CN112725107B - Mixed fermentation process based on Klatong yeast and Saccharomyces cerevisiae - Google Patents
Mixed fermentation process based on Klatong yeast and Saccharomyces cerevisiae Download PDFInfo
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- CN112725107B CN112725107B CN202110191367.6A CN202110191367A CN112725107B CN 112725107 B CN112725107 B CN 112725107B CN 202110191367 A CN202110191367 A CN 202110191367A CN 112725107 B CN112725107 B CN 112725107B
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- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 144
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- 230000004151 fermentation Effects 0.000 title claims abstract description 113
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- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 description 1
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- 241000894007 species Species 0.000 description 1
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Classifications
-
- 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
-
- 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
-
- 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
-
- 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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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Mycology (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Botany (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a mixed fermentation process based on Clarithromycin yeast and Saccharomyces cerevisiae, and belongs to the technical field of microorganisms. The mixed fermentation process based on the klalomycetes and the saccharomyces cerevisiae is characterized in that the klalomycetes and the saccharomyces cerevisiae are subjected to mixed fermentation. The mixed bacteria fermentation product of the invention has unique flavor compared with a single bacteria fermentation product, can produce a wine beverage with unique flavor, fragrance and taste, enriches consumer products and enlarges consumption selection.
Description
Technical Field
The invention belongs to the technical field of beverage fermentation, and particularly relates to a mixed fermentation process based on Klatanomyces 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 report of fermentation by using the Cladonia yeast Saccharomycopsis crataegensis is very rare in the field, but no related report is found in the field at present regarding the mixed fermentation process of the Cladonia yeast Saccharomycopsis crataegensis and the saccharomyces cerevisiae.
Disclosure of Invention
Based on the above-mentioned needs and blank in the art, the invention provides a mixed fermentation process based on Klatong yeast Saccharomycopsis crataegensis and Saccharomyces cerevisiae Saccharomyces cerevisiae, which is remarkably improved in a plurality of aroma substances affecting the flavor of alcoholic beverages compared with the single-strain fermentation of Saccharomyces cerevisiae.
The technical scheme of the invention is as follows:
a mixed fermentation process based on Klatong yeast and Saccharomyces cerevisiae is characterized in that the Klatong yeast and Saccharomyces cerevisiae are subjected to mixed fermentation.
Preferably, the klandia clarituximab strain Saccharomycopsis crataegensis YC30; the preservation number of the Clarithromycin yeast Saccharomycopsis crataegensis strain YC30 is CCTCC M2021089.
Preferably, the Saccharomyces cerevisiae refers to Saccharomyces cerevisiae strain F33.
The mixed bacteria fermentation process based on the Klatanomyces 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: the strain Clarithromycin yeast Saccharomycopsis crataegensis YC30, or the strain Saccharomyces cerevisiae 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%, 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.
The activated strain refers to: activated klandia t Saccharomycopsis crataegensis strain YC30, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating the activated strain YC30 of the Clarithromycin yeast Saccharomycopsis crataegensis to a substrate for fermentation for 0-96h, preferably 0h, 24h, 48h and 96h, and then inoculating the activated strain F33 of the Saccharomyces cerevisiae for continuous fermentation;
preferably, the fermentation temperature is 18 ℃ ± 2 ℃;
preferably, after activationThe total inoculum size of the strain YC30 of Clarithromycin strain Saccharomycopsis crataegensis and the activated Saccharomyces cerevisiae strain F33 was 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 production method of grape wine is characterized in that grape juice is taken as a substrate, and Klatong yeast and Saccharomyces cerevisiae are subjected to mixed fermentation;
the Clarithromycin yeast Saccharomyces cerevisiae Saccharomycopsis crataegensis strain YC30; the preservation number of the Clarithromycin yeast Saccharomycopsis crataegensis strain YC30 is CCTCC M2021089.
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: the strain Clarithromycin yeast Saccharomycopsis crataegensis YC30, or the strain Saccharomyces cerevisiae 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 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 klandia t Saccharomycopsis crataegensis strain YC30, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating the activated strain YC30 of the Clarithromycin yeast Saccharomycopsis crataegensis to a substrate for fermentation for 0-96h, preferably 0h, 24h, 48h or 96h, and then inoculating the activated strain F33 of the Saccharomyces cerevisiae for continuous fermentation;
preferably, the fermentation temperature is 18 ℃ ± 2 ℃;
preferably, the total access of activated Saccharomyces cerevisiae strain Saccharomycopsis crataegensis YC30 and 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, klatong yeast Saccharomycopsis crataegensis and Saccharomyces cerevisiae are adopted for mixed fermentation, and among tens of produced aroma substances, the yield of most aroma substances is improved by 64.7% by 1-methylnaphthalene, 1.33 times by 2-ethylhexanol, 14 times by 1-pentanol, 7.25 times by n-butanol, 31.67 times by ethyl lactate, 1.35 times by acetaldehyde, 26.33 times by diethyl succinate, 1.1 times by 3-hydroxybutyrate, 15.7 times by 2-methylbutanoic acid, 45.6 times by isopentyl caproate, 51.75 times by ethyl acetate, 1.17 times by n-hexanol, 9.52 times by 2, 4-di-tert-butylphenol, 20.36 times by caproic acid, 19.23 times by 9-decenoic acid, 14.25 times by linalool, 2, 3-butanediol, 52.67 times by benzyl alcohol, 88.14% by 1, 3-propanediol monoethyl ether, 43.77 times by ethyl myristate, 53.5 times by large Ma Shitong, 11.8 times by ethyl palmitate, 11.75 times by 2.17 times by 2, 8.17 times by 2.17 times by 2, 8.8 times by 2.84 times by 2, 8.8% by 2, 8% by 2.8% by 2-ethyl isovalerate, 2.8; at the same time, aroma substances which cannot be produced by the single-strain fermentation of Saccharomyces cerevisiae are also produced, for example: butyl decalactone, 2-methylnaphthalene, ethyl 2-furoate, nonanal, 3-methyl-3-buten-1-ol, isobutyl acetate, isoamyl octanoate, n-heptanol, ethyl valerate, butyric acid, 2, 3-butanedione, 3-hydroxy-2-butanone, 1-decanol, isovaleraldehyde, dodecanol, 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 strain Clarithromycin yeast Saccharomycopsis crataegensis YC30 used in the experimental example was a new strain screened by the applicant laboratory, and the preservation information is as follows:
naming: YC30
Classification name: claritonas sp
Latin name: saccharomycopsis crataegensis
Deposit number: CCTCC M2021089
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.
Group 1 example, mixed bacteria fermentation Process of the invention
The embodiment of the group provides a mixed fermentation process based on Klatanomyces and Saccharomyces cerevisiae. All embodiments of this group share the following common features: mixing and fermenting the Klatong yeast and the saccharomyces cerevisiae.
Those skilled in the art can practice the fermentation and production of Clarithromycin yeast in combination with Saccharomyces cerevisiae in accordance with the teachings of the present invention, and all such activities are 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 Clarithromycin yeast refers to Clarithromycin yeast strain Saccharomycopsis crataegensis YC30; the preservation number of the Clarithromycin yeast Saccharomycopsis crataegensis strain YC30 is CCTCC M2021089.
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., and these strains can be used for mixed fermentation with the Klatong yeast Saccharomycopsis crataegensis strain YC30 of the present invention, so as to obtain similar technical effects as those of the present invention.
In some embodiments, the mixed fermentation process based on klatomyces and saccharomyces cerevisiae comprises the following steps: and inoculating the activated strain and the activated strain into a fermentation substrate for fermentation.
Preferably, the strain refers to: the strain Clarithromycin yeast Saccharomycopsis crataegensis YC30, or the strain Saccharomyces cerevisiae 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%, 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.
In other embodiments, the activated strain refers to: activated klandia t Saccharomycopsis crataegensis strain YC30, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating the activated strain YC30 of the Clarithromycin yeast Saccharomycopsis crataegensis to a substrate for fermentation for 0-96h, preferably 0h, 24h, 48h or 96h, and then inoculating the activated strain F33 of the Saccharomyces cerevisiae for continuous fermentation;
preferably, the fermentation temperature is 18 ℃ ± 2 ℃;
preferably, the total access of activated Saccharomyces cerevisiae strain Saccharomycopsis crataegensis YC30 and 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 Klatong yeast and Saccharomyces cerevisiae with grape juice as substrate.
In some preferred embodiments, the kluyveromyces Saccharomycopsis crataegensis refers to the strain kluyveromyces Saccharomycopsis crataegensis, YC30; the preservation number of the Clarithromycin yeast Saccharomycopsis crataegensis strain YC30 is CCTCC M2021089.
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: the strain Clarithromycin yeast Saccharomycopsis crataegensis YC30, or the strain Saccharomyces cerevisiae 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%, 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.
In specific embodiments, the activated strain refers to: activated klandia t Saccharomycopsis crataegensis strain YC30, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating the activated strain YC30 of the Clarithromycin yeast Saccharomycopsis crataegensis to a substrate for fermentation for 0-96h, preferably 0h, 24h, 48h or 96h, and then inoculating the activated strain F33 of the Saccharomyces cerevisiae for continuous fermentation;
when the activated strain YC30 of the Clarithromycin yeast Saccharomycopsis crataegensis is inoculated to a substrate for fermentation for 0h, the strain YC30 of the Clarithromycin yeast Saccharomycopsis crataegensis after surface activation and the strain F33 of the Saccharomyces cerevisiae after activation can be simultaneously inoculated to the substrate for fermentation until the weight loss of the substrate is continuously 3 days without changing any more to terminate the fermentation.
Preferably, the fermentation temperature is 18 ℃ ± 2 ℃;
preferably, the total access of activated Saccharomyces cerevisiae strain Saccharomycopsis crataegensis YC30 and 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 pressing is added simultaneouslySulfur dioxide or K 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.
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: butyl decalactone, 2-methylnaphthalene, ethyl 2-furoate, nonanal, 3-methyl-3-buten-1-ol, isobutyl acetate, isoamyl octanoate, n-heptanol, ethyl valerate, butyric acid, 2, 3-butanedione, 3-hydroxy-2-butanone, 1-decanol, isovaleraldehyde, dodecanol, ethyl 3-phenylpropionate, while being much higher in the content of aroma substances than the single fermented wine: 1-methylnaphthalene, 2-ethylhexanol, 1-pentanol, n-butanol, ethyl lactate, acetaldehyde, diethyl succinate, ethyl 3-hydroxybutyrate, 2-methylbutanoate, isoamyl hexanoate, ethyl acetate, n-hexanol, 2, 4-di-tert-butylphenol, hexanoic acid, 9-decenoic acid, linalool, 2, 3-butanediol, benzyl alcohol, 1, 3-propanediol monoethyl ether, ethyl myristate, large Ma Shitong, phenethyl acetate, ethyl palmitate, 4-vinyl-2-methoxyphenol, octanol, citronellol, ethyl n-hexanoate, isoamyl acetate, octanoic acid, isobutanol, 1-diethoxyethane, ethyl octanoate, glacial acetic acid, isoamyl alcohol.
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 and the isolated and screened strain of Clarithromycin yeast Saccharomycopsis crataegensis, YC30, 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. The pressed grape juice contains 432g/dm of sugar 3 Acidity 4.65g/dm 3 (tartaric acid) pH 4.21.
3. Fermentation operation
2 strains: saccharomyces cerevisiae F33 strain, saccharomyces cerevisiae Saccharomycopsis crataegensis strain YC30 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 40mLYPD culture medium and a 250mL triangular flask filled with 150mLYPD 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 40mLYPD culture medium and the 250mL triangular flask filled with 150mLYPD culture medium respectively, repeating the culture, and completing the 2 nd passage activation, thus obtaining the bacterial liquid after activation. Firstly, inoculating the activated Clarithromycin strain YC30 into the collected grape juice, and after 0 hour (namely YC30 and F33 are simultaneously inoculated), or 24 hours, or 48 hours, or 96 hours, inoculating the Clarituxia strain YC Saccharomycopsis crataegensis into the S.cerevisiae strain F33 according to the inoculation proportion of the YC30/F33 volume ratio of 2:1, wherein the total inoculation amount of the strains is controlled to be 6 multiplied by 10 6 CFU, using S.cerevisiae F33 pure fermented grape juice as blank control, fermenting at 18+ -2deg.C until grape juice loses weightThe fermentation was terminated three days after which no further changes were made. 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 (camasa, 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 table 1 above refers to the lowest concentration lower limit value at which a person can sniff the substance, and the fragrance description and threshold are based on the reports of the relevant documents in which the fragrance description and threshold of the fragrance substance are described, and the fragrance substance having no fragrance description in the table is a document in which the relevant document about the fragrance description 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_YC30" indicates data of mixed-strain fermentation using a commercial strain of Saccharomyces cerevisiae F33 and a strain of Saccharomyces clavulans Saccharomycopsis crataegensis YC30.
F33 is data obtained by fermenting only the Saccharomyces cerevisiae F33;
Sc+48H2F 33 refers to data obtained by inoculating a Clarithromycin yeast strain YC30, fermenting for 48 hours, and then inoculating Saccharomyces cerevisiae F33, and continuing fermentation;
Sc+24H2F33 refers to data obtained by inoculating the Clarithromycin yeast strain YC30 for fermentation for 24 hours and then inoculating the Saccharomyces cerevisiae F33 for continuous fermentation;
Sc+96H2F33 refers to data obtained by inoculating the Clarithromycin yeast strain YC30, fermenting for 96 hours, and then inoculating the Saccharomyces cerevisiae F33 for continuous fermentation;
Sc+F33 refers to data obtained by fermentation by simultaneous access of the Clarithromycin yeast strain YC30 and Saccharomyces cerevisiae F33.
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 (30)
1. The mixed fermentation process based on the Klatong yeast and the saccharomyces cerevisiae is characterized by comprising the following steps of: activating the strain, and inoculating the activated strain into a substrate to be fermented for fermentation; the activated strain is inoculated with a substrate to be fermented for fermentation, which means that: the activated Clarithromycin yeast is first treatedSaccharomycopsis crataegensisInoculating strain YC30 to substrate, fermenting for 24-96 hr, inoculating activated Saccharomyces cerevisiaeThe strain F33 continues fermentation; the Claritomyces spSaccharomycopsis crataegensisThe preservation number of the strain YC30 is CCTCC M2021089; the fermentation temperature is 18+/-2 ℃, the total inoculation amount of the activated Clarithromycin strain Saccharomycopsis crataegensis YC30 and the activated Saccharomyces cerevisiae strain F33 is 10 6 -10 7 CFU; stopping fermentation when the substrate weight loss is not changed for 3 days; the substrate is grape juice;
the mixed bacteria fermentation process further comprises the following steps: preparing grape juice;
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 grape is ripe.
2. The process according to claim 1, wherein the activated strain is a strain inoculated in a medium for cultivation.
3. A Cladonia-based yeast according to claim 2Saccharomycopsis crataegensisAnd Saccharomyces cerevisiae, and is characterized in that the culture temperature is 24-30 ℃; the culture time is 20-30h, and the rotating speed is 120-250rpm.
4. A kltonomyces-based yeast according to claim 3Saccharomycopsis crataegensisAnd Saccharomyces cerevisiae, and is characterized in that the culture temperature is 28 ℃; the incubation time was 24 hours and the rotational speed was 150rpm.
5. A Cladonia-based yeast according to claim 2Saccharomycopsis crataegensisAnd Saccharomyces cerevisiae, which is a special fermentation processCharacterized in that the inoculation refers to inoculating the initial strain preservation solution into a culture medium according to the inoculation amount of 2-4% of the volume ratio.
6. The Claritomyces-based yeast of claim 5Saccharomycopsis crataegensisAnd 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.
7. A Cladonia-based yeast according to any one of claims 2, 5 and 6Saccharomycopsis crataegensisAnd a mixed fermentation process of saccharomyces cerevisiae, wherein the culture medium is YPD culture medium.
8. The Cladonia-based yeast of claim 7Saccharomycopsis crataegensisAnd 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% of Clatong yeast extract powder, 1.0-3.0% of peptone, 1.0-5.0% of glucose and the balance of water.
9. The Cladonia-based yeast of claim 7Saccharomycopsis crataegensisAnd 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 Cladonia yeast extract powder, 2% of peptone, 2% of glucose and the balance of water.
10. A Cladonia-based yeast according to claim 5 or 6Saccharomycopsis crataegensisAnd 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%.
11. The Cladonia-based yeast of claim 10Saccharomycopsis crataegensisAnd Saccharomyces cerevisiae, characterized in that the initialThe strain preservation solution means that the strain is preserved in 25% glycerol/YPD medium by volume.
12. A kltonomyces-based yeast according to claim 1 or 2Saccharomycopsis crataegensisAnd a mixed fermentation process of Saccharomyces cerevisiae, wherein the activated strain is performed 1-3 times.
13. A Cladonia-based yeast according to claim 12Saccharomycopsis crataegensisAnd a mixed fermentation process of saccharomyces cerevisiae, wherein the activated strain is performed 2 times.
14. The mixed fermentation process based on the Klatong yeast and the saccharomyces cerevisiae according to claim 1, wherein the activated strain is inoculated into a substrate to be fermented for fermentation, and the fermentation is as follows: the activated Clarithromycin yeast is first treatedSaccharomycopsis crataegensisAfter the strain YC30 is inoculated to a substrate for fermentation for 24 hours, 48 hours or 96 hours, the activated saccharomyces cerevisiae strain F33 is inoculated for continuous fermentation.
15. The process of claim 1, wherein the activated yeast is a combination of Saccharomyces cerevisiae and Saccharomyces cerevisiaeSaccharomycopsis crataegensisThe total inoculum size of strain YC30 and activated Saccharomyces cerevisiae strain F33 was 6X 10 6 CFU。
16. A method of producing wine comprising: preparing grape juice, activating a strain, inoculating the activated strain into a substrate grape juice to be fermented, and fermenting; the activated strain is inoculated with a substrate to be fermented for fermentation, which means that: the activated Clarithromycin yeast is first treatedSaccharomycopsis crataegensisInoculating the strain YC30 to a substrate for fermentation for 48-96 hours, and inoculating the activated saccharomyces cerevisiae strain F33 for continuous fermentation; the Claritomyces spSaccharomycopsis crataegensisThe preservation number of the strain YC30 is CCTCC M2021089;
the fermentation temperature is 18+/-2 ℃; the total access of activated Saccharomyces cerevisiae strain Saccharomycopsis crataegensis YC30 and activated Saccharomyces cerevisiae strain F33 was 10 6 -10 7 CFU; stopping fermentation when the substrate weight loss 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 Pectase; 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 grape is ripe.
17. A wine production method according to claim 16, wherein said activating strain is a strain inoculated in a culture medium for cultivation.
18. A process for the production of wine according to claim 17, wherein the cultivation temperature is between 24 and 30 ℃; the culture time is 20-30h, and the rotating speed is 120-250rpm.
19. A process for the production of wine according to claim 18 wherein the cultivation temperature is 28 ℃; the incubation time was 24 hours and the rotational speed was 150rpm.
20. A method according to claim 17, wherein the inoculation is to inoculate a stock solution of the initial strain in a culture medium in an amount of 3% by volume.
21. A method of producing wine according to claim 17 or 18 wherein said medium is YPD medium.
22. A method of producing wine according to claim 21 wherein the YPD medium comprises the following components in mass to 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.
23. A method of producing wine according to claim 22 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.
24. A wine production method according to claim 20 wherein said initial strain preservation fluid means that the strain is preserved in 15-35% glycerol/YPD medium by volume.
25. A method of producing wine according to claim 20 wherein said initial strain-preserving fluid means strain is preserved in 25% glycerol/YPD medium by volume.
26. A wine production method according to claim 16 or 17, wherein said activating strain is performed 1-3 times.
27. A wine production method according to claim 26 wherein said activating strain is performed 2 times.
28. A method of producing wine according to claim 16 wherein the activated klandia yeastSaccharomycopsis crataegensisThe total access of strain YC30 and activated Saccharomyces cerevisiae strain F33 was 6X 10 6 CFU。
29. A method of producing wine according to claim 16 wherein 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.
30. Wine produced by the method according to any one of claims 16-29.
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| CN106987531A (en) * | 2017-05-05 | 2017-07-28 | 湖南农业大学 | One plant of grape wine laminating adhesive spore yeast and its application in monoterpene production |
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| JP2004254554A (en) * | 2003-02-25 | 2004-09-16 | Mitsubishi Chemicals Corp | Method of producing optically active 2-methoxy-1-(4-trifluoromethyl-phenyl)ethanol |
| CN106987531A (en) * | 2017-05-05 | 2017-07-28 | 湖南农业大学 | One plant of grape wine laminating adhesive spore yeast and its application in monoterpene production |
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