Hansenula polymorpha X4 and application of hansenula polymorpha X4 in preparation of rose enzyme

Abstract

The invention discloses hansenula polymorpha X4 and application thereof in preparation of rose enzyme, and belongs to the technical field of microbial fermentation and food processing. Aiming at the problems of low raw material utilization rate, insufficient enzyme aroma production efficiency, single fruit wine flavor and the like in the existing rose processing, the invention discloses a method for preparing a rose enzyme liquid by using an autonomously screened hanseniaspora uvarium X4 strain through a two-stage fermentation process for the first time, so that the aroma and antioxidant activity of the rose enzyme are improved. The obtained enzyme liquid is further blended with rice wine base liquid, concentrated grape juice and cold-extracted oolong tea liquid, a natural additive-free composite fruit wine product is developed, and the finished product is rich in polyphenol substances, organic acid, free amino acid and high-concentration volatile ester and alcohol components and shows unique flavor. The invention provides a new way for efficient utilization of rose resources and development of fruit wine with natural flavor.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial fermentation and food processing, and particularly relates to application of a grape Hanseniaspora uvarum strain in preparation of rose enzyme. BACKGROUND

[0002] Large-scale planting of rose flowers has highlighted the problem of seasonal overcapacity, and a large amount of unused fresh flowers are wasted due to preservation difficulties. Traditional rose processing mainly involves drying or essential oil extraction, but has defects such as high energy consumption and low added value. The use of biological fermentation technology to prepare rose enzyme can not only efficiently convert excess raw materials, but also retain active ingredients, and thus becomes a new solution. However, existing rose enzyme production processes mostly rely on fermentation of lactic acid bacteria or Saccharomyces cerevisiae, and the efficiency of key floral substances (such as phenylethanol and esters) is low, and the stability of enzyme products is prone to decline due to microbial residues. At the same time, the fruit wine market has long been plagued by flavor homogenization, and most of the products on the market rely on single fruit juice fermentation substrate. In order to make up for the lack of aroma, synthetic fragrances are often added, resulting in a harsh taste and a bitter aftertaste. If natural rose enzyme and composite substrate (such as rice wine and tea soup) are prepared, the fruit wine can be endowed with multi-level floral fragrance, and the use of artificial additives can be avoided, but the compatibility of enzyme and base wine and the flavor synergistic mechanism in the related technical system still lack systematic research. SUMMARY

[0003] The technical problem to be solved by the present application is to provide a grape Hanseniaspora uvarum X4 strain with high ester yield and its application in preparation of rose enzyme.

[0004] The technical solution of the present application is a grape Hanseniaspora uvarum X4 strain, which is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20251121.

[0005] The application of the grape Hanseniaspora uvarum X4 strain in fermentation of rose petals to produce rose enzyme.

[0006] A preparation method of rose enzyme, comprising the following steps:

[0007] S1. Fresh heavy-petaled red rose petals are washed and crushed to a particle size of ≤2 mm, mixed with 3 kg of water per kg of petals to form a rose slurry;

[0008] S2. The grape Hanseniaspora uvarum X4 strain is inoculated into YPD liquid medium and shaken to obtain a seed liquid;

[0009] S3. Access 5% seed liquid to the rose slurry obtained from S1, and ferment at 25℃ for 5-7 days in a closed state, with stirring twice a day;

[0010] S4. Filter the fermentation product of S3, then adjust the pH to 4.0-4.5, and oxygen fermentation at 15℃ for 3 days;

[0011] S5. Centrifuge the fermentation product of S4, take the supernatant, and filter to obtain rose enzyme.

[0012] Further, in S2, the conditions of the shaking culture are as follows: 28℃, 150rpm shaking culture for 24h.

[0013] A compound fruit wine, which is composed of the following raw materials in volume percentage: rice wine base wine 60-70%, concentrated grape juice 15-20%, oolong tea extract 5-8%, and rose enzyme liquid obtained by the above preparation method 2-20%.

[0014] Further, the rice wine base wine is fermented from glutinous rice, and the alcohol content is 6-12%vol.

[0015] Further, the soluble solids of the concentrated grape juice are 65°Bx.

[0016] Further, the tea polyphenol in the oolong extract is ≥200mg / L.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] 1. The present application selects a high-yield ester grape Hanseniaspora uvarum X4, and uses it to ferment to produce rose enzyme, which can significantly improve the total ester content, phenolic compounds and in vitro antioxidant activity level in rose flowers, and endow the enzyme with unique rich aroma and health care function.

[0019] 2. The present application establishes a two-stage fermentation process for preparing rose enzyme, which further significantly improves the total ester content, phenolic compounds and in vitro antioxidant activity level.

[0020] 3. The enzyme liquid is compounded with rice wine, oolong tea and grape juice to develop a new fruit wine with balanced aroma and taste, which provides an innovative path for high-value utilization of rose flowers and development of natural flavor fruit wine.

[0021] Preservation information:

[0022] Hanseniaspora uvarum X4, deposited in China Center for Type Culture Collection, preservation number: CCTCC NO: M 20251121, preservation date: May 20, 2025, preservation address: Wuhan University, No. 299, Bajiyilu, Wuchang District, Wuhan, Hubei Province. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 A morphological characteristic diagram of the Hanseniaspora uvarum X4 strain provided in the present application is provided.

[0024] Figure 2 A ITS phylogenetic tree construction diagram of the Hanseniaspora uvarum X4 strain provided in the present application is provided.

[0025] Figure 3 A schematic diagram of the Hanseniaspora uvarum X4 strain provided in the present application growing in an ester-producing solid culture medium is provided.

[0026] Figure 4 A radar chart of the electronic tongue in Example 2. DETAILED DESCRIPTION

[0027] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from commercial channels unless otherwise specified.

[0028] Example 1 Isolation, identification and preservation of Hanseniaspora uvarum X4

[0029] (1) Yeast strain isolation and purification

[0030] Take 10 g of green plum sample, add 90 mL of 0.1% physiological saline under sterile conditions, homogenize, and stand for 30 min, then take the supernatant for 10-fold gradient dilution. Select 10 -3 ~ 10 -5 Diluted liquid is spread on YPD solid medium containing 0.1 g / L chloramphenicol, and cultured at 28°C for 48 h. Single colonies with significant morphological differences are picked and purified by plate streaking method for 3 generations until the colony morphology is stable. The purified strain is inoculated into YPD slant and stored at 4°C for short-term preservation.

[0031] (2) Strain identification and preservation

[0032] Morphological identification: After 24 h of activation in YPD liquid medium, a ring of bacterial liquid is picked with a sterile inoculation ring and streaked on a YPD plate, which is then cultured at 28±1°C for 48 h, and its colony characteristics are recorded in terms of size, color, texture, edge shape, etc. The colony of strain X4 is shown in FIG. 1. Figure 1The colony characteristics of the non-Saccharomyces cerevisiae X4 strain according to the present application are as follows: the colony is milky white, spherical, opaque, with a raised center, smooth surface, regular edge and easy to pick up on YPD medium.

[0033] Molecular identification: the ITS molecular biology identification was performed on the strain, and primers ITS1 and ITS4 were used for amplification; the purified PCR product was subjected to sequence determination, and the results were submitted to the NCBI database for homology comparison, and the sequences with higher homology were selected, and the sequences were analyzed by using MEGA analysis software, and a phylogenetic tree was constructed, and the results are shown in Figure 2 The X4 strain has the highest homology with Hanseniaspora uvarum. According to the morphological characteristics and molecular biology identification results of the strain X4 in the present application, it is determined that the strain of the present application is Hanseniaspora uvarum, and is named as Hanseniaspora uvarum X4, and was preserved in the China Center for Type Culture Collection located in Wuhan University on May 20, 2025, with the preservation number of CCTCC NO: M 20251121.

[0034] (3) Ester production capacity determination

[0035] The ester production capacity of the strain was determined by using ester production solid medium. The preparation method of the ester production solid medium can be as follows: 20 g of glucose, 20 g of proteose peptone / L, 10 g of yeast extract powder, 15 mL of tributyrin, 0.04 g of bromocresol purple, 20 g of agar and 1 L of water were mixed, and sterilized by high-pressure steam at 121 ℃ for 20 min. The yeast strain was inoculated on the ester production solid medium, and cultured at 30 ℃ for 3 days, and then the colony color was observed. As shown in Figure 3 The X4 produced a deep red hydrolysis circle with a diameter of ≥15 mm on the medium containing tributyrin, proving that it has high esterase activity.

[0036] Example 2 Preparation of rose enzyme

[0037] (1) Raw material treatment: fresh heavy red rose petals (without pesticide residues) were washed and crushed to a particle size of ≤2 mm, and then slurried at a ratio of petals: water = 1:3 (w / v);

[0038] (2) Strain activation: the Hanseniaspora uvarum X4 strain was inoculated in YPD liquid medium, and cultured at 28 ℃ with 150 rpm shaking for 24 h;

[0039] (3) Main fermentation: 5% (v / v) of the bacterial liquid was inoculated into the rose slurry, and fermented at 25 ℃ for 5 days in a sealed state, with stirring twice a day;

[0040] (4) Secondary fermentation: after filtration, the pH was adjusted to 4.0-4.5, and oxygen fermentation was carried out at 15 ℃ for 3 days;

[0041] (5) Filtration: centrifugation to take supernatant, and filtration to obtain rose enzyme.

[0042] Comparative Example 1

[0043] The difference from Example 1 is that fermentation treatment is not performed, i.e. steps (2)-(4) are removed.

[0044] Comparative Example 2

[0045] The difference from Example 1 is that after the main fermentation is completed, filtration is directly performed, and secondary fermentation is not performed, i.e. step (4) is removed.

[0046] Example 3 Active Substance Content Detection

[0047] 1. Analysis Method

[0048] (1) SOD enzyme activity determination

[0049] The SOD enzyme activity determination refers to the national standard GB / T 5009.171-2003.

[0050] (2) Total ester determination

[0051] The saponification reflux method is used to determine the total ester content of the fermentation liquor.

[0052] (3) Total phenol and total flavonoid content determination

[0053] The Folin phenol method is used to determine the total phenol content, and the specific operation method is as follows: 0.1 mL of sample solution is taken, 0.5 mL of 10% (v / v) Folin phenol reagent is added, and after oscillation and mixing, it is reacted at room temperature for 3-8 min, then 0.4 mL of 7.5% (m / v) sodium carbonate solution is added, and after standing for 60 min, the absorbance value is determined at 765 nm. According to the same method, the absorbance values of gallic acid solutions of different concentrations are determined, and a standard curve is drawn. The sample absorbance value is substituted into the gallic acid standard curve to calculate the total phenol content in the sample, and the result is expressed as gallic acid equivalent (mg / L).

[0054] The aluminum chloride colorimetric method is used to determine the total flavonoid content, and the specific operation method is as follows: 0.1 mL of sample solution is taken, 2.0 mL of 70% (v / v) methanol solution and 0.1 mL of 5% (m / v) sodium nitrite solution are added, and after oscillation and mixing, it is reacted at room temperature for 6 min, then 0.2 mL of 10% (m / v) aluminum chloride solution is added, and after oscillation and mixing, it is reacted at room temperature for 5 min, finally 0.6 mL of 70% (v / v) methanol solution is added, and the absorbance value is determined at 420 nm. According to the same method, the absorbance values of rutin solutions of different concentrations are determined, and a standard curve is drawn. The sample absorbance value is substituted into the rutin standard curve to calculate the total flavonoid content in the sample, and the result is expressed as rutin equivalent (mg / L).

[0055] (4) Anti-oxidative activity assay

[0056] The in vitro anti-oxidative activity was evaluated by DPPH and ABTS free radical scavenging rate.

[0057] DPPH free radical scavenging rate: 99.20 mg of DPPH was dissolved in 25.00 mL of anhydrous ethanol to obtain a DPPH free radical stock solution, which was mixed well and stored at 4°C in the dark. A certain amount of the stock solution was diluted with anhydrous ethanol to obtain a DPPH working solution with an absorbance of 0.5-0.8 at 517 nm. Sample group A1: 100 μL of sample solution was mixed with 100 μL of DPPH working solution; blank group A2: 100 μL of sample solution was mixed with 100 μL of anhydrous ethanol; control group A3: 100 μL of ultrapure water was mixed with 100 μL of DPPH working solution, and the absorbance was measured at 517 nm after the reaction of each group in the dark for 30 min. Each group had 3 replicates, and the free radical scavenging activity was determined as follows:

[0058]

[0059] ABTS free radical scavenging rate: 7.00 mM of ABTS solution and 2.45 mM of potassium persulfate solution were prepared with ultrapure water, mixed well, and reacted at room temperature in the dark for 12-16 h to obtain an ABTS free radical stock solution, which was stored at 4°C in the dark. A small amount of the stock solution was diluted with ultrapure water to obtain an ABTS working solution with an absorbance of 0.70±0.02 at 734 nm. Sample group A1: sample solution (20 μL) was mixed with ABTS working solution (180 μL); control group A2: ultrapure water (20 μL) was mixed with ABTS working solution (180 μL), and the absorbance was measured at 734 nm after the reaction in the dark for 6 min. The formula is as follows:

[0060]

[0061] 2. Result analysis

[0062] The SOD enzyme activity of Comparative Example 1 was 6.23 U / mL, and the SOD enzyme activity of Comparative Example 2 and Example 2 was significantly increased. During the fermentation process, the microorganisms continuously multiplied, and the SOD enzyme activity increased and accumulated. The total ester content of Example 2 was 3.82 g / L, which was higher than that of Comparative Example 1 and Comparative Example 2. Compared with Comparative Example 1 and Comparative Example 2, the total phenol and total flavonoid contents in Example 2 were increased to 110.23 mg / L and 88.39 mg / L, respectively. Through two-stage fermentation, the DPPH and ABTS scavenging abilities of the rose enzyme of Example 2 were significantly improved.

[0063] From the above results, it can be seen that the fermentation of rose enzyme by Hanseniaspora uvarum X4 can significantly increase the total ester content, phenolic compounds and in vitro antioxidant activity in rose flowers, and the quality of rose enzyme can be further improved by two-stage fermentation.

[0064] Table 1: Physicochemical indicators

[0065]

[0066] Note: The results are shown in the form of mean ± standard deviation (n = 3); significant difference between different samples P < 0.05

[0067] The above a-c represent.

[0068] Example 3: Preparation of compound fruit wine

[0069] (1) Formula (per 1000L): rice wine base wine (alcohol content 6-12%vol): 800L; concentrated grape juice (soluble solids 65°Bx): 50kg; cold-brewed oolong tea liquid (tea polyphenols ≥200mg / L): 100L; rose enzyme liquid obtained in Example 2: 5kg;

[0070] (2) Mixing and blending: mix and blend the components at 10°C in a stirring environment.

[0071] (3) Sterilization: sterilize by membrane filtration (0.45μm).

[0072] Example 4: Detection of compound fruit wine

[0073] 1. Analysis method

[0074] (1) Detection of basic physicochemical indicators

[0075] Use a handheld digital refractometer and a pH meter to measure the soluble solids content and pH value, respectively; the total acid and reducing sugar content is determined according to the national standard GB / T 15038-2006.

[0076] (2) Detection of phenolic substance composition and content

[0077] Mix the sample with ethyl acetate in a centrifuge tube, ultrasonic for 30min, repeat the above operation 3 times to collect all the organic layers, rotary evaporation at 35°C under reduced pressure until the organic liquid is evaporated, dissolve the extracted polyphenol sample in 5mL of methanol, and put it in a-20°C refrigerator for use.

[0078] Pass the sample through a 0.22μm organic filter membrane for HPLC analysis. The detection conditions are as follows:

[0079] Chromatographic column: ODS-4 (particle size 5μm, The column temperature was 30℃; mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was methanol, with 5-40% A for 0-35 min, 95% A for 35-55 min, and 5% A for 55-60 min; the flow rate was 0.8 mL / min; the UV detection wavelengths were 280 nm, 320 nm, and 360 nm; and the injection volume was 20 μL. Characterization of phenolic compounds involved determining the peak times of the target substances using standard solution chromatograms and identifying the peaks of different substances in the sample chromatograms. The content of monomeric phenols was quantitatively determined using a standard curve of standard samples.

[0080] (3) Organic acid detection

[0081] The sample was filtered through a 0.22 μm filter membrane and then analyzed by HPLC. The detection conditions are as follows.

[0082] Column: C18-H (particle size 5μm) The column temperature was 30℃; mobile phase A: 0.009 mol / L concentrated sulfuric acid, mobile phase B: methanol, A:B = 97.5:2.5; flow rate: 0.8 mL / min; UV detection wavelength: 210 nm; injection volume: 20 μL. Organic acids were characterized by determining the peak times of the target substance using standard solution chromatograms and identifying the peaks of different substances in the sample chromatograms. The organic acid content was quantitatively determined using a standard curve of the standard samples.

[0083] (4) Detection of free amino acids

[0084] The sample (100 μL) was mixed with 400 μL of 20% sulfosalicylic acid and hydrolyzed pre-column at room temperature for 1 h. The derivative was filtered through a 0.22 μm nylon membrane for subsequent determination. The automated amino acid analyzer was set as follows.

[0085] Mobile phase flow rate: 180 μL / min; column temperature: 40℃; reaction solution flow rate: 180 μL / min; reaction temperature: 70℃; detection wavelength: 550 nm and 440 nm; injection volume: 20 μL.

[0086] (5) Electronic tongue taste detection

[0087] Taste analysis was performed using the TS-5000Z electronic tongue sensory system to detect sour, sweet, bitter, salty, umami, and astringent flavors in the enzyme-infused wine. 35 mL of the enzyme-infused wine was placed in the sample cell and continuously measured at 25°C for 120 seconds, followed by another 30 seconds to assess the aftertaste. Each sample was measured four times, and the three most stable data points were selected for further analysis.

[0088] (6) Detection of volatile components

[0089] Sample treatment: 2 mL sample was accurately weighed in a 20 mL headspace bottle, 20 μL (20 μg / mL) 3-octanol and 0.2 g sodium chloride were added, and then the bottle was immediately sealed and placed in a 45 °C water bath for magnetic stirring for 30 min. After the solid-phase microextraction sampling handle and solid-phase microextraction head were installed, the headspace bottle was inserted, the fiber probe was pushed out, and the magnetic stirring was continued in the 45 °C water bath. After 30 min of headspace extraction, the fiber head was pulled back in and inserted into the GC-MS sampling port at 240 °C for desorption for 5 min.

[0090] GC instrument parameters: inlet temperature: 240 °C; column: DB-WAX (0.25 μm x 0.25 mm x 60 m); helium flow rate: 1.0 mL / min; temperature program: 40 °C for 3 min, increased to 120 °C at a rate of 4 °C / min, increased to 210 °C at a rate of 6 °C / min and maintained for 3 min, and then increased to 240 °C at a rate of 10 °C / min and maintained for 3 min; linear velocity: 1.0 mL / min; injection mode: splitless.

[0091] MS instrument parameters: ion source temperature: 230 °C; interface temperature: 240 °C; acquisition mode: Scan.

[0092] Qualitative and quantitative analysis: The identification of volatile components was determined based on the preliminary results obtained from the NIST 20s database search, and the volatile components were qualitatively analyzed by comparing their retention indices (RI) with the data reported in the literature.

[0093] 2. Results analysis

[0094] The pH value and total acid content of the compound fruit wine of Example 3 were 3.43 and 5.98 g / L, respectively. Acidity, as one of the key factors affecting the flavor of fruit wine, is crucial for maintaining the flavor and taste of the wine body at an appropriate level. The soluble solids and reducing sugar contents were 18.73° and 39.7 g / L, respectively (Table 2).

[0095] Table 2 Physicochemical indicators

[0096]

[0097] Note: The results are shown as mean ± standard deviation (n = 3)

[0098] Seven phenolic compounds were detected in the compound fruit wine of Example 3 (Table 3). Among these phenolic compounds, the contents of epicatechin (190.34 mg / L) and gallic acid (59.60 mg / L) were the highest, followed by catechin (18.13 mg / L) and rutin (13.19 mg / L).

[0099] Table 3 Phenolic compound content (mg / L)

[0100]

[0101] Note: Results are shown as mean ± standard deviation (n = 3)

[0102] Eight organic acid species were detected in the compound fruit wine of Example 3 (Table 4), among which acetic acid, lactic acid, and succinic acid were the main organic acid components, with concentrations of 2044.16 mg / L, 673.79 mg / L, and 675.3 mg / L, respectively. The high concentration of acetic acid may be closely related to the metabolic activity of yeast during the base wine fermentation process. The high concentration of lactic acid is believed to have a positive impact on the soft taste of the wine body, while succinic acid is characterized by its salty and bitter taste.

[0103] Table 4 Organic acid content (mg / L)

[0104]

[0105] Note: Results are shown as mean ± standard deviation (n = 3)

[0106] Ten free amino acids were detected in the compound fruit wine of Example 3 (Table 5), with a total mass concentration of 50.20 mg / L. Further analysis of the distribution of taste amino acids showed that the content of umami amino acids was the highest, reaching 38.69 mg / L. Umami amino acids such as glutamic acid and aspartic acid are important flavor components in fruit wine, which can significantly enhance the umami flavor and taste of fruit wine.

[0107] Table 5 Free amino acid content (mg / L)

[0108]

[0109] Note: Results are shown as mean ± standard deviation (n = 3)

[0110] Sixty volatile compounds were detected in the compound fruit wine of Example 3 (Table 6), among which esters (2839.07 μg / L) and alcohols (4474.31 μg / L) were dominant. The concentrations of 3-methyl-1-butanol (3071.85 μg / L), ethyl acetate (1250 μg / L), and phenylethyl alcohol (916.02 μg / L) were relatively high, followed by 3-methyl-2-butanol acetate (369.75 μg / L), 2-methyl-1-propanol (365.19 μg / L), ethyl octanoate (234.96 μg / L), linalool acetate (207.88 μg / L), ethyl lactate (131.11 μg / L), and ethyl butyrate (112.49 μg / L). In addition, Example 2 contained abundant terpenes and ketones, which were usually associated with fruity aromas and played an important role in the flavor of the wine body, although their content was not high. These compounds included (+)-a-terpineol (57.16 μg / L), linalool (20.09 μg / L), damascenone (18.91 μg / L), dihydroterpineol (17.19 μg / L), a-damascenone (12.74 μg / L), β-ionone (10.38 μg / L), and a-ionone (9.88 μg / L).

[0111] Table 6. Volatile compound content in Example 2 (μg / L)

[0112]

[0113]

[0114]

[0115]

[0116] Note: The results are shown as mean ± standard deviation (n = 3)

[0117] The compound fruit wine of Example 3 had a low sourness intensity and a high umami intensity. Figure 4 In fruit wine, umami can increase the richness of the wine body, and sourness is one of the important indicators affecting the fullness of the wine body.

[0118] In summary, the independently screened Hanseniaspora uvarum X4 strain was used to prepare rose enzyme liquid through a two-stage fermentation process, which improved the antioxidant activity of the rose enzyme. The enzyme liquid was further blended with rice wine base, concentrated grape juice, and cold-brewed oolong tea liquid to develop a fruit wine product. The finished product is rich in polyphenols, organic acids, free amino acids, and high concentrations of volatile esters and alcohols, and exhibits a unique flavor.

Claims

1. A strain of Hanseniaspora uvarum X4, which is deposited in China Center for Type Culture Collection, and the deposit number is CCTCC NO: M 20251121.

2. The use of the Hanseniaspora uvarum X4 strain of claim 1 in the production of rose enzyme from fermented rose petals.

3. A method for preparing rose ferment, characterized in that, The method comprises the following steps: S1. Fresh heavy red rose petals are washed and crushed to a particle size of ≤2 mm, and then mixed with 3 kg of water per kg of petals to obtain rose slurry; S2. The Hanseniaspora uvarum X4 strain of claim 1 is inoculated into YPD liquid medium and shaken to obtain seed liquid; S3. 5% of the seed liquid is inoculated into the rose slurry obtained in S1, and then fermented at 25°C for 5-7 days with stirring twice a day; S4. The fermentation product of S3 is filtered, and then the pH is adjusted to 4.0-4.5, and then oxygen fermentation is carried out at 15°C for 3 days; S5. The fermentation product of S4 is centrifuged, and then the supernatant is filtered to obtain rose enzyme.

4. The production method according to claim 3, characterized by, In S2, the conditions for shaking culture are as follows: 28°C, 150 rpm shaking culture for 24 h.

5. A composite fruit wine, characterized by, The rice wine base wine is fermented from glutinous rice, and the alcohol content is 6-12%vol.

6. The compound fruit wine according to claim 5, characterized by, The concentrated grape juice has a soluble solid content of 65°Bx.

7. The compound fruit wine according to claim 5, characterized by, The tea polyphenol content in the oolong tea extract is ≥200 mg / L.

8. The compound fruit wine according to claim 5, characterized by, ​

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