CN113150896B - Mixed fermentation process based on cryptococcus gracilis and saccharomyces cerevisiae - Google Patents
Mixed fermentation process based on cryptococcus gracilis and saccharomyces cerevisiae Download PDFInfo
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- CN113150896B CN113150896B CN202110188934.2A CN202110188934A CN113150896B CN 113150896 B CN113150896 B CN 113150896B CN 202110188934 A CN202110188934 A CN 202110188934A CN 113150896 B CN113150896 B CN 113150896B
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Classifications
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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
-
- 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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- 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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- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Mycology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Botany (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a mixed fermentation process based on cryptococcus gracilis and saccharomyces cerevisiae, and belongs to the technical field of microorganisms. The mixed fermentation process based on the cryptococcus albus and the saccharomyces cerevisiae is characterized in that the cryptococcus albus 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 cryptococcus albus and saccharomyces cerevisiae.
Background
Wine fermentation is a complex biochemical process in which cryptococcus albus plays a very critical role, for example, in the conversion of sugars 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 cryptococcus albuminosus, and the different cryptococcus albuminosus has 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 cryptococcus albus 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 cryptococcus albus Cryptococcus albidus is very rare in the field at present, but about the mixed fermentation process of the cryptococcus albus Cryptococcus albidus and the saccharomyces cerevisiae, no related report is found in the field at present.
Disclosure of Invention
Based on the above-mentioned needs and blank in the art, the invention provides a mixed fermentation process based on the cryptococcus albuminosus Cryptococcus albidus and the saccharomyces cerevisiae Saccharomyces cerevisiae, which is remarkably improved on a plurality of aroma substances affecting the flavor of alcoholic beverages compared with the single-strain fermentation of the saccharomyces cerevisiae.
The technical scheme of the invention is as follows:
a mixed fermentation process based on cryptococcus albus and saccharomyces cerevisiae is characterized in that the cryptococcus albus and the saccharomyces cerevisiae are subjected to mixed fermentation.
Preferably, the cryptococcus albus refers to cryptococcus albus Cryptococcus albidus strain YN14; the preservation number of the cryptococcus albus Cryptococcus albidus strain YN14 is CCTCC M2021081.
Preferably, the Saccharomyces cerevisiae refers to Saccharomyces cerevisiae strain F33.
The mixed fermentation process based on the cryptococcus gracilis 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 cryptococcus albus Cryptococcus albidus strain YN14, or the 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% cryptococcus albus 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 cryptococcus gracilis Cryptococcus albidus strain YN14 or activated Saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating activated cryptococcus albus Cryptococcus albidus strain YN14 to a substrate for fermentation for 0-96h, preferably 48h, and inoculating activated saccharomyces cerevisiae strain F33 for continuous fermentation;
preferably, the fermentation temperature is 18 ℃ ± 2 ℃;
preferably, the total inoculum size of the activated cryptococcus albus Cryptococcus albidus strain YN14 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 used as a substrate, and cryptococcus albuminosus and saccharomyces cerevisiae are subjected to mixed fermentation;
the cryptococcus albicans refers to a cryptococcus albicans Cryptococcus albidus strain YN14; the preservation number of the cryptococcus albus Cryptococcus albidus strain YN14 is CCTCC M2021081.
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 cryptococcus albus Cryptococcus albidus strain YN14, or the 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% cryptococcus albus 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 cryptococcus gracilis Cryptococcus albidus strain YN14 or activated Saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating activated cryptococcus albus Cryptococcus albidus strain YN14 to a substrate for fermentation for 0-96h, preferably 48h, and inoculating activated saccharomyces cerevisiae strain F33 for continuous fermentation;
preferably, the fermentation temperature is 18 ℃ ± 2 ℃;
preferably, the total access of activated cryptococcus albus Cryptococcus albidus strain YN14 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, the cryptococcus albuminosus Cryptococcus albidus and the saccharomyces cerevisiae are adopted for mixed fermentation, the yield of most aroma substances is improved in tens of produced aroma substances, for example, ethyl acetate is improved by about 15%, octanoic acid is improved by 80%, n-decanoic acid is improved by 48%, 9-decenoic acid is improved by 1.17 times, glacial acetic acid is improved by 53%, 2-methylbutyric acid is improved by nearly 1 time, 1-pentanol is improved by 33%, dodecanol is improved by 26.7%, n-butanol is improved by 41.3%, isobutanol is improved by 42%, n-hexanol is improved by 29%, benzyl alcohol is improved by 47%, dimethyl silanediol is improved by 28.6%, 42.5% improvement in isovaleraldehyde, 52% improvement in n-hexanal, 53.8% improvement in 1, 3-propanediol monoethyl ether, 18% improvement in trans-4-decenoic acid ethyl ester, 19.2% improvement in 9-hexadecenoic acid ethyl ester, 40% improvement in 3-hydroxy-2-butanone, 55% improvement in 4-hydroxybutyrate acetyl ester, 22.7% improvement in 1, 3-propanediol diacetate, 2% improvement in Ma Shitong, 61% improvement in linalool, 1.67% improvement in citronellol, 26.3% improvement in linalool, 3% improvement in 2, 6-bis (t-butyl) -4-hydroxy-4-methyl-2, 5-cyclohexadien-1-one, and 41% improvement in 4-vinyl-2-methoxyphenol; at the same time, aroma substances which cannot be produced by the single-strain fermentation of Saccharomyces cerevisiae are also produced, for example: isobutyl acetate, phenethyl acetate, 2-benzyl propionic acid, 3-methyl-1-pentanol, 1-decanol, 2-ethylhexanol, propylene glycol, 3-furanmethanol, sec-octanol, nonanal, ethyl pyruvate, 2, 3-butanedione, m-xylene, 1,2,4, 5-tetramethylene, cypress brain, alpha-terpineol. 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 cryptococcus albus Cryptococcus albidus strain YN14 used in the experimental example is a new strain screened by the applicant laboratory, and the preservation information is as follows:
naming: YN14
Classification name: cryptococcus albidus
Latin name: cryptococcus albidus
Deposit number: CCTCC M2021081
Preservation mechanism: china center for type culture Collection
Preservation date: 2021, 1-15;
preservation agency address: in the Jiuqiu 299 Wuhan university school (first He-shou face 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 cryptococcus gracilis and saccharomyces cerevisiae. All embodiments of this group share the following common features: mixing and fermenting the cryptococcus albus and the saccharomyces cerevisiae.
According to the teaching of the present invention, any action of fermentation and production by combining the cryptococcus albuminosus with Saccharomyces cerevisiae falls 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 cryptococcus albus refers to cryptococcus albus Cryptococcus albidus strain YN14; the preservation number of the cryptococcus albus Cryptococcus albidus strain YN14 is CCTCC M2021081.
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 cryptococcus albus Cryptococcus albidus strain YN14 of the present invention to obtain similar technical effects as the present invention.
In some embodiments, the mixed fermentation process based on cryptococcus gracilis 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 cryptococcus albus Cryptococcus albidus strain YN14, or the 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% cryptococcus albus 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 cryptococcus gracilis Cryptococcus albidus strain YN14 or activated Saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating activated cryptococcus albus Cryptococcus albidus strain YN14 to a substrate for fermentation for 0-96h, preferably 48h, and inoculating activated saccharomyces cerevisiae strain F33 for continuous fermentation;
preferably, the fermentation temperature is 18 ℃ ± 2 ℃;
preferably, the total access of activated cryptococcus albus Cryptococcus albidus strain YN14 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 the cryptococcus albus and the saccharomyces cerevisiae by taking grape juice as a substrate.
In some preferred embodiments, the cryptococcus albus Cryptococcus albidus is cryptococcus albus Cryptococcus albidus strain YN14; the preservation number of the cryptococcus albus Cryptococcus albidus strain YN14 is CCTCC M2021081.
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 cryptococcus albus Cryptococcus albidus strain YN14, or the 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% cryptococcus albus 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 cryptococcus gracilis Cryptococcus albidus strain YN14 or activated Saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating activated cryptococcus albus Cryptococcus albidus strain YN14 to a substrate for fermentation for 0-96h, preferably 48h, and inoculating activated saccharomyces cerevisiae strain F33 for continuous fermentation;
when activated cryptococcus albus Cryptococcus albidus strain YN14 is inoculated to a substrate for fermentation for 0h, the activated cryptococcus albus Cryptococcus albidus strain YN14 and the activated saccharomyces cerevisiae strain F33 can be simultaneously inoculated to the substrate for fermentation until the weight loss of the substrate is continuous for 3 days and the weight loss of the substrate is not changed any more to stop fermentation.
Preferably, the fermentation temperature is 18 ℃ ± 2 ℃;
preferably, the total access of activated cryptococcus albus Cryptococcus albidus strain YN14 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 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: isobutyl acetate, phenethyl acetate, 2-benzyl propionic acid, 3-methyl-1-pentanol, 1-decanol, 2-ethylhexanol, propylene glycol, 3-furanmethanol, sec-octanol, nonanal, ethyl pyruvate, 2, 3-butanedione, meta-xylene, 1,2,4, 5-tetramethylbenzene, cypress brain, alpha-terpineol, and at the same time far higher aroma contents than the single-fermented wine: ethyl acetate, octanoic acid, n-decanoic acid, 9-decenoic acid, glacial acetic acid, 2-methylbutanoic acid, 1-pentanol, dodecanol, n-butanol, isobutanol, n-hexanol, benzyl alcohol, dimethylsilanediol, isopentane, n-hexanal, 1, 3-propanediol monoethyl ether, trans-4-decenoic acid ethyl ester, 9-hexadecenoic acid ethyl ester, 3-hydroxy-2-butanone, 4-hydroxybutyric acid acetyl ester, 1, 3-propanediol diacetate, up Ma Shitong, linalool, citronellol, linalool, 2, 6-di (tert-butyl) -4-hydroxy-4-methyl-2, 5-cyclohexadien-1-one, 4-vinyl-2-methoxyphenol.
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 Cryptococcus albus Cryptococcus albidus strain YN14 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, cryptococcus albus Cryptococcus albidus strain YN14, were all stored in 25% glycerol/YPD medium by volume prior to use. YPD medium was 1% Cryptococcus gracilis extract, 2% peptone, and 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 activated cryptococcus albus Cryptococcus albidus strain YN14 into collected grape juice, after 48 hours, inoculating S.cerevisiae F33 strain according to the inoculation proportion of YN14/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 grapesThe wine samples were centrifuged at 7500rpm for 8 minutes and the supernatants were 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 22.0for 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
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_Y14" indicates data of mixed-strain fermentation using a commercial strain of Saccharomyces cerevisiae F33 and a strain of Cryptococcus albicans Cryptococcus albidus YN14.
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 (44)
1. A mixed fermentation process based on cryptococcus gracilis and saccharomyces cerevisiae is 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 is inoculated with a substrate to be fermented for fermentation, which means that: activated cryptococcus albus is first treatedCryptococcus albidusInoculating the strain YN14 to a substrate for fermentation for 48-96 hours, and inoculating activated saccharomyces cerevisiae strain F33 for continuous fermentation; the cryptococcus albusCryptococcus albidusThe preservation number of the strain YN14 is CCTCC M2021081.
2. The mixed fermentation process based on cryptococcus gracilis and saccharomyces cerevisiae according to claim 1, wherein the activated strain is cultured by inoculating the strain into a culture medium.
3. The mixed fermentation process based on cryptococcus gracilis and saccharomyces cerevisiae according to claim 2, wherein the culture temperature is 24-30 ℃; the culture time is 20-30h, and the rotating speed is 120-250rpm.
4. A mixed fermentation process based on cryptococcus gracilis and saccharomyces cerevisiae according to claim 3, wherein the culture temperature is 28 ℃; the incubation time was 24 hours and the rotational speed was 150rpm.
5. The mixed fermentation process based on cryptococcus gracilis and saccharomyces cerevisiae according to claim 2, wherein the inoculation is to inoculate the initial strain preservation solution in a culture medium according to an inoculation amount of 2-4% by volume.
6. The mixed fermentation process based on the cryptococcus gracilis and saccharomyces cerevisiae according to claim 5, wherein the inoculation is to inoculate the initial strain preservation solution in a culture medium according to an inoculation amount of 3% by volume.
7. The mixed fermentation process based on cryptococcus gracilis and Saccharomyces cerevisiae according to any one of claims 2,5 and 6, wherein the culture medium is YPD culture medium.
8. The mixed fermentation process based on the cryptococcus gracilis and saccharomyces cerevisiae as claimed in claim 7, wherein the YPD culture medium comprises the following components in mass volume ratio: 0.5-3.5% of cryptococcus albus extract powder, 1.0-3.0% of peptone, 1.0-5.0% of glucose and the balance of water.
9. The mixed fermentation process based on the cryptococcus gracilis and saccharomyces cerevisiae as claimed in claim 8, wherein the YPD culture medium comprises the following components in mass-volume ratio: 1% of cryptococcus albus extract powder, 2% of peptone, 2% of glucose and the balance of water.
10. The mixed fermentation process based on cryptococcus gracilis and Saccharomyces cerevisiae according to claim 5 or 6, wherein the initial strain preservation solution means that the strain is preserved in a glycerol/YPD medium with a volume ratio of 15-35%.
11. The mixed fermentation process based on cryptococcus gracilis and saccharomyces cerevisiae according to claim 10, wherein the initial strain preservation solution refers to the strain preservation in a 25% glycerol/YPD medium by volume.
12. The process according to claim 1 or 2, wherein the activation of the strain is carried out 1-3 times.
13. The mixed fermentation process based on cryptococcus gracilis and saccharomyces cerevisiae according to claim 12, wherein said activating strain is performed 2 times.
14. The mixed fermentation process based on cryptococcus gracilis and saccharomyces cerevisiae according to claim 1, wherein the activated strain is inoculated with a substrate to be fermented for fermentation, which is characterized in that: activated cryptococcus albus is first treatedCryptococcus albidusThe strain YN14 is inoculated to a substrate for fermentation for 48 hours, and then the activated saccharomyces cerevisiae strain F33 is inoculated for continuous fermentation.
15. The mixed fermentation process based on cryptococcus gracilis and saccharomyces cerevisiae according to claim 1 or 14, wherein the fermentation temperature is 18 ℃ ± 2 ℃.
16. The process according to claim 1 or 14, wherein the activated cryptococcus albus is a mixed fermentation process of cryptococcus albus and saccharomyces cerevisiaeCryptococcus albidusStrain YN14 and, after activation, saccharomyces cerevisiae strain F33 were inoculated in a total inoculum size of 10 6 -10 7 CFU。
17. The mixed fermentation process based on cryptococcus gracilis and Saccharomyces cerevisiae according to claim 16, wherein the activated cryptococcus gracilisCryptococcus albidusStrain YN14 and activated Saccharomyces cerevisiae strain F33 were used in a total inoculum size of 6X 10 6 CFU。
18. A mixed fermentation process based on cryptococcus gracilis and saccharomyces cerevisiae according to claim 1 or 14, wherein the fermentation is terminated after the substrate weight loss is no longer changed for 3 consecutive days.
19. The mixed fermentation process based on cryptococcus gracilis and saccharomyces cerevisiae according to claim 18, wherein the substrate is grape juice.
20. A method of producing wine comprising: activating the strain, and inoculating the activated strain into substrate grape juice to be fermented for fermentation; the activated strain is inoculated with substrate grape juice to be fermented for fermentation, which means that: activated cryptococcus albus is first treatedCryptococcus albidusInoculating the strain YN14 to a substrate for fermentation for 48-96 hours, and inoculating activated saccharomyces cerevisiae strain F33 for continuous fermentation; the cryptococcus albusCryptococcus albidusThe preservation number of the strain YN14 is CCTCC M2021081.
21. A wine production method according to claim 20, wherein said activating strain is a strain inoculated in a culture medium for cultivation.
22. A process for the production of wine according to claim 21, wherein the cultivation temperature is between 24 and 30 ℃; the culture time is 20-30h, and the rotating speed is 120-250rpm.
23. A process for the production of wine according to claim 22 wherein the cultivation temperature is 28 ℃; the incubation time was 24 hours and the rotational speed was 150rpm.
24. A method according to claim 21, wherein the inoculation is performed by inoculating the initial strain stock solution into the culture medium at an inoculation rate of 3% by volume.
25. A method of producing wine according to claim 21 or 24 wherein said medium is YPD medium.
26. A method of producing wine according to claim 25 wherein the YPD medium comprises the following components in mass to volume ratio: 0.5-3.5% of cryptococcus albus extract powder, 1.0-3.0% of peptone, 1.0-5.0% of glucose and the balance of water.
27. A method of producing wine according to claim 26 wherein the YPD medium comprises the following components in mass to volume ratio: 1% of cryptococcus albus extract powder, 2% of peptone, 2% of glucose and the balance of water.
28. A wine production method according to claim 24 wherein said initial strain preservation fluid means that the strain is preserved in 15-35% glycerol/YPD medium by volume.
29. A method of producing wine according to claim 28 wherein said initial strain-preserving fluid means strain is preserved in 25% glycerol/YPD medium by volume.
30. A wine production method according to claim 20 or 21, wherein said activating strain is performed 1-3 times.
31. A wine production method according to claim 30 wherein said activating strain is performed 2 times.
32. A wine production method according to claim 20, wherein said activated strain is inoculated with a substrate must to be fermented for fermentation: activated cryptococcus albus is first treatedCryptococcus albidusThe strain YN14 is inoculated to a substrate for fermentation for 48 hours, and then the activated saccharomyces cerevisiae strain F33 is inoculated for continuous fermentation.
33. A process for producing wine according to claim 20 or 32 wherein the fermentation temperature is 18 ℃ ± 2 ℃.
34. A method of producing wine according to claim 20 or 32 wherein the activated cryptococcus albus isCryptococcus albidusThe total access of strain YN14 and activated Saccharomyces cerevisiae strain F33 is 10 6 -10 7 CFU。
35. The method of claim 34, wherein the activated cryptococcus albus is a white yeastCryptococcus albidusStrain YN14 and activated Saccharomyces cerevisiae strain F33 with a total access of 6×10 6 CFU。
36. A method of producing wine according to claim 20 or 32 wherein fermentation is terminated after the substrate loss has been unchanged for 3 consecutive days.
37. A method of wine production according to any one of claims 20-24, 26-29, 31, 32, 35 and further comprising: preparing grape juice;
the preparation of grape juice refers to: squeezing grape fruit at low temperature.
38. A method of producing wine according to claim 37 wherein the harvested grape fruit particles are pressed with ice using an air bag press.
39. A method of producing wine according to claim 38 wherein the pressing is accompanied by the addition of sulphur dioxide or K 2 S 2 O 5 And, pectase.
40. A method of producing wine according to claim 39 wherein 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.
41. A process for producing wine according to claim 40 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.
42. A method of producing wine according to claim 38 wherein the grape fruit particles are of uniform size.
43. The method according to claim 38 or 42, wherein the grape fruit particles are grape fruit particles harvested after ripening of the grape and after a temperature drop below-8 ℃ for 24 hours.
44. Wine produced by the method according to any one of claims 20 to 43.
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| Title |
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| 3株非酿酒酵母在模拟葡萄汁中的生长特性与发酵香气;原苗苗等;《食品工业科技》;20180116(第09期);第111-118、123页 * |
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