Artificial pit mud wet culture method

Abstract

The invention relates to the technical field of wine brewing, and provides an artificial pit mud wet culture method. The method comprises the following steps: S1, collecting a sample from old pit mud, and separating to obtain an anaerobic strain; s2, respectively carrying out activation culture and adaptive pre-culture on the separated strains; s3, preparing a pit mud matrix, and performing nutrition activation treatment; s4, uniformly planting the activated mixed flora on the substrate, and slightly stirring to ensure uniform distribution; s5, dynamically controlling fermentation in stages in the fermentation process until the pit mud reaches a mature state; and S6, performing real-time monitoring and component detection on the fermentation process. Core microorganisms such as anaerobic bacillus, lactic acid bacteria and methane-oxidizing bacteria are separated and activated from the aged pit mud, the technical effect of rapidly establishing a high-activity flora ecosystem is achieved through the technical scheme of activation culture and adaptive pre-culture, the defects that the flora colonization speed is low, and the microbial ecosystem is unstable are overcome, and the method is suitable for large-scale popularization and application. The mature period of the new pit mud is obviously shortened.

Description

Technical Field

[0001] The invention relates to the technical field of winemaking, in particular to an artificial pit mud wet culture method. Background Art

[0002] In the production of Luzhou-flavor liquor, traditional cellar mud is an irreplaceable core material for fermentation in the production process. The microbial community in the cellar mud, including anaerobic Bacillus, lactic acid bacteria, methanotrophic bacteria, caproic acid bacteria and butyric acid bacteria, is responsible for fermenting in an anaerobic environment to produce aroma components such as ethyl caproate, which directly affects the flavor and quality of the liquor. Traditional high-quality cellar mud relies on years of accumulation of old cellar mud to form a stable ecosystem through natural fermentation and microbial proliferation. However, with the expansion of the production scale of modern wineries, new cellars need to be put into use as soon as possible, which has created a contradiction between production needs and the natural fermentation cycle and process of traditional cellar mud.

[0003] In traditional technology, the formation of mud in newly built cellars depends on natural fermentation. This process requires the microbial community in the cellar to form a stable microbial ecosystem through long-term gradual proliferation, colonization and competition. In the natural environment, microbial colonization, the formation of dominant flora and the accumulation of metabolites require a long period of time, so new mud often takes several years or even longer to achieve the fermentation effect of old mud. For expanded or newly built wineries, relying on this natural fermentation method is difficult to meet the needs of rapid production of new cellars, which directly affects production progress and efficiency.

[0004] The core flavor components of Luzhou-flavor liquor come from the metabolites of microorganisms in the pit mud, especially the aroma components generated by caproic acid bacteria and butyric acid bacteria under specific fermentation conditions. Traditional pit mud fermentation relies on natural accumulation, and the generation process of its aroma components is slow, usually taking several years to achieve the ideal flavor requirements of Luzhou-flavor liquor. At the same time, due to the instability of the microbial community, the aroma components generated by the newly built pit mud during the fermentation process fluctuate greatly, resulting in unstable flavor of the finished liquor. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides an artificial pit mud wet culture method, which solves the problems of long pit mud culture period, unstable fermentation and high labor cost in newly built pits.

[0006] To achieve the above object, the present invention is implemented by the following technical scheme: an artificial pit mud wet culture method comprises the following steps;

[0007] S1. Collect samples from old cellar mud and isolate anaerobic bacteria;

[0008] S2, performing activation culture and adaptive pre-culture on the isolated strains respectively;

[0009] S3, preparing pit mud substrate and performing nutrient activation treatment;

[0010] S4. Evenly plant the activated mixed bacterial flora on the substrate and stir gently to ensure uniform distribution;

[0011] S5. Dynamically control the fermentation in stages during the fermentation process until the pit mud reaches a mature state;

[0012] S6. Real-time monitoring and ingredient detection of the fermentation process.

[0013] Preferably, the step S1 specifically includes:

[0014] S1.1. Collect samples from old cellar mud, place them in anaerobic sealed bags, and store them at 4°C to avoid contamination by bacteria and loss of active microorganisms in the samples;

[0015] S1.2. The dilution concentration of the old pit mud sample ranges from 10^-3 to 10^-7 to ensure the adaptability of selective culture;

[0016] S1.3, isolating anaerobic Bacillus, lactic acid bacteria and methanotrophic bacteria, and culturing them;

[0017] S1.4. The isolated target strain was purified multiple times and identified by 16SrRNA gene sequencing to ensure that it met the fermentation characteristics requirements.

[0018] Preferably, in step S1.3, the culturing temperature range of anaerobic bacillus is 35-39°C, the culturing temperature range of lactic acid bacteria is 33-37°C, and the culturing temperature range of methane oxidizing bacteria is 28-32°C.

[0019] Preferably, the step S2 specifically includes:

[0020] S2.1, prepare deionized water 97.848%-98.592%, glucose 0.8%-1.2%, amino acid 0.4%-0.6%, polypeptide 0.15%-0.25%, organic zinc 0.04%-0.06%, organic selenium 0.01%-0.03% and magnesium chloride 0.008%-0.012%;

[0021] S2.2, adding glucose, amino acids, polypeptides, organic zinc, organic selenium and magnesium chloride to deionized water in sequence and mixing them to obtain a high-nutrient activation medium;

[0022] S2.3, inoculating the target bacteria into the activation culture medium respectively;

[0023] S2.4. The temperature range of activation culture is 35-39°C, the culture time is 18-30 hours, the time interval for each determination of the number of viable bacteria can be adjusted to 4-8 hours, and the activity should reach 10^7-10^9 CFU / mL.

[0024] Preferably, the step S3 specifically includes:

[0025] S3.1. Prepare 72% to 78% wet yellow mud, 6% to 8% Daqu powder, 3% to 5% soybean powder, 3% to 5% distiller's grains, 1% to 2% wine tails and 0.5% to 1% compound nutrient;

[0026] S3.2, add Daqu powder, soybean powder, distiller's grains, wine tails and compound nutrients into the wet yellow mud in sequence and mix evenly to obtain a pit mud matrix;

[0027] S3.3, adding 0.5% to 1.5% glucose solution to the pit mud matrix and mixing;

[0028] S3.4. Control the activation culture temperature range to 35-39°C and the culture time to 18-30h;

[0029] S3.5. The time interval for each determination of the number of viable bacteria can be adjusted to 4 to 8 hours, and the activity should reach 10^7 to 10^9 CFU / mL.

[0030] Preferably, the ingredients of the composite nutrient in step S3.1 are 0.3% to 0.7% organic zinc, 0.1% to 0.3% selenium, 0.5% to 1.5% polypeptide and 1% to 3% amino acid mixture.

[0031] Preferably, the mixed bacterial community in step S4 is 30% to 50% anaerobic Bacillus, 20% to 40% lactic acid bacteria, 15% to 25% methanotrophic bacteria, 5% to 15% caproic acid bacteria and 5% to 15% butyric acid bacteria.

[0032] Preferably, the spraying amount of the mixed bacterial flora in step S4 can be adjusted to 0.5% to 1.5% of the total weight of the substrate to ensure an appropriate initial bacterial flora density.

[0033] Preferably, the step S5 specifically includes:

[0034] S5.1. In the initial activation stage, the temperature is controlled at 36-38°C, the humidity is controlled at 80%-90%, and the nitrogen replacement frequency is every 2-4 days;

[0035] S5.2, during the propagation stage, the temperature is controlled at 34-36°C, the humidity is controlled at 78%-88%, and the stirring frequency is every 6-12 days;

[0036] S5.3, During the propagation stage, the pH value is controlled between 4.2 and 5.0. If the pH value is lower than 4.2, 0.2% lactic acid solution is added to maintain a stable bacterial environment;

[0037] S5.4, aroma generation stage, the temperature is controlled at 30-34°C, the humidity is controlled at 72%-82%, and the amount of wine tail added is 0.5%-1.5%;

[0038] S5.5, during the stabilization phase, the temperature is controlled at 29-33°C, the humidity is controlled at 68%-78%, and the frequency of nitrogen replacement is every 25-30 days.

[0039] Preferably, the monitoring interval in step S6 is 4 to 8 hours, and the density of anaerobic bacillus and lactic acid bacteria is required to be maintained at 10^7 to 10^9 CFU / mL.

[0040] The present invention provides an artificial pit mud wet culture method, which has the following beneficial effects:

[0041] 1. The present invention achieves the technical effect of rapidly establishing a highly active bacterial community ecosystem by separating and activating core microorganisms such as anaerobic bacillus, lactic acid bacteria and methanotrophic bacteria from old cellar mud through activation culture and adaptive pre-culture technical solutions, solves the problems of slow colonization of bacterial community and unstable microbial ecosystem, and significantly shortens the maturation period of new cellar mud.

[0042] 2. The present invention achieves the technical effect of rapidly proliferating the bacterial flora and optimizing the generation of aroma by dynamically controlling the temperature and humidity in stages during the fermentation process and accurately adjusting the environmental conditions according to the needs of microorganisms in different fermentation stages. Compared with the single control scheme of constant temperature and humidity in the prior art, it solves the problem of long fermentation cycle and significantly improves the production speed of newly built cellars.

[0043] 3. The present invention achieves the technical effect of significantly improving the metabolic activity and aroma generation ability of microorganisms by adding a composite nutrient to the pit mud matrix to provide the nutrients required by the microorganisms, thereby solving the problems of slow aroma generation speed and insufficient aroma components;

[0044] 4. The present invention monitors and regulates temperature and humidity, pH value and bacterial density, thereby reducing the frequency of manual intervention, achieving the technical effect of reducing manual operation costs and improving production efficiency, and solving the problems of frequent manual operation, low control accuracy and high production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The figure is a flow chart of the method of the present invention. DETAILED DESCRIPTION

[0046] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] Please see attached Figure 1 , the embodiment of the present invention provides an artificial pit mud wet culture method, comprising the following steps;

[0048] S1. Collect samples from old cellar mud and isolate anaerobic bacteria;

[0049] S2, performing activation culture and adaptive pre-culture on the isolated strains respectively;

[0050] S3, preparing pit mud substrate and performing nutrient activation treatment;

[0051] S4. Evenly plant the activated mixed bacterial flora on the substrate and stir gently to ensure uniform distribution;

[0052] S5. Dynamically control the fermentation in stages during the fermentation process until the pit mud reaches a mature state;

[0053] S6. Real-time monitoring and ingredient detection of the fermentation process.

[0054] Specifically, samples were collected from old cellar mud, placed in anaerobic sealed bags, and kept at 4°C to avoid contamination by foreign bacteria and loss of active microorganisms in the samples. The dilution concentration of the old cellar mud samples was set in the range of 10^-3 to 10^-7 to ensure the adaptability of selective culture. Anaerobic bacillus, lactic acid bacteria and methanotrophic bacteria were separated and cultured. The culture temperature range of anaerobic bacillus was 35 to 39°C, the culture temperature range of lactic acid bacteria was 33 to 37°C, and the culture temperature range of methanotrophic bacteria was 28 to 32°C. The isolated target strains were purified multiple times and identified by 16SrRNA gene sequencing to ensure that they met the requirements of fermentation characteristics.

[0055] Prepare 97.848% to 98.592% deionized water, 0.8% to 1.2% glucose, 0.4% to 0.6% amino acid, 0.15% to 0.25% polypeptide, 0.04% to 0.06% organic zinc, 0.01% to 0.03% organic selenium and 0.008% to 0.012% magnesium chloride, add glucose, amino acid, polypeptide, organic zinc, organic selenium and magnesium chloride to the deionized water in turn and mix to obtain a high-nutrient activation medium, sterilize the prepared medium with high pressure and cool for standby use, inoculate the target strains in the activation medium respectively, the temperature range of the activation culture is 35 to 39° C., the culture time is 18 to 30 hours, the time interval for each determination of the number of viable bacteria can be adjusted to 4 to 8 hours, and the activity should reach 10^7 to 10^9 CFU / mL;

[0056] Prepare 72% to 78% wet yellow mud, 6% to 8% Daqu powder, 3% to 5% soybean powder, 3% to 5% distiller's grains, 1% to 2% wine tailings, and 0.5% to 1% compound nutrient, add Daqu powder, soybean powder, distiller's grains, wine tailings, and compound nutrient into the wet yellow mud in sequence and mix evenly to obtain a cellar mud matrix, add 0.5% to 1.5% glucose solution into the cellar mud matrix and mix, and culture at 35 to 39° C. for 18 to 30 hours to ensure uniform distribution and full activation of nutrients in the matrix;

[0057] The compound nutrient includes 0.3% to 0.7% organic zinc, 0.1% to 0.3% selenium, 0.5% to 1.5% polypeptide and 1% to 3% amino acid mixture. The compound nutrient provides a variety of trace elements and nitrogen sources to meet the metabolic needs of the bacteria and can significantly improve the activity of the bacterial flora. The polypeptide and amino acid can effectively promote the proliferation of the bacteria and ensure the stability of the bacterial flora during the fermentation process. The organic zinc and selenium can enhance the tolerance of the bacterial flora, improve the adaptability of the bacteria to acidic and anaerobic environments, and prolong the active period of the microorganisms.

[0058] 30% to 50% of anaerobic bacillus, 20% to 40% of lactic acid bacteria, 15% to 25% of methanotrophic bacteria, 5% to 15% of caproic acid bacteria and 5% to 15% of butyric acid bacteria are mixed to obtain a mixed bacterial community, and 5% to 15% of caproic acid bacteria and butyric acid bacteria are added, and the mixed bacterial community is cultured in an anaerobic environment at 35°C for 24 hours to ensure the initial stability of the mixed bacterial community, and the spraying amount of the mixed bacterial community is 0.5% to 1.5% of the total weight of the pit mud substrate to ensure an appropriate initial bacterial community density;

[0059] In the initial activation stage, the temperature is controlled at 36-38°C, the humidity is controlled at 80%-90%, and the anaerobic environment is ensured by nitrogen replacement every 2-4 days. This stage provides a suitable environment for the early proliferation of the bacterial flora and ensures rapid colonization.

[0060] During the propagation stage, the temperature is controlled at 34-36°C, the humidity is controlled at 78%-88%, and light stirring is performed on the 7th and 12th days to make the bacterial flora more evenly distributed in the matrix. At the same time, the pH value is tested every 6-12 days and controlled between 4.2 and 5.0. If the pH value is lower than 4.2, 0.2% lactic acid solution is added to ensure a stable environment for bacterial flora propagation.

[0061] During the aroma generation stage, the temperature is controlled at 30-34°C, the humidity is controlled at 72%-82%, and 0.5%-1.5% of wine tailings are added on the 20th day to promote the generation of aroma components such as ethyl caproate, ensuring that the cellar mud forms a strong aroma characteristic.

[0062] During the stabilization stage, the temperature is controlled at 29-33°C, the humidity is controlled at 68%-78%, and nitrogen replacement is performed on the 28th day to ensure an anaerobic environment. Stop stirring to stabilize the microecological structure of the bacterial flora;

[0063] The fermentation process is monitored in real time with a monitoring interval of 4 to 8 hours. The density of anaerobic Bacillus and lactic acid bacteria is required to be maintained at 10^7 to 10^9 CFU / mL. Trend analysis is performed and fermentation parameters are adjusted in real time to ensure the stability and controllability of the fermentation process.

[0064] Embodiment 1;

[0065] Purpose: This experiment sets the ingredients and process parameters within the standard range to verify the optimal fermentation effect.

[0066] Sample collection and bacterial isolation;

[0067] Collect samples from pit mud older than 50 years old and dilute them to a concentration of 10^-5;

[0068] Using selective medium to separate anaerobic Bacillus, lactic acid bacteria and methanotrophic bacteria, anaerobic Bacillus was cultured at 37°C, lactic acid bacteria at 35°C and methanotrophic bacteria at 30°C for 48 hours respectively;

[0069] The purified target strain was obtained by plate separation method, and its species was confirmed by 16S rRNA gene sequencing;

[0070] Activation culture of bacteria;

[0071] Prepare standard activation culture medium: deionized water 97.9%, glucose 1.0%, amino acid 0.5%, polypeptide 0.2%, organic zinc 0.05%, organic selenium 0.02%, magnesium chloride 0.01%;

[0072] The isolated strains were inoculated into the culture medium and cultured at 37°C for 24 hours. The number of viable bacteria was measured every 8 hours, and the number of viable bacteria reached 10^8 CFU / mL;

[0073] Preparation of pit mud matrix;

[0074] Prepare the cellar mud matrix, the ingredients of which are: 78% wet yellow mud, 7% Daqu powder, 4% soybean powder, 5% distiller's grains, and 1.0% compound nutrient;

[0075] Add 1% glucose solution and let stand at 35°C for 24 hours to ensure that the nutrients are fully activated;

[0076] The activated mixed bacteria were evenly sprayed on the surface of the cellar mud substrate, with a spraying amount of 1.0% of the substrate weight, and the initial temperature was set at 37°C and the humidity was 85%;

[0077] During the fermentation process, the temperature and humidity are adjusted dynamically in stages, and the temperature is lowered on the 5th, 10th and 15th days respectively to gradually adapt to the growth needs of different bacterial communities. The fermentation cycle is 30 days.

[0078] Summarize;

[0079] Under the conditions within the standard range, the fermentation effect is good, the number of viable bacteria is high, the fermentation cycle is reasonable, and the final aroma component (ethyl caproate) reaches 70 mg / kg, which proves that the present invention can quickly and efficiently cultivate pit mud under the conditions within this range.

[0080] Embodiment 2;

[0081] Purpose: This experiment was conducted to observe the effects of low values ​​of ingredients and parameters on fermentation performance under conditions below the standard range;

[0082] Sample collection and bacterial isolation;

[0083] The sampling and strain separation were the same as in Example 1;

[0084] Activation culture of bacteria;

[0085] Prepare activation medium below the range: deionized water 98.2%, glucose 0.8%, amino acid 0.4%, polypeptide 0.15%, organic zinc 0.04%, organic selenium 0.01%, magnesium chloride 0.008%;

[0086] The activation temperature was 35°C, the culture was carried out for 18 hours, and the final viable count was 10^7 CFU / mL;

[0087] Matrix preparation;

[0088] Matrix ingredients: wet yellow mud 76%, Daqu powder 6%, soybean powder 3%, distiller's grains 3%, compound nutrient 0.5%;

[0089] Add 0.5% glucose solution and let stand at 32°C for 24 hours;

[0090] Bacterial colonization and fermentation control;

[0091] The initial bacterial spraying amount was 0.5% of the substrate weight, and the initial temperature was set at 35°C and the humidity was 80%;

[0092] The temperature and humidity were dynamically adjusted in stages, but due to the low initial number of live bacteria, the overall fermentation period was extended to 40 days, and the final aroma component concentration was 50 mg / kg.

[0093] Summarize;

[0094] Lower composition and parameter values ​​reduce the activation effect, slow bacterial proliferation, extend the fermentation cycle, and produce insufficient aroma components. This result shows that it is difficult to achieve fast and efficient fermentation under conditions below the standard range.

[0095] Embodiment 3;

[0096] Purpose: To test the fermentation effect under conditions higher than the standard range and observe the effect of high parameter values ​​on fermentation;

[0097] Sample collection and bacterial isolation;

[0098] The sampling and separation steps are the same as in Example 1;

[0099] Activation culture of bacteria;

[0100] Prepare activation medium above the range: deionized water 97.4%, glucose 1.2%, amino acid 0.6%, polypeptide 0.25%, organic zinc 0.06%, organic selenium 0.03%, magnesium chloride 0.012%;

[0101] The activation temperature was 39°C, and the culture time was 30 hours. The number of viable bacteria reached 10^9 CFU / mL, but the bacterial flora stability was not as good as that under standard conditions.

[0102] Matrix preparation;

[0103] Matrix ingredients: wet yellow mud 78%, Daqu powder 8%, soybean powder 5%, distiller's grains 5%, compound nutrient 1.5%;

[0104] 1.5% glucose solution was added to the matrix and allowed to stand at 36°C for 30 h;

[0105] Bacterial colonization and fermentation control;

[0106] The initial temperature was 39°C and the humidity was 90%. The temperature and humidity were controlled in stages, but high temperature caused the stability of the bacterial flora to decrease;

[0107] The fermentation period was shortened to 25 days, but the aroma components fluctuated greatly, with a final concentration of 60 mg / kg.

[0108] Summarize;

[0109] Although high parameter values ​​speed up fermentation, high temperature and high humidity conditions have a certain impact on the stability of the bacterial flora, resulting in unstable aroma components. The results show that exceeding the standard range is not conducive to the best fermentation effect.

[0110] Embodiment 4;

[0111] Purpose: The effect of the combination of components below the range value and parameters above the range value;

[0112] Sample collection and bacterial isolation;

[0113] The same sampling and strain isolation methods as in Example 1;

[0114] Activation culture of bacteria;

[0115] Prepare activation culture medium below the standard: deionized water 98.3%, glucose 0.8%, amino acid 0.4%, polypeptide 0.15%, organic zinc 0.04%, organic selenium 0.01%, magnesium chloride 0.008%.

[0116] The activation temperature is 39°C, the culture time is 30 hours, and the viable count is 10^8 CFU / mL;

[0117] Matrix preparation;

[0118] Wet yellow mud 76%, Daqu powder 6%, soybean powder 3%, distiller's grains 3%, compound nutrient 0.5%;

[0119] 0.5% glucose solution was added to the matrix and allowed to stand at 35°C for 24 h;

[0120] Bacterial colonization and fermentation control;

[0121] The initial temperature was 38°C, the humidity was 90%, the fermentation period was extended to 35 days, the aroma production was insufficient, and the final concentration was 55 mg / kg.

[0122] Summarize;

[0123] The combination of low activation ingredients and high temperature and high humidity conditions affected the proliferation and stability of the flora, the fermentation period was extended to 35 days, and the aroma production was insufficient. This result shows that unreasonable condition combinations have a negative impact on the colonization of the flora and the fermentation effect.

[0124] Embodiment 5;

[0125] Purpose: To test the effect of the combination of components above the range value and parameters below the range value;

[0126] Sample collection and bacterial isolation;

[0127] Same as Example 1;

[0128] Activation culture of bacteria;

[0129] Prepare activation medium higher than the standard: deionized water 97.5%, glucose 1.2%, amino acid 0.6%, peptide 0.25%, organic zinc 0.06%, organic selenium 0.03%, magnesium chloride 0.012%;

[0130] The activation temperature is 35°C, and after 18 hours of culture, the number of viable bacteria reaches 10^9 CFU / mL;

[0131] Matrix preparation;

[0132] Wet yellow mud 79%, Daqu powder 8%, soybean powder 5%, distiller's grains 5%, compound nutrient 1.5%;

[0133] 1.5% glucose solution was added to the matrix and allowed to stand at 34°C for 24 h;

[0134] Bacterial colonization and fermentation control;

[0135] Initial temperature 35°C, humidity 80%;

[0136] The fermentation period is 32 days, and the aroma component concentration is 65 mg / kg;

[0137] Summarize;

[0138] Under the combination of high-activation ingredients and low-temperature and low-humidity environment, the bacterial activity is high, but the fermentation rate is slightly slower, and the fermentation period is extended to 32 days. This result shows that even if the ingredient ratio is relatively high, reasonable temperature and humidity control is still required to maintain bacterial activity and aroma production.

[0139] Comparative experiment

[0140] Comparative experiment 1;

[0141] Experimental design ideas;

[0142] This experiment verifies the role of compound nutrients and evaluates their effects on fermentation rate, bacterial proliferation, and aroma production;

[0143] Experimental groups;

[0144] Experimental group A: complete protocol.

[0145] Experimental group B: no compound nutrients were added, only basic nutrients were contained.

[0146] Experimental data table;

[0147]

[0148] Results Analysis

[0149] The compound nutrient significantly improved the activity of the bacterial flora and the production of aroma, and shortened the fermentation cycle. This shows that the compound nutrient plays a key role in the colonization of the bacterial flora and the production of aroma components.

[0150] Comparative experiment 2:

[0151] Experimental design ideas;

[0152] This experiment evaluated the effect of staged control on bacterial flora adaptability and aroma production by comparing constant temperature and humidity control;

[0153] Experimental groups;

[0154] Experimental group A: complete protocol.

[0155] Experimental group C: constant temperature and humidity control.

[0156] Experimental data table;

[0157]

[0158] Result analysis;

[0159] Staged dynamic control significantly improved the fermentation efficiency. The constant temperature and humidity environment failed to meet the needs of the bacterial flora at different stages, resulting in a long fermentation cycle, indicating that staged control is of great significance for rapid cultivation.

[0160] Comparative experiment 3;

[0161] Experimental design ideas;

[0162] This experiment compares the traditional process without activation treatment with the process of the present invention including activation treatment to verify the effect of activation treatment on bacterial proliferation and fermentation effect;

[0163] Experimental groups

[0164] Experimental group A: complete protocol.

[0165] Experimental group E: no activation pretreatment.

[0166] Experimental data table;

[0167]

[0168] Result analysis;

[0169] Activation pretreatment significantly improved the initial bacterial activity and rapidly increased the final live bacterial count and aroma component content, indicating that activation pretreatment plays a key role in the cellar mud culture cycle and fermentation quality.

[0170] Comprehensive experimental data table

[0171]

[0172] in conclusion;

[0173] Cultivation cycle;

[0174] Example 1 of the present invention demonstrates the best effect within the standard range, shortening the culture period to less than 30 days, which is suitable for rapid production requirements;

[0175] The examples below or above the range values ​​(Examples 2, 3, 4, and 5) showed different degrees of fermentation delay or instability. Although some parameters beyond the range (such as 25 days in Example 3) could shorten the cycle, the final live bacteria stability and aroma generation did not reach the optimal effect;

[0176] Comparative experiments show that the traditional solution lacks innovative ingredients or appropriate processes, resulting in a cycle extension of 45-90 days. In contrast, the solution of the present invention shortens the cycle to less than 30 days.

[0177] Initial and final viable counts;

[0178] In the comparative experiments of the standard embodiment of the present invention and the innovative components, the initial viable bacterial count was significantly increased to more than 10^8 CFU / mL, the proliferation rate was fast, and the final viable bacterial count reached 10^10 CFU / mL;

[0179] In the examples below or above the range values ​​(Examples 2, 3, 4, and 5), the stability and proliferation rate of the bacterial flora were affected to a certain extent, and the number of viable bacteria at the end stage was low;

[0180] The traditional scheme without activation treatment has low viable bacteria count and long proliferation cycle;

[0181] Aroma component generation

[0182] The standard range (Example 1) of the present invention and the comparison experimental group of dynamic control showed a higher aroma component generation effect, and the final ethyl acetate content was 70-75 mg / kg;

[0183] In the experimental groups lacking compound nutrients or activation pretreatment, aroma production was significantly insufficient, only 30-40 mg / kg;

[0184] In the examples (Examples 3 and 5) that were outside the standard range under high temperature or high humidity conditions, the aroma generation was slightly lower, indicating that the standard parameter range was more conducive to the stability of aroma generation;

[0185] The number of manual operations and production costs;

[0186] The standard embodiment of the present invention and the experimental group containing innovative ingredients significantly reduced the number of manual operations and production costs. Through automated control and strain activation treatment, the number of operations was reduced to less than 10 times, and the production cost was reduced.

[0187] The traditional scheme and the experimental group lacking innovative components had frequent manual operations and high monitoring costs, which significantly increased production costs and labor times;

[0188] in conclusion;

[0189] The embodiment of the present invention within the standard parameter range (Example 1) significantly improves fermentation efficiency, aroma generation and bacterial proliferation, reduces culture cycle and production cost, and can effectively meet the needs of wineries to quickly, efficiently and low-cost put new cellars into production.

[0190] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An artificial pit mud wet culture method, characterized in that: The steps include: S1. Collect samples from old cellar mud and isolate anaerobic bacteria; S2, performing activation culture and adaptive pre-culture on the isolated strains respectively; S3, preparing pit mud substrate and performing nutrient activation treatment; S4. Evenly plant the activated mixed bacterial flora on the substrate and stir gently to ensure uniform distribution; S5. Dynamically control the fermentation in stages during the fermentation process until the pit mud reaches a mature state; S6. Real-time monitoring and ingredient detection of the fermentation process.

2. The artificial pit mud wet culture method according to claim 1, characterized in that: The step S1 specifically includes: S1.

1. Collect samples from old pit mud, place them in anaerobic sealed bags, and store them at 4°C to avoid contamination by bacteria and loss of active microorganisms in the samples; S1.

2. The dilution concentration of the old pit mud sample ranges from 10^-3 to 10^-7 to ensure the adaptability of selective culture; S1.3, isolating anaerobic Bacillus, lactic acid bacteria and methanotrophic bacteria, and culturing them; S1.

4. The isolated target strain was purified multiple times and identified by 16SrRNA gene sequencing to ensure that it met the fermentation characteristics requirements.

3. The artificial pit mud wet culture method according to claim 1, characterized in that: In step S1.3, the anaerobic bacillus culture temperature range is 35-39°C, the lactic acid bacteria culture temperature range is 33-37°C, and the methane oxidizing bacteria culture temperature range is 28-32°C.

4. The artificial pit mud wet culture method according to claim 1, characterized in that: The step S2 specifically includes: S2.1, prepare deionized water 97.848%-98.592%, glucose 0.8%-1.2%, amino acid 0.4%-0.6%, polypeptide 0.15%-0.25%, organic zinc 0.04%-0.06%, organic selenium 0.01%-0.03% and magnesium chloride 0.008%-0.012%; S2.2, adding glucose, amino acids, polypeptides, organic zinc, organic selenium and magnesium chloride to deionized water in sequence and mixing them to obtain a high-nutrient activation medium; S2.3, inoculating the target bacteria into the activation culture medium respectively; S2.

4. The temperature range of activation culture is 35-39°C, the culture time is 18-30 hours, the time interval for each determination of the number of viable bacteria can be adjusted to 4-8 hours, and the activity should reach 10^7-10^9 CFU / mL.

5. The artificial pit mud wet culture method according to claim 1, characterized in that: The step S3 specifically includes: S3.

1. Prepare 72% to 78% wet yellow mud, 6% to 8% Daqu powder, 3% to 5% soybean powder, 3% to 5% distiller's grains, 1% to 2% wine tailings and 0.5% to 1% compound nutrient; S3.2, add Daqu powder, soybean powder, distiller's grains, wine tails and compound nutrients into the wet yellow mud in sequence and mix evenly to obtain a pit mud matrix; S3.3, adding 0.5% to 1.5% glucose solution to the pit mud matrix and mixing; S3.

4. Control the activation culture temperature range to 35-39°C and the culture time to 18-30h; S3.

5. The time interval between each determination of the number of viable bacteria can be adjusted to 4 to 8 hours, and the activity should reach 10^7 to 10^9 CFU / mL.

6. The artificial pit mud wet culture method according to claim 1, characterized in that: The ingredients of the composite nutrient in step S3.1 are 0.3% to 0.7% organic zinc, 0.1% to 0.3% selenium, 0.5% to 1.5% polypeptide and 1% to 3% amino acid mixture.

7. The artificial pit mud wet culture method according to claim 1, characterized in that: The mixed bacterial community in step S4 is 30% to 50% of anaerobic bacillus, 20% to 40% of lactic acid bacteria, 15% to 25% of methanotrophic bacteria, 5% to 15% of caproic acid bacteria and 5% to 15% of butyric acid bacteria.

8. The artificial pit mud wet culture method according to claim 1, characterized in that: The spraying amount of the mixed bacterial flora in step S4 can be adjusted to 0.5% to 1.5% of the total weight of the substrate to ensure an appropriate initial bacterial flora density.

9. The artificial pit mud wet culture method according to claim 1, characterized in that: The step S5 specifically includes: S5.

1. In the initial activation stage, the temperature is controlled at 36-38°C, the humidity is controlled at 80%-90%, and the nitrogen replacement frequency is every 2-4 days; S5.2, during the propagation stage, the temperature is controlled at 34-36°C, the humidity is controlled at 78%-88%, and the stirring frequency is every 6-12 days; S5.3, During the propagation stage, the pH value is controlled between 4.2 and 5.

0. If the pH value is lower than 4.2, 0.2% lactic acid solution is added to maintain a stable bacterial environment; S5.4, aroma generation stage, the temperature is controlled at 30-34°C, the humidity is controlled at 72%-82%, and the amount of wine tail added is 0.5%-1.5%; S5.5, during the stabilization phase, the temperature is controlled at 29-33°C, the humidity is controlled at 68%-78%, and the frequency of nitrogen replacement is every 25-30 days.

10. The artificial pit mud wet culture method according to claim 1, characterized in that: The monitoring interval in step S6 is 4 to 8 hours, and the density of anaerobic bacillus and lactic acid bacteria is required to be maintained at 10^7 to 10^9 CFU / mL.

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