Composite microbial agent and application thereof in regulation and control of saccharification pile fermentation temperature
By using complex microbial agents, including mixed culture medium of multiple strains, the problem of slow fermentation speed of saccharification reactors in low temperature climates is solved, and more efficient saccharification reactor fermentation is achieved, improving the production efficiency and drying effect of soy sauce-flavored wine making .
Patent Information
- Application Number
- CN202411316267.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-20
- Publication Date
- 2024-12-27
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The fermentation rate of the saccharification heap is slow in low temperature climates, which leads to difficulty in producing sauce-flavored wine, affecting production efficiency and cost, and insufficient drying site.
Complex microbial agents, including Bacillus coliformis, Bacillus subtilis, Pichia Kudriazwi, Candida tropicalis, Saccharomyces cerevisiae, Acetaminophyllis, and Ordinary Acetaminophyllis, by mixing medium and specific Preparation of culture conditions, used for saccharification stacking and fermentation, increase the fermentation temperature and shorten the time.
The fermentation time of the saccharified heap is significantly shortened, the accumulation temperature is increased, the normal production progress is ensured, the production cost is reduced, and the drying effect is improved.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention belongs to the fields of biotechnology and fermentation technology, and relates to a composite microbial agent and application thereof in regulating the fermentation temperature of a saccharification pile. Background Art
[0002] The production of Maotai liquor lasts for a year, from autumn to winter, spring to summer. The first and second rounds of production are carried out in winter, which is a cold weather. The saccharification piles heat up slowly, and the production area is occupied by a large number of saccharification piles, resulting in a serious shortage of available drying space. The mash is spread thick, and the moisture and acidity of the mash surface cannot evaporate properly, making it difficult for employees to operate, which seriously affects the saccharification effect of the piles.
[0003] The slow heating of the saccharification pile in low temperature climate has become a common and difficult problem in the production of Maotai-flavor liquor, and has become an urgent problem that restricts the normal production of Maotai-flavor liquor. By using the principle of microorganisms, by creating an environment suitable for the growth and reproduction of microorganisms and improving the vitality and quantity of microorganisms in the initial saccharification pile, the fermentation time of the saccharification pile in low temperature climate can be shortened, which can promote the low-temperature round production of Maotai-flavor liquor to be organized according to the normal production schedule, shorten the round working time, reduce production costs, ensure sufficient drying space, improve the drying effect, and reduce human intervention in the stacking process of the saccharification pile, which is of great significance for reducing the output of primary and secondary liquors, achieving more reasonable rounds of liquor production, and achieving high quality and high yield. Summary of the invention
[0004] In order to solve the above problems, the present invention uses Bacillus coagulans, Bacillus subtilis, Pichia kudriavzevii, Candida tropicalis, Saccharomyces cerevisiae, Thermoactinomyces daqus and Thermoactinomyces vulgaris to prepare a composite microbial agent, and uses the composite microbial agent for saccharification pile fermentation, thereby increasing the pile fermentation temperature and shortening the pile time.
[0005] On the one hand, the present invention provides a composite microbial agent, comprising: a composite bacillus, a composite yeast and a composite actinomycete with a live cell count ratio of 3 to 4:1 to 2:1; the composite bacillus comprises Bacillus coagulans and Bacillus subtilis; the composite yeast comprises Pichia kudriavzevii, Candida tropicalis and Saccharomyces cerevisiae; the composite actinomycete comprises Thermoactinomyces daqus and Thermoactinomyces vulgaris.
[0006] In some embodiments, the composite Bacillus comprises Bacillus coagulans and Bacillus subtilis at a viable bacterial ratio of 1:0.8-1.2; the composite yeast comprises Pichiakudriavzevii, Candida tropicalis and Saccharomyces cerevisiae at a viable bacterial ratio of 1:0.8-1.2:0.8-1.2; the composite actinomycetes comprises Thermoactinomyces daqus and Thermoactinomyces vulgaris at a viable bacterial ratio of 1:0.8-1.2.
[0007] In one aspect, the present invention provides an application of the composite microbial agent, which is any one of the following a1-a3:
[0008] a1. Shorten the saccharification and fermentation time;
[0009] a2. Preparation of products for shortening saccharification and fermentation time;
[0010] a3. Increase the temperature of saccharification pile entering the cellar;
[0011] a4. Preparation of products for increasing the temperature of saccharification piles entering the cellar;
[0012] a5. Prepare freeze-dried bacterial agent.
[0013] In one aspect, the present invention provides a method for preparing the composite microbial agent, comprising the following steps:
[0014] (1) Preparation of composite Bacillus bacterial solution
[0015] activating Bacillus coagulans and Bacillus subtilis respectively; and then inoculating them into MRS culture medium to obtain Bacillus coagulans bacterial liquid and Bacillus subtilis bacterial liquid;
[0016] The Bacillus coagulans bacterial solution and the Bacillus subtilis bacterial solution are mixed to prepare a composite Bacillus bacterial solution;
[0017] (2) Preparation of composite yeast solution
[0018] activating Pichia kudriavzevii, Candida tropicalis and Saccharomyces cerevisiae respectively; and then inoculating them into YPD medium to obtain Pichia kudriavzevii, Candida tropicalis and Saccharomyces cerevisiae bacterial liquid;
[0019] The Pichia kudriavzevii bacterial solution, the Candida tropicalis bacterial solution and the Saccharomyces cerevisiae bacterial solution are mixed to prepare a composite yeast bacterial solution;
[0020] (3) Preparation of composite actinomycete solution
[0021] The thermophilic Daqu actinomycetes and the common thermophilic actinomycetes were activated respectively, and then inoculated into Gao's No. 1 liquid culture medium to obtain the thermophilic Daqu actinomycetes liquid and the common thermophilic actinomycetes liquid;
[0022] The high-temperature Daqu actinomycete liquid and the common high-temperature actinomycete liquid are mixed to prepare the composite actinomycete liquid.
[0023] (4) Preparation of bacterial solution of composite bacterial agent
[0024] The prepared composite bacillus bacterial solution, composite yeast bacterial solution and composite actinomycete bacterial solution are mixed at a live bacteria count ratio of 3 to 4:1 to 2:1.
[0025] In some embodiments, the activation in step (1) is performed at 35°C-39°C for 10 to 14 hours; in some embodiments, the activation in step (2) is performed at 28°C-32°C for 10 to 14 hours; in some embodiments, the activation in step (3) is performed at 24°C-30°C for 2 to 5 days.
[0026] In some embodiments, in step (1), the Bacillus coagulans solution and the Bacillus subtilis solution are mixed at a live bacteria count ratio of 1:0.8-1; in step (2), the Pichia kudriavzevii solution, the Candida tropicalis solution and the Saccharomyces cerevisiae solution are mixed at a live bacteria count ratio of 1:0.8-1.2:0.8-1.2; in step (3), the thermophilic actinomycetes solution and the common thermophilic actinomycetes are mixed at a live bacteria count ratio of 1:0.8-1.2.
[0027] In one aspect, the present invention provides a freeze-dried bacterial agent, comprising the composite microbial agent and skimmed milk powder; in some embodiments, the total number of viable bacteria in the freeze-dried bacterial agent is 2 to 3×10 10 Pieces / g.
[0028] On the one hand, the present invention provides a method for preparing the freeze-dried bacterial agent, comprising the following steps: centrifuging and washing the composite microbial agent, resuspending it with a skimmed milk powder aqueous solution, and freeze-drying to obtain the freeze-dried bacterial agent.
[0029] In one aspect, the present invention provides an application of the freeze-dried bacterial agent, which is any one of the following b1-b2:
[0030] b1. Shorten the saccharification fermentation time;
[0031] b2. Increase the temperature of the saccharification pile entering the cellar.
[0032] On the one hand, the present invention provides a method for shortening the fermentation time of a saccharification pile or increasing the temperature of a saccharification pile entering a cellar, comprising the following steps: inoculating the composite microbial agent or the freeze-dried agent into the mash.
[0033] In some embodiments, the inoculum amount is 5-15 kg / pile; in some embodiments, the inoculum amount is 10-15 kg / pile.
[0034] The beneficial effects of the present invention include:
[0035] The invention uses bacillus coagulans, bacillus subtilis, pichia kudriavzevii, candida tropicalis, saccharomyces cerevisiae, thermophilic actinomycetes and common thermophilic actinomycetes to prepare a composite microbial agent, and uses the composite microbial agent for saccharification pile fermentation, thereby increasing the pile fermentation temperature and shortening the pile time.
[0036] Specific:
[0037] When 10 kg of the composite microbial agent prepared by the invention is inoculated, the medium temperature start-up time of the saccharification pile is shortened by more than 30%, the average stacking time of the saccharification pile is shortened by more than 24 hours, and the average medium temperature data of the saccharification pile is above 38°C when it is put into the cellar.
[0038] Biomaterials
[0039] The strains used in the specific embodiments of the present invention are as follows:
[0040] Bacillus coagulans CICC 24625 was purchased from China Industrial Microbiological Culture Collection Center;
[0041] Bacillus subtilis CICC 24542 was purchased from China Industrial Microbiological Culture Collection Center;
[0042] Pichia kudriavzevii CICC 33179 was purchased from China Industrial Microbiological Culture Collection Center;
[0043] Candida tropicalis CICC31488 was purchased from China Industrial Microbiological Culture Collection Center;
[0044] Saccharomyces cerevisiae CICC 1307 was purchased from China Industrial Microbiological Culture Collection Center;
[0045] Thermoactinomyces daqus CICC 10681 was purchased from China Industrial Microbiological Culture Collection Center;
[0046] Thermoactinomyces vulgaris CICC 24227 was purchased from China Industrial Microbiological Culture Collection Center. DETAILED DESCRIPTION
[0047] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.
[0048] As used in the present invention, the expressions “includes,” “comprising,” “including,” and “having” are open-ended and mean including the listed elements, steps or components but not excluding other unlisted elements, steps or components.
[0049] Unless otherwise indicated, any numerical value or numerical range, such as a concentration or concentration range, is in any case to be understood as being modified by the term "about". Thus, numerical values generally include ±10% of the stated value. For example, 2×10 10 The number of viable bacteria / g includes 2×10 9 / g to 2×10 11 Pieces / g.
[0050] As used herein, the use of numerical ranges expressly includes all possible subranges, all individual numerical values within that range, including integers and fractions within that range, unless the context clearly dictates otherwise.
[0051] MRS medium formula (g / L): tryptone 10, beef extract 10, yeast extract 5, glucose 20, potassium hydrogen phosphate dihydrate 2, ammonium citrate trihydrate 2, magnesium sulfate 0.2, manganese sulfate 0.05, sodium acetate 5, ox bile salt 0.3.
[0052] YPD medium formula (g / L): yeast extract 10, tryptone 20, glucose 20.
[0053] Gao's medium No. 1 formula (g / L): potassium nitrate 1.0, potassium dihydrogen phosphate 0.5, magnesium sulfate 0.5, ferrous sulfate 0.01, sodium chloride 0.5, soluble starch 20.0.
[0054] Example 1: Preparation of microbial inoculants
[0055] The steps for preparing the microbial agent are as follows:
[0056] 1. Preparation of composite Bacillus liquid
[0057] Bacillus coagulans CICC 24625 and Bacillus subtilis CICC 24542 were inoculated from glycerol tubes into MRS medium and cultured at 37°C for 12 h for activation; then inoculated into MRS medium at a 10% (v / v) inoculum volume and cultured at 37°C for 12 h until the number of cells under microscopic examination reached 2.0×10 8 Bacillus coagulans CICC 24625 bacterial solution and Bacillus subtilis CICC 24542 bacterial solution were mixed according to the ratio of viable bacteria to prepare a composite Bacillus bacterial solution.
[0058] 2. Preparation of compound yeast solution
[0059] Pichia kudriavzevii CICC 33179, Candida tropicalis CICC31488 and Saccharomyces cerevisiae CICC1307 were inoculated from glycerol tubes into YPD medium and cultured at 30°C for 12 h for activation; then inoculated into YPD medium at a 10% (v / v) inoculum and cultured at 30°C for 12 h until the number of cells under microscopic examination reached 2.0×10 8 The bacterial solution of Pichia kudriavzevii CICC 33179, the bacterial solution of Candida tropicalis CICC31488 and the bacterial solution of Saccharomyces cerevisiae CICC 1307 were mixed according to the ratio of viable bacteria count of 1:1:1 to prepare a composite yeast bacterial solution.
[0060] 3. Preparation of composite actinomycete solution
[0061] Thermostatic actinomycetes CICC 10681 and common thermostatic actinomycetes CICC 24227 were inoculated into Gao's No. 1 liquid medium for activation, and an appropriate amount of glass beads were added. The medium was cultured in a shaking incubator at 28°C and 100 r / min for 3 days. Then, the inoculation amount was 10% (v / v) and inoculated into Gao's No. 1 liquid medium, and the culture was continued until the bacterial solution OD 600 Reach 0.6 (2.0×10 8 The prepared high-temperature Daqu actinomycete CICC 10681 bacterial solution and the common high-temperature actinomycete CICC 24227 were mixed according to the viable bacterial count of 1:1 to prepare a composite actinomycete bacterial solution.
[0062] 4. Preparation of composite bacterial agent
[0063] The prepared composite bacillus liquid, composite yeast liquid and composite actinomycete liquid were mixed in a ratio of 4:2:1 for viable bacteria, washed by centrifugation, resuspended with a 10% w / v skim milk powder aqueous solution, and freeze-dried to prepare a composite microbial agent (the total number of viable bacteria reached 2.0×10 10 / g).
[0064] Comparative Example 1: No addition of composite actinomycete solution
[0065] On the basis of Example 1, in step 4, when preparing the composite bacterial agent, no composite actinomycete solution is added, and the ratio of the composite Bacillus solution and the composite yeast solution and the total number of live bacteria in the composite microbial agent are kept unchanged.
[0066] Comparative Example 2: No compound yeast solution added
[0067] On the basis of Example 1, in step 4, when preparing the composite bacterial agent, no composite yeast solution is added, and the ratio of the composite Bacillus solution to the composite actinomycete solution and the total number of live bacteria in the composite microbial agent are kept unchanged.
[0068] Comparative Example 3: Using only the compound Bacillus liquid
[0069] On the basis of Example 1, the composite microbial inoculant was prepared using the composite Bacillus bacterial solution, keeping the total number of viable bacteria unchanged.
[0070] Comparative Example 4: No addition of Bacillus coagulans CICC 24625
[0071] On the basis of Example 1, when preparing the composite Bacillus culture liquid in step 1, the Bacillus coagulans CICC24625 culture liquid was not added to keep the total number of viable bacteria of the composite Bacillus culture liquid consistent, and the remaining steps were consistent with Example 1 to prepare the composite microbial agent.
[0072] Comparative Example 5: No addition of Pichia kudriavzevii CICC 33179
[0073] On the basis of Example 1, when preparing the composite yeast solution in step 2, the Pichia kudriavzevii CICC 33179 solution was not added, the ratio of Candida tropicalis CICC31488 and Saccharomyces cerevisiae CICC 1307 was kept consistent, and the total number of viable bacteria in the composite yeast solution was kept consistent, and the remaining steps were consistent with Example 1 to prepare a composite microbial agent.
[0074] Example 2: Optimization of the dosage of composite microbial agent
[0075] The composite microbial agent prepared in Example 1 was used as an inoculant, and three saccharification piles were inoculated with 5kg, 10kg, and 15kg, respectively, and the stacking time when the saccharification piles reached the cellar standard was detected.
[0076] The results are shown in Table 1. The results show that when the inoculation amount of the composite microbial agent is 10 kg, the stacking time is most appropriate.
[0077] Table 1 Inoculation amount
[0078]
[0079] Example 3: Application of composite microbial inoculants in shortening the fermentation time of sauce-flavor saccharification piles in low-temperature climates
[0080] 1. Stacking time detection
[0081] The composite microbial agent prepared in Example 1 and Comparative Examples 1 to 3 was inoculated at a dosage of 10 kg, and no composite microbial agent was added as a control to detect the stacking time under different stacking numbers in the same class (10 classes).
[0082] The test results of the stacking time are shown in Table 2. The results show that the composite microbial agent prepared in Example 1 can significantly shorten the time, while when Comparative Examples 1 to 3 lack one or more of the composite actinomycete solution and the composite yeast solution, and Comparative Examples 4 to 5 lack the key strains (Bacillus coagulans CICC 24625 and Pichia kudriavzevii CICC33179), the composite microbial agent cannot shorten the stacking time.
[0083] Table 2 Different stacking times of composite microbial agents
[0084]
[0085] 2. Temperature detection of saccharification pile startup
[0086] The start-up temperature of the saccharification pile is based on the change of the medium temperature of the saccharification pile as the indicator data. The medium temperature of the saccharification pile refers to the center temperature of the saccharification pile, which is measured by the temperature measuring rod at a height of 80-100 cm from the bottom of the saccharification pile and a parallel depth of 150 cm.
[0087] The composite microbial agent prepared in Example 1 was inoculated in multiple shifts in the workshop for test comparison to detect the effect of inoculation on the start-up temperature duration of the saccharification pile. The results are shown in Table 3.
[0088] Table 3 Effect of inoculation on the start-up temperature and duration of saccharification pile
[0089]
[0090] The results show that when the composite microbial agent is not inoculated, the startup temperature change range of the saccharification pile is significantly increased, and it takes 72 hours; after inoculating 10kg of the composite microbial agent, the startup temperature change range of the saccharification pile is significantly shortened, and it only takes about 24 hours. That is, the composite microbial agent can effectively increase the activity of microorganisms and greatly shorten the startup time of the saccharification pile in low temperature weather, from the original 72 hours to about 24 hours.
[0091] 3. Saccharification pile in the cellar
[0092] The central temperature of the saccharification pile when entering the cellar refers to the central temperature of the saccharification pile when it enters the cellar, which is measured by the temperature measuring rod at a height of 80-100cm from the bottom of the saccharification pile and a parallel depth of 150cm.
[0093] The composite microbial agent prepared in Example 1 was inoculated in multiple teams to ensure that the starting time, starting temperature, completion time and air temperature were basically the same. The average temperature of the saccharification pile entering the pit was compared between the saccharification pile inoculated with 10 kg of the composite microbial agent and the one not inoculated. The results are shown in Table 4.
[0094] Table 4 Effect of inoculation on the temperature of saccharification pile entering the pit
[0095]
[0096]
[0097] The results showed that the average temperature of the saccharification pile entering the cellar of the uninoculated group was 33.58℃, while the average temperature of the saccharification pile entering the cellar with 10kg of inoculant added was 38.24℃, indicating that inoculation with composite microbial agents can effectively increase the temperature of the saccharification pile entering the cellar and solve the problem of poor fermentation in the core of the saccharification pile.
[0098] The above results show that the composite microbial agent prepared by the present invention is used for saccharification pile fermentation, which increases the fermentation temperature and shortens the fermentation time.
[0099] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.
Claims
1. A composite microbial agent, characterized in that: Contains: composite bacillus, composite yeast and composite actinomycetes with a live cell count ratio of 3 to 4:1 to 2:1; The composite Bacillus comprises Bacillus coagulans and Bacillus subtilis; the composite yeast comprises Pichiakudriavzevii, Candida tropicalis and Saccharomyces cerevisiae; and the composite actinomycetes comprises Thermoactinomyces daqus and Thermoactinomyces vulgaris.
2. The composite microbial agent according to claim 1, characterized in that: The composite bacillus comprises Bacillus coagulans and Bacillus subtilis in a ratio of 1:0.8 to 1.2 of viable bacteria; The composite yeast comprises Pichia kudriavzevii, Candida tropicalis and Saccharomyces cerevisiae mixed at a live bacteria count ratio of 1:0.8-1.2:0.8-1.2; The composite actinomycetes include thermoactinomyces daqus and thermoactinomyces vulgaris, which are compounded at a live bacteria ratio of 1:0.8-1.
2.
3. The use of the composite microbial agent according to claim 1 or 2, which is any one of the following a1-a3: a1. Shorten the saccharification and fermentation time; a2. Preparation of products for shortening saccharification and fermentation time; a3. Increase the temperature of saccharification pile entering the cellar; a4. Preparation of products for increasing the temperature of saccharification piles entering the cellar; a5. Prepare freeze-dried bacterial agent.
4. The method for preparing the composite microbial agent according to claim 1 or 2, characterized in that: The following steps are involved: (1) Preparation of composite Bacillus spore solution activating Bacillus coagulans and Bacillus subtilis respectively; and then inoculating them into MRS culture medium to obtain Bacillus coagulans bacterial liquid and Bacillus subtilis bacterial liquid; The Bacillus coagulans bacterial solution and the Bacillus subtilis bacterial solution are mixed to prepare a composite Bacillus bacterial solution; (2) Preparation of composite yeast solution activating Pichia kudriavzevii, Candida tropicalis and Saccharomyces cerevisiae respectively; and then inoculating them into YPD medium to obtain Pichia kudriavzevii, Candida tropicalis and Saccharomyces cerevisiae bacterial liquid; The Pichia kudriavzevii bacterial solution, the Candida tropicalis bacterial solution and the Saccharomyces cerevisiae bacterial solution are mixed to prepare a composite yeast bacterial solution; (3) Preparation of composite actinomycete solution The thermophilic Daqu actinomycetes and the common thermophilic actinomycetes were activated respectively, and then inoculated into Gao's No. 1 liquid culture medium to obtain the thermophilic Daqu actinomycetes liquid and the common thermophilic actinomycetes liquid; The high-temperature Daqu actinomycete liquid and the common high-temperature actinomycete liquid are mixed to prepare the composite actinomycete liquid. (4) Preparation of bacterial solution of composite bacterial agent The prepared composite bacillus bacterial solution, composite yeast bacterial solution and composite actinomycete bacterial solution are mixed at a live bacteria count ratio of 3 to 4:1 to 2:
1.
5. The preparation method according to claim 4, characterized in that: The activation in step (1) is performed at 35°C-39°C for 10-14h; Preferably, the activation in step (2) is carried out at 28°C-32°C for 10-14h; Preferably, the activation in step (3) is carried out at 24°C-30°C for 2 to 5 days.
6. The preparation method according to claim 4, characterized in that: In step (1), the Bacillus coagulans liquid and the Bacillus subtilis liquid are mixed at a ratio of 1:0.8 to 1 in terms of viable bacterial count; In step (2), the Pichia kudriavzevii solution, the Candida tropicalis solution and the Saccharomyces cerevisiae solution are mixed at a live cell count ratio of 1:0.8-1.2:0.8-1.2; In step (3), the high-temperature Daqu actinomycete liquid and the common high-temperature actinomycetes are mixed at a live bacteria count ratio of 1:0.8 to 1.
2.
7. A freeze-dried bacterial agent, characterized in that: Comprising the composite microbial agent as claimed in claim 1 or 2 and skimmed milk powder; Preferably, the total number of viable bacteria in the freeze-dried bacterial preparation is 2 to 3×10 10 Pieces / g.
8. The method for preparing the freeze-dried bacterial agent according to claim 7, characterized in that: The method comprises the following steps: washing the composite microbial agent according to claim 1 or 2 by centrifugation, resuspending the composite microbial agent with a skimmed milk powder aqueous solution, and freeze-drying the composite microbial agent to obtain the freeze-dried agent.
9. The use of the freeze-dried bacterial agent according to claim 4, which is any one of the following b1-b2: b1. Shorten the saccharification and fermentation time; b2. Increase the temperature of the saccharification pile entering the cellar.
10. A method for shortening the fermentation time of saccharification pile or increasing the temperature of saccharification pile entering the cellar, characterized in that: The method comprises the following steps: inoculating the composite microbial agent according to claim 1 or 2, or the freeze-dried microbial agent according to claim 4 into the mash; Preferably, the inoculation amount is 5 to 15 kg / pile; Preferably, the inoculation amount is 10-15 kg / pile.