CN115926914B - Application of Kluyveromyces marxianus in rice-flavor white spirit - Google Patents
Application of Kluyveromyces marxianus in rice-flavor white spirit Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The application relates to the technical field of microorganisms, in particular to application of Kluyveromyces marxianus in rice-flavor white spirit, and the beneficial effects of the application are as follows: the Kluyveromyces marxianus with high yield of ethyl acetate and ethyl lactate is adopted for producing the rice-flavor small starter base wine, so that the content of ethyl acetate and ethyl lactate in the rice-flavor small starter base wine is effectively improved, the taste of the wine is enriched, and the finally produced small starter base wine has elegant honey aroma, soft and soft taste, sweet taste and pleasant aftertaste. And obtaining an accelerant for promoting the production of the yeast Y245 through screening: the application adopts the traditional method to obtain the yeast and the yeast promoter, does not need to carry out yeast transformation in a genetic engineering mode, has low technical difficulty, is convenient to operate, and can be used for a long time under a proper cultivation environment, thereby greatly reducing the production cost.
Description
[ Field of technology ]
The invention relates to the technical field of microorganisms, in particular to application of Kluyveromyces marxianus in rice-flavor white spirit.
[ Background Art ]
The ester has aromatic smell, is a key substance for forming the typical style of wine body, and in rice-flavor white wine, the main aroma substances are tetraesters in the white wine, wherein ethyl acetate, ethyl caproate, ethyl lactate and ethyl butyrate are the main substances. At present, two approaches for generating esters are mainly adopted, namely, the esters are generated through organic chemical reaction; secondly, the ester is produced by a certain biochemical reaction of a class of microorganisms such as Hansenula polymorpha and the like which have esterification capability on alcohol and acid. Esters are important bases for quality assessment of white wine, have important practical application value for research of the white wine, the content of the esters can influence the coordination of taste and the wine, and the too low ethyl acetate can lead the wine body to have insufficient fragrance; a proper amount of ethyl lactate can increase the thick feel of the wine, and an excessively low content can make the wine body light and tasteless. Therefore, by a series of measures such as breeding high-quality strains, scientific blending and the like through an improvement process, the content proportion of various esters in the white spirit is regulated, so that the production of high-quality wine with outstanding style and better taste is a imperative thing.
Ethyl lactate and ethyl acetate are main aroma components of rice-flavor type, white spirit of dried and fermented soybean-flavor type. The quality of the white spirit and the function of the functional bacteria in the brewing process are inseparable, and the functional bacteria are utilized to improve the yield of the white spirit and the generation of flavor substances, so that the white spirit is increasingly and widely focused by related researchers. In the prior art, a plurality of reports on separating high-yield lipid microorganisms from wine bodies or brewing environments exist, for example, in the text of research on fermentation characteristics of Saccharomyces cerevisiae Y1 in white spirit Daqu, an esterifying enzyme preparation is prepared by using monascus strains with higher ester production capacity, which are separated from Fenjiu Daqu, and meanwhile, the catalytic conditions of esterifying enzyme are optimized, and finally, the yield of ethyl lactate can reach 3.38g/L. In the section I of breeding of Saccharomyces cerevisiae for producing ethyl lactate, the lactic dehydrogenase gene of lactobacillus plantarum is introduced into white spirit yeast by a genetic modification method, and gene recombination is carried out, so that recombinant strains with the production amounts of lactic acid and ethyl lactate of 12.64g/L and 162.75mg/L respectively are finally obtained. In the research on the ester production condition of high-yield ethyl acetate yeast, a strain with high yield of ethyl acetate is screened from distiller's yeast, the total ester yield in corn saccharification liquid is 2.152g/L, and the ester yield of the strain is improved to 4.812g/L after condition optimization. In the text of "influence of acetyl-CoA content on the synthesis of ethyl acetate by Saccharomyces cerevisiae", the yield of ethyl acetate of a recombinant strain obtained by modifying the knock-out acetyl-CoA hydrolase gene (ACH) over-expression acetyl-CoA synthetase gene (ACS) over-expression ATF1 gene of Saccharomyces cerevisiae is improved by 530.43% at maximum.
Strains were obtained by screening proprietary Jinzhen limited: kluyveromyces marxianus Y245 finds that the strain has the effect of reducing n-propanol, and after the applicant is purchased by the patentee, the strain is widely applied to white spirit production, and experiments carried out by the applicant show that the strain has good effect on flavoring of rice-flavor white spirit, is an important way for improving the content of ethyl acetate and ethyl lactate in the rice-flavor Xiaoqu white spirit, and has important significance for improving the quality of the rice-flavor white spirit.
[ Invention ]
The invention aims at researching the application of Kluyveromyces marxianus Y245, and can effectively improve the contents of ethyl acetate and ethyl lactate in wine bodies in the production process of rice-flavor small starter wine base, so that the quality of the wine base is improved, and the yeast has an aroma-enhancing effect on rice-flavor white spirit.
The invention comprises application of Kluyveromyces marxianus (Kluyveromycesmarxianus) Y245 in flavoring of rice-flavor base wine, wherein the Kluyveromyces marxianus (Kluyveromycesmarxianus) Y245 is preserved in China center for type culture Collection, the preservation date is 2021, 3 months and 15 days, and the preservation number is CCTCCNO: m2021225.
The invention also comprises an accelerator applied to the aroma enhancement of the Kluyveromyces marxianus (Kluyveromycesmarxianus) Y245 in the rice-flavor raw wine, wherein the accelerator is a climbing rock aroma extracting solution, a mulberry leaf extracting solution and/or a mint extracting solution.
The invention also comprises a distiller's yeast applied to the Kluyveromyces marxianus (Kluyveromycesmarxianus) Y245 for flavoring rice-flavor base wine, and the preparation method of the distiller's yeast comprises the following steps: inoculating Kluyveromyces marxianus (Kluyveromyces marxianus) Y245 into YPD culture medium for primary culture, then picking up Kluyveromyces marxianus (Kluyveromycesmarxianus) Y245 colony, inoculating into rice yeast juice for culture to obtain secondary yeast liquid, pouring the secondary yeast liquid into sterilized bran to obtain bran yeast, inoculating rhizopus into rice powder to obtain pure rhizopus, and mixing the bran yeast and the pure rhizopus according to a mass ratio of 1:10 to obtain the finished distiller's yeast.
Furthermore, kluyveromyces marxianus (Kluyveromyces marxianus) Y245 promoter is also added into the YPD culture medium and the rice koji juice, and the promoter is a climbing rock aroma extracting solution, a mulberry leaf extracting solution and/or a mint extracting solution.
Further, the mass ratio of the climbing rock aroma extracting solution, the mulberry leaf extracting solution and the mint extracting solution is as follows: 3:2-3:1-2.
Further, the preparation method of the climbing rock aroma extracting solution, the mulberry leaf extracting solution and the mint extracting solution comprises the following steps: mixing herba Euphorbiae Humifusae, folium Mori and herba Menthae with water at a mass ratio of 1:1, boiling, keeping boiling for 10min, and cooling to room temperature to obtain corresponding plant extractive solution.
The invention also comprises a method for producing rice-flavor white spirit by using the distiller's yeast, which comprises the following steps: soaking rice, steaming with a horizontal rice steamer, cooling to 26-32deg.C, adding distiller's yeast according to 0.6-0.8% of inoculation amount, stirring distiller's yeast and rice, saccharifying, fermenting, and distilling to obtain rice-flavor Chinese liquor.
The specific processing method comprises the following steps:
preparation of primary medium yeast: placing yeast soaked powder, peptone, glucose, D (+) -maltose and agar into a triangular flask, adding water, stirring uniformly, sealing, placing into a sterilizing pot for sterilization, pouring into a culture dish after cooling slightly (not scalding hands but not cooling to normal temperature), and obtaining YPD culture medium after solidifying and drying water; and (3) streaking and inoculating a pure culture of Kluyveromyces marxianus (Kluyveromycesmarxianus) Y245 with the preservation number of CCTCCNO. M2021225 into a culture medium, and culturing for 24-36h in a 30 ℃ incubator to obtain the first-stage culture medium yeast.
Preparing secondary yeast liquid: taking rice yeast juice, adding appropriate amount of water, sealing, sterilizing, cooling to obtain rice Qu Zhiye, adding primary culture medium yeast (Kluyveromycesmarxianus) Y245 into rice yeast juice, shake culturing in shaking table for 24 hr, and standing for 24 hr to obtain secondary yeast liquid.
Preparation of pure wheat bran yeast: weighing a certain amount of bran, placing the bran into a sterilizing pot for sterilization, shoveling the raw materials out after sterilization is finished, spreading the materials for cooling, cooling to 28-37 ℃, uniformly pouring the cultured secondary yeast liquid into the raw materials, adding water, stirring and uniformly mixing, placing the uniformly mixed materials into a dustpan, and transferring the dustpan into a culture room for culture according to culture parameters. The pure wheat bran yeast is prepared by taking pure yeast fragrance and no peculiar smell, and the surface of the wheat bran is light white as qualified yeast, and is prepared after drying.
Preparing a finished yeast: and uniformly mixing the pure wheat bran yeast and the pure rhizopus in proportion to obtain the finished yeast.
Preparation of rice-flavor raw wine: soaking rice in water at below 45deg.C for 20-25min, steaming with horizontal rice steamer, cooling to 26-32deg.C, adding yeast at an inoculation amount of 0.6-0.8%, stirring, saccharifying with disk saccharifying machine, saccharifying at below 45deg.C for 19-24 hr, adding water, transferring to fermentation tank, controlling the temperature of fermentation tank to 26-28deg.C, and fermenting for 13 days to obtain rice-flavored wine base.
Preferably, in the preparation step of the YPD medium, the proportion of yeast extract, peptone, glucose, D (+) -maltose and agar is 1% of yeast extract, 2% of peptone, 2% of glucose, 2% of D (+) -maltose and 2% of agar.
Preferably, in the step of preparing the secondary yeast solution, the rice yeast juice is syrup produced in the brewing saccharification process.
Preferably, in the preparation step of the secondary yeast solution, the concentration of the rice yeast solution is adjusted to be 14g/L.
Preferably, in the preparation step of the pure rhizopus, the raw material treatment parameters are that the rice is crushed by a 40-mesh sieve, and the water content of the material is 25-28%.
Preferably, in the preparation step of the pure rhizopus, the culture parameters are that the temperature is 28-37 ℃ and the culture time is more than or equal to 36 hours.
Preferably, in the preparation step of the finished yeast, the proportion of each component is as follows: rhizopus is pure wheat bran yeast=10:1.
The preparation method of the more preferable distiller's yeast comprises the following steps: preparing plant extract: mixing and boiling the climbing rock fragrance, the mulberry leaf and the mint with water according to the mass ratio of 1:1, keeping boiling for 10min after boiling, cooling to room temperature to obtain corresponding plant extract, and mixing the climbing rock fragrance extract, the mulberry leaf extract and the mint extract according to the mass ratio of 3:2:1 to obtain mixed plant liquid. Weighing 10g of yeast extract powder, 20g of peptone, 20g of glucose, 20g of D (+) -maltose and 20g of agar; mixing the mixed plant liquid and water according to the volume ratio of 1:2 respectively to prepare mixed liquid, then adding 1L of mixed liquid into the solid substance, sterilizing for 30min, pouring into a plate, and airing to obtain the YPD-plant extract plate. And (3) inoculating the yeast Y245 into a YPD-plant extract flat plate for streaking culture, then picking a Y245 yeast colony, inoculating the Y245 yeast colony into a rice yeast juice plant extract with the sugar degree of 14-16 degrees Bx, performing shake culture for 12 hours, and performing stationary culture for 12 hours to obtain solid yeast Y245 yeast seeds as liquid yeast seeds in bran materials for expanding culture. The preparation method of the rice yeast juice plant extract comprises the following steps: mixing rice koji juice and mixed plant liquid according to the volume ratio of 2:1. The prepared solid yeast Y245 starter was mixed with rhizopus (available from Angel Yeast Co., ltd.) at a ratio of 1:10 to obtain a finished starter.
The application has the beneficial effects that: the Kluyveromyces marxianus with high yield of ethyl acetate and ethyl lactate is adopted for producing the rice-flavor small starter base wine, so that the content of ethyl acetate and ethyl lactate in the rice-flavor small starter base wine is effectively improved, the taste of the wine is enriched, and the finally produced small starter base wine has elegant honey aroma, soft and soft taste, sweet taste and pleasant aftertaste. And obtaining an accelerant for promoting the production of the yeast Y245 through screening: the application adopts the traditional method to obtain the yeast and the yeast promoter, does not need to carry out yeast transformation in a genetic engineering mode, has low technical difficulty, is convenient to operate, and can be used for a long time under a proper cultivation environment, thereby greatly reducing the production cost.
[ Description of the drawings ]
FIG. 1 is a schematic plate morphology of Kluyveromyces marxianus.
[ Detailed description ] of the invention
All of the features disclosed in this specification, or all of the steps in a method or process disclosed, may be combined in any combination, except for mutually exclusive features and/or steps.
Any feature disclosed in this specification (including any accompanying claims, abstract) may be provided with respect to each feature disclosed herein, unless otherwise indicated, as an example of a generic series of equivalent or similar features.
Example 1:
The high-yield ethyl acetate and ethyl lactate yeast is classified and named as Kluyveromyces marxianus (Kluyveromyces marxianus) Y245 (hereinafter referred to as yeast Y245), and is preserved in China center for type culture Collection, with a preservation date of 2021, 3 months and 15 days, and a preservation number of CCTCCNO: m2021225, introduced from Jinba limited.
The influence of the saccharomycete on the flavor substances in the wine is studied, and the method is specifically as follows:
1. pilot experiment:
(1) And (3) selecting Y245 yeast colonies on a YPD plate (the YPD plate comprises 10g of yeast extract powder, 20g of peptone, 20g of glucose, 20g of D (+) -maltose and 20g of agar, adding 1L of purified water, mixing the materials, sterilizing the materials for 30min, pouring the materials into a culture dish, airing the materials to obtain the product), inoculating the Y245 yeast colonies into rice yeast juice with the sugar degree of 14-16 degrees Bx, culturing the rice yeast colonies for 12h in a shaking table, and performing stationary culture for 12h to obtain solid yeast Y245 yeast seeds serving as liquid yeast seeds in bran materials by expanding culture.
(2) Mixing the solid yeast Y245 starter prepared in the step (1) with rhizopus (purchased from Angel Yeast Co., ltd.) according to a ratio of 1:10 to obtain a finished yeast, inoculating the finished yeast into 200g of cooked rice according to a lower yeast amount of 1%, uniformly mixing, saccharifying for 24 hours in a constant temperature incubator at 30 ℃, adding water with a mass 1.3 times of that of the base material, uniformly mixing, continuously fermenting in the constant temperature incubator at 30 ℃ for 13 days, distilling after fermenting for 13 days, removing 25ml of wine heads, inoculating 100ml of raw wine, and detecting indexes such as total acid, total ester and the like of the raw wine. The experimental group is provided with two parallel groups, namely rhizopus+Y245 (1) and rhizopus+Y245 (2)
Control group: the results of performing the pilot tests according to the experimental procedures of step (1) and step (2) using commercially available yeasts and rhizopus (both from Angel Yeast Co., ltd.) and detecting the indexes of total acids, total esters, etc. of the raw wine are shown in Table 1:
TABLE 1 Main index data of Small test base wine
As can be seen from Table 1, the experimental group inoculated with yeast Y245 in the test base liquor has significantly improved ethyl acetate and ethyl lactate content and significantly reduced fusel oil content, which indicates that the replacement of commercial Angel yeast with yeast Y245 has the effects of increasing the content of esters and reducing the fusel oil content.
2. Large scale experiment:
(1) Solid yeast Y245 was mixed with rhizopus (available from Angel Yeast Co., ltd.) at a ratio of 1:10 to give a finished yeast.
(2) And carrying out batch feeding verification in a brewing workshop according to the process of 20 tons/batch, the lower bending temperature is 28-32 ℃, the lower bending amount is 0.6-0.8% (weight percent), the saccharification temperature is less than or equal to 45 ℃, the water adding proportion is 130% (weight percent), the fermentation temperature is 26-28 ℃ and the fermentation time is 13 days. Repeat 4 batches and name: rhizopus+y245 (1), rhizopus+y245 (2), rhizopus+y245 (3), and rhizopus+y245 (4).
In addition, a control group was also provided, and the experimental procedure of the control group was identical to those of (1) and (2), except that the yeast was Angel yeast, which was commercially available.
When the fermentation of the wine of the batch is finished, the operation of 'slow steaming distillation, medium temperature flow wine and rear-end collision' is carried out, the wine receiving temperature and the wine degree are strictly carried out according to the process standard, the flow wine temperature is less than 30 ℃, the wine degree of the first section is kept at 55+/-1%vol, the wine degree of the middle section is kept at 30+/-1%vol, the raw wine is singly stored, 500ml of sample is taken for detecting total acid, total ester and gas chromatography, and the obtained large test results are shown in Table 2:
TABLE 2 Main index data of Large test base wine
Proved by large tests, the yeast Y245 strain is used for replacing other yeast strains sold in the market in the rice flavor brewing process, and the yeast Y245 strain has the functions of high-yield ethyl acetate and ethyl lactate, and the contents of the yeast Y245 strain and the ethyl lactate are obviously improved in the large test wine base, which is far higher than the original standard of the original wine base of a company, and meanwhile, the yeast Y245 strain has a certain fusel oil reducing effect. Therefore, in the processing of the rice-flavor white spirit, the yeast Y245 has a good aroma raising function, can effectively improve the ester content of the rice-flavor white spirit and reduce the content of fusel oil.
Example 2:
Through the verification of the embodiment 1, the yeast Y245 strain has the fragrance-improving effect on the rice-flavor white spirit, so that the strain is used for fragrance-improving processing of the rice-flavor white spirit, and the specific process is as follows:
1. preparation of primary medium yeast:
Weighing 10g of yeast extract powder, 20g of peptone, 20g of glucose, 20g of D (+) -maltose and 20g of agar, adding 1L of purified water, stirring uniformly, sealing, placing into a sterilizing pot, starting to count time for 30min after the sterilizing pot is first steam-discharged, closing, pouring into a culture dish after slightly cooling, and standing for three days to observe that no water drops exist on the surface of the culture medium, thus obtaining the yeast culture medium. And (3) streaking and inoculating a pure culture of Kluyveromyces marxianus (Kluyveromyces marxianus) Y245 with the preservation number of CCTCC No. M2021225 into a culture medium, and culturing for 24-36h in a 30 ℃ incubator to obtain the first-stage culture medium yeast.
2. Preparation of secondary yeast liquid
Measuring Qu Zhi ml of saccharified rice, measuring sugar degree with a sugar degree refractometer, calculating and adding appropriate amount of water to adjust sugar degree to 14 degrees Bx, sealing, sterilizing for 10min, discharging, cooling to obtain rice Qu Zhiye, adding Kluyveromyces marxianus (Kluyveromyces marxianus) Y245 cultured in YPD culture medium into the rice juice, shake culturing at 30deg.C in shaking table for 24 hr, and standing for 24 hr to obtain secondary yeast liquid.
3. Preparation of pure wheat bran yeast
Weighing 20kg of bran, placing the bran into a sterilizing pot for sterilization, after sterilization, shoveling and cooling the raw materials, when the temperature is reduced to 28-37 ℃, uniformly pouring 20ml of secondary yeast liquid into the raw materials, adding 10kg of water, stirring and uniformly mixing, placing the uniformly mixed material into a dustpan, transferring into a culture room, controlling the temperature in the room to be 25-38 ℃, culturing, and turning over the materials and discharging moisture according to the temperature change in the room. The pure wheat bran yeast is prepared by taking pure yeast fragrance and no peculiar smell, and the surface of the wheat bran is light white as qualified yeast, and is prepared after drying.
4. Preparation of pure rhizopus
Selecting 110kg of fresh rice, crushing, sieving with a 40-mesh sieve, sterilizing the rice flour for 2+/-0.5 hours, shoveling out, spreading for cooling, adding 0.22kg of rhizopus (purchased from Angel Yeast Co., ltd.) and 19.25kg of water when the temperature is reduced to about 28-37 ℃, uniformly mixing, loading into a culture chamber, and detecting that the water content of the material is 25-28%, wherein the temperature in the culture chamber is controlled to be 25-32 ℃ and the humidity is controlled to be more than 60%rh. The temperature of the product is controlled to be 28-37 ℃ in the culture process, and the culture time is more than or equal to 36 hours. When the temperature reaches 33-35 ℃, spreading and culturing can be carried out. After the culture is continued for about 16 hours, the product can be crushed when the temperature is reduced to below 33 ℃, and the product is dried until the moisture is less than or equal to 12 percent. Drying to obtain pure rhizopus.
5. Preparation of finished koji
Weighing 10.9kg of pure wheat bran yeast prepared in the step (3), and uniformly mixing 109.1kg of pure rhizopus prepared in the step (4) to prepare the finished yeast.
6. Preparation of rice-flavor Xiaoqu wine base
Soaking 20 tons of rice for 20-25min at a water temperature below 45 ℃, steaming the rice by a horizontal rice steaming machine, cooling the rice to 26-32 ℃, adding 120kg of finished yeast by a yeast making machine according to an inoculum size of 0.6% by weight, uniformly stirring the yeast and the rice by a harrow roller, conveying the yeast and the rice to a disk saccharification machine by a conveying belt for bacteria cultivation and saccharification, controlling the temperature below 45 ℃, adding 26 tons of tap water according to a water adding ratio of 130% after bacteria cultivation and saccharification for 19-24h, uniformly mixing and subpackaging the mixture into a fermentation tank, controlling the temperature in the tank to 26-28 ℃ within 24 hours, controlling the fermentation period to 26-30 ℃, and carrying out fermentation period to 13 days to obtain the rice fragrance type small yeast wine base by slow steam distillation.
Comparative example
A commercially available Angel yeast is used for replacing the yeast Y245 of the application to produce distiller's yeast, and then rice-flavor small starter base wine produced by synchronous brewing is carried out. The component content in the wine base is detected, and the final detection result is shown in table 3:
TABLE 3 Rice-flavored Xiaoqu wine base experimental results produced by different Yeast strains
| Sample of | Yield of wine | Ethyl acetate (g/L) | Ethyl lactate (g/L) |
| Examples | 60.39％ | 0.749 | 1.636 |
| Comparative example | 61.03％ | 0.162 | 0.295 |
As can be seen from Table 3, the yield of the rice-flavor type small starter base wine produced by using the yeast Y245 is equivalent to that of the comparative example using Angel yeast, but the ethyl acetate and ethyl lactate contents are remarkably improved, wherein the ethyl acetate content is improved by 362.35% compared with the comparative example and the ethyl lactate content is improved by 454.58% compared with the comparative example, so that the yeast Y245 can effectively improve the taste and coordination of the wine body, and the wine base is elegant in honey aroma, soft in inlet, sweet and cool in mouth, and pleasant in aftertaste.
Example 3:
this example investigated the effect of different plant extracts on the ester production effect of yeast Y245.
In the process of brewing Xiaoqu wine, some plants are added to prepare distiller's yeast, but in actual production, it is found that, because the components of the plants are complex and the active components are not clear, some plants have the effect of promoting the growth of microorganisms, but some plants have the effect of inhibiting the growth of microorganisms, and in the process of using the plants, if the plant components are simply combined, the metabolic performance of microorganisms is limited, so that the wine of the raw wine is affected. Therefore, the influence of several plant extracts on the ester production effect of yeast Y245 is mainly studied in the embodiment, and the method is as follows:
1. preparing a plant extract: mixing herba Polygoni Hydropiperis, herba Euphorbiae Humifusae, folium Mori, folium Osmanthi Fragrantis and herba Menthae with water according to mass ratio of 1:1, boiling, keeping boiling for 10min, and cooling to room temperature to obtain corresponding plant extractive solution.
2. Preparation of YPD-plant extract plates containing plant extract:
Weighing 10g of yeast extract powder, 20g of peptone, 20g of glucose, 20g of D (+) -maltose and 20g of agar; mixing plant extract and water according to a volume ratio of 1:2 respectively to obtain mixed solution, adding 1L of mixed solution into the solid substance, sterilizing for 30min, pouring into a plate, and air-drying to obtain YPD-plant extract plate.
3. And (3) activating and culturing:
inoculating yeast Y245 into the YPD-plant extract flat plate in the step 2 for streaking culture, then picking Y245 yeast colony, inoculating into rice yeast juice-plant extract with the sugar degree of 14-16 degrees Bx, performing shake culture for 12 hours, standing and culturing for 12 hours, and performing expansion culture in bran materials as liquid yeast to obtain solid yeast Y245 yeast. The preparation method of the rice koji juice-plant extract comprises the following steps: mixing rice koji juice and plant extract at a volume ratio of 2:1.
4. Preparing a finished starter:
Mixing the solid yeast Y245 starter prepared in the step 3 with rhizopus (purchased from Angel Yeast Co., ltd.) at a ratio of 1:10 to obtain the final starter.
5. Fermentation experiment:
inoculating the finished yeast into 200g of rice according to the lower yeast amount of 1%, uniformly mixing, placing in a constant temperature incubator at 30 ℃ for saccharification for 24 hours, adding water with the mass 1.3 times of the mass of the base material, uniformly mixing, continuously placing in the constant temperature incubator at 30 ℃ for fermentation for 13 days, distilling after fermentation for 13 days, removing 25ml of wine heads, inoculating 100ml of raw wine, and detecting indexes such as total acid, total ester and the like of the raw wine. The experimental groups were set up with 3 replicates and the average was recorded. The control group adopts the same experimental method, except that the flat culture medium in the step 2 and the rice juice in the step 3 do not contain plant extract components, but are replaced by purified water with the same amount, and the specific experimental results are shown in table 4:
TABLE 4 influence of different plant extracts on the ester yield of wine base
| Test item | Polygonum hydropiper (L.) kuntze group | Rock climbing incense group | Mulberry leaf group | Sweet osmanthus tree leaf group | Peppermint group | Control group |
| Ethyl lactate g/L | 0.034 | 0.312 | 0.265 | 0.087 | 0.195 | 0.189 |
| Ethyl acetate g/L | 0.473 | 0.946 | 0.924 | 0.531 | 0.931 | 0.885 |
As can be seen from Table 4, the herba polygoni hydropiperis and the osmanthus fragrans leaves have a certain inhibition effect on the ester production of the yeast Y245, the use of the herba polygoni hydropiperis and the osmanthus fragrans leaves does not improve the ester production of the rice-flavor white spirit, the effect of improving the fragrance of the rice-flavor white spirit is not achieved, the climbing rock and the mulberry leaves can effectively improve the ester production of the yeast Y245, the effect is most obvious, the mint group can improve the ester production of the yeast Y245, and the effect is not obvious as that of the mulberry leaves and the climbing rock.
Example 4:
According to the research result of the embodiment 3, plant extracts of climbing rock, mulberry leaf and peppermint are selected for proportioning experiments, and the proportion of the extract with the best ester production capacity of the yeast Y245 is found out, wherein the proportion is as follows:
The method is characterized in that an orthogonal experiment is designed by the mass ratio of climbing rock fragrance, mulberry leaf and mint, a small experiment is carried out by replacing a single plant extract with a compound mixture of the plant extract, and the ethyl acetate yield is used as a screening index, and specifically comprises the following steps:
TABLE 5 orthogonal Experimental factor level of the ethyl lactate production capability of Yeast Y245
The results of the orthogonal analysis of the above ratios are shown in table 6:
TABLE 6 orthogonal experiments and results
From the table, in the experiment of group 8, the ethyl lactate content of the wine body is highest and reaches 1.187g/L, namely the climbing rock extract: mulberry leaf extract: the mass ratio of the mint extract is 3:2:1; the yield of the ethyl lactate in the experiment of the 9 th group can also reach more than 1.0g/L, namely the Polygonum tinctorium extract: mulberry leaf extract: the mass ratio of the mint extract is 3:3:2. From the extremely poor, the extremely poor of the climbing-rock-flavor extracting solution > the mint extracting solution > the mulberry leaf extracting solution, thereby indicating that the climbing-rock-flavor extracting solution has the greatest influence on the ethyl acetate content in the wine body, and then the mint has the least influence on the mulberry leaves.
Therefore, according to the experiment, the optimal proportion of the plant extract capable of effectively improving the rice-flavor raw wine is obtained as follows: climbing rock aroma extracting solution: mulberry leaf extract: the mass ratio of the mint extract is 3:2-3:1-2.
Example 5:
the influence of different distiller's yeast on brewing of rice-flavor white spirit is studied:
1. different finished starter is prepared:
① The preparation method of the finished koji by adopting the proportion of the plant extract of the experiment of the 8 th group of the example 4 comprises the following steps:
Preparing a plant extract: mixing and boiling the climbing rock fragrance, the mulberry leaf and the mint with water according to the mass ratio of 1:1, keeping boiling for 10min after boiling, cooling to room temperature to obtain corresponding plant extract, and mixing the climbing rock fragrance extract, the mulberry leaf extract and the mint extract according to the mass ratio of 3:2:1 to obtain mixed plant liquid. Weighing 10g of yeast extract powder, 20g of peptone, 20g of glucose, 20g of D (+) -maltose and 20g of agar; mixing the mixed plant liquid and water according to the volume ratio of 1:2 respectively to prepare mixed liquid, then adding 1L of mixed liquid into the solid substance, sterilizing for 30min, pouring into a plate, and airing to obtain the YPD-plant extract plate. Culturing yeast Y245 in YPD-plant extract plate by streaking, inoculating Y245 yeast colony into rice yeast juice-plant extract with sugar degree of 14-16 degree Bx, shake culturing for 12 hr, standing for 12 hr, and culturing as liquid yeast in bran material to obtain solid yeast Y245 yeast. The preparation method of the rice koji juice-plant extract comprises the following steps: mixing rice koji juice and mixed plant liquid according to the volume ratio of 2:1. The prepared solid yeast Y245 starter was mixed with rhizopus (available from Angel Yeast Co., ltd.) at a ratio of 1:10 to give a finished starter for the experimental group.
② Solid yeast Y245 and rhizopus (available from Angel Yeast Co., ltd.) were mixed in a ratio of 1:10 to give a final yeast, which was used as a final yeast for control group 1.
③ A finished yeast was prepared by mixing commercially available Angel Yeast with Rhizopus (available from Angel Yeast Co., ltd.) at a ratio of 1:10 and was recorded as a control group 2 finished yeast.
2. Large scale fermentation experiment:
And carrying out batch feeding verification in a brewing workshop according to the process of 20 tons/batch, the lower bending temperature is 28-32 ℃, the lower bending amount is 0.6-0.8% (weight percent), the saccharification temperature is less than or equal to 45 ℃, the water adding proportion is 130% (weight percent), the fermentation temperature is 26-28 ℃ and the fermentation time is 13 days. Experiments were repeated with 10 batches, and the average was taken and recorded.
When the fermentation of the wine of the batch is finished, the operation of 'slow steaming distillation, medium temperature flow wine and rear-end collision' is carried out, the wine receiving temperature and the wine degree are strictly carried out according to the process standard, the flow wine temperature is less than 30 ℃, the wine degree of the first section is kept at 55+/-1%vol, the wine degree of the middle section is kept at 30+/-1%vol, the raw wine is singly stored, 500ml of sample is taken for detecting total acid, total ester and gas chromatography, and the obtained large test results are shown in table 7:
TABLE 7 Main index data of different distiller's yeast Daqu base wines
Through large test, the plant extract is adopted: the climbing rock aroma extracting solution, the mulberry leaf extracting solution and the mint extracting solution are mixed according to the mass ratio of 3:2:1 and then added into a yeast making culture medium (comprising a YPD culture medium and a rice yeast juice culture medium) of the yeast Y245 strain, so that the ester production capacity of the yeast Y245 strain can be remarkably improved, a good aroma enhancement effect is further achieved on rice aroma type white spirit, and meanwhile, the further fusel oil reducing effect is achieved.
In conclusion, a large number of production tests prove that the introduced yeast strain Y245 has good ester yield and good flavoring effect in the brewing process of the rice-flavor white spirit, and the plant extract composition with the enhancing effect on the ester yield of the yeast strain Y245 and the proportion thereof are found out through the verification of the plant extract.
The above examples merely represent a few embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the present invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of the invention should be assessed as that of the appended claims.
Claims (3)
1. The distiller's yeast for rice-flavored base wine flavoring is characterized in that the preparation method of the distiller's yeast comprises the following steps: inoculating Kluyveromyces marxianus (Kluyveromyces marxianus) Y245 into YPD culture medium for primary culture, then picking up Kluyveromyces marxianus (Kluyveromyces marxianus) Y245 colony, inoculating into rice yeast juice for culture to obtain secondary yeast liquid, pouring the secondary yeast liquid into sterilized bran to prepare bran yeast, inoculating rhizopus into rice powder to prepare pure rhizopus, and mixing the bran yeast and pure rhizopus according to a mass ratio of 1:10 to prepare finished distiller's yeast;
the preservation number of the Kluyveromyces marxianus is CCTCC NO: m202122;
The YPD culture medium and the rice yeast juice are also added with a kluyveromyces marxianus (Kluyveromycesmarxianus) Y245 promoter, wherein the promoter comprises a climbing rock aroma extracting solution, a mulberry leaf extracting solution and a mint extracting solution according to the mass ratio: 3:2-3:1-2.
2. The distiller's yeast according to claim 1, wherein the preparation method of the climbing rock aroma extract, the mulberry leaf extract and the peppermint extract comprises the following steps: mixing herba Euphorbiae Humifusae, folium Mori and herba Menthae with water at a mass ratio of 1:1, boiling, keeping boiling for 10min, and cooling to room temperature to obtain corresponding plant extractive solution.
3. A method for producing rice-flavor white spirit by using distiller's yeast according to claim 1, wherein the method comprises the following steps: soaking rice, steaming with a horizontal rice steamer, cooling to 26-32deg.C, adding distiller's yeast according to 0.6-0.8% of inoculation amount, stirring distiller's yeast and rice, saccharifying, fermenting, and distilling to obtain rice-flavor Chinese liquor.
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