharomyces cerevisiae and culture on a shaker;

[0054] The details are as follows:

[0055] Pichia kluyveri ZFM-0524 was inoculated into 10 ml of YPD liquid medium and cultured at 30°C and 180 rpm for 24 h to obtain seed solution;

[0056] The seed solution was inoculated into YPD liquid medium at a volume of 1% (1%), and cultured in a shaking incubator at 30°C and 180 rpm until the viable count of Pichia kluyveri ZFM-0524 reached 10 7 -10 8 CFU / mL (culture time is about 24h);

[0057] Then, the yeast was washed three times with 0.9% sterile saline to remove the culture medium components; the yeast was activated after removal of impurities (10 7 -10 8 CFU / mL).

[0058] S8. Inoculation and fermentation: The activated yeast obtained in step S7 is used to inoculate and ferment the cooled sterile bayberry juice obtained in step S6 (in a fermentation bottle) to ensure that the initial viable count of the bayberry juice after inoculation is 10 6CFU / mL, the bottle cap was half-tightened to seal the bottle mouth, and then the fermentation bottle was placed in a constant temperature incubator and the temperature was controlled at 16-20℃. The fermentation cycle was about 10 days.

[0059] S9. Exhaust treatment: Unscrew the fermentation bottle every 24 hours to discharge the CO2 gas produced by alcohol fermentation in the fermentation bottle;

[0060] The purpose of venting is to reduce the air pressure in the bottle, prevent the bottle from bursting, maintain a stable fermentation environment and promote gas exchange.

[0061] S10. Termination of fermentation: Monitor the weight change and Brix change of bayberry wine every 24 hours during fermentation. The fermentation is terminated when there is no significant change in Brix for 3 consecutive days (this condition is met after 10 days of fermentation). Then, the temperature is lowered to 4°C and maintained for about 12 hours to terminate the fermentation.

[0062] The change in sugar content (°Brix value) was measured using a saccharimeter.

[0063] S11 wine clarification: The bayberry wine obtained after fermentation was terminated in step S10 and centrifuged at 1000rpm for 20min at 4 °C. The supernatant obtained by centrifugation (the clarified liquid) was used as the product of bayberry wine and stored at -20 °C.

[0064] The bayberry wine obtained above was tested by conventional gas chromatography, and the alcohol content was about 5±1%, and the pH value was about 3.5.

[0065] Control: Commercial active dry yeast EC1118 (identified as Saccharomyces cerevisiae) purchased from Angel Company was used for fermentation as a control.

[0066] That is, relative to Example 3:

[0067] Cancel S7;

[0068] S8 was changed to: the cooled sterile bayberry juice obtained in step S6 was inoculated and fermented with commercial active dry yeast EC1118 (≥5×109 CFU / g) to make the viable bacteria count of the bayberry juice after inoculation about 10 6 CFU / mL, the fermentation period was 9 days, and the rest were the same as in Example 3.

[0069] Experiment 1: Analysis of aroma components in bayberry wine (this detection method is a conventional technique)

[0070] The bayberry wine obtained in step S11 of Example 3 (commercial bayberry wine, obtained by fermentation with Pichia kluyveri ZFM-0524 for 10 days), the bayberry wine obtained in the control (obtained by fermentation with commercial active dry yeast EC1118 for 9 days), and the cooled sterile bayberry juice (Juice) obtained in step S6 of Example 3 were used as samples, and the aroma components of the samples were measured. The specific measurement method is as follows:

[0071] (1) Extraction of aroma components using headspace solid phase microextraction technology

[0072] A 10 mL sample was mixed with 1 g of sodium chloride and 10 μL of 4-methyl-2-pentanol (approximately 8.11 mg / mL) and placed in a 20 mL headspace vial. The mixture was completely sealed and headspace extracted for volatile components in the wine. The mixture was heated at 50°C for 20 minutes. After equilibration, an SPME fiber coated with divinylbenzene / carboxymethyl / polydimethylsiloxane (50 / 30 μm DVB / CAR / PDMS) was inserted into the headspace vial and extracted at 50°C for 40 minutes.

[0073] (2) Analysis of aroma components using GC-MS

[0074] The extraction head was removed and inserted into the gas chromatograph injection port. After 15 minutes of decomposition, the extraction head was removed and GC-MS analysis was started.

[0075] Gas chromatography conditions: The chromatographic column was a DB-WAX column (0.5 μm, 60 mm × 0.25 mm, Agilent), the carrier gas was high-purity helium (purity ≥99.999%), the flow rate was 1 mL / min, the inlet temperature was 250°C, and splitless injection was used. The temperature program was as follows: the initial column temperature was 35°C, held for 3 min, then increased to 160°C at a rate of 2°C / min, then to 200°C at a rate of 3°C / min, and finally to 240°C at a rate of 4°C / min, held for 5 min, and splitless injection was used.

[0076] Mass spectrometry conditions: full scan mode was used to collect signals, the ion source was EI, the ion source temperature was 230°C, the electron energy was 70 eV, the quadrupole temperature was 150°C, the scan frequency was 4.58 s, and the scan mass range was 30.00-500.00 amu.

[0077] A standard database was used for analysis, with qualitative analysis performed through library searches and the retention index (RI) and retention time (RT) of the mass spectrometric fragment ion peaks of the volatile components. Subsequently, the concentrations of the volatile compounds were calculated based on the area ratio of the identified compounds to the internal standard, and calibration was performed using calibration curves of various standards. (The instrument used was an Agilent 7890A-5975C, Santa Clara, CA). The names and contents of the aroma compounds are shown in Table 1.

[0078] Table 1 Comparison of aroma components of bayberry juice and bayberry wine fermented by EC1118 and ZFM-0524

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] Note: ND means not detected; the aroma descriptions in the above table were all found on the websites Flavornet and vcf-online.

[0087] As shown in Table 1, a total of 76 volatile compounds were detected in bayberry juice, including 13 alcohols, 18 esters, 5 acids, 7 aldehydes, 6 ketones, 18 terpenes, and 9 others. A total of 68 volatile compounds were detected in bayberry wine fermented with Saccharomyces cerevisiae EC1118, including 13 alcohols, 23 esters, 7 acids, 3 ketones, 13 terpenes, and 9 others. In contrast, a total of 65 volatile compounds were detected in bayberry wine fermented with Pichia kluyveri ZFM-0524, including 12 alcohols, 26 esters, 6 acids, 1 aldehyde, 3 ketones, 10 terpenes, and 7 others. Compared with the control juice, the volatile content in fermented bayberry wine increased significantly, ranging from 5.50 to 8.09 times. This is attributed to the release of more volatile compounds through a series of chemical reactions during yeast metabolism during fermentation. Comparing the total concentrations of volatile compounds in different fermented bayberry wines, the bayberry wine fermented by Pichia kluyveri ZFM-0524 had the highest total concentration of volatile substances, which was 464.09 mg / L. Compared with the control group Saccharomyces cerevisiae EC1118, its total concentration increased by about 32%, further enhancing the aroma complexity of the bayberry wine.

[0088] Esters are important aroma compounds in fruit wine, contributing to pleasant floral and fruity aromas and forming a crucial component of wine aroma. Compared to the control juice, Pichia kluyveri ZFM-0524 increased the total amount of esters by 14.27-fold, and compared to the control yeast EC1118, Pichia kluyveri ZFM-0524 increased the total amount of esters by 5.34-fold, reaching 371.17 mg / L. Ethyl acetate, isoamyl acetate, and phenylethyl acetate were the predominant esters in bayberry wine, with ethyl acetate being the most abundant. The ethyl acetate content in ZFM-0524 samples was 6.71-fold higher than that in the control EC1118 and 18.20-fold higher than that in the fruit juice. Isoamyl acetate contributes to rich fruity aromas, such as apple and banana. The content of isoamyl acetate in ZFM-0524 samples was 28.48-fold higher than that in the control EC1118, demonstrating the robust production of isoamyl acetate by Pichia kluyveri ZFM-0524. The ZFM-0524 wine sample contained 13,669.20 μg / L of phenylethyl acetate, while the control group, EC1118, contained only 118.31 μg / L, a 115-fold increase. This ester imparts aromas such as rose and honey to bayberry wine, enhancing the wine's floral notes. Furthermore, the fermentation of Pichia kluyveri ZFM-0524 produced higher levels of esters such as hexyl acetate, propyl acetate, isobutyl acetate, 3-(methylthio)propyl acetate, (Z)-3-hexenyl acetate, ethyl nonanoate, methyl benzoate, ethyl benzoate, 3-phenylpropyl acetate, and γ-nonalactone than the control group, Saccharomyces cerevisiae EC1118. These esters significantly enhance the fruity and floral aroma of bayberry wine, increasing the complexity and uniqueness of the wine's aroma and ultimately improving the sensory quality of the final product. In addition, esters such as butyl acetate, (Z)-2-pentenyl acetate, heptyl acetate, benzyl acetate, neryl acetate and phenylethyl propionate are secondary metabolites unique to the fermentation of Pichia kluyveri ZFM-0524. These specific esters impart multiple aroma characteristics to the wine, including the pear aroma of butyl acetate, the floral aroma of heptyl acetate, the rich jasmine aroma of benzyl acetate, the citrus and floral aromas of neryl acetate, and the fruity aroma of phenylethyl propionate, which together constitute a complex aroma that is different from traditional fermented wines.

[0089] In summary, it can be concluded that the use of Pichia kluyveri ZFM-0524 in the fermentation of bayberry wine can improve the flavor quality of bayberry wine.

[0090] Comparison: Pichia kluyveri PK-21, Pichia kluyveri PKL-A, and strain HSP11 were used instead of Pichia kluyveri ZFM-0524 of the present invention, and bayberry wine was prepared according to Example 3; similarly, no significant change in sugar content (°Brix) for 3 consecutive days was considered the fermentation endpoint, and the fermentation time was about 9-10 days.

[0091] The aroma components of the resulting bayberry wine were analyzed using the aforementioned method. The results showed no significant differences in the contents of hexyl acetate and (Z)-3-hexenyl acetate compared to the control EC1118. Furthermore, the following components, (Z)-2-pentenyl acetate, benzyl acetate, neryl acetate, and phenylethyl propionate, were not detected. Benzyl acetate, neryl acetate, and phenylethyl propionate, among others, contribute to the wine's unique and irreplaceable floral and fruity aroma.

[0092] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples and is subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention are considered to be within the scope of protection of the present invention.

Claims

1. A strain of Pichia kluyveri ZFM-0524, characterized in that The accession number is CCTCC NO:M2025281.

2. The use of Pichia kluyveri ZFM-0524 according to claim 1, characterized in that: The Pichia kluyveri ZFM-0524 is used for the fermentation of bayberry wine.

3. The use of Pichia kluyveri ZFM-0524 according to claim 2, characterized in that: It can not only increase the content of volatile aroma substances in bayberry wine, but also produce unique ester substances that can impart floral and fruity aroma.

4. The use of Pichia kluyveri ZFM-0524 according to claim 2, characterized in that: The volatile aroma substances with increased content are: ethyl acetate, isoamyl acetate, phenylethyl acetate, hexyl acetate, propyl acetate, isobutyl acetate, (Z)-3-hexenyl acetate, ethyl nonanoate, methyl benzoate, ethyl benzoate, 3-(methylthio)propyl acetate, 3-phenylpropyl acetate and γ-nonalactone; The unique ester substances capable of imparting floral and fruity aroma are: butyl acetate, heptyl acetate, benzyl acetate, neryl acetate, and phenylethyl propionate.

5. A method for producing bayberry wine, characterized in that: The method comprises fermenting bayberry juice with the Pichia kluyveri ZFM-0524 as claimed in claim 1 to obtain bayberry wine.

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