CN112980705B - Combined fermentation process based on klatomyces, hansenula polymorpha and saccharomyces cerevisiae - Google Patents
Combined fermentation process based on klatomyces, hansenula polymorpha and saccharomyces cerevisiae Download PDFInfo
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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
- 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
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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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- 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
- C12G1/04—Sulfiting the must; Desulfiting
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- 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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- 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
- C12G2200/00—Special features
- C12G2200/05—Use of particular microorganisms in the preparation of wine
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Abstract
The invention discloses a combined fermentation process based on klatomyces, hansenula polymorpha and saccharomyces cerevisiae, and belongs to the technical field of microorganisms. The combined fermentation process based on the klandia, the grape juice Hansenula polymorpha and the saccharomyces cerevisiae is characterized in that the klandia, the grape juice Hansenula polymorpha and the saccharomyces cerevisiae are adopted for fermentation. The invention combines the Clarithromycin yeast, the grape juice Hansenula polymorpha and the Saccharomyces cerevisiae to perform combined fermentation, and the fermentation product presents a unique flavor compared with a single-fungus fermentation product, so that a wine beverage with unique flavor, fragrance and taste can be produced, the consumer product is enriched, and the consumption selection is enlarged.
Description
Technical Field
The invention belongs to the technical field of food fermentation, and particularly relates to a combined fermentation process based on klandia clarithromycin, hansenula polymorpha and saccharomyces cerevisiae.
Background
Wine fermentation is a complex biochemical process in which klatomyces plays a very critical role, for example, the conversion of sugar into 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 activity and fermentation behavior of different klatomyces, and the different klatomyces 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 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 native klatomyces 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 reports of fermentation using klandia Saccharomy copsiscrataegensis are very rare in the art, but no related report is found in the art at present regarding the combined fermentation process of klandia Saccharomy copsiscrataegensis, hansenula grape Hanseniaspora uvarum and saccharomyces cerevisiae.
Disclosure of Invention
Based on the above-mentioned needs and blank in the art, the invention provides a combined fermentation process based on Klatong yeast Saccharomy copsiscrataegensis, hansenula polymorpha Hanseniaspora uvarum and Saccharomyces cerevisiae, which has very remarkable improvement on a plurality of aroma substances affecting the flavor of alcoholic beverages compared with the conventional Saccharomyces cerevisiae fermentation.
The technical scheme of the invention is as follows:
A combined fermentation process based on klandia and grape juice Hansenula polymorpha is characterized in that the fermentation is carried out by using the klandia, the grape juice Hansenula polymorpha and the saccharomyces cerevisiae.
The Clarithromycin yeast Saccharomyces cerevisiae Saccharomy copsiscrataegensis strain YC30; the preservation number of the Clarithromycin strain Saccharomy copsiscrataegensis YC30 is CCTCC M2021089.
The Hansenula polymorpha refers to Hansenula polymorpha Hanseniaspora uvarum strain QTX22; the preservation number of the Hansenula polymorpha Hanseniaspora uvarum strain QTX22 is CCTCC M2021083;
preferably, the saccharomyces cerevisiae is saccharomyces cerevisiae strain F33.
The fermentation process for adding amino acid to a substrate and based on klatothrough yeast comprises the following steps: activating the strain, and inoculating the activated strain into a substrate to be fermented for fermentation;
Preferably, the activated strain refers to a strain inoculated in a culture medium for cultivation.
The strain is selected from the group consisting of: klatomyces Saccharomy copsiscrataegensis strain YC30, hansenula grape juice Hanseniaspora uvarum strain QTX22, saccharomyces cerevisiae strain F33;
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%, preferably 1%, of a klatose yeast extract, 1.0-3.0%, preferably 2%, of peptone, 1.0-5.0%, preferably 2%, glucose, the remainder being water;
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.
Preferably, the activated strain means: activated klatomyces Saccharomy copsiscrataegensis strain YC30, or activated hansenula polymorpha Hanseniaspora uvarum strain QTX22, or activated saccharomyces cerevisiae strain F33;
Preferably, the fermentation temperature is 18 ℃ ± 2 ℃;
Preferably, the inoculum size of the activated strain in the substrate is 10 6-107 CFU, preferably 6×10 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 method for producing grape wine is characterized in that grape juice is taken as a substrate, and Clarithromycin yeast and Hansenula polymorpha are inoculated for fermentation.
The Clarithromycin yeast Saccharomyces cerevisiae Saccharomy copsiscrataegensis strain YC30; the preservation number of the Clarithromycin yeast Saccharomy copsiscrataegensis strain YC30 is CCTCC M2021089;
preferably, the Hansenula polymorpha refers to Hansenula polymorpha Hanseniaspora uvarum strain QTX22; the preservation number of the Hansenula polymorpha Hanseniaspora uvarum strain QTX22 is CCTCC M2021083;
Preferably, the wine production method comprises the following steps: and inoculating the activated strain and the activated strain into a substrate to be fermented for fermentation.
The strain is selected from the group consisting of: klatomyces Saccharomy copsiscrataegensis strain YC30, hansenula grape juice Hanseniaspora uvarum strain QTX22, 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%, preferably 1%, of a klatose yeast extract, 1.0-3.0%, preferably 2%, of peptone, 1.0-5.0%, preferably 2%, glucose, the remainder being water;
preferably, the initial strain preservation solution refers to a strain preserved in 15-35% glycerol/YPD medium, preferably 25% glycerol/YPD medium by volume;
More preferably, the activated strain is performed 1-3 times, preferably 2 times;
Preferably, the activated strain means: activated klatomyces Saccharomy copsiscrataegensis strain YC30, or activated hansenula polymorpha Hanseniaspora uvarum strain QTX22, or activated saccharomyces cerevisiae strain F33;
Preferably, the activated klatomyces Saccharomy copsiscrataegensis strain YC30, the activated Hansenula polymorpha Hanseniaspora uvarum strain QTX22 and the activated Saccharomyces cerevisiae strain F33 are simultaneously inoculated into a substrate to be fermented for fermentation;
Preferably, the fermentation temperature is 18 ℃ ± 2 ℃;
preferably, the total access of activated klandia glabrata Saccharomy copsiscrataegensis strain YC30 and activated hansenula polymorpha Hanseniaspora uvarum strain QTX22 is 10 6-107 CFU, preferably 6 x 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 pressing is performed with the addition of sulfur dioxide or K 2S2O5, and, pectase;
preferably, the addition amount of sulfur dioxide or K 2S2O5 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, the substrate is simultaneously inoculated with the klpastoris Saccharomy copsiscrataegensis, the hansenula polymorpha Hanseniaspora uvarum and the saccharomyces cerevisiae strain F33 for combined fermentation, and compared with the single use of the commercial saccharomyces cerevisiae for fermentation, the yield of a plurality of aroma substances is improved, for example, the yield of glacial acetic acid is improved by 49%, the yield of octanoic acid is improved by 11 times, the yield of ethyl caproate is improved by 12.6 times, the yield of citronellol is improved by 11.5 times, the yield of octanol is improved by 1 time, the yield of 4-vinyl-2-methoxyphenol is improved by 72%, the yield of ethyl palmitate is improved by 4.67 times, the yield of ethyl acetate is improved by 10.86 times, and the yield of ethyl myristate is improved by 25.8%; 36 times of 1, 3-propylene glycol monoethyl ether, 1.58 times of benzyl alcohol, 41.8 times of linalool, 9 times of 9-decenoic acid, 2 times of caproic acid, 8.64 times of 2, 4-di-tert-butylphenol, 8.1 times of ethyl acetate, 12.4 times of 2-methylbutyric acid, 17.3 times of diethyl succinate, 32 times of ethyl lactate, 18 times of alpha-terpineol and 1.58 times of 2-ethylhexanol; the combined fermentation process of the present invention also produces aroma substances that cannot be produced by fermentation using commercial Saccharomyces cerevisiae alone, for example: ethyl isobutyrate, linalool, ethyl 2-furoate, isoamyl octanoate, butyric acid, 3-hydroxy-2-butanone, (2S-cis) -tetrahydro4-methyl-2- (2-methyl-1-propenyl) -2H-pyran, 1-decanol, isovaleraldehyde, dodecanol, trans-3-hexen-1-ol, ethyl 3-phenylpropionate. 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 Clarithromycin yeast Saccharomy copsiscrataegensis strain YC30 used in the experimental examples was a new strain screened by the applicant laboratory, and the preservation information is as follows:
naming: YC30
Classification name: claritonas sp
Latin name: saccharomy copsiscrataegensis A
Deposit number: CCTCC M2021089
Preservation mechanism: china center for type culture Collection
Preservation date: 2021, 1-15;
the Hansenula polymorpha Hanseniaspora uvarum strain QTX22 used in the experimental example is a new strain screened by the applicant laboratory, and the preservation information is as follows:
naming: QTX22
Classification name: hansenula polymorpha of grape juice
Latin name: hanseniaspora uvarum A
Deposit number: CCTCC M2021083
Preservation mechanism: china center for type culture Collection
Preservation date: 2021, 1-15;
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.
Example 1 group, substrate-added amino acid fermentation Process of the invention
The present set of examples provides a combined fermentation process based on klandia and hansenula polymorpha. All embodiments of this group share the following common features: fermenting with Cladonia yeast, hansenula polymorpha, and Saccharomyces cerevisiae.
Those skilled in the art can, in light of the teachings of the present invention, utilize Cladonia, hansenula polymorpha and Saccharomyces cerevisiae for fermentation and production activities that fall within the scope of the present invention. Target products of fermentation include, but are not limited to: alcoholic beverages, dairy products, pasta, and the like.
Such dairy products include, but are not limited to, yogurt, fermented milk, milk drinks, and the like; the flour product includes but is not limited to: bread, cake, steamed stuffed bun, steamed bread, steamed roll, etc.
In a preferred embodiment, the Clarithromycin yeast refers to Clarithromycin yeast Saccharomy copsiscrataegensis strain YC30; the preservation number of the Clarithromycin strain Saccharomy copsiscrataegensis YC30 is CCTCC M2021089.
In other embodiments, the hansenula polymorpha refers to hansenula polymorpha Hanseniaspora uvarum strain QTX22; the preservation number of the Hansenula polymorpha Hanseniaspora uvarum strain QTX22 is CCTCC M2021083.
In some preferred embodiments, the Saccharomyces cerevisiae refers to Saccharomyces cerevisiae strain F33, commercially available.
In a further embodiment, the process for fermenting a substrate with amino acids and based on klatomyces comprises the steps of: activating the strain, and inoculating the activated strain into a substrate to be fermented for fermentation;
preferably, the activated strain refers to a strain inoculated in a culture medium for culture;
In a preferred embodiment, the strain is selected from the group consisting of: klatomyces Saccharomy copsiscrataegensis strain YC30, hansenula grape juice Hanseniaspora uvarum strain QTX22, saccharomyces cerevisiae strain F33;
The invention not only verifies the combined fermentation effect of the klthrough yeast Saccharomy copsiscrataegensis strain, the Hansenula polymorpha Hanseniaspora uvarum strain and the Saccharomyces cerevisiae strain, but also verifies the single fermentation effect of the commercial Saccharomyces cerevisiae strain, compared with the single fermentation of the commercial Saccharomyces cerevisiae strain, the combined fermentation of the klthrough yeast Saccharomy copsiscrataegensis strain and the Hansenula polymorpha Hanseniaspora uvarum strain can obtain higher aroma substance yield, so that according to the teaching of the invention, a person skilled in the art can select other commercial strains to combine with the klthrough yeast Saccharomy copsiscrataegensis strain and the Hansenula polymorpha Hanseniaspora uvarum strain for fermentation, and besides F33, various commercial strains such as ,Saccharomyces cerevisiae V1116、Saccharomyces cerevisiae VL1、Saccharomyces cerevisiae X16、Saccharomyces cerevisiae ST and the like exist on the market at present, and can be used for inoculating the substrate added with amino acid for fermentation, and the similar technical effect is expected.
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%, preferably 1%, of a klatose yeast extract, 1.0-3.0%, preferably 2%, of peptone, 1.0-5.0%, preferably 2%, glucose, the remainder being water;
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.
Preferably, the activated strain means: activated klatomyces Saccharomy copsiscrataegensis strain YC30, or activated hansenula polymorpha Hanseniaspora uvarum strain QTX22, or activated saccharomyces cerevisiae strain F33;
Preferably, the fermentation temperature is 18 ℃ ± 2 ℃;
Preferably, the inoculum size of the activated strain in the substrate is 10 6-107 CFU, preferably 6×10 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: taking grape juice as a substrate, and inoculating the Clarithromycin yeast, the Hansenula polymorpha and the Saccharomyces cerevisiae to ferment.
In a preferred embodiment, the Clarithromycin yeast refers to Clarithromycin yeast Saccharomy copsiscrataegensis strain YC30; the preservation number of the Clarithromycin strain Saccharomy copsiscrataegensis YC30 is CCTCC M2021089.
In other embodiments, the hansenula polymorpha refers to hansenula polymorpha Hanseniaspora uvarum strain QTX22; the preservation number of the Hansenula polymorpha Hanseniaspora uvarum strain QTX22 is CCTCC M2021083.
In some preferred embodiments, the saccharomyces cerevisiae refers to saccharomyces cerevisiae strain F33.
In other embodiments, the method of producing wine comprises: adding amino acid, activated strain and activated strain into grape juice, and inoculating the strain after activation into a fermentation substrate for fermentation;
In some embodiments, the strain is selected from the group consisting of: klatomyces Saccharomy copsiscrataegensis strain YC30, hansenula grape juice Hanseniaspora uvarum strain QTX22, 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%, preferably 1%, of a klatose yeast extract, 1.0-3.0%, preferably 2%, of peptone, 1.0-5.0%, preferably 2%, glucose, the remainder being water;
preferably, the initial strain preservation solution refers to a strain preserved in 15-35% glycerol/YPD medium, preferably 25% glycerol/YPD medium by volume;
More preferably, the activated strain is performed 1-3 times, preferably 2 times;
preferably, the activated strain means: activated klatomyces Saccharomy copsiscrataegensis strain YC30, or activated saccharomyces cerevisiae strain F33, or activated hansenula polymorpha Hanseniaspora uvarum strain QTX22;
Preferably, the fermentation temperature is 18 ℃ ± 2 ℃;
preferably, the total access of activated klatomyces Saccharomy copsiscrataegensis strain YC30 and activated hansenula polymorpha Hanseniaspora uvarum strain QTX22 and activated saccharomyces cerevisiae strain F33 is 10 6-107 CFU, preferably 6 x 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 pressing is performed with the addition of sulfur dioxide or K 2S2O5, and, pectase;
preferably, the addition amount of sulfur dioxide or K 2S2O5 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: ethyl isobutyrate, linalool, ethyl 2-furoate, isoamyl octanoate, butyric acid, 3-hydroxy-2-butanone, (2S-cis) -tetrahydro4-methyl-2- (2-methyl-1-propenyl) -2H-pyran, 1-decanol, isovaleraldehyde, dodecanol, trans-3-hexen-1-ol, ethyl 3-phenylpropionate.
Experimental example, mixed bacteria fermentation process and fermentation data of the invention
1. Strain
The strains used in this experiment were: commercial Saccharomyces cerevisiae F33, clarithromycin strain Saccharomy copsiscrataegensis YC30 isolated and screened according to the present invention, hansenula polymorpha Hanseniaspora uvarum grape juice QTX22.
2. Grape juice
The Weidale ice grape raw material is planted in Yinchuan Yongning county Yuquan Yinchun of Ningxia Hui nationality, ningxia Bagues United states wine village Co., ltd (E106.02 degrees, N38.24 degrees). 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 2S2O5) and 20mg/L pectase (more than or equal to 500U/mg) are added simultaneously to inhibit bacteria and improve juice yield. The pressed grape juice had a sugar content of 432g/dm 3, an acidity of 4.65g/dm 3 (tartaric acid) and a pH of 4.21.
3. Fermentation operation
3 Strains: saccharomyces cerevisiae F33 strain, clarithromycin strain Saccharomy copsiscrataegensis strain YC30, hansenula grape strain Hanseniaspora uvarum strain QTX22 were all stored in 25% glycerol/YPD medium by volume prior to use. YPD medium was 1% Kratoria 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. The activated klthrough yeast Saccharomy copsiscrataegensis strain YC30, activated grape juice Hansenula polymorpha Hanseniaspora uvarum strain QTX22 and activated saccharomyces cerevisiae strain F33 are simultaneously inoculated into the collected grape juice, the total inoculation amount of the strains is controlled to be 6 multiplied by 10 6 CFU, the grape juice which is purely fermented by S.cerevisiae F33 is used as a blank control, the fermentation temperature is 18+/-2 ℃, and the fermentation is stopped when the grape juice weight loss is not changed any more three days continuously. All wine samples were centrifuged at 7500rpm for 8 minutes and the supernatant was stored at 4 ℃.
The combined fermentation of the klandia Saccharomy copsiscrataegensis strain YC30, the Hansenula polymorpha Hanseniaspora uvarum strain QTX22 and the Saccharomyces cerevisiae strain F33 is carried out for 3 times of parallel fermentation treatment, and the single-strain fermentation of S.cerevisiae F33 is carried out for 3 times of parallel fermentation treatment.
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/L of 4-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: inertCap WAX polarity chromatography 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
The fermented product obtained by each parallel fermentation treatment in the 3 rd part is used as different samples to respectively measure the aroma substance content, and each sample is respectively measured in parallel for 3 times to obtain an average value. 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); 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 aroma content of grape juice fermentation products
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. The meaning of each label in the header is listed below:
Sc+f33+hu: the average value of the aroma substance content of fermentation products obtained by 3 parallel fermentation treatments of combined fermentation of a klatomyces Saccharomy copsiscrataegensis strain YC30, a Hansenula polymorpha Hanseniaspora uvarum strain QTX22 and a Saccharomyces cerevisiae strain F33 in grape juice is measured respectively;
F33: S.Morevisiae F33 single bacteria fermentation is carried out for 3 times, and the average value of the aroma substance content is measured respectively for the fermentation products obtained by parallel fermentation treatment.
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 single-bacteria fermentation is higher than the fermentation of adding amino acid on certain aroma components, which is a normal phenomenon in the field.
Claims (34)
1. A combined fermentation process based on klatomyces lanuginosus and hansenula polymorpha, characterized by comprising the following steps: activating the strain, and inoculating the activated strain into a substrate to be fermented for fermentation; the activated strain refers to that the strain is inoculated in a culture medium for culture; the strain is selected from the group consisting of: the Clarithromycin yeast Saccharomycopsis crataegensis strain YC30 with the preservation number of CCTCC M2021089, the Hansenula polymorpha Hanseniaspora uvarum strain QTX22 with the preservation number of CCTCC M2021083 and the Saccharomyces cerevisiae F33 are prepared; the culture temperature is 24-30 ℃, the culture time is 20-30h, and the rotating speed is 120-250rpm; the substrate is grape juice.
2. The combined fermentation process based on klandia and hansenula grape juice according to claim 1, wherein the cultivation temperature is 28 ℃; the incubation time was 24 hours and the rotational speed was 150rpm.
3. The combined fermentation process based on klandia and hansenula grape juice according to claim 1, wherein the inoculation is to inoculate the initial strain preservation solution in the culture medium according to the inoculation amount of 2-4% of the volume ratio.
4. A combined fermentation process based on klandia and hansenula grape juice according to claim 3, characterized in that the volume ratio is 3%.
5. A combined fermentation process based on klatomyces lanuginosus and hansenula polymorpha according to claim 3, characterized in that the medium is YPD medium; the YPD medium comprises the following components in mass volume ratio: 0.5-3.5% of Clatong yeast extract powder, 1.0-3.0% of peptone, 1.0-5.0% of glucose and the balance of water.
6. The combined fermentation process based on klandia and hansenula grape juice according to claim 5, wherein the YPD medium comprises the following components in mass-volume ratio: 1% of Cladonia yeast extract powder, 2% of peptone, 2% of glucose and the balance of water.
7. A combined fermentation process based on klandia and hansenula grape juice according to claim 3, wherein the initial strain preservation solution means that the strain is preserved in 15-35% glycerol/YPD medium by volume.
8. The combined fermentation process of claim 7, wherein the initial strain-preserving fluid is strain-preserved in 25% glycerol/YPD medium by volume.
9. The combined fermentation process based on klandia and hansenula polymorpha according to claim 1, wherein the activated strain is performed 1-3 times.
10. The combined fermentation process based on klandia and hansenula grape juice according to claim 9, wherein the activation strain is performed 2 times.
11. The combined fermentation process based on klandia and hansenula grape juice as claimed in claim 1, wherein the fermentation temperature is 18 ℃ ± 2 ℃.
12. The combined fermentation process based on klandia and hansenula grape juice according to claim 1, wherein the inoculation amount of the activated strain in the substrate is 10 6-107 CFU.
13. The combined fermentation process based on klandia and hansenula grape juice according to claim 12, wherein the inoculation amount of the activated strain in the substrate is 6 x 10 6 CFU.
14. The combined fermentation process based on klandia and hansenula polymorpha according to claim 1, wherein the fermentation is terminated after the substrate weight loss is no longer changed for 3 consecutive days.
15. A wine production method is characterized in that grape juice is taken as a substrate, and an activated strain are inoculated into the substrate to be fermented for fermentation; the strain is selected from the group consisting of: the Clarithromycin yeast Saccharomycopsis crataegensis strain YC30 with the preservation number of CCTCC M2021089, the Hansenula polymorpha Hanseniaspora uvarum strain QTX22 with the preservation number of CCTCC M2021083 and the Saccharomyces cerevisiae F33 are prepared; the activated strain refers to that the strain is inoculated in a culture medium for culture; the culture temperature is 24-30 ℃; the culture time is 20-30h, and the rotating speed is 120-250rpm.
16. A wine production method according to claim 15, wherein said cultivation temperature is 28 ℃; the incubation time was 24 hours and the rotational speed was 150rpm.
17. A method according to claim 15, wherein the inoculation is to inoculate a stock solution of the initial strain in a culture medium in an amount of 3% by volume.
18. A method of producing wine according to claim 15 wherein said medium is YPD medium; the YPD medium comprises the following components in mass volume ratio: 0.5-3.5% of Clatong yeast extract powder, 1.0-3.0% of peptone, 1.0-5.0% of glucose and the balance of water.
19. A method of producing wine according to claim 18 wherein the YPD medium comprises the following components in mass to volume ratio: 1% of Cladonia yeast extract powder, 2% of peptone, 2% of glucose and the balance of water.
20. The method according to claim 17, wherein the initial strain-preserving fluid is a strain preserved in 15-35% glycerol/YPD medium by volume.
21. The method according to claim 20, wherein the initial strain-preserving fluid is a strain preserved in 25% glycerol/YPD medium by volume.
22. A wine production method according to claim 15, wherein said activating strain is performed 1-3 times.
23. A process for the production of wine according to claim 15, characterised in that said fermentation temperature is 18 ℃ ± 2 ℃.
24. The method according to claim 15, wherein the total access of activated strain YC30 of Cladonia yeast Saccharomycopsis crataegensis and activated strain QTX22 of Hansenula polymorpha Hanseniaspora uvarum is 10 6-107 CFU.
25. The process for producing wine according to claim 24, wherein the total access of activated hansenula polymorpha Hanseniaspora uvarum strain QTX22 is 6 x 10 6 CFU.
26. A process for the production of wine according to claim 15 wherein the fermentation is terminated after the substrate weight loss has not changed for 3 consecutive days.
27. A method of wine production according to any one of claims 15-26 and further comprising: preparing grape juice;
The preparation of grape juice refers to: squeezing grape fruit at low temperature.
28. A method of producing wine according to claim 27 wherein the harvested grape fruit particles are pressed with ice using an air bag press.
29. A method of producing wine according to claim 27 wherein the pressing is accompanied by the addition of sulphur dioxide or K 2S2O5 and, pectase.
30. A process for the production of wine according to claim 29, wherein sulphur dioxide or K 2S2O5 is added in an amount of 30-100mg/L; the addition amount of pectase is 10-30mg/L.
31. A process for the production of wine according to claim 30 wherein sulfur dioxide or K 2S2O5 is added in an amount of 50mg/L; the addition amount of pectase is 20mg/L.
32. A method of producing wine according to claim 27 wherein the grape fruit particles are of uniform size.
33. A method according to claim 27, wherein the grape fruit particles are collected after ripening of the grape and when the temperature drops below-8 ℃ for 24 hours.
34. Wine produced by the method according to any one of claims 15 to 33.
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