CN112812980B - Mixed fermentation process based on Hansenula polymorpha and Saccharomyces cerevisiae - Google Patents
Mixed fermentation process based on Hansenula polymorpha and Saccharomyces cerevisiae Download PDFInfo
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Abstract
The invention discloses a mixed fermentation process based on Hansenula polymorpha and Saccharomyces cerevisiae, and belongs to the technical field of microorganisms. The mixed fermentation process is characterized in that Hansenula polymorpha and Saccharomyces cerevisiae in grape juice are subjected to mixed fermentation; the Hansenula polymorpha refers to Hansenula polymorpha Hanseniaspora uvarum strain QTX22; the preservation number of the Hansenula polymorpha Hanseniaspora uvarum strain QTX22 is CCTCC M2021083. The mixed bacteria fermentation product of the invention has unique flavor compared with the single bacteria fermentation product, and can produce a wine beverage with unique flavor, fragrance and taste, enrich the consumer product and enlarge 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 Hansenula polymorpha and Saccharomyces cerevisiae.
Background
Wine fermentation is a complex biochemical process in which Hansenula polymorpha plays a very critical role in fermentation, for example, the conversion of sugar into ethanol, carbon dioxide and other thousands of secondary metabolites. A great deal of scientific researches show that the quality of the wine is highly dependent on the metabolic activity and fermentation behavior of Hansenula polymorpha in different grape juice, and the Hansenula polymorpha in different grape juice has important contributions to the chemical composition, the organoleptic properties, the flavor characteristics and the like of the wine. Saccharomyces cerevisiae is the most widely used strain in wine industrial production so far, and has the advantages of ensuring the risk of deterioration in the fermentation process of wine, having good fermentation power, along with the problems of single flavor characteristic, serious homogenization phenomenon and the like. Therefore, in order to pursue style characterization of the wine, the aroma characteristics are more representative, diversified and complex, and brewers often adopt a method of mixed fermentation of saccharomyces cerevisiae and non-saccharomyces cerevisiae, especially some native grape juice Hansenula polymorpha 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 prior art currently reports about mixed fermentation of Hansenula polymorpha Hanseniaspora uvarum and Saccharomyces cerevisiae in grape juice: the invention patent application 201911057302.1 discloses a flavoring brewing process of fresh grape brandy, which improves the aroma components and the content of fermentation esters of grape varieties in the fermented raw wine through mixed fermentation of Hansenula polymorpha (Hanseniaspora uvarum) strain Yun268 and Saccharomyces cerevisiae (Saccharomyces cerevisiae) of grape juice with the preservation number of CCTCC NO: M2013658.
However, the types of aroma substances generated by the mixed fermentation in the prior art are limited, so in order to further improve the flavor and aroma of alcoholic beverages, more mixed fermentation processes capable of generating more types of aroma substances are needed to be developed in the field.
Disclosure of Invention
Based on the above requirements and blank in the art, the invention provides a mixed fermentation process based on Hansenula polymorpha Hanseniaspora uvarum and Saccharomyces cerevisiae Saccharomyces cerevisiae, which has very remarkable improvement on a plurality of aroma substances affecting the flavor of alcoholic beverages compared with single-fungus fermentation of Saccharomyces cerevisiae F33.
The technical scheme of the invention is as follows:
a mixed fermentation process based on Hansenula polymorpha and Saccharomyces cerevisiae is characterized in that Hansenula polymorpha and Saccharomyces cerevisiae are subjected to mixed fermentation;
the Hansenula polymorpha refers to Hansenula polymorpha Hanseniaspora uvarum strain QTX22; the preservation number of the Hansenula polymorpha Hanseniaspora uvarum strain QTX22 is CCTCC M2021083.
The saccharomyces cerevisiae refers to saccharomyces cerevisiae strain F33.
The mixed fermentation process based on the Hansenula polymorpha and the Saccharomyces cerevisiae comprises the following steps of: and inoculating the activated strain and the activated strain into a fermentation substrate for fermentation.
Preferably, the strain refers to: hansenula polymorpha Hanseniaspora uvarum strain QTX22 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 grape juice has Hansenula polymorpha Hanseniaspora uvarum strain QTX22 or activated Saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating activated Hansenula polymorpha Hanseniaspora uvarum strain QTX22 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 Hansenula polymorpha Hanseniaspora uvarum strain QTX22 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 Hansenula polymorpha and Saccharomyces cerevisiae in the grape juice are subjected to mixed fermentation;
the Hansenula polymorpha refers to Hansenula polymorpha Hanseniaspora uvarum strain QTX22; the preservation number of the Hansenula polymorpha Hanseniaspora uvarum strain QTX22 is CCTCC M2021083.
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: hansenula polymorpha Hanseniaspora uvarum strain QTX22 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 grape juice has Hansenula polymorpha Hanseniaspora uvarum strain QTX22 or activated Saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating activated Hansenula polymorpha Hanseniaspora uvarum strain QTX22 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 must has a Hansenula polymorpha Hanseniaspora uvarum strain QTX22 and an activated Saccharomyces cerevisiae strain F33 of 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.
The invention adopts hansenula polymorpha Hanseniaspora uvarum and saccharomyces cerevisiae to carry out mixed fermentation, and the yield of most of aroma substances is improved in dozens of produced aroma substances, for example, the yield of isoamyl acetate is improved by 158 percent, the yield of methyl acetate is improved by about 61 percent, the yield of methyl acetate is improved by 180 percent, the yield of octanoic acid is improved by about 46 percent, the yield of n-decanoic acid is improved by 167 percent, the yield of 9-decenoic acid is improved by about 54 percent, the yield of 2-methylbutanoic acid is improved by 42 percent, the yield of 2-oxo-octanoic acid is improved by 37 percent, the yield of dimethyl silanediol is improved by nearly 2 times, the yield of acetaldehyde is improved by 38 percent, the n-hexanal is improved by nearly 138%, the vinyl ethyl ether is improved by 22%, the lauric acid ethyl ester is improved by 68%, the heptanoic acid ethyl ester is improved by 21%, the n-hexanoic acid ethyl ester is improved by 34%, the butyric acid ethyl ester is improved by 73%, the myristic acid ethyl ester is improved by 1 time, the 3-phenylpropionic acid ethyl ester is improved by 39%, the trans-4-decenoic acid ethyl ester is improved by 18%, the 9-hexadecenoic acid ethyl ester is improved by 50%, the diisobutyl phthalate is improved by 65%, the 2, 4-trimethylpentanediol isobutyl ester is improved by 36%, the 2, 4-trimethyl-1, 3-pentanediol monoisobutyrate is improved by 71%, and the vinyl acetate is improved by 43%; 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, lauric acid, 2-benzyl propionic acid, 3-methyl-1-pentanol, 2-ethylhexanol, propylene glycol, (Z) -3-methyl-2-hepten-1-ol, 2-tridecanone, isopentyl decanoate, m-xylene, alpha-terpineol, levo-beta-pinene, 2, 4-di-tert-butylphenol. 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 Hansenula polymorpha Hanseniaspora uvarum strain QTX22 used in the experimental example is a new strain screened by the applicant laboratory, and the preservation information is as follows:
naming: QTX22
Classification name: hansenula polymorpha of grape juice
Latin name: hanseniaspora uvarum
Deposit number: CCTCC M2021083
Preservation mechanism: china center for type culture Collection
Preservation address: in the Jiuqiu 299 Wuhan university school (first He-shou face of Wuhan university) in Wuhan City, hubei province, wuhan university collection center
Preservation date: 2021, 1-15;
saccharomyces cerevisiae F33 is a commercial strain available from Laffort, inc.
The grape variety used was Wedelian iced grape, purchased from Ningxia Bug Ge Zuimei International wine village Co.
Group 1 example, mixed bacteria fermentation Process of the invention
The embodiment of the group provides a mixed fermentation process based on Hansenula polymorpha and Saccharomyces cerevisiae. All embodiments of this group share the following common features: mixing fermentation is carried out on Hansenula polymorpha and Saccharomyces cerevisiae in grape juice; the Hansenula polymorpha refers to Hansenula polymorpha Hanseniaspora uvarum strain QTX22; the preservation number of the Hansenula polymorpha Hanseniaspora uvarum strain QTX22 is CCTCC M2021083.
According to the teaching of the present invention, any fermentation and production activities of Hansenula polymorpha strain Hanseniaspora uvarum QTX22 in combination with Saccharomyces cerevisiae falls within the scope of the present invention. Target products of fermentation include, but are not limited to: alcoholic beverages, fermented milk, bread, etc.
In a specific embodiment, the saccharomyces cerevisiae refers to saccharomyces cerevisiae strain F33.
According to the teaching of the invention, a person skilled in the art can select other commercial strains for mixed fermentation, and besides F33, various commercial strains exist on the market at present, for example Saccharomyces cerevisiae V1116, saccharomyces cerevisiae VL1, saccharomyces cerevisiae X16 and the like, and all the strains can be used for mixed fermentation with the Hansenula polymorpha Hanseniaspora uvarum strain QTX22 of the grape juice of the invention, so that similar technical effects as the invention are obtained.
In some embodiments, the mixed fermentation process based on Hansenula polymorpha and Saccharomyces cerevisiae in grape juice comprises the following steps: and inoculating the activated strain and the activated strain into a fermentation substrate for fermentation.
Preferably, the strain refers to: hansenula polymorpha Hanseniaspora uvarum strain QTX22 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 grape juice has Hansenula polymorpha Hanseniaspora uvarum strain QTX22 or activated Saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating activated Hansenula polymorpha Hanseniaspora uvarum strain QTX22 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 must has a Hansenula polymorpha Hanseniaspora uvarum strain QTX22 and an activated Saccharomyces cerevisiae strain F33 of 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: taking grape juice as a substrate, and carrying out mixed fermentation on Hansenula polymorpha and Saccharomyces cerevisiae in the grape juice; the Hansenula polymorpha refers to Hansenula polymorpha Hanseniaspora uvarum strain QTX22; the preservation number of the Hansenula polymorpha Hanseniaspora uvarum strain QTX22 is CCTCC M2021083.
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: hansenula polymorpha Hanseniaspora uvarum strain QTX22 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 grape juice has Hansenula polymorpha Hanseniaspora uvarum strain QTX22 or activated Saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating activated Hansenula polymorpha Hanseniaspora uvarum strain QTX22 to a substrate, fermenting for 0-96h, preferably 48h, and inoculating activated Saccharomyces cerevisiae strain F33 for continuous fermentation;
when activated Hansenula polymorpha Hanseniaspora uvarum strain QTX22 is inoculated to a substrate for fermentation for 0h, the activated Hansenula polymorpha Hanseniaspora uvarum strain QTX22 and activated Saccharomyces cerevisiae strain F33 can be simultaneously inoculated to the substrate for fermentation until the weight loss of the substrate is continuously 3 days and the fermentation is stopped.
Preferably, the fermentation temperature is 18 ℃ ± 2 ℃;
preferably, the total access of activated must has a Hansenula polymorpha Hanseniaspora uvarum strain QTX22 and an activated Saccharomyces cerevisiae strain F33 of 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, lauric acid, 2-benzyl propionic acid, 3-methyl-1-pentanol, 2-ethylhexanol, propylene glycol, (Z) -3-methyl-2-hepten-1-ol, 2-tridecanone, isopentyl decanoate, meta-xylene, alpha-terpineol, levo-beta-pinene, 2, 4-di-tert-butylphenol, while being much higher than the monoallyl fermented wine in terms of the content of aroma substances: isoamyl acetate, methyl acetate, octanoic acid, n-decanoic acid, 9-decenoic acid, 2-methylbutanoic acid, 2-oxooctanoic acid, dimethylsilanediol, acetaldehyde, n-hexanal, vinyl ethyl ether, ethyl laurate, ethyl heptanoate, ethyl n-hexanoate, ethyl butyrate, ethyl myristate, ethyl 3-phenylpropionate, ethyl trans-4-decenoate, ethyl 9-hexadecenoate, diisobutyl phthalate, isobutyl 2, 4-trimethylpentanediol, 2, 4-trimethyl-1, 3-pentanediol monoisobutyrate, vinyl acetate.
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 Hansenula polymorpha Hanseniaspora uvarum strain QTX22 isolated and screened for grape juice according to 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, hansenula polymorpha Hanseniaspora uvarum strain, QTX22 strain, were all used before useStored in 25% glycerol/YPD medium by volume. 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. Firstly, inoculating the activated Hansenula polymorpha Hanseniaspora uvarum strain QTX22 into the collected grape juice, and after 48 hours, inoculating the S.cerevisiae F33 strain according to the inoculation proportion that the volume ratio of QTX22/F33 is 2:1, wherein the total inoculation amount of the strain is controlled to be 6 multiplied by 10 6 CFU, using s.cerevisiae F33 pure fermented grape juice as blank control, fermenting at 18±2 ℃ until grape juice weight loss is no longer changed for three consecutive days, stopping fermentation. All 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 top refers to data obtained by single-strain fermentation using a commercial strain, saccharomyces cerevisiae F33, and "F33_QTX22" refers to data obtained by mixed-strain fermentation using a commercial strain, saccharomyces cerevisiae F33 and Hansenula polymorpha strain Hanseniaspora uvarum strain QTX22.
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 (28)
1. A mixed fermentation process based on Hansenula polymorpha and Saccharomyces cerevisiae is characterized in that Hansenula polymorpha and Saccharomyces cerevisiae are subjected to mixed fermentation;
hansenula polymorpha refers to Hansenula polymorpha in grape juiceHanseniaspora uvarumStrain QTX22; hansenula polymorpha of grape juiceHanseniaspora uvarumThe preservation number of the strain QTX22 is CCTCC M2021083; the saccharomyces cerevisiae refers to saccharomyces cerevisiae strain F33;
the mixed bacteria fermentation process comprises the following steps: activating the strain, and inoculating the activated strain into a substrate to be fermented for fermentation;
the activated strain refers to: activated Hansenula polymorpha in grape juiceHanseniaspora uvarumStrain QTX22, or activated saccharomyces cerevisiae strain F33;
the activated substrate to be fermented is inoculated for fermentation, which means that: firstly, activated grape juice Hansenula polymorpha isHanseniaspora uvarumThe strain QTX22 is inoculated on a substrate for fermentation for 48-96 hours, and then the activated saccharomyces cerevisiae strain F33 is inoculated for continuous fermentation;
the fermentation temperature is 18+/-2 ℃;
activated Hansenula polymorpha in grape juiceHanseniaspora uvarumThe total inoculum size of the strain QTX22 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 mixed bacteria fermentation process further comprises the following steps: preparing grape juice;
the preparation of grape juice refers to: squeezing grape fruit at low temperature;
squeezing the collected grape fruits with ice by using an air bag squeezer;
squeezing 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 uniform in size;
the grape fruit particles are the grape fruit particles which are harvested when the temperature is reduced to below-8 ℃ for 24 hours after the grape is ripe.
2. The mixed fermentation process based on Hansenula polymorpha and Saccharomyces cerevisiae according to claim 1, wherein the activated fermentation of the substrate to be fermented is carried out by: firstly, activated grape juice Hansenula polymorpha isHanseniaspora uvarumThe strain QTX22 is inoculated to a substrate for fermentation for 48 hours, and then the activated saccharomyces cerevisiae strain F33 is inoculated for continuous fermentation;
activated Hansenula polymorpha in grape juiceHanseniaspora uvarumThe total access of strain QTX22 and activated Saccharomyces cerevisiae strain F33 was 6X 10 6 CFU；
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.
3. The mixed fermentation process based on Hansenula polymorpha and Saccharomyces cerevisiae according to claim 1, wherein the activated strain is a strain inoculated in a culture medium for cultivation.
4. A mixed fermentation process based on hansenula polymorpha and saccharomyces cerevisiae according to claim 3, characterized in that said culture temperature is 24-30 ℃; the culture time is 20-30h, and the rotating speed is 120-250rpm.
5. The mixed fermentation process based on Hansenula polymorpha and Saccharomyces cerevisiae according to claim 4, wherein the cultivation temperature is 28 ℃; the incubation time was 24 hours and the rotational speed was 150rpm.
6. The mixed fermentation process based on Hansenula polymorpha and Saccharomyces cerevisiae according to claim 1, wherein the inoculation is to inoculate the initial strain preservation solution in a culture medium according to an inoculation amount of 2-4% by volume.
7. The mixed fermentation process based on Hansenula polymorpha and Saccharomyces cerevisiae according to claim 6, wherein the inoculation is to inoculate the initial strain preservation solution in a culture medium according to an inoculation amount of 3% by volume.
8. The mixed fermentation process based on Hansenula polymorpha and Saccharomyces cerevisiae according to claim 6 or 7, wherein the medium is YPD medium.
9. The mixed fermentation process based on Hansenula polymorpha and Saccharomyces cerevisiae according to claim 8, wherein the YPD 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.
10. The mixed fermentation process based on Hansenula polymorpha and Saccharomyces cerevisiae according to claim 9, wherein the YPD 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.
11. The mixed fermentation process based on Hansenula polymorpha and Saccharomyces cerevisiae according to claim 6 or 7, wherein the initial strain preservation solution means that the strain is preserved in a glycerol/YPD medium with a volume ratio of 15-35%.
12. The mixed fermentation process based on Hansenula polymorpha and Saccharomyces cerevisiae according to claim 11, wherein the initial strain preservation solution refers to the strain preservation in a 25% glycerol/YPD medium by volume.
13. A mixed fermentation process based on hansenula polymorpha and saccharomyces cerevisiae according to claim 1 or 3, characterized in that said activating strain is carried out 1-3 times.
14. A mixed fermentation process based on hansenula polymorpha and saccharomyces cerevisiae according to claim 1 or 3, characterized in that said activating strain is carried out 2 times.
15. A production method of grape wine is characterized in that grape juice is taken as a substrate, and Hansenula polymorpha and Saccharomyces cerevisiae in the grape juice are subjected to mixed fermentation;
hansenula polymorpha refers to Hansenula polymorpha in grape juiceHanseniaspora uvarumStrain QTX22; hansenula polymorpha of grape juiceHanseniaspora uvarumThe preservation number of the strain QTX22 is CCTCC M2021083;
the wine production method comprises the following steps: activating the strain, and inoculating the activated strain into a substrate to be fermented for fermentation; comprising the following steps: activating the strain, and inoculating the activated strain into a substrate to be fermented for fermentation;
the activated strain refers to: activated Hansenula polymorpha in grape juiceHanseniaspora uvarumStrain QTX22, or activated saccharomyces cerevisiae strain F33;
the activated substrate to be fermented is inoculated for fermentation, which means that: firstly, activated grape juice Hansenula polymorpha isHanseniaspora uvarumThe strain QTX22 is inoculated on a substrate for fermentation for 48-96 hours, and then the activated saccharomyces cerevisiae strain F33 is inoculated for continuous fermentation;
the fermentation temperature is 18+/-2 ℃;
activated Hansenula polymorpha in grape juiceHanseniaspora uvarumThe total access of the strain QTX22 and the activated Saccharomyces cerevisiae strain F33 is 10 6 -10 7 CFU；
Stopping fermentation when the substrate weight loss is not changed for 3 days;
the wine production method further comprises the following steps: preparing grape juice;
the preparation of grape juice refers to: squeezing grape fruit at low temperature;
squeezing the collected grape fruits with ice by using an air bag squeezer;
squeezing 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 uniform in size;
the grape fruit particles are the grape fruit particles which are harvested when the temperature is reduced to below-8 ℃ for 24 hours after the grape is ripe.
16. A process for the production of wine according to claim 15, characterised in that said activated fermentation of the substrate to be fermented is: firstly, activated grape juice Hansenula polymorpha isHanseniaspora uvarumThe strain QTX22 is inoculated to a substrate for fermentation for 48 hours, and then the activated saccharomyces cerevisiae strain F33 is inoculated for continuous fermentation;
activated Hansenula polymorpha in grape juiceHanseniaspora uvarumThe total access of strain QTX22 and activated Saccharomyces cerevisiae strain F33 was 6X 10 6 CFU；
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.
17. A wine production method according to claim 15, wherein said activating strain is a strain inoculated in a culture medium for cultivation.
18. A wine production method according to claim 17, wherein said cultivation temperature is 24-30 ℃; the culture time is 20-30h, and the rotating speed is 120-250rpm.
19. A wine production process according to claim 18, wherein said cultivation temperature is 28 ℃; the incubation time was 24 hours and the rotational speed was 150rpm.
20. 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.
21. A method of producing wine according to claim 20 wherein said medium is YPD medium.
22. A method of producing wine according to claim 21 wherein the YPD medium comprises the following components in mass to volume ratio: 0.5-3.5% yeast extract powder, 1.0-3.0% peptone, 1.0-5.0% glucose, and water in balance.
23. A method of producing wine according to claim 22 wherein the YPD medium comprises the following components in mass to volume ratio: 1% of yeast extract powder, 2% of peptone, 2% of glucose and the balance of water.
24. A wine production method according to claim 20 wherein said initial strain preservation fluid means that the strain is preserved in 15-35% glycerol/YPD medium by volume.
25. A method of producing wine according to claim 24 wherein said initial strain-preserving fluid means strain is preserved in 25% glycerol/YPD medium by volume.
26. A wine production method according to claim 15 or 17, wherein said activating strain is performed 1-3 times.
27. A wine production method according to claim 26 wherein said activating strain is performed 2 times.
28. Wine produced by the method according to any one of claims 15 to 27.
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