CN112940884B - Mixed bacteria fermentation process based on Curvibasidium pallidicorallinum - Google Patents

Mixed bacteria fermentation process based on Curvibasidium pallidicorallinum Download PDF

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CN112940884B
CN112940884B CN202110189033.5A CN202110189033A CN112940884B CN 112940884 B CN112940884 B CN 112940884B CN 202110189033 A CN202110189033 A CN 202110189033A CN 112940884 B CN112940884 B CN 112940884B
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pallidicorallinum
curvibasidium
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王晓菁
葛谦
吴燕
开建荣
赵子丹
张锋锋
孙翔宇
马婷婷
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Ningxia Institute of Quality Standards and Testing Technology for Agro Products of Ningxia Agricultural Product Quality Monitoring Center
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    • C12G1/0203—Preparation of must from grapes; Must treatment and fermentation by microbiological or enzymatic treatment
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Abstract

The invention discloses a mixed bacteria fermentation process based on Curvibasidium pallidicorallinum, and belongs to the technical field of microorganisms. The mixed bacteria fermentation process based on Curvibasidium pallidicorallinum is characterized in that: mixing Curvinyidieumparllicocellinum with Saccharomyces cerevisiae, and fermenting. The product obtained by the mixed bacteria fermentation process of the invention has unique flavor compared with a single bacteria fermentation product, and can produce a wine beverage with unique flavor, fragrance and taste, enrich consumer products and enlarge consumption selection.

Description

Mixed bacteria fermentation process based on Curvibasidium pallidicorallinum
Technical Field
The invention belongs to the technical field of beverage fermentation, and particularly relates to a mixed bacteria fermentation process based on Curvibasidium pallidicorallinum.
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.
There are reports in the prior art that saccharomyces cerevisiae and non-saccharomyces cerevisiae are subjected to mixed fermentation to prepare alcoholic beverages, for example: the invention patent application 202011019509.2 discloses a method for preparing fruit wine by mixing rhodotorula mucilaginosa Bei-29 and saccharomyces cerevisiae for fermentation, which can improve the content of carotenoids (astaxanthin, beta-carotene, zeaxanthin and lycopene) in the fermented base wine, and improve the components and the content of norisoprenoids, terpene aroma substances and fruit aroma esters.
In order to improve the flavor and aroma of alcoholic beverages, more mixed fermentation processes are needed to be developed in the field. However, there has not been a report in the art on the existence of a fermentation process using yeast Curvibasidium pallidicorallinum, and more on the mixed fermentation of yeast Curvibasidium pallidicorallinum and Saccharomyces cerevisiae.
Disclosure of Invention
Based on the above-mentioned needs and blank in the art, the invention provides a mixed fermentation process based on yeast Curvibasidium pallidicorallinum and Saccharomyces cerevisiae Saccharomyces cerevisiae, which is remarkably improved in a plurality of aroma substances affecting the flavor of alcoholic beverages compared with single-bacteria fermentation of Saccharomyces cerevisiae F33.
The technical scheme of the invention is as follows:
a mixed fermentation process based on Curvibasidium pallidicorallinum, which is characterized in that Curvibasidium pallidicorallinum and saccharomyces cerevisiae are subjected to mixed fermentation.
Preferably, the Curvibasidium pallidicorallinum strain Curvibasidium pallidicorallinum QTX; the preservation number of the strain Curvibasidium pallidicorallinum QTX is CCTCC M2021082.
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 Curvibasidium pallidicorallinum strain QTX11, 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 Curvibasidium pallidicorallinum strain QTX11, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating activated yeast Curvibasidium pallidicorallinum strain QTX11 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 yeast Curvibasidium pallidicorallinum strain QTX11 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 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 Curvibasidium pallidicorallinum strain QTX11; the preservation number of the yeast Curvibasidium pallidicorallinum strain QTX11 is CCTCC M2021082.
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 Curvibasidium pallidicorallinum strain QTX11, 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 Curvibasidium pallidicorallinum strain QTX11, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating activated yeast Curvibasidium pallidicorallinum strain QTX11 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 yeast Curvibasidium pallidicorallinum strain QTX11 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 Curvibasidium pallidicorallinum and saccharomyces cerevisiae are adopted for mixed fermentation, and among dozens of aroma substances produced, the yield of most aroma substances is improved, for example, the yield of 9-decenoic acid is improved by 54%, the yield of glacial acetic acid is improved by about 84%, the yield of 2-methylbutanoic acid is improved by 89%, the yield of 1-pentanol is improved by about 60%, the yield of dodecanol is improved by 40%, the yield of isobutanol is improved by about 57%, the yield of n-hexanol is improved by 56%, the yield of 2-heptanol is improved by about 10 times, the yield of benzyl alcohol is improved by 32%, the yield of isovaleraldehyde is improved by 67%, the yield of 3-hydroxy-2-butanone is improved by about 4 times, the yield of vinyl acetate is improved by about 3 times, the yield of Ma Shitong is improved by 1.3 times, and the yield of citronellol is improved by 76% and the yield of 2, 6-di (tert-butyl) -4-hydroxy-4-methyl-2, 5-cyclohexadien-1-one is improved by 1 times; 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, 2-propyl-1-heptanol, (Z) -3-methyl-2-hepten-1-ol, nonanal, ethyl octanoate, ethyl decanoate, ethyl pyruvate, 2, 3-butanedione, 2-methylpyrazine, 3, 5-trimethyl-1-hexene, alpha-terpineol, 2, 3-pentanedione, 1, 7-trimethyl-propylene [2.2.1] hept-2-ene, isophorone, (+) -alfalfa alkene. 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 Curvibasidium pallidicorallinum strain QTX11 used in the experimental example was a new strain screened in the applicant laboratory and deposited as follows:
naming: QTX11
Classification name: yeast
Latin name: curvibasidium pallidicorallinum
Deposit number: CCTCC M2021082
Preservation mechanism: china center for type culture Collection
Preservation date: 2021, 1-15;
saccharomyces cerevisiae F33 is a commercial strain available from Laffort, inc.
The grape variety used was Wedelian iced grape, purchased from Ningxia Bug Ge Zuimei International wine village Co.
Group 1 example, mixed bacteria fermentation Process of the invention
The embodiment of the group provides a mixed bacteria fermentation process based on Curvibasidium pallidicorallinum. All embodiments of this group share the following common features: and (3) carrying out mixed fermentation on Curvibasidium pallidicorallinum and saccharomyces cerevisiae.
Those skilled in the art can use Curvibasidium pallidicorallinum in combination with Saccharomyces cerevisiae to perform fermentation and production according to the teachings of the present invention, and any 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 Curvibasidium pallidicorallinum refers to the road strain Curvibasidium pallidicorallinum QTX; the preservation number of the strain Curvibasidium pallidicorallinum QTX is CCTCC M2021082.
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 Curvibasidium pallidicorallinum strain QTX11 of the present invention to obtain similar technical effects as those of the present invention.
In some embodiments, the yeast 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 Curvibasidium pallidicorallinum strain QTX11, 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 Curvibasidium pallidicorallinum strain QTX11, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating activated yeast Curvibasidium pallidicorallinum strain QTX11 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 yeast Curvibasidium pallidicorallinum strain QTX11 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 Curvibasidium pallidicorallinum with Saccharomyces cerevisiae with grape juice as substrate.
In some preferred embodiments, the yeast refers to yeast Curvibasidium pallidicorallinum strain QTX11; the preservation number of the yeast Curvibasidium pallidicorallinum strain QTX11 is CCTCC M2021082.
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 Curvibasidium pallidicorallinum strain QTX11, 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 Curvibasidium pallidicorallinum strain QTX11, or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating activated yeast Curvibasidium pallidicorallinum strain QTX11 to a substrate for fermentation for 0-96h, preferably 48h, and inoculating activated Saccharomyces cerevisiae strain F33 for continuous fermentation;
when the activated yeast Curvibasidium pallidicorallinum strain QTX11 is inoculated to the substrate for fermentation for 0h, the surface activated yeast Curvibasidium pallidicorallinum strain QTX11 and the activated Saccharomyces cerevisiae strain F33 can be simultaneously inoculated to the substrate for fermentation until the substrate weight loss is continuously changed for 3 days, and the fermentation is stopped.
Preferably, the fermentation temperature is 18 ℃ ± 2 ℃;
preferably, the total access of activated yeast Curvibasidium pallidicorallinum strain QTX11 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, 2-propyl-1-heptanol, (Z) -3-methyl-2-hepten-1-ol, nonanal, ethyl octanoate, ethyl decanoate, ethyl pyruvate, 2, 3-butanedione, 2-methylpyrazine, 3, 5-trimethyl-1-hexene, α -terpineol, 2, 3-pentanedione, 1, 7-trimethyl- α - [2.2.1] hept-2-ene, isophorone, (+) -alfalfa alkene, while being much higher in the content of the following aroma substances than the single fermented wine: 9-decenoic acid, glacial acetic acid, 2-methylbutanoic acid, 1-pentanol, dodecanol, isobutanol, n-hexanol, 2-heptanol, benzyl alcohol, isovaleraldehyde, 3-hydroxy-2-butanone, vinyl acetate, large Ma Shitong, citronellol, 2, 6-di (tert-butyl) -4-hydroxy-4-methyl-2, 5-cyclohexadien-1-one.
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 Curvibasidium pallidicorallinum strain QTX11 of the 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, yeast Curvibasidium pallidicorallinum strain QTX11 was kept 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 40mLYPD culture medium and a 250mL triangular flask filled with 150mLYPD culture medium respectively, wherein the culture temperature is 28 ℃, the rotation speed is 150rpm, the culture time is 24 hours, the bacterial liquid activated for the 1 st time is obtained, then inoculating the bacterial liquid according to the inoculum size of 3% into the 50mL triangular flask filled with 40mLYPD culture medium and the 250mL triangular flask filled with 150mLYPD culture medium respectively, repeating the culture, and completing the 2 nd passage activation, thus obtaining the bacterial liquid after activation. Firstly, inoculating QTX11 of activated yeast Curvibasidium pallidicorallinum strain into collected grape juice, after 48 hours, inoculating S.cerevisiae F33 strain according to the inoculation proportion that the volume ratio of QTX11/F33 is 2:1, and controlling the total inoculation amount of the strainMade into 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 (camasa, norway).
The final statistical treatment gave the following Table 1, in units of μg/L, meaning: the aroma content per liter of wine.
TABLE 1
The fragrance threshold in 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_QTX1" indicates data of mixed-strain fermentation using commercial strains of Saccharomyces cerevisiae F33 and Saccharomyces Curvibasidium pallidicorallinum strain QTX11.
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 (27)

1. Based onCurvibasidium pallidicorallinumThe mixed bacteria fermentation process is characterized by comprising the following steps of: preparing grape juice, activating a strain, inoculating the activated strain into a substrate to be fermented, and fermenting; the activated strain is inoculated with a substrate to be fermented for fermentation, which means that: firstly, activating the yeastCurvibasidium pallidicorallinumThe strain QTX11 is inoculated to a substrate for fermentation for 48-96 hours, and then the activated saccharomyces cerevisiae strain F33 is inoculated for continuous fermentation; the yeastCurvibasidium pallidicorallinumThe preservation number of the strain QTX11 is CCTCC M2021082; the fermentation temperature is 18+/-2 ℃; activated yeastCurvibasidium pallidicorallinumThe total inoculum size of the strain QTX11 and the activated Saccharomyces cerevisiae strain F33 was 10 6 -10 7 CFU; stopping fermentation when the substrate weight loss is not changed for 3 days; the substrate is grape juice; the preparation of grape juice refers to: squeezing the harvested grape fruit with ice by using an air bag squeezerSqueezing while adding sulfur dioxide and pectase, or K 2 S 2 O 5 And pectinase; sulfur dioxide or K 2 S 2 O 5 The addition amount of the (B) is 30-100mg/L; the addition amount of pectase is 10-30mg/L; the grape fruit particles are uniformly sized grape fruit particles harvested when the temperature is reduced to below-8 ℃ for 24 hours after the grape is ripe.
2. The base of claim 1Curvibasidium pallidicorallinumThe mixed bacteria fermentation process is characterized in that the activated strain is inoculated in a culture medium for culture.
3. The base of claim 2Curvibasidium pallidicorallinumThe mixed bacteria fermentation process 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 base according to claim 3Curvibasidium pallidicorallinumThe mixed bacteria fermentation process is characterized in that the culture temperature is 28 ℃; the incubation time was 24 hours and the rotational speed was 150rpm.
5. The base of claim 2Curvibasidium pallidicorallinumThe mixed bacteria fermentation process is characterized in that the inoculation refers to inoculating an initial strain preservation solution into a culture medium according to an inoculation amount of 2-4% by volume.
6. The base of claim 5Curvibasidium pallidicorallinumThe mixed bacteria fermentation process is characterized in that the inoculation refers to inoculating an initial strain preservation solution into a culture medium according to an inoculation amount of 3% by volume.
7. The basis of any one of claims 2,5, 6Curvibasidium pallidicorallinumThe mixed bacteria fermentation process is characterized in that the culture medium is YPD culture medium.
8. The base of claim 7Curvibasidium pallidicorallinumThe mixed bacteria fermentation process 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. The base of claim 8Curvibasidium pallidicorallinumThe mixed bacteria fermentation process 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. The basis of claim 5 or 6Curvibasidium pallidicorallinumThe mixed fermentation process is characterized in that the initial strain preservation solution refers to the strain preservation in a glycerol/YPD culture medium with the volume ratio of 15-35%.
11. The base of claim 10Curvibasidium pallidicorallinumThe mixed fermentation process is characterized in that the initial strain preservation solution refers to the strain preservation in a glycerol/YPD culture medium with the volume ratio of 25%.
12. The basis of claim 1 or 2Curvibasidium pallidicorallinumThe mixed bacteria fermentation process is characterized in that the activated strain is carried out for 1-3 times.
13. The base of claim 12Curvibasidium pallidicorallinumThe mixed fermentation process is characterized in that the activated strain is carried out for 2 times.
14. A method of producing wine comprising: preparing grape juice, activating a strain, inoculating the activated strain into a substrate grape juice to be fermented, and fermenting; the activated strain is inoculated with substrate grape juice to be fermented for fermentation, which means that: firstly, activating the yeastCurvibasidium pallidicorallinumBacterial strain QTX11 inoculated on the bottomFermenting for 48-96h, and inoculating activated Saccharomyces cerevisiae strain F33 for continuous fermentation; the yeastCurvibasidium pallidicorallinumThe preservation number of the strain QTX11 is CCTCC M2021082; the preparation of grape juice refers to: squeezing the harvested grape fruit with ice using an air bag squeezer, and adding sulfur dioxide and pectase, or K 2 S 2 O 5 And pectinase; sulfur dioxide or K 2 S 2 O 5 The addition amount of the (B) is 30-100mg/L; the addition amount of pectase is 10-30mg/L; the grape fruit particles are uniformly sized grape fruit particles harvested when the temperature is reduced to below-8 ℃ for 24 hours after the grape is ripe; the fermentation temperature is 18+/-2 ℃; activated yeastCurvibasidium pallidicorallinumThe total access of the strain QTX11 and the activated Saccharomyces cerevisiae strain F33 is 10 6 -10 7 CFU; the fermentation was terminated after the substrate loss was no longer changed for 3 consecutive days.
15. A wine production method according to claim 14, wherein said activating strain is a strain inoculated in a culture medium for cultivation.
16. A process for the production of wine according to claim 15, wherein the cultivation temperature is 24-30 ℃; the culture time is 20-30h, and the rotating speed is 120-250rpm.
17. A process for producing wine according to claim 16 wherein the cultivation temperature is 28 ℃; the incubation time was 24 hours and the rotational speed was 150rpm.
18. A method according to claim 15, wherein the inoculation is to inoculate a stock solution of the initial strain in a culture medium in an amount of 3% by volume.
19. A method of producing wine according to claim 15 or 18 wherein said medium is YPD medium.
20. A method of producing wine according to claim 19 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.
21. A method of producing wine according to claim 20 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.
22. A wine production method according to claim 18 wherein said initial strain preservation fluid means that the strain is preserved in 15-35% glycerol/YPD medium by volume.
23. A method of producing wine according to claim 22 wherein said initial strain-preserving fluid means strain is preserved in 25% glycerol/YPD medium by volume.
24. A wine production method according to claim 14 or 15, wherein said activating strain is performed 1-3 times.
25. A wine production method according to claim 24 wherein said activating strain is performed 2 times.
26. A method of producing wine according to claim 14 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.
27. Wine produced by the method according to any one of claims 14 to 26.
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