CN113150898B - Mixed fermentation process based on yeast Naganishia albida and saccharomyces cerevisiae - Google Patents
Mixed fermentation process based on yeast Naganishia albida and saccharomyces cerevisiae Download PDFInfo
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- CN113150898B CN113150898B CN202110189024.6A CN202110189024A CN113150898B CN 113150898 B CN113150898 B CN 113150898B CN 202110189024 A CN202110189024 A CN 202110189024A CN 113150898 B CN113150898 B CN 113150898B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G1/00—Preparation of wine or sparkling wine
- C12G1/02—Preparation of must from grapes; Must treatment and fermentation
- C12G1/0203—Preparation of must from grapes; Must treatment and fermentation by microbiological or enzymatic treatment
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/18—Baker's yeast; Brewer's yeast
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G2200/00—Special features
- C12G2200/05—Use of particular microorganisms in the preparation of wine
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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Abstract
The invention relates to a mixed fermentation process based on yeast Naganishia albida and saccharomyces cerevisiae, belonging to the technical field of microorganisms. The mixed fermentation process based on the yeast Naganishia albida and the saccharomyces cerevisiae is characterized in that the yeast Naganishia albida and the saccharomyces cerevisiae are subjected to mixed fermentation; the yeast Naganishia albida refers to a yeast Naganishia albida strain YC22; the preservation number of the yeast Naganishia albida strain YC22 is CCTCC M2021085. Compared with the single-fungus fermentation of the Saccharomyces cerevisiae F33, the method has very remarkable promotion on a plurality of aroma substances which can influence the flavor of the alcoholic beverage, thereby bringing about unique flavor, aroma and taste of the fermented beverage.
Description
Technical Field
The invention belongs to the technical field of beverage fermentation, and particularly relates to a mixed fermentation process based on yeast Naganishia albida and saccharomyces cerevisiae.
Background
Wine fermentation is a complex biochemical process in which yeast plays a very critical role, for example, in the conversion of sugar to ethanol, carbon dioxide and other thousands of secondary metabolites. A great deal of scientific researches show that the quality of the wine is highly dependent on the metabolic activities and fermentation behaviors of different yeasts, and the different yeasts have important contributions to the chemical composition, the organoleptic properties, the flavor characteristics and the like of the wine. Saccharomyces cerevisiae is the most widely used strain in wine industrial production so far, and has the advantages of ensuring the risk of deterioration in the fermentation process of wine, having good fermentation power, along with the problems of single flavor characteristic, serious homogenization phenomenon and the like. Therefore, in order to pursue style characterization of the wine, the aroma characteristics are more representative, diversified and complex, and brewers often adopt a method of mixed fermentation of 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 the wine.
Non-saccharomyces cerevisiae has become an option to improve wine quality. Numerous studies have shown that non-Saccharomyces cerevisiae is capable of producing enzymes and some of the secondary metabolites we desire, thereby improving wine aroma and flavor characteristics, and controlling the growth of some undesirable species in wine, but has the disadvantage of inadequate fermentation kinetics. The mixing fermentation of non-Saccharomyces cerevisiae and Saccharomyces cerevisiae can not only improve the fragrance diversity and complexity of the wine, but also make up for the problem of insufficient fermentation power of non-Saccharomyces cerevisiae, and is an effective method for improving the fragrance quality of the wine.
The related report of the fermentation by using yeast Naganishia albida is very rare in the field, but regarding the mixed fermentation process of yeast Naganishia albida and saccharomyces cerevisiae, no related report is found in the field.
Disclosure of Invention
Based on the above-mentioned needs and blank in the art, the invention provides a mixed fermentation process based on yeast Naganishia albida and Saccharomyces cerevisiae Saccharomyces cerevisiae, which has very remarkable promotion on a plurality of aroma substances affecting the flavor of alcoholic beverages compared with the single-bacteria fermentation of Saccharomyces cerevisiae.
The technical scheme of the invention is as follows:
a mixed fermentation process based on yeast Naganishia albida and Saccharomyces cerevisiae is characterized in that yeast Naganishia albida and Saccharomyces cerevisiae are subjected to mixed fermentation.
Preferably, the yeast Naganishia albida refers to a yeast Naganishia albida strain YC22; the preservation number of the yeast Naganishia albida strain YC22 is CCTCC M2021085.
Preferably, the Saccharomyces cerevisiae refers to Saccharomyces cerevisiae strain F33.
The mixed fermentation process based on the yeast Naganishia albida and the saccharomyces cerevisiae comprises the following steps of: and inoculating the activated strain and the activated strain into a fermentation substrate for fermentation.
Preferably, the strain refers to: yeast Naganishia albida strain YC22, 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 cultivation time is 20-30h, preferably 24h, and the rotation speed is 120-250rpm, preferably 150rpm;
preferably, the inoculation refers to inoculating the initial strain preservation solution into a culture medium according to an inoculation amount of 2-4% by volume, preferably 3%;
preferably, the medium is YPD medium; preferably, the YPD medium comprises the following components in mass-volume ratio: 0.5-3.5% yeast extract powder preferably 1%, 1.0-3.0% peptone preferably 2%, 1.0-5.0% glucose preferably 2%, and water for the rest;
preferably, the initial strain preservation solution refers to strain preservation in 15-35% glycerol/YPD medium, preferably 25% glycerol/YPD medium;
more preferably, the activation strain is performed 1-3 times, preferably 2 times.
The activated strain refers to: activated yeast Naganishia albida strain YC22, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating the activated yeast Naganishia albida strain YC22 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 yeast Naganishia albida strain YC22 and the activated Saccharomyces cerevisiae strain F33 is 10 6 -10 7 CFU, preferably 6X 10 6 CFU；
Preferably, the fermentation is terminated until the substrate weight loss is no longer changed for 3 consecutive days;
preferably, the substrate is grape juice.
A wine production method is characterized in that grape juice is taken as a substrate, and yeast Naganishia albida and saccharomyces cerevisiae are subjected to mixed fermentation;
the yeast refers to a yeast Naganishia albida strain YC22; the preservation number of the yeast Naganishia albida strain YC22 is CCTCC M2021085.
The saccharomyces cerevisiae refers to saccharomyces cerevisiae strain F33.
The wine production method comprises the following steps: and inoculating the activated strain and the activated strain into a fermentation substrate for fermentation.
Preferably, the strain refers to: yeast Naganishia albida strain YC22, 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 cultivation time is 20-30h, preferably 24h, and the rotation speed is 120-250rpm, preferably 150rpm;
preferably, the inoculation refers to inoculating the initial strain preservation solution into a culture medium according to an inoculation amount of 3% by volume;
preferably, the medium is YPD medium; preferably, the YPD medium comprises the following components in mass-volume ratio: 0.5-3.5% yeast extract powder preferably 1%, 1.0-3.0% peptone preferably 2%, 1.0-5.0% glucose preferably 2%, and water for the rest;
preferably, the initial strain preservation solution refers to strain preservation in 15-35% glycerol/YPD medium, preferably 25% glycerol/YPD medium;
more preferably, the activation strain is performed 1-3 times, preferably 2 times.
The activated strain refers to: activated yeast Naganishia albida strain YC22, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating the activated yeast Naganishia albida strain YC22 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 access of the activated yeast Naganishia albida strain YC22 and the activated Saccharomyces cerevisiae strain F33 is 10 6 -10 7 CFU, preferably 6X 10 6 CFU；
Preferably, the fermentation is terminated after the substrate weight loss has not changed for 3 consecutive days.
The wine production method further comprises the following steps: preparing grape juice;
the preparation of grape juice refers to: squeezing grape fruit at low temperature;
preferably, the harvested grape fruit particles are pressed with ice by using an air bag press;
preferably, the sulfur dioxide or K is added at the same time by pressing 2 S 2 O 5 And, pectase;
preferably sulfur dioxide or K 2 S 2 O 5 The addition amount of (C) is 30-100mg/L, preferably 50mg/L; the addition amount of pectase is 10-30mg/L, preferably 20mg/L;
preferably, the grape fruit particles are uniform-sized fruit particles;
preferably, the grape fruit particles are grape fruit particles harvested after the grape is ripe and the temperature is reduced to below-8 ℃ for 24 hours.
The wine is characterized by being produced by the wine production method.
According to the invention, yeast Naganishia albida and saccharomyces cerevisiae are adopted for mixed fermentation, and among dozens of aroma substances produced, the yield of most aroma substances is improved, for example, about 47% of 9-decenoic acid is improved, 50% of glacial acetic acid is improved, 34% of isobutanol is improved, 33% of n-hexanol is improved, 3R, 3-butanediol is improved by 1.71 times, 3.23 times of 1, 3-propanediol monoethyl ether is improved, 3.8 times of 3-hydroxy-2-butanone is improved, 2.73 times of vinyl acetate is improved, 1.5 times of Ma Shitong is improved, 1.8 times of myrcene is improved, 14% of linalool is improved, 36.4% of citronellol is improved, 53% of linalool is improved, and 31% of 2, 6-di (tert-butyl) -4-hydroxy-4-methyl-2, 5-cyclohexadiene-1-ketone is improved; at the same time, aroma substances which cannot be produced by the single-strain fermentation of Saccharomyces cerevisiae are also produced, for example: 2-benzyl propionic acid, 3-methyl-1-pentanol, 2-ethylhexanol, propylene glycol, 3, 4-dimethyl-2-hexanol, sec-octanol, (Z) -3-methyl-2-hepten-1-ol, nonanal, ethyl octanoate, ethyl decanoate, ethyl pyruvate, acetone, 2, 3-butanedione, isopentyl hexanoate, isopentyl decanoate, 3, 5-trimethyl-1-hexene, alpha-terpineol, 2, 3-pentanedione, 1, 7-trimethyl [2.2.1] hept-2-ene, isophorone. The production of the aroma substances or the improvement of the output of the aroma substances can generate certain influence on the flavor and aroma of the beverage obtained by fermentation, so that the mixed bacteria fermentation product presents more unique aroma compared with a single bacteria fermentation product, and the wine beverage with unique flavor, aroma and taste can be produced, so that the consumer product is enriched, and the consumption selection is enlarged.
Detailed Description
The following describes the invention in more detail with reference to specific examples, but is not intended to limit the scope of the invention.
Sources and documentations of biological materials
The yeast Naganishia albida strain YC22 used in the experimental example is a new strain screened by the applicant laboratory, and the preservation information is as follows:
naming: YC22
Classification name: yeast
Latin name: naganishia albida
Deposit number: CCTCC M2021085
Preservation mechanism: china center for type culture Collection
Preservation date: 2021, 1-15;
deposit unit address: in the Jiuqiu 299 Wuhan university (first appendage of Wuhan university) in Wuhan City, hubei province, wuhan university collection center.
Saccharomyces cerevisiae F33 is a commercial strain available from Laffort, inc.
The grape variety used was Wedelian iced grape, purchased from Ningxia Bug Ge Zuimei International wine village Co.
Group 1 example, mixed bacteria fermentation Process of the invention
The embodiment of the group provides a mixed fermentation process based on yeast Naganishia albida and saccharomyces cerevisiae. All embodiments of this group share the following common features: mixing yeast Naganishia albida and Saccharomyces cerevisiae for fermentation.
Those skilled in the art can use yeast Naganishia albida in combination with Saccharomyces cerevisiae to perform fermentation and production according to the teachings of the present invention, and any such actions fall within the scope of the present invention. Target products of fermentation include, but are not limited to: alcoholic beverages, fermented milk, bread, etc.
In a preferred embodiment, the yeast Naganishia albida refers to the yeast Naganishia albida strain YC22; the preservation number of the yeast Naganishia albida strain YC22 is CCTCC M2021085.
In a specific embodiment, the saccharomyces cerevisiae refers to 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, and besides F33, there are various commercial strains on the market, for example Saccharomyces cerevisiae V1116, saccharomyces cerevisiae VL1, saccharomyces cerevisiae X16, etc., which can be used for mixed fermentation with the yeast Naganishia albida strain YC22 of the present invention, so as to obtain similar technical effects as those of the present invention.
In some embodiments, the yeast Naganishia albida and saccharomyces cerevisiae-based mixed fermentation process comprises the following steps: and inoculating the activated strain and the activated strain into a fermentation substrate for fermentation.
Preferably, the strain refers to: yeast Naganishia albida strain YC22, 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 cultivation time is 20-30h, preferably 24h, and the rotation speed is 120-250rpm, preferably 150rpm;
preferably, the inoculation refers to inoculating the initial strain preservation solution into a culture medium according to an inoculation amount of 2-4% by volume, preferably 3%;
preferably, the medium is YPD medium; preferably, the YPD medium comprises the following components in mass-volume ratio: 0.5-3.5% yeast extract powder preferably 1%, 1.0-3.0% peptone preferably 2%, 1.0-5.0% glucose preferably 2%, and water for the rest;
preferably, the initial strain preservation solution refers to strain preservation in 15-35% glycerol/YPD medium, preferably 25% glycerol/YPD medium;
more preferably, the activation strain is performed 1-3 times, preferably 2 times.
In other embodiments, the activated strain refers to: activated yeast Naganishia albida strain YC22, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating the activated yeast Naganishia albida strain YC22 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 access of the activated yeast Naganishia albida strain YC22 and the activated Saccharomyces cerevisiae strain F33 is 10 6 -10 7 CFU, preferably 6X 10 6 CFU；
Preferably, the fermentation is terminated until the substrate weight loss is no longer changed for 3 consecutive days;
preferably, the substrate is grape juice.
Group 2 example, method of producing wine according to the invention
The present set of embodiments provides a wine production method. The present set of embodiments all share the following common features: mixing and fermenting yeast Naganishia albida and Saccharomyces cerevisiae with grape juice as substrate.
In some preferred embodiments, the yeast Naganishia albida refers to the yeast Naganishia albida strain YC22; the preservation number of the yeast Naganishia albida strain YC22 is CCTCC M2021085.
In a specific embodiment, the saccharomyces cerevisiae refers to saccharomyces cerevisiae strain F33.
In other specific embodiments, the wine production method comprises: and inoculating the activated strain and the activated strain into a fermentation substrate for fermentation.
Preferably, the strain refers to: yeast Naganishia albida strain YC22, 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 cultivation time is 20-30h, preferably 24h, and the rotation speed is 120-250rpm, preferably 150rpm;
preferably, the inoculation refers to inoculating the initial strain preservation solution into a culture medium according to an inoculation amount of 2-4% by volume, preferably 3%;
preferably, the medium is YPD medium; preferably, the YPD medium comprises the following components in mass-volume ratio: 0.5-3.5% yeast extract powder preferably 1%, 1.0-3.0% peptone preferably 2%, 1.0-5.0% glucose preferably 2%, and water for the rest;
preferably, the initial strain preservation solution refers to strain preservation in 15-35% glycerol/YPD medium, preferably 25% glycerol/YPD medium;
more preferably, the activation strain is performed 1-3 times, preferably 2 times.
In specific embodiments, the activated strain refers to: activated yeast Naganishia albida strain YC22, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating the activated yeast Naganishia albida strain YC22 to a substrate for fermentation for 0-96h, preferably 48h, and then inoculating the activated saccharomyces cerevisiae strain F33 for continuous fermentation;
when the activated yeast Naganishia albida strain YC22 is inoculated to a substrate for fermentation for 0h, the surface activated yeast Naganishia albida strain YC22 and the activated Saccharomyces cerevisiae strain F33 can be simultaneously inoculated to the substrate for fermentation until the substrate weight loss is continuously changed for 3 days, and the fermentation is stopped.
Preferably, the fermentation temperature is 18 ℃ ± 2 ℃;
preferably, the total access of the activated yeast Naganishia albida strain YC22 and the activated Saccharomyces cerevisiae strain F33 is 10 6 -10 7 CFU, preferably 6X 10 6 CFU；
Preferably, the fermentation is terminated after the substrate weight loss has not changed for 3 consecutive days.
In a further embodiment, the wine production method further comprises: preparing grape juice;
the preparation of grape juice refers to: squeezing grape fruit at low temperature;
preferably, the harvested grape fruit particles are pressed with ice by using an air bag press;
preferably, the sulfur dioxide or K is added at the same time by pressing 2 S 2 O 5 And, pectase;
preferably, the addition amount of sulfur dioxide or K2S2O5 is 30-100mg/L, preferably 50mg/L; the addition amount of pectase is 10-30mg/L, preferably 20mg/L;
preferably, the grape fruit particles are uniform-sized fruit particles;
preferably, the grape fruit particles are grape fruit particles harvested after the grape is ripe and the temperature is reduced to below-8 ℃ for 24 hours.
Group 3 example, wine of the invention
The present set of embodiments provides a wine. All embodiments of this group share the following common features: the wine produced by the method of any one of examples in group 2.
The wine of the invention produces aroma substances which cannot be produced by the following single-fungus fermented wine: 2-benzyl propionic acid, 3-methyl-1-pentanol, 2-ethylhexanol, propylene glycol, 3, 4-dimethyl-2-hexanol, sec-octanol, (Z) -3-methyl-2-hepten-1-ol, nonanal, ethyl octanoate, ethyl decanoate, ethyl pyruvate, acetone, 2, 3-butanedione, isopentyl hexanoate, isopentyl decanoate, 3, 5-trimethyl-1-hexene, alpha-terpineol, 2, 3-pentanedione, 1, 7-trimethyl-e [2.2.1] hept-2-ene, isophorone, while being much higher in the content of the following aroma substances than the single fermented wine: 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, large Ma Shitong, myrcene, linalool, citronellol, linalool, 2, 6-di (tert-butyl) -4-hydroxy-4-methyl-2, 5-cyclohexadien-1-one.
Experimental example, mixed bacteria fermentation process and fermentation data of the invention
1. Strain
The strains used in this experiment were: commercial s.cerevisiae F33 and the isolated and screened yeast Naganishia albida strain YC22 of the present invention.
2. Grape juice
The Weidale iced grape raw material was planted in Yinchuan Yongning county, yinchuang county, ningxia Bagues United states wine village, inc. (E106.02, N38.24). The grape vine is planted in 2013, the grape vine is cultivated by adopting a small shed frame, the plant row spacing is 1.0m multiplied by 2.0m, the grape vine is harvested in 2017, the grape vine is not harvested after being ripe, the grape vine is harvested when the temperature is reduced to be lower than-8 ℃ continuously for 24 hours, small fruit grains are removed, and the ice grape fruits with the same size are randomly selected and squeezed at low temperature. The harvested iced grapes are pressed with ice through an air bag press, and sulfur dioxide (50 mg/L K) 2 S 2 O 5 ) And 20mg/L pectase (more than or equal to 500U/mg), inhibit bacteria and increase juice yield. Obtained by squeezingGlucose content of grape juice 432g/dm 3 Acidity 4.65g/dm 3 (tartaric acid) pH 4.21.
3. Fermentation operation
2 strains: saccharomyces cerevisiae F33 strain, saccharomyces Naganishia albida strain YC22 were all stored in 25% glycerol/YPD medium by volume prior to use. YPD medium was 1% yeast extract, 2% peptone, 2% glucose. Inoculating the bacterial liquid according to the inoculum size of 3% of the volume ratio into a 50mL triangular flask filled with 40mL of YPD culture medium and a 250mL triangular flask filled with 150mL of YPD culture medium respectively, wherein the culture temperature is 28 ℃, the rotation speed is 150rpm, the culture time is 24 hours, the bacterial liquid activated for the 1 st time is obtained, then inoculating the bacterial liquid according to the inoculum size of 3% into the 50mL triangular flask filled with 40mL of YPD culture medium and the 250mL triangular flask filled with 150mL of YPD culture medium respectively, and repeating the culture to finish the 2 nd passage activation, thus obtaining the bacterial liquid after activation. Firstly, inoculating the activated yeast Naganishia albida strain YC22 into the collected grape juice, after 48 hours, inoculating the strain S.cerevisiae F33 according to the inoculation proportion of YC22/F33 of 2:1, wherein the total inoculation amount of the strain is controlled to be 6 multiplied by 10 6 CFU, using s.cerevisiae F33 pure fermented grape juice as blank control, fermenting at 18±2 ℃ until grape juice weight loss is no longer changed for three consecutive days, stopping fermentation. All wine samples were centrifuged at 7500rpm for 8 minutes and the supernatant was stored at 4 ℃.
4. Method for quantifying aroma substances
A headspace-solid phase microextraction method-gas phase mass spectrometry (HS-SPME-GC/MS) is adopted. An accurate measurement of 8mL of wine sample was added to a headspace bottle containing 1.5g NaCl, while 394.08. Mu.g/L4-methyl-1-pentanol (internal standard) was capped and sealed. The CAR/DVB/PDMS extraction fiber is inserted, the extraction fiber is desorbed for 3min at 250 ℃ at the GC inlet after being adsorbed for 30min at 45 ℃ for GC-MS analysis. Chromatographic column: an InertCap WAX polar column (60 m 0.25mm,0.25 μm); the temperature-raising program is as follows: keeping the temperature at 40 ℃ for 5min, raising the temperature to 120 ℃ at 3 ℃/min, raising the temperature to 230 ℃ at 8 ℃/min, and keeping the temperature for 10min; the carrier gas (He) flow rate was 0.8mL/min, without split flow. An electron bombardment ion source; electron energy 70eV; the temperature of the transmission line is 275 ℃; the ion source temperature is 230 ℃; the activation voltage is 1.5V; filament flow 0.25mA; mass scanning range m/z is 33-450. Compound quantitative analysis was performed using an external standard quantitative method.
5. Data analysis method
All samples were averaged 3 times in parallel. 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); partial least squares analysis was performed by un crambler 9.7 (CAMO ASA, norway).
The final statistical treatment gave the following Table 1, in units of μg/L, meaning: the aroma content per liter of wine.
TABLE 1
The fragrance threshold in the above table 1 means the lowest concentration lower limit value at which a human can sniff the substance, and the fragrance description and threshold are based on the reports of the related documents in which the fragrance description and threshold of the fragrance substance are described, and the fragrance substance without fragrance description and threshold in the table is a document in which the related document about the fragrance description and threshold of the substance is not searched. "F33" in the table indicates data of single-strain fermentation using a commercial strain of Saccharomyces cerevisiae F33, and "F33_YC22" indicates data of mixed-strain fermentation using a commercial strain of Saccharomyces cerevisiae F33 and a yeast Naganishia albida strain YC22.
As known in the fermentation field, the factors influencing the fermentation are numerous and complex, and the components of the fermented product can be changed due to the changes of factors such as raw material batch, raw material components, fermentation conditions, temperature, time and the like, so that the situation that single-bacteria fermentation is higher than mixed-bacteria fermentation on certain aroma components can also occur, which belongs to the normal phenomenon in the field.
Claims (16)
1. Based on Cryptococcus shallowusNaganishia albida) And Saccharomyces cerevisiae, and is characterized in that,
comprising the following steps: preparing grape juice, activating strain, activating the activated cryptococcus shallowiiNaganishia albida) The strain YC22 and the saccharomyces cerevisiae strain F33 are inoculated into a substrate to be fermented for fermentation; the Cryptococcus shallowus isNaganishia albida) The preservation number of the strain YC22 is CCTCC M2021085;
the fermentation by inoculating the substrate to be fermented means: activated cryptococcus shallowii is first treatedNaganishia albida) Inoculating the strain YC22 to substrate grape juice for fermentation for 48-96h, and inoculating activated saccharomyces cerevisiae strain F33 for continuous fermentation;
the fermentation temperature is 18+/-2 ℃;
activated cryptococcus shallowiiNaganishia albida) The total inoculum size of strain YC22 and activated Saccharomyces cerevisiae strain F33 was 10 6 -10 7 CFU；
Stopping fermentation when the weight loss of the substrate grape juice is not changed for 3 days;
the preparation of grape juice refers to: squeezing grape fruit particles with uniform size with air bag squeezer with ice, and adding sulfur dioxide and pectase or K 2 S 2 O 5 And pectinase;
sulfur dioxide or K 2 S 2 O 5 The addition amount of the (B) is 30-100mg/L; the addition amount of pectase is 10-30mg/L;
the grape fruit particles are the grape fruit particles which are harvested when the temperature is reduced to below-8 ℃ for 24 hours after the ripening of the Weibull iced grape.
2. A method based on Cryptococcus shallowus as defined in claim 1Naganishia albida) The mixed fermentation process of the saccharomyces cerevisiae is characterized in that the cryptococcus shallowii is%Naganishia albida) The bacterial strain YC22 is inoculated on a substrate for fermentation for 48 hours, and the leucococcus shallowii is used for preparing the bacterial strain YC22Naganishia albida) The total inoculum size of strain YC22 and activated Saccharomyces cerevisiae strain F33 was 6X 10 6 CFU; sulfur dioxide or K 2 S 2 O 5 The addition amount of (2) is 50mg/L; the addition amount of pectase is 20mg/L.
3. A method based on Cryptococcus shallowus as defined in claim 1Naganishia albida) And a mixed fermentation process of saccharomyces cerevisiae, which is characterized in that the activated strain is inoculated in a culture medium for culture.
4. A method based on Cryptococcus shallowus as claimed in claim 3Naganishia albida) And a mixed fermentation process of Saccharomyces cerevisiae, wherein the culture temperature is 24-30 ℃; the culture time is 20-30h, and the rotating speed is 120-250rpm.
5. A method based on Cryptococcus shallowus as claimed in claim 3Naganishia albida) And a mixed fermentation process of saccharomyces cerevisiae, wherein the culture temperature is 28 ℃; the incubation time was 24 hours and the rotational speed was 150rpm.
6. A method based on Cryptococcus shallowus as claimed in claim 3Naganishia albida) And a mixed fermentation process of saccharomyces cerevisiae, which is characterized in that the inoculation means that the initial strain preservation solution is inoculated into a culture medium according to the inoculation amount of 2-4% of the volume ratio.
7. The method of claim 6 based on Cryptococcus shallowusNaganishia albida) And a mixed fermentation process of saccharomyces cerevisiae, which is characterized in that the inoculation means that an initial strain preservation solution is inoculated into a culture medium according to an inoculation amount of 3% by volume.
8. A method according to any one of claims 3, 6 and 7 based on cryptococcus shallowiiNaganishia albida) And a mixed fermentation process of saccharomyces cerevisiae, wherein the culture medium is YPD culture medium.
9. The method of claim 8 based on Cryptococcus shallowusNaganishia albida) And a saccharomyces cerevisiae mixed fermentation process, which is characterized in that the YPD culture medium comprises the following components in mass volume ratio: 0.5-3.5% yeast extract powder, 1.0-3.0% peptone, 1.0-5.0% glucose, and water in balance.
10. A method based on Cryptococcus shallowus according to claim 9Naganishia albida) And a saccharomyces cerevisiae mixed fermentation process, which is characterized in that the YPD culture medium comprises the following components in mass volume ratio: 1% of yeast extract powder, 2% of peptone, 2% of glucose and the balance of water.
11. A method according to claim 6 or 7 based on Cryptococcus shallowusNaganishia albida) And a mixed fermentation process of saccharomyces cerevisiae, wherein the initial strain preservation solution means that the strain is preserved in a glycerol/YPD culture medium with the volume ratio of 15-35%.
12. A method based on Cryptococcus shallowus as defined in claim 11Naganishia albida) And a mixed fermentation process of saccharomyces cerevisiae, wherein the initial strain preservation solution means that the strain is preserved in a glycerol/YPD culture medium with a volume ratio of 25%.
13. A method based on Cryptococcus shallowus as defined in claim 11Naganishia albida) And a mixed fermentation process of Saccharomyces cerevisiae, wherein the activated strain is performed 1-3 times.
14. A method based on Cryptococcus shallowus as defined in claim 13Naganishia albida) And a mixed fermentation process of saccharomyces cerevisiae, wherein the activated strain is performed 2 times.
15. A process for the production of wine, characterised in that it comprises the steps of any one of claims 1 to 14The preparation method is based on Cryptococcus shallowusNaganishia albida) And mixing fermentation of Saccharomyces cerevisiae with grape juice as substrate.
16. Wine produced by the wine production method according to claim 15.
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