CN113150899B - 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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- CN113150899B CN113150899B CN202110189032.0A CN202110189032A CN113150899B CN 113150899 B CN113150899 B CN 113150899B CN 202110189032 A CN202110189032 A CN 202110189032A CN 113150899 B CN113150899 B CN 113150899B
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- laurentii
- saccharomyces cerevisiae
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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
Abstract
The invention relates to 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 yeast Papiliotrema laurentii and the saccharomyces cerevisiae is characterized in that the yeast Papiliotrema laurentii 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 yeast Papiliotrema laurentii and saccharomyces cerevisiae.
Background
Wine fermentation is a complex biochemical process in which yeast plays a very critical role, for example, in the conversion of sugar to ethanol, carbon dioxide and other thousands of secondary metabolites. A great deal of scientific researches show that the quality of the wine is highly dependent on the metabolic activities and fermentation behaviors of different yeasts, and the different yeasts have important contributions to the chemical composition, the organoleptic properties, the flavor characteristics and the like of the wine. Saccharomyces cerevisiae is the most widely used strain in wine industrial production so far, and has the advantages of ensuring the risk of deterioration in the fermentation process of wine, having good fermentation power, along with the problems of single flavor characteristic, serious homogenization phenomenon and the like. Therefore, in order to pursue style characterization of the wine, the aroma characteristics are more representative, diversified and complex, and brewers often adopt a method of mixed fermentation of saccharomyces cerevisiae and non-saccharomyces cerevisiae, especially some indigenous yeasts with strong adaptability and representativeness, so as to improve and enhance the flavor quality of the wine.
Non-saccharomyces cerevisiae has become an option to improve wine quality. Numerous studies have shown that non-Saccharomyces cerevisiae is capable of producing enzymes and some of the secondary metabolites we desire, thereby improving wine aroma and flavor characteristics, and controlling the growth of some undesirable species in wine, but has the disadvantage of inadequate fermentation kinetics. The mixing fermentation of non-Saccharomyces cerevisiae and Saccharomyces cerevisiae can not only improve the fragrance diversity and complexity of the wine, but also make up for the problem of insufficient fermentation power of non-Saccharomyces cerevisiae, and is an effective method for improving the fragrance quality of the wine.
The related report of the fermentation by using yeast Papiliotrema laurentii is very rare in the field, but regarding the mixed fermentation process of yeast Papiliotrema laurentii and saccharomyces cerevisiae, no related report is found in the field.
Disclosure of Invention
Based on the above-mentioned needs and blank in the art, the invention provides a mixed fermentation process based on yeast Papiliotrema laurentii and Saccharomyces cerevisiae Saccharomyces cerevisiae, which has very remarkable promotion on a plurality of aroma substances affecting the flavor of alcoholic beverages compared with the single-bacteria fermentation of Saccharomyces cerevisiae.
The technical scheme of the invention is as follows:
a mixed fermentation process based on yeast Papiliotrema laurentii and Saccharomyces cerevisiae is characterized in that yeast Papiliotrema laurentii and Saccharomyces cerevisiae are subjected to mixed fermentation.
Preferably, the yeast Papiliotrema laurentii refers to yeast Papiliotrema laurentii strain HSP22; 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: and inoculating the activated strain and the activated strain into a fermentation substrate for fermentation.
Preferably, the strain refers to: yeast Papiliotrema laurentii 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 cultivation time is 20-30h, preferably 24h, and the rotation speed is 120-250rpm, preferably 150rpm;
preferably, the inoculation refers to inoculating the initial strain preservation solution into a culture medium according to an inoculation amount of 2-4% by volume, preferably 3%;
preferably, the medium is YPD medium; preferably, the YPD medium comprises the following components in mass-volume ratio: 0.5-3.5% yeast extract powder preferably 1%, 1.0-3.0% peptone preferably 2%, 1.0-5.0% glucose preferably 2%, and water for the rest;
preferably, the initial strain preservation solution refers to strain preservation in 15-35% glycerol/YPD medium, preferably 25% glycerol/YPD medium;
more preferably, the activation strain is performed 1-3 times, preferably 2 times.
The activated strain refers to: activated yeast Papiliotrema laurentii strain HSP22, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating activated yeast Papiliotrema laurentii strain HSP22 to a substrate, fermenting 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 activated yeast Papiliotrema laurentii strain HSP22, 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.
A production method of grape wine is characterized in that grape juice is taken as a substrate, and yeast and saccharomyces cerevisiae are subjected to mixed fermentation;
the yeast refers to yeast Papiliotrema laurentii strain HSP22; the preservation number of the yeast Papiliotrema laurentii strain HSP22 is CCTCC M2021086.
The saccharomyces cerevisiae refers to saccharomyces cerevisiae strain F33.
The wine production method comprises the following steps: and inoculating the activated strain and the activated strain into a fermentation substrate for fermentation.
Preferably, the strain refers to: yeast Papiliotrema laurentii 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 cultivation time is 20-30h, preferably 24h, and the rotation speed is 120-250rpm, preferably 150rpm;
preferably, the inoculation refers to inoculating the initial strain preservation solution into a culture medium according to an inoculation amount of 3% by volume;
preferably, the medium is YPD medium; preferably, the YPD medium comprises the following components in mass-volume ratio: 0.5-3.5% yeast extract powder preferably 1%, 1.0-3.0% peptone preferably 2%, 1.0-5.0% glucose preferably 2%, and water for the rest;
preferably, the initial strain preservation solution refers to strain preservation in 15-35% glycerol/YPD medium, preferably 25% glycerol/YPD medium;
more preferably, the activation strain is performed 1-3 times, preferably 2 times.
The activated strain refers to: activated yeast Papiliotrema laurentii strain HSP22, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating activated yeast Papiliotrema laurentii strain HSP22 to a substrate, fermenting 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 yeast Papiliotrema laurentii strain HSP22 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, yeast Papiliotrema laurentii and saccharomyces cerevisiae are adopted for mixed fermentation, and among tens of aroma substances produced, the yield of most aroma substances is improved, for example, ethyl acetate is improved by about 17.6%, octanoic acid is improved by 58%, n-decanoic acid is improved by 44%, butyric acid is improved by 1 time, 9-decenoic acid is improved by 1.26 times, glacial acetic acid is improved by 65%, 2-methylbutanoic acid is improved by 6.1 times, 1-pentanol is improved by 5.9 times, dodecanol is improved by 60%, n-butanol is improved by 4.3 times, isobutanol is improved by 58%, n-hexanol is improved by 23%, benzyl alcohol is improved by 37%, dihydrolinalool is improved by 17.5%, isovaleraldehyde is improved by 1.2 times, 1, 3-propanediol monoethyl ether is improved by 2.76 times, nonanoic acid is improved by 50%, 9-hexadecenoic acid ethyl ester is improved by 50%, 3-hydroxy-2-decanoate is improved by 73%, methyl decanoate is improved by 42%, 4-hydroxybutyrate is improved by 45%,1, 3-propanediol diacetate is improved by 1.9 times, 3-propanediol diacetate is improved by 54% and linalool is improved by 3.33%, linalool is improved by 2.12% by 3 times, 2.12% by 2.3 times, linalool is improved by 3.12% by 32% by 2; 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, 2-ethylhexanol, propylene glycol, 3-furanmethanol, sec-octanol, ethyl pyruvate, 2, 3-butanedione, 2-methylpyrazine, meta-xylene, alpha-terpineol, 1, 7-trimethyl-2, 1-hept-2-ene, isophorone. The production of the aroma substances or the improvement of the output of the aroma substances can generate certain influence on the flavor and aroma of the beverage obtained by fermentation, so that the mixed bacteria fermentation product presents more unique aroma compared with a single bacteria fermentation product, and the wine beverage with unique flavor, aroma and taste can be produced, so that the consumer product is enriched, and the consumption selection is enlarged.
Detailed Description
The following describes the invention in more detail with reference to specific examples, but is not intended to limit the scope of the invention.
Sources and documentations of biological materials
The yeast Papiliotrema laurentii strain HSP22 used in the experimental examples was a new strain screened in the applicant laboratory and deposited as follows:
naming: HSP22
Classification name: yeast
Latin name: papiliotrema laurentii
Deposit number: CCTCC M2021086
Preservation mechanism: china center for type culture Collection
Preservation date: 2021, 1-15;
deposit unit address: in the Jiuqiu 299 Wuhan university (first appendage of Wuhan university) in Wuhan City, hubei province, wuhan university collection center.
Saccharomyces cerevisiae F33 is a commercial strain available from Laffort, inc.
The grape variety used was Wedelian iced grape, purchased from Ningxia Bug Ge Zuimei International wine village Co.
Group 1 example, mixed bacteria fermentation Process of the invention
The embodiment of the group provides a mixed fermentation process based on yeast Papiliotrema laurentii and saccharomyces cerevisiae. All embodiments of this group share the following common features: mixing yeast Papiliotrema laurentii and Saccharomyces cerevisiae for fermentation.
Those skilled in the art can use yeast Papiliotrema laurentii in combination with Saccharomyces cerevisiae to perform fermentation and production according to the teachings of the present invention, and any such actions fall within the scope of the present invention. Target products of fermentation include, but are not limited to: alcoholic beverages, fermented milk, bread, etc.
In a preferred embodiment, the yeast Papiliotrema laurentii refers to yeast Papiliotrema laurentii strain HSP22; the preservation number of the yeast Papiliotrema laurentii strain HSP22 is CCTCC M2021086.
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 yeast Papiliotrema laurentii strain HSP22 of the present invention to obtain similar technical effects as those of the present invention.
In some embodiments, the yeast Papiliotrema laurentii and saccharomyces cerevisiae-based mixed fermentation process comprises the following steps: and inoculating the activated strain and the activated strain into a fermentation substrate for fermentation.
Preferably, the strain refers to: yeast Papiliotrema laurentii 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 cultivation time is 20-30h, preferably 24h, and the rotation speed is 120-250rpm, preferably 150rpm;
preferably, the inoculation refers to inoculating the initial strain preservation solution into a culture medium according to an inoculation amount of 2-4% by volume, preferably 3%;
preferably, the medium is YPD medium; preferably, the YPD medium comprises the following components in mass-volume ratio: 0.5-3.5% yeast extract powder preferably 1%, 1.0-3.0% peptone preferably 2%, 1.0-5.0% glucose preferably 2%, and water for the rest;
preferably, the initial strain preservation solution refers to strain preservation in 15-35% glycerol/YPD medium, preferably 25% glycerol/YPD medium;
more preferably, the activation strain is performed 1-3 times, preferably 2 times.
In other embodiments, the activated strain refers to: activated yeast Papiliotrema laurentii strain HSP22, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating activated yeast Papiliotrema laurentii strain HSP22 to a substrate, fermenting 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 yeast Papiliotrema laurentii strain HSP22 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 yeast Papiliotrema laurentii and Saccharomyces cerevisiae with grape juice as substrate.
In some preferred embodiments, the yeast Papiliotrema laurentii refers to yeast Papiliotrema laurentii strain HSP22; the preservation number of the yeast Papiliotrema laurentii strain HSP22 is CCTCC M2021086.
In a specific embodiment, the saccharomyces cerevisiae refers to saccharomyces cerevisiae strain F33.
In other specific embodiments, the wine production method comprises: and inoculating the activated strain and the activated strain into a fermentation substrate for fermentation.
Preferably, the strain refers to: yeast Papiliotrema laurentii 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 cultivation time is 20-30h, preferably 24h, and the rotation speed is 120-250rpm, preferably 150rpm;
preferably, the inoculation refers to inoculating the initial strain preservation solution into a culture medium according to an inoculation amount of 2-4% by volume, preferably 3%;
preferably, the medium is YPD medium; preferably, the YPD medium comprises the following components in mass-volume ratio: 0.5-3.5% yeast extract powder preferably 1%, 1.0-3.0% peptone preferably 2%, 1.0-5.0% glucose preferably 2%, and water for the rest;
preferably, the initial strain preservation solution refers to strain preservation in 15-35% glycerol/YPD medium, preferably 25% glycerol/YPD medium;
more preferably, the activation strain is performed 1-3 times, preferably 2 times.
In specific embodiments, the activated strain refers to: activated yeast Papiliotrema laurentii strain HSP22, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating activated yeast Papiliotrema laurentii strain HSP22 to a substrate, fermenting for 0-96h, preferably 48h, and inoculating activated Saccharomyces cerevisiae strain F33 for continuous fermentation;
when activated yeast Papiliotrema laurentii strain HSP22 is inoculated to a substrate for fermentation for 0h, surface activated yeast Papiliotrema laurentii strain HSP22 and activated Saccharomyces cerevisiae strain F33 can be simultaneously inoculated to the substrate for fermentation until the substrate weight loss is no longer changed for 3 days to terminate fermentation.
Preferably, the fermentation temperature is 18 ℃ ± 2 ℃;
preferably, the total access of activated yeast Papiliotrema laurentii strain HSP22 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, 2-ethylhexanol, propylene glycol, 3-furanmethanol, sec-octanol, ethyl pyruvate, 2, 3-butanedione, 2-methylpyrazine, meta-xylene, alpha-terpineol, 1, 7-trimethyl, and [2.2.1] hept-2-ene, isophorone, and at the same time is much higher than the single fermented wine in terms of the content of aroma substances: ethyl acetate, octanoic acid, n-decanoic acid, butyric acid, 9-decenoic acid, glacial acetic acid, 2-methylbutanoic acid, 1-pentanol, dodecanol, n-butanol, isobutanol, n-hexanol, benzyl alcohol, dihydrolinalool, isopentyl aldehyde, 1, 3-propanediol monoethyl ether, ethyl nonanoate, ethyl 9-hexadecenoate, 3-hydroxy-2-butanone, methyl decanoate, acetyl 4-hydroxybutyrate, 1, 3-propanediol diacetate, vinyl acetate, naphthalene, large Ma Shitong, linalool, citronellol, linalool, 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 yeast Papiliotrema laurentii strain of the invention HSP22.
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, yeast Papiliotrema laurentii strain, HSP22, was all stored in 25% glycerol/YPD medium by volume prior to use. YPD medium was 1% yeast extract, 2% peptone, 2% glucose. Inoculating the bacterial liquid according to the inoculum size of 3% of the volume ratio into a 50mL triangular flask filled with 40mL of YPD culture medium and a 250mL triangular flask filled with 150mL of YPD culture medium respectively, wherein the culture temperature is 28 ℃, the rotation speed is 150rpm, the culture time is 24 hours, the bacterial liquid activated for the 1 st time is obtained, then inoculating the bacterial liquid according to the inoculum size of 3% into the 50mL triangular flask filled with 40mL of YPD culture medium and the 250mL triangular flask filled with 150mL of YPD culture medium respectively, and repeating the culture to finish the 2 nd passage activation, thus obtaining the bacterial liquid after activation. The activated yeast Papiliotrema laurentii strain HSP22 is firstly inoculated into the collected grape juice, after 48 hours, the strain S.cerevisiae F33 is inoculated according to the inoculation proportion that the volume ratio of the HSP22 to the F33 is 2:1, and the total inoculation amount of the strain is controlled to be 6 multiplied by 10 6 CFU, using s.cerevisiae F33 pure fermented grape juice as blank control, fermenting at 18±2 ℃ until grape juice weight loss is no longer changed for three consecutive days, stopping fermentation. All wine samples were centrifuged at 7500rpm for 8 minutes and the supernatant was stored at 4 ℃.
4. Method for quantifying aroma substances
A headspace-solid phase microextraction method-gas phase mass spectrometry (HS-SPME-GC/MS) is adopted. An accurate measurement of 8mL of wine sample was added to a headspace bottle containing 1.5g NaCl, while 394.08. Mu.g/L4-methyl-1-pentanol (internal standard) was capped and sealed. The CAR/DVB/PDMS extraction fiber is inserted, the extraction fiber is desorbed for 3min at 250 ℃ at the GC inlet after being adsorbed for 30min at 45 ℃ for GC-MS analysis. Chromatographic column: an InertCap WAX polar column (60 m 0.25mm,0.25 μm); the temperature-raising program is as follows: keeping the temperature at 40 ℃ for 5min, raising the temperature to 120 ℃ at 3 ℃/min, raising the temperature to 230 ℃ at 8 ℃/min, and keeping the temperature for 10min; the carrier gas (He) flow rate was 0.8mL/min, without split flow. An electron bombardment ion source; electron energy 70eV; the temperature of the transmission line is 275 ℃; the ion source temperature is 230 ℃; the activation voltage is 1.5V; filament flow 0.25mA; mass scanning range m/z is 33-450. Compound quantitative analysis was performed using an external standard quantitative method.
5. Data analysis method
All samples were averaged 3 times in parallel. Single-factor analysis of variance (ANOVA) and Duncan's multi-range test (P < 0.05) were performed using SPSS 22.0for 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_HSP22" indicates data of mixed-strain fermentation using a commercial strain of Saccharomyces cerevisiae F33 and a yeast Papiliotrema laurentii strain of HSP22.
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. Yeast-basedPapiliotrema laurentiiAnd a saccharomyces cerevisiae mixed fermentation process, which 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: firstly, activating the yeastPapiliotrema laurentiiInoculating the strain HSP22 to a substrate for fermentation for 48-96 hours, and inoculating activated saccharomyces cerevisiae strain F33 for continuous fermentation; the yeastPapiliotrema laurentiiThe preservation number of the strain HSP22 is CCTCC M2021086.
2. A yeast-based according to claim 1Papiliotrema laurentiiAnd a mixed fermentation process of saccharomyces cerevisiae, which is characterized in that the activated strain is inoculated in a culture medium for culture.
3. A yeast-based according to claim 2Papiliotrema laurentiiAnd 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 fermentation-based process according to claim 3Mother and motherPapiliotrema laurentiiAnd 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 yeast-based according to claim 2Papiliotrema laurentiiAnd a mixed fermentation process of saccharomyces cerevisiae, which is characterized in that the inoculation means that the initial strain preservation solution is inoculated into a culture medium according to the inoculation amount of 2-4% of the volume ratio.
6. A yeast-based according to claim 5Papiliotrema laurentiiAnd 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 yeast-based according to any one of claims 2, 5, 6Papiliotrema laurentiiAnd a mixed fermentation process of saccharomyces cerevisiae, wherein the culture medium is YPD culture medium.
8. A yeast-based according to claim 7Papiliotrema laurentiiAnd a saccharomyces cerevisiae mixed fermentation process, which is characterized in that the YPD culture medium comprises the following components in mass volume ratio: 0.5-3.5% yeast extract powder, 1.0-3.0% peptone, 1.0-5.0% glucose, and water in balance.
9. A yeast-based according to claim 8Papiliotrema laurentiiAnd a saccharomyces cerevisiae mixed fermentation process, which is characterized in that the YPD culture medium comprises the following components in mass volume ratio: 1% of yeast extract powder, 2% of peptone, 2% of glucose and the balance of water.
10. A yeast-based according to claim 5 or 6Papiliotrema laurentiiAnd Saccharomyces cerevisiae, wherein the initial strain preservation solution is prepared by culturing strain in 15-35% glycerol/YPDIn the base.
11. A yeast-based according to claim 10Papiliotrema laurentiiAnd a mixed fermentation process of saccharomyces cerevisiae, wherein the initial strain preservation solution means that the strain is preserved in a glycerol/YPD culture medium with a volume ratio of 25%.
12. A yeast-based according to claim 1 or 2Papiliotrema laurentiiAnd a mixed fermentation process of Saccharomyces cerevisiae, wherein the activated strain is performed 1-3 times.
13. A yeast-based according to claim 12Papiliotrema laurentiiAnd a mixed fermentation process of saccharomyces cerevisiae, wherein the activated strain is performed 2 times.
14. A yeast-based according to claim 1Papiliotrema laurentiiAnd a mixed fermentation process of saccharomyces cerevisiae, which is characterized in that the activated strain is inoculated with a substrate to be fermented for fermentation, and the fermentation process comprises the following steps: firstly, activating the yeastPapiliotrema laurentiiThe strain HSP22 is inoculated to a substrate for fermentation for 48 hours, and then the activated saccharomyces cerevisiae strain F33 is inoculated for continuous fermentation.
15. A yeast-based according to claim 1 or 14Papiliotrema laurentiiAnd Saccharomyces cerevisiae, and is characterized in that the fermentation temperature is 18+/-2 ℃.
16. A yeast-based according to claim 1 or 14Papiliotrema laurentiiAnd Saccharomyces cerevisiae, characterized in that the activated yeastPapiliotrema laurentiiThe total inoculum size of strain HSP22 and activated Saccharomyces cerevisiae strain F33 was 10 6 -10 7 CFU。
17. A yeast-based according to claim 16Papiliotrema laurentiiAnd Saccharomyces cerevisiae, characterized in that the activated yeastPapiliotrema laurentiiThe total inoculum size of strain HSP22 and activated Saccharomyces cerevisiae strain F33 was 6×10 6 CFU。
18. A yeast-based according to claim 1 or 14Papiliotrema laurentiiAnd a mixed fermentation process of saccharomyces cerevisiae, which is characterized in that the fermentation is stopped after the substrate weight loss is continuously changed for 3 days.
19. A yeast-based according to claim 18Papiliotrema laurentiiAnd a mixed fermentation process of saccharomyces cerevisiae, which is characterized in that 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: firstly, activating the yeastPapiliotrema laurentiiInoculating the strain HSP22 to a substrate for fermentation for 48-96 hours, and inoculating activated saccharomyces cerevisiae strain F33 for continuous fermentation; the yeastPapiliotrema laurentiiThe preservation number of the strain HSP22 is CCTCC M2021086.
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% yeast extract powder, 1.0-3.0% peptone, 1.0-5.0% glucose, and water in balance.
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 yeast 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 method of producing wine according to claim 20, which comprisesCharacterized in that the activated strain is inoculated with substrate grape juice to be fermented for fermentation, which means that: firstly, activating the yeastPapiliotrema laurentiiThe strain HSP22 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 yeast is activatedPapiliotrema laurentiiThe total access of strain HSP22 and activated Saccharomyces cerevisiae strain F33 was 10 6 -10 7 CFU。
35. A method of producing wine according to claim 34 wherein the yeast is activatedPapiliotrema laurentiiThe total access of strain HSP22 and activated Saccharomyces cerevisiae strain F33 was 6X 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 pressing is accompanied by the addition of sulphur dioxide orK 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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| CN110923080A (en) * | 2019-10-30 | 2020-03-27 | 镇江瑞德酒业有限公司 | Flavor enhancing brewing process for fresh grape brandy |
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| Mar＇ıa Victoria Mestre Furlani，等.Selection of non-Saccharomyces yeasts to be used in grape musts with high alcoholic potential: a strategy to obtain wines with reduced ethanol content.《FEMS Yeast Research》.2017,第1-9页. * |
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