CN111778181A - Method for preparing pit mud caproic acid bacterial liquid - Google Patents
Method for preparing pit mud caproic acid bacterial liquid Download PDFInfo
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Abstract
The invention particularly relates to a method for preparing a pit mud caproic acid bacterial liquid, which takes old pit mud as a sample, and utilizes an RCM culture medium to optimize caproic acid producing functional bacteria in the pit mud to obtain a caproic acid high-yield strain, and specifically comprises the following steps: selecting pit mud, performing water-bath heat treatment on the pit mud, screening caproic acid bacteria, performing low-temperature anaerobic preservation on the strains, researching the growth rule of the strains, performing enlarged culture on the strains and filling the pits for maintenance. The pit mud caproic acid bacteria has relatively strong caproic acid producing capacity and strong reproductive capacity, and the optimal inoculation time is determined by analyzing a strain growth curve in the process of enlarged culture, so that the maximum quantity and activity of bacteria are ensured; meanwhile, in the expanding culture process, the brewing production raw materials are mainly utilized to prepare a microorganism culture medium, namely the daqu powder, the cellar mud powder, the yellow serofluid, the wine heads and the like, so that the adaptability of the caproic acid bacteria is enhanced; the method is applied to the pit, can improve the quantity of caproic acid bacteria in pit mud and improve the quality of the pit mud.
Description
Technical Field
The invention belongs to the field of white spirit brewing, and particularly relates to a method for preparing pit mud caproic acid bacterial liquid.
Background
Liquor brewing is a traditional industry of Chinese nationality. In the production of the Luzhou-flavor liquor, the older the pit, the better the quality of the liquor produced. In addition, the fermented grains near the bottom or wall of the cellar pool produce wine with high ester content (especially ethyl caproate and ethyl butyrate) and good wine quality; as for the same pit, the liquor produced in the bottom layer is better than that produced in the middle layer, and the liquor produced in the middle layer is better than that produced in the upper layer, so that the quality of the strong aromatic white liquor is closely related to pit mud of the pit. Researches show that the old pit mud contains various anaerobic functional bacteria, mainly anaerobic bacillus, which participate in the fermentation and aroma generation of the white spirit. Since ethyl caproate is a substance which forms the main body fragrance of the strong aromatic white spirit, the caproic acid bacteria become main functional bacteria of the pit mud.
The pit mud is one of the important conditions in the production process of the strong aromatic Daqu liquor, and plays an important role in the formation of trace aroma components in the liquor and the quantitative ratio relation of the trace aroma components. The strong-flavor liquor is characterized in that the cellar aroma is the style reflected by main body aromatic ethyl caproate in the liquor, and is formed by metabolizing caproic acid functional bacteria in cellar mud to generate caproic acid and esterifying the caproic acid with ethanol. The content of caproic acid bacteria in the existing pit mud is very limited, and the quality of the Luzhou-flavor liquor can be directly influenced by the content of the caproic acid bacteria.
Disclosure of Invention
In order to solve the problem of low caproic acid bacteria content in pit mud in the prior art, the invention provides a method for preparing a pit mud caproic acid bacteria liquid, which can obtain high-yield caproic acid bacteria in pit mud, improve the quantity of beneficial bacteria contained in unit volume of a caproic acid bacteria maintenance liquid, and improve the quantity of caproic acid bacteria in pit mud and the quality of pit mud when applied to a pit.
The technical problem to be solved by the invention is realized by the following technical scheme:
a manufacturing method of pit mud caproic acid bacterial liquid takes old pit mud as a sample, and utilizes an RCM culture medium to optimize caproic acid producing functional bacteria in the pit mud to obtain caproic acid high-yield strains, and specifically comprises the following steps:
the method comprises the following steps: selecting pit mud;
step two: carrying out water bath heat treatment on pit mud;
step three: screening caproic acid bacteria;
step four: performing low-temperature anaerobic preservation on the strains;
step five: researching the growth rule of strains;
step six: carrying out strain expansion culture;
step seven: and (6) filling the cellar for maintenance.
Preferably, the selected pit mud specifically comprises: sampling pit wall mud below a high-quality pit yellow water line, performing multi-point sampling at a position 15-20cm away from the pit bottom on the pit wall, kneading and uniformly mixing the pit wall mud after sampling, and performing vacuum sealing preservation at the temperature of 2-6 ℃.
Preferably, the pit mud water bath heat treatment specifically comprises:
weighing a certain amount of old pit mud, adding the old pit mud into sterile water filled with glass beads, and continuously oscillating until the sample is uniformly dispersed to form pit mud suspension; and (3) absorbing the pit mud suspension into a test tube, carrying out water bath heat treatment at the temperature of 75-85 ℃ to eliminate non-spore trophosome, and intermittently shaking in the water bath process to avoid local heating.
Preferably, the screening for caproic acid bacteria is specifically:
putting the pit mud suspension into a triangular flask filled with an RCM culture medium, sealing the liquid level of the culture medium by using liquid paraffin, culturing at the constant temperature of 35 ℃ for 11 days, and observing the gas production condition of the culture solution;
and selecting a culture solution with high gas production rate for transfer culture, observing the gas production condition of the bacteria, and carrying out qualitative and quantitative analysis on the caproic acid, preferably selecting the composite caproic acid bacteria with high caproic acid yield.
Preferably, the transfer culture is specifically: absorbing the culture solution, inoculating into RCM culture medium test tube, sealing the liquid surface with liquid paraffin, and deep culturing at constant temperature of 35 deg.C for 11 d.
Preferably, the low-temperature anaerobic preservation of the strain is as follows: the strain is subjected to test tube submerged culture by isolating liquid paraffin from air, and then stored in a refrigerator at 2-6 deg.C.
Preferably, the study on the growth rule of the strains specifically comprises the following steps:
transferring the strain to an RCM culture medium by adopting aseptic operation at an inoculum size of 5%, and culturing at constant temperature of 35 ℃ for 12 d;
using a blank culture medium as a control, detecting the gas production condition of the thalli, the OD600nm value and the thalli number every day, and drawing a strain growth curve by taking the culture time as an abscissa and the thalli number and the OD600nm value as ordinates;
and determining the optimal inoculation time according to the microbial growth curve, ensuring the maximum thallus quantity and activity and providing a basis for enlarged culture.
Preferably, the strain expansion culture specifically comprises:
gradually expanding the strain with natural culture medium mainly comprising brewing material, sterilizing the culture medium at 121-126 deg.C for 30-40min, cooling to room temperature, adding distiller's yeast, stirring, gradually expanding culture with 10% inoculum size, sealing the culture medium with small amount of distiller's yeast, expanding culture with cellar mud caproic acid bacteria liquid, culturing at 35 deg.C for 5-6 days, and regulating the bacteria number to 9.6 × 108-10.0×108one/mL.
Preferably, the natural culture medium comprises 5% of yeast powder, 4% of alcohol, 4% of soybean meal, 1% of pit mud powder, 0.5% of yellow serofluid and the balance of water.
Preferably, the cellar filling and maintaining specifically comprises:
after the residual grains in the cellar pool of the brewing workshop are cleaned, punching the four walls of the cellar pool at an angle of 30 degrees by using bamboo sticks, wherein the hole diameter is 6-10mm, the hole depth is 5cm, and the hole distance is 6-8 cm;
uniformly stirring the pit mud caproic acid bacterial liquid, pouring from top to bottom along the wall of the pit, stopping pouring for a while after one time, and pouring again after the bacterial liquid permeates;
and (3) smoothing the wall of the cellar by using a wooden trowel, and uniformly scattering 1.0-1.5kg of fine powder of high-quality Daqu to obtain the solid fermented grains.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention discloses a method for preparing pit mud caproic acid bacterial liquid, which is characterized in that on the basis of a microbiological experiment technology, high-quality pit mud is used as a test sample, caproic acid producing functional bacteria are obtained through enrichment, separation and optimization, a high-bacterial-number culture solution is prepared, and the bacterial liquid is applied to pit maintenance, artificial pit mud preparation and the like, so that the pit mud quality can be improved, and an inferior pit can be improved;
2. in the process of strain amplification culture, the optimal inoculation time is determined by analyzing a growth curve, so that the maximum quantity and activity of thalli are ensured; in the strain expanding culture process, brewing production raw materials are mainly utilized to prepare a microorganism culture medium, namely daqu powder, pit mud powder, yellow serofluid, wine heads and the like, so as to enhance the adaptability of caproic acid bacteria; the natural culture medium used for the expanding culture of the caproic acid bacteria is subjected to high-temperature moist heat sterilization treatment after being prepared in proportion, so that the mixed bacteria contained in the raw materials are favorably eliminated, the dominant flora is established in the culture process of the caproic acid bacteria, and the quality of the bacteria liquid is better ensured;
3. according to the invention, through the research on the ratio of different nutrients in a natural culture medium, an optimal culture scheme is explored, so that the quantity of beneficial bacteria in the culture solution is increased.
Drawings
FIG. 1 is a flow chart of a method for preparing a cellar mud caproic acid bacterial liquid according to the embodiment.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited thereto.
This embodiment provides a method for preparing a cellar mud caproic acid bacterial liquid, which uses old cellar mud as a sample, and uses an RCM culture medium to optimize caproic acid-producing functional bacteria in the cellar mud to obtain caproic acid high-yield strains with high growth speed and strong fertility, and in the strain propagation process, a natural culture medium mainly containing brewing raw materials is selected to perform step-by-step propagation so as to maximize the concentration of bacteria (9.6 × 10)8-10.0×108one/mL). The pit mud caproic acid bacterium liquid is applied to pit maintenance, the number of pit mud caproic acid bacteria in a pit can be increased, and the quality of pit mud is improved. The flow chart of the preparation method of the pit mud caproic acid bacterial liquid refers to figure 1.
In the embodiment, the pit wall mud under the yellow water line of the high-quality pit is selected for sampling, the pit wall is positioned 15-20cm away from the pit bottom for sampling, and the pit mud is kneaded and homogenized to be operated next step. If necessary, the product is preferably stored under vacuum at 2-6 deg.C.
Weighing 10g of old cellar mud by an electronic balance, adding into 100mL of sterile water containing a small amount of glass beads (the sterile water needs to be subjected to damp-heat sterilization treatment at 121 ℃ for 20 min), and continuously oscillating for 30min at 180r/min to uniformly disperse the sample. 10mL of pit mud suspension is sucked into a test tube by aseptic operation, the non-spore trophosome is eliminated by heat treatment in a water bath at 75-85 ℃ for 10min, and the non-spore trophosome is intermittently shaken in the water bath process to avoid local heating.
And (3) aseptically sucking 10mL of pit mud suspension into 120mL of aseptic RCM culture medium (120 mL of 100mL of small triangular flask liquid), sealing the liquid level of the culture medium by using 3mL of liquid paraffin, culturing at the constant temperature of 35 ℃ for 11d, and observing the gas production condition of the culture solution. Selecting culture solution with large gas production for transfer culture, namely sucking 2mL of culture solution and inoculating the culture solution into a test tube containing 20mL of sterile RCM medium, sealing the liquid level with 500 μ L of liquid paraffin, and carrying out submerged constant-temperature culture at 35 ℃ for 11 d. And (3) observing the gas production condition of the bacteria and carrying out qualitative and quantitative analysis on the caproic acid, wherein the caproic acid complex bacteria with high caproic acid yield are preferred. The strain is subjected to test tube submerged culture by isolating liquid paraffin from air, and then stored in a refrigerator at 2-6 deg.C.
Qualitative analysis of caproic acid
And (3) fully and uniformly mixing the caproic acid bacteria culture solution, accurately sucking 4mL of bacteria solution, then adding 1mL of 5% copper acetate solution, then adding 1mL of n-butanol, fully oscillating for 1min, standing to enable the solution to be layered, and observing the color of the n-butanol layer. If the bacteria liquid is blue-green, the bacteria liquid contains caproic acid, and the deeper the color, the higher the caproic acid content.
② quantitative analysis of caproic acid
Diluting liquid: 0.4% sodium acetate; 0.1% yeast extract; 0.05% ammonium sulphate; 0.04% dipotassium hydrogen phosphate; 0.02% magnesium sulfate; 1% ethanol.
1% standard adipic acid solution: accurately sucking 1mL of caproic acid (the content is more than or equal to 99.0 percent and the relative density is 0.926g/mL), adding the diluent to the volume of 100mL, and adjusting the pH value to 6.8 by using 20 percent NaOH solution when the volume is increased to 95 mL.
Preparing 8.0mL of a 1% standard adipic acid solution and a diluent into different concentrations of the adipic acid solution, adding 2.0mL of a 5% copper acetate solution and 5.0mL of n-butanol, performing a color development test, simultaneously performing blank control, fully shaking for 1min, standing to layer the solution, and absorbing the n-butanol layer solution to determine the OD value at 660 nm. And (3) drawing a standard curve by taking the volume of the 1% standard caproic acid liquid as an abscissa and the OD value as an ordinate, and then determining the concentration of caproic acid in the sample liquid according to the standard curve.
The strain is transferred to RCM medium by aseptic operation with the inoculation amount of 5%, and is cultivated for 12 days at the constant temperature of 35 ℃. And (3) using a blank culture medium as a control, detecting the gas production condition of the thalli, the OD600nm value and the thalli number every day, and drawing a strain growth curve by taking the culture time as an abscissa and the thalli number and the OD600nm value as ordinates. And determining the optimal inoculation time as 3d of culture according to the growth curve of the microorganisms, wherein the maximum thallus number and activity can be ensured, and a basis is provided for enlarged culture.
In order to research the tolerance of the strain to yellow serofluid and ethanol, the strain is subjected to a yellow serofluid tolerance test and an ethanol tolerance test by using an RCM test tube culture medium (20 mL/tube), and is cultured at a constant temperature of 35 ℃ and a high liquid level by adopting an inoculation amount of 5%. See table below for details.
Table 1: influence of yellow serofluid on the number of caproic acid bacteria
| Concentration of yellow serofluid (%) | 0 | 0.5 | 1 | 2 | 3 | 4 | 5 | 6 |
| Bacterial count (× 10)8one/mL) | 6.2 | 6.8 | 6.3 | 5.9 | 5.6 | 0.15 | 0.13 | 0.11 |
As can be seen from Table 1, the strain grows well in 0% -3% yellow serofluid environment, more than 4% (including 4%) yellow serofluid can inhibit the growth of the strain, and the number of the strain is only 0.11 × 108Per mL-0.15 × 108The optimum amount of yellow serofluid added is 0.5%, and the number of bacteria is maximum (6.8 × 10)8one/mL).
Table 2: effect of ethanol on the number of caproic acid bacteria
| Ethanol concentration (%) | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| Bacterial count (× 10)8one/mL) | 6.4 | 6.8 | 6.5 | 7.5 | 6.6 | 6.8 | 6.9 | 6.7 |
As can be seen from Table 2, the strains grow well in 0% -7% ethanol environment and are not inhibited significantly by the increase of ethanol concentration, the optimal ethanol addition amount of the strains is 3%, the maximum number of the strains is 7.5 × 108one/mL.
In conclusion, the strain can tolerate 3% yellow serofluid and 7% ethanol, and the optimal addition amount of the yellow serofluid is 0.5% and the optimal addition amount of the ethanol is 3%.
Then, under the conditions of 0.5% yellow serofluid and 3% ethanol (equal to 4% of alcohol head), single-factor tests are carried out on the addition amounts of yeast powder, grain stillage powder, soybean meal powder and pit mud powder (all pass through a 20-mesh sieve) in the natural culture medium for enlarged culture, and the caproic acid bacteria culture process is gradually optimized.
(1) Daqu powder addition test
Adding 0%, 1%, 2%, 3%, 4%, 5% Daqu powder based on 1% mud powder, 0.5% grain lees powder, 0.5% yellow serofluid, and 4% distiller's yeast, respectively, making culture medium containing Daqu powder with different concentrations, and sterilizing at 121 deg.C for 30 min. Cooling, inoculating caproic acid bacteria in 10% inoculation amount, sealing with alcohol, and standing at 35 deg.C for submerged culture. And detecting the number of bacteria after the bacteria stop producing gas.
Table 3: influence of yeast powder concentration in culture medium on bacterial count
| Concentration of Yeast powder (%) | 0 | 1 | 2 | 3 | 4 | 5 |
| Bacterial count (× 10)8one/mL) | 3.0 | 5.0 | 6.4 | 6.7 | 7.2 | 7.7 |
As can be seen from Table 3, the number of the bacterial species increased with the increase of the concentration of the koji powder, and the number of the bacterial species was the largest in the koji powder medium with a concentration of 5% (7.7 × 10)8one/mL). The Daqu powder contains rich nutrition, and the number of bacteria is obviously low due to insufficient nutrition in the culture solution with little or no addition of Daqu powder. After considering the economic cost, the culture medium containing 5% of yeast powder is preferred.
(2) Grain stillage powder addition test
Based on 1% mud powder, 5% yeast powder, 0.5% yellow serofluid and 4% distiller's head, respectively adding 0%, 0.5%, 1%, 1.5%, 2%, 2.5% grain stillage powder, making culture medium containing grain stillage powder with different concentrations, and sterilizing at 121 deg.C for 30 min. Cooling, inoculating caproic acid bacteria in 10% inoculation amount, sealing with alcohol, and standing at 35 deg.C for submerged culture. And detecting the number of bacteria after the bacteria stop producing gas.
Table 4: influence of grain lees powder concentration in culture medium on bacterial count
| Concentration of grain stillage powder (%) | 0 | 0.5 | 1 | 1.5 | 2 | 2.5 |
| Bacterial count (× 10)8one/mL) | 7.8 | 7.0 | 6.8 | 6.8 | 6.5 | 5.6 |
Table 4 shows that the number of bacteria showed a decreasing trend with increasing concentration of the grain stillage powder, and the number of bacteria was the largest in the culture medium without grain stillage powder (7.8 × 10)8one/mL) and the bacteria content in the culture medium containing 2.5 percent of grain stillage powder is the minimum (5.6 × 10)8one/mL). For the strain, the culture medium without grain stillage powder is more beneficial to growth.
(3) Test for addition amount of soybean meal
Based on 1% mud powder, 5% yeast powder, 0.5% yellow serofluid and 4% distiller's yeast, 0%, 1%, 2%, 3%, 4% and 5% soybean meal powder are respectively added to prepare culture media containing soybean meal powder with different concentrations, and the culture media are sterilized for 30min at 121 ℃. Cooling, inoculating caproic acid bacteria in 10% inoculation amount, sealing with alcohol, and standing at 35 deg.C for submerged culture. And detecting the number of bacteria after the bacteria stop producing gas.
Table 5: effect of the concentration of Soybean meal in the Medium on the number of bacteria
Table 5 shows that the number of bacteria increases and then decreases with the increase of the concentration of the soybean meal in the culture medium, and the number of bacteria reaches the maximum value in the culture medium with 4 percent of soybean meal (1.0 × 10)9one/mL). I.e. a culture medium containing 4% of soybean meal is most suitable.
(4) Cellar mud powder addition test
Adding 0%, 1%, 2%, 3%, 4% and 5% cellar mud powder based on 5% yeast powder, 4% soybean meal powder, 0.5% yellow serofluid and 4% distiller's head respectively, making culture medium containing cellar mud powder with different concentrations, and sterilizing at 121 deg.C for 30 min. Cooling, inoculating caproic acid bacteria in 10% inoculation amount, sealing with alcohol, and standing at 35 deg.C for submerged culture. And detecting the number of bacteria after the bacteria stop producing gas.
Table 6: influence of pit mud powder concentration in culture medium on bacterial count
| Pit mud powder concentration (%) | 0 | 1 | 2 | 3 | 4 | 5 |
| Bacterial count (× 10)8one/mL) | 9.0 | 9.6 | 9.5 | 9.5 | 9.5 | 9.0 |
Table 6 shows that the influence of pit mud powder contents with different concentrations in the culture medium on the number of bacteria is not obvious, and the number is maintained at 9.0 × 10 after the bacteria stop producing gas8Per mL-9.6 × 108one/mL, the difference is very small. The bacterial count of the culture medium containing 1% of pit mud powder is slightly higher than that of the culture medium with other concentration. Therefore, a culture medium containing 1% of cellar mud powder is preferably used for strain propagation.
By researching the addition of natural culture medium nutrients, the optimum addition of nutrients of the optimized strain is 5% of yeast powder, 4% of wine head, 4% of soybean meal, 1% of mud powder and 0.5% of yellow serofluid, and the number of bacteria in culture solution can reach 9.6 × 108Per mL-10 × 108one/mL.
The strain is expanded and cultured step by utilizing a natural culture medium which takes a brewing raw material as a main component, namely 5 percent of yeast powder, 4 percent of wine head, 4 percent of soybean meal powder, 1 percent of mud powder, 0.5 percent of yellow serofluid and the balance of purified water. The embodiment researches the ratio of different nutrients of the natural culture medium, and the culture scheme is obtained after exploration, so that the quantity of beneficial bacteria in the culture solution is increased. Carrying out high-temperature damp-heat sterilization treatment on a culture medium at the temperature of 121-The number of bodies can reach 9.6 × 108-10.0×108one/mL. The natural culture medium used for the propagation of the caproic acid bacteria is subjected to moist heat sterilization treatment at the high temperature of 121 ℃ for 30min after being prepared in proportion, so that the mixed bacteria contained in the raw materials are favorably eliminated, the dominant flora is established in the culture process of the caproic acid bacteria, and the quality of the bacteria liquid is better ensured.
And punching the four walls of the pit at an angle of 30 degrees by using a bamboo stick, wherein the aperture is 6-10mm, the hole depth is 5cm, and the hole distance is 6-8 cm. And (3) uniformly stirring 20L of pit mud caproic acid bacterial liquid, pouring from top to bottom along the wall of the pit, stopping pouring for a while after one time, and pouring again after the bacterial liquid permeates. And (3) smoothing the wall of the cellar by using a wooden trowel, and uniformly scattering 1.0-1.5kg of fine powder of high-quality Daqu to obtain the solid fermented grains.
Note: the RCM medium of this example was: 0.3% of yeast extract, 1% of beef extract, 1% of peptone, 0.1% of soluble starch, 0.5% of glucose, 0.05% of cysteine hydrochloride, 0.3% of sodium chloride and 0.3% of sodium acetate, wherein the pH is natural, the beef extract is sterilized at 121 ℃ for 30min, and 2% of absolute ethyl alcohol is added after cooling.
The natural culture medium used for the expanding culture in this example was: 1% of cellar mud powder, 5% of Daqu powder, 4% of soybean meal powder, 0.5% of yellow serofluid, and purified water for dilution, wherein the pH value is natural, the sterilization is carried out at 121 ℃ for 30min, and 4% of alcohol is added after cooling.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (10)
1. The method for preparing the cellar mud caproic acid bacterial liquid is characterized in that old cellar mud is used as a sample, and RCM culture medium is used for optimizing caproic acid producing functional bacteria in the cellar mud to obtain caproic acid high-yield strains, and specifically comprises the following steps:
the method comprises the following steps: selecting pit mud;
step two: carrying out water bath heat treatment on pit mud;
step three: screening caproic acid bacteria;
step four: performing low-temperature anaerobic preservation on the strains;
step five: researching the growth rule of strains;
step six: carrying out strain expansion culture;
step seven: and (6) filling the cellar for maintenance.
2. The method for preparing the pit mud caproic acid bacterial liquid according to claim 1, wherein the selection of the pit mud specifically comprises the following steps: sampling pit wall mud below a high-quality pit yellow water line, performing multi-point sampling at a position 15-20cm away from the pit bottom on the pit wall, kneading and uniformly mixing the pit wall mud after sampling, and performing vacuum sealing preservation at the temperature of 2-6 ℃.
3. The method for preparing the pit mud caproic acid bacterial liquid according to claim 1, wherein the pit mud water-bath heat treatment specifically comprises:
weighing a certain amount of old pit mud, adding the old pit mud into sterile water filled with glass beads, and continuously oscillating until the sample is uniformly dispersed to form pit mud suspension; and (3) absorbing the pit mud suspension into a test tube, carrying out water bath heat treatment at the temperature of 75-85 ℃ to eliminate non-spore trophosome, and intermittently shaking in the water bath process to avoid local heating.
4. The method for preparing the pit mud caproic acid bacteria liquid as claimed in claim 1, wherein the screening caproic acid bacteria is specifically:
putting the pit mud suspension into a triangular flask filled with an RCM culture medium, sealing the liquid level of the culture medium by using liquid paraffin, culturing at the constant temperature of 35 ℃ for 11 days, stopping the gas production process of the culture liquid after 11 days, and observing the gas production condition at the culture stage;
and selecting a culture solution with high gas production rate for transfer culture, observing the gas production condition of the bacteria, and carrying out qualitative and quantitative analysis on the caproic acid, preferably selecting the composite caproic acid bacteria with high caproic acid yield.
5. The method for preparing the pit mud caproic acid bacterial liquid according to claim 4, wherein the transfer culture specifically comprises: absorbing the culture solution, inoculating into RCM culture medium test tube, sealing the liquid surface with liquid paraffin, and deep culturing at constant temperature of 35 deg.C for 11 d.
6. The method for preparing the pit mud caproic acid bacterial liquid according to claim 1, wherein the low-temperature anaerobic preservation of the bacterial strain is specifically as follows: the strain is subjected to test tube submerged culture by isolating liquid paraffin from air, and then stored in a refrigerator at 2-6 deg.C.
7. The method for preparing the pit mud caproic acid bacterial liquid according to claim 1, wherein the research on the growth rule of the bacterial is as follows:
transferring the strain to an RCM culture medium by adopting aseptic operation at an inoculum size of 5%, and culturing at constant temperature of 35 ℃ for 12 d;
using a blank culture medium as a control, detecting the gas production condition of the thalli, the OD600nm value and the thalli number every day, and drawing a strain growth curve by taking the culture time as an abscissa and the thalli number and the OD600nm value as ordinates;
and determining the optimal inoculation time according to the microbial growth curve, ensuring the maximum thallus quantity and activity and providing a basis for enlarged culture.
8. The method for preparing the pit mud caproic acid bacterial liquid according to claim 1, wherein the strain expanded culture specifically comprises:
gradually expanding the strain with natural culture medium mainly comprising brewing material, sterilizing the culture medium at 121-126 deg.C for 30-40min, cooling to room temperature, adding distiller's yeast, stirring, gradually expanding culture with 10% inoculum size, sealing the culture medium with small amount of distiller's yeast, expanding culture with cellar mud caproic acid bacteria liquid, culturing at 35 deg.C for 5-6 days, and regulating the bacteria number to 9.6 × 108-10.0×108one/mL.
9. The method for preparing the cellar mud caproic acid bacteria liquid as claimed in claim 8, wherein the natural culture medium comprises 5% of yeast powder, 4% of wine head, 4% of soybean meal, 1% of cellar mud powder, 0.5% of yellow serofluid and the balance of water.
10. The method for preparing the pit mud caproic acid bacterial liquid according to claim 1, wherein the pit filling and maintenance specifically comprises the following steps:
after the residual grains in the cellar pool of the brewing workshop are cleaned, punching the four walls of the cellar pool at an angle of 30 degrees by using bamboo sticks, wherein the hole diameter is 6-10mm, the hole depth is 5cm, and the hole distance is 6-8 cm;
uniformly stirring the pit mud caproic acid bacterial liquid, pouring from top to bottom along the wall of the pit, stopping pouring for a while after one time, and pouring again after the bacterial liquid permeates;
and (3) smoothing the wall of the cellar by using a wooden trowel, and uniformly scattering 1.0-1.5kg of fine powder of high-quality Daqu to obtain the solid fermented grains.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113151061A (en) * | 2021-03-24 | 2021-07-23 | 湖北工业大学 | Glucose-inhibited oxytoca |
| CN113186121A (en) * | 2021-04-06 | 2021-07-30 | 江南大学 | Caproic acid bacteria capable of utilizing various substrates and application thereof |
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| CN106434125A (en) * | 2016-10-17 | 2017-02-22 | 湖北枝江酒业股份有限公司 | Wine making multi-bacteria functional bacterial liquid, and production method and application thereof |
| CN107034108A (en) * | 2017-05-09 | 2017-08-11 | 江南大学 | It is a kind of that the method for improving the refreshing cleanliness of aromatic Chinese spirit is conserved by pit mud |
| CN110804576A (en) * | 2019-12-24 | 2020-02-18 | 新疆伊力特实业股份有限公司 | Preparation method of composite caproic acid bacterial liquid |
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| CN107034108A (en) * | 2017-05-09 | 2017-08-11 | 江南大学 | It is a kind of that the method for improving the refreshing cleanliness of aromatic Chinese spirit is conserved by pit mud |
| CN110804576A (en) * | 2019-12-24 | 2020-02-18 | 新疆伊力特实业股份有限公司 | Preparation method of composite caproic acid bacterial liquid |
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| CN113151061A (en) * | 2021-03-24 | 2021-07-23 | 湖北工业大学 | Glucose-inhibited oxytoca |
| CN113151061B (en) * | 2021-03-24 | 2022-06-17 | 湖北工业大学 | Glucose-inhibited oxytoca |
| CN113186121A (en) * | 2021-04-06 | 2021-07-30 | 江南大学 | Caproic acid bacteria capable of utilizing various substrates and application thereof |
| WO2022213898A1 (en) * | 2021-04-06 | 2022-10-13 | 江南大学 | Clostridium butyricum capable of utilizing various substrates and use thereof |
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