CN117887597A - Issatchenkia terrestris and application thereof in wine - Google Patents
Issatchenkia terrestris and application thereof in wine Download PDFInfo
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- 238000000855 fermentation Methods 0.000 claims abstract description 77
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- 102000006995 beta-Glucosidase Human genes 0.000 claims abstract description 15
- 108010047754 beta-Glucosidase Proteins 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 15
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Classifications
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- 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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- 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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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a strain of Issatchenkia terrestris and application thereof in grape wine, wherein the strain is preserved in China Center for Type Culture Collection (CCTCC) in the year of 2023, month of 05 and 22; address: wu Changou eight ways of Wuhan City in Hubei province, wuhan university; post code: 430072, the preservation number is CCTCC NO: M2023794, latin name ISSATCHENKIA TERRICOLA, the strain SIVE4101 has better low-temperature tolerance in the process of fermenting the wine, and high yield of beta-glucosidase, can improve the content of phenethyl acetate and terpene compounds in the wine, and enriches the fragrance of the wine. The main fermentation is carried out and the malic acid is converted into lactic acid, so that time and labor are saved, the aculeate stimulation of the wine can be reduced, and the wine body is softer and more coordinated.
Description
Technical Field
The invention relates to the technical field of wine fermentation, in particular to a strain of Issatchenkia terrestris and application thereof in wine.
Background
The aroma of wine comprises variety aroma and fermentation aroma, and the aroma substances exist in free state and combined state. It has been found that the content of flavour precursors in the bound form is much richer than the content of flavour precursors in the free form. Since most of the aroma substances exist in the form of glycoside bound state, have no aroma and cannot be perceived, in order to enhance and improve the aroma of wine, the bound aroma precursor substances in the grape must be converted into free aroma substances, while beta-glucosidase is a key enzyme for the release of bound aroma. Yeast is the power generated by fermenting aroma, and non-Saccharomyces cerevisiae synthesizes some enzymes with higher activity, so that non-volatile precursor substances in raw materials can be converted into aroma compounds such as esters, higher alcohols and the like, the odor activity value of volatile compounds is improved, the content of acid substances is changed, and the content of bad odor active compounds is reduced. In recent years, research shows that the content of various enzymes of different types of non-Saccharomyces cerevisiae is greatly different.
In the brewing process, under the condition that the raw materials are certain, the factors influencing the quality of the wine are not saccharomycetes, and the fermentation temperature has larger influence on the quality of the wine. The low-temperature fermentation can better retain the aroma substances with low volatile points of terpenes, and the aromatic compounds can be better synthesized under the action of low temperature. For example: the low-temperature environment is beneficial to the generation of volatile esters and C6-C10 medium-chain fatty acid esters and the reduction of the content of higher alcohols and volatile acids, thereby improving the flavor and quality of the wine. Therefore, low temperature fermentation technology is widely used in the production of wine. However, in practice, low temperature fermentation is difficult to achieve. Under the action of low temperature, the cell activity of the yeast is reduced, and the phenomena of growth retardation, stop and the like are easy to occur, which is unfavorable for the smooth proceeding of the fermentation process and seriously can cause the phenomenon of stopping the fermentation. Therefore, in order to improve the accumulation of low-volatile point aroma substances in the product wine, yeast screening suitable for low-temperature fermentation has important significance for industry.
In the wine, taste and aroma have the same important function, the wine mainly contains tartaric acid, malic acid and citric acid, the existence of the acidic substances is an important component for supporting the wine body structure of the wine, the wine body structure of the wine influences the taste coordination and balance of the wine, and the too high or too low acidity can influence the taste of the wine, so in the traditional process, after the alcoholic fermentation of the wine is finished, lactobacillus is added into the wine, and the lactic acid with sharp sour taste is converted into lactic acid with soft taste by utilizing the metabolism of the lactobacillus, so that the acidity of the wine is reduced; the method can improve the stability of the fruit wine to microorganisms, change the content and proportion of trace element components in the wine, and is beneficial to improving the flavor of the wine; however, the inoculation of lactobacillus for fermentation has the characteristics of time and labor waste, and the lactobacillus is sensitive to SO 2, SO that SO 2 cannot be supplemented at the end of fermentation, SO that the mixed bacteria diseases are easy to infect in the post-fermentation, and the operation is difficult to control, SO that the lactobacillus diseases are caused; the schizosaccharomyces pombe (Schisosaccharomyces pombe) is decomposed into ethanol and CO 2 under anaerobic conditions, the yeast has strong SO 2 resistance, but has slower propagation and fermentation speed and higher fermentation optimum temperature, and the fruit wine obtained by pure fermentation of the schizosaccharomyces pombe has the taste of gynostemma and poor wine quality.
In the application document with the application number of CN201010202066.0 and the subject name of Liu Shengyi Sasa yeast strain capable of simultaneously degrading malic acid and citric acid in fruit wine, a Liu Shengyi Sasa yeast strain capable of converting malic acid and citric acid is disclosed, the maximum SO 2 -resistant concentration of the strain is 450mg/L, the maximum alcohol-resistant degree of the strain is 5%, and the optimal growth temperature is 30 ℃; that is, the strain cannot be adapted to lower temperature fermentation.
In the patent document with the application number of CN201911331670.0 and the subject name of a strain of Issatchenkia glabra and the application of the strain in citric acid degradation, the strain is obtained on fresh raspberries in a raspberry orchard, has excellent citric acid degradation capability, and mainly aims at biological acid degradation of high-citric acid fruits; i.e. the strain has a degrading ability to citric acid.
Therefore, the prior microbial acid reduction mainly aims at malic acid and citric acid, and can not realize fermentation at low temperature; the tartaric acid in grape fruits accounts for 42.8-77.0% of the total acid, is typical tartaric acid fruits, and secondly is malic acid, and accounts for 10.3-41.6% of the total acid, and the content of citric acid is only 0.02-0.03% of the total acid; the existence of tartaric acid in the wine can affect the structure of the wine body, so that the breeding of non-saccharomyces cerevisiae which is suitable for low-temperature fermentation and has the aroma-producing performance and proper acid-reducing function is particularly important.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a strain of Issatchenkia terrestris and application thereof in wine, wherein the strain is preserved in China center for type culture collection (CCTCC for short) at 22 nd month of 2023; address: wu Changou eight ways of Wuhan City in Hubei province, wuhan university; post code: 430072 with the preservation number of CCTCC NO: M2023794 and Latin name ISSATCHENKIA TERRICOLA, the strain SIVE4101 has good low-temperature tolerance in the process of wine fermentation, and the tolerance range is 8-38 ℃, namely, the strain can be fermented smoothly at the low temperature of 6-20 ℃; the strain SIVE4101 has the performance of high-yield beta-glucosidase, can improve the content of phenethyl acetate and terpene compounds in the wine, and enriches the fragrance of the wine; in addition, the strain SIVE4101 has the acid reducing effect in the process of alcoholic fermentation, reduces the process of adding lactobacillus to carry out malic acid fermentation, saves the fermentation time, can also reduce the aculeate stimulation of the wine, and ensures that the wine body is softer and more coordinated.
The technical scheme of the invention is as follows:
The strain of Issatchenkia terrestris with the strain number SIVE4101, which is preserved in China center for type culture collection (CCTCC for short) at the month 22 of 2023; address: wu Changou eight ways of Wuhan City in Hubei province, wuhan university; post code: 430072 with the preservation number of CCTCC NO: M2023794 and Latin name ISSATCHENKIA TERRICOLA.
Preferably, liu Shengyi Saccharomyces SIVE4101 is obtained by screening grape mash in the earlier stage of fermentation, and the nucleotide sequence of 26S rDNA D1/D2 region is shown in SEQ ID NO:1 is shown in the specification; the Liu Shengyi Saccharomyces SIVE4101 has good SO 2 tolerance and temperature tolerance, wherein the SO 2 tolerance is up to 1000mg/L; the temperature tolerance is 8-38 ℃; the SO 2 tolerance and the temperature tolerance of the strain SIVE4101 enable the strain to be capable of smoothly metabolizing and producing beta-glucosidase during low-temperature alcohol fermentation, SO that the strain can act on combined aroma substances metabolizing and producing by saccharomyces cerevisiae during the alcohol fermentation, the content of phenethyl acetate and terpene compounds in wine is improved, and the quality of finished wine is further improved; in addition, the strain SIVE4101 can produce acid reducing effect in the alcoholic fermentation process, so that the wine body structure of the wine is stable, and the taste coordination and balance are better.
Preferably, the Liu Shengyi Saccharomyces SIVE4101 has a glucose tolerance of 100-400g/L, a fructose tolerance of 100-400g/L, an osmotic pressure tolerance of 0.8-2.0mol/L based on KCl concentration, a pH tolerance of not less than 3.0 and an ethanol tolerance of up to 8% (v/v).
The Liu Shengyi Saccharomyces SIVE4101 is applied to the fermentation of wine, the fermentation temperature is 20+/-1 ℃, and the enzyme activity of beta-glucosidase is 88.33U/L in 72 hours; the Liu Shengyi Saccharomyces SIVE4101 can be well adapted to the low-temperature fermentation temperature in the fermentation process, and can be used for producing beta-glucosidase in high yield, so that the contents of phenethyl acetate and terpene compounds in the wine can be improved, and the fragrance of the wine is enriched; in addition, the acid in the grape can be degraded while the alcohol is fermented, so that time and labor are saved, the aculeate stimulation of the grape wine can be reduced, and the wine body is softer and more coordinated;
in the alcoholic fermentation process, the enzyme activity of the beta-glucosidase produced by the Issatchenkia glabra SIVE4101 in 72 hours is 88.33U/L, and the method has the advantage of high enzyme activity; along with the fermentation process, a large amount of combined aroma substances can be generated in the wine, aroma in the combined aroma substances cannot be released, and the high activity of the beta-glucosidase can effectively convert the combined state into a free state, so that the aroma in the wine is plump.
Preferably, in the application, liu Shengyi Saccharomyces SIVE4101 is independently fermented, the content of the ethyl benzene acetate is 237.89mg/L, and the ethyl benzene acetate has sweet fragrance and can optimize the fragrance of the wine.
Preferably, in the application, liu Shengyi Saccharomyces SIVE4101 is fermented independently, the total content of terpene compounds is 1.84mg/L, and the terpene compounds have the characteristic of low threshold value and are easy to sense.
Preferably, in the above application, liu Shengyi Saccharomyces SIVE4101 is added in an amount of 0.5g of yeast fresh cells per liter of grape juice.
Preferably, in the above application, liu Shengyi Saccharomyces SIVE4101 is prepared by: inoculating 1-ring inclined plane Liu Shengyi Saccharomycetes SIVE4101 into YPD liquid culture medium, culturing at 28deg.C for 18-24 hr with shaking table 200r/min, centrifuging, and collecting fresh thallus; EC1118, the preparation method is: inoculating 1-ring inclined plane EC1118 into YPD liquid culture medium, culturing at 28deg.C with shaking table 200r/min for 18-24 hr, centrifuging, and collecting fresh thallus.
Preferably, liu Shengyi Saccharomyces SIVE4101 and EC1118 are mixed in wine for fermentation, and the weight ratio of Liu Shengyi Saccharomyces SIVE4101 to EC1118 is 1:1; after fermentation, the content of phenethyl acetate and terpene aroma substances in the obtained wine is improved.
Compared with the prior art, the invention has the beneficial effects that:
1. The invention provides a strain of Issatchenkia terrestris, which is preserved in China center for type culture collection (CCTCC for short) at 22 nd year 2023; address: wu Changou eight ways of Wuhan City in Hubei province, wuhan university; post code: 430072, the preservation number is CCTCC NO: M2023794, latin name ISSATCHENKIA TERRICOLA, the strain SIVE4101 has good low temperature tolerance, and can be successfully fermented at 20+/-1 ℃ to realize low temperature fermentation of grape wine; the low-temperature fermentation can better retain the aroma substances with low volatile points of terpenes, and the aromatic compounds can be better synthesized under the action of low temperature.
2. The Liu Shengyi Saccharomyces SIVE4101 provided by the invention can be used for producing beta-glucosidase in a high yield in the fermentation process, so that the contents of phenethyl acetate and terpene compounds in the wine can be improved, and the fragrance of the wine is enriched.
3. When the Liu Shengyi Saccharomyces SIVE4101 provided by the invention is applied to wine fermentation, malic acid can be converted into lactic acid during alcohol fermentation, so that time and labor are saved, the aculeate stimulation of wine can be reduced, and the wine body is softer and more coordinated.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the description of the embodiments or the prior art will be briefly described below, and it will be obvious to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.
FIG. 1 shows the cell morphology of Liu Shengyi Saccharomyces SIVE4101 under electron microscopy.
FIG. 2 shows the colony morphology of Liu Shengyi Saccharomyces SIVE4101 on YPD solid medium.
FIG. 3 shows the growth and enzyme activity curves of Liu Shengyi Saccharomyces SIVE4101 in YPD liquid medium.
FIG. 4 is a photograph showing the growth of SIVE4101 and EC1118 on WL plates.
Fig. 5 is Liu Shengyi the change in soluble solids during fermentation of the torulopsis SIVE4101 and EC 1118.
FIG. 6 is a GC-MS ion diagram of Liu Shengyi Saccharomyces SIVE 4101.
Detailed Description
In order to better understand the technical solutions of the present invention, the following description will clearly and completely describe the technical solutions of the embodiments of the present invention in conjunction with the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
Example 1 isolation and characterization
Crushing Chardonnay grape, propagating in a constant temperature incubator at 28 ℃ for 24 hours, taking 1ml of grape juice, adding 99ml of sterile water to obtain bacterial suspension with a dilution gradient of 10 -2, continuously diluting to 10 -3,10-4,10-5, respectively sucking 100 μl of bacterial liquids with different concentrations, coating the bacterial liquids on YPD solid culture medium plates, culturing for 3-5 days at 28 ℃, picking up different forms of single colonies, scribing and purifying on WL nutrient agar plates, and culturing for 5-7 days at 28 ℃; single colony is selected, the pure colony is transferred to YPD inclined plane for preservation, and the target strain is obtained through a series of primary screening and secondary screening.
The identification proves that the strain belongs to Liu Shengyi Saccharomyces, liu Shengyi Saccharomyces ISSATCHENKIA TERRICOLA, is named SIVE4101, and has the nucleotide sequence of 26S rDNA D1/D2 region shown in SEQ ID NO:1 is shown in the specification; the strain is preserved in China center for type culture collection (CCTCC for short) at 22 months of 2023; address: wu Changou eight ways of Wuhan City in Hubei province, wuhan university; post code: 430072, the preservation number is CCTCC NO: M2023794, latin name ISSATCHENKIA TERRICOLA, and the cell morphology of Liu Shengyi Saccharomyces SIVE4101 under an electron microscope is shown in figure 1.
SEQ ID NO：1
ACTCCGCCAGTGCGCATCCTTGACGATCGCAGACCTCAGTCCCCGCTCGCGGCATCTGAGAGAGGCTATAACACTCCGAAGAGCCACGTTCCTCCCCCCATTCTCCCGCGGCAAAAACTGATGCTGGCCCAGAAACCGCACAGAGCGCCGCCTACAAGAAGCAACGGTGCGCAGTCCCCATGTCGAGCCCAATACCCTTCCCTTTCAACAATTTCACGTGCTGTTTCACTCTCTTTTCAAAGTGCTTTTCATCTTTCCTTCACAGTACTTGTTCGCTATCGGTCTCTCGCCAGTATTTAGCCTTAGATGGAATTTACCACCCACTTAGAGCTGCATTCCCAAACAACTCGACTCGTCAGAAGGGTCTTAAAGCTTGGACGCGTGCCGCACGGGGCTCTCACCCTCAATGGCGCCCTGTTCCAAGGGACTTAGACACACGCCGCCACAAAGACTCCAACCTGCAATTTACAACTCGCCGAGGCGATTTCAAATCTGAGCTCTTGCCGCTTCACTCGCCGCTACTGAGGCAATCCCTGTTGGTTTCTTTTCCTCCGCATT
The Liu Shengyi Saccharomyces SIVE4101 was inoculated onto YPD solid medium plates by streaking, and the results are shown in FIG. 2.
Example 2 Liu Shengyi test of tolerance of Saccharomyces SIVE4101
(1) SO 2 tolerance
The Liu Shengyi Saccharomyces SIVE4101 deposited on the inclined plane of example 1 was inoculated in equal amounts into a tube with Du's tube containing 10ml of YPD liquid medium with different SO 2 concentrations, the mass concentration of SO 2 was 300mg/L, 400mg/L, 500mg/L, 600mg/L, 700mg/L, 800mg/L,700mg/L, 1000mg/L, 1. Mu.l of the inoculating loop was scraped for 1 loop, and then incubated at constant temperature at 28℃for 7 days, and the growth conditions were recorded by observation, and the results are shown in Table 1;
TABLE 1 Liu Shengyi Saccharomyces SIVE4101 SO 2 tolerance results
(2) Temperature tolerance
The Liu Shengyi Saccharomyces SIVE4101 deposited on the inclined surface of example 1 was inoculated in an equal amount into a tube with Du's tube containing 10ml of YPD liquid medium, 1 loop was scraped off with an inoculating loop in an amount of 1. Mu.l, and the tube was cultured at different temperatures for 7 days under constant temperature, the temperature was set to 6 ℃, 8 ℃,10 ℃, 35 ℃, 38 ℃, 40 ℃, 45 ℃, and the growth conditions were observed and recorded, and the results are shown in Table 2;
TABLE 2 results of temperature tolerance of Issatchenkia terrestris SIVE4101
Remarks: after one week, the gas production condition was observed, "+" represents gas production in Du's tube and "-" does not occur in Du's tube.
(3) Glucose tolerance
The Liu Shengyi Saccharomyces SIVE4101 deposited on the inclined plane of example 1 was inoculated in equal amounts into a tube with Du's tube containing 10ml of YPD liquid medium with different glucose mass concentrations, the glucose mass concentrations were 100g/L, 200g/L, 300g/L, 400g/L, 1. Mu.l of inoculating loop was scraped to obtain 1 loop, then incubated at 28℃for 7 days at constant temperature, and the growth conditions were observed and recorded, and the results are shown in Table 3;
TABLE 3 Liu Shengyi Saccharomyces SIVE4101 glucose tolerance results
(4) Fructose tolerance
The Liu Shengyi Saccharomyces SIVE4101 deposited on the inclined plane of example 1 was inoculated in equal amounts into test tubes with Du's tubes containing 10ml of YPD liquid medium with different fructose mass concentrations of 100g/L, 200g/L, 300g/L, 400g/L, 1. Mu.l of a microliter inoculating loop was scraped to obtain 1 loop, and then incubated at 28℃for 7 days at constant temperature, and the growth conditions were observed and recorded, and the results are shown in Table 4;
Table 4 Liu Shengyi results of fructose tolerance of Saccharomyces SIVE4101
(5) Osmotic pressure tolerance
The Liu Shengyi Saccharomyces SIVE4101 deposited on the inclined surface of example 1 was inoculated in equal amounts into test tubes with Du's tubes containing 10ml of YPD liquid medium with different osmotic pressures, KCl concentration was 0.8mol/L, 1.0mol/L, 1.8mol/L, 2.0mol/L, 2.2mol/L, 1. Mu.l inoculating loop was scraped for 1 loop, and then incubated at 28℃for 7 days under constant temperature conditions, and the growth conditions were observed and recorded, and the results are shown in Table 5;
TABLE 5 results of osmotic pressure tolerance of Issatchenkia continental SIVE4101
(6) PH tolerance
The Liu Shengyi Saccharomyces SIVE4101 deposited on the inclined surface of example 1 was inoculated in equal amounts into test tubes with Dunaliella tertiolecta tubes containing 10ml of YPD liquid medium at different pH values at pH values of 2.5, 3.0, 3.5, 4.0, 4.5 in an amount of 1. Mu.l of an inoculating loop to scrape 1 loop, then incubated at 28℃for 7 days, and the growth conditions were recorded by observation, and the results are shown in Table 6;
table 6 Liu Shengyi Saccharomyces SIVE4101 pH tolerance results
(7) Ethanol tolerance
The Liu Shengyi Saccharomyces SIVE4101 deposited on the inclined plane of example 1 was inoculated in equal amounts into test tubes with Du's tubes containing 10ml of YPD liquid medium with different alcohol concentrations of 4% (v/v), 6% (v/v), 8% (v/v), 10% (v/v), 12% (v/v), 14% (v/v), 16% (v/v) with 1. Mu.l of inoculating loops scraped 1 loop, and then incubated at 28℃for 7 days, and the growth conditions were observed and recorded, and the results are shown in Table 7;
TABLE 7 Liu Shengyi Saccharomyces SIVE4101 ethanol tolerance results
As shown in tables 1-7, liu Shengyi Saccharomyces SIVE4101 obtained by screening in the invention has the highest SO 2 tolerance of 1000mg/L, the temperature tolerance of 8-38 ℃, the glucose tolerance of 100-400g/L, the fructose tolerance of 100-400g/L, the osmotic pressure tolerance of 0.8-2.0mol/L based on KCl concentration, the pH tolerance of more than or equal to 3.0 and the highest ethanol tolerance of 8% (v/v), and the strain SIVE4101 provided by the invention has higher sugar, osmotic pressure, pH and SO 2 tolerance; the temperature tolerance capability of the fermentation starter can maintain the fermentation of the wine under the condition of low temperature (20+/-1 ℃), and the maximum glucose is not more than 200g/L, so that Liu Shengyi Saccharomyces SIVE4101 can be well suitable for the brewing environment of alcoholic fermentation.
EXAMPLE 3 Liu Shengyi Saccharomyces SIVE4101 growth curve and beta-glucosidase Activity
The fermentation process of the wine is a main process for producing alcohol and aroma substances, the aroma substances mainly comprise combined aroma substances and free aroma substances, and the combined aroma substances are mainly in the wine; however, during the evaluation, only free aroma substances can be identified, that is, even if there are a large amount of combined aroma substances in the wine, such aroma does not function in the sensory evaluation; beta-glucosidase is an enzyme capable of hydrolyzing a combined aroma substance into a free aroma substance, and the presence of the enzyme can influence the amount of the free aroma substance in the wine, so that the growth curve and the beta-glucosidase activity of Liu Shengyi Saccharomyces SIVE4101 provided by the invention are tested in the following process;
Experiment design:
Liu Shengyi Saccharomyces SIVE culture 4101:
Activation of strains: adding Liu Shengyi Saccharomyces SIVE4101 on the inclined plane of 1 ring of example 1 into YPD liquid, and shake culturing at 28deg.C under 200r/min for 24 hr;
Inoculating and culturing: 200 μl of the activated Liu Shengyi Saccharomycetes SIVE4101 strain suspension was weighed and added to 100ml YPD liquid medium, and shake-cultured at 28deg.C under 200r/min conditions, with 3 replicates per group.
Control EC1118 treatment was incubated with Liu Shengyi sa SIVE 4101.
Drawing a growth curve under different temperature conditions:
After inoculation, 3 samples were taken at 0, 4, 8, 12, 16, 20, 24, 48, 72 hours each; and (3) measuring: measuring absorbance value of the bacterial suspension at 600nm wavelength, drawing a growth curve by taking culture time as an abscissa and absorbance value as an ordinate, and drawing a growth curve as shown in figure 3;
Beta-glucosidase activity assay:
Sampling at 24 hours, 48 hours and 72 hours after inoculation respectively, measuring the activity of the beta-glucosidase, taking the culture time as an abscissa and the enzyme activity as an ordinate, and drawing an enzyme activity curve as shown in figure 3;
fig. 3 is a graph showing two vertical axes, and referring to fig. 3, it is shown that the growth curve of Liu Shengyi torula yeast SIVE4101 can approach the equilibrium period about 18 hours, secondary metabolites begin to accumulate, the enzyme activity after 72 hours of culture is up to 88.33U/L, the enzyme activity after 72 hours of control group EC1118 reaches 69.04U/L, and compared with the control group EC1118, the enzyme activity of Liu Shengyi torula yeast SIVE4101 of the present invention is 19.29U/L higher than that of the control group EC1118 at 72 hours, and it is found that the yeast SIVE4101 has the advantage of higher enzyme activity compared with the control group EC1118, liu Shengyi torula yeast.
Example 4SIVE 4101A counter experiment with EC1118
Preparation of bacterial suspension: adding Liu Shengyi Saccharomyces SIVE4101 on the inclined plane of 1 ring of example 1 into 100ml YPD liquid, shake culturing at 28deg.C under 200r/min for 6-8 hr, and performing the same treatment on control group EC 1118;
and (3) culturing in a counter manner: respectively taking 100 mu l of the activated EC1118 and Liu Shengyi Saccharomyces SIVE4101 bacterial suspensions, respectively coating on different WL solid plates, ventilating and airing in a super clean bench, taking 5 mu l of the EC1118 bacterial suspension, and inoculating on a plate coated with the terrestrial Issatchenkia SIVE4101 bacterial suspension in advance by adopting a five-point inoculation method; taking 5 μl Liu Shengyi Saccharomyces SIVE4101 bacterial suspension, inoculating on a plate coated with EC1118 bacterial suspension in advance by five-point inoculation method, culturing at 28deg.C for 5 days, and observing the growth condition, and the result is shown in figure 4;
In FIG. 4, the left image is a photograph of a plate previously coated with a suspension of Issatchenkia armigera SIVE4101 by the EC1118 five-point inoculation method, and the right image is a photograph of a plate previously coated with a suspension of EC1118 by the Liu Shengyi Sachenkia armigera SIVE4101 five-point inoculation method; as can be seen from FIG. 4, both the yeast strains can grow well, regardless of the EC1118 spot-jointed on the plate of Liu Shengyi Saccharomyces SIVE4101 or the Liu Shengyi Saccharomyces SIVE4101 spot-jointed on the plate of EC1118, which indicates that the two yeast strains can coexist with each other and provides preconditions for mixed fermentation of SIVE4101 and EC 1118.
Example 5 Liu Shengyi aroma characteristics of Saccharomyces SIVE4101
Crushing the grape stem, squeezing juice, and sterilizing at 90deg.C for 20min; split 800ml of grape juice into 1000ml blue cap bottles, 3 replicates per group, each inoculated according to the following protocol:
Experiment ① 0.5.5 g Liu Shengyi Saccharomyces SIVE4101 of example 1;
Experiment ② 0.25.25 g Liu Shengyi Saccharomyces SIVE4101 of example 1, after 48 hours of fermentation 0.25g EC1118 was added;
Experiment ③ 0.5g EC1118;
In the inoculation scheme, liu Shengyi Saccharomycetes SIVE4101 and EC1118 are added according to the amount of fresh thallus, and the method for obtaining the fresh thallus is as follows:
The preparation method of the fresh cell of the Issatchenkia glabra SIVE4101 comprises the following steps: inoculating 1-ring inclined plane Liu Shengyi Saccharomycetes SIVE4101 into YPD liquid culture medium, culturing at 28deg.C for 18 hr with shaking table 200r/min, centrifuging, and collecting fresh thallus;
The preparation method of the EC1118 fresh thallus comprises the following steps: inoculating 1-ring inclined plane EC1118 into YPD liquid culture medium, culturing at 28deg.C with shaking table 200r/min for 18 hr, centrifuging, and collecting fresh thallus;
Sealing with a check valve sealing plug containing 5-6ml glycerol, fermenting at 20deg.C, wherein the content of soluble solids changes during fermentation are shown in figure 5, the physical and chemical index measurement indexes of sugar alcohol and organic acid after fermentation are shown in tables 8 and 9, and the detection results of GC-MS are shown in tables 10 and 6;
As can be seen from fig. 5, liu Shengyi, the fermentation of the torula pastoris SIVE4101 is slower than that of the control group, which is the commonality of all non-saccharomyces cerevisiae, the fermentation is carried out for about 18 days, the soluble solids are not changed any more, the EC1118 is fermented for about 8 days under the same fermentation conditions, the soluble solids are not changed any more, the SIVE4101 and the EC1118 are mixed-fermented for about 11 days, and the soluble solids are not changed any more; (the soluble solids are not changing, marked as end of alcoholic fermentation);
TABLE 8 sugar alcohol content in Small Mandson grape juice and wine
As can be seen in combination with table 8, the soluble solids of Liu Shengyi torulopsis SIVE4101 mainly included glucose and fructose remaining from fermentation, whereas the control group EC1118 left only a small amount of fructose, and the sequential inoculation fermentation of SIVE4101 and EC1118 left only a small amount of fructose, and the fermentation was more thorough than the EC1118 alone, and the ethanol yield was also higher for the corresponding sequential inoculation group.
TABLE 9 composition of organic acids in Small Mandson grape juice and wine
As can be seen by combining Table 9, liu Shengyi Saccharomyces SIVE4101 has obvious acid reducing capability, and the content of citric acid, malic acid, succinic acid and acetic acid is lower than that of the control group whether the fermentation is carried out singly or the fermentation is carried out sequentially; and Liu Shengyi Saccharomyces SIVE4101 has significantly higher lactic acid production capacity than the control group; this is probably because the yeast Issatchenkia glabra SIVE4101 converts malic acid into lactic acid during alcoholic fermentation, which is time-saving and labor-saving, reduces the feeling of sharp acid stimulation of wine, and makes the wine softer and more harmonious.
TABLE 10GC-MS detection results
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As is clear from Table 10, 79 kinds of components were detected in total in the fermentation product obtained by experiment ① (fermentation by strain SIVE 4101), and the total content was 322.79mg/L; 75 components are detected in the fermentation product obtained by experiment ③ (EC 1118 fermentation), and the total content is 185.75mg/L; 76 components are detected in the fermentation product obtained by experiment ③ (the strain SIVE4101 and the EC1118 are sequentially inoculated and fermented), and the total content is 247.93mg/L; it can be seen that strain SIVE4101 is rich in aroma content by fermentation (experiment ①), the total content is 1.74 times that of EC1118 (experiment ③), the total content is 1.33 times that of the control group by sequential inoculation (experiment ②);
Among the fermentation products, the greatest difference is phenethyl acetate, and the content of the phenethyl acetate in the experiment ① (strain SIVE4101 fermentation) is 237.89mg/L, which is 65.9 times that of the experiment ③ (EC 1118 fermentation); the phenethyl acetate in sequential inoculation (experiment ②) is also greatly improved, which is 22 times that of experiment ③ (EC 1118 fermentation), and the phenethyl acetate has sweet fragrance and can optimize the fragrance of the wine;
The terpene aroma content in experiment ① (strain SIVE4101 fermentation) was 1.84mg/L, 1.57 times that in experiment ③ (EC 1118 fermentation); terpene compounds in sequential inoculation (experiment ②) are also improved to a certain extent, and the terpene compounds have the characteristic of low threshold value and are easy to be perceived;
In addition, the ingredients in experiment ① (strain SIVE4101 fermentation) and sequential inoculation (experiment ②) were significantly higher than in experiment ③ (EC 1118 fermentation) were: ethyl acetate, isobutyl acetate, isoamyl acetate, 2-phenylethyl propionate, ethyl palmitate, and ethyl 9-hexadecenoate.
Although the present invention has been described in detail by way of reference to preferred embodiments, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (7)
1. The strain is characterized by being SIVE4101, and the strain is preserved in China Center for Type Culture Collection (CCTCC) at 22 nd year 2023; address: wu Changou eight ways of Wuhan City in Hubei province, wuhan university; post code: 430072 with the preservation number of CCTCC NO: M2023794 and Latin name ISSATCHENKIA TERRICOLA.
2. The Liu Shengyi sarsasa of claim 1, wherein the terrestrial light sarsasa SIVE4101 is obtained from a pre-mash screen of naturally fermented grapes, and the nucleotide sequence of the 26s rDNA D1/D2 region is as shown in SEQ ID NO:1 is shown in the specification; the Liu Shengyi Saccharomyces SIVE4101 and SO 2 tolerance is up to 1000mg/L; the temperature tolerance is 8-38 ℃.
3. The Liu Shengyi torula according to claim 1, wherein said Liu Shengyi torula SIVE4101 has a glucose tolerance of between 100 and 400g/L, a fructose tolerance of between 100 and 400g/L, an osmotic pressure tolerance of between 0.8 and 2.0mol/L in KCl concentration, a pH tolerance of 3.0 or higher, and an ethanol tolerance of up to 8% (v/v).
4. Use of the terrestrial Issatchenkia of any of claims 1 to 3 in the fermentation of wine, characterized in that the fermentation temperature of Liu Shengyi Saccharomyces SIVE4101 is 20±1 ℃, and the enzyme activity of β -glucosidase is 88.33U/L at 72 hours.
5. The method according to claim 4, wherein Liu Shengyi g of fresh yeast cells are added per liter of grape juice in an amount of SIVE 4101.
6. The use according to claim 5, wherein Liu Shengyi sarcandra SIVE4101 is prepared by: inoculating 1-ring inclined plane Liu Shengyi Saccharomycetes SIVE4101 into YPD liquid culture medium, culturing at 28deg.C for 18-24 hr with shaking table 200r/min, centrifuging, and collecting fresh thallus; EC1118, the preparation method is: inoculating 1-ring inclined plane EC1118 into YPD liquid culture medium, culturing at 28deg.C with shaking table 200r/min for 18-24 hr, centrifuging, and collecting fresh thallus.
7. Use of Liu Shengyi sarcandra and EC1118 according to any of claims 1-3 in wine, characterized in that when Liu Shengyi sarcandra and EC1118 are mixed fermented, liu Shengyi sarcandra SIVE4101 is added and fermented for 48 hours before EC1118 is added; liu Shengyi Saccharomyces SIVE4101 and EC1118 in a weight ratio of 1:1; after fermentation, the content of phenethyl acetate and terpene aroma substances in the obtained wine is improved.
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