Cellar mud cultivation enhanced microbial inoculum, its production method, usage method and application

Abstract

The present invention discloses a pit mud culture strengthening bacterial agent, its preparation method, usage method and application, belonging to the technical field of microorganisms. The present invention uses Syntrophococcus fermenticellae, which can promote the growth of other Clostridium microorganisms, to establish a co-culture system with four Clostridiales, and regulates the abundance of each bacterium, thereby preparing a pit mud culture strengthening bacterial agent. This bacterial agent can be used for the biological maintenance of pit mud to improve the quality of pit mud, significantly promote the production of flavor substances such as short-chain fatty acids and fatty acid ethyl esters, and improve the quality of Baijiu.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a pit mud culture strengthening bacterium agent, a preparation method, a usage method and an application thereof. Background Art

[0002] The strong-flavor Baijiu is one of the products with the highest annual sales and the widest popularity in the traditional brewing beverages in China. The flavor of the strong-flavor Baijiu is closely related to the brewing mud pit. During the long-term continuous batch fermentation process of the mud pit, the quality of the produced Baijiu improves as the pit mud gradually matures. Therefore, there is a saying in the industry that "good wine depends entirely on the old pit". However, the process of pit mud maturation usually takes 10 years, 20 years or even longer, and it is also easily disturbed during the maturation process, resulting in the destruction of the stability of the pit mud microbial community and the degradation of the pit mud quality. All kinds of difficulties and problems in the process of Baijiu maturation restrict the improvement of the production capacity of high-quality Baijiu. How to accelerate the process of pit mud maturation and stabilize the pit mud quality has become the focus of attention in the industry.

[0003] In the traditional strong-flavor Baijiu brewing system, the pit mud in the pit is rich in microorganisms, which plays a key role in the enrichment of short-chain fatty acids and volatile flavor substances in the pit. These microorganisms do not exist independently. They will interact with each other and form a complex interaction network, gradually forming a stable dynamic equilibrium community structure during the long-term brewing process, and the pit mud is gradually domesticated and matured. Existing research has shown that the relative abundance of microorganisms belonging to the genus Clostridium in the pit mud is very significant, and they have important functions in the community. Microorganisms of this genus can produce a variety of flavor substance precursors that are important for the flavor of Baijiu, such as acetic acid, butyric acid and caproic acid. By the interaction between microorganisms, improving the metabolism of key microorganisms in the community to make them produce more target metabolites is also one of the methods to increase the content of fatty acid esters in Baijiu and improve the quality of Baijiu. Therefore, studying the interaction of microorganisms is of great significance for clarifying the microecology of pit mud.

[0004] The role of syntrophic microorganisms in the community cannot be ignored. For example, syntrophy can broaden the ecological niche of other microorganisms and affect the metabolic profile of microorganisms. At present, the research on the interaction of the microbial community in the strong-flavor Baijiu pit mud has not yet formed a complete system. Among them, the impact of the presence of syntrophic microorganisms in the pit mud on other microorganisms in the brewing system is not fully understood. Therefore, using their syntrophic characteristics to prepare a bacterium agent for co-culturing syntrophic microorganisms and other acid-producing target microorganisms, and increasing the accumulation of target microorganism products, is helpful to realize the regulation of pit mud quality and improve the output of high-quality Baijiu. Summary of the Invention

[0005] The present invention provides a pit mud culture enhancing bactericide, its preparation method, usage method and application, aiming to solve the problems occurring in the above-mentioned existing pit mud maintenance and aging technologies. The present invention co-cultures a bacterium of the genus Syntrophococcus screened from pit mud with other microorganisms of the genus Clostridium, which can promote the growth of beneficial functional microorganisms such as the genus Clostridium and promote the formation of short-chain fatty acids; furthermore, after the bactericide prepared by co-culturing the Syntrophococcus with other microorganisms of the genus Clostridium is inoculated into the pit mud, it can regulate the microbial community of the pit mud, realize the biological maintenance of the pit mud, and improve the quality of the pit mud.

[0006] To achieve the above object, the present invention first provides a pit mud culture enhancing bactericide, which is prepared from the following microorganisms as raw materials: Novisyntrophococcus fermenticellae of the genus Novisyntrophococcus, preservation number CICC No. 24502; Clostridium fermenticellae of the genus Clostridium, preservation number CGMCC No. 17037; Terrisporobacter petrolearius of the genus Terrisporobacter, preservation number JCM No. 19845; Clostridium scatologenes of the genus Clostridium, preservation number DSM No. 757; Clostridium sporogenes of the genus Clostridium, preservation number DSM No. 795.

[0007] It is found through experiments that regulating the abundance of each bacterium can further increase the content of short-chain fatty acids and fatty acid ethyl esters in the pit mud after maintenance. Therefore, in some embodiments of the present invention, in the above-mentioned pit mud culture enhancing bactericide, the relative abundances of the microorganisms are: Novisyntrophococcus fermenticellae: 40-50%; Terrisporobacter petrolearius: 5-10%; Clostridium scatologenes: 5-10%; Clostridium fermenticellae: 15-20%; Clostridium sporogenes: 15-20%.

[0008] The present invention also provides a preparation method of the above-mentioned pit mud culture enhancing bactericide, which includes the following steps:

[0009] A. Separately take Novisyntrophococcus fermenticellae, Clostridium fermenticellae, Terrisporobacter petrolearius, Clostridium scatologenes, and Clostridium sporogenes, and inoculate them separately into an enriched liquid medium. Cultivate until the bacteria grow to the logarithmic phase, with an OD 600 value of 0.8 - 1.2, to obtain five kinds of bacterial solutions;

[0010] B. According to the relative abundances of the above-mentioned microorganisms, take the five kinds of bacterial solutions obtained in step A for compounding to obtain a compound bacterial solution;

[0011] C. Inoculate the compound bacterial solution into the enriched liquid medium and continue to cultivate. After the growth state of the compound bacteria is stable, the enhanced microbial inoculum for pit mud cultivation is obtained.

[0012] Among them, in the preparation method of the above-mentioned enhanced microbial inoculum for pit mud cultivation, the formula of the enriched liquid medium is: CaCl2·2H2O 0.05 - 0.07 g / L, NH4Cl 0.40 - 0.60 g / L, MgSO4·7H2O 0.40 - 0.60 g / L, K2HPO4·3H2O 0.50 - 0.70 g / L, NaCl 0.40 - 0.60 g / L, FeSO4·7H2O 0.001 - 0.002 g / L, tryptone 0.80 - 1.00 g / L, yellow water 15 - 20 mL / L, trace element mother liquor 1 mL / L, sodium acetate 20.0 - 25.0 g / L, glucose 20.0 - 25.0 g / L, and the solvent is water; the formula of the trace element mother liquor is MnSO4·H2O 1.0 g / L, CoCl2·6H2O 0.2 g / L, ZnSO4·7H2O 0.2 mg / L, CuCl2·2H2O 20 mg / L, NiCl2·6H2O 20 mg / L, Na2MoO4·2H2O 20 mg / L, Na2SeO4 20 mg / L, Na2WO4 20 mg / L, and the solvent is water.

[0013] Among them, in the preparation method of the above-mentioned enhanced microbial inoculum for pit mud cultivation, in step A, the cultivation conditions are: cultivate under an anaerobic environment at 36 - 38 °C, and the volume ratio of the mixed gas is H2:CO2:N2 = 10%:10 - 20%:70 - 80%.

[0014] Among them, in the preparation method of the above-mentioned enhanced microbial inoculum for pit mud cultivation, in step C, at the time of inoculation, the volume ratio of the compound bacterial solution to the enriched liquid medium is 1 - 1.5:10.

[0015] Among them, in the preparation method of the above-mentioned pit mud culture-enhanced bacterial agent, in step C, the conditions for continuous cultivation are as follows: anaerobic, continuous subculture 6-8 times at 36-38 °C, and the volume ratio of the mixed gas is H2:CO2:N2 = 10%:10-20%:70-80%.

[0016] The present invention also provides a method for using the above-mentioned pit mud culture-enhanced bacterial agent for biological maintenance of pit mud, which includes the following steps:

[0017] a. Inoculate the pit mud culture-enhanced bacterial agent into an enhanced liquid medium for scale-up cultivation to obtain an enhanced bacterial agent seed liquid;

[0018] b. Mix the enhanced bacterial agent seed liquid with new pit mud and seal it in a pit for cultivation to obtain the new pit mud after cultivation with the enhanced bacterial agent.

[0019] Among them, in the method for using the above-mentioned pit mud culture-enhanced bacterial agent for biological maintenance of pit mud, in step a, during inoculation, the volume ratio of the pit mud culture-enhanced bacterial agent to the enhanced liquid medium is 15-20%.

[0020] Among them, in the method for using the above-mentioned pit mud culture-enhanced bacterial agent for biological maintenance of pit mud, in step a, the conditions for scale-up cultivation are as follows: the temperature is controlled at 36-38 °C, the anaerobic environment is maintained with high-purity nitrogen at a flow rate of 0.8-1.0 L / min, and the cultivation time is 3-5 d.

[0021] Among them, in the method for using the above-mentioned pit mud culture-enhanced bacterial agent for biological maintenance of pit mud, in step b, the mass ratio of the enhanced bacterial agent seed liquid to the new pit mud is 1-1.5:10.

[0022] Among them, in the method for using the above-mentioned pit mud culture-enhanced bacterial agent for biological maintenance of pit mud, in step b, the conditions for sealed cultivation are as follows: cultivation at 29-31 °C for 55-65 d.

[0023] The present invention also provides the application of the above-mentioned pit mud culture-enhanced bacterial agent in liquor brewing, short-chain fatty acid fermentation production, pit mud ripening and maintenance.

[0024] In the present invention, Novisyntrophococcus fermenticellae and Clostridium fermenticellae can be isolated from aged pit mud or directly use the preserved strains. When isolated from pit mud, the general operation is as follows: microbial enrichment is carried out according to the mass-to-volume ratio of aged pit mud continuously used in the production of Luzhou-flavor liquor to the enriched liquid medium of 1-1.5 g:50 mL. The conditions for microbial enrichment are: time 15-20 d, anaerobic culture, temperature 37 °C, and the volume ratio of the mixed gas is H2:CO2:N2 = 10%:10-20%:70-80%. Then, Novisyntrophococcus fermenticellae and Clostridium fermenticellae are isolated therefrom.

[0025] Advantages of the present invention:

[0026] The present invention uses the syntrophic bacterium Novisyntrophococcus fermenticellae screened from pit mud and the microorganisms in the order Clostridiales, Terrisporobacter petrolearius, Clostridium scatologenes, Clostridium fermenticellae, and Clostridium sporogenes to establish a co-culture system, and regulates the abundance of each bacterium, significantly increasing the contents of short-chain fatty acids and fatty acid ethyl esters. After preparing the co-culture system into a pit mud culture-enriched bacterial agent, the bacterial agent can be used for the biological maintenance of pit mud. Compared with new pit mud, the quality of pit mud is significantly improved, and further, the production of flavor substances such as short-chain fatty acids and fatty acid ethyl esters is significantly promoted, which is of great significance for improving the quality of liquor. Specific embodiments

[0027] The following will explain the solution of the present invention in combination with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0028] In the examples, the formula of the enriched liquid medium used is as follows: 0.05 g of CaCl₂·2H₂O, 0.40 g of NH₄Cl, 0.40 g of MgSO₄·7H₂O, 0.50 g of K₂HPO₄·3H₂O, 0.40 g of NaCl, 0.002 g of FeSO₄·7H₂O, 1.00 g of tryptone, 15 mL of yellow water, 1 mL of trace element mother liquor, 20.0 g of sodium acetate, 20.0 g of glucose, and the balance is water; among them, the formula of the trace element mother liquor (1 L) is 1.0 g of MnSO₄·H₂O, 0.2 g of CoCl₂·6H₂O, 0.2 mg of ZnSO₄·7H₂O, 20 mg of CuCl₂·2H₂O, 20 mg of NiCl₂·6H₂O, 20 mg of Na₂MoO₄·2H₂O, 20 mg of Na₂SeO₄, 20 mg of Na₂WO₄, and the balance is water.

[0029] In the examples, Novisyntrophococcus fermenticellae, with the preservation number of CICC No. 24502; Clostridium fermenticellae, with the preservation number of CGMCC No. 17037; Terrisporobacter petrolearius, with the preservation number of JCM No. 19845; Clostridium scatologenes, with the preservation number of DSM No. 757; Clostridium sporogenes, with the preservation number of DSM No. 795.

[0030] Example 1: Exploration of the microbial species in the pit mud culture enrichment bacterium agent

[0031] 1.1. Mix Novisyntrophococcus fermenticellae with four Clostridiales bacteria, namely Clostridium fermenticellae, Terrisporobacter petrolearius, Clostridium scatologenes, and Clostridium sporogenes, at a ratio of 1:1, and inoculate them into the enriched liquid medium at a ratio of 1:10 (V / V) for co-culture in pairs. The culture conditions are anaerobic culture at 37°C, and the volume ratio of the mixed gas is H₂:CO₂:N₂ = 1:1:8. After 96 h, measure the content of short-chain fatty acids in the bacterial liquid.

[0032] The results showed that co-culturing N.fermenticellae with C.fermenticellae, C.sporogenes increased the caproic acid production of C.fermenticellae (from 4.56 g / L to 6.21 g / L) and C.sporogenes (from 6.64 g / L to 7.96 g / L); co-culturing N.fermenticellae with T.petrolearius, C.scatologenes increased the butyric acid production of T.petrolearius (from 1.33 g / L to 2.56 g / L) and C.scatologenes (from 1.47 g / L to 2.65 g / L).

[0033] 1.2 Based on the characteristic that butyric acid can be used as a substrate for caproic acid synthesis in the fatty acid metabolic pathway, the above five bacteria were mixed in the ratio of 50% N.fermenticellae and 12.5% for each of the remaining four Clostridiales bacteria, and inoculated into an enhanced liquid medium at a ratio of 1:10 (V / V) for co-culture. The culture conditions were anaerobic culture at 37°C, and the volume ratio of the mixed gas was H2:CO2:N2 = 1:1:8. After 96 h, the butyric acid and caproic acid contents in the bacterial liquid were measured.

[0034] 1.3 Comparing the short-chain fatty acid results of the five-bacteria co-culture with the acid production amounts of the two-bacteria co-culture and single-bacteria culture, the results are shown in Table 1. The caproic acid content produced by the five-bacteria co-culture system was significantly higher than that of the two-bacteria co-culture and single-bacteria culture. It was proved that in the case of N.fermenticellae providing acetic acid and T.petrolearius and C.scatologenes providing butyric acid, the caproic acid-producing bacteria C.fermenticellae and C.sporogenes in the system could significantly increase the caproic acid production, which was beneficial to the subsequent biological maintenance of pit mud after the inoculant was put into use.

[0035] Table 1 Contents of butyric acid and caproic acid in the bacterial liquid after co-culture (unit: g / L bacterial liquid)

[0036]

[0037]

[0038] Example 2: Exploration of the strain ratio of the enhanced inoculant for pit mud culture

[0039] 2.1. Microbial enrichment of pit mud using fortified liquid medium. Take 10 g of pit mud from a 30-year-old cellar of Luzhou-flavor liquor, inoculate it into 500 mL of fortified liquid medium, and conduct enrichment culture for 15 d under anaerobic conditions at 37 °C. The ratio of the anaerobic mixed gas is H2:CO2:N2 = 1:1:8. Isolate and obtain Novisyntrophococcus fermenticellae and Clostridium fermenticellae from the pit mud and store them at -80 °C.

[0040] 2.2. Take Novisyntrophococcus fermenticellae, Clostridium fermenticellae, Terrisporobacter petrolearius, Clostridium scatologenes, and Clostridium sporogenes stored in a -80 °C refrigerator respectively, and inoculate them into fortified liquid medium for activation according to a ratio of 1:10 (V / V). Culture anaerobically at 37 °C, and the volume ratio of the mixed gas is H2:CO2:N2 = 1:1:8. Monitor the growth of single bacteria in the medium (OD 600 ) during the culture process until the bacteria in the medium grow to the logarithmic phase and OD 600 value > 1.0.

[0041] 2.3. Mix the five bacterial solutions according to the relative abundance ratio of Novisyntrophococcus fermenticellae: 60%; Clostridium fermenticellae: 10%; Clostridium sporogenes: 10%; Terrisporobacter petrolearius: 10%; Clostridium scatologenes: 10%. Then, inoculate the mixed bacterial solution into fortified liquid medium according to a ratio of 1:10 (V / V) and conduct continuous anaerobic culture at 37 °C. The volume ratio of the anaerobic mixed gas is H2:CO2:N2 = 1:1:8, and subculture continuously for 6 times to obtain a stable compound microbial agent.

[0042] 2.4. Conduct enlarged culture of the compound microbial agent in an anaerobic fermenter. Put the fortified liquid medium into a 3 L anaerobic fermenter, and inoculate the compound microbial agent into the fermenter according to a ratio of 15% (V / V). Control the fermentation temperature at 37 °C, maintain the anaerobic environment with high-purity nitrogen at a flow rate of 0.8 L / min, and the culture time is 3 d to obtain the seed liquid of the compound microbial agent.

[0043] 2.5. Mix the obtained seed liquid with the newly prepared pit mud from the winery at a mass ratio of 1:10, seal it in a pit, and culture it at 30 °C for 60 days to obtain the newly prepared pit mud after culturing with the composite bacterial agent.

[0044] 2.6. Detect the caproic acid content of the newly prepared pit mud after enhanced culturing with the composite bacterial agent. It is found that the caproic acid content of the newly prepared pit mud cultured in equal proportion is 6.88 g / kg (dry weight of pit mud), which is higher than that of the unprepared pit mud, but the increase range is limited.

[0045] Example 3: Construction and application of enhanced bacterial agent for pit mud culture

[0046] 3.1. Respectively take Novisyntrophococcus fermenticellae, Clostridium fermenticellae, Terrisporobacter petrolearius, Clostridium scatologenes and Clostridium sporogenes stored in a -80 °C refrigerator, inoculate them into the enhanced liquid medium at a ratio of 1:10 (V / V) for activation, culture anaerobically at 37 °C, and the ratio of the mixed gas is H2:CO2:N2 = 1:1:8. During the culture process, monitor the growth of single bacteria in the medium (OD 600 ), until the bacteria in the medium grow to the logarithmic phase, OD 600 value > 1.0.

[0047] 3.2. Mix the five bacterial solutions according to the relative abundance ratio of Novisyntrophococcus fermenticellae: 40%; Clostridium fermenticellae: 20%; Clostridium sporogenes: 20%; Terrisporobacter petrolearius: 5%; Clostridium scatologenes: 5%. Then, according to the ratio of 1:10 (V / V), connect the mixed bacterial solution into the enhanced liquid medium and continuously culture anaerobically at 37 °C. The volume ratio of the anaerobic mixed gas is H2:CO2:N2 = 1:1:8, and continuously passage 6 times to obtain the enhanced bacterial agent for pit mud culture.

[0048] 3.3. Amplify and culture the enhanced bacterial agent in an anaerobic fermenter. Put the enhanced liquid medium into a 3 L anaerobic fermenter, and connect the enhanced bacterial agent into the fermenter at a ratio of 15% (V / V). The fermentation temperature is controlled at 37 °C, and the anaerobic environment is maintained with high-purity nitrogen with a flow rate of 0.8 L / min. The culture time is 3 days to obtain the seed liquid of the enhanced bacterial agent.

[0049] 3.4. Mix the obtained seed liquid with the newly prepared pit mud from the winery at a mass ratio of 1:10, seal it in the pit, and incubate it at 30 °C for 60 days to obtain the newly prepared pit mud after being cultured with the enhanced bacterial agent. Add water to the control group's newly prepared pit mud until the moisture content is similar to that of the newly prepared pit mud after enhanced culture, and then seal and culture it at 30 °C for 60 days in the pit in the same way.

[0050] 3.5. Measure the bacterial microbial community structure and the four major acids of the newly prepared pit mud after enhanced culture (Group E1) and the control group's newly prepared pit mud (Group W1). The results are shown in Table 2. In the newly prepared pit mud cultured with the enhanced bacterial agent, the relative abundance of the microorganism Caproiciproducens increased from 15.4% to 20.2% compared with the non-enhanced newly prepared pit mud; the relative abundance of the microorganism Clostridium increased from 2.5% to 16.3%; the contents of lactic acid and acetic acid decreased compared with the non-enhanced newly prepared pit mud; the content of caproic acid increased significantly, indicating that the enhanced bacterial agent has a good effect on the aging of pit mud and the biological maintenance of pit mud.

[0051] Example 4: Construction and application of an enhanced bacterial agent for pit mud culture

[0052] 4.1. Respectively take Novisyntrophococcus fermenticellae, Clostridium fermenticellae, Terrisporobacter petrolearius, Clostridium scatologenes, and Clostridium sporogenes stored in a -80 °C refrigerator, inoculate them into the enhanced liquid medium at a ratio of 1.2:10 (V / V) for activation, and culture them anaerobically at 36 °C. The ratio of the mixed gas is H2:CO2:N2 = 1:1.5:7.5. Monitor the growth of single bacteria in the medium (OD 600 ) during the culture process until the bacteria in the medium grow to the logarithmic phase, and OD 600 value > 0.8.

[0053] 4.2 Mix the five kinds of bacterial solutions according to the relative abundance ratio of Novisyntrophococcus fermenticellae: 45%; Clostridium fermenticellae: 17.5%; Clostridium sporogenes: 17.5%; Terrisporobacter petrolearius: 5%; Clostridium scatologenes: 5%, and then inoculate them into the enhanced liquid medium at a ratio of 1.2:10 (V / V) and continuously culture anaerobically at 37°C. The ratio of the mixed gas is H2:CO2:N2 = 1:1.5:7.5. Continuously passage 7 times to obtain the enhanced microbial inoculum for pit mud culture.

[0054] 4.3 Amplify and culture the enhanced microbial inoculum in an anaerobic fermenter. Put the enhanced liquid medium into a 3L anaerobic fermenter and inoculate the enhanced microbial inoculum into the fermenter at a ratio of 18% (V / V). Control the fermentation temperature at 36°C, and maintain the anaerobic environment with high-purity nitrogen gas at a flow rate of 0.9L / min. The culture time is 4d to obtain the seed liquid of the enhanced microbial inoculum.

[0055] 4.4 Mix the obtained seed liquid with the new pit mud prepared by the winery at a mass ratio of 1.2:10, seal it in the pit, and culture it at 31°C for 55d to obtain the new pit mud after culturing with the enhanced microbial inoculum. Add water to the control group of new pit mud to make the moisture content similar to that of the enhanced cultured new pit mud, and then put it into the pit and seal it at 31°C for 55d.

[0056] 4.5 Measure the bacterial microbial community structure and the four major acids of the new pit mud after enhanced culture (E2 group) and the control group of new pit mud (W2 group). The results are shown in Table 2. The results show that in the new pit mud cultured with the enhanced microbial inoculum, the relative abundance of microorganisms of the genus Caproiciproducens increased from 15.2% to 18.7% compared with the non-enhanced new pit mud; the relative abundance of microorganisms of the genus Clostridium increased from 2.5% to 17.2%; the contents of lactic acid and acetic acid decreased compared with the non-enhanced new pit mud; the content of caproic acid increased significantly, indicating that the enhanced microbial inoculum has a good effect on the aging of pit mud and the biological maintenance of pit mud.

[0057] Example 5: Construction and application of the enhanced microbial inoculum for pit mud culture

[0058] 5.1. Respectively take Novisyntrophococcus fermenticellae, Clostridium fermenticellae, Terrisporobacter petrolearius, Clostridium scatologenes, and Clostridium sporogenes stored in a -80°C refrigerator, inoculate them into an enhanced liquid medium for activation at a ratio of 1.5:10 (V / V), and culture anaerobically at 38°C. The ratio of the anaerobic gas mixture is H2:CO2:N2 = 1:2:7. During the culture process, monitor the growth of single bacteria in the medium (OD 600 ), until the bacteria in the medium grow to the logarithmic phase, and the OD 600 value > 1.1.

[0059] 5.2. Mix the five bacterial solutions according to the relative abundance ratio of Novisyntrophococcus fermenticellae: 50%; Clostridium fermenticellae: 15%; Clostridium sporogenes: 15%; Terrisporobacter petrolearius: 10%; Clostridium scatologenes: 10%, and then inoculate them into an enhanced liquid medium at a ratio of 1.5:10 (V / V) and continuously culture anaerobically at 38°C. The ratio of the anaerobic gas mixture is H2:CO2:N2 = 1:2:7. Continuously passage 8 times to obtain the enhanced microbial inoculant for pit mud culture.

[0060] 5.3. Amplify and culture the enhanced microbial inoculant in an anaerobic fermenter. Put the enhanced liquid medium into a 3L anaerobic fermenter and inoculate the enhanced microbial inoculant into the fermenter at a ratio of 20% (V / V). Control the fermentation temperature at 38°C, maintain the anaerobic environment with high-purity nitrogen at a flow rate of 1.0 L / min, and the culture time is 5 days to obtain the seed liquid of the enhanced microbial inoculant.

[0061] 5.4. Mix the obtained seed liquid with the new pit mud prepared by the winery according to a mass ratio of 1.5:10, seal it in the pit, and culture it at 29°C for 65 days to obtain the new pit mud after culturing with the enhanced microbial inoculant. Add water to the control group of new pit mud until the moisture content is similar to that of the newly pit mud with enhanced culture, and then put it into the pit and seal it at 29°C for 65 days.

[0062] 5.5. The bacterial microbial community structure and the determination of the four major acids were carried out on the newly strengthened pit mud (Group E3) and the control group of newly pit mud (Group W3) after intensive cultivation. The results are shown in Table 2. The results show that in the newly pit mud cultivated with the strengthening bacterium agent, the relative abundance of the microorganisms of the genus Caproiciproducens increased from 16.8% to 22.4% compared with the non-strengthened pit mud; the relative abundance of the microorganisms of the genus Clostridium increased from 3.9% to 15.8%; the contents of lactic acid and acetic acid decreased compared with the non-strengthened pit mud; the content of caproic acid increased significantly, indicating that the strengthening bacterium agent has good effects on the ripening of pit mud and the biological maintenance of pit mud.

[0063] Table 2 Comparison of the contents of the four major acids in pit mud after inoculating the strengthening bacterium agent for pit mud cultivation (unit: g / kg dry weight of pit mud)

[0064] Index <![CDATA[Group W1]]> <![CDATA[Group E1]]> <![CDATA[Group W2]]> <![CDATA[Group E2]]> <![CDATA[Group W3]]> <![CDATA[Group E3]]> Lactic acid 8.62 7.45 8.01 7.32 8.29 7.02 Acetic acid 3.84 3.66 3.47 3.26 3.35 2.97 Butyric acid 1.18 1.09 1.29 1.54 1.12 1.24 Caproic acid 5.69 8.61 5.48 7.34 5.30 7.58

[0065] Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. The pit mud culture intensifying bacterial agent is characterized in that: The microbial agent is prepared from the following microorganisms as raw materials: Novisyntrophococcus fermenticellae of the genus Novisyntrophococcus, with the preservation number of CICC No. 24502; Clostridium fermenticellae of the genus Clostridium, with the preservation number of CGMCC No. 17037; Terrisporobacter petrolearius of the genus Terrisporobacter, with the preservation number of JCM No. 19845; Clostridium scatologenes of the genus Clostridium, with the preservation number of DSM No. 757; Clostridium sporogenes of the genus Clostridium, with the preservation number of DSM No.

795. The relative abundances of the microorganisms are as follows: Novisyntrophococcus fermenticellae: 40 - 50%; Terrisporobacter petrolearius: 5 - 10%; Clostridium scatologenes: 5 - 10%; Clostridium fermenticellae: 15 - 20%; Clostridium sporogenes: 15 - 20%.

2. The preparation method of the pit mud culture intensifying bacterial agent according to claim 1, characterized in that: It includes the following steps: A. Separately take Novisyntrophococcus fermenticellae, Clostridium fermenticellae, Terrisporobacter petrolearius, Clostridium scatologenes and Clostridium sporogenes, and inoculate them separately into an enhanced liquid medium. Cultivate until the bacteria grow to the logarithmic phase, and the OD 600 value is 0.8 - 1.2 to obtain five kinds of bacterial solutions; B. According to the relative abundances of the microorganisms described in claim 1, take the five kinds of bacterial solutions obtained in step A for compounding to obtain a compound bacterial solution. C. Inoculate the compound bacterial solution into an enhanced liquid medium for continuous cultivation. After the growth state of the compound bacteria is stable, the pit mud culture enhanced microbial agent is obtained.

3. The preparation method of the pit mud culture-enhanced bacterial agent according to claim 2, characterized in that: The formula of the enhanced liquid medium is as follows: CaCl2·2H2O 0.05 - 0.07 g / L, NH4Cl 0.40 - 0.60 g / L, MgSO4·7H2O 0.40 - 0.60 g / L, K2HPO4·3H2O 0.50 - 0.70 g / L, NaCl 0.40 - 0.60 g / L, FeSO4·7H2O 0.001 - 0.002 g / L, tryptone 0.80 - 1.00 g / L, yellow water 15 - 20 mL / L, trace element mother liquor 1 mL / L, sodium acetate 20.0 - 25.0 g / L, glucose 20.0 - 25.0 g / L, and the solvent is water; the formula of the trace element mother liquor is MnSO4·H2O 1.0 g / L, CoCl2·6H2O 0.2 g / L, ZnSO4·7H2O 0.2 mg / L, CuCl2·2H2O 20 mg / L, NiCl2·6H2O 20 mg / L, Na2MoO4·2H2O 20 mg / L, Na2SeO4 20 mg / L, Na2WO4 20 mg / L, and the solvent is water.

4. The preparation method of the pit mud culture intensifying microbial inoculum according to claim 2, characterized in that: In step A, the conditions for cultivation are: cultivation in an anaerobic environment at 36 - 38 °C, and the volume ratio of the mixed gas is H2:CO2:N2 = 10%:10 - 20%:70 - 80%.

5. The preparation method of the pit mud culture strengthening bacterial agent according to any one of claims 2 to 4, characterized in that: In step C, at least one of the following is satisfied: During inoculation, the volume ratio of the complex bacterial liquid to the fortified liquid medium is 1-1.5:10; The conditions for continuous cultivation are: anaerobic, continuous subculture 6-8 times at 36-38 °C, and the volume ratio of the mixed gas is H2:CO2:N2 = 10%:10-20%:70-80%.

6. The method for biological maintenance of pit mud using the pit mud culture-enhanced bacterium agent described in claim 1 or the pit mud culture-enhanced bacterium agent prepared by the method described in any one of claims 2 to 5, characterized in that: It includes the following steps: a. Inoculate the pit mud culture fortified bactericide into the fortified liquid medium, and perform enlarged cultivation to obtain the fortified bactericide seed liquid; b. Mix the fortified bactericide seed liquid with the new pit mud and seal it in a pit pond for cultivation to obtain the new pit mud after culturing with the fortified bactericide.

7. The method according to claim 6, characterized in that: In step a, at least one of the following is satisfied: During inoculation, the volume ratio of the pit mud culture fortified bactericide to the fortified liquid medium is 15-20%; The conditions for enlarged cultivation are: the temperature is controlled at 36-38 °C, the anaerobic environment is maintained by high-purity nitrogen with a flow rate of 0.8-1.0 L / min, and the cultivation time is 3-5 days.

8. The method according to claim 6, wherein: In step b, at least one of the following is satisfied: The mass ratio of the fortified bactericide seed liquid to the new pit mud is 1-1.5:10; The conditions for sealed cultivation are: cultivate at 29-31 °C for 55-65 days.

9. The application of the pit mud culture fortified bactericide described in claim 1, or the pit mud culture fortified bactericide prepared by the method described in any one of claims 2-5, in liquor brewing, short-chain fatty acid fermentation production, pit mud aging and maintenance.

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