CN113150902B - Fermentation process for adding amino acid into substrate and based on Hansenula polymorpha of grape juice - Google Patents

Fermentation process for adding amino acid into substrate and based on Hansenula polymorpha of grape juice Download PDF

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CN113150902B
CN113150902B CN202110314715.4A CN202110314715A CN113150902B CN 113150902 B CN113150902 B CN 113150902B CN 202110314715 A CN202110314715 A CN 202110314715A CN 113150902 B CN113150902 B CN 113150902B
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strain
substrate
fermentation
hansenula polymorpha
grape juice
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CN113150902A (en
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葛谦
岳田利
袁亚宏
王周利
蔡瑞
郭春锋
胡仲秋
刘斌
张艳
李彩虹
闫玥
张静
苟春林
赵丹青
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Northwest A&F University
Ningxia Institute of Quality Standards and Testing Technology for Agro Products of Ningxia Agricultural Product Quality Monitoring Center
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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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    • 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
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

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Abstract

The invention relates to a fermentation process for adding amino acid into a substrate and based on Hansenula polymorpha of grape juice, belonging to the technical field of microorganisms. The fermentation process for adding amino acid to a substrate and based on Hansenula polymorpha in grape juice is characterized in that the amino acid is added to the substrate and the Hansenula polymorpha in grape juice is used for fermentation. The fermentation product obtained by adding amino acid into a substrate and fermenting with Hansenula polymorpha serving as a grape juice has unique fragrance compared with a single-fungus fermentation product without the amino acid, and can produce a wine beverage with unique flavor, fragrance and taste, enrich consumer products and enlarge consumption selection.

Description

Fermentation process for adding amino acid into substrate and based on Hansenula polymorpha of grape juice
Technical Field
The invention belongs to the technical field of beverage fermentation, and particularly relates to a fermentation process for adding amino acid into a substrate and based on Hansenula polymorpha of grape juice.
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.
Hansenula polymorpha (Hanseniaspora uvarum) is characterized in that: the cells are oval and sharp at both ends. The colony is not sticky and can grow anaerobically. The main uses of the current common Hansenula polymorpha in grape juice include: and (5) researching and fermenting. For example:
the invention patent application 201910635939.8 provides a Hansenula polymorpha strain, the preservation number of which is CCTCCNO: m2019304, the strain can convert inorganic selenium into organic selenium, and the content of the organic selenium is up to 2332mg/kg.
The invention patent application 201911406443.X discloses a strain A14 of Hansenula polymorpha (Hanseniaspora uvarum) with grape juice with a collection number of CGMCC No.18666, which has high geraniol production capacity and can ferment for 48 hours with the concentration of 63.48 mug/L.
The invention patent application 202010265892.3 discloses a composite strain for fermented red date wine, which comprises Hansenula polymorpha (Hanseniaspora uvarum) in grape juice, solves the problems of excessive methanol, high residual sugar, high acidity, single flavor and the like, and maintains the rich nutritive value of the fermented red date wine product.
However, hansenula polymorpha (Hanseniaspora uvarum) is a grape juice which can produce aroma substances such as o-cresol, 6-methylheptanol, 1, 3-pentanediol, and cyclooctanol, and has not been reported in the art. However, no report is known in the art concerning the process of adding amino acids to a substrate and fermenting with Hansenula polymorpha Hanseniaspora uvarum.
Disclosure of Invention
Based on the above-mentioned needs and blank in the art, the invention provides a fermentation process for adding amino acid into a substrate and based on Hansenula polymorpha Hanseniaspora uvarum, which has very remarkable improvement on a plurality of aroma substances affecting the flavor of alcoholic beverages compared with the conventional Saccharomyces cerevisiae fermentation.
The technical scheme of the invention is as follows:
a fermentation process for adding amino acid to a substrate and based on Hansenula polymorpha in grape juice is characterized in that the amino acid is added to the substrate and the Hansenula polymorpha in grape juice is used for 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 addition concentration of the amino acid is 0.6-1.4g/L;
preferably, the amino acid is selected from: leucine, threonine, isoleucine, valine.
The fermentation process for adding amino acid to a substrate and based on Hansenula polymorpha in grape juice comprises the following steps of: amino acid, an activated strain and an activated strain are added into a substrate, and the substrate to be fermented is inoculated into the activated strain for fermentation;
preferably, the activated strain refers to a strain inoculated in a culture medium for culture;
preferably, the concentration of the added amino acid in the substrate is selected from 0.6g/L, 1g/L and 1.4g/L.
The strain is selected from the group consisting of: grape juice has Hansenula polymorpha Hanseniaspora uvarum strain QTX22, saccharomyces cerevisiae strain F33, saccharomyces cerevisiae strain X16 and Saccharomyces cerevisiae strain ST;
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% preferably 1% of grape juice Hansenula polymorpha extract powder, 1.0-3.0% preferably 2% of peptone, 1.0-5.0% preferably 2% glucose, and the balance being water;
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.
Preferably, the activated strain means: activated grape juice has Hansenula polymorpha Hanseniaspora uvarum strain QTX22, or activated Saccharomyces cerevisiae strain F33, or activated Saccharomyces cerevisiae strain X16, or activated Saccharomyces cerevisiae strain ST;
preferably, the fermentation temperature is 18 ℃ ± 2 ℃;
preferably, the inoculation amount of the activated strain in the substrate 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, amino acid is added into the substrate, and then grape juice Hansenula polymorpha is inoculated for 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;
preferably, the wine production method comprises the following steps: adding amino acid, activated strain and activated strain into grape juice, and inoculating the strain after activation into a fermentation substrate for fermentation;
preferably, the concentration of the amino acid added into the grape juice is 0.6-1.4g/L; preferably 0.6g/L, 1g/L, 1.4g/L;
preferably, the amino acid added to the grape juice is selected from: leucine, threonine, valine, isoleucine.
The strain is selected from the group consisting of: grape juice has Hansenula polymorpha Hanseniaspora uvarum strain QTX22, saccharomyces cerevisiae strain F33, saccharomyces cerevisiae strain X16 and Saccharomyces cerevisiae strain ST;
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% preferably 1% of grape juice Hansenula polymorpha extract powder, 1.0-3.0% preferably 2% of peptone, 1.0-5.0% preferably 2% glucose, and the balance being water;
preferably, the initial strain preservation solution refers to a strain preserved in 15-35% glycerol/YPD medium, preferably 25% glycerol/YPD medium by volume;
more preferably, the activated strain is performed 1-3 times, preferably 2 times;
preferably, the activated strain means: activated grape juice has Hansenula polymorpha Hanseniaspora uvarum strain QTX22, or activated Saccharomyces cerevisiae strain F33, or activated Saccharomyces cerevisiae strain X16, or activated Saccharomyces cerevisiae strain ST;
preferably, the fermentation temperature is 18 ℃ ± 2 ℃;
preferably, the total access of activated Hansenula polymorpha Hanseniaspora uvarum strain QTX22 is 10 6 -10 7 CFU, preferably 6X 10 6 CFU；
Preferably, the fermentation is terminated after the substrate weight loss has not changed for 3 consecutive days.
The wine production method further comprises the following steps: preparing grape juice;
the preparation of grape juice refers to: squeezing grape fruit at low temperature;
preferably, the harvested grape fruit particles are pressed with ice by using an air bag press;
preferably, the sulfur dioxide or K is added at the same time by pressing 2 S 2 O 5 And, pectase;
preferably sulfur dioxide or K 2 S 2 O 5 The addition amount of (C) is 30-100mg/L, preferably 50mg/L; the addition amount of pectase is 10-30mg/L, preferably 20mg/L;
preferably, the grape fruit particles are uniform-sized fruit particles;
preferably, the grape fruit particles are grape fruit particles harvested after the grape is ripe and the temperature is reduced to below-8 ℃ for 24 hours.
The wine is characterized by being produced by the wine production method.
According to the invention, the substrate is added with amino acid and then is fermented by adding Hansenula polymorpha Hanseniaspora uvarum with grape juice, and compared with the single use of Hansenula polymorpha Hanseniaspora uvarum or commercial Saccharomyces cerevisiae for fermentation, or the substrate is fermented by using commercial Saccharomyces cerevisiae after adding amino acid, the yield of a plurality of aroma substances in the produced nearly hundred aroma substances is improved, for example, when leucine is added, the yield of 7-tridecanol is improved by 30%, the yield of cyclohexanol is improved by 41%, the yield of 2-nonone is improved by 1.34 times, and the yield of 2, 4-hexadienoic acid ethyl ester is improved by 33%; when threonine is added, 7-tridecanol is 1630 times higher, cyclohexanol is 268 times higher, 3-octanol is 262 times higher, 3-furanmethanol is 264 times higher, benzyl alcohol is 21 times higher, 1-pentanol is 12 times higher, 4-penten-1-ol is 108 times higher, ethylbenzene is 7.9 times higher, methyl 2-hydroxybutyrate is 375 times higher, 3-furfural is 87 times higher, isobutyraldehyde is 123 times higher, 2-methylbutyraldehyde is 174 times higher, isooctanoic acid is 1.08 times higher, isophorone is 37.4 times higher, 2-nonanone is 201 times higher, 2, 3-dihydro-3, 5 dihydroxy-6-methyl-4 (H) -pyran-4-one is 76 times higher, 3-nonanone is 14 times higher, 1-fluorohexane is 187 times higher, diethoxydimethylsilane is 34% higher, 54% ethyl phenylacetate, 1460% ethyl 2, 4-hexadienoate, 295% isobutyl acetate, 1.88% isoamyl acetate, 7.2% cyclohexanol, 24% 3-octanol, 34% gamma-butyrolactone, 94% 2-nonanone, 73% 2-methyl-1-butanol, 90% alpha-terpineol, 1609% 7-tridecanol, 269% cyclohexanol, 275% 3-octanol, 266% 3-furanmethanol, 85% sec-octanol, 211% phenol, 2.97% trans linalool oxide, the meta-xylene is 51 times, 4-isopropyl toluene is 146 times, 4-methyl-1, 3-pentadiene is 59 times, methyl 2-hydroxybutyrate is 340 times, gamma-butyrolactone is 327 times, 3-furfural is 106 times, isobutyraldehyde is 156 times, 2-methyl butyraldehyde is 3.52 times, (+) -limonene is 87 times, 1, 7-trimethyl ring [2.2.1] hept-2-ene 16.7%, myrcene is 1.11 times, 2-nonene is 90 times, 2, 3-dihydro-3, 5 dihydroxy-6-methyl-4 (H) -pyran-4-one is 56 times, 3-nonene is 45 times, 1-fluorohexane is 207 times, L (-) -ethyl lactate is 113 times, ethyl phenylacetate is 103 times, ethyl butyrate is 36% and 2, 4-hexadienoic acid ethyl ester is 289 times, isobutyl acetate is 373 times, isoamyl acetate is 3.19 times; the fermentation process of the invention also produces aroma substances which cannot be produced by fermentation with Hansenula polymorpha Hanseniaspora uvarum or commercial Saccharomyces cerevisiae alone, for example: when leucine is added, 3-heptanol, o-cresol is produced; when threonine is added, 6-methylheptanol, 1, 3-pentanediol, 3-heptanol, cyclooctanol, 2-methylpyrazine, 3-methyltridecane, 2, 4-dimethyl-1, 3-dioxane is produced; when valine is added, propyl acetate, ethyl propionate, 3-methyltridecane, 2-methylpyrazine, 3-heptanol, 1, 3-pentanediol, 6-methylheptanol are produced; when isoleucine was added, ethyl isobutyrate, ethyl 2-furoate, ethyl 2-methylbutyrate, 3-methyltridecane, dimethanol formal, P-cymene, 2, 3-dihydrobenzofuran, o-cresol, cyclooctanol, 3-heptanol, 6-methylheptanol were produced. 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: hanseniaspora uvarum QTX22 and 5222
Classification name: hansenula polymorpha of grape juice
Latin name: hanseniaspora uvarum
Deposit number: CCTCC NO: M2021083
Preservation mechanism: china center for type culture Collection
Preservation date: 2021, 1-15;
preservation agency address: university of Chinese, wuhan and Wuhan
Saccharomyces cerevisiae F33, X16, ST are commercial strains, available from Laffort, inc.
The grape variety used was Wedelian iced grape, purchased from Ningxia Bug Ge Zuimei International wine village Co.
Example 1 group, substrate-added amino acid fermentation Process of the invention
The present set of examples provides a fermentation process wherein amino acids are added to a substrate and based on Hansenula polymorpha in must. All embodiments of this group share the following common features: amino acids are added to the substrate and fermentation is carried out using Hansenula polymorpha in the grape juice.
Any action of adding amino acids to the substrate and fermenting and producing with Hansenula polymorpha must falls within the scope of the invention, as suggested by the present invention. Target products of fermentation include, but are not limited to: alcoholic beverages, dairy products, pasta, and the like.
Such dairy products include, but are not limited to, yogurt, fermented milk, milk drinks, and the like; the flour product includes but is not limited to: bread, cake, steamed stuffed bun, steamed bread, steamed roll, etc.
In a preferred embodiment, 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 some embodiments, the amino acid is added at a concentration of 0.6-1.4g/L;
in specific embodiments, the amino acid is selected from the group consisting of: leucine, threonine, isoleucine, valine.
In a further embodiment, the fermentation process for adding amino acids to a substrate and based on Hansenula polymorpha in grape juice comprises the steps of: amino acid, an activated strain and an activated strain are added into a substrate, and the substrate to be fermented is inoculated into the activated strain for fermentation;
preferably, the activated strain refers to a strain inoculated in a culture medium for culture;
preferably, the concentration of the added amino acid in the substrate is selected from 0.6g/L, 1g/L and 1.4g/L.
In a preferred embodiment, the strain is selected from the group consisting of: grape juice has Hansenula polymorpha Hanseniaspora uvarum strain QTX22, saccharomyces cerevisiae strain F33, saccharomyces cerevisiae strain X16 and Saccharomyces cerevisiae strain ST;
in addition to verifying the fermentation effect of Hansenula polymorpha Hanseniaspora uvarum strain in substrates added with amino acids, and simultaneously verifying the fermentation effect of 3 commercial Saccharomyces cerevisiae strains in substrates added with amino acids, the fermentation of the substrates added with amino acids can obtain higher aroma substance yield compared with the fermentation of the substrates without amino acids, so that according to the teaching of the invention, a person skilled in the art can select other commercial strains to perform the fermentation of the substrates added with amino acids, and besides F33, various commercial strains exist in the market at present, such as Saccharomyces cerevisiae V1116, saccharomyces cerevisiae VL1 and the like, and the strains can be used for inoculating the substrates added with amino acids to perform the fermentation, and the similar technical effects as those of the invention are expected.
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% preferably 1% of grape juice Hansenula polymorpha extract powder, 1.0-3.0% preferably 2% of peptone, 1.0-5.0% preferably 2% glucose, and the balance being water;
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.
Preferably, the activated strain means: activated grape juice has Hansenula polymorpha Hanseniaspora uvarum strain QTX22, or activated Saccharomyces cerevisiae strain F33, or activated Saccharomyces cerevisiae strain X16, or activated Saccharomyces cerevisiae strain ST;
preferably, the fermentation temperature is 18 ℃ ± 2 ℃;
preferably, the inoculation amount of the activated strain in the substrate 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: taking grape juice as a substrate, adding amino acid into the substrate, and inoculating Hansenula polymorpha into the grape juice for fermentation.
In a preferred embodiment, 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 other embodiments, the method of producing wine comprises: adding amino acid, activated strain and activated strain into grape juice, and inoculating the strain after activation into a fermentation substrate for fermentation;
preferably, the concentration of the amino acid added into the grape juice is 0.6-1.4g/L; preferably 0.6g/L, 1g/L, 1.4g/L;
preferably, the amino acid added to the grape juice is selected from: leucine, threonine, valine, isoleucine.
In some embodiments, the strain is selected from the group consisting of: grape juice has Hansenula polymorpha Hanseniaspora uvarum strain QTX22, saccharomyces cerevisiae strain F33, saccharomyces cerevisiae strain X16 and Saccharomyces cerevisiae strain ST;
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% preferably 1% of grape juice Hansenula polymorpha extract powder, 1.0-3.0% preferably 2% of peptone, 1.0-5.0% preferably 2% glucose, and the balance being water;
preferably, the initial strain preservation solution refers to a strain preserved in 15-35% glycerol/YPD medium, preferably 25% glycerol/YPD medium by volume;
more preferably, the activated strain is performed 1-3 times, preferably 2 times;
preferably, the activated strain means: activated grape juice has Hansenula polymorpha Hanseniaspora uvarum strain QTX22, or activated Saccharomyces cerevisiae strain F33, or activated Saccharomyces cerevisiae strain X16, or activated Saccharomyces cerevisiae strain ST;
preferably, the fermentation temperature is 18 ℃ ± 2 ℃;
preferably, the total access of activated Hansenula polymorpha Hanseniaspora uvarum strain QTX22 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 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.
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: o-cresol, 6-methylheptanol, 1, 3-pentanediol, cyclooctanol, 2-methylpyrazine, 3-methyltridecane, 2, 4-dimethyl-1, 3-dioxan, propyl acetate, ethyl propionate, 3-heptanol, ethyl isobutyrate, ethyl 2-furoate, ethyl 2-methylbutyrate, dimethanol formal, P-cymene, 2, 3-dihydrobenzofuran.
Experimental example, mixed bacteria fermentation process and fermentation data of the invention
1. Strain
The strains used in this experiment were: the strain S.cerevisiae X16, S.cerevisiae ST, F33 and Hansenula polymorpha Hanseniaspora uvarum, QTX22.
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. Substrate amino acid addition procedure
Preparing a plurality of pressed grape juice for parallel treatment:
(1) The labeling of grape juice awaiting fermentation by the individual addition of the strain is as follows:
CK-F33: inoculating only the grape juice fermented by the F33 strain;
CK-ST: inoculating only ST strain fermented grape juice;
CK-X16: inoculating only the grape juice fermented by the X16 strain;
CK-Hu: inoculating only Hu strain fermented grape juice;
(2) The grape juice added with amino acid is marked respectively as follows:
L-0.6-Hu: taking grape juice added with 0.6g/L leucine as a substrate, and inoculating Hu strain for fermentation;
L-0.6-F33: taking grape juice added with 0.6g/L leucine as a substrate, and inoculating F33 strain for fermentation;
L-0.6-ST: taking grape juice added with 0.6g/L leucine as a substrate, and inoculating ST strain for fermentation;
L-0.6-X16: taking grape juice added with 0.6g/L leucine as a substrate, and inoculating an X16 strain for fermentation;
L-1-Hu: taking grape juice added with 1g/L leucine as a substrate, and inoculating Hu strain for fermentation;
L-1-F33: taking grape juice added with 1g/L leucine as a substrate, and inoculating F33 strain for fermentation;
L-1-ST: taking grape juice added with 1g/L leucine as a substrate, and inoculating ST strain for fermentation;
L-1-X16: taking grape juice added with 1g/L leucine as a substrate, and inoculating an X16 strain for fermentation;
L-1.4-Hu: taking grape juice added with 1.4g/L leucine as a substrate, and inoculating Hu strain for fermentation;
L-1.4-F33: taking grape juice added with 1.4g/L leucine as a substrate, and inoculating F33 strain for fermentation;
L-1.4-ST: taking grape juice added with 1.4g/L leucine as a substrate, and inoculating ST strain for fermentation;
L-1.4-X16: taking grape juice added with 1.4g/L leucine as a substrate, and inoculating an X16 strain for fermentation;
S-0.6-Hu: taking grape juice added with 0.6g/L threonine as a substrate, and inoculating Hu strain for fermentation;
S-0.6-F33: taking grape juice added with 0.6g/L threonine as a substrate, and inoculating F33 strain for fermentation;
S-0.6-ST: taking grape juice added with 0.6g/L threonine as a substrate, and inoculating ST strain for fermentation;
S-0.6-X16: taking grape juice added with 0.6g/L threonine as a substrate, and inoculating an X16 strain for fermentation;
S-1-Hu: taking grape juice added with 1g/L threonine as a substrate, and inoculating Hu strain for fermentation;
S-1-F33: taking grape juice added with 1g/L threonine as a substrate, and inoculating F33 strain for fermentation;
S-1-ST: taking grape juice added with 1g/L threonine as a substrate, and inoculating ST strain for fermentation;
S-1-X16: taking grape juice added with 1g/L threonine as a substrate, and inoculating an X16 strain for fermentation;
S-1.4-Hu: taking grape juice added with 1.4g/L threonine as a substrate, and inoculating Hu strain for fermentation;
S-1.4-F33: taking grape juice added with 1.4g/L threonine as a substrate, and inoculating F33 strain for fermentation;
S-1.4-ST: taking grape juice added with 1.4g/L threonine as a substrate, and inoculating ST strain for fermentation;
S-1.4-X16: taking grape juice added with 1.4g/L threonine as a substrate, and inoculating an X16 strain for fermentation;
YL-0.6-Hu: taking grape juice added with 0.6g/L isoleucine as a substrate, and inoculating Hu strain for fermentation;
YL-0.6-F33: taking grape juice added with 0.6g/L isoleucine as a substrate, and inoculating F33 strain for fermentation;
YL-0.6-ST: taking grape juice added with 0.6g/L isoleucine as a substrate, and inoculating ST strain for fermentation;
YL-0.6-X16: taking grape juice added with 0.6g/L isoleucine as a substrate, and inoculating an X16 strain for fermentation;
YL-1-Hu: taking grape juice added with 1g/L isoleucine as a substrate, and inoculating Hu strain for fermentation;
YL-1-F33: taking grape juice added with 1g/L isoleucine as a substrate, and inoculating F33 strain for fermentation;
YL-1-ST: taking grape juice added with 1g/L isoleucine as a substrate, and inoculating ST strain for fermentation;
YL-1-X16: taking grape juice added with 1g/L isoleucine as a substrate, and inoculating an X16 strain for fermentation;
YL-1.4-Hu: taking grape juice added with 1.4g/L isoleucine as a substrate, and inoculating Hu strain for fermentation;
YL-1.4-F33: taking grape juice added with 1.4g/L isoleucine as a substrate, and inoculating F33 strain for fermentation;
YL-1.4-ST: taking grape juice added with 1.4g/L isoleucine as a substrate, and inoculating ST strain for fermentation;
YL-1.4-X16: taking grape juice added with 1.4g/L isoleucine as a substrate, and inoculating an X16 strain for fermentation;
X-0.6-Hu: taking grape juice added with 0.6g/L valine as a substrate, and inoculating Hu strain for fermentation;
X-0.6-F33: taking grape juice added with 0.6g/L valine as a substrate, and inoculating F33 strain for fermentation;
X-0.6-ST: taking grape juice added with 0.6g/L valine as a substrate, and inoculating ST strain for fermentation;
X-0.6-X16: taking grape juice added with 0.6g/L valine as a substrate, and inoculating an X16 strain for fermentation;
X-1-Hu: taking grape juice added with 1g/L valine as a substrate, and inoculating Hu strain for fermentation;
X-1-F33: taking grape juice added with 1g/L valine as a substrate, and inoculating F33 strain for fermentation;
X-1-ST: taking grape juice added with 1g/L valine as a substrate, and inoculating ST strain for fermentation;
x-1 to X16: taking grape juice added with 1g/L valine as a substrate, and inoculating an X16 strain for fermentation;
X-1.4-Hu: taking grape juice added with 1.4g/L valine as a substrate, and inoculating Hu strain for fermentation;
X-1.4-F33: taking grape juice added with 1.4g/L valine as a substrate, and inoculating F33 strain for fermentation;
X-1.4-ST: taking grape juice added with 1.4g/L valine as a substrate, and inoculating ST strain for fermentation;
X-1.4-X16: grape juice added with 1.4g/L valine is taken as a substrate, and then the X16 strain is inoculated for fermentation.
The fermentation products represented by each of the above markers are fermentation products obtained by performing 3 times of parallel fermentation respectively in the respective meanings.
4. Fermentation operation
4 strains: saccharomyces cerevisiae S.cerevisiae X16 strain, saccharomyces cerevisiae S.cerevisiae ST strain, saccharomyces cerevisiae S.cerevisiae F33 strain, hansenula polymorpha Hanseniaspora uvarum strain QTX22 was kept in 25% glycerol/YPD medium by volume prior to use. YPD medium is 1% glucose extract with Hansenula polymorpha extract powder, 2% peptone, and 2% glucose. Inoculating the bacterial liquid according to the inoculum size of 3% of the volume ratio into a 50mL triangular flask filled with 40mL of YPD culture medium and a 250mL triangular flask filled with 150mL of YPD culture medium respectively, wherein the culture temperature is 28 ℃, the rotation speed is 150rpm, the culture time is 24 hours, the bacterial liquid activated for the 1 st time is obtained, then inoculating the bacterial liquid according to the inoculum size of 3% into the 50mL triangular flask filled with 40mL of YPD culture medium and the 250mL triangular flask filled with 150mL of YPD culture medium respectively, and repeating the culture to finish the 2 nd passage activation, thus obtaining the bacterial liquid after activation. The activated grape juice Hansenula polymorpha Hanseniaspora uvarum strain QTX22 is respectively inoculated into the collected grape juice added with amino acids with different gradient concentrations, and the total inoculation amount of the strain is controlled to be 10 6 -10 7 CFU is preferably 6×10 6 CFU, the fermentation temperature is 18+/-2 ℃ by taking the grape juice without adding amino acid, which is obtained by single-strain fermentation in 4 strains of Saccharomyces cerevisiae S.cerevisiae X16 strain, saccharomyces cerevisiae S.cerevisiae ST strain, saccharomyces cerevisiae S.cerevisiae F33 strain and Hansenula polymorpha Hanseniaspora uvarum strain QTX22 as blank control, and the fermentation is stopped when the grape juice weight loss is not changed for three consecutive days. All wine samples were centrifuged at 7500rpm for 8 minutes and the supernatant was stored at 4 ℃.
5. 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.
6. Data analysis method
The 3 parallel fermentation products corresponding to the marks are taken as 3 samples to respectively measure the aroma substance content, and each sample is respectively measured in parallel for 3 times to obtain an average value. 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 tables 1-4, where the data are given in μg/L, meaning: the aroma content per liter of wine.
TABLE 1 aroma content of grape juice fermentation product with leucine added
TABLE 2 aroma content of threonine-supplemented grape juice fermentation products
TABLE 3 fragrance content of valine added grape juice fermentation products
TABLE 4 aroma content of grape juice fermentation product with added isoleucine
The fragrance threshold values in tables 1 to 4 above refer to the lowest concentration lower limit value at which a person can sniff the substance, and the fragrance descriptions and threshold values are based on the reports of the relevant documents in which the fragrance descriptions and threshold values of the fragrance substances are described, and fragrance substances without fragrance descriptions and threshold values are documents in which the relevant documents in which the fragrance descriptions and threshold values of the substance are not searched. The meaning of each symbol in the header is as described in Experimental example 3.
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 single-bacteria fermentation is higher than the fermentation of adding amino acid on certain aroma components, which is a normal phenomenon in the field.

Claims (50)

1. A fermentation process for adding amino acid to a substrate and based on Hansenula polymorpha in grape juice is characterized in that the amino acid is added to the substrate and the Hansenula polymorpha in grape juice is adopted for 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 addition concentration of the amino acid is 0.6-1.4g/L; the amino acid is selected from: leucine, threonine, isoleucine, valine.
2. A fermentation process for adding amino acids to a substrate based on hansenula polymorpha according to claim 1, characterized in that it comprises the following steps: amino acid is added into the substrate, the strain is activated, and the activated strain is inoculated into the substrate to be fermented for fermentation.
3. A fermentation process for adding amino acids to a substrate based on hansenula polymorpha according to claim 2, wherein the activated strain is a strain inoculated in a culture medium for cultivation.
4. The fermentation process for adding amino acids to a substrate according to claim 2, wherein the concentration of the amino acids added to the substrate is selected from the group consisting of 0.6g/L, 1g/L, 1.4g/L.
5. A fermentation process for adding amino acids to a substrate based on hansenula polymorpha according to claim 3, wherein the cultivation temperature is 24-30 ℃; the culture time is 20-30h, and the rotating speed is 120-250rpm.
6. The process for the fermentation of Hansenula polymorpha with substrate addition of amino acids according to claim 5, wherein the cultivation temperature is 28 ℃; the incubation time was 24 hours and the rotational speed was 150rpm.
7. A fermentation process for adding amino acids to a substrate based on hansenula polymorpha according to claim 3, wherein the inoculation is performed by inoculating the stock solution of the initial strain in a culture medium in an amount of 2-4% by volume.
8. A fermentation process for adding amino acids to a substrate according to claim 3, wherein the inoculation is to inoculate a stock solution of the initial strain in a culture medium in an amount of 3% by volume.
9. A fermentation process for adding amino acids to a substrate according to claim 3 or 7, based on hansenula polymorpha in must, wherein the medium is YPD medium.
10. The process for the fermentation of Hansenula polymorpha with substrate addition of amino acids according to claim 9, wherein the YPD medium comprises the following components in mass/volume ratio: 0.5-3.5% of grape juice Hansenula polymorpha extract powder, 1.0-3.0% of peptone, 1.0-5.0% of glucose and the balance of water.
11. The process for the fermentation of Hansenula polymorpha with substrate addition of amino acids according to claim 10, wherein the YPD medium comprises the following components in mass/volume ratio: 1% of grape juice has Hansenula polymorpha extract powder, 2% of peptone, 2% of glucose and the balance of water.
12. The process for fermentation of Hansenula polymorpha with substrate addition of amino acids according to claim 7, wherein said initial strain preservation solution is a strain preserved in 15-35% glycerol/YPD medium by volume.
13. The process for fermentation of Hansenula polymorpha with substrate addition of amino acids according to claim 12, wherein said initial strain preservation solution is a strain preserved in 25% glycerol/YPD medium by volume.
14. A fermentation process for adding amino acids to a substrate based on hansenula polymorpha according to claim 2, wherein the activation strain is performed 1-3 times.
15. A fermentation process for adding amino acids to a substrate based on hansenula polymorpha according to claim 14, wherein the activation strain is performed 2 times.
16. A substrate-added amino acid and glucose-based hansenula polymorpha fermentation process according to claim 2, characterized in that the fermentation temperature is 18 ℃ ± 2 ℃.
17. A fermentation process for the addition of amino acids to a substrate based on Hansenula polymorpha according to claim 2, wherein the inoculation amount of the activated strain in the substrate is 10 6 -10 7 CFU。
18. The process for the fermentation of Hansenula polymorpha with substrate addition of amino acids according to claim 17, wherein the inoculation of the activated strain in the substrate is 6X 10 6 CFU。
19. A fermentation process for adding amino acids to a substrate based on hansenula polymorpha according to claim 2, characterized in that the fermentation is terminated after the loss of weight of the substrate has not changed for 3 consecutive days.
20. Fermentation process of Hansenula polymorpha, based on the addition of amino acids to a substrate according to any one of claims 1 to 8 and 10 to 19, characterised in that the substrate is grape juice.
21. A production method of grape wine is characterized in that grape juice is taken as a substrate, amino acid is added into the substrate, and then grape juice Hansenula polymorpha is inoculated for 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.
22. A method of producing wine according to claim 21 comprising: amino acid, an activated strain and an activated strain are added into grape juice, and the activated strain is inoculated into a substrate to be fermented for fermentation.
23. The method of claim 22, wherein the concentration of amino acid added to the must is 0.6-1.4g/L.
24. The method of claim 23, wherein the concentration of added amino acid in the must is 0.6g/L, 1g/L or 1.4g/L.
25. A method of producing wine according to any one of claims 21-24 wherein said amino acids added to said must are selected from the group consisting of: leucine, threonine, valine, isoleucine.
26. A wine production method according to claim 22, wherein said activating strain is a strain inoculated in a culture medium for cultivation.
27. A wine production process according to claim 26, wherein said cultivation temperature is 24-30 ℃; the culture time is 20-30h, and the rotating speed is 120-250rpm.
28. A wine production process according to claim 27, wherein said cultivation temperature is 28 ℃; the incubation time was 24 hours and the rotational speed was 150rpm.
29. A method according to claim 26, wherein the inoculation is performed by inoculating the initial strain stock solution into the culture medium at an inoculation rate of 3% by volume.
30. A method of producing wine according to claim 26 or 29 wherein said medium is YPD medium.
31. A method of producing wine according to claim 30 wherein the YPD medium comprises the following components in mass to volume ratio: 0.5-3.5% of grape juice Hansenula polymorpha extract powder, 1.0-3.0% of peptone, 1.0-5.0% of glucose and the balance of water.
32. A method of producing wine according to claim 31 wherein the YPD medium comprises the following components in mass to volume ratio: 1% of grape juice has Hansenula polymorpha extract powder, 2% of peptone, 2% of glucose and the balance of water.
33. A wine production method according to claim 29, wherein said initial strain preservation solution is a strain preserved in 15-35% glycerol/YPD medium by volume.
34. The method according to claim 33, wherein the initial strain-preserving fluid is a strain preserved in 25% glycerol/YPD medium by volume.
35. A wine production method according to claim 22, wherein said activating strain is performed 1-3 times.
36. A wine production method according to claim 35 wherein said activating strain is performed 2 times.
37. A wine production method according to claim 21, wherein said fermentation temperature is 18 ℃ ± 2 ℃.
38. A wine production method according to claim 22, wherein said fermentation temperature is 18 ℃ ± 2 ℃.
39. The method according to claim 22, wherein the activated must has Hansenula polymorphaHanseniaspora uvarumThe total access of the strain QTX22 is 10 6 -10 7 CFU。
40. The method of claim 39, wherein the activated must has Hansenula polymorphaHanseniaspora uvarumThe total access of the strain QTX22 was 6X 10 6 CFU。
41. A process for the production of wine according to claim 21 wherein fermentation is terminated after the substrate loss has been unchanged for 3 consecutive days.
42. A process for the production of wine according to claim 22 wherein fermentation is terminated after the substrate loss has been unchanged for 3 consecutive days.
43. A method of wine production according to any one of claims 21-24, 26-29, 31-42 and further comprising: preparing grape juice;
the preparation of grape juice refers to: squeezing grape fruit at low temperature.
44. The method of claim 43, wherein the harvested grape fruit particles are pressed with ice using an air bag press.
45. A process for producing wine according to claim 44 wherein the pressing is accompanied by addition of sulfur dioxide or K 2 S 2 O 5 And pectinase.
46. A method according to claim 45, 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.
47. A method according to claim 46, 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.
48. The method of claim 43, wherein the grape fruit particles are uniform sized fruit particles.
49. The method of claim 48, wherein the grape fruit particles are grape fruit particles harvested after ripening of the grape and after a temperature drop of about 24 hours to about 8deg.C or less.
50. A wine produced by the wine production method of any one of claims 21 to 49.
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