CN115058378B - Directional domestication method for microorganisms in Daqu starter house environment - Google Patents

Abstract

The invention discloses a directional domestication method of microorganisms in a yeast room environment, which adopts finished yeast of a mature yeast room as a microorganism source, uses a specific culture medium to directionally culture aspergillus, rhizopus and the like required by the yeast room, uses culture bacteria liquid to spray on a wood board added with the yeast room, and the microorganisms in the bacteria liquid propagate on the wood board and are dissipated into the air to inoculate the yeast in the yeast room. The yeast house domesticated by the method can remarkably improve the content of corresponding microorganisms of the yeast, and is beneficial to improving the quality of the yeast.

Description

Directional domestication method for microorganisms in Daqu starter house environment

Technical Field

The invention relates to a directional domestication method of microorganisms in a Daqu room environment, and belongs to the field of white spirit brewing.

Background

"Yeast is the bone of wine", daqu is one of the main sources of wine-making microorganisms in the production of wine-making. The Daqu starter culture process is a process of directionally inoculating environmental microorganisms by taking starter blocks as a culture medium.

The microorganisms in the environment of the starter propagation room are important components of microbial flora for brewing white wine and are also one of main sources of microorganisms required for white wine fermentation. Therefore, whether the microbial community in the yeast house contains microorganisms required for brewing white wine and the proportion of the microorganisms are important to the quality of the yeast and the importance of the microorganisms. In general, the yeast room with longer use time, especially the relatively independent yeast cultivation section in the yeast making workshop, can maintain a relatively stable microbial environment in the yeast room, and provide suitable microorganisms for fermenting Daqu. However, the environmental microorganisms in the new starter propagation room are not optimized, and more microorganisms are not needed for starter propagation requirements, so that even if the same process conditions are implemented, the mass of starter propagation blocks still has large difference, and a long time is needed to meet the starter propagation environmental requirements. This process can be greatly accelerated by suitable microbiological domestication methods.

Disclosure of Invention

The invention provides a directional domestication method of microorganisms in a Daqu starter room environment, which aims at improving the microorganisms in the starter room by adopting a proper inoculation mode and a microorganism source. The method is scientific, rapid and accurate, and can be used for domesticating microorganisms in the Daqu starter house.

The invention relates to a directional domestication method of Daqu starter house environment microorganisms, which adopts finished Daqu starter produced in a mature starter house which is continuously produced for more than 3 years as a microorganism source, carries out microorganism domestication on a new starter house and a starter house with abnormal starter house environment (according to QB/T4257-2011 brewing Daqu general analysis method, starter Fang Tanghua force is less than or equal to 800U) in light industry standard by two stages, and comprises the following steps:

step 1: selection of species

The yeast Pi Houbao produced by a yeast room (the force of the yeast Fang Tanghua is more than or equal to 1000U) which is continuously used for producing the yeast for more than 3 years is uniform, the yeast core has no residual moisture and color, qu Xiangnong is a pure finished product yeast, and the yeast skin (the surface layer of the yeast is 0-6 mm) and the yeast core (the center position of the yeast is 0-20 mm) are separated according to the microbial structure characteristics of different parts of the finished product yeast, and are respectively crushed into yeast skin powder A and yeast core powder B. The study shows that the microbial communities of the Daqu skin and the firering are different, and the yeast skin dominant beneficial bacteria are mold and yeast, and the yeast heart dominant beneficial bacteria are bacillus.

Step 2: preparation and enrichment of bacterial liquid

2a, culturing bacterial liquid A: mixing 5 parts of water and 1 part of wheat flour, steaming for 1 hour, adding amylase accounting for 2-5% of the wheat flour, gradually adding normal-temperature water, regulating the temperature to about 60 ℃, then adding saccharifying enzyme accounting for 1-2% of the wheat flour, saccharifying, and regulating to a yeast skin microorganism culture solution with the Baume degree of 7.0+/-0.5 by using sterile water; inoculating 1-2% of yeast powder A into the culture solution, placing in a shaking incubator at 35 ℃ for 180 turns/min, culturing for 36-48 hours, and filtering the culture solution by using gauze to obtain bacterial solution A.

2B, culturing bacterial liquid B: taking 87 parts by mass of sterile water, 10 parts by mass of yeast extract, 2 parts by mass of yeast extract and 1 part by mass of sodium chloride, uniformly mixing, regulating the pH value to 4-5 by using a lactic acid solution with the mass fraction of 10%, placing the mixture into an incubator with the temperature of 40-45 ℃ for culturing for 36-48 hours, and filtering the culture solution by using gauze to obtain bacterial liquid B.

The inoculum size A, B is different from the inoculum size used. The bacterial liquid A is used for culturing mould and saccharomycetes, and the bacterial liquid B is used for culturing high-temperature-resistant bacillus.

Step 3: inoculating culture

3a, song Fang Zengshi: firstly, humidifying the yeast room, and sprinkling clean water on the wall surface and the ground of the yeast room to fully absorb water and saturate the yeast room, and keeping the yeast Fang Shidu to be more than or equal to 85 percent.

3b, inoculating and culturing the bacterial liquid A: the bacterial liquid A is uniformly brushed on the wall surface of a curved room by using a roller brush, and the relative humidity of air is kept above 70% by using an air humidifier, and the culture is carried out for 36-48 hours.

3c, inoculating and culturing the bacterial liquid B: and uniformly brushing the bacterial liquid B on the wall surface of the curved room by using a roller brush, and maintaining the relative humidity of air to be above 70% by using an air humidifier, and culturing for 36-48 hours.

3d, sowing the yeast core powder B: the room is sprayed with the yeast core powder B (the particle size is 60 meshes & gt 80%), and the spraying amount per square meter is more than or equal to 10g according to the area of the yeast room. Bacillus is weaker in transmission capacity than fungi, and bacillus inoculation is further enhanced by sowing of yeast powder.

The domestication process comprises two stages, wherein the first stage uses bacterial liquid A to cultivate the wall body, the second stage uses bacterial liquid B to cultivate the wall body, and the second stage is inoculated and cultivated after three days of inoculation in the first stage are completed. And (3) inoculating the bacterial liquid A according to the inoculation time, namely, completing the inoculation of the bacterial liquid A on the 0 th day, and inoculating the bacterial liquid B on the 3 rd day.

Preferably, the whole starter propagation building or a plurality of starter propagation buildings are adopted for starter propagation domestication, and the doors and windows of the starter propagation building are kept in a closed state (note: better effect can be achieved by the starter propagation building domestication together).

Preferably, the domestication of the microorganisms in the starter culture room is carried out for 1-4 rounds, and the number of the air microorganisms in the starter culture room (total number of bacterial colonies) after the domestication is taken as a reference index, so that the number of the air microorganisms in the starter culture room reaches more than 75 percent.

Step 4: starter propagation production

And (3) carrying out conventional starter propagation production within three days after starter propagation (starter propagation heart powder is sowed according to inoculation time, namely, the day 0 and starter propagation production is carried out within 3 days) so as to avoid the secondary degradation of the microbial environment.

Compared with the prior art, the invention has the following beneficial effects:

the microorganism domestication method used by the method can rapidly achieve the purpose of improving the environment of the starter room and increase the starter making yield of the new starter room.

Drawings

FIG. 1 is a flow chart of the starter propagation domestication process of the invention.

FIG. 2 is a graph showing comparison of the number of airborne microorganisms in the starter house before and after acclimatization.

FIG. 3 shows the change of the structural components of eukaryotic microorganisms before and after the domestication of the new starter house.

FIG. 4 shows the change in structural composition of prokaryotic microorganisms before and after domestication in a newly developed room.

FIG. 5 is a graph showing comparison of the number of airborne microorganisms in a starter house for three modes of acclimation.

FIG. 6 is a comparison of structural components of eukaryotic microorganisms in a new starter house under different modes of acclimation.

FIG. 7 is a comparison of the structural components of prokaryotic microorganisms in a new starter house under different modes of acclimation.

Detailed Description

The present invention will be further described with reference to the drawings and specific examples in the following, but should not be construed as limiting the invention. Simple modifications and substitutions of the method, steps or conditions of the invention without departing from the spirit and nature of the invention are intended to be within the scope of the invention; the technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.

Example 1: domestication method

1. Preparation of bacterial liquid

And (3) separating and crushing the yeast skin and the yeast core to prepare yeast skin powder and yeast core powder respectively according to the microbial structural characteristics of different parts of the finished yeast.

Preparing bacterial liquid: the inoculum size A, B is different from the inoculum size used. The bacterial liquid A is used for culturing aspergillus and saccharomycetes, and the bacterial liquid B is used for culturing high-temperature-resistant bacillus.

The culture method of the bacterial liquid A comprises the following steps: 5 parts of water and 1 part of wheat flour are mixed and cooked for about 1 hour, 5% amylase is added, then cold water is added to adjust the temperature to about 60 ℃, then 2% saccharifying enzyme is added, and after saccharification, clear water is used to adjust the yeast skin microorganism culture solution with the sugar degree of 7. Inoculating 2% of the yeast powder, placing in a shake incubator at 35 ℃ for 180 revolutions per minute, and culturing for 48 hours. The culture broth was filtered using gauze to prepare a bacterial solution a.

The culture method of the bacterial liquid B comprises the following steps: taking 87 parts of sterile water, 10 parts of yeast powder, 2 parts of yeast extract and 1 part of sodium chloride according to mass ratio, uniformly mixing, regulating the PH value to 4-5 by using a lactic acid solution with mass fraction of 10%, and placing the mixture in a 45 ℃ incubator for culturing for 48 hours. The culture broth was filtered using gauze to prepare a bacterial solution B.

2. Inoculation of

Firstly, humidifying the yeast room, spraying clean water on the wall surface and the ground of the yeast room to fully absorb water and saturate the yeast room, and keeping the relative humidity of air above 70% in the inoculation and culture process.

Inoculating and culturing bacterial liquid: the bacterial liquid is uniformly brushed on the wall surface of the curved room by using a roller brush, and the bacterial liquid A is inoculated first and then the bacterial liquid B is inoculated after three days.

Sowing the yeast core powder B: after inoculation of the bacterial liquid A and the bacterial liquid B is completed, the room is sprayed with the yeast core powder B (the size of the particles is required to be finely ground to be 60 meshes & gt 80%), and the total amount is calculated to be more than 10g per square meter according to the area of the yeast room.

Example 2: microorganism content comparison of the curved chamber before and after domestication

To verify the effect of the number of microorganisms in the starter house before and after acclimation, a microbial sedimentation test was performed using PCA medium before and after acclimation. PCA medium was used for colony count calculation, reflecting the total amount of microorganisms in the air. The Bengalia culture medium was used for eukaryotic microbial enumeration, reflecting the mould and yeast content in air. After two media are prepared, the flat plate is sterilized and poured, then the surface sedimentation time of a yeast room is half an hour, the PCA medium is placed in a 36 ℃ incubator for culture, the Bengalia red medium is placed in a 28 ℃ incubator for culture, and after the settled microorganisms in the medium form colonies, the number of microbial colonies is counted. As can be seen from fig. 2, the culture acclimation can significantly raise the microorganism content in the air.

Example 3: comparison of microorganism composition in the curved room before and after domestication

To verify the effect of acclimation on the composition of colonies of microorganisms in the air in the yeast room, air samplers were used to extract microorganisms in the air before and after acclimation, and then high throughput sequencing techniques were used to detect ITS2 region and 16SV3-V4 region of the samples, respectively, and the structural composition of eukaryotic and prokaryotic microorganisms in the air in the yeast room was analyzed (FIGS. 3, 4). And respectively selecting air samples before and after the domestication of the normal and newly built houses for analysis.

Aspergillus in Daqu can decompose starch and protein, so that macromolecular substances are decomposed into micromolecular saccharides and proteins which can be utilized by microorganisms, and the method has important significance for fermentation bacteria of Daqu and fermented grains. The main eukaryotic microorganism in the air in the control koji room is Aspergillus (Aspergillus) microorganism with a proportion of 53.57%, the new koji room is raised to 34% after domestication before the domestication of Aspergillus (Aspergillus) with a proportion of 5.82%. The domesticated yeast house is favorable for inoculating aspergillus with Daqu. Other functional bacteria such as Rhizopus (Rhizopus), saccharomyces (Saccharomyces), issatchenkia (Issatchenkia) and the like show the same trend.

Bacterial flora in Daqu is an important source of organic acids and aroma substances, wherein lactobacillus and bacillus play a major role. The air in the control starter culture room contains mainly prokaryotic microorganism Lactobacillus (Lactobacillus) 39.10%, and the ratio of Lactobacillus (Lactobacillus) in the new starter culture room is 4.50% before domestication, and the ratio is raised to 27.03% after domestication. The ratio of Bacillus in the control koji house is 23.31%, the ratio of Bacillus in the new-region chamber before domestication is 2.11%, and the ratio of Bacillus is raised to 16.71% after domestication.

Comparative example 1:

the following three modes are adopted to carry out domestication on the microbial environment of the yeast room respectively, and the advantages and disadvantages of the scheme are compared.

Mode one: directly using the yeast powder to spread the yeast house.

Selecting high-quality mature Daqu, crushing by using a crusher, finely grinding the Daqu to 60 meshes of more than 80%, and directly sowing the powder in a starter room according to the inoculation amount of 10 g/square meter.

Mode two: directly inoculating the bacterial liquid without sowing yeast powder.

And (3) separating and crushing the yeast skin and the yeast heart to prepare yeast skin powder and yeast heart powder respectively from the high-quality finished product Daqu produced in the mature yeast house. Bacterial liquid A and bacterial liquid B are prepared according to the patent culture mode. Firstly, humidifying the yeast room, spraying clean water on the wall surface and the ground of the yeast room to fully absorb water and saturate the yeast room, and keeping the relative humidity of air above 70% in the inoculation and culture process.

Mode three: inoculating the bacterial liquid and sowing bacterial powder.

And (3) separating and crushing the yeast skin and the yeast core to prepare yeast skin powder and yeast core powder respectively according to the microbial structural characteristics of different parts of the finished yeast. Bacterial liquid A and bacterial liquid B are prepared according to the patent culture mode. Firstly, humidifying the yeast room, spraying clean water on the wall surface and the ground of the yeast room to fully absorb water and saturate the yeast room, and keeping the relative humidity of air above 70% in the inoculation and culture process. After the cultivation, 10 g/square meter of fungus powder B was scattered.

The three domestication modes are adopted to perform two continuous domestication respectively.

And after the culture is finished, selecting a PCA culture medium and a Bengalia culture medium for microbial settlement test, placing the PCA culture medium in a 36 ℃ incubator for culture, placing the Bengalia culture medium in a 28 ℃ incubator for culture, and counting the number of microbial colonies after the settled microorganisms in the culture medium form colonies.

And respectively sampling air microorganisms in the room environment by adopting a high-throughput sequencing technology before and after domestication, detecting ITS2 region and 16SV3-V4 region of a sample genome, and analyzing the structural composition of eukaryotic and prokaryotic microorganisms in the room air.

Fig. 5 shows the results of the microbial count in the sedimentation test, wherein the first mode directly uses the yeast powder to spread the yeast room, so that the microbial count in the air environment of the yeast room can be improved, but the effect is obvious without the second mode and the third mode, and the effect of humidifying culture is obvious. The necessity of a mode of culturing the bacterial liquid will be described.

Fig. 6 and 7 show structural analysis of air microbial communities before and after acclimation in three acclimation modes. Comparison of the environmental microorganism components in the yeast room after the culture in the three modes can find that the mode two and the mode three can obviously improve the proportion of Aspergillus (Aspergillus) and saccharomycete (Saccharomyces) of eukaryotic microorganisms, and the mode three can further improve the proportion of Lactobacillus (Bacillus) Bacillus by sowing the yeast powder B on the basis of the mode two. And (3) injection: mould (Aspergillus), yeast (Saccharomyces), lactobacillus (Lactobacillus) are the main microorganisms in Daqu, and have important roles in the process of making yeast.

In summary, the microbial environment domestication result of the third mode is closest to the control starter room used for a long time.

Claims (3)

1. A directional domestication method of microorganisms in a Daqu room environment is characterized by comprising the following steps:

step 1: selection of species

Separating the yeast skin and the yeast core of the finished Daqu, and respectively pulverizing into yeast skin powder A and yeast core powder B;

step 2: preparation and enrichment of bacterial liquid

2a, culturing bacterial liquid A: mixing 5 parts of water and 1 part of wheat flour, steaming for 1 hour, adding amylase accounting for 2-5% of the mass of the wheat flour, gradually adding normal-temperature water to adjust the temperature to 60 ℃, then adding saccharifying enzyme accounting for 1-2% of the mass of the wheat flour, saccharifying, and adjusting to a yeast skin microorganism culture solution with the Baume degree of 7.0+/-0.5 by using sterile water; inoculating 1-2% of yeast powder A into the culture solution, placing the culture solution into a shake incubator at 35 ℃ for 180 turns/min, culturing for 36-48 hours, and filtering the culture solution by using gauze to obtain bacterial solution A;

2B, culturing bacterial liquid B: taking 87 parts by mass of sterile water, 10 parts by mass of yeast extract B, 2 parts by mass of yeast extract and 1 part by mass of sodium chloride, uniformly mixing, regulating the pH value to 4-5 by using a lactic acid solution with the mass fraction of 10%, placing the mixture into an incubator with the temperature of 40-45 ℃ for culturing for 36-48 hours, and filtering the culture solution by using gauze to obtain bacterial solution B;

step 3: inoculating culture

Firstly, humidifying a yeast room, and splashing clean water on the wall surface and the ground of the yeast room to fully absorb water and saturate the yeast room, wherein the yeast Fang Shidu is kept to be more than or equal to 85%; culturing the wall body by using the bacterial liquid A and the bacterial liquid B respectively so as to realize domestication of microorganisms in the curved room;

step 4: starter propagation production

Conventional starter propagation production is carried out within three days after starter propagation domestication, so that the microbial environment is prevented from being degraded again;

in the step 3, domestication of the microorganisms in the yeast room comprises two stages, wherein the first stage uses the bacterial liquid A to cultivate the wall body, the second stage uses the bacterial liquid B to cultivate the wall body, and inoculation cultivation of the second stage is performed after three days of inoculation in the first stage is completed;

inoculating and culturing bacterial liquid A: brushing bacterial liquid A on the wall surface of a curved room uniformly by using a roller brush, and culturing for 36-48 hours by using an air humidifier to keep the relative humidity of air above 70%;

inoculating and culturing the bacterial liquid B: uniformly brushing the bacterial liquid B on the wall surface of a curved room by using a roller brush, and culturing for 36-48 hours by using an air humidifier to keep the relative humidity of air above 70%;

and the domestication process of the microorganisms in the yeast room further comprises sowing yeast core powder B after the second stage is finished, specifically, throwing the yeast core powder B in a room, and calculating the throwing amount per square meter according to the area of the yeast room to be more than or equal to 10g.

2. The directional domestication method according to claim 1, wherein:

in the step 1, the finished product yeast is a finished product yeast with uniform yeast Pi Houbao produced by yeast room of more than 3 years by continuous yeast production and residual moisture and variegated yeast core.

3. The directional domestication method according to claim 1, wherein:

the process of acclimatizing the microorganisms in the starter propagation is repeated 1-4 times.