An abnormal williamsmycoderma yeast and application thereof, wolfberry wine and preparation method thereof

Abstract

The application discloses an abnormal williamsonia fermentum, application of the abnormal williamsonia fermentum, a medlar wine and a preparation method of the medlar wine, and belongs to the technical field of medlar wine preparation. The biological preservation number of the abnormal williamsonia fermentum is CGMCC No. 25068. The abnormal williamsonia fermentum can be used for preparing fruit wine, in particular, medlar wine. The preparation method of the corresponding medlar wine comprises the following steps: inoculating seed liquid of the abnormal williamsonia fermentum and seed liquid of brewing yeast into fermentation liquid prepared from medlar, and fermenting. The method can effectively solve the problems of insufficient aroma and non-outstanding typicality of the medlar wine caused by single fermentation. Compared with pure strain fermentation of the brewing yeast, the content of ester substances in the medlar wine brewed by mixed fermentation is increased by 85.8%, and the flavor quality of the medlar wine is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of wolfberry wine preparation technology, and more specifically, to an abnormal Vickham yeast and its application, wolfberry wine and its preparation method. Background Technology

[0002] Goji berries, a food with both medicinal and nutritional value, are rich in amino acids, organic acids, vitamins, polysaccharides, crude protein, and dietary fiber. Currently, goji berry products are mainly dried, but this single drying method leads to insufficient processing conversion, resulting in resource waste and severely hindering the development of the Ningxia goji berry industry. Deep processing goji berries into goji berry wine not only broadens the goji berry processing industry but also solves the resource waste caused by goji berry hoarding and unsold stock.

[0003] Goji berry wine is an alcoholic beverage made from dried or fresh goji berries through yeast fermentation. It is characterized by its low alcohol content, rich flavor, and high nutritional value. Using goji berries in fruit wine production not only preserves their nutritional components but also extends shelf life, expands the goji berry industry chain, and improves economic benefits. It is an effective way to drive the development of the goji berry industry and increase the income of goji berry growers. However, due to the low content of aroma components in goji berries, the resulting fruit wine suffers from insufficient aroma and a lack of distinctive character.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide an abnormal Vickham yeast that can be used to prepare fruit wine.

[0006] The second objective of this invention is to provide an application of the aforementioned abnormal Vickham yeast in the preparation of fruit wine.

[0007] The third objective of this invention is to provide a method for preparing wolfberry wine that is simple, easy to operate, and can solve the problems of insufficient aroma and lack of typicality in existing wolfberry wines.

[0008] The fourth objective of this invention is to provide a wolfberry wine prepared by the above-described preparation method, which has a superior flavor quality.

[0009] This application can be implemented as follows: In the first aspect, this application provides an abnormal *V. wickhamnium* yeast, with the biological preservation number CGMCC No. 25068. This abnormal *V. wickhamnium* yeast was deposited on June 13, 2022, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] Secondly, this application provides the use of the abnormal Vickham yeast as described in the above embodiments in the preparation of fruit wine.

[0011] In an optional implementation, the fruit wine is wolfberry wine.

[0012] Thirdly, this application provides a method for preparing wolfberry wine, comprising the following steps: inoculating the seed liquid of brewing yeast and the seed liquid of abnormal Vickham yeast of the aforementioned embodiments into the fermentation liquid obtained from wolfberry, and fermenting.

[0013] In an optional implementation, the brewing yeast has the preservation number CGMCC No. 23603.

[0014] In an optional implementation, the seed culture of *V. aberrantis* and the seed culture of *Saccharomyces cerevisiae* are inoculated into the fermentation broth simultaneously or at intervals.

[0015] In an optional implementation, when using an interval inoculation method, the seed culture of *V. aberrant* *V. erg.* is inoculated first, followed by the seed culture of *Saccharomyces cerevisiae*.

[0016] In an optional implementation, the interval is 6-18 hours.

[0017] In a preferred embodiment, the interval is 12 hours.

[0018] In an optional implementation, the inoculum size of the *V. aberrantus* seed culture is 1 × 10⁻⁶. 6 Up to 1×10 8 CFU / mL.

[0019] In an optional implementation, the seed culture of *V. aberrantis* yeast is prepared by the following method: Inoculate abnormal Vickham yeast into YPD liquid medium and incubate at 28-32℃ for 42-54 hours.

[0020] In an optional embodiment, the inoculation amount of the brewer's yeast seed culture is 1×10⁻⁶. 6 Up to 1×10 7 CFU / m.

[0021] In an optional embodiment, the seed culture of brewer's yeast is prepared by the following method: Inoculate the yeast with YPD liquid medium and incubate at 28-32℃ for 36-48 hours.

[0022] In an optional implementation, fermentation is carried out for at least 5 days at 18-22°C.

[0023] In an optional embodiment, the preparation of the fermentation liquid of wolfberry includes: mixing wolfberry with water and juicing, and adjusting the sugar content.

[0024] In an optional implementation, the goji berries are dried goji berries.

[0025] In an optional embodiment, dried goji berries are mixed with water at a mass ratio of 1:8-12.

[0026] In an optional implementation, sucrose or white sugar is used to adjust the sugar content.

[0027] In an optional embodiment, the sugar content of the wolfberry extract after sugar content adjustment is 150-250 g / L.

[0028] In an optional embodiment, after adjusting the sugar content, pectinase and potassium metabisulfite are added to the wolfberry extract.

[0029] In an optional embodiment, the amount of pectinase added is 20-40 U / g, and / or the amount of potassium metabisulfite added is 80-120 mg / L.

[0030] In an optional embodiment, the fermentation broth after fermentation is further subjected to solid-liquid separation, and the liquid after solid-liquid separation is allowed to stand at 3-5°C for 12-16 days.

[0031] Fourthly, this application provides a wolfberry wine, prepared by any of the preparation methods described in the foregoing embodiments.

[0032] The beneficial effects of this application include: The method provided in this application, which involves inoculating wolfberry juice with *V. aberrantus* yeast (Y11) (CGMCC No. 25068) and *Saccharomyces cerevisiae* (M-23-7-14) (CGMCC No. 23603) to prepare wolfberry wine, can enhance the taste, aroma, and quality of wolfberry wine. Compared with single-strain fermentation with *Saccharomyces cerevisiae*, it can increase the content of esters in wolfberry wine by 85.8%, effectively solving the problems of insufficient aroma and lack of typicality in wolfberry wine caused by single fermentation. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 Biological preservation information and survival certificate of the abnormal Wickham yeast provided for this application; Figure 2This is a graph showing the alcohol tolerance results of the abnormal Wickham yeast in Example 2 of this application. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0036] The following provides a detailed description of the abnormal Vickham yeast and its application, as well as the wolfberry wine and its preparation method provided in this application.

[0037] The abnormal Vicham yeast provided in this application was screened from the natural fermentation broth of Ningqi No. 5 wolfberry juice, specifically obtained through the following screening method: (1) Microbial enrichment culture Add 10 g of dried wolfberry fruit to a shake flask containing 100 mL of enrichment medium and incubate at 30 ℃ and 220 r / min for 24 h.

[0038] (2) Strain isolation and screening The fermentation broth obtained in step (1) above was serially diluted with sterile water to 10. -7 Select 10 -5 10 -6 10 -7 Three dilution gradients were evenly spread on YPD medium and incubated upside down at 30°C for 72 h. Suitable colonies were selected for 18S rDNA identification.

[0039] The strain was identified as *Vichamus aberrantus*. Wickerhamomyces anomalus The specific name is Y11, and it was deposited on June 13, 2022, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 25068. For biopreservation information and viability proof of the *V. anomala* used in this application, please refer to [link to biopreservation information]. Figure 1 .

[0040] The colonies of this strain are creamy white, smooth, moist, and glossy, and can tolerate a maximum alcohol content of 8% and a maximum sugar content of 260 g·L⁻¹. -1 .

[0041] In addition, this application also provides the application of the above-mentioned abnormal Vickham yeast in the preparation of fruit wines, such as wolfberry wine, grape wine or apple wine.

[0042] In a preferred embodiment, *Vichamus aberrantus* yeast is used to prepare wolfberry wine.

[0043] Through comparison by the inventors, the specific abnormal Vickham yeast provided in this application has a significant impact on the aroma of wolfberry wine, which can increase the content of aroma components in wolfberry wine, enhance the aroma of wolfberry wine, and solve the current problems of insufficient aroma and lack of typicality in wolfberry wine.

[0044] Accordingly, this application provides a method for preparing wolfberry wine, which includes the following steps: inoculating the seed liquid of brewing yeast and the above-mentioned abnormal Vickham yeast seed liquid into the fermentation liquid obtained from wolfberry, and fermenting.

[0045] For reference, in this application, the preparation of the fermentation liquid of wolfberry includes: mixing wolfberry with water and juicing, and adjusting the sugar content.

[0046] Among them, wolfberry refers to dried wolfberry fruit (moisture content not exceeding 13%), and dried wolfberry fruit and water can be mixed in a mass ratio of 1:8-12 (such as 1:10).

[0047] The sugar used to adjust the sugar content can be sucrose or white sugar to avoid affecting the color of the wolfberry wine itself. The sugar content of the wolfberry liquid after adjustment can be controlled at 150-250g / L, such as 150g / L, 180g / L, 200g / L, 220g / L or 250g / L, etc., with 200g / L being preferred.

[0048] It should be noted that the sugar content in the fermentation broth directly affects the alcohol content of the goji berry wine. The higher the sugar content, the higher the alcohol content. However, if the sugar content is too high, the yeast cannot tolerate it, and fermentation cannot be effective. This application controls the sugar content within the above-mentioned range, which on the one hand ensures yeast tolerance, and on the other hand ensures that a goji berry wine with a suitable alcohol content is obtained, resulting in a better taste.

[0049] Furthermore, after adjusting the sugar content, pectinase and potassium metabisulfite are added to the wolfberry liquid.

[0050] The amount of pectinase added can be 20-40 U / g, such as 20 U / g, 25 U / g, 30 U / g, 35 U / g, or 40 U / g, or any other value within the range of 20-40 U / g. The "g" in the above pectinase dosage unit is based on dried goji berries.

[0051] By adding pectinase, the goji berries can be enzymatically hydrolyzed during fermentation, dissolving the pectin on their surface and fully releasing the nutrients contained within the goji berries. Some of the released nutrients can be utilized by yeast, which is beneficial to the growth and metabolism of yeast and promotes the fermentation reaction.

[0052] The amount of potassium metabisulfite added can be 80-120 mg / L, such as 80 mg / L, 90 mg / L, 100 mg / L, 110 mg / L or 120 mg / L, or any other value within the range of 80-120 mg / L.

[0053] Adding potassium metabisulfite can have a certain antibacterial and antioxidant effect on wolfberry wine.

[0054] In this application, the brewing yeast used is the brewing yeast with preservation number CGMCC No.23603 (M-23-7-14).

[0055] The inventors discovered through research that by combining the specific abnormal Vickham yeast provided in this application with the above-mentioned preferred brewing yeast, the taste and aroma of wolfberry wine can be enhanced and the quality of wolfberry wine can be improved; compared with single-strain fermentation of brewing yeast, the content of esters in wolfberry wine can be increased by 85.8%.

[0056] Furthermore, by combining other abnormal Vicham yeast strains with the aforementioned preferred brewing yeast under the same conditions as described in this application, a comparison showed that this method was significantly less effective in improving the aroma of goji berry wine than the specific abnormal Vicham yeast strain provided in this application. This indicates that even strains of the same genus can have different effects on the aroma of goji berry wine.

[0057] In this application, the seed culture of *V. aberrantis* and the seed culture of *Saccharomyces cerevisiae* are simultaneously inoculated into the fermentation broth, or they can be inoculated into the fermentation broth at intervals, with the interval inoculation method being preferred.

[0058] When using an intermittent inoculation method, inoculate the seed culture of abnormal Vickham yeast first, followed by the seed culture of Saccharomyces cerevisiae.

[0059] The above inoculation method primarily considers the difference in alcohol tolerance between *V. aberrantis* and *Saccharomyces cerevisiae*. *V. aberrantis* has poor alcohol tolerance, so an intermittent inoculation method before *Saccharomyces cerevisiae* produces alcohol is preferred, thereby enhancing the physiological activity of *V. aberrantis*. Adding *V. aberrantis* at the initial stage of fermentation allows it to secrete extracellular enzymes (such as β-glucosidase). When the β-D-glucosidic bonds in the raw materials are specifically hydrolyzed by this enzyme, free aroma compounds are released from the glycosides, thus improving the flavor quality and value of the raw materials. *Saccharomyces cerevisiae* generally lacks the ability to synthesize β-glucosidase, while *V. aberrantis* does. Therefore, using *V. aberrantis*, which produces β-glucosidase, in alcohol production can assist *Saccharomyces cerevisiae* in promoting the production of aroma compounds and improving the quality of the alcohol.

[0060] For reference, the interval can be 6-18 hours, such as 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h or 16h, or any other value within the range of 6-18 hours. The preferred interval is 12 hours.

[0061] In some implementations, the inoculum size of the *V. aberrantus* seed culture is 1 × 10⁻⁶. 6 Up to 1×10 8 CFU / mL, such as 1×10 6 CFU / mL, 5×10 6 CFU / mL, 1×10 7 CFU / mL, 5×10 7 CFU / mL or 1×10 8 CFU / mL, etc., can also be 1×10 6 Up to 1×10 8 Any other value within the range of CFU / mL.

[0062] The above-mentioned abnormal Vickham yeast seed culture can be exemplarily prepared by the following method: Inoculate *V. aberrantis* into YPD liquid medium and culture at 28-32°C (preferably 30°C) for 42-54 h (preferably 48 h). These conditions promote rapid growth of *V. aberrantis*, ensuring a high biomass.

[0063] The YPD liquid culture medium consists of 2% glucose, 2% tryptone, and 1% yeast extract, and is sterilized at 115°C for 20 minutes. Specific preparation methods can be found in existing technologies and will not be elaborated upon here.

[0064] The inoculum size of the Saccharomyces cerevisiae seed culture is 1×10⁻⁶. 6 Up to 1×10 7 CFU / m, such as 1×10 6 CFU / mL, 5×10 6 CFU / mL or 1×10 7 CFU / mL, etc., can also be 1×10 6 Up to 1×10 7 Any other value within the range of CFU / m.

[0065] The above-mentioned brewer's yeast seed culture can be exemplarily prepared by the following method: Saccharomyces cerevisiae was inoculated into YPD liquid medium and cultured at 28-32℃ (preferably 30℃) for 36-48 h (preferably 42 h). These conditions are conducive to the rapid growth of Saccharomyces cerevisiae, ensuring a high biomass.

[0066] After inoculation, ferment at 18-22℃ (preferably 20℃) for at least 5 days.

[0067] The above temperature conditions are the optimal fermentation temperatures for the fermentation strains used in this application.

[0068] Furthermore, after fermentation, the fermentation broth is subjected to solid-liquid separation (such as filtration), and the separated liquid is then allowed to stand at 3-5℃ for 12-16 days.

[0069] Low-temperature settling promotes the gradual formation of some flavor compounds. It should be noted that the temperature during the low-temperature settling process is relatively low, allowing the yeast to enter a semi-dormant state (while fermentation can still occur). However, excessively high temperatures can cause the brewing yeast to autolyze (break down), resulting in an unpleasant odor and affecting the quality of the goji berry wine. Accordingly, this application also provides a wolfberry wine, which is prepared by the above-mentioned preparation method. The wolfberry wine has a high content of esters and good flavor quality.

[0070] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0071] The following tests involve the following detection methods: Alcohol content: Analyzed using the density bottle method, referring to the national standard GB / T 15038-2006 General Analysis Methods for Wines and Fruit Wines.

[0072] Total acid: Accurately transfer 1 mL of sample into a 150 mL beaker using a pipette, add 50 mL of distilled water, place a rotor in the beaker, and then slowly add 0.1 mol / L NaOH standard solution dropwise onto a thermostatic magnetic stirrer until the pH reaches 8.2. Record the reading. The total acid content (calculated as lemon) is calculated as follows: ; In the formula, v0 is the amount of NaOH consumed in the blank titration, mL; v1 is the amount of NaOH consumed in the titration, mL; v2 is the volume of the sample taken, mL; C is the concentration of standard NaOH, mol / L; and 64 is the molar mass of citric acid, g / mol.

[0073] Total sugar: Accurately pipette 5 mL of sample and 5 mL of 6 mol / L hydrochloric acid into a 100 mL volumetric flask, incubate at 68°C for 15 min, cool, adjust pH to 7, and dilute to 100 mL. Accurately pipette 5.00 mL each of Fehling's A and B solutions and 5.00 mL of sample hydrolysate into a 100 mL Erlenmeyer flask, add 1.00 mL less glucose standard solution than the pre-titration value, shake well, and heat to boiling on a hot plate. Continue titrating with glucose standard solution to the endpoint. Record the volume (V) of glucose standard solution consumed. Near the endpoint, the amount of glucose standard solution added should be controlled between 0.5 and 1.0 mL. The total sugar content is calculated as follows: The total sugar content in the sample is calculated using the following formula: X=[(V0 V) ×n×c ×1000] / 5 ; In the formula: X is the total sugar content in the sample, g / L; V0 is the volume of glucose standard solution consumed in the blank test, mL; V is the volume of glucose standard solution consumed in the sample determination, mL; c is the concentration of glucose standard solution, g / mL; n is the dilution factor of the sample.

[0074] Analysis of flavor compounds: The flavor compounds in wolfberry wine were analyzed using headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS). The analytes were qualitatively identified by comparison with mass spectra from the NIST 05 mass spectral library. The relative contents of each substance were calculated using a semi-quantitative method after adding 2-octanol as an internal standard to the wine sample.

[0075] Headspace solid-phase microextraction (SPE) conditions: A 50 μm / 30 μm SPME fiber extraction head for CAR / DVB / PDMS was used for flavor extraction; 6.0 mL of sample and 60 μL of internal standard 2-octanol (5 mg / L) were accurately transferred into a 10 mL extraction flask, and extraction was performed at 50 °C for 30 min. Flavor analysis was performed using GC-MS equipped with a DB-Wax column (30 m × 0.25 mm × 0.25 μm). Helium was used as the carrier gas at a flow rate of 1.5 mL / min. The injection port and detector temperatures were 250 °C, and the ion source temperature was 260 °C. MS conditions: EI was used as the ionization source. Temperature program: Initial temperature 50 °C, hold for 2 min, then increase to 230 °C at a rate of 15 °C / min, hold for 15 min.

[0076] Example 1 This embodiment provides a wolfberry wine, the preparation method of which is as follows: Step (1): Mix the washed dried goji berries with sterile water at a mass ratio of 1:10, juice them, add sucrose to adjust the sugar content to 200g / L, add 30U / g pectinase and 100mg / L potassium metabisulfite to obtain the fermentation liquid. Step (2): Inoculate the seed culture of *Vichamomilla malariae* Y11 (CGMCC No. 25068) into the fermentation broth obtained in step (1), with an inoculation amount of 5 × 10⁻⁶. 6 CFU / mL; The seed culture of the above-mentioned abnormal Vicham yeast was obtained by inoculating the abnormal Vicham yeast into YPD liquid medium and culturing it at 30°C for 48 h.

[0077] Step (3): Inoculate the seed culture of Saccharomyces cerevisiae M-23-7-14 (CGMCC No. 23603) into the fermentation broth obtained in step (2) at an inoculation amount of 5 × 10⁻⁶. 6 CFU / mL; The above-mentioned seed culture of Saccharomyces cerevisiae was obtained by inoculating Saccharomyces cerevisiae into YPD liquid medium and culturing at 30°C for 42 h.

[0078] Step (4): Ferment the inoculated liquid obtained in step (3) at 20°C for 6 days, then filter it and let the filtrate stand at 4°C for 14 days.

[0079] Example 2 Alcohol tolerance of Abnormal Virbac yeast Y11 (CGMCC No. 25068) The alcohol tolerance of the *Bacillus venereum* strain provided in this application was investigated using the following experimental method: The ethanol volume fraction of YPD liquid culture medium was adjusted to 0% (i.e., no ethanol added), 3%, 6%, 9%, 12%, 15%, 18%, and 21% with anhydrous ethanol. Yeast activation solution was inoculated at a 5% inoculum size and cultured at 30℃ for 48 h. The absorbance was measured at 600 nm, and a curve was plotted to obtain the highest ethanol tolerance concentration of the strain. The ethanol tolerance of the strain was then evaluated, and the results are as follows: Figure 2 As shown.

[0080] Depend on Figure 2 It can be seen that as the alcohol concentration increases, the growth of the strain is inhibited. When the alcohol concentration is 8%, the abnormal Vickham yeast can still maintain growth, but when the alcohol concentration is 10%, the abnormal Vickham yeast can hardly grow. Overall, the abnormal Vickham yeast provided in this application has good tolerance to alcohol.

[0081] Example 3 The effect of inoculation method on the fermentation of wolfberry wine Step (1): Wash the dried goji berries, rehydrate the dried goji berries with purified water at a ratio of 1:10 and juice them to obtain goji berry juice; add white sugar to adjust the total sugar content of the goji berry juice to 200g / L, add 30U / g of pectinase and 100mg / L of potassium metabisulfite to obtain the fermentation liquid.

[0082] Step (2): Inoculate the seed culture of abnormal Vicham yeast Y11 (preservation number CGMCC No. 25068) into the wolfberry juice to be fermented obtained in step (1): The specific process is as follows: Step (2.1): Inoculate abnormal Vickham yeast into YPD liquid medium and culture at 30℃ and 22 rpm for 48h to obtain abnormal Vickham yeast seed liquid; Step (2.2): Under aseptic conditions, the abnormal Vicham yeast seed culture obtained in step (2.1) is inoculated into the wolfberry juice to be fermented obtained in step (1), with an inoculation amount of 1×10⁻⁶. 6 CFU / mL.

[0083] Step (3): Inoculate the brewing yeast seed liquid into the fermentation liquid obtained in step (2). The specific process is as follows: Step (3.1): Inoculate Saccharomyces cerevisiae M-23-7-14 (preservation number CGMCC No.23603) into YPD liquid medium and culture at 30℃ and 220 rpm for 48 h to obtain Saccharomyces cerevisiae seed liquid.

[0084] Step (3.2): Under aseptic conditions, inoculate the brewing yeast seed culture obtained in step (3.1) into the fermentation broth obtained in step (2), with an inoculation amount of 1×10⁻⁶. 7 The concentration of CFU / mL was 0 h, and the inoculation time with *Vichamus aberrantus* was 12 h.

[0085] Step (4): The fermentation liquid obtained in step (3) is fermented, filtered and settled at low temperature to obtain mixed fermentation wolfberry wine based on abnormal Wickham yeast. The fermentation parameters are as follows: fermentation temperature is 20℃, fermentation time is 6 days; standing temperature is 4℃, standing time is 14 days.

[0086] After fermentation, the physicochemical properties of the wolfberry wine were measured, and the results are shown in Table 1.

[0087] Table 1. Routine physicochemical properties of wolfberry wine under different inoculation methods

[0088] As shown in Table 1, the relative content of flavor compounds is higher in mixed-culture fermentation compared to pure-culture fermentation. In particular, the relative content of flavor compounds in wolfberry wine is the highest when the sequential inoculation interval is 12 hours, which confirms the application potential of the abnormal Vickham yeast provided in this application in enhancing the aroma of wolfberry wine.

[0089] Example 4 The effect of mixed fermentation of *Vaccinium bacillus* Y11 and *Saccharomyces cerevisiae* M-23-7-14 on the flavor of wolfberry wine This embodiment describes a mixed-culture fermentation experiment conducted under the process conditions described in this application: a fermentation temperature of 20°C, an initial sugar concentration of 200 g / L, and an inoculation method of first inoculating with *V. aberrantus* (Y11) followed by *Saccharomyces cerevisiae* (Saccharomyces cerevisiae) at 12-hour intervals. The inoculation amount of *V. aberrantus* Y11 was 1 × 10⁻⁶. 6 CFU / mL, the inoculum size of Saccharomyces cerevisiae M-23-7-14 was 1×10⁻⁶. 7 CFU / mL.

[0090] The effects of mixed-strain fermentation and single-strain fermentation on the ester content in wolfberry wine were investigated, and the results are shown in Table 2.

[0091] Table 2. Ester content in wolfberry wine (µg / L)

[0092] Note: "nd" indicates that it was not detected. As shown in Table 2, the content of esters in mixed-culture fermented wolfberry wine is 7297.46 μg / L, which is 85.8% higher than that in pure yeast fermentation. Among them, isoamyl acetate increased by 65.9%, ethyl acetate by 118.8%, and ethyl hexanoate by 166.7%. The increase in these esters makes an important contribution to enhancing the floral and fruity aroma of wolfberry wine.

[0093] Therefore, the mixed-culture fermentation provided in this application can significantly increase the content of esters in wolfberry wine and improve its flavor and quality.

[0094] Comparative Example 1 The effect of mixed fermentation of abnormal Vicham yeast Y20 and Saccharomyces cerevisiae on the flavor of wolfberry wine This embodiment describes a mixed fermentation experiment using another strain of *V. aberrant* *Bacillus* Y20 and *Saccharomyces cerevisiae* under the process conditions described in this application. Specifically, the fermentation temperature was 20°C, the initial sugar concentration was 200 g / L, and the inoculation method involved first inoculating *V. aberrant* *Bacillus* Y20, followed by *Saccharomyces cerevisiae* 12 hours later. The inoculation amount of *V. aberrant* *Bacillus* Y20 was 1 × 10⁻⁶. 6 CFU / mL, the inoculum size of Saccharomyces cerevisiae M-23-7-14 was 1×10⁻⁶. 7 CFU / mL.

[0095] The effect of abnormal Vicham yeast Y20 on the ester content in wolfberry wine was investigated, and the results are shown in Table 3.

[0096] Table 3. Ester content (µg / L) in wolfberry wine fermented with a mixed strain of abnormal Vicham yeast Y20

[0097] Note: "nd" indicates that it was not detected. As shown in Table 3, the content of esters in wolfberry wine obtained by mixed fermentation using abnormal Vicham yeast Y20 was 6285.87 μg / L, which was 60.1% higher than that obtained by pure yeast fermentation, but significantly lower than that obtained by mixed fermentation using abnormal Vicham yeast Y11.

[0098] In summary, this application provides a method for preparing wolfberry wine by inoculating wolfberry juice with abnormal Vicham's yeast Y11 (accession number CGMCC No. 25068) and brewer's yeast, which can enhance the taste, aroma, and quality of wolfberry wine. Compared with single-strain fermentation with brewer's yeast, this invention increases the content of esters in wolfberry wine by 85.8%, effectively solving the problems of insufficient aroma and lack of typicality in wolfberry wine caused by single fermentation.

[0099] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing wolfberry wine, characterized in that, Includes the following steps: The seed culture of brewer's yeast and the seed culture of abnormal Vickham's yeast were inoculated into the fermentation liquid made from wolfberry and fermented. Fermentation was carried out at 18-22℃ for at least 5 days. The fermented liquid was then separated into solid and liquid, and the liquid after solid-liquid separation was allowed to stand at 3-5℃ for 12-16 days. The preservation number of the brewing yeast is CGMCC No. 23603; The biopreservation number of the abnormal Vicham yeast is: CGMCC No. 25068; The seed culture of the abnormal Vickham yeast and the seed culture of the brewer's yeast were inoculated into the fermentation broth alternately. The inoculation method was to inoculate the seed culture of the abnormal Vickham yeast first, followed by the seed culture of the brewer's yeast, with an interval of 6-18 hours.

2. The preparation method according to claim 1, characterized in that, The interval is 12 hours.

3. The preparation method according to claim 1, characterized in that, The inoculation volume of the abnormal Vicham yeast seed culture was 1×10⁻⁶. 6 Up to 1×10 8 CFU / mL.

4. The preparation method according to claim 1, characterized in that, The seed culture of the abnormal Wickham yeast was prepared by the following method: Inoculate abnormal Vicham yeast into YPD liquid medium and incubate at 28-32℃ for 42-54 h.

5. The preparation method according to claim 1, characterized in that, The inoculation amount of the brewer's yeast seed culture is 1×10⁻⁶. 6 Up to 1×10 7 CFU / mL.

6. The preparation method according to claim 1, characterized in that, The seed culture of the brewing yeast is prepared by the following method: Inoculate the yeast with YPD liquid medium and incubate at 28-32℃ for 36-48 hours.

7. The preparation method according to claim 1, characterized in that, The preparation of the fermentation liquid of goji berries includes: mixing goji berries with water and juicing, and adjusting the sugar content.

8. The preparation method according to claim 7, characterized in that, The goji berries mentioned are dried goji berries.

9. The preparation method according to claim 8, characterized in that, The dried goji berries are mixed with water at a mass ratio of 1:8-12.

10. The preparation method according to claim 7, characterized in that, The sugar content can be adjusted using sucrose or white sugar.

11. The preparation method according to claim 10, characterized in that, The sugar content of the wolfberry liquid after adjustment is 150-250g / L.

12. The preparation method according to claim 11, characterized in that, After adjusting the sugar content, the process also includes adding pectinase and potassium metabisulfite to the wolfberry liquid.

13. The preparation method according to claim 12, characterized in that, The amount of pectinase added is 20-40 U / g and / or the amount of potassium metabisulfite added is 80-120 mg / L.

14. A type of wolfberry wine, characterized in that, It is prepared by the preparation method according to any one of claims 1-13.

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