CN113150899A - Mixed fermentation process based on yeast Papiliotrema laurentii and saccharomyces cerevisiae - Google Patents
Mixed fermentation process based on yeast Papiliotrema laurentii and saccharomyces cerevisiae Download PDFInfo
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- CN113150899A CN113150899A CN202110189032.0A CN202110189032A CN113150899A CN 113150899 A CN113150899 A CN 113150899A CN 202110189032 A CN202110189032 A CN 202110189032A CN 113150899 A CN113150899 A CN 113150899A
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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)
- 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)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a mixed fermentation process based on yeast Papiliotrema laurentii and saccharomyces cerevisiae, belonging to the technical field of microorganisms. The mixed fermentation process based on the yeasts Papiliotrema laurentii and the Saccharomyces cerevisiae is characterized in that the yeasts Papiliotrema laurentii and the Saccharomyces cerevisiae are subjected to mixed fermentation. Compared with a single-bacterium fermented product, the mixed-bacterium fermented product has more unique fragrance, can produce a wine with unique flavor, fragrance and taste, fills the consumer category and expands the 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 yeast Papiliotrema laurentii and saccharomyces cerevisiae.
Background
Wine fermentation is a complex biochemical process in which yeast plays a very critical role, such as the conversion of sugars to ethanol, carbon dioxide and other secondary metabolites in the thousands. A large number of scientific studies show that the quality of the wine is highly dependent on the metabolic activity and fermentation behavior of different yeasts, and the different yeasts have important contributions to the chemical composition, the sensory characteristics, the flavor characteristics and the like of the wine. Saccharomyces cerevisiae is the strain which is widely applied in the industrial production of wine so far, has the advantages of ensuring the risk of deterioration in the fermentation process of wine, and having good fermentation power, but has the problems of single flavor characteristic, serious homogenization phenomenon and the like of wine. Therefore, in order to pursue wine style specialization and make the aroma characteristics more representative, diverse and complex, brewers often adopt a method of mixing and fermenting saccharomyces cerevisiae and non-saccharomyces cerevisiae, especially some indigenous yeasts with strong adaptability and representativeness, so as to improve and enhance the flavor quality of wine.
Non-saccharomyces cerevisiae has become an option for improving the quality of wine. Numerous studies have shown that non-saccharomyces cerevisiae is able to produce enzymes as well as some of our desired secondary metabolites, thus improving wine aroma and flavor characteristics, and is able to control the growth of some undesirable species in wine, but has the disadvantage of insufficient fermentation power. The mixed fermentation of the non-saccharomyces cerevisiae and the saccharomyces cerevisiae can improve the aroma diversity and complexity of the wine, can make up for the problem of insufficient fermentation power of the non-saccharomyces cerevisiae, and is an effective method for improving the aroma quality of the wine.
The related reports of fermentation by using the yeast Papiliotrema laurentii are extremely rare at present in the field, and the related reports on the mixed fermentation process of the yeast Papiliotrema laurentii and saccharomyces cerevisiae are not seen at present in the field.
Disclosure of Invention
Based on the above needs and blanks in the art, the invention provides a mixed fermentation process based on the yeasts Papiliotrema laurentii and the Saccharomyces cerevisiae, and compared with single-strain fermentation of the Saccharomyces cerevisiae, the process has the advantage that various aroma substances which affect the flavor of wine are remarkably improved.
The technical scheme of the invention is as follows:
a mixed fermentation process based on yeast Papiliotrema laurentii and saccharomyces cerevisiae is characterized in that the yeast Papiliotrema laurentii and the saccharomyces cerevisiae are subjected to mixed fermentation.
Preferably, the yeast papiiotrema laurentii refers to the yeast papiiotrema laurentii strain HSP 22; the preservation number of the yeast Papiliotrema laurentii strain HSP22 is CCTCC M2021086.
Preferably, the Saccharomyces cerevisiae refers to Saccharomyces cerevisiae strain F33.
The mixed fermentation process based on the yeast and the saccharomyces cerevisiae comprises the following steps: the activated strain and the activated strain are inoculated into a substrate for secondary fermentation for fermentation.
Preferably, said strain refers to: yeast papiioterma laurentiii strain HSP22, or, saccharomyces cerevisiae strain F33;
preferably, the activated strain refers to a strain inoculated in a culture medium for culture;
preferably, the culture temperature is 24-30 ℃, preferably 28 ℃; the culture time is 20-30h, preferably 24h, the rotation speed is 120-250rpm, preferably 150 rpm;
preferably, the inoculation refers to inoculating the initial strain stock solution into the culture medium according to the inoculation amount of 2-4%, preferably 3% in volume ratio;
preferably, the medium is a YPD medium; preferably, the YPD medium comprises the following components in mass-to-volume ratio: 0.5-3.5%, preferably 1%, yeast extract, 1.0-3.0%, preferably 2%, peptone, 1.0-5.0%, preferably 2%, glucose, and water in balance;
preferably, the initial strain stock solution refers to the strain stock in 15-35% by volume, preferably 25% glycerol/YPD medium;
more preferably, the activation of the strain is performed 1-3 times, preferably 2 times.
The activated strain refers to: activated yeast strain papiioterma laurentiii HSP22, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate into the secondary fermentation refers to: firstly inoculating activated yeast Papiliotrema laurentiii strain HSP22 to a substrate for fermentation for 0-96h, preferably 48h, and then inoculating activated saccharomyces cerevisiae strain F33 for continuous fermentation;
preferably, the fermentation temperature is 18 ℃ +/-2 ℃;
preferably, the total inoculum size of the activated yeast strain, strain PAPilliotrema laurentii HSP22, and activated Saccharomyces cerevisiae strain F33 is 106-107CFU, preferably 6X 106CFU；
Preferably, the fermentation is terminated when the substrate weight loss does not change for 3 consecutive days;
preferably, the substrate is grape juice.
A wine production method is characterized in that grape juice is used as a substrate, and yeast and saccharomyces cerevisiae are subjected to mixed fermentation;
the yeast refers to yeast strain papiiotema laurentiii HSP 22; the preservation number of the yeast Papiliotrema laurentii strain HSP22 is CCTCC M2021086.
The saccharomyces cerevisiae refers to saccharomyces cerevisiae strain F33.
The production method of the wine comprises the following steps: the activated strain and the activated strain are inoculated into a substrate for secondary fermentation for fermentation.
Preferably, said strain refers to: yeast papiioterma laurentiii strain HSP22, or, saccharomyces cerevisiae strain F33;
preferably, the activated strain refers to a strain inoculated in a culture medium for culture;
preferably, the culture temperature is 24-30 ℃, preferably 28 ℃; the culture time is 20-30h, preferably 24h, the rotation speed is 120-250rpm, preferably 150 rpm;
preferably, the inoculation refers to inoculating the initial strain preservation solution into the culture medium according to the inoculation amount of 3% by volume;
preferably, the medium is a YPD medium; preferably, the YPD medium comprises the following components in mass-to-volume ratio: 0.5-3.5%, preferably 1%, yeast extract, 1.0-3.0%, preferably 2%, peptone, 1.0-5.0%, preferably 2%, glucose, and water in balance;
preferably, the initial strain stock solution refers to the strain stock in 15-35% by volume, preferably 25% glycerol/YPD medium;
more preferably, the activation of the strain is performed 1-3 times, preferably 2 times.
The activated strain refers to: activated yeast strain papiioterma laurentiii HSP22, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate into the secondary fermentation refers to: firstly inoculating activated yeast Papiliotrema laurentiii strain HSP22 to a substrate for fermentation for 0-96h, preferably 48h, and then inoculating activated saccharomyces cerevisiae strain F33 for continuous fermentation;
preferably, the fermentation temperature is 18 ℃ +/-2 ℃;
preferably, the total inoculation amount of the activated yeast strain Papiliotrema laurentii HSP22 and the activated saccharomyces cerevisiae strain F33 is 106-107CFU, preferably 6X 106CFU；
Preferably, the fermentation is terminated when the substrate weight loss is not changed for 3 consecutive days.
The wine production method further comprises the following steps: preparing grape juice;
the preparation of the grape juice comprises the following steps: squeezing grape fruit grains at low temperature;
preferably, the harvested grape fruit particles are pressed with ice by an air bag press;
preferably, pressing is performed while adding sulfur dioxide or K2S2O5And, pectinase;
preferably, sulfur dioxide or K2S2O5The addition amount of (B) is 30-100mg/L, preferably 50 mg/L; the addition amount of pectinase is 10-30mg/L, preferably 20 mg/L;
preferably, the grape fruit pieces are uniformly sized fruit pieces;
preferably, the grape fruit particles are grape fruit particles collected when the temperature of the mature grapes continuously drops below 24 hours to 8 ℃.
The wine is characterized by being produced by the wine production method.
The invention adopts the yeast Papiliotrema laurentii and the saccharomyces cerevisiae for mixed fermentation, and the yield of most of aroma substances in dozens of aroma substances is improved, for example, the ethyl acetate is improved by about 17.6 percent, the caprylic acid is improved by 58 percent, the n-capric acid is improved by 44 percent, the butyric acid is improved by 1 time, the 9-decenoic acid is improved by 1.26 times, the glacial acetic acid is improved by 65 percent, the 2-methylbutyric acid is improved by 6.1 times, the 1-pentanol is improved by 5.9 times, the dodecanol is improved by 60 percent, the n-butyl alcohol is improved by 4.3 times, the isobutanol is improved by 58 percent, the n-hexanol is improved by 23 percent, the benzyl alcohol is improved by 37 percent, the dihydrolinalool is improved by 17.5 percent, the isovaleraldehyde is improved by 1.2 times, the 1, 3-propylene glycol monoethyl ether is improved by 2.76 times, the ethyl nonanoate is improved by 50 percent, the ethyl 9-hexadecanoate is improved by 50 percent, and the 3-hydroxy-2-butanone is improved by 73 percent, the methyl decanoate is improved by 42 percent, the acetyl 4-hydroxybutyrate is improved by 45 percent, the 1, 3-propylene glycol diacetate is improved by 1 time, the vinyl acetate is improved by 54 percent, the naphthalene is improved by 61 percent, the damascenone is improved by 2.3 times, the linalool is improved by 1.12 times, the citronellol is improved by 1.12 times, the linalool is improved by 32 percent, and the 4-vinyl-2-methoxyphenol is improved by 33 percent; meanwhile, aroma substances which cannot be produced by single-strain fermentation of saccharomyces cerevisiae are also produced, such as: isobutyl acetate, phenethyl acetate, 2-benzylpropionic acid, 3-methyl-1-pentanol, 2-ethylhexanol, propylene glycol, 3-furanmethanol, sec-octanol, ethyl pyruvate, 2, 3-butanedione, 2-methylpyrazine, m-xylene, α -terpineol, 1,7, 7-trimethylbisazulene [2.2.1] hept-2-ene, isophorone. The production of the aroma substances or the increase of the yield of the aroma substances can exert certain influence on the flavor and aroma of the beverage obtained by fermentation, so that the mixed fermentation product presents more unique aroma compared with a single-bacterium fermentation product, a wine beverage with unique flavor, aroma and taste can be produced, the consumer class is enriched, and the consumption selection is expanded.
Detailed Description
The following detailed description of the present invention will be made with reference to specific examples, but the scope of the present invention is not limited thereto.
Sources and documentations of biological materials
The yeast strain HSP22 used in the experimental examples, papilliotrema laurentii, was a new strain screened by the applicant's laboratory, with the following accession information:
naming: HSP22
And (4) classification name: yeast
The name of Latin is: papiliotrema laurentii
The preservation number is as follows: CCTCC M2021086
The preservation organization: china center for type culture Collection
The preservation date is as follows: 1 month 15 days 2021;
the address of the depository: wuhan university school (the first small facing of Wuhan university), Wuhan university collection, eight Wuhan district 299, Wuhan City, Hubei province.
Saccharomyces cerevisiae F33 was a commercial strain purchased from Lafford (Laffort) France.
The grape variety used was a wital ice grape, purchased from Ningxia Bagges drunk intersomatic wine village, Inc.
Group 1 example, Mixed fermentation Process of the invention
The embodiment of the group provides a mixed fermentation process based on the yeasts Papiliotrema laurentii and saccharomyces cerevisiae. All embodiments of this group share the following common features: the yeast Papiliotrema laurentii and the saccharomyces cerevisiae are subjected to mixed fermentation.
Any fermentation and production behavior of the yeast Papiliotrema laurentii in combination with Saccharomyces cerevisiae falls within the scope of the present invention as will be appreciated by those skilled in the art in light of the teachings of the present invention. Target products of fermentation include, but are not limited to: wine drink, fermented milk, bread, etc.
In a preferred embodiment, the yeast papiiotrema laurentii is yeast papiiotrema laurentii strain HSP 22; the preservation number of the yeast Papiliotrema laurentii strain HSP22 is CCTCC M2021086.
In a specific embodiment, the Saccharomyces cerevisiae is Saccharomyces cerevisiae strain F33.
According to the teaching of the present invention, other commercial strains can be selected by those skilled in the art for mixed fermentation, and besides F33, various commercial strains exist in the market, such as Saccharomyces cerevisiae V1116, Saccharomyces cerevisiae VL1, Saccharomyces cerevisiae X16, etc., and these strains can be used for mixed fermentation with the yeast PapiliotRelaxii strain HSP22 of the present invention, so as to obtain similar technical effects as the present invention.
In some embodiments, the mixed fermentation process based on the yeasts papilliottema laurentii and saccharomyces cerevisiae comprises the following steps: the activated strain and the activated strain are inoculated into a substrate for secondary fermentation for fermentation.
Preferably, said strain refers to: yeast papiioterma laurentiii strain HSP22, or, saccharomyces cerevisiae strain F33;
preferably, the activated strain refers to a strain inoculated in a culture medium for culture;
preferably, the culture temperature is 24-30 ℃, preferably 28 ℃; the culture time is 20-30h, preferably 24h, the rotation speed is 120-250rpm, preferably 150 rpm;
preferably, the inoculation refers to inoculating the initial strain stock solution into the culture medium according to the inoculation amount of 2-4%, preferably 3% in volume ratio;
preferably, the medium is a YPD medium; preferably, the YPD medium comprises the following components in mass-to-volume ratio: 0.5-3.5%, preferably 1%, yeast extract, 1.0-3.0%, preferably 2%, peptone, 1.0-5.0%, preferably 2%, glucose, and water in balance;
preferably, the initial strain stock solution refers to the strain stock in 15-35% by volume, preferably 25% glycerol/YPD medium;
more preferably, the activation of the strain is performed 1-3 times, preferably 2 times.
In other embodiments, the activated strain refers to: activated yeast strain papiioterma laurentiii HSP22, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate into the secondary fermentation refers to: firstly inoculating activated yeast Papiliotrema laurentiii strain HSP22 to a substrate for fermentation for 0-96h, preferably 48h, and then inoculating activated saccharomyces cerevisiae strain F33 for continuous fermentation;
preferably, the fermentation temperature is 18 ℃ +/-2 ℃;
preferably, the total inoculation amount of the activated yeast strain Papiliotrema laurentii HSP22 and the activated saccharomyces cerevisiae strain F33 is 106-107CFU, preferably 6X 106CFU；
Preferably, the fermentation is terminated when the substrate weight loss does not change for 3 consecutive days;
preferably, the substrate is grape juice.
Group 2 example, Process for the production of wine according to the invention
The present group of embodiments provides a method of producing wine. The present group of embodiments all have the following common features: mixing yeast Papiliotrema laurentii and Saccharomyces cerevisiae for fermentation with grape juice as substrate.
In some preferred embodiments, the yeast papiiotrema laurentii refers to yeast papiiotrema laurentii strain HSP 22; the preservation number of the yeast Papiliotrema laurentii strain HSP22 is CCTCC M2021086.
In a specific embodiment, the Saccharomyces cerevisiae is Saccharomyces cerevisiae strain F33.
In other specific embodiments, the wine production method comprises: the activated strain and the activated strain are inoculated into a substrate for secondary fermentation for fermentation.
Preferably, said strain refers to: yeast papiioterma laurentiii strain HSP22, or, saccharomyces cerevisiae strain F33;
preferably, the activated strain refers to a strain inoculated in a culture medium for culture;
preferably, the culture temperature is 24-30 ℃, preferably 28 ℃; the culture time is 20-30h, preferably 24h, the rotation speed is 120-250rpm, preferably 150 rpm;
preferably, the inoculation refers to inoculating the initial strain stock solution into the culture medium according to the inoculation amount of 2-4%, preferably 3% in volume ratio;
preferably, the medium is a YPD medium; preferably, the YPD medium comprises the following components in mass-to-volume ratio: 0.5-3.5%, preferably 1%, yeast extract, 1.0-3.0%, preferably 2%, peptone, 1.0-5.0%, preferably 2%, glucose, and water in balance;
preferably, the initial strain stock solution refers to the strain stock in 15-35% by volume, preferably 25% glycerol/YPD medium;
more preferably, the activation of the strain is performed 1-3 times, preferably 2 times.
In specific embodiments, the activated strain refers to: activated yeast strain papiioterma laurentiii HSP22, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate into the secondary fermentation refers to: firstly inoculating activated yeast Papiliotrema laurentiii strain HSP22 to a substrate for fermentation for 0-96h, preferably 48h, and then inoculating activated saccharomyces cerevisiae strain F33 for continuous fermentation;
when the time for fermenting by inoculating the activated yeast Papiliotrema laurentii strain HSP22 to the substrate is 0h, the activated yeast Papiliotrema laurentii strain HSP22 and the activated saccharomyces cerevisiae strain F33 can be simultaneously inoculated to the substrate for fermenting until the weight loss of the substrate is not changed for 3 days continuously, and the fermentation is stopped.
Preferably, the fermentation temperature is 18 ℃ +/-2 ℃;
preferably, the total inoculation amount of the activated yeast strain Papiliotrema laurentii HSP22 and the activated saccharomyces cerevisiae strain F33 is 106-107CFU, preferably 6X 106CFU；
Preferably, the fermentation is terminated when the substrate weight loss is not changed for 3 consecutive days.
In a further embodiment, the method of wine production further comprises: preparing grape juice;
the preparation of the grape juice comprises the following steps: squeezing grape fruit grains at low temperature;
preferably, the harvested grape fruit particles are pressed with ice by an air bag press;
preferably, pressing is performed while adding sulfur dioxide or K2S2O5And, pectinase;
preferably, the addition amount of sulfur dioxide or K2S2O5 is 30-100mg/L, preferably 50 mg/L; the addition amount of pectinase is 10-30mg/L, preferably 20 mg/L;
preferably, the grape fruit pieces are uniformly sized fruit pieces;
preferably, the grape fruit particles are grape fruit particles collected when the temperature of the mature grapes continuously drops below 24 hours to 8 ℃. Group 3 examples, wine of the invention
The present group of embodiments provides a wine. All embodiments of this group share the following common features: produced by the wine production method of any one of the group 2 examples.
The wine of the invention produces the following aroma substances which cannot be produced by the wine fermented by single bacteria: isobutyl acetate, phenethyl acetate, 2-benzylpropionic acid, 3-methyl-1-pentanol, 2-ethylhexanol, propylene glycol, 3-furancarbinol, sec-octanol, ethyl pyruvate, 2, 3-butanedione, 2-methylpyrazine, m-xylene, alpha-terpineol, 1,7, 7-trimethyldiol, and isophorone, while being much higher in the content of the following aroma substances than in a single-bacterium fermented wine: ethyl acetate, octanoic acid, n-decanoic acid, butyric acid, 9-decenoic acid, glacial acetic acid, 2-methylbutyric acid, 1-pentanol, dodecanol, n-butanol, isobutanol, n-hexanol, benzyl alcohol, dihydrolinalool, isovaleraldehyde, 1, 3-propanediol monoethyl ether, ethyl nonanoate, ethyl 9-hexadecenoic acid, 3-hydroxy-2-butanone, methyl decanoate, acetyl 4-hydroxybutyrate, 1, 3-propanediol diacetate, vinyl acetate, naphthalene, damascenone, linalool, citronellol, linalool, 4-vinyl-2-methoxyphenol.
Experimental example, mixed bacteria fermentation process and fermentation data of the invention
1. Bacterial strains
The strains used in this experiment were: commercial s.cerevisiae F33 and the isolated and selected yeast papiioterma laurentiii strain HSP22 of the present invention.
2. Grape juice
The Weidai ice grape raw material is planted in Yuquan Yingning county, Yinchuan city, Ningxia Bagges Zuius boundary wine village GmbH (E106.02 degree, N38.24 degree). The grape vines are planted in 2013, small-canopy-frame cultivation is adopted, the plant row spacing is 1.0m multiplied by 2.0m, the grape vines are harvested in 2017, the grape vines are not harvested after being mature, the grape vines are harvested when the temperature is reduced to be below 24 hours-8 ℃, small fruit grains are removed, and ice grape fruits with the same size are randomly selected and squeezed at low temperature. Pressing harvested ice grape with ice by air bag press while adding sulfur dioxide (50mg/L K)2S2O5) And 20mg/L of pectinase (more than or equal to 500U/mg), inhibits bacteria and improves the juice yield. The squeezed grape juice has sugar content of 432g/dm3Acidity of 4.65g/dm3(tartaric acid) pH 4.21.
3. Fermentation operation
2 strains: saccharomyces cerevisiae strain F33, yeast strain papiiotema laurentiii HSP22 were all stored in 25% by volume glycerol/YPD medium prior to use. YPD medium is 1% yeast extract powder, 2% peptone and 2% glucose. Inoculating to 50mL triangular flask containing 40mL YPD medium and 250mL triangular flask containing 150mL YPD medium respectively according to 3% inoculum size of volume ratio, culturing at 28 deg.C, 150rpm for 24 hr to obtain 1 st activated bacterial liquid, inoculating to 50mL triangular flask containing 40mL YPD medium and 250mL YPD medium respectively according to 3% inoculum sizeAnd repeating the culture in a mL triangular flask to complete the 2 nd passage activation to obtain activated bacteria liquid. Inoculating activated yeast Papiliotema laurentiii strain HSP22 into collected grape juice, inoculating into S.cerevisiae F33 strain at an inoculation ratio of HSP22/F33 of 2:1 after 48 hours, wherein the total inoculation amount of the strain is controlled at 6 × 106CFU, using S.cerevisiae F33 pure fermented grape juice as blank control, fermenting at 18 + -2 deg.C, and stopping fermentation when grape juice weight loss is not changed for three consecutive days. All wine samples were centrifuged at 7500 rpm for 8 minutes and the supernatant was stored at 4 ℃.
4. Method for quantifying aroma substance
Headspace-solid phase microextraction-gas mass spectrometry (HS-SPME-GC/MS) was used. An 8mL sample of wine was accurately weighed into a headspace bottle containing 1.5g NaCl, while 394.08. mu.g/L4-methyl-1-pentanol (internal standard) was capped and sealed. Inserting CAR/DVB/PDMS extraction fiber, adsorbing at 45 deg.C for 30min, desorbing at 250 deg.C for 3 min, and performing GC-MS analysis. A chromatographic column: InertCap WAX polar chromatography column (60m × 0.25mm, 0.25 μm); the temperature rising procedure is as follows: maintaining at 40 deg.C for 5min, heating to 120 deg.C at 3 deg.C/min, heating to 230 deg.C at 8 deg.C/min, and maintaining for 10 min; the flow rate of the carrier gas (He) was 0.8mL/min, and was not split. Electron bombardment ion source; electron energy 70 eV; the transmission line temperature was 275 ℃; the ion source temperature is 230 ℃; the activation voltage is 1.5V; the filament flow is 0.25 mA; the mass scanning range m/z is 33-450. Compound quantitative analysis was performed using external standard quantitation method.
5. Data analysis method
All samples were averaged in 3 replicates respectively. Single-factor analysis of variance (ANOVA) and Duncan's multi-range test (P <0.05) were performed using SPSS 22.0for Windows (SPSS inc., Chicago, IL, US); unscamblebler 9.7(CAMO ASA, Norway) was analyzed by partial least squares.
The final statistical interpretation gives the following Table 1, where the data are in μ g/L, meaning: the content of the aroma substances in each liter of wine.
TABLE 1
The aroma threshold value in the above table 1 refers to the lower limit value of the lowest concentration at which a human can smell the substance, and the aroma description and the threshold value are subject to the relevant literature reports that the aroma description and the threshold value of the aroma substance are recorded, and the aroma substance without the aroma description and the threshold value in the table is the literature that the relevant literature about the aroma description and the threshold value of the substance is not searched. "F33" in the header refers to data on single fermentation using the commercial strain Saccharomyces cerevisiae F33 and "F33 _ HSP 22" refers to data on mixed fermentation using the commercial strains Saccharomyces cerevisiae F33 and the yeast Papiliotrema laurentii strain HSP 22.
As is well known in the field of fermentation, factors influencing fermentation are numerous and complex, and the components of a fermented product can change due to the change of factors such as raw material batches, raw material components, fermentation conditions, temperature, time and the like, so that the condition that single-strain fermentation is higher than mixed-strain fermentation on certain aroma components can occur, which is a normal phenomenon in the field.
Claims (10)
1. A mixed fermentation process based on yeast Papiliotrema laurentii and saccharomyces cerevisiae is characterized in that the yeast Papiliotrema laurentii and the saccharomyces cerevisiae are subjected to mixed fermentation.
2. The mixed fermentation process based on the yeast Papiliotrema laurentii and the saccharomyces cerevisiae as claimed in claim 1, wherein the yeast Papiliotrema laurentii refers to a yeast Papiliotrema laurentii strain HSP 22; the preservation number of the yeast Papiliotrema laurentii strain HSP22 is CCTCC M2021086; preferably, the Saccharomyces cerevisiae refers to Saccharomyces cerevisiae strain F33.
3. The mixed fermentation process based on the yeast Papiliotrema laurentii and the saccharomyces cerevisiae as claimed in claim 1, which comprises the following steps: the activated strain and the activated strain are inoculated into a substrate for secondary fermentation for fermentation.
Preferably, said strain refers to: yeast papiioterma laurentiii strain HSP22, or, saccharomyces cerevisiae strain F33;
preferably, the activated strain refers to a strain inoculated in a culture medium for culture;
preferably, the culture temperature is 24-30 ℃, preferably 28 ℃; the culture time is 20-30h, preferably 24h, the rotation speed is 120-250rpm, preferably 150 rpm;
preferably, the inoculation refers to inoculating the initial strain stock solution into the culture medium according to the inoculation amount of 2-4%, preferably 3% in volume ratio;
preferably, the medium is a YPD medium; preferably, the YPD medium comprises the following components in mass-to-volume ratio: 0.5-3.5%, preferably 1%, yeast extract, 1.0-3.0%, preferably 2%, peptone, 1.0-5.0%, preferably 2%, glucose, and water in balance;
preferably, the initial strain stock solution refers to the strain stock in 15-35% by volume, preferably 25% glycerol/YPD medium;
more preferably, the activation of the strain is performed 1-3 times, preferably 2 times.
4. The mixed fermentation process based on the yeasts Papiliotrema laurentii and Saccharomyces cerevisiae according to claim 3, wherein the activated strains refer to: activated yeast strain papiioterma laurentiii HSP22, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate into the secondary fermentation refers to: firstly inoculating activated yeast Papiliotrema laurentiii strain HSP22 to a substrate for fermentation for 0-96h, preferably 48h, and then inoculating activated saccharomyces cerevisiae strain F33 for continuous fermentation;
preferably, the fermentation temperature is 18 ℃ +/-2 ℃;
preferably, the total inoculum size of the activated yeast strain, strain PAPilliotrema laurentii HSP22, and activated Saccharomyces cerevisiae strain F33 is 106-107CFU, preferably 6X 106CFU；
Preferably, the fermentation is terminated when the substrate weight loss does not change for 3 consecutive days;
preferably, the substrate is grape juice.
5. A wine production method is characterized in that grape juice is used as a substrate, and yeast Papiliotrema laurentii and saccharomyces cerevisiae are subjected to mixed fermentation;
the yeast refers to yeast strain papiiotema laurentiii HSP 22; the preservation number of the yeast Papiliotrema laurentii strain HSP22 is CCTCC M2021086.
6. A wine production method according to claim 5, characterised in that the Saccharomyces cerevisiae is Saccharomyces cerevisiae strain F33.
7. A wine production process according to claim 5 or 6, comprising: the activated strain and the activated strain are inoculated into a substrate for secondary fermentation for fermentation.
Preferably, said strain refers to: yeast papiioterma laurentiii strain HSP22, or, saccharomyces cerevisiae strain F33;
preferably, the activated strain refers to a strain inoculated in a culture medium for culture;
preferably, the culture temperature is 24-30 ℃, preferably 28 ℃; the culture time is 20-30h, preferably 24h, the rotation speed is 120-250rpm, preferably 150 rpm;
preferably, the inoculation refers to inoculating the initial strain preservation solution into the culture medium according to the inoculation amount of 3% by volume;
preferably, the medium is a YPD medium; preferably, the YPD medium comprises the following components in mass-to-volume ratio: 0.5-3.5%, preferably 1%, yeast extract, 1.0-3.0%, preferably 2%, peptone, 1.0-5.0%, preferably 2%, glucose, and water in balance;
preferably, the initial strain stock solution refers to the strain stock in 15-35% by volume, preferably 25% glycerol/YPD medium;
more preferably, the activation of the strain is performed 1-3 times, preferably 2 times.
8. A wine production process according to claim 7, wherein the activated strain is: activated yeast strain papiioterma laurentiii HSP22, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate into the secondary fermentation refers to: firstly inoculating activated yeast Papiliotrema laurentiii strain HSP22 to a substrate for fermentation for 0-96h, preferably 48h, and then inoculating activated saccharomyces cerevisiae strain F33 for continuous fermentation;
preferably, the fermentation temperature is 18 ℃ +/-2 ℃;
preferably, the total inoculation amount of the activated yeast strain Papiliotrema laurentii HSP22 and the activated saccharomyces cerevisiae strain F33 is 106-107CFU, preferably 6X 106CFU；
Preferably, the fermentation is terminated when the substrate weight loss is not changed for 3 consecutive days.
9. A wine production process according to any one of claims 5 to 8, further comprising: preparing grape juice;
the preparation of the grape juice comprises the following steps: squeezing grape fruit grains at low temperature;
preferably, the harvested grape fruit particles are pressed with ice by an air bag press;
preferably, pressing is performed while adding sulfur dioxide or K2S2O5And, pectinase;
preferably, sulfur dioxide or K2S2O5The addition amount of (B) is 30-100mg/L, preferably 50 mg/L; the addition amount of pectinase is 10-30mg/L, preferably 20 mg/L;
preferably, the grape fruit pieces are uniformly sized fruit pieces;
preferably, the grape fruit particles are grape fruit particles collected when the temperature of the mature grapes continuously drops below 24 hours to 8 ℃.
10. Wine produced by the wine production method of any one of claims 5 to 9.
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