CN112852569B - Mixed fermentation process based on rhodosporidium rhodochrous and saccharomyces cerevisiae - Google Patents

Mixed fermentation process based on rhodosporidium rhodochrous and saccharomyces cerevisiae Download PDF

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CN112852569B
CN112852569B CN202110189046.2A CN202110189046A CN112852569B CN 112852569 B CN112852569 B CN 112852569B CN 202110189046 A CN202110189046 A CN 202110189046A CN 112852569 B CN112852569 B CN 112852569B
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rhodosporidium
saccharomyces cerevisiae
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苟春林
葛谦
李振永
李娴
刘俭
俞鸿千
张维军
马婷婷
张伟
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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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Abstract

The invention discloses a mixed fermentation process based on rhodosporidium and saccharomyces cerevisiae in Lusita, belonging to the technical field of microorganisms. The mixed fermentation process based on the rhodosporidium roursonii and the saccharomyces cerevisiae is characterized in that the rhodosporidium roursonii and the saccharomyces cerevisiae are subjected to mixed fermentation; the rhodosporidium roseum of the Lissajous is the rhodosporidium roseum Rhodosporidiobolus lusitaniae strain QTX26 of the Lissajous; the collection number of the rhodosporidium rhodochrous Rhodosporidiobolus lusitaniae strain QTX26 is CCTCC M2021088. Compared with the single-fungus fermentation of the Saccharomyces cerevisiae F33, the method has very remarkable promotion on a plurality of aroma substances which can influence the flavor of the alcoholic beverage, thereby bringing about unique flavor, aroma and taste of the fermented beverage.

Description

Mixed fermentation process based on rhodosporidium rhodochrous and saccharomyces cerevisiae
Technical Field
The invention belongs to the technical field of beverage fermentation, and particularly relates to a mixed fermentation process based on rhodosporidium and saccharomyces cerevisiae.
Background
Wine fermentation is a complex biochemical process in which yeast plays a very critical role, for example, in the conversion of sugar to ethanol, carbon dioxide and other thousands of secondary metabolites. A great deal of scientific researches show that the quality of the wine is highly dependent on the metabolic activities and fermentation behaviors of different yeasts, and the different yeasts have important contributions to the chemical composition, the organoleptic properties, the flavor characteristics and the like of the wine. Saccharomyces cerevisiae is the most widely used strain in wine industrial production so far, and has the advantages of ensuring the risk of deterioration in the fermentation process of wine, having good fermentation power, along with the problems of single flavor characteristic, serious homogenization phenomenon and the like. Therefore, in order to pursue style characterization of the wine, the aroma characteristics are more representative, diversified and complex, and brewers often adopt a method of mixed fermentation of saccharomyces cerevisiae and non-saccharomyces cerevisiae, especially some indigenous yeasts with strong adaptability and representativeness, so as to improve and enhance the flavor quality of the wine.
Non-saccharomyces cerevisiae has become an option to improve wine quality. Numerous studies have shown that non-Saccharomyces cerevisiae is capable of producing enzymes and some of the secondary metabolites we desire, thereby improving wine aroma and flavor characteristics, and controlling the growth of some undesirable species in wine, but has the disadvantage of inadequate fermentation kinetics. The mixing fermentation of non-Saccharomyces cerevisiae and Saccharomyces cerevisiae can not only improve the fragrance diversity and complexity of the wine, but also make up for the problem of insufficient fermentation power of non-Saccharomyces cerevisiae, and is an effective method for improving the fragrance quality of the wine.
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 is no report in the art that a fermentation process using rhodosporidium rhodosporum Rhodosporidiobolus lusitaniae has been found, and there is no report in the mixed fermentation of rhodosporidium rhodosporum Rhodosporidiobolus lusitaniae 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 rhodosporidium Rhodosporidiobolus lusitaniae 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-strain fermentation of saccharomyces cerevisiae F33.
The technical scheme of the invention is as follows:
a mixed fermentation process based on rhodosporidium roursoni and saccharomyces cerevisiae is characterized in that rhodosporidium roursoni and saccharomyces cerevisiae are subjected to mixed fermentation.
Preferably, the rhodosporidium roursonii refers to rhodosporidium roursonii Rhodosporidiobolus lusitaniae strain QTX26; the collection number of the rhodosporidium rhodochrous Rhodosporidiobolus lusitaniae strain QTX26 is CCTCC M2021088.
Preferably, the Saccharomyces cerevisiae refers to Saccharomyces cerevisiae strain F33.
The mixed bacteria fermentation process based on the rhodosporidium and 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: lu Xida rhodosporidium Rhodosporidiobolus lusitaniae strain QTX26 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 rhodosporidium rhodochrous strain Rhodosporidiobolus lusitaniae QTX26 or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating activated rhodosporidium rouxii strain QTX26 of rhodosporidium rouxii Rhodosporidiobolus lusitaniae 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 activated Brevibacterium roseosporus strain QTX26, brevibacterium roseum Rhodosporidiobolus lusitaniae, and of 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 used as a substrate, and rhodosporidium and saccharomyces cerevisiae of the Lusitoni are subjected to mixed fermentation;
the rhodosporidium roseum of the Lissajous is the rhodosporidium roseum Rhodosporidiobolus lusitaniae strain QTX26 of the Lissajous; the collection number of the rhodosporidium rhodochrous Rhodosporidiobolus lusitaniae strain QTX26 is CCTCC M2021088.
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: lu Xida rhodosporidium Rhodosporidiobolus lusitaniae strain QTX26 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 rhodosporidium rhodochrous strain Rhodosporidiobolus lusitaniae QTX26 or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating activated rhodosporidium rouxii strain QTX26 of rhodosporidium rouxii Rhodosporidiobolus lusitaniae 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 Brevibacterium roseosporus strain QTX26, rhodosporidium rhodochrous Rhodosporidiobolus lusitaniae, 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, rhodosporidium roseum Rhodosporidiobolus lusitaniae and saccharomyces cerevisiae are adopted for mixed fermentation, the yield of most of aroma substances is improved in tens of produced aroma substances, for example, glacial acetic acid is improved by 24%, 2R, 3R) - (-) -2, 3-butanediol is improved by about 65%, n-octanal is improved by 1 time, ethyl pelargonate is improved by about 50%, 2, 4-trimethylpentanediol isobutyl ester is improved by 41%, vinyl acetate is improved by about 43%, and 2, 6-di (tert-butyl) -4-hydroxy-4-methyl-2, 5-cyclohexadien-1-one is improved by nearly 1 time; 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-ethylhexanol, 1, 4-butanediol, 2, 7-dimethyl-1-octanol, nonanal, ethyl pyruvate, methyl nonylketone, isoamyl decanoate, p-xylene, alpha-terpineol, levo-beta-pinene, (+) -alfalfa-ene. 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 rhodosporidium strain Rhodosporidiobolus lusitaniae QTX used in the experimental example is a new strain screened by the applicant laboratory, and the preservation information is as follows:
naming: QTX26
Classification name: lu Xida rhodosporidium and rhodosporidium parvum
Latin name: rhodosporidiobolus lusitaniae
Deposit number: CCTCC M2021088
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 fermentation process based on rhodosporidium and saccharomyces cerevisiae. All embodiments of this group share the following common features: and (3) carrying out mixed fermentation on rhodosporidium and saccharomyces cerevisiae.
According to the teaching of the invention, any fermentation and production activities of rhodosporidium and saccharomyces cerevisiae are all within the scope of the invention. Target products of fermentation include, but are not limited to: alcoholic beverages, fermented milk, bread, etc.
In a preferred embodiment, the rhodosporidium rouxii, of the present invention, refers to rhodosporidium rouxii strain Rhodosporidiobolus lusitaniae QTX26; the collection number of the rhodosporidium rhodochrous Rhodosporidiobolus lusitaniae strain QTX26 is CCTCC M2021088.
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 rhodosporidium necatrix Rhodosporidiobolus lusitaniae strain QTX26 of the invention to obtain similar technical effects as the invention.
In some embodiments, the mixed fermentation process based on rhodosporidium rhodochrous and 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: lu Xida rhodosporidium Rhodosporidiobolus lusitaniae strain QTX26 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 rhodosporidium rhodochrous strain Rhodosporidiobolus lusitaniae QTX26 or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating activated rhodosporidium rouxii strain QTX26 of rhodosporidium rouxii Rhodosporidiobolus lusitaniae 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 Brevibacterium roseosporus strain QTX26, rhodosporidium rhodochrous Rhodosporidiobolus lusitaniae, 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: taking grape juice as a substrate, and carrying out mixed fermentation on rhodosporidium and saccharomyces cerevisiae.
In some preferred embodiments, the rhodosporidium rouxii, of the present invention, refers to rhodosporidium rouxii strain Rhodosporidiobolus lusitaniae QTX26; the collection number of the rhodosporidium rhodochrous Rhodosporidiobolus lusitaniae strain QTX26 is CCTCC M2021088.
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: lu Xida rhodosporidium Rhodosporidiobolus lusitaniae strain QTX26 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 rhodosporidium rhodochrous strain Rhodosporidiobolus lusitaniae QTX26 or activated saccharomyces cerevisiae strain F33;
preferably, the fermentation of the activated substrate for fermentation refers to: inoculating activated rhodosporidium rouxii strain QTX26 of rhodosporidium rouxii Rhodosporidiobolus lusitaniae to a substrate for fermentation for 0-96h, preferably 48h, and inoculating activated saccharomyces cerevisiae strain F33 for continuous fermentation;
when the time for fermenting by inoculating activated rhodosporidium roursonii Rhodosporidiobolus lusitaniae strain QTX26 to a substrate is 0h, the activated rhodosporidium roursonii Rhodosporidiobolus lusitaniae strain QTX26 and the activated Saccharomyces cerevisiae strain F33 can be simultaneously inoculated to the substrate for fermenting 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 Brevibacterium roseosporus strain QTX26, rhodosporidium rhodochrous Rhodosporidiobolus lusitaniae, 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-ethylhexanol, 1, 4-butanediol, 2, 7-dimethyl-1-octanol, nonanal, ethyl pyruvate, methylnonylketone, isoamyl decanoate, paraxylene, alpha-terpineol, levo-beta-pinene, (+) -alfalfa-ene, and at the same time far higher aroma contents than the monoallyl fermented wine: glacial acetic acid, (2 r,3 r) - (-) -2, 3-butanediol, n-octanal, ethyl nonanoate, isobutyl 2, 4-trimethylpentanediol, vinyl acetate, 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 eF33 and rhodosporidium strain Rhodosporidiobolus lusitaniae strain QTX26 isolated and screened in accordance with the present 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/LK) is added simultaneously 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 F33Strain, rhodosporidium solani Rhodosporidiobolus lusitaniae strain QTX26, 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 activated rhodosporidium necatrix Rhodosporidiobolus lusitaniae strain QTX26 into collected grape juice, after 48 hours, inoculating S.cerevisiae F33 strain according to the inoculation proportion of QTX26/F33 of 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 (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 top refers to data obtained by single-strain fermentation using a commercial strain, saccharomyces cerevisiae F33, and "F33_QTX26" refers to data obtained by mixed-strain fermentation using a commercial strain, saccharomyces cerevisiae F33 and Rhodosporidium rouxii Rhodosporidiobolus lusitaniae strain QTX26.
As known in the fermentation field, the factors influencing the fermentation are numerous and complex, and the components of the fermented product can be changed due to the changes of factors such as raw material batch, raw material components, fermentation conditions, temperature, time and the like, so that the situation that single-bacteria fermentation is higher than mixed-bacteria fermentation on certain aroma components can also occur, which belongs to the normal phenomenon in the field.

Claims (44)

1. A mixed fermentation process based on rhodosporidium and saccharomyces cerevisiae in the Lissa is characterized by comprising the following steps: activating the strain, and inoculating the activated strain into a substrate to be fermented for fermentation; the activated strain is inoculated with a substrate to be fermented for fermentation, which means that: firstly, activated rhodosporidium of LissataRhodosporidiobolus lusitaniaeThe strain QTX26 is inoculated to a substrate for fermentation for 48-96 hours, and then the activated saccharomyces cerevisiae strain F33 is inoculated for continuous fermentation; brevibacterium roseum of Liujia rhodosporidiumRhodosporidiobolus lusitaniaeThe preservation number of the strain QTX26 is CCTCC M2021088.
2. The mixed fermentation process based on rhodosporidium and saccharomyces cerevisiae, as claimed in claim 1, wherein the activated strain is obtained by inoculating the strain into a culture medium for culture.
3. The mixed fermentation process based on rhodosporidium and saccharomyces cerevisiae, as claimed in claim 2, wherein the culture temperature is 24-30 ℃; the culture time is 20-30h, and the rotating speed is 120-250rpm.
4. A mixed fermentation process based on rhodosporidium and saccharomyces cerevisiae, as claimed in claim 3, characterized in that the culture temperature is 28 ℃; the incubation time was 24 hours and the rotational speed was 150rpm.
5. A mixed fermentation process based on rhodosporidium and saccharomyces cerevisiae as claimed in claim 3, wherein the inoculation means that the initial strain preservation solution is inoculated in the culture medium according to the inoculation amount of 2-4% of the volume ratio.
6. The mixed fermentation process based on rhodosporidium and saccharomyces cerevisiae as claimed in claim 5, wherein the inoculation is to inoculate the initial strain preservation solution in a culture medium according to an inoculum size of 3% by volume.
7. The mixed fermentation process based on rhodosporidium and saccharomyces cerevisiae as claimed in any one of claims 2,5 and 6, wherein the culture medium is YPD culture medium.
8. The mixed fermentation process based on rhodosporidium and saccharomyces cerevisiae, as claimed in claim 7, 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.
9. The mixed fermentation process based on rhodosporidium and saccharomyces cerevisiae, as claimed in claim 7, 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.
10. The mixed fermentation process based on rhodosporidium and saccharomyces cerevisiae as claimed in claim 5 or 6, wherein the initial strain preservation solution is that the strain is preserved in a glycerol/YPD culture medium with a volume ratio of 15-35%.
11. The mixed fermentation process based on rhodosporidium and saccharomyces cerevisiae, as claimed in claim 10, wherein the initial strain preservation solution refers to the strain preservation in a 25% glycerol/YPD culture medium by volume.
12. The process according to claim 1 or 2, wherein the activation of the strain is carried out 1-3 times.
13. The process for mixed fermentation based on rhodosporidium and saccharomyces cerevisiae, according to claim 12, wherein said activating strain is carried out 2 times.
14. The mixed fermentation process based on rhodosporidium and saccharomyces cerevisiae, as claimed in claim 1, wherein the activated strain is inoculated with a substrate to be fermented for fermentation, which is characterized in that: firstly, activated rhodosporidium of LissataRhodosporidiobolus lusitaniaeThe strain QTX26 is inoculated on a substrate for fermentation for 48 hours, and then the activated saccharomyces cerevisiae strain F33 is inoculated for continuous fermentation.
15. The mixed fermentation process based on rhodosporidium and saccharomyces cerevisiae, according to claim 1 or 14, wherein the fermentation temperature is 18 ℃ ± 2 ℃.
16. The mixed fermentation process based on rhodosporidium and saccharomyces cerevisiae as claimed in claim 1 or 14, wherein the activated rhodosporidium and saccharomyces cerevisiae is characterized in thatRhodosporidiobolus lusitaniaeThe total inoculum size of the strain QTX26 and the activated Saccharomyces cerevisiae strain F33 was 10 6 -10 7 CFU。
17. The mixed fermentation process based on rhodosporidium and saccharomyces cerevisiae as claimed in claim 16, wherein the activated rhodosporidium and saccharomyces cerevisiae is characterized in thatRhodosporidiobolus lusitaniaeThe total inoculum size of strain QTX26 and, after activation, of Saccharomyces cerevisiae strain F33 was 6X 10 6 CFU。
18. The process for mixed fermentation based on rhodosporidium and saccharomyces cerevisiae, according to claim 1 or 14, characterized in that the fermentation is terminated after the loss of substrate weight is no longer changed for 3 consecutive days.
19. The process for mixed fermentation based on rhodosporidium and saccharomyces cerevisiae, as claimed in claim 18, wherein the substrate is grape juice.
20. A method of producing wine comprising: activating the strain, and inoculating the activated strain into substrate grape juice to be fermented for fermentation; the activated strain is inoculated with substrate grape juice to be fermented for fermentation, which means that: firstly, activated rhodosporidium of LissataRhodosporidiobolus lusitaniaeThe strain QTX26 is inoculated to a substrate for fermentation for 48-96 hours, and then the activated saccharomyces cerevisiae strain F33 is inoculated for continuous fermentation; brevibacterium roseum of Liujia rhodosporidiumRhodosporidiobolus lusitaniaeThe preservation number of the strain QTX26 is CCTCC M2021088.
21. A wine production method according to claim 20, wherein said activating strain is a strain inoculated in a culture medium for cultivation.
22. A process for the production of wine according to claim 21, wherein the cultivation temperature is between 24 and 30 ℃; the culture time is 20-30h, and the rotating speed is 120-250rpm.
23. A process for the production of wine according to claim 22 wherein the cultivation temperature is 28 ℃; the incubation time was 24 hours and the rotational speed was 150rpm.
24. A method according to claim 21, wherein the inoculation is performed by inoculating the initial strain stock solution into the culture medium at an inoculation rate of 3% by volume.
25. A method of producing wine according to claim 21 or 24 wherein said medium is YPD medium.
26. A method of producing wine according to claim 25 wherein the YPD medium comprises the following components in mass to volume ratio: 0.5-3.5% yeast extract powder, 1.0-3.0% peptone, 1.0-5.0% glucose, and water in balance.
27. A method of producing wine according to claim 25 wherein the YPD medium comprises the following components in mass to volume ratio: 1% of yeast extract powder, 2% of peptone, 2% of glucose and the balance of water.
28. A wine production method according to claim 24 wherein said initial strain preservation fluid means that the strain is preserved in 15-35% glycerol/YPD medium by volume.
29. A method of producing wine according to claim 24 wherein said initial strain-preserving fluid means strain is preserved in 25% glycerol/YPD medium by volume.
30. A wine production method according to claim 20 or 21, wherein said activating strain is performed 1-3 times.
31. A wine production method according to claim 30 wherein said activating strain is performed 2 times.
32. A wine production method according to claim 20, wherein said activated strain is inoculated with a substrate must to be fermented for fermentation: firstly, activated rhodosporidium of LissataRhodosporidiobolus lusitaniaeThe strain QTX26 is inoculated on a substrate for fermentation for 48 hours, and then the activated saccharomyces cerevisiae strain F33 is inoculated for continuous fermentation.
33. A process for producing wine according to claim 20 or 32 wherein the fermentation temperature is 18 ℃ ± 2 ℃.
34. A method of producing wine according to claim 20 or 32 wherein the activated rhodosporidium of lucerneRhodosporidiobolus lusitaniaeThe total access of the strain QTX26 and the activated Saccharomyces cerevisiae strain F33 is 10 6 -10 7 CFU。
35. A process for producing wine according to claim 34 wherein, activated rhodosporidium of LecitonRhodosporidiobolus lusitaniaeThe total access of strain QTX26 and activated Saccharomyces cerevisiae strain F33 was 6X 10 6 CFU。
36. A method of producing wine according to claim 20 or 32 wherein fermentation is terminated after the substrate loss has been unchanged for 3 consecutive days.
37. A method of wine production according to any one of claims 20-24, 26-29, 31, 32, 35 and further comprising: preparing grape juice;
the preparation of grape juice refers to: squeezing grape fruit at low temperature.
38. A method of producing wine according to claim 37 wherein the harvested grape fruit particles are pressed with ice using an air bag press.
39. A method of producing wine according to claim 38 wherein the pressing is accompanied by the addition of sulphur dioxide or K 2 S 2 O 5 And, pectase.
40. A method of producing wine according to claim 39 wherein sulfur dioxide or K 2 S 2 O 5 The addition amount of the (B) is 30-100mg/L; addition of pectaseThe addition amount is 10-30mg/L.
41. A process for producing wine according to claim 40 wherein sulfur dioxide or K 2 S 2 O 5 The addition amount of (2) is 50mg/L; the addition amount of pectase is 20mg/L.
42. A method of producing wine according to claim 38 wherein the grape fruit particles are of uniform size.
43. The method according to claim 38 or 42, wherein the grape fruit particles are grape fruit particles harvested after ripening of the grape and after a temperature drop below-8 ℃ for 24 hours.
44. Wine produced by the method according to any one of claims 20 to 43.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5877013A (en) * 1997-07-31 1999-03-02 Food Industry Research And Development Institute Rhodosporidium D-amino acid oxidase
CN110923080A (en) * 2019-10-30 2020-03-27 镇江瑞德酒业有限公司 Flavor enhancing brewing process for fresh grape brandy

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5877013A (en) * 1997-07-31 1999-03-02 Food Industry Research And Development Institute Rhodosporidium D-amino acid oxidase
CN110923080A (en) * 2019-10-30 2020-03-27 镇江瑞德酒业有限公司 Flavor enhancing brewing process for fresh grape brandy

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