CN112812915A - Mixed fermentation process based on candida zephyrae and saccharomyces cerevisiae - Google Patents
Mixed fermentation process based on candida zephyrae and saccharomyces cerevisiae Download PDFInfo
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Abstract
The invention discloses a mixed fermentation process based on candida zephyranthi and saccharomyces cerevisiae, and belongs to the technical field of microorganisms. The mixed fermentation process based on candida zephyrae and saccharomyces cerevisiae is characterized in that the candida zephyrae and the saccharomyces cerevisiae are subjected to mixed fermentation; the Candida zephyrae refers to Candida zemplina strain YC 32; the preservation number of the Candida zempilina strain YC32 is CCTCC M2021090. Compared with single-strain fermentation of saccharomyces cerevisiae F33, the method has the advantages that various aroma substances which can influence the flavor of the wine are remarkably improved, so that the unique flavor, aroma and taste of the fermented beverage are produced.
Description
Technical Field
The invention belongs to the technical field of beverage fermentation, and particularly relates to a mixed fermentation process based on candida zephyranthes and saccharomyces cerevisiae.
Background
Wine fermentation is a complex biochemical process, and candida zephyrae plays a very critical role in fermentation processes, such as the conversion of sugars to ethanol, carbon dioxide and other secondary metabolites in the thousands. A large number of scientific researches show that the quality of the wine is highly dependent on the metabolic activity and fermentation behavior of different candida zephyranthes, and the different candida zephyranthes 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 have representative, diversified and complicated aroma characteristics, brewing workers often adopt a method of mixing and fermenting saccharomyces cerevisiae and non-saccharomyces cerevisiae, particularly some candida rugosa 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.
Reports on fermentation processes of Candida zemplina are rare in the field, and the reports on mixed fermentation processes of Candida zemplina and Saccharomyces cerevisiae in the field are never reported.
Disclosure of Invention
Based on the above needs and blanks in the field, the invention provides a mixed fermentation process based on Candida zempilina and Saccharomyces cerevisiae, which has a very significant improvement on various aroma substances affecting the flavor of wine compared with single-strain fermentation of Saccharomyces cerevisiae.
The technical scheme of the invention is as follows:
a mixed fermentation process based on Candida zephyrae and Saccharomyces cerevisiae is characterized in that the Candida zephyrae and the Saccharomyces cerevisiae are subjected to mixed fermentation.
Preferably, the Candida zempinella is Candida zempilina strain YC 32; the preservation number of the Candida zempilina strain YC32 is CCTCC M2021090.
Preferably, the Saccharomyces cerevisiae refers to Saccharomyces cerevisiae strain F33.
The mixed fermentation process based on candida zephyranthi 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: candida zemplina strain YC32, Candida zemplina, 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: activated Candida zemplina strain YC32, or activated Saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate into the secondary fermentation refers to: firstly inoculating the activated Candida zemplina strain YC32 to a substrate for fermentation for 0-96h, preferably 48h, and then inoculating the activated Saccharomyces cerevisiae strain F33 for continuous fermentation;
preferably, the fermentation temperature is 18 ℃ +/-2 ℃;
preferably, the total inoculum size of the activated Candida zemplina strain YC32, 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 Candida zephysalsa and saccharomyces cerevisiae are subjected to mixed fermentation;
the Candida zephyrae refers to Candida zemplina strain YC 32; the preservation number of the Candida zempilina strain YC32 is CCTCC M2021090.
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: candida zemplina strain YC32, Candida zemplina, 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: activated Candida zemplina strain YC32, or activated Saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate into the secondary fermentation refers to: firstly inoculating the activated Candida zemplina strain YC32 to a substrate for fermentation for 0-96h, preferably 48h, and then inoculating the activated Saccharomyces cerevisiae strain F33 for continuous fermentation;
preferably, the fermentation temperature is 18 ℃ +/-2 ℃;
preferably, the total inoculum size of the activated Candida zemplina strain YC32, 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 Candida zemplina of Jeep and Saccharomyces cerevisiae to carry out mixed fermentation, and the output of most aroma substances in dozens of aroma substances generated is improved, for example, caprylic acid is improved by 16.6%, 9-decenoic acid is improved by about 46.8%, glacial acetic acid is improved by 53%, isobutanol is improved by about 46.6%, hexanol is improved by 34.8%, 2R,3R) - (-) -2, 3-butanediol is improved by 1.2 times, 1, 3-propanediol monoethyl ether is improved by 3.4 times, 3-hydroxy-2-butanone is improved by 3.4 times, vinyl acetate is improved by nearly 1.2 times, damascenone is improved by 1.8 times, myrcene is improved by 1.6 times, citronellol is improved by 78%, 2, 6-di (tert-butyl) -4-hydroxy-4-methyl-2, the 5-cyclohexadiene-1-one is improved by 86 percent; meanwhile, aroma substances which cannot be produced by single-strain fermentation of saccharomyces cerevisiae are also produced, such as: isobutyl acetate, phenethyl acetate, 2-benzylpropionic acid, 3-methyl-1-pentanol, 2-ethylhexanol, 3-furancarbinol, sec-octanol, ethyl octanoate, ethyl decanoate, ethyl pyruvate, methylheptenone, 2, 3-butanedione, m-xylene, 3,5, 5-trimethyl-1-hexene, 2, 3-pentanedione, 1,7, 7-trimethylbis [2.2.1] hept-2-ene, isophorone, (+) -medicarene. 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-bacterium fermentation product, 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 Candida zemplina strain YC32 used in the experimental examples was a new strain selected in the laboratory of the applicant and the deposit information is as follows:
naming: YC32
And (4) classification name: candida zephyrae
The name of Latin is: candida zemplinina
The preservation number is as follows: CCTCC M2021090
The preservation organization: china center for type culture Collection
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 candida zephyranthi and saccharomyces cerevisiae. All embodiments of this group share the following common features: and (3) performing mixed fermentation on candida zephyranthi and saccharomyces cerevisiae.
Any fermentation and production behavior using Candida zephysa in combination with Saccharomyces cerevisiae would be within the scope of the present invention as taught by one of skill in the art. Target products of fermentation include, but are not limited to: wine drink, fermented milk, bread, etc.
In a preferred embodiment, the Candida zephyrae is Candida zemplina strain YC 32; the preservation number of the Candida zempilina strain YC32 is CCTCC M2021090.
In a specific embodiment, the Saccharomyces cerevisiae is Saccharomyces cerevisiae strain F33.
Those skilled in the art can select other commercial strains for mixed fermentation according to the teaching of the present invention, and besides F33, there are many commercial strains on the market, such as Saccharomyces cerevisiae V1116, Saccharomyces cerevisiae VL1, Saccharomyces cerevisiae X16, etc., which can be used for mixed fermentation with Candida zemplina strain YC32 of the present invention to obtain similar technical effects as the present invention.
In some embodiments, the mixed fermentation process based on candida zephyrae 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: candida zemplina strain YC32, Candida zemplina, 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: activated Candida zemplina strain YC32, or activated Saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate into the secondary fermentation refers to: firstly inoculating the activated Candida zemplina strain YC32 to a substrate for fermentation for 0-96h, preferably 48h, and then inoculating the activated Saccharomyces cerevisiae strain F33 for continuous fermentation;
preferably, the fermentation temperature is 18 ℃ +/-2 ℃;
preferably, the total inoculum size of the activated Candida zemplina strain YC32, 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 candida zephyranthes and saccharomyces cerevisiae.
In some preferred embodiments, the Candida zempilina is Candida zempilina strain YC 32; the preservation number of the Candida zempilina strain YC32 is CCTCC M2021090.
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: candida zemplina strain YC32, Candida zemplina, 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: activated Candida zemplina strain YC32, or activated Saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate into the secondary fermentation refers to: firstly inoculating the activated Candida zemplina strain YC32 to a substrate for fermentation for 0-96h, preferably 48h, and then inoculating the activated Saccharomyces cerevisiae strain F33 for continuous fermentation;
when the time for fermentation is 0h when the activated Candida zemplina strain YC32 is inoculated to the substrate, the activated Candida zemplina strain YC32 and the activated Saccharomyces cerevisiae strain F33 can be inoculated to the substrate simultaneously for fermentation until the substrate weight loss is not changed any more for 3 days.
Preferably, the fermentation temperature is 18 ℃ +/-2 ℃;
preferably, the total inoculum size of the activated Candida zemplina strain YC32, 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: isobutyl acetate, phenethyl acetate, 2-benzylpropionic acid, 3-methyl-1-pentanol, 2-ethylhexanol, 3-furancarbinol, sec-octanol, ethyl octanoate, ethyl decanoate, ethyl pyruvate, methylheptenone, 2, 3-butanedione, m-xylene, 3,5, 5-trimethyl-1-hexene, 2, 3-pentanedione, 1,7, 7-trimethylbis [2.2.1] hept-2-ene, isophorone, (+) -medicarene, while being much higher in the content of the following aroma substances than in a single-bacterium fermented wine: octanoic acid, 9-decenoic acid, glacial acetic acid, isobutanol, n-hexanol, (2R,3R) - (-) -2, 3-butanediol, 1, 3-propanediol monoethyl ether, 3-hydroxy-2-butanone, vinyl acetate, damascenone, myrcene, citronellol, 2, 6-di (tert-butyl) -4-hydroxy-4-methyl-2, 5-cyclohexadiene-1-one.
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 the isolated and selected Candida zempilina strain YC32 of the present 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, Candida zemplina strain YC32 were all stored in 25% by volume glycerol/YPD medium prior to 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. Inoculating activated Candida zemplina strain YC32 into collected grape juice, inoculating into S.cerevisiae F33 strain at YC32/F33 volume ratio of 2:1 after 48 hr, wherein the total inoculation amount of the strain is controlled at 6 × 106CFU, grape juice pure fermented with S.cerevisiae F33As a blank control, the fermentation temperature is 18 +/-2 ℃, and the fermentation is stopped when the weight loss of the grape juice is not changed for three consecutive days. All wine samples were centrifuged at 7500rpm for 8 minutes and the supernatant was stored 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 3min, 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 22.0 for 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. "F33" in the top list refers to data obtained from a single fermentation using the commercial strain Saccharomyces cerevisiae F33, and "F33 _ YC 32" refers to data obtained from a mixed fermentation using the commercial strains Saccharomyces cerevisiae F33 and Candida zemplina strain YC 32.
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 Candida zephyrae and Saccharomyces cerevisiae is characterized in that the Candida zephyrae and the Saccharomyces cerevisiae are subjected to mixed fermentation.
2. The mixed fermentation process based on Candida zephyrae and Saccharomyces cerevisiae as claimed in claim 1, wherein the Candida zephyrina is Candida zemplina strain YC 32; the preservation number of the Candida zempilina strain YC32 is CCTCC M2021090; preferably, the Saccharomyces cerevisiae refers to Saccharomyces cerevisiae strain F33.
3. The mixed fermentation process based on candida zephyrae and saccharomyces cerevisiae as claimed in claim 1, which is 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: candida zemplina strain YC32, Candida zemplina, 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 mixed fermentation process based on Candida zephyrae and Saccharomyces cerevisiae according to claim 3, wherein the activated strains refer to: activated Candida zemplina strain YC32, or activated Saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate into the secondary fermentation refers to: firstly inoculating the activated Candida zemplina strain YC32 to a substrate for fermentation for 0-96h, preferably 48h, and then inoculating the activated Saccharomyces cerevisiae strain F33 for continuous fermentation;
preferably, the fermentation temperature is 18 ℃ +/-2 ℃;
preferably, the total inoculum size of the activated Candida zemplina strain YC32, 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 Candida zephysalsa and saccharomyces cerevisiae are subjected to mixed fermentation;
the Candida zephyrae refers to Candida zemplina strain YC 32; the preservation number of the Candida zempilina strain YC32 is CCTCC M2021090.
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: candida zemplina strain YC32, Candida zemplina, 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: activated Candida zemplina strain YC32, or activated Saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate into the secondary fermentation refers to: firstly inoculating the activated Candida zemplina strain YC32 to a substrate for fermentation for 0-96h, preferably 48h, and then inoculating the activated Saccharomyces cerevisiae strain F33 for continuous fermentation;
preferably, the fermentation temperature is 18 ℃ +/-2 ℃;
preferably, the total inoculum size of the activated Candida zemplina strain YC32, 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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| VASILEIOS ENGLEZOS，等: "Aroma profile and composition of Barbera wines obtained by mixed fermentations of Starmerella bacillaris (synonym Candida zemplinina) and Saccharomyces cerevisiae", 《LWT - FOOD SCIENCE AND TECHNOLOGY》 * |
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