CN109234179B - Yeast separation medium in fermented grains and preparation method thereof - Google Patents
Yeast separation medium in fermented grains and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a yeast isolated culture medium in the technical field of microbial fermentation engineering, in particular to a yeast isolated culture medium in fermented grains and a preparation method thereof. Discloses a yeast separation culture medium in fermented grains, the formula of the culture medium comprises the following components: 5-10g/L of yeast powder, 10-20g/L of peptone, 5-20g/L of glucose, 15-22g/L of agar, 100 mL/L of Daqu-fermented grain leaching liquor and the balance of water. Aiming at the fermentation characteristics of fermented grains in different periods, the pH value of the culture medium is properly adjusted, which is beneficial to the systematic separation of various yeasts in the brewing process. The culture medium has the advantages of rich composition, strong pertinence and wide screening range.
Description
Technical Field
The invention relates to a yeast isolated culture medium in the technical field of microbial fermentation engineering, in particular to a yeast isolated culture medium in fermented grains and a preparation method thereof.
Background
The yeast is widely existed in yeast making, stacking, cellar, air and airing, and is an important microorganism in the production process of white spirit. The functional bacteria are divided into functional bacteria for producing liquor and functional bacteria for producing fragrance according to functions, and the growth and metabolism conditions of the functional bacteria have important influence on the yield and flavor of the white spirit. In order to research the action mechanism of different yeasts in the production of white spirit, the yeasts need to be fully separated and screened firstly.
The existing culture medium for separating the yeast comprises a yeast leaching peptone glucose culture medium (YPD culture medium), a potato glucose agar culture medium (PDA culture medium), a WL nutrient agar culture medium (WL culture medium), a Bengal red culture medium and the like, wherein the YPD culture medium and the WL culture medium have higher use frequency and more prominent separation effect. So far, there has been no report on yeast separation in fermented grains by adding a Daqu-fermented grain leaching solution to a culture medium.
Disclosure of Invention
The invention aims to provide a yeast separating culture medium.
The invention further aims to provide a yeast separating culture medium with strong pertinence.
The invention further aims to provide a culture medium capable of separating yeasts in fermented grains in different periods.
The present invention further provides a method for preparing the koji-fermented grain leaching solution contained in the culture medium.
It is a further object of the present invention to provide a method for preparing the above culture medium.
The Daqu-fermented grain leaching liquor is added into the culture medium, so that the growth environment of brewing microorganisms can be better simulated, and the separation of the fermented grain microorganisms is promoted. When the bacteria in the strong aromatic fermented grains are separated, researchers try to prepare a Daqu-fermented grain leaching solution by shaking fermented grains and boiled distilled water and add the Daqu-fermented grain leaching solution to different bacterial culture media, but the separation effect is not obvious. The Daqu-fermented grains leaching solution is not added with Daqu for production, and is not boiled at high temperature.
In order to solve the technical problems, the technical scheme adopted by the invention specifically comprises the following contents:
a culture medium for separating yeasts in fermented grains comprises the following components: 5-10g/L of yeast extract, 10-20g/L of peptone, 5-20g/L of glucose, 15-22g/L of agar, 100 mL/L of Daqu-fermented grain leaching liquor and 800mL/L of water in balance.
As a preferred embodiment, the formulation of the medium comprises the following components: 5-10g/L of yeast extract, 10-15g/L of peptone, 10-20g/L of glucose, 18-20g/L of agar, 200 mL/L of Daqu-fermented grain leaching liquor and 700mL/L of water in balance.
As a more preferred embodiment, the formulation of the culture medium comprises the following components: 5-7g/L of yeast extract, 10-15g/L of peptone, 15-20g/L of glucose, 18-20g/L of agar and 700mL/L of Daqu-fermented grain leaching liquor. In the invention, the addition of the Daqu-fermented grain leaching liquor is particularly important for screening, and the culture medium generally used for yeast separation is a general culture medium, is not added with special substances, cannot better simulate the original growth environment of the yeast, and increases the separation difficulty.
In order to further optimize the components of the culture medium, a proper amount of Daqu-fermented grain leaching liquor is added into the common yeast isolated culture medium, which is beneficial to the separation of microorganisms in the fermented grains.
As a preferred embodiment, the inventor also adjusts the pH of the culture medium in a targeted manner according to the fermentation characteristics of fermented grains, and is favorable for separating the yeasts in the fermented grains in different periods.
As an alternative embodiment, the pH of the culture medium is between 3.0 and 6.0. In the present invention, as an alternative embodiment, the culture medium is used for isolating yeast in a fermented grain sample.
The timing and batch of the fermented grains are not particularly limited, and the fermented grains may be stacked fermented grains, fermented grains placed in a cellar, or fermented grains in a cellar.
In a preferred embodiment, the culture medium further comprises 0.1-1.0g/L of chloramphenicol. The growth of bacteria is inhibited and the separation effect of the saccharomycetes is improved by adding chloramphenicol into the saccharomycetes separation medium.
As an optional scheme, the preparation method of the Daqu-fermented grain leaching liquor comprises the following steps:
(1) weighing fermented grains and Daqu, and mixing;
(2) adding pure water into the fermented grain-Daqu mixture and boiling;
(3) cooling and filtering to obtain filtrate for later use.
In a preferred embodiment, in the step (2), the boiling time is 5 to 30 min.
In a more preferred embodiment, in the step (2), the boiling time is 10 to 15 min.
In some embodiments, the culture medium to which the boiled koji-fermented grains leaching solution is added is more capable of promoting the growth of yeast than the culture medium to which the non-boiled koji-fermented grains leaching solution is added.
As an optional implementation mode, in the step (1), the mass ratio of the Daqu to the fermented grains is 1:1-1: 20.
In a more preferred embodiment, in the step (1), the mass ratio of the Daqu to the fermented grains is 1:3 to 1: 10.
As an alternative embodiment, in the step (2), the mass ratio of the mixture to the water is 1:2 to 1: 20.
As a preferred embodiment, the mass ratio of the mixture to water is from 1:4 to 1: 10.
In a preferred embodiment, in the step (3), filtration is performed with gauze after boiling.
On the other hand, the invention also provides a preparation method of the yeast separating medium, which comprises the following steps:
(1) weighing the components according to the formula, adding yeast powder, peptone, glucose, agar powder and Daqu-fermented grain leaching liquor into a container, adjusting the pH value with an acid solution, supplementing the rest with water, and sterilizing;
(2) dissolving chloramphenicol in ethanol, and shaking to obtain chloramphenicol mother liquor;
(3) and (3) cooling the sterilized mixture obtained in the step (1) to 50-60 ℃, and uniformly mixing the sterilized mixture with the chloramphenicol mother liquor obtained in the step (2).
Preferably, in the step (1), the acid solution is hydrochloric acid solution, glacial acetic acid solution or lactic acid solution.
Further preferably, in step (1), the medium is sterilized at 121 ℃ for 15 to 20 min.
In addition, the invention also provides a method for separating the yeast, wherein the method comprises the step of preparing a series of diluted bacterial liquids from fermented grains by adopting a plate dilution method, and coating the diluted bacterial liquids in the culture medium for culture, so that the effect of separating the yeast is achieved. The rest of the culture conditions and methods are the same as those of the yeast in the prior art.
In still another aspect, the invention provides the application of the culture medium in yeast separation of fermented grains.
The invention has the beneficial effects that:
(1) according to the invention, the Daqu-fermented grain leaching liquor is added into the separation culture medium of the yeast for the first time, and the culture medium can better simulate the growth environment of brewing microorganisms and promote the separation of the fermented grain microorganisms. In the experimental process, the inventor tries to prepare a Daqu-fermented grain leaching liquor by oscillating fermented grains and boiled distilled water for separating yeasts, but the separation effect is not obvious. In addition, the inventors tried to use the koji-fermented grains leachate which was not boiled at high temperature for the separation of yeast, and the separation effect was not remarkable, and therefore, in the present invention, high-temperature boiling and a koji-fermented grains leachate which is common to koji and distilled grains were necessary.
(2) Compared with the conventional yeast separation culture medium, the culture medium disclosed by the invention solves the problem of poor separation effect of the yeast in the fermented grains, better simulates the growth environment of the yeast, is more favorable for systematic separation of the yeast in the fermented grains sample, has strong pertinence to yeast separation, is suitable for the fermented grains in different stages in the brewing process, and has the advantage of wide screening range.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical means of the present invention more clearly understood, the present invention may be implemented in accordance with the content of the description, and in order to make the above and other objects, features, and advantages of the present invention more clearly understandable, the following specific preferred embodiments are described in detail.
Drawings
FIG. 1 shows the influence of different contents of Daqu-fermented grains leaching liquor in a culture medium on the growth and separation of yeast.
FIG. 2 is a graph showing the effect of different pH of the culture medium on the growth and separation of yeast in a sample of stacked fermented grains.
Detailed Description
The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.
The term "fermented grains at different periods" used in the present invention refers to fermented grains samples at any stage in the whole brewing process.
"pH-native" in the context of the present invention means that the pH of the medium is not adjusted.
In the present invention, "pH adjusted" means that the pH of the medium is adjusted by adding an acid solution.
The 'stacked fermented grains' in the invention are fermented grains samples stacked and fermented on the airing chamber.
Wherein the fermented grains entering the cellar refer to fermented grain samples which are prepared to be transferred into the cellar after the stacking fermentation is finished.
The fermented grains in the cellar are fermented grain samples for anaerobic fermentation in the cellar.
Wherein the fermented grains discharged from the cellar are fermented grains samples prepared for opening the cellar for cooking after fermentation in the cellar is finished.
The Daqu used in the Daqu-fermented grain leaching liquor in the embodiment of the invention is a starter for producing white spirit, is also a saccharifying agent, and can provide certain flavor substances for the white spirit, including low-temperature Daqu, medium-temperature Daqu and high-temperature Daqu. The used Daqu is obtained from Daqu obtained by fermenting wheat in the production process of liquor, and specifically is high-temperature Daqu used in the production process of Maotai-flavor liquor.
The fermented grains used in the Daqu-fermented grain leaching liquor are grains which are cooked and fermented in the production process of the white spirit, are important carriers in the brewing process, and can provide certain flavor substances for the white spirit, wherein the grains comprise sorghum, corn, wheat, rice, sticky rice, rice hulls and bran. In the embodiment, the fermented grains used in the Daqu-fermented grain leaching liquor are selected from fermented grains in different rounds of cellars formed by fermenting steamed sorghum in the production process of Maotai-flavor liquor, and particularly fermented grains in a third round of cellars.
Example 1 Yeast isolated Medium formulation and method of preparation
TABLE 1 composition and content of culture Medium
| Components | Medium 1 | Medium 2 | Medium 3 | Medium 4 | Medium 5 |
| Yeast cream | 5g | 5g | 5g | 7g | 7g |
| Peptone | 10g | 10g | 10g | 10g | 15g |
| Glucose | 10g | 10g | 15g | 20g | 20g |
| Agar-agar | 18g | 18g | 20g | 20g | 20g |
| Leach liquor | 100mL | 150mL | 200mL | 200mL | 200mL |
Note: the pH of the culture medium is natural.
1. The preparation method of the leaching liquor comprises the following steps:
(1) weighing and mixing the Daqu and the fermented grains according to the ratio of 1: 5;
(2) adding pure water into the Daqu-fermented grain mixture and water according to the mass ratio of 1:7, and boiling for 10-15 min;
(3) after cooling, filtering with gauze to obtain filtrate for later use.
2. The preparation method of the yeast isolated culture medium comprises the following steps:
(1) weighing the components according to the formula in the table 1, adding yeast powder, peptone, glucose, agar powder and leaching liquor into a container, supplementing water to 1L, mixing, and sterilizing at 121 ℃ for 15-20 min;
(2) dissolving chloramphenicol in 100% ethanol, shaking to obtain chloramphenicol mother liquor with concentration of 60 g/L;
(3) and (3) cooling the sterilized mixture obtained in the step (1) to 50-60 ℃, adding the chloramphenicol mother liquor obtained in the step (2), and uniformly mixing to dilute the chloramphenicol in the culture medium to 0.2 g/L.
In order to evaluate the influence of different formulas in the culture medium of the invention on the growth of saccharomycetes, fermented grains entering a cellar are prepared into a series of diluted bacterial liquids by adopting a plate dilution method, the diluted bacterial liquids are respectively coated on the culture medium shown in Table 1 for culture, the dilution gradient of each group is three and is 10-4、10-5、10-6(dilution factor).
After 48h of culture, 5 groups of culture media were selected to have a dilution gradient of 10-5The growth of yeast is shown in Table 2.
TABLE 2 influence of the formulation of the culture medium of the present invention on the growth of yeast in fermented grains
| Growth of yeasts | Medium 1 | Medium 2 | Medium 3 | Medium 4 | Medium 5 |
| Number of yeast (CFU/g) | 89×105 | 99×105 | 103×105 | 115×105 | 110×105 |
| Presence or absence of miscellaneous bacteria | A little bit | Is free of | Is free of | Is free of | Is free of |
Note: and the fermented grains are fermented grain samples entering the cellar.
The experimental results are as follows: the yeast in the fermented grains grow on the 5 culture media with slightly different quantities, wherein the culture media 1 and 2 are lower in quantity, the yeast on the culture media 3, 4 and 5 are higher in quantity, and no obvious difference exists on the whole, which indicates that the culture media in the formula range can be used for separating the yeast in the fermented grains.
EXAMPLE 2 Effect of addition of leach liquor on Yeast separation in fermented grains
In order to evaluate the influence of the addition of the leaching liquor in the culture medium of the invention on the growth of yeast, the invention adopts a plate dilution method to prepare fermented grains into a series of diluted bacterial liquids, and the diluted bacterial liquids are respectively coated on the culture media shown in Table 3 for culture, the dilution gradient of each group is three and is 10-3、10-4、10-5(dilution factor).
TABLE 3 composition and content of culture Medium
| Components | Medium 6 | Culture Medium 7 | Culture medium 8 | Comparison group |
| Yeast cream | 5.0g | 5.0g | 5.0g | 5.0g |
| Peptone | 10.0g | 10.0g | 10.0g | 10.0g |
| Glucose | 10.0g | 10.0g | 10.0g | 10.0g |
| Agar-agar | 20.0g | 20.0g | 20.0g | 20.0g |
| Leach liquor | 700mL | 500mL | 200mL | 0 |
After 48h incubation, a dilution gradient of 10 was selected-4The growth of yeast is shown in FIG. 1, Table 4 and Table5, respectively.
TABLE 4 influence of extract content on growth of yeast in fermented grains A
| Growth of yeasts | Medium 6 | Culture Medium 7 | Culture medium 8 | Comparison group |
| Number of yeast (CFU/g) | 48×104 | 129×104 | 42×104 | 9×104 |
| Presence or absence of miscellaneous bacteria | A little bit | Is free of | Is free of | Is free of |
Note: the fermented grain A is a stacked fermented grain sample.
TABLE 5 Effect of the Medium of the present invention on the growth of Yeast in fermented grains B
Note: the fermented grain B is a fermented grain sample in the cellar.
The four culture mediums have natural pH and the content of chloramphenicol is 0.2 g/L.
The preparation method, substances and contents of the leaching solution are the same as those in example 1.
The yeast isolation medium was the same as in example 1 except for the components and their contents shown in Table 3, other components and their contents, and the method for preparing the medium.
The experimental results are as follows: the yeast in the fermented grains A and B grow on the 4 culture mediums, but the number of the yeast is different, wherein the number of the yeast in the culture medium added with the leaching liquor is higher than that of the yeast in the control group, which indicates that certain nutrient substances in the leaching liquor are beneficial to the growth and separation of the yeast in the fermented grains.
And the yeast in the fermented grains A and B grows on the culture medium 7 in the best condition, so that the growth and separation of the yeast can be remarkably promoted. However, when the addition amount of the leaching solution is more than 700mL, the culture medium is not easy to solidify and has darker color, so that when the content of the leaching solution is within 200 to 700mL, the growth and separation of the yeast are more facilitated.
EXAMPLE 3 Effect of culture Medium pH on Yeast separation in Stacking fermented grains samples
In order to evaluate the influence of the difference of the pH of the culture medium on the separation of the yeast, the invention adopts a plate dilution method to prepare a series of diluted bacterial liquids from the accumulated fermented grains, and the diluted bacterial liquids are respectively coated on the culture media shown in the table 6 for culture, wherein the dilution gradients of the bacterial liquids inoculated in the two groups of culture media are 3 and 10 respectively-4、10-5、10-6. The medium was identical in composition and content and preparation method to medium 8 in example 2, except that the pH was different.
Note: wherein "pH is natural" means that the pH of the medium is adjusted without adding an acid solution.
"pH adjusted" means that the pH of the medium is adjusted to 5.0 by adding an acid solution (hydrochloric acid solution having a concentration of 1 mol/L) to the medium.
The stacking fermentation process is used for screening and enriching a large amount of microorganisms in fields, air and yeast, the colony structure of the fermented grains is complex in the early stacking stage, and the colony structure gradually tends to be stable in the later stacking stage due to acclimation for a certain time.
After 48h incubation, a dilution gradient of 10 was selected-4The growth of yeast is shown in FIG. 2 and Table 6.
TABLE 6 influence of the pH of the culture Medium on the growth of yeasts in the Stacking of fermented grains C
| Growth of yeasts | Natural pH | Adjusted pH |
| Number of yeast (CFU/g) | 21×104 | 27×104 |
| Presence or absence of miscellaneous bacteria | Is provided with | Is free of |
The experimental result shows that the yeast in the accumulated fermented grains C grow on the two culture media, but a large amount of mould grows on the culture medium without adjusting the pH, and the quantity of the yeast is slightly low; and the culture medium after the pH is adjusted to 5.0 has no mould growth, and the growth quantity of the microzyme is large.
Therefore, the yeast in the stacked fermented grain sample can be effectively separated, and the pH value of the culture medium also needs to meet certain conditions.
Example 4 influence of different ratios of Daqu and fermented grains in the culture Medium on the separation of Yeast
In order to test the influence of different proportions of the Daqu and the fermented grains on yeast separation, the fermented grains are prepared into a series of diluted bacterial liquids by a plate dilution method, the diluted bacterial liquids are respectively coated on culture media shown in Table 7 for culture, the dilution gradient of the bacterial liquids inoculated by 4 groups of culture media is 3 and 10 respectively-2、10-3、10-4. The components and contents of each group of culture medium are the same as those of the culture medium 8 in the example 2 except that the ratio of the Daqu to the fermented grains is different.
After 48h of culture, each group of culture medium was selected with a dilution gradient of 10-2The growth of yeast is shown in Table 7.
TABLE 7 influence of different ratios of Daqu and fermented grains on yeast growth of fermented grain sample in cellar
The experimental results are as follows: the yeast in the fermented grains grows in the culture medium, but the quantity of the yeast is the least when the mass ratio of the Daqu to the fermented grains in the leaching solution is 1: 25. And when the yeast is Daqu: after the ratio of the fermented grains is more than 1:1, the mixture of the Daqu and the fermented grains is easy to be cooked into paste, and the prepared culture medium forms a grey brown color, which is not beneficial to the observation of culture colonies in the culture medium. Therefore, the mass ratio of the Daqu to the fermented grains in the leaching solution is within the range of 1:1 to 1:20, and the growth and separation of the yeast in the fermented grains are facilitated.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Claims (21)
1. The yeast separation culture medium is characterized by comprising the following components:
5-10g/L of yeast extract,
10-20g/L of peptone,
5-20g/L of glucose,
15-22g/L of agar is added,
the Daqu-fermented grain leaching liquor is 150-500 mL/L, and the culture medium further contains 0.1-1.0g/L of chloramphenicol;
the balance of water;
the pH of the culture medium is 3.0-6.0;
the preparation method of the Daqu-fermented grain leaching liquor comprises the following steps:
(1) weighing fermented grains and Daqu, mixing,
(2) adding pure water into the fermented grain-Daqu mixture, boiling,
(3) cooling and filtering to obtain filtrate for later use;
in the step (1), the mass ratio of the Daqu to the fermented grains is 1:1-1: 20;
in the step (2), the mass ratio of the mixture to water is 1: 7.
2. The yeast isolation medium of claim 1, wherein the yeast isolation medium comprises:
5-10g/L of yeast extract,
10-15g/L of peptone,
10-20g/L of glucose,
18-20g/L of agar is added,
150-500 mL/L of Daqu-fermented grain leaching liquor.
3. The yeast isolation medium of claim 1, wherein in the yeast isolation medium:
5-7g/L of yeast extract,
10-15g/L of peptone,
15-20g/L of glucose,
18-20g/L of agar is added,
150-500 mL/L of Daqu-fermented grain leaching liquor.
4. The isolated yeast culture medium of claim 1, wherein the Daqu in the Daqu-fermented grain leaching solution is selected from Daqu fermented from wheat used in a liquor production process.
5. The yeast separation medium according to claim 1, wherein the Daqu in the Daqu-fermented grain leaching liquor is selected from high-temperature Daqu used in the production process of Maotai-flavor liquor.
6. The isolated yeast culture medium of claim 1, wherein the fermented grains in the Daqu-fermented grain leach liquor are fermented from grains boiled in a white spirit production process.
7. The isolated yeast culture medium of claim 1, wherein the fermented grains in the Daqu-fermented grain leaching solution are fermented from cooked sorghum in the production process of Maotai-flavor liquor.
8. The isolated yeast culture medium of claim 1, wherein the fermented grains in the Daqu-fermented grain leaching solution are selected from fermented grains in different rounds of fermentation in the production process of Maotai-flavor liquor.
9. The isolated yeast culture medium of claim 1, wherein the fermented grains in the Daqu-fermented grain leaching solution are selected from fermented grains in 1 st to 3 th rounds in the production process of Maotai-flavor liquor.
10. The isolated yeast culture medium of claim 1, wherein the fermented grains in the Daqu-fermented grain leaching solution are selected from 3 rd round fermented grains in the production process of Maotai-flavor liquor.
11. The isolated yeast culture medium according to claim 1, wherein the boiling time in step (2) is 5-30 min.
12. The isolated yeast culture medium according to claim 1, wherein the boiling time in step (2) is 10-15 min.
13. The isolated yeast culture medium according to claim 1, wherein in the step (1), the mass ratio of the Daqu to the fermented grains is 1:3-1: 10.
14. The isolated yeast culture medium according to claim 1, wherein in the step (1), the mass ratio of the Daqu to the fermented grains is 1:4-1: 10.
15. A method for preparing the yeast isolation medium of any one of claims 1 to 14, comprising the steps of:
(1) adding yeast powder, peptone, glucose, agar powder, and Daqu-fermented grains leaching solution into a container, adjusting pH with acid solution, supplementing water, and sterilizing;
(2) dissolving chloramphenicol in ethanol, and shaking to obtain chloramphenicol mother liquor;
(3) and (3) cooling the sterilized mixture obtained in the step (1) to 50-60 ℃, and uniformly mixing the sterilized mixture with the chloramphenicol mother liquor obtained in the step (2).
16. The method according to claim 15, wherein the acid solution in the step (1) is a hydrochloric acid solution, a glacial acetic acid solution or a lactic acid solution.
17. The method according to claim 15, wherein the medium is sterilized at 121 ℃ for 15 to 20min in the step (1).
18. The method for isolating yeast using the culture medium according to any one of claims 1 to 14, wherein the yeast is isolated by plate dilution.
19. Use of the culture medium of any one of claims 1 to 14 for isolating yeast from fermented grains.
20. The use according to claim 19, wherein the fermented grains for separation are selected from any stage of a brewing process.
21. The use according to claim 19, wherein the fermented grains for separation are selected from one or more of stacked fermented grains, fermented grains for cellar entry and fermented grains for cellar entry.
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