CN112812914A - Mixed fermentation process based on pichia kluyveri and saccharomyces cerevisiae - Google Patents
Mixed fermentation process based on pichia kluyveri and saccharomyces cerevisiae Download PDFInfo
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Abstract
The invention discloses a mixed fermentation process based on pichia kluyveri and saccharomyces cerevisiae, belonging to the technical field of microorganisms. The mixed fermentation process based on the pichia kluyveri and the saccharomyces cerevisiae is characterized in that: performing mixed fermentation on the pichia kluyveri and the saccharomyces cerevisiae; the Pichia kluyveri refers to Pichia kluyveri Kluyveri strain HSP 11; the preservation number of the Pichia kluyveri strain HSP11 is CCTCC M2021087. Compared with single-bacterium fermented products, the product obtained by the fermentation process has more unique fragrance, can produce a wine with unique flavor, fragrance and taste, fills the consumer category and expands the consumption selection.
Description
Technical Field
The invention belongs to the technical field of beverage fermentation, and particularly relates to a mixed fermentation process based on pichia kluyveri and saccharomyces cerevisiae.
Background
Wine fermentation is a complex biochemical process in which yeast plays a very critical role, such as the conversion of sugars to ethanol, carbon dioxide and other secondary metabolites in the thousands. A large number of scientific studies show that the quality of the wine is highly dependent on the metabolic activity and fermentation behavior of different yeasts, and the different yeasts have important contributions to the chemical composition, the sensory characteristics, the flavor characteristics and the like of the wine. Saccharomyces cerevisiae is the strain which is widely applied in the industrial production of wine so far, has the advantages of ensuring the risk of deterioration in the fermentation process of wine, and having good fermentation power, but has the problems of single flavor characteristic, serious homogenization phenomenon and the like of wine. Therefore, in order to pursue wine style specialization and make the aroma characteristics more representative, diverse and complex, brewers often adopt a method of mixing and fermenting saccharomyces cerevisiae and non-saccharomyces cerevisiae, especially some indigenous yeasts with strong adaptability and representativeness, so as to improve and enhance the flavor quality of wine.
Non-saccharomyces cerevisiae has become an option for improving the quality of wine. Numerous studies have shown that non-saccharomyces cerevisiae is able to produce enzymes as well as some of our desired secondary metabolites, thus improving wine aroma and flavor characteristics, and is able to control the growth of some undesirable species in wine, but has the disadvantage of insufficient fermentation power. The mixed fermentation of the non-saccharomyces cerevisiae and the saccharomyces cerevisiae can improve the aroma diversity and complexity of the wine, can make up for the problem of insufficient fermentation power of the non-saccharomyces cerevisiae, and is an effective method for improving the aroma quality of the wine.
In the prior art, some reports exist at present for preparing wine drink by carrying out mixed fermentation on saccharomyces cerevisiae and non-saccharomyces cerevisiae, such as: the invention patent application 202010852525.3 discloses a method for preparing a compound with a preservation number of CCTCC NO: m2020372 Pichia kluyveri strain LI-27-1 and Saccharomyces cerevisiae mixed bacteria fermentation to degrade L-malic acid in wine.
In order to improve the flavor and aroma of wine, more mixed fermentation processes need to be developed in the field.
Disclosure of Invention
Based on the above needs in the field, the invention provides a mixed fermentation process based on pichia kluyveri strain HSP11 and saccharomyces cerevisiae F33, which has a very significant improvement on various aroma substances that can affect the flavor of wine compared with single-strain fermentation of saccharomyces cerevisiae F33.
The technical scheme of the invention is as follows:
a mixed fermentation process based on Pichia kluyveri and Saccharomyces cerevisiae is characterized in that mixed fermentation is carried out on the Pichia kluyveri and the Saccharomyces cerevisiae;
the Pichia kluyveri refers to Pichia kluyveri Kluyveri strain HSP 11; the preservation number of the Pichia kluyveri strain HSP11 is CCTCC M2021087.
The saccharomyces cerevisiae refers to saccharomyces cerevisiae strain F33.
The mixed fermentation process based on the pichia kluyveri and the saccharomyces cerevisiae comprises the following steps: the activated strain and the activated strain are inoculated into a substrate for secondary fermentation for fermentation.
Preferably, said strain refers to: pichia kluyveri strain HSP11, Pichia kluyveri, or Saccharomyces cerevisiae strain F33;
preferably, the activated strain refers to a strain inoculated in a culture medium for culture;
preferably, the culture temperature is 24-30 ℃, preferably 28 ℃; the culture time is 20-30h, preferably 24h, the rotation speed is 120-250rpm, preferably 150 rpm;
preferably, the inoculation refers to inoculating the initial strain stock solution into the culture medium according to the inoculation amount of 2-4%, preferably 3% in volume ratio;
preferably, the medium is a YPD medium; preferably, the YPD medium comprises the following components in mass-to-volume ratio: 0.5-3.5%, preferably 1%, yeast extract, 1.0-3.0%, preferably 2%, peptone, 1.0-5.0%, preferably 2%, glucose, and water in balance;
preferably, the initial strain stock solution refers to the strain stock in 15-35% by volume, preferably 25% glycerol/YPD medium;
more preferably, the activation of the strain is performed 1-3 times, preferably 2 times.
The activated strain refers to: the activated Pichia kluyveri strain HSP11 of Pichia kluyveri or the activated Saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate into the secondary fermentation refers to: firstly, inoculating an activated Pichia kluyveri strain HSP11 to a substrate for fermentation for 0-96h, preferably 48h, and then inoculating an activated Saccharomyces cerevisiae strain F33 for continuous fermentation;
preferably, the fermentation temperature is 18 ℃ +/-2 ℃;
preferably, the total inoculum size of the activated Pichia kluyveri strain HSP11 and the activated Saccharomyces cerevisiae strain F33 is 106-107CFU, preferably 6X 106CFU；
Preferably, the fermentation is terminated when the substrate weight loss does not change for 3 consecutive days;
preferably, the substrate is grape juice.
A production method of wine is characterized in that grape juice is used as a substrate, and mixed fermentation is carried out on Pichia kluyveri and saccharomyces cerevisiae;
the Pichia kluyveri refers to Pichia kluyveri Kluyveri strain HSP 11; the preservation number of the Pichia kluyveri strain HSP11 is CCTCC M2021087.
The saccharomyces cerevisiae refers to saccharomyces cerevisiae strain F33.
The production method of the wine comprises the following steps: the activated strain and the activated strain are inoculated into a substrate for secondary fermentation for fermentation.
Preferably, said strain refers to: pichia kluyveri strain HSP11, Pichia kluyveri, or Saccharomyces cerevisiae strain F33;
preferably, the activated strain refers to a strain inoculated in a culture medium for culture;
preferably, the culture temperature is 24-30 ℃, preferably 28 ℃; the culture time is 20-30h, preferably 24h, the rotation speed is 120-250rpm, preferably 150 rpm;
preferably, the inoculation refers to inoculating the initial strain preservation solution into the culture medium according to the inoculation amount of 3% by volume;
preferably, the medium is a YPD medium; preferably, the YPD medium comprises the following components in mass-to-volume ratio: 0.5-3.5%, preferably 1%, yeast extract, 1.0-3.0%, preferably 2%, peptone, 1.0-5.0%, preferably 2%, glucose, and water in balance;
preferably, the initial strain stock solution refers to the strain stock in 15-35% by volume, preferably 25% glycerol/YPD medium;
more preferably, the activation of the strain is performed 1-3 times, preferably 2 times.
The activated strain refers to: the activated Pichia kluyveri strain HSP11 of Pichia kluyveri or the activated Saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate into the secondary fermentation refers to: firstly, inoculating an activated Pichia kluyveri strain HSP11 to a substrate for fermentation for 0-96h, preferably 48h, and then inoculating an activated Saccharomyces cerevisiae strain F33 for continuous fermentation;
preferably, the fermentation temperature is 18 ℃ +/-2 ℃;
preferably, the total inoculation amount of the activated Pichia kluyveri strain HSP11 and the activated Saccharomyces cerevisiae strain F33 is 106-107CFU, preferably 6X 106CFU；
Preferably, the fermentation is terminated when the substrate weight loss is not changed for 3 consecutive days.
The wine production method further comprises the following steps: preparing grape juice;
the preparation of the grape juice comprises the following steps: squeezing grape fruit grains at low temperature;
preferably, the harvested grape fruit particles are pressed with ice by an air bag press;
preferably, pressing is performed while adding sulfur dioxide or K2S2O5And, pectinase;
preferably, sulfur dioxide or K2S2O5The addition amount of (B) is 30-100mg/L, preferably 50 mg/L; the addition amount of pectinase is 10-30mg/L, preferably 20 mg/L;
preferably, the grape fruit pieces are uniformly sized fruit pieces;
preferably, the grape fruit particles are grape fruit particles collected when the temperature of the mature grapes continuously drops below 24 hours to 8 ℃.
The wine is characterized by being produced by the wine production method.
The invention adopts Pichia kluyveri bacterial strain HSP11 and Saccharomyces cerevisiae F33 to carry out mixed fermentation, among dozens of generated aroma substances, the yield of most aroma substances is improved, for example, the yield of dimethyl silanediol is improved by more than 1 time, (2R,3R) - (-) -2, 3-butanediol is improved by about 45 percent, isovaleraldehyde is improved by 28 percent, 1, 3-propanediol monoethyl ether is improved by about 49 percent, ethyl myristate is improved by more than 1 time, ethyl nonanoate is improved by about 80 percent, 2, 3-dihydro-3, 5 dihydroxy-6-methyl-4 (H) -pyran-4-one is improved by about 44 percent, 2,2, 4-trimethylpentanediol isobutyl ester is improved by about 36 percent, and 4-isopropyltoluene is improved by nearly one time; meanwhile, aroma substances which cannot be produced by single-bacterium fermentation are also produced, such as: 3-methyl-1-pentanol, 2-ethylhexanol, propylene glycol, (Z) -3-methyl-2-hepten-1-ol, 2, 3-butanedione, isoamyl decanoate, p-xylene, (+) -medicatene. The production of the aroma substances or the increase of the yield of the aroma substances can exert certain influence on the flavor and aroma of the beverage obtained by fermentation, so that the mixed fermentation product presents more unique aroma compared with a single fermentation product, and a wine beverage with unique flavor, aroma and taste can be produced, the consumer class is enriched, and the consumption selection is expanded.
Detailed Description
The following detailed description of the present invention will be made with reference to specific examples, but the scope of the present invention is not limited thereto.
Sources and documentations of biological materials
The Pichia kluyveri strain HSP11 used in the experimental examples is a new strain screened by the applicant's laboratory, and the preservation information is as follows:
naming: HSP11
And (4) classification name: pichia kluyveri
The name of Latin is: pichia kluyveri
The preservation number is as follows: CCTCC NO: M2021087
The preservation organization: china center for type culture Collection
And (4) storage address: wuhan university school of eight-channel 299 # Wuhan university in Wuchang district of Wuhan city, Hubei province (first attached to small opposite surface of Wuhan university), Wuhan university collection center
The preservation date is as follows: 1 month 15 days 2021;
saccharomyces cerevisiae F33 was a commercial strain purchased from Lafford (Laffort) France.
The grape variety used was a wital ice grape, purchased from Ningxia Bagges drunk intersomatic wine village, Inc.
Group 1 example, Mixed fermentation Process of the invention
The embodiment of the group provides a mixed fermentation process based on pichia kluyveri and saccharomyces cerevisiae. All embodiments of this group share the following common features: performing mixed fermentation on the pichia kluyveri and the saccharomyces cerevisiae; the Pichia kluyveri refers to Pichia kluyveri Kluyveri strain HSP 11; the preservation number of the Pichia kluyveri strain HSP11 is CCTCC M2021087.
Any fermentation and production behavior of Pichia kluyveri, HSP11, in combination with Saccharomyces cerevisiae falls within the scope of the present invention as one skilled in the art can appreciate in light of the present disclosure. Target products of fermentation include, but are not limited to: wine drink, fermented milk, bread, etc.
In a specific embodiment, the Saccharomyces cerevisiae is Saccharomyces cerevisiae strain F33.
According to the teaching of the present invention, those skilled in the art can select other commercial strains for mixed fermentation, besides F33, there are many commercial strains on the market, such as Saccharomyces cerevisiae V1116, Saccharomyces cerevisiae VL1, Saccharomyces cerevisiae X16, etc., and these strains can be used for mixed fermentation with Pichia kluyveri Kluyveri Pichia kluyveri strain HSP11 of the present invention to obtain similar technical effects as the present invention.
In some embodiments, the mixed fermentation process based on pichia kluyveri and saccharomyces cerevisiae comprises the following steps: the activated strain and the activated strain are inoculated into a substrate for secondary fermentation for fermentation.
Preferably, said strain refers to: pichia kluyveri strain HSP11, Pichia kluyveri, or Saccharomyces cerevisiae strain F33;
preferably, the activated strain refers to a strain inoculated in a culture medium for culture;
preferably, the culture temperature is 24-30 ℃, preferably 28 ℃; the culture time is 20-30h, preferably 24h, the rotation speed is 120-250rpm, preferably 150 rpm;
preferably, the inoculation refers to inoculating the initial strain stock solution into the culture medium according to the inoculation amount of 2-4%, preferably 3% in volume ratio;
preferably, the medium is a YPD medium; preferably, the YPD medium comprises the following components in mass-to-volume ratio: 0.5-3.5%, preferably 1%, yeast extract, 1.0-3.0%, preferably 2%, peptone, 1.0-5.0%, preferably 2%, glucose, and water in balance;
preferably, the initial strain stock solution refers to the strain stock in 15-35% by volume, preferably 25% glycerol/YPD medium;
more preferably, the activation of the strain is performed 1-3 times, preferably 2 times.
In other embodiments, the activated strain refers to: the activated Pichia kluyveri strain HSP11 of Pichia kluyveri or the activated Saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate into the secondary fermentation refers to: firstly, inoculating an activated Pichia kluyveri strain HSP11 to a substrate for fermentation for 0-96h, preferably 48h, and then inoculating an activated Saccharomyces cerevisiae strain F33 for continuous fermentation;
preferably, the fermentation temperature is 18 ℃ +/-2 ℃;
preferably, the total inoculation amount of the activated Pichia kluyveri strain HSP11 and the activated Saccharomyces cerevisiae strain F33 is 106-107CFU, preferably 6X 106CFU；
Preferably, the fermentation is terminated when the substrate weight loss does not change for 3 consecutive days;
preferably, the substrate is grape juice.
Group 2 example, Process for the production of wine according to the invention
The present group of embodiments provides a method of producing wine. The present group of embodiments all have the following common features: taking grape juice as a substrate, and carrying out mixed fermentation on pichia kluyveri and saccharomyces cerevisiae; the Pichia kluyveri refers to Pichia kluyveri Kluyveri strain HSP 11; the preservation number of the Pichia kluyveri strain HSP11 is CCTCC M2021087.
In a specific embodiment, the Saccharomyces cerevisiae is Saccharomyces cerevisiae strain F33.
In other specific embodiments, the wine production method comprises: the activated strain and the activated strain are inoculated into a substrate for secondary fermentation for fermentation.
Preferably, said strain refers to: pichia kluyveri strain HSP11, Pichia kluyveri, or Saccharomyces cerevisiae strain F33;
preferably, the activated strain refers to a strain inoculated in a culture medium for culture;
preferably, the culture temperature is 24-30 ℃, preferably 28 ℃; the culture time is 20-30h, preferably 24h, the rotation speed is 120-250rpm, preferably 150 rpm;
preferably, the inoculation refers to inoculating the initial strain stock solution into the culture medium according to the inoculation amount of 2-4%, preferably 3% in volume ratio;
preferably, the medium is a YPD medium; preferably, the YPD medium comprises the following components in mass-to-volume ratio: 0.5-3.5%, preferably 1%, yeast extract, 1.0-3.0%, preferably 2%, peptone, 1.0-5.0%, preferably 2%, glucose, and water in balance;
preferably, the initial strain stock solution refers to the strain stock in 15-35% by volume, preferably 25% glycerol/YPD medium;
more preferably, the activation of the strain is performed 1-3 times, preferably 2 times.
In specific embodiments, the activated strain refers to: the activated Pichia kluyveri strain HSP11 of Pichia kluyveri or the activated Saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate into the secondary fermentation refers to: firstly, inoculating an activated Pichia kluyveri strain HSP11 to a substrate for fermentation for 0-96h, preferably 48h, and then inoculating an activated Saccharomyces cerevisiae strain F33 for continuous fermentation;
when the time for fermenting the activated Pichia kluyveri strain HSP11 inoculated to the substrate is 0h, the activated Pichia kluyveri strain HSP11 and the activated Saccharomyces cerevisiae strain F33 can be simultaneously inoculated to the substrate for fermenting until the substrate weight loss is continuous for 3 days and is not changed any more, and the fermentation is terminated.
Preferably, the fermentation temperature is 18 ℃ +/-2 ℃;
preferably, the total inoculation amount of the activated Pichia kluyveri strain HSP11 and the activated Saccharomyces cerevisiae strain F33 is 106-107CFU, preferably 6X 106CFU；
Preferably, the fermentation is terminated when the substrate weight loss is not changed for 3 consecutive days.
In a further embodiment, the method of wine production further comprises: preparing grape juice;
the preparation of the grape juice comprises the following steps: squeezing grape fruit grains at low temperature;
preferably, the harvested grape fruit particles are pressed with ice by an air bag press;
preferably, pressing is performed while adding sulfur dioxide or K2S2O5And, pectinase;
preferably, the addition amount of sulfur dioxide or K2S2O5 is 30-100mg/L, preferably 50 mg/L; the addition amount of pectinase is 10-30mg/L, preferably 20 mg/L;
preferably, the grape fruit pieces are uniformly sized fruit pieces;
preferably, the grape fruit particles are grape fruit particles collected when the temperature of the mature grapes continuously drops below 24 hours to 8 ℃. Group 3 examples, wine of the invention
The present group of embodiments provides a wine. All embodiments of this group share the following common features: produced by the wine production method of any one of the group 2 examples.
The wine of the invention produces the following aroma substances which cannot be produced by the wine fermented by single bacteria: 3-methyl-1-pentanol, 2-ethylhexanol, propylene glycol, (Z) -3-methyl-2-hepten-1-ol, 2, 3-butanedione, isoamyl decanoate, p-xylene, (+) -medicatene, while being much higher in the content of the following aroma substances than in single-strain fermented wines: dimethylsilanediol, (2R,3R) - (-) -2, 3-butanediol, isovaleraldehyde, 1, 3-propanediol monoethyl ether, ethyl myristate, ethyl nonanoate, 2, 3-dihydro-3, 5 dihydroxy-6-methyl-4 (H) -pyran-4-one, 2,2, 4-trimethylpentanediol isobutyl ester, 4-isopropyltoluene.
Experimental example, mixed bacteria fermentation process and fermentation data of the invention
1. Bacterial strains
The strains used in this experiment were: commercial s.cerevisiae F33 and Pichia kluyveri strain HSP11 isolated and screened according to the invention.
2. Grape juice
The Weidai ice grape raw material is planted in Yuquan Yingning county, Yinchuan city, Ningxia Bagges Zuius boundary wine village GmbH (E106.02 degree, N38.24 degree). The grape vines are planted in 2013, small-canopy-frame cultivation is adopted, the plant row spacing is 1.0m multiplied by 2.0m, the grape vines are harvested in 2017, the grape vines are not harvested after being mature, the grape vines are harvested when the temperature is reduced to be below 24 hours-8 ℃, small fruit grains are removed, and ice grape fruits with the same size are randomly selected and squeezed at low temperature. Pressing harvested ice grape with ice by air bag press while adding sulfur dioxide (50mg/L K)2S2O5) And 20mg/L of pectinase (more than or equal to 500U/mg), inhibits bacteria and improves the juice yield. The squeezed grape juice has sugar content of 432g/dm3Acidity of 4.65g/dm3(tartaric acid) pH 4.21.
3. Fermentation operation
2 strains: saccharomyces cerevisiae F33 strain, and Pichia kluyveri HSP11 strain were all stored in 25% glycerol/YPD medium by volume before use. YPD medium is 1% yeast extract powder, 2% peptone and 2% glucose. Respectively inoculating the strain with 3% of volume ratio into a 50mL triangular flask containing 40mL YPD medium and a 250mL triangular flask containing 150mL YPD medium, culturing at 28 deg.C and 150rpm for 24h to obtain the 1 st activated bacterial liquid, respectively inoculating the strain with 3% of volume ratio into a 50mL triangular flask containing 40mL YPD medium and a 250mL triangular flask containing 150mL YPD medium, and repeating the above culture to complete the 2 nd passage activation, thus obtaining the activated bacterial liquid. Firstly, inoculating activated Pichia kluyveri strain HSP11 into collected grape juice, and after 48 hours, inoculating S. cerevisiae F33 strain according to the inoculation ratio of HSP11/F33 in a volume ratio of 2:1, wherein the total inoculation amount of the strain is controlled at 6 x 106CFU, using S.cerevisiae F33 pure fermented grape juice as blank control, fermenting at 18 + -2 deg.C, and stopping fermentation when grape juice weight loss is not changed for three consecutive days. All wine samples were centrifuged 8 min at 7500rpmAnd (4) taking the supernatant and storing at 4 ℃.
4. Method for quantifying aroma substance
Headspace-solid phase microextraction-gas mass spectrometry (HS-SPME-GC/MS) was used. An 8mL sample of wine was accurately weighed into a headspace bottle containing 1.5g NaCl, while 394.08. mu.g/L4-methyl-1-pentanol (internal standard) was capped and sealed. Inserting CAR/DVB/PDMS extraction fiber, adsorbing at 45 deg.C for 30min, desorbing at 250 deg.C for 3 min, and performing GC-MS analysis. A chromatographic column: InertCap WAX polar chromatography column (60m × 0.25mm, 0.25 μm); the temperature rising procedure is as follows: maintaining at 40 deg.C for 5min, heating to 120 deg.C at 3 deg.C/min, heating to 230 deg.C at 8 deg.C/min, and maintaining for 10 min; the flow rate of the carrier gas (He) was 0.8mL/min, and was not split. Electron bombardment ion source; electron energy 70 eV; the transmission line temperature was 275 ℃; the ion source temperature is 230 ℃; the activation voltage is 1.5V; the filament flow is 0.25 mA; the mass scanning range m/z is 33-450. Compound quantitative analysis was performed using external standard quantitation method.
5. Data analysis method
All samples were averaged in 3 replicates respectively. Single-factor analysis of variance (ANOVA) and Duncan's multi-range test (P <0.05) were performed using SPSS 22.0for Windows (SPSS inc., Chicago, IL, US); unscamblebler 9.7(CAMO ASA, Norway) was analyzed by partial least squares.
The final statistical interpretation gives the following Table 1, where the data are in μ g/L, meaning: the content of the aroma substances in each liter of wine.
TABLE 1
The aroma threshold value in the above table 1 refers to the lower limit value of the lowest concentration at which a human can smell the substance, and the aroma description and the threshold value are subject to the relevant literature reports that the aroma description and the threshold value of the aroma substance are recorded, and the aroma substance without the aroma description and the threshold value in the table is the literature that the relevant literature about the aroma description and the threshold value of the substance is not searched. In the table, "F33" refers to data obtained by single fermentation using a commercial strain Saccharomyces cerevisiae F33, and "F33 _ HSP 11" refers to data obtained by mixed fermentation using commercial strains Saccharomyces cerevisiae F33 and Pichia kluyveri Kluyveromyces strain HSP 11.
As is well known in the field of fermentation, factors influencing fermentation are numerous and complex, and the components of a fermented product can change due to the change of factors such as raw material batches, raw material components, fermentation conditions, temperature, time and the like, so that the condition that single-strain fermentation is higher than mixed-strain fermentation on certain aroma components can occur, which is a normal phenomenon in the field.
Claims (10)
1. A mixed fermentation process based on Pichia kluyveri and Saccharomyces cerevisiae is characterized in that mixed fermentation is carried out on the Pichia kluyveri and the Saccharomyces cerevisiae;
the Pichia kluyveri refers to Pichia kluyveri Kluyveri strain HSP 11; the preservation number of the Pichia kluyveri strain HSP11 is CCTCC M2021087.
2. The mixed fermentation process based on Pichia kluyveri and Saccharomyces cerevisiae of claim 1, wherein the Saccharomyces cerevisiae is Saccharomyces cerevisiae strain F33.
3. The pichia kluyveri and saccharomyces cerevisiae based mixed fermentation process according to claim 1, characterized by comprising the following steps: the activated strain and the activated strain are inoculated into a substrate for secondary fermentation for fermentation.
Preferably, said strain refers to: pichia kluyveri strain HSP11, Pichia kluyveri, or Saccharomyces cerevisiae strain F33;
preferably, the activated strain refers to a strain inoculated in a culture medium for culture;
preferably, the culture temperature is 24-30 ℃, preferably 28 ℃; the culture time is 20-30h, preferably 24h, the rotation speed is 120-250rpm, preferably 150 rpm;
preferably, the inoculation refers to inoculating the initial strain stock solution into the culture medium according to the inoculation amount of 2-4%, preferably 3% in volume ratio;
preferably, the medium is a YPD medium; preferably, the YPD medium comprises the following components in mass-to-volume ratio: 0.5-3.5%, preferably 1%, yeast extract, 1.0-3.0%, preferably 2%, peptone, 1.0-5.0%, preferably 2%, glucose, and water in balance;
preferably, the initial strain stock solution refers to the strain stock in 15-35% by volume, preferably 25% glycerol/YPD medium;
more preferably, the activation of the strain is performed 1-3 times, preferably 2 times.
4. The pichia kluyveri and saccharomyces cerevisiae based mixed fermentation process of claim 3, wherein the activated strain is: the activated Pichia kluyveri strain HSP11 of Pichia kluyveri or the activated Saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate into the secondary fermentation refers to: firstly, inoculating an activated Pichia kluyveri strain HSP11 to a substrate for fermentation for 0-96h, preferably 48h, and then inoculating an activated Saccharomyces cerevisiae strain F33 for continuous fermentation;
preferably, the fermentation temperature is 18 ℃ +/-2 ℃;
preferably, the total inoculum size of the activated Pichia kluyveri strain HSP11 and the activated Saccharomyces cerevisiae strain F33 is 106-107CFU, preferably 6X 106CFU；
Preferably, the fermentation is terminated when the substrate weight loss does not change for 3 consecutive days;
preferably, the substrate is grape juice.
5. A production method of wine is characterized in that grape juice is used as a substrate, and mixed fermentation is carried out on Pichia kluyveri and saccharomyces cerevisiae;
the Pichia kluyveri refers to Pichia kluyveri Kluyveri strain HSP 11; the preservation number of the Pichia kluyveri strain HSP11 is CCTCC M2021087.
6. A wine production method according to claim 5, characterised in that the Saccharomyces cerevisiae is Saccharomyces cerevisiae strain F33.
7. A wine production process according to claim 5 or 6, comprising: the activated strain and the activated strain are inoculated into a substrate for secondary fermentation for fermentation.
Preferably, said strain refers to: pichia kluyveri strain HSP11, Pichia kluyveri, or Saccharomyces cerevisiae strain F33;
preferably, the activated strain refers to a strain inoculated in a culture medium for culture;
preferably, the culture temperature is 24-30 ℃, preferably 28 ℃; the culture time is 20-30h, preferably 24h, the rotation speed is 120-250rpm, preferably 150 rpm;
preferably, the inoculation refers to inoculating the initial strain preservation solution into the culture medium according to the inoculation amount of 3% by volume;
preferably, the medium is a YPD medium; preferably, the YPD medium comprises the following components in mass-to-volume ratio: 0.5-3.5%, preferably 1%, yeast extract, 1.0-3.0%, preferably 2%, peptone, 1.0-5.0%, preferably 2%, glucose, and water in balance;
preferably, the initial strain stock solution refers to the strain stock in 15-35% by volume, preferably 25% glycerol/YPD medium;
more preferably, the activation of the strain is performed 1-3 times, preferably 2 times.
8. A wine production process according to claim 7, wherein the activated strain is: the activated Pichia kluyveri strain HSP11 of Pichia kluyveri or the activated Saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate into the secondary fermentation refers to: firstly, inoculating an activated Pichia kluyveri strain HSP11 to a substrate for fermentation for 0-96h, preferably 48h, and then inoculating an activated Saccharomyces cerevisiae strain F33 for continuous fermentation;
preferably, the fermentation temperature is 18 ℃ +/-2 ℃;
preferably, the total inoculation amount of the activated Pichia kluyveri strain HSP11 and the activated Saccharomyces cerevisiae strain F33 is 106-107CFU, preferably 6X 106CFU；
Preferably, the fermentation is terminated when the substrate weight loss is not changed for 3 consecutive days.
9. A wine production process according to any one of claims 5 to 8, further comprising: preparing grape juice;
the preparation of the grape juice comprises the following steps: squeezing grape fruit grains at low temperature;
preferably, the harvested grape fruit particles are pressed with ice by an air bag press;
preferably, pressing is performed while adding sulfur dioxide or K2S2O5And, pectinase;
preferably, sulfur dioxide or K2S2O5The addition amount of (B) is 30-100mg/L, preferably 50 mg/L; the addition amount of pectinase is 10-30mg/L, preferably 20 mg/L;
preferably, the grape fruit pieces are uniformly sized fruit pieces;
preferably, the grape fruit particles are grape fruit particles collected when the temperature of the mature grapes continuously drops below 24 hours to 8 ℃.
10. Wine produced by the wine production method of any one of claims 5 to 9.
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