Preparation and application of non-saccharomyces and immobilized yeast capsules thereof

Abstract

This invention provides a non-Saccharomyces anomalus yeast, with accession number CGMCC No. 25528, deposited at the China General Microbiological Culture Collection Center on August 12, 2022. A method for preparing immobilized yeast capsules of this non-Saccharomyces anomalus yeast is also provided. The immobilized yeast capsules prepared under optimal conditions exhibit high hardness, good permeability, and improved ethanol tolerance, allowing for simultaneous inoculation and fermentation with Saccharomyces cerevisiae. Furthermore, this invention provides the application of immobilized yeast capsules in winemaking. Using the immobilized yeast capsules of this invention in winemaking can significantly increase the total amount of volatile substances in the wine, enhance the complexity of the aroma, and thus improve the quality of the wine.

Description

Technical Field

[0001] This invention belongs to the field of winemaking technology, specifically relating to the preparation and application of a non-brewing yeast and its immobilized yeast capsules. Background Technology

[0002] Currently, wineries across China and even globally exhibit highly homogenized winemaking processes, with fermentation using only a single commercial brewing yeast, resulting in a lack of distinctive characteristics and individuality in their wines. In recent years, research has shown that non-brewing yeasts play a crucial role in winemaking. These non-brewing yeasts have transformed from an unpopular yeast associated with spoiled wines into microorganisms that can improve the aromatic properties of wines. The synergistic effect of non-brewing yeasts with brewing yeast strains, and their continuous fermentation, has become a hot topic in wine research. Currently, approximately 20 genera of non-Saccharomyces yeasts have been discovered and studied. The main non-Saccharomyces yeasts associated with winemaking include: *Schizosaccharomyces*, *Hanseniaspora*, *Pichia*, *Issatchenkia*, *Kloeckera*, *Candida*, *Metschnikowia*, *Hansenula*, *Cryptococcus*, *Zygosaccharomyces*, and *Rhodotorula*. These non-yeast strains can increase acidity, aromatic complexity, glycerol content, ethanol reduction, mannitol, anthocyanin, and polysaccharide concentrations, and even... Non-Sacchariculture yeasts can produce bioactive compounds: ① Organic acids produced by non-Sacchariculture yeasts alter pH, increasing anthocyanin color. Their released metabolites or high expression of hydroxycinnamic acid decarboxylase activity can promote the formation of stable pigments and enhance the formation of pyranocyanin and polymeric pigments. For example, *Schizosacchariformis* increases the release of extracellular pyruvate; the higher the extracellular release of pyruvate, the higher the production of stable pyranocyanin. ② Non-Sacchariculture yeasts can also release yeast polysaccharides and accelerate the lees aging (AOL) process, improving wine structure. ③ The presence of non-Sacchariculture yeasts leads to a decrease in alcohol concentration, while increasing the concentrations of terpenes, esters, higher alcohols, glycerol, acetaldehyde, acetic acid, and succinic acid. ④ The presence of specific enzymes in non-Sacchariculture yeasts, such as glycosidases not encoded by them, can cause non-volatile precursor enzymes to release aroma substances, thus affecting flavor compounds. ⑤ Non-Sacchariculture yeasts can also produce toxic agents to prevent wine spoilage microorganisms and their unpleasant flavors. Therefore, the important role of non-Sacchariculture yeasts in wine production is becoming increasingly prominent.

[0003] Currently, commercially available non-brewing yeast strains include Metschnikowia pulcherrima, Torulaspora delbrueckii, Lachancea thermotolerans, Pichia kluyvery, and Schizosaccharomyces pombe, but Wickerhamomyces anomalus has not yet been commercially applied to wine.

[0004] In the co-fermentation process between non-Saccharomyces cerevisiae and Saccharomyces cerevisiae, firstly, there are direct interactions between yeast cells, such as physical contact and quorum sensing between microorganisms. The content of volatile substances in the co-fermentation of thermotolerant Kluyveromyces and Saccharomyces cerevisiae is significantly different compared with that in non-contact co-fermentation, indicating that cell-cell contact alters yeast metabolism. Some quorum sensing molecules, such as tyrosol, tryptophan, and phenylethyl alcohol, also participate in yeast-yeast interactions and affect yeast growth. Secondly, yeast strains produce killing factors that kill yeasts that are sensitive to them. Albergaria et al. (2010) found that 2-10 kDa proteins in the supernatant of Saccharomyces cerevisiae CCMI 885 fermentation have antibacterial and even bactericidal effects on some strains of Kluyveromyces, Saccharomyces sporulationans, and Hansenula polymorpha. In addition, during ethanol fermentation, as the ethanol concentration increases, it stresses yeast cell growth. Yeast cells will produce corresponding stress responses to cope with this stress in order to survive and grow. This coping mechanism is the development of ethanol tolerance in yeast cells. Non-Saccharomyces cerevisiae have the disadvantage of poor ethanol tolerance, generally below 5% ethanol concentration. Therefore, effective measures are needed to control the direct interaction between non-sacchariculture yeast and sacchariculture yeast during co-fermentation, and the ethanol tolerance of non-sacchariculture yeast also needs to be improved. Summary of the Invention

[0005] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a non-brewing yeast, namely *Wickerhamomyces anomalus*, with accession number CGMCC No. 25528, deposited at the China General Microbiological Culture Collection Center on August 12, 2022; providing a method for preparing immobilized yeast capsules that can improve the yeast's ethanol tolerance; and providing an application of immobilized yeast capsules in winemaking that can increase the total amount of volatile substances in the wine and improve the quality of the wine.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a non-Saccharomyces yeast, wherein the non-Saccharomyces yeast is Wickerhamomyces anomalus, with accession number CGMCC No. 25528, deposited by the China General Microbiological Culture Collection Center, with a deposit date of August 12, 2022; the 26S rDNA nucleotide sequence of the non-Saccharomyces yeast is shown in SEQ ID NO:1.

[0007] Purification of Wickerhamomyces anomalus: Grape skins were soaked in sterile distilled water and shaken to obtain a shaking solution. 100 μL of the shaking solution was evenly spread on YPD solid medium and cultured at 28°C for 2 days to obtain a mother plate. The colonies in the mother plate were streaked and inoculated into new YPD solid medium and cultured until a single colony appeared in the new YPD solid medium. The strain of the single colony is Wickerhamomyces anomalus. Wickerhamomyces anomalus was stored in cryovials and mixed well for later use.

[0008] The YPD solid culture medium is composed of the following raw materials in the indicated mass fractions: 2% glucose, 2% peptone, 1% yeast extract and 2% agar, with the remainder being water, and is then sterilized at 121°C for 20 minutes.

[0009] The YPD liquid culture medium is composed of the following raw materials in the indicated mass fractions: 2% glucose, 2% peptone and 1% yeast extract, with the remainder being water, and is then sterilized at 121°C for 20 minutes.

[0010] The volume ratio of glycerol to YPD liquid culture medium in the cryovial is 1:1, and the volume fraction of glycerol in the cryovial is 60%.

[0011] Culture of *Wickham's Abnormal* yeast: Preserved *Wickham's Abnormal* yeast was inoculated into YPD liquid medium and activated for 48 hours at 28°C and 200 rpm / min. The activated *Wickham's Abnormal* yeast was then inoculated into acclimatization medium at a volume fraction of 2% and cultured for at least 48 hours at 28°C and 200 rpm / min until the total number of *Wickham's Abnormal* yeast clusters per milliliter of solution was ≥10. 8 Stop culturing when CFU is activated;

[0012] The volume ratio of YPD liquid medium to grape juice in the acclimatization medium is 1:2.

[0013] This invention provides a method for preparing immobilized yeast capsules using the above-mentioned non-Saccharomyces cerevisiae, the method being as follows:

[0014] S1. Preparation of bacterial suspension: Under aseptic conditions, 200 μL of frozen non-Saccharomyces cerevisiae was inoculated into 20 mL of YPD liquid medium and cultured at 28℃ for 48 h to obtain a first-generation culture. This was repeated three times for activation. The resulting culture was then centrifuged at 4℃ and 4500 r / min. The collected bacterial cells were diluted with sterile physiological saline to prepare a 10... 10 The bacterial suspension with cfu / mL was prepared and stored at 4°C for later use. The operation steps for repeating the three passages were as follows: 200 μL of the first-generation culture was inoculated into 20 mL of YPD liquid medium and cultured at 28°C for 48 h to obtain the second-generation culture. 200 μL of the second-generation culture was then inoculated into 20 mL of YPD liquid medium and cultured under the same conditions to obtain the third-generation culture.

[0015] S2. Preparation of immobilized yeast capsules: Take 10 mL of the refrigerated bacterial suspension from S1 and allow it to return to room temperature. Add the room-temperature bacterial suspension to 1 L of 2% sodium alginate solution and stir until the total number of bacterial clusters per milliliter of solution is 10. 8 CFU was then added dropwise through a 3.3mm inner diameter tubing to 2L of 2% (w / w) sterile calcium chloride solution using a constant flow pump, and stirred with a magnetic stirrer. The mixture was allowed to solidify for 0.5 hours to form immobilized capsule particles. These particles were washed three times with sterile saline solution and then transferred to 3L of 1.5% (w / w) chitosan solution. The mixture was shaken for 3 hours to promote the formation of a polyelectrolyte composite polymer film between the chitosan and the sodium alginate of the immobilized capsules under electrostatic action, enhancing the mechanical stability of the capsules. The capsules were then washed three more times with sterile saline solution until the chitosan solution was completely removed, yielding immobilized yeast capsules. The viscosity of the sodium alginate solution was 115 mPa·s, and the molecular weight of the chitosan solution was 250,000 Da.

[0016] Preferably, the ethanol tolerance of the immobilized yeast capsule in S2 is 9% vol.

[0017] Preferably, the average particle size of the immobilized yeast capsules in S2 is 2.301–3.829 mm.

[0018] This invention also provides an application of the above-mentioned immobilized yeast capsule, which can be used in winemaking to increase the content of volatile substances; the winemaking method is as follows:

[0019] S1. Remove the stems and crush the grapes, put them directly into the fermentation tank or press them and then put them into the fermentation tank. Add sulfurous acid to make the sulfurous acid concentration 60 mg / L, let it stand for 4 hours, then add pectinase to make the final concentration of pectinase 20 mg / L, and then ferment at 4℃ for 48 hours to obtain grape fermentation liquid.

[0020] S2. After the grape fermentation broth obtained in S1 has returned to room temperature, inoculate with a volume of 10... 6 Immobilized yeast capsules at cfu / mL were inoculated into the grape fermentation broth and fermented for 24 hours, followed by inoculation with 10 6 The activated commercial brewing yeast (CFU / mL) was thoroughly stirred and allowed to ferment statically at 18°C ​​or 26°C. The fermentation tank was sealed with a fermentation plug. When the ethanol concentration reached 9% vol, the mixture of immobilized yeast capsules and grape seeds was separated by filtration through a stainless steel sieve to obtain grape must. The grape must was fermented under the action of free yeast in the liquid until the residual sugar content in the wine was <2 g / L. Fermentation was then terminated, and the residue was filtered to obtain the wine liquid. The commercial brewing yeast was brewing yeast CEC01, which was purchased from Angel Yeast Co., Ltd.

[0021] Preferably, when the grapes mentioned in S1 are grape varieties for making red wine, the grapes are destemmed, crushed, and directly loaded into the fermentation tank, with a volume fraction of 80%; when the grapes mentioned in S1 are grape varieties for making white wine, the grapes are destemmed, crushed, pressed, and then loaded into the fermentation tank, with a volume fraction of 90%.

[0022] Preferably, when the grapes mentioned in S1 are grape varieties for making red wine, the temperature of static fermentation in S2 is 26°C; when the grapes mentioned in S1 are grape varieties for making white wine, the temperature of static fermentation in S2 is 18°C.

[0023] Preferably, when the grapes mentioned in S1 are grape varieties for making red wine, the mixture of immobilized yeast capsules and grape seeds is separated by filtering through a stainless steel sieve in S2; when the grapes mentioned in S1 are grape varieties for making white wine, the mixture of immobilized yeast capsules is separated by filtering through a stainless steel sieve in S2.

[0024] Preferably, when the grapes in S1 are grape varieties for making red wine, the total volatile matter content of the wine liquid in S2 is 56746.86 μg / L; when the grapes in S1 are grape varieties for making white wine, the total volatile matter content of the wine liquid in S2 is 274466.80 μg / L.

[0025] The total volatile matter content of dry red wine made using immobilized yeast capsules increased by 8591.96 μg / L, and the total volatile matter content of dry white wine made using immobilized yeast capsules increased by 106124.73 μg / L. Therefore, the total volatile matter content of wine made using immobilized yeast capsules is significantly increased, and there are also more major contributors to the aroma of the wine samples, which can enhance the complexity of the aroma and improve the quality of the wine.

[0026] A sodium alginate solution with a mass fraction of 2% and a viscosity of 115 mPa·s, a calcium chloride solution with a mass fraction of 2% and a chitosan solution with a mass fraction of 1.5%, wherein the chitosan in the chitosan solution has a molecular weight of 250,000 Da, are the optimal conditions for preparing immobilized yeast capsules. Immobilized yeast capsules prepared under these conditions possess both high hardness and good permeability. The surface of the immobilized yeast capsule particles is generally smooth and uniform, and the three-dimensional network structure formed by sodium alginate and chitosan is also uniform and porous. Non-brewing yeast is well immobilized, the cross-section is smooth, and the immobilized non-brewing yeast cells can be seen inside. The immobilized yeast capsule structure is uniform and stable, and it encapsulates yeast cells well, creating conditions for its subsequent application in wine.

[0027] The ethanol tolerance of immobilized yeast capsules increased from 6% vol to 9% vol compared to that of abnormal Wickham yeast, an increase of 33.33%, and they can be inoculated and fermented simultaneously with Saccharomyces cerevisiae.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] 1. The non-Saccharomyces cerevisiae used in this invention is Wickerhamomyces anomalus. The conditions for preparing the immobilized yeast capsules are: a sodium alginate solution with a mass fraction of 2% and a viscosity of 115 mPa·s, a calcium chloride solution with a mass fraction of 2% and a chitosan solution with a mass fraction of 1.5%. The chitosan in the chitosan solution has a molecular weight of 250,000 Da. The resulting immobilized yeast capsules have the advantages of high hardness and good permeability. Furthermore, the ethanol tolerance of the immobilized yeast capsules is improved to 9% vol, allowing for simultaneous inoculation and fermentation with Saccharomyces cerevisiae.

[0030] 2. The immobilized yeast capsules prepared by this invention have an average particle size of 2.301 mm to 3.829 mm. The surface of the capsule particles is relatively smooth and uniform, and the cross-section is smooth and shows the immobilized non-brewing cells inside. The capsule structure is uniform and stable, and it encapsulates the yeast cells well, which creates conditions for its subsequent application in winemaking.

[0031] 3. The total amount of volatile substances in wine brewed using immobilized yeast capsules in this invention is significantly increased, with a marked increase in terpenes and acetates. Wine brewed using immobilized yeast capsules contains more major aroma-contributing substances. The application of immobilized yeast capsules in winemaking enhances the complexity of the wine's aroma and improves the quality of the wine.

[0032] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0033] Figure 1 This shows the distribution of each wine sample and the main aroma substances in main components 1 and 2 in Example 1.

[0034] Figure 2 (A)-(C) are SEM images of the immobilized yeast capsules prepared in Example 1.

[0035] Figure 3 This describes the effect of ethanol concentration on the catalytic performance of immobilized yeast capsules in Example 3.

[0036] Figure 4 This illustrates the effect of sodium alginate solutions of different viscosities on the performance of immobilized yeast capsules in Example 4.

[0037] Figure 5 This describes the effect of different mass fractions of sodium alginate solution on the hardness of immobilized yeast capsules in Example 4.

[0038] Figure 6 This describes the effect of sodium alginate solutions of different mass fractions on the permeability of immobilized yeast capsules in Example 4.

[0039] Figure 7 This describes the effect of different mass fractions of calcium chloride solution on the performance of immobilized yeast capsules in Example 4.

[0040] Figure 8 This describes the effect of chitosan solutions with different molecular weights on the hardness of immobilized yeast capsules in Example 4.

[0041] Figure 9 This describes the effect of chitosan solutions with different molecular weights on the permeability of immobilized yeast capsules in Example 4.

[0042] Figure 10 This describes the effect of chitosan solutions with different molecular weights and mass fractions on the particle size of immobilized yeast capsules in Example 4.

[0043] Figure 11 This describes the effect of different mass fractions of chitosan solution on the performance of immobilized yeast capsules in Example 4. Detailed Implementation

[0044] Example 1

[0045] This embodiment provides a non-Saccharomyces yeast, namely Wickerhamomyces anomalus, with accession number CGMCC No. 25528, deposited at the China General Microbiological Culture Collection Center on August 12, 2022.

[0046] Isolation and purification of Wickerhamomyces anomalus: Grape skins were soaked in sterile distilled water and shaken to obtain a shaking solution. 100 μL of the shaking solution was evenly spread on YPD solid medium and cultured at 28°C for 2 days to obtain a master plate. The colonies in the master plate were streaked and inoculated into new YPD solid medium and cultured until a single colony appeared in the new YPD solid medium. The cluster of this single colony is Wickerhamomyces anomalus. Wickerhamomyces anomalus was stored in cryovials and mixed well for later use.

[0047] The YPD solid culture medium is composed of the following raw materials in the indicated mass fractions: 2% glucose, 2% peptone, 1% yeast extract and 2% agar, with the remainder being water, and is then sterilized at 121°C for 20 minutes.

[0048] The YPD liquid culture medium is composed of the following raw materials in the indicated mass fractions: 2% glucose, 2% peptone and 1% yeast extract, with the remainder being water, and is then sterilized at 121°C for 20 minutes.

[0049] The volume ratio of glycerol to YPD liquid culture medium in the cryovial is 1:1, and the volume fraction of glycerol in the cryovial is 60%.

[0050] The abnormal Wickhamomyces anomalus was identified by sequencing the D1 / D2 region of its 26S rDNA gene. The 26S rDNA nucleotide sequence of the non-Saccharomyces yeast is shown in SEQ ID NO:2. The 26S rDNA nucleotide sequence using primers is shown in SEQ ID NO:2 and SEQ ID NO:3.

[0051] Culture of *Wickham's Abnormal* yeast: Preserved *Wickham's Abnormal* yeast was inoculated into YPD liquid medium and activated for 48 hours at 28°C and 200 rpm / min. The activated *Wickham's Abnormal* yeast was then inoculated into acclimatization medium at a volume fraction of 2% and cultured for at least 48 hours at 28°C and 200 rpm / min until the total number of *Wickham's Abnormal* yeast clusters per milliliter of solution was ≥10. 8 Stop culturing when CFU is activated;

[0052] The volume ratio of YPD liquid medium to grape juice in the acclimatization medium is 1:2.

[0053] This embodiment provides a method for preparing immobilized yeast capsules from the above-mentioned non-Saccharomyces cerevisiae, the method being as follows:

[0054] S1. Preparation of bacterial suspension: Under aseptic conditions, 200 μL of frozen non-Saccharomyces cerevisiae was inoculated into 20 mL of YPD liquid medium and cultured at 28℃ for 48 h to obtain the first-generation culture. This process was repeated three times for activation. The resulting culture was then centrifuged at 4℃ and 4500 r / min. The collected cells were diluted with sterile physiological saline to prepare a bacterial suspension of 10¹⁰ cfu / mL. The prepared bacterial suspension was then refrigerated at 4℃ for later use. The procedure for repeating the three-times-passage culture was as follows: 200 μL of the first-generation culture was inoculated into 20 mL of YPD liquid medium and cultured at 28℃ for 48 h to obtain the second-generation culture. 200 μL of the second-generation culture was then inoculated into 20 mL of YPD liquid medium and cultured under the same conditions to obtain the third-generation culture.

[0055] S2. Preparation of immobilized yeast capsules: Take 10 mL of the refrigerated bacterial suspension from S1 and allow it to return to room temperature. Add the room-temperature bacterial suspension to 1 L of 2% sodium alginate solution and stir until the total number of bacterial clusters per milliliter of solution is 10. 8 CFU was then added dropwise through a 3.3mm inner diameter tubing to 2L of 2% (w / w) sterile calcium chloride solution using a constant flow pump, and stirred with a magnetic stirrer. The mixture was allowed to solidify for 0.5 hours to form immobilized capsule particles. These particles were washed three times with sterile saline solution and then transferred to 3L of 1.5% (w / w) chitosan solution. The mixture was shaken for 3 hours to promote the formation of a polyelectrolyte composite polymer film between the chitosan and the sodium alginate of the immobilized capsules under electrostatic action, enhancing the mechanical stability of the capsules. The capsules were then washed three more times with sterile saline solution until the chitosan solution was completely removed, yielding immobilized yeast capsules. The viscosity of the sodium alginate solution was 115 mPa·s, and the molecular weight of the chitosan solution was 250,000 Da.

[0056] This embodiment also provides an application of the above-mentioned immobilized yeast capsules in red wine brewing, the brewing method being as follows:

[0057] S1. Remove the stems from the most mature, intact, and disease-free parts of Cabernet Sauvignon grapes and crush them to obtain grape juice. Put the grape juice into a fermentation tank at 80% by volume, add sulfite to make the sulfite concentration 60 mg / L, let it stand for 4 hours, then add pectinase to make the final pectinase concentration 20 mg / L, and then ferment at 4℃ for 48 hours to obtain grape fermentation liquid.

[0058] S2. After the grape fermentation broth obtained in S1 has returned to room temperature...

[0059] a. Press the immobilized yeast capsules at a ratio of 10 6 An inoculum of cfu / mL was added to the grape fermentation broth, and fermentation was carried out for 24 hours. Then, an inoculum of 10... 6 The activated commercial brewer's yeast at cfu / mL was designated as group I-Wa1; the commercial brewer's yeast was brewer's yeast CEC01, which was purchased from Angel Yeast Co., Ltd.

[0060] b. Inoculate the abnormal Wickham yeast at a rate of 10 6 CFU / mL was inoculated into the grape fermentation broth, and fermentation was carried out for 24 hours. Then, the inoculation volume was 10. 6 The activated commercial brewer's yeast CEC01 with cfu / mL was designated as group F-Wa1.

[0061] c. The control group received only 10 doses. 6 The activated commercial brewer's yeast CEC01 with cfu / mL was designated as group Sc1.

[0062] After thorough stirring, the mixture is allowed to ferment at a temperature of 26°C. The fermentation tank is sealed with a fermentation plug. The mixture is stirred or circulated twice a day, morning and evening, to ensure that the grape skins are fully immersed. When the ethanol concentration reaches 9% vol, the mixture of immobilized yeast capsules and grape seeds is separated by filtration through a stainless steel sieve to obtain grape must. The grape must is fermented under the action of free yeast in the liquid until the residual sugar content of the wine is <2 g / L. Fermentation is then terminated, and the residue is filtered to obtain dry red wine liquid. The residue includes grape skins and lees.

[0063] The obtained dry red wine liquid was used for index testing, and the test results are shown in Table 1:

[0064] Table 1 Results of the test of indicators of the wine

[0065]

[0066]

[0067]

[0068] The total volatile matter content of dry red wine brewed using immobilized yeast capsules increased from 48154.90 μg / L to 56746.86 μg / L, an increase of 8591.96 μg / L. The concentration of terpenes in the Sc1 group of wine samples inoculated only with activated commercial brewing yeast CEC01 was 182.41 μg / L, while the concentration of terpenes in the I-Wa1 group of wine samples with the same inoculation amount of immobilized yeast capsules was 205.09 μg / L, an increase of 22.68 μg / L. Acetates, including ethyl acetate, isoamyl acetate, hexyl acetate, and phenylethyl acetate, showed a concentration of 3001.46 μg / L in the Sc1 group of wine samples inoculated only with activated commercial brewing yeast CEC01, compared to 4088.63 μg / L in the I-Wa1 group of wine samples with the same inoculation amount of immobilized yeast capsules, an increase of 1087.17 μg / L.

[0069] Because the aromas in wine are quite complex, the aroma activity value (OAV) was used to assess the overall contribution of aroma compounds to the wine's aroma, and the detected substances were described in terms of odor. To more intuitively illustrate the contribution of each aroma compound in the wine sample, principal component analysis was performed on 15 aroma compounds with an OAV greater than 1.

[0070] The first two principal components account for 92.78% of the total variance, which can well explain the differences between sample factors. The variance contribution rate of the first principal component (F1) is 51.71%, and that of the second principal component (F2) is 41.07%. The distribution of each wine sample and the main aroma compounds on principal components 1 and 2 is as follows: Figure 1 As shown, the co-fermentation of immobilized yeast capsules and commercial yeast CEC01 (I-Wa1), the co-fermentation of free abnormal Wickham yeast and commercial yeast (F-Wa1), and the control group with only commercial brewing yeast (Sc1) were significantly distributed in different regions, indicating that the use of abnormal Wickham yeast and immobilized yeast capsules significantly altered the quality of the wine. In particular, I-Wa1 was located at the positive end of F1 and the negative end of F2, loading significantly more compounds than the F-Wa1 and Sc1 groups, mainly including: isoamyl alcohol, ethyl butyrate, hexyl acetate, linalool, phenylethyl alcohol, ethyl 2-methylbutyrate, ethyl isovalerate, and isoamyl acetate. Principal component analysis showed that the I-Wa1 group had more major aroma contributors, and the co-fermentation of immobilized yeast capsules and commercial yeast CEC01 enhanced the aroma complexity of the wine and improved its quality.

[0071] The surface morphology and cross-sectional morphology of the immobilized yeast capsules prepared in this embodiment were characterized using scanning electron microscopy. The results are as follows: Figure 2 As shown. Figure 2From left to right, the images show the overall surface morphology, surface tissue structure, and cross-sectional morphology of the immobilized yeast capsule. Figure 2 As can be seen, the average particle size of the immobilized yeast capsules ranges from 2.301 mm to 3.829 mm. The immobilized yeast capsules prepared under the following conditions—2% sodium alginate solution with a viscosity of 115 mPa·s, 2% calcium chloride solution, and 1.5% chitosan solution (where the chitosan has a molecular weight of 250,000 Da)—have relatively smooth and uniform particle surfaces. The three-dimensional network structure formed by sodium alginate and chitosan is also uniform and porous. It can be seen that *Aberrant Wickham's yeast* is well immobilized, with a smooth cross-section revealing the immobilized *Aberrant Wickham's yeast* cells inside. The prepared immobilized yeast capsules have a uniform and stable structure and good encapsulation of yeast cells, creating conditions for their subsequent application in winemaking.

[0072] Example 2

[0073] This embodiment provides a non-Saccharomyces yeast, namely Wickerhamomyces anomalus, with accession number CGMCC No. 25528, deposited at the China General Microbiological Culture Collection Center on August 12, 2022.

[0074] Isolation and purification of Wickerhamomyces anomalus: Grape skins were soaked in sterile distilled water and shaken to obtain a shaking solution. 100 μL of the shaking solution was evenly spread on YPD solid medium and cultured at 28°C for 2 days to obtain a master plate. The colonies in the master plate were streaked and inoculated into new YPD solid medium and cultured until a single colony appeared in the new YPD solid medium. The cluster of this single colony is Wickerhamomyces anomalus. Wickerhamomyces anomalus was stored in cryovials and mixed well for later use.

[0075] The YPD solid culture medium is composed of the following raw materials in the indicated mass fractions: 2% glucose, 2% peptone, 1% yeast extract and 2% agar, with the remainder being water, and is then sterilized at 121°C for 20 minutes.

[0076] The YPD liquid culture medium is composed of the following raw materials in the indicated mass fractions: 2% glucose, 2% peptone and 1% yeast extract, with the remainder being water, and is then sterilized at 121°C for 20 minutes.

[0077] The volume ratio of glycerol to YPD liquid culture medium in the cryovial is 1:1, and the volume fraction of glycerol in the cryovial is 60%.

[0078] Culture of *Wickham's Abnormal* yeast: Preserved *Wickham's Abnormal* yeast was inoculated into YPD liquid medium and activated for 48 hours at 28°C and 200 rpm / min. The activated *Wickham's Abnormal* yeast was then inoculated into acclimatization medium at a volume fraction of 2% and cultured for at least 48 hours at 28°C and 200 rpm / min until the total number of *Wickham's Abnormal* yeast clusters per milliliter of solution was ≥10. 8 Stop culturing when CFU is activated;

[0079] The volume ratio of YPD liquid medium to grape juice in the acclimatization medium is 1:2.

[0080] This embodiment provides a method for preparing immobilized yeast capsules from the above-mentioned non-Saccharomyces cerevisiae, the method being as follows:

[0081] S1. Preparation of bacterial suspension: Under aseptic conditions, 200 μL of frozen non-Saccharomyces cerevisiae was inoculated into 20 mL of YPD liquid medium and cultured at 28℃ for 48 h to obtain the first-generation culture. This process was repeated three times for activation. The resulting culture was then centrifuged at 4℃ and 4500 r / min. The collected cells were diluted with sterile physiological saline to prepare a bacterial suspension of 10¹⁰ cfu / mL. The prepared bacterial suspension was then refrigerated at 4℃ for later use. The procedure for repeating the three-times-passage culture was as follows: 200 μL of the first-generation culture was inoculated into 20 mL of YPD liquid medium and cultured at 28℃ for 48 h to obtain the second-generation culture. 200 μL of the second-generation culture was then inoculated into 20 mL of YPD liquid medium and cultured under the same conditions to obtain the third-generation culture.

[0082] S2. Preparation of immobilized yeast capsules: Take 10 mL of the refrigerated bacterial suspension from S1 and allow it to return to room temperature. Add the room-temperature bacterial suspension to 1 L of 2% sodium alginate solution and stir until the total number of bacterial clusters per milliliter of solution is 10. 8 CFU was then added dropwise through a 3.3mm inner diameter tubing to 2L of 2% (w / w) sterile calcium chloride solution using a constant flow pump, and stirred with a magnetic stirrer. The mixture was allowed to solidify for 0.5 hours to form immobilized capsule particles. These particles were washed three times with sterile saline solution and then transferred to 3L of 1.5% (w / w) chitosan solution. The mixture was shaken for 3 hours to promote the formation of a polyelectrolyte composite polymer film between the chitosan and the sodium alginate of the immobilized capsules under electrostatic action, enhancing the mechanical stability of the capsules. The capsules were then washed three more times with sterile saline solution until the chitosan solution was completely removed, yielding immobilized yeast capsules. The viscosity of the sodium alginate solution was 115 mPa·s, and the molecular weight of the chitosan solution was 250,000 Da.

[0083] This embodiment also provides an application of the above-mentioned immobilized yeast capsules in white wine brewing, the brewing method being as follows:

[0084] S1. Remove the stems from the most mature, intact, and disease-free parts of the longan grapes, crush them, and press them to obtain grape juice. Put the grape juice into a fermentation tank at a volume fraction of 90%, add sulfite to make the sulfite concentration 60 mg / L, let it stand for 4 hours, then add pectinase to make the final pectinase concentration 20 mg / L, and then ferment at 4℃ for 48 hours to obtain grape fermentation liquid.

[0085] S2. After the grape fermentation broth obtained in S1 has returned to room temperature...

[0086] a. Press the immobilized yeast capsules at a ratio of 10 6 Inoculate the grape fermentation broth at an inoculum concentration of cfu / mL, ferment for 24 hours, and then inoculate at a concentration of 10... 6 The activated commercial brewer's yeast CEC01 was inoculated at an inoculation rate of cfu / mL and designated as group I-Wa2.

[0087] b. Add the abnormal Wickham yeast at 10 6 Inoculate the grape fermentation broth at an inoculum concentration of cfu / mL, ferment for 24 hours, and then inoculate at a concentration of 10... 6 The activated commercial brewer's yeast CEC01 was inoculated at an inoculation rate of cfu / mL and designated as group F-Wa2.

[0088] c. The control group received only 10 6 The activated commercial brewer's yeast CEC01 was inoculated at an inoculum volume of cfu / mL and designated as group Sc2.

[0089] After thorough stirring, the mixture is allowed to ferment at a temperature of 18°C. The fermentation tank is sealed with a fermentation plug. The mixture is stirred or circulated twice a day, morning and evening, to ensure that the grape skins are fully immersed. When the ethanol concentration reaches 9% vol, the mixture of immobilized yeast capsules and grape seeds is separated by filtration through a stainless steel sieve to obtain grape must. The grape must is fermented under the action of free yeast in the liquid until the residual sugar content in the wine is <2 g / L. Fermentation is then terminated, and the residue is filtered to obtain dry white wine liquid. The residue includes lees.

[0090] The obtained dry white wine liquid was used for index determination, and the results are shown in Table 2:

[0091] Table 2 Results of wine index determination

[0092]

[0093]

[0094]

[0095] The total volatile matter content of dry white wine brewed using immobilized yeast capsules increased from 168,342.07 μg / L to 274,466.80 μg / L, representing an increase of 106,124.73 μg / L. The concentration of terpenes in the Sc2 group of wine samples inoculated only with activated commercial brewing yeast CEC01 was 227.85 μg / L, while the concentration of terpenes in the I-Wa2 group of wine samples inoculated with the same amount of immobilized yeast capsules was 395 μg / L. The concentration of acetate esters was 0.97 μg / L, an increase of 168.12 μg / L. Acetyl esters included ethyl acetate, isoamyl acetate, hexyl acetate, and phenylethyl acetate. The concentration of acetate esters in the Sc2 group of wine samples inoculated only with activated commercial brewing yeast CEC01 was 30438.52 μg / L, while the concentration of acetate esters in the I-Wa2 group of wine samples with the same inoculation amount of immobilized yeast capsules was 60571.72 μg / L, an increase of 30133.2 μg / L. The co-fermentation of immobilized yeast capsules and commercial yeast enhanced the complexity of the wine's aroma and improved its quality.

[0096] Example 3

[0097] The ethanol tolerance of the immobilized Saccharomyces cerevisiae prepared in Examples 1-2 was determined.

[0098] Different volumes of anhydrous ethanol were added to 50 mL of sterile modified YPD liquid medium to make ethanol concentrations of 3%, 6%, 9%, 12%, and 15% vol, respectively. The immobilized yeast capsules prepared in Examples 1-2 were inoculated into the above modified YPD liquid medium containing different ethanol concentrations at an inoculation rate of 2%. The medium was cultured at 28°C and 180 rpm / min for 48 h. Then, the cell biomass (OD600), sugar consumption, and number of viable cells in the capsules of abnormal Wickham yeast that had extravasated into the culture medium were measured. The modified YPD liquid medium was composed of the following raw materials in the following mass fractions: 10% glucose, 2% peptone, and 1% yeast extract.

[0099] from Figure 3It can be seen that with increasing ethanol concentration, cell biomass, glucose consumption, and the number of viable cells within the capsule all show a decreasing trend. The cell biomass decreased at the fastest rate, reaching almost its lowest point at a 9% vol ethanol concentration. This means that at a 9% vol ethanol concentration, the growth and reproduction of *Wickham's abnormal* yeast are strongly inhibited, with only minimal reproductive activity. The number of viable cells within the capsule indicates that a 12% vol ethanol concentration has a strong inhibitory effect on the growth of yeast cells within the immobilized yeast capsule, while at a 9% vol ethanol concentration, the growth of viable cells within the capsule is partially inhibited, and its cell biomass is comparable to that in the culture medium at a 6% vol ethanol concentration.

[0100] The ethanol tolerance of immobilized yeast capsules of *Saccharomyces cerevisiae* increased from 6% vol to 9% vol, an improvement of 33.3%. Immobilization of non-brewing yeast enhances its tolerance to alcohol and makes it more adaptable to the environment of winemaking.

[0101] Example 4

[0102] This example is a screening of the preparation conditions for immobilized yeast capsules in Examples 1 and 2:

[0103] (1) Screening of the viscosity of sodium alginate solution.

[0104] The viscometer measured the viscosities of three sodium alginate solutions to be 85 mPa·s, 115 mPa·s, and 225 mPa·s, respectively, and labeled them as type 85, type 115, and type 225. Immobilized yeast capsules were prepared by adding 2% (w / w) calcium chloride solution and 1.5% (w / w) chitosan solution to the three sodium alginate solutions of different viscosities with the bacterial suspension. The chitosan in the chitosan solution had a molecular weight of 250,000 Da.

[0105] The hardness and permeability of the prepared immobilized yeast capsules were measured, and the results are as follows: Figure 4 As shown.

[0106] Depend on Figure 4 It can be seen that there is no significant difference in hardness and permeability between immobilized yeast capsules prepared with sodium alginate solutions of type 85 and type 115 (p>0.05), while the permeability of these two groups is significantly higher than that of immobilized yeast capsules prepared with sodium alginate solution of type 225 (p<0.05).

[0107] Immobilized yeast capsules aim to achieve sufficient substance exchange while retaining cells. Hardness is a crucial performance indicator; insufficient hardness during fermentation will cause the capsules to break easily, failing to achieve the intended purpose of yeast immobilization. Permeability represents the ability to transfer substances; often, increased permeability comes at the cost of reduced hardness. Based on the conversion relationship between viscosity and molecular weight, higher viscosity corresponds to a larger molecular weight. Sodium alginate has a large molecular weight, similar to Ca... 2+ The more binding sites a gel has, the tighter its cross-linked structure becomes, thus increasing its hardness. However, an overly dense gel structure can also lead to decreased permeability. Therefore, both hardness and permeability must be considered when evaluating the performance of immobilized yeast capsules.

[0108] Therefore, considering all indicators, the immobilized yeast capsules prepared by type 85 and type 115 have better performance. The immobilized yeast capsules prepared by type 225 sodium alginate solution have the largest molecular weight and the highest hardness and lowest permeability, and their performance is not as good as the immobilized yeast capsules prepared by type 85 and type 115.

[0109] (2) Screening of the mass fraction of sodium alginate solution.

[0110] In Comparative Example 1, the immobilized yeast capsules prepared with sodium alginate solutions of types 85 and 115 showed similar hardness and permeability. Therefore, the hardness and permeability of immobilized yeast capsules prepared with sodium alginate solutions of different mass fractions (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, and 3.5%) were measured, and the results are as follows. Figure 5 and Figure 6 As shown.

[0111] When the mass fraction of sodium alginate solution during preparation is 0.5% or 1.0%, the immobilized yeast capsules are poorly formed and have too low hardness due to the low mass fraction of sodium alginate solution, making it impossible to measure them using a texture analyzer.

[0112] Depend on Figure 5 and Figure 6 It was found that when the mass fraction of sodium alginate solution was 2%, the immobilized yeast capsules prepared with both type 85 and type 115 sodium alginate solutions reached their maximum hardness and permeability. Immobilized yeast capsules prepared with higher sodium alginate solution mass fractions exhibited stronger mechanical properties; further increasing the sodium alginate solution mass fraction significantly decreased both the hardness and permeability of the immobilized yeast capsules (p < 0.05).

[0113] Further comparative analysis of sodium alginate solutions of type 856 and type 115 showed that there was no significant difference in the performance of immobilized yeast capsules prepared by the two solutions when the sodium alginate solution mass fraction was 2% (p>0.05); however, under other mass fraction conditions, the immobilized yeast capsules prepared by sodium alginate solution of type 115 had significantly better hardness than those prepared by sodium alginate solution of type 85 (p<0.05).

[0114] Meanwhile, the immobilized yeast capsules prepared with sodium alginate solution of type 115 have a regular round shape. Therefore, under the conditions of calcium chloride solution with a mass fraction of 2% and chitosan solution with a mass fraction of 1.5%, the immobilized yeast capsules prepared with sodium alginate solution with a mass fraction of 2% and a viscosity of 115 mPa·s have the best hardness and permeability. The molecular weight of chitosan in the chitosan solution is 250,000 Da.

[0115] (3) Screening of the mass fraction of calcium chloride solution.

[0116] The hardness and permeability of immobilized yeast capsules prepared with different mass fractions (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, and 3.0%) of calcium chloride solution were determined under the conditions of sodium alginate solution with a mass fraction of 2% and a viscosity of 115 mPa·s and chitosan solution with a mass fraction of 1.5%. The effect of different mass fractions of calcium chloride solution on the hardness and permeability of the prepared immobilized yeast capsules was studied. The chitosan in the chitosan solution had a molecular weight of 250,000 Da.

[0117] The hardness and permeability of the prepared immobilized yeast capsules were measured, and the results are as follows: Figure 7 As shown.

[0118] Depend on Figure 7 It can be seen that as the mass fraction of calcium chloride solution increases, the overall trend of hardness and permeability of immobilized yeast capsules is first to rise and then to fall, reaching the highest point when the mass fraction is 2%. When the mass fraction of calcium chloride solution is 2%, the hardness and permeability are significantly better than those under other concentration conditions (p < 0.05).

[0119] Because of the crosslinking agent Ca 2+ The concentration of Ca has a significant impact on the properties of gel particles. 2+ It combines with sodium alginate to form a gel network structure, Ca 2+ Higher concentrations of Ca result in a denser structure, increased hardness, and decreased permeability. 2+ When the concentration is too high, the structure becomes too compact, which will affect the penetration of the remaining sodium alginate molecules and the binding of the carboxyl groups on the sodium alginate molecules with the primary amino groups on the chitosan molecules, thus preventing the permeability from increasing further.

[0120] Therefore, under the conditions of sodium alginate solution with a mass fraction of 2% and a viscosity of 115 mPa·s and chitosan solution with a mass fraction of 1.5%, the immobilized yeast capsules prepared with a calcium chloride solution mass fraction of 2% exhibit the best hardness and permeability. The chitosan in the chitosan solution has a molecular weight of 250,000 Da.

[0121] (4) Screening of chitosan molecular weight.

[0122] The hardness and permeability of immobilized yeast capsules prepared by preparing chitosan solutions with a mass fraction of 2% sodium alginate and a viscosity of 115 mPa·s and a mass fraction of 2% calcium chloride were determined under the conditions of chitosan solutions with a mass fraction of 2% for three different molecular weights.

[0123] The hardness and permeability of the prepared immobilized yeast capsules were measured, and the results are as follows: Figure 8 and Figure 9 As shown.

[0124] At the same mass fraction, the hardness and permeability of immobilized yeast capsules prepared from chitosan solutions with molecular weight of 100,000 Da were significantly lower than those prepared from chitosan solutions with molecular weights of 250,000 and 700,000 Da (p < 0.05). This is because as the molecular weight increases, the number of protonated amino groups in the chitosan molecular chain increases, resulting in more gel binding sites with sodium alginate, which in turn increases the density and hardness of the immobilized yeast capsule membrane.

[0125] Depend on Figure 8 It can be seen that as the mass fraction of the chitosan solution increases, the hardness of the immobilized yeast capsules reaches its maximum value when the mass fraction of the chitosan solution prepared from chitosan with a molecular weight of 250,000 Da and 700,000 Da increases to 1.5%, and then decreases.

[0126] Depend on Figure 9 It can be seen that the permeability of immobilized yeast capsules prepared from chitosan solutions containing 250,000 Da molecular weight chitosan gradually decreases with increasing concentration, while the effect of the concentration of chitosan solutions containing 750,000 Da molecular weight chitosan on the permeability of immobilized yeast capsules is disordered. This is because the chitosan solution containing 750,000 Da molecular weight chitosan has a large molecular weight and high viscosity, resulting in poor flowability and slow diffusion rate. The dense gel film formed by the combination with the carboxyl groups of sodium alginate prevents chitosan molecules from penetrating into the interior, thus leading to the irregular change in permeability. Furthermore, due to its high viscosity, it is difficult to achieve industrial-scale preparation. Comprehensive analysis indicates that chitosan solutions containing 250,000 Da molecular weight chitosan are most suitable for preparing immobilized yeast capsules.

[0127] Depend on Figure 10Particle size analysis showed that the larger the molecular weight of chitosan, the smaller the particle size of the prepared immobilized yeast capsules, and the stronger their compactness. At different mass fractions, there was no significant difference in permeability (p>0.05) between chitosan solutions prepared with 100,000 Da molecular weight chitosan. The increase in hardness was relatively slow, because the molecular weight was too small, and small differences in mass fraction had little effect on its hardness and permeability. Furthermore, the smaller the molecular weight of chitosan, the thicker the gel film formed, which reduces the yeast's proliferation space and increases mass transfer resistance, also contributing to its poor permeability.

[0128] (5) Screening of chitosan solution mass fraction.

[0129] Comparative Example 4 shows that the chitosan solution prepared from chitosan with a molecular weight of 250,000 Da is suitable for the preparation of immobilized yeast capsules. The permeability of the obtained immobilized yeast capsules decreases with the increase of the mass fraction of chitosan solution, but the hardness does not continue to increase. Therefore, under the conditions of sodium alginate solution with a mass fraction of 2% and a viscosity of 115 mPa·s and calcium chloride solution with a mass fraction of 2%, the mass fraction of chitosan solution prepared from chitosan with a molecular weight of 250,000 Da was increased to prepare immobilized yeast capsules.

[0130] The hardness and permeability of the prepared immobilized yeast capsules were measured, and the results are as follows: Figure 11 As shown.

[0131] Depend on Figure 11 It can be seen that, starting from the absence of chitosan, the permeability of immobilized yeast capsules continuously decreased with the increase of chitosan solution mass fraction, reaching a minimum at a chitosan solution mass fraction of 2.5%, and the decrease was no longer significant thereafter. The hardness was better at a chitosan solution mass fraction of 1.5%, and was significantly higher than that without chitosan (p < 0.05). Excessively high chitosan solution mass fraction can affect the exchange of substances between microorganisms and nutrients, thus affecting their activity.

[0132] Therefore, considering both hardness and permeability, the immobilized yeast capsules prepared with a chitosan solution containing 1.5% chitosan have better performance under the conditions of sodium alginate solution with a mass fraction of 2% and a viscosity of 115 mPa·s and calcium chloride solution with a mass fraction of 2%. The chitosan in the chitosan solution has a molecular weight of 250,000 Da.

[0133] In summary, the optimal preparation conditions for immobilized yeast capsules are: a sodium alginate solution with a mass fraction of 2% and a viscosity of 115 mPa·s, a calcium chloride solution with a mass fraction of 2% and a chitosan solution with a mass fraction of 1.5%, wherein the chitosan in the chitosan solution has a molecular weight of 250,000 Da.

[0134] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing immobilized yeast capsules using non-Saccharomyces cerevisiae, characterized in that, The non-Saccharomyces cerevisiae yeast is *Wickhamia lanceolata*. Wickerhamomyces anomalus The accession number is CGMCC No. 25528, the depositary institution is the China General Microbiological Culture Collection Center, and the deposit date is August 12, 2022; the 26S rDNA nucleotide sequence of the non-Saccharomyces cerevisiae is shown in SEQ ID NO: 1, and the method is as follows: S1. Preparation of bacterial suspension: Under aseptic conditions, frozen non-Saccharomyces cerevisiae was inoculated into YPD liquid medium and cultured at 28℃ for 48 h. Activation was achieved by three repeated subculturings. The culture was centrifuged at 4℃ and 4500 r / min. The collected bacterial cells were diluted with sterile physiological saline to prepare 10... 10 The bacterial suspension of cfu / mL was refrigerated at 4°C for later use. S2. Preparation of immobilized yeast capsules: Take 10 mL of the refrigerated bacterial suspension from S1, allow it to return to room temperature naturally, and add it to 1 L of 2% sodium alginate solution. Stir well to ensure a total bacterial count of 10⁻⁶ per mL of solution. 8 CFU was added dropwise to 2L of 2% (w / w) sterile calcium chloride solution, stirred evenly, and solidified for 0.5h to form immobilized capsule particles. The particles were washed three times with sterile saline, then transferred to 3L of 1.5% (w / w) chitosan solution, shaken for 3h, and washed three more times with sterile saline to obtain immobilized yeast capsules. The viscosity of the sodium alginate solution was 115 mPa·s, and the molecular weight of the chitosan in the chitosan solution was 250,000 Da.

2. The method for preparing immobilized yeast capsules using non-Saccharomyces cerevisiae according to claim 1, characterized in that, The YPD liquid culture medium described in S1 is composed of the following raw materials in the indicated mass fractions: 2% glucose, 2% peptone and 1% yeast extract, with the remainder being water, and then sterilized at 121°C for 20 minutes.

3. The method for preparing immobilized yeast capsules using non-Saccharomyces cerevisiae according to claim 1, characterized in that, The ethanol tolerance of the immobilized yeast capsules described in S2 is 9% vol.

4. The method for preparing immobilized yeast capsules using non-Saccharomyces cerevisiae according to claim 2, characterized in that, The average particle size of the immobilized yeast capsules described in S2 is 2.301 mm to 3.829 mm.

5. An application of an immobilized yeast capsule prepared by the method according to any one of claims 1-4, characterized in that, The immobilized yeast capsules are used in winemaking to increase the content of volatile substances; The method of winemaking is as follows: S1. Remove the stems from the grapes and crush them. Put them directly into the fermentation tank or press them and then put them into the fermentation tank to obtain grape juice. Put the grape juice into the fermentation tank, add sulfite to make the sulfite concentration 60mg / L, and then let it stand for 4 hours. Then add pectinase to make the final pectinase concentration 20mg / L, and then ferment at 4℃ for 48 hours to obtain grape fermentation liquid. S2. After the grape fermentation broth obtained in S1 has returned to room temperature, inoculate with a volume of 10... 6 Immobilized yeast capsules at a concentration of CFU / mL were inoculated into the grape fermentation broth. After fermentation for 24 hours, a second inoculation was performed at a concentration of 10 CFU / mL. 6 Commercially activated brewing yeast (cfu / mL) was thoroughly stirred and then allowed to ferment statically at 18°C ​​or 26°C. The fermentation tank was sealed with a fermentation plug. When the ethanol concentration reached 9% vol, the mixture of immobilized yeast capsules and grape seeds was separated by filtration to obtain grape must. The grape must was fermented under the action of free yeast in the liquid until the residual sugar content in the wine was <2 g / L. Fermentation was then terminated, and the wine was obtained after filtering the residue.

6. The application according to claim 5, characterized in that, When the grapes mentioned in S1 are grape varieties for making red wine, the grapes are destemmed, crushed, and directly loaded into the fermentation tank, with a volume fraction of 80%; when the grapes mentioned in S1 are grape varieties for making white wine, the grapes are destemmed, crushed, pressed, and then loaded into the fermentation tank, with a volume fraction of 90%.

7. The application according to claim 5, characterized in that, When the grapes mentioned in S1 are grape varieties for making red wine, the temperature for static fermentation mentioned in S2 is 26°C; when the grapes mentioned in S1 are grape varieties for making white wine, the temperature for static fermentation mentioned in S2 is 18°C.

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