CN115895953A - Pit mud culture strengthening microbial inoculum and preparation method, use method and application thereof - Google Patents
Pit mud culture strengthening microbial inoculum and preparation method, use method and application thereof Download PDFInfo
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Abstract
The invention discloses a pit mud culture strengthening microbial inoculum and a preparation method, a use method and application thereof, belonging to the technical field of microorganisms. The method utilizes the Novisynophococcus formentiicelle which can promote the growth of other clostridium microorganisms to establish a co-culture system with four clostridium subjects and regulates and controls the abundance of each bacterium to prepare the pit mud culture strengthening microbial inoculum which can be used for biological maintenance of pit mud so as to improve the quality of the pit mud, remarkably promote the generation of flavor substances such as short-chain fatty acid, fatty acid ethyl ester and the like and improve the quality of white spirit.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a pit mud culture strengthening microbial inoculum, and a preparation method, a use method and application thereof.
Background
The strong aromatic Chinese spirits are one of the products with highest annual sales volume and most famous degree in the traditional brewed drinks in China. The flavor of the Luzhou-flavor liquor is closely related to that of a mud cellar for brewing the liquor. The mud cellar pool is in long-term continuous batch fermentation process, and the white spirit quality of its output promotes along with cellar mud is ageing gradually, so the industry element has "good wine relies on cellar pool old" the saying. However, the aging process of the pit mud usually takes 10 years, 20 years or even longer, and the aging process is also susceptible to interference, so that the stability of the microbial community of the pit mud is damaged, and the quality of the pit mud is degraded. Various difficulties and problems in the white spirit aging process restrict the promotion of high-quality white spirit productivity, and how to accelerate the cellar mud aging process and stabilize the quality of the cellar mud becomes a focus problem of industry attention.
In the traditional strong aromatic Chinese spirits brewing system, pit mud in the pit is rich in microorganisms, and plays a key role in enriching short-chain fatty acids and volatile flavor substances in the pit. The microorganisms do not exist independently, and can mutually influence each other to form a complex interaction network, a stable dynamic equilibrium community structure is gradually formed in the long-term brewing process, and the pit mud is gradually domesticated and aged. It has been shown that microorganisms of the genus clostridium occupy a very significant relative abundance in pit mud and have important functions in communities. This genus of microorganisms is capable of producing a variety of flavor precursors such as acetic acid, butyric acid and caproic acid, which contribute significantly to the flavor of white wine. The metabolism of key microorganisms in the community is improved through interaction among the microorganisms, so that more target metabolites are produced, and the method is also one of the methods for improving the content of fatty acid esters in the white spirit and improving the quality of the white spirit. Therefore, the research on the microbial interaction has important significance for clarifying the microbial ecology of the pit mud.
The role of the cross-feeding microorganisms in the community is not negligible. For example, cross-breeding can broaden the niche of other microorganisms, affect the metabolic profile of the microorganisms, and the like. At present, researches on interaction of microbial communities of pit mud of Luzhou-flavor liquor relatively do not form a complete system. The influence that the existence of the intercropping microorganisms in the pit mud may have on other microorganisms in the brewing system is not completely clear. Therefore, by utilizing the mutual-nutrient characteristic, the microbial inoculum for co-culturing the mutual-nutrient microorganisms and other acid-producing target microorganisms is prepared, the accumulation of target microorganism products is increased, the quality of pit mud is favorably regulated and controlled, and the yield of high-quality white spirit is improved.
Disclosure of Invention
The invention provides a pit mud culture strengthening microbial inoculum and a preparation method, a use method and application thereof, and aims to solve the problems in the existing pit mud maintenance and aging technology. The method utilizes the screened and obtained mutual campylococcus bacteria from the pit mud to carry out co-culture with other clostridium microorganisms, can promote the growth of beneficial functional microorganisms such as clostridium and the like, and promote the formation of short-chain fatty acid; the microbial inoculum prepared by co-culturing the intercropping coccus and other clostridium microorganisms is inoculated into the pit mud, so that the microbial community of the pit mud can be regulated, the biological maintenance of the pit mud is realized, and the quality of the pit mud is improved.
In order to realize the purpose, the invention firstly provides a pit mud culture strengthening microbial inoculum which is prepared by taking the following microorganisms as raw materials: novisyntropophococcus fermenticelle of the genus Novitrophophococci having a accession number of CICC No.24502; clostridium permmenticelle of Clostridium, with a preservation number of CGMCC No.17037; terrisporobacter petrolerius of terrestribacillus, accession No. JCM No.19845; clostridium scoriogens with the accession number DSM No.757; clostridium sporogenes of Clostridium genus, deposited under DSM No.795.
Experiments show that the content of short-chain fatty acid and fatty acid ethyl ester in the pit mud after maintenance can be further improved by regulating and controlling the abundance of each bacterium. Therefore, in some embodiments of the present invention, in the above pit mud culture enhancing microbial inoculum, the relative abundance of the microorganisms is: noviostrophophococcus fermenticelle: 40 to 50 percent; terrispobacter petrilerius: 5 to 10 percent; clostridium scoriogenes: 5 to 10 percent; clostridium fermenticelle: 15 to 20 percent; clostridium sporogenes:15 to 20 percent.
The invention also provides a preparation method of the pit mud culture strengthening microbial inoculum, which comprises the following steps:
A. inoculating Novitrophorococcus fermenticella, clostridium fermenticella, terrisporabacter petri, clostridium scoriogenes and Clostridium sporogenes into intensified liquid culture medium, culturing until the bacteria grow to logarithmic phase, OD 600 The value is 0.8-1.2, five kinds of bacteria liquid are obtained;
B. compounding the five bacterial liquids obtained in the step A according to the relative abundance of the microorganisms to obtain a composite bacterial liquid;
C. inoculating the compound bacterial liquid into an enhanced liquid culture medium for continuous culture, and obtaining the pit mud culture enhanced microbial inoculum after the growth state of the compound bacteria is stable.
In the preparation method of the pit mud culture strengthening microbial inoculum, the formula of the strengthening liquid culture medium is as follows: caCl 2 ·2H 2 O 0.05～0.07g/L，NH 4 Cl 0.40～0.60g/L，MgSO 4 ·7H 2 O 0.40～0.60g/L，K 2 HPO 4 ·3H 2 O0.50～0.70g/L，NaCl 0.40～0.60g/L，FeSO 4 ·7H 2 0.001-0.002 g/L of O, 0.80-1.00 g/L of tryptone, 15-20 mL/L of yellow water, 1mL/L of microelement mother liquor, 20.0-25.0 g/L of sodium acetate, 20.0-25.0 g/L of glucose and water as a solvent; the formula of the microelement mother solution is MnSO 4 ·H 2 O 1.0g/L，CoCl 2 ·6H 2 O 0.2g/L，ZnSO 4 ·7H 2 O0.2mg/L，CuCl 2 ·2H 2 O 20mg/L，NiCl 2 ·6H 2 O 20mg/L，Na 2 MoO 4 ·2H 2 O 20mg/L，Na 2 SeO 4 20mg/L，Na 2 WO 4 20mg/L and the solvent is water.
In the step A, the culture conditions are as follows: culturing at 36-38 deg.C under anaerobic condition with mixed gas volume ratio of H 2 ：CO 2 ：N 2 ＝10％：10～20％：70～80％。
In the preparation method of the pit mud culture strengthening microbial inoculum, in the step C, during inoculation, the volume ratio of the compound bacterial liquid to the strengthening liquid culture medium is 1-1.5: 10.
in the preparation method of the pit mud culture strengthening microbial inoculum, in the step C, the conditions for continuously culturing are as follows: continuously passaging for 6-8 times at 36-38 ℃ under anaerobic condition, wherein the volume ratio of mixed gas is H 2 ：CO 2 ：N 2 ＝10％：10～20％：70～80％。
The invention also provides a method for biologically curing pit mud by using the pit mud culture strengthening microbial inoculum, which comprises the following steps:
a. inoculating the pit mud culture strengthening microbial inoculum into a strengthening liquid culture medium, and carrying out amplification culture to obtain a strengthening microbial inoculum seed solution;
b. and mixing the enhanced microbial inoculum seed liquid with the new pit mud, sealing and putting into a pit pool for culturing to obtain the new pit mud cultured by the enhanced microbial inoculum.
In the step a, during inoculation, the volume ratio of the pit mud culture strengthening microbial inoculum to the strengthening liquid culture medium is 15-20%.
In the step a, the condition of amplification culture is as follows: the temperature is controlled to be 36-38 ℃, the anaerobic environment is maintained by high-purity nitrogen with the flow rate of 0.8-1.0L/min, and the culture time is 3-5 days.
In the step b, the mass ratio of the enhanced microbial inoculum seed liquid to the new pit mud is 1-1.5: 10.
in the step b, the sealed culture conditions are as follows: culturing at 29-31 deg.c for 55-65 days.
The invention also provides application of the pit mud culture strengthening bacterium agent in brewing of white spirit, fermentation production of short-chain fatty acid, aging and maintenance of pit mud.
In the invention, novisinterveccus fermentum and Clostridium fermentum can be separated from aged pit mud, and can also be directly used as preserved products. When the pit mud is obtained by separation, the operation is generally as follows: the mass volume ratio of aged pit mud to intensified liquid culture medium used for continuous production of strong aromatic white spirit is 1-1.5 g: and (3) carrying out microbial enrichment by 50mL, wherein the conditions of microbial enrichment are as follows: the time is 15 to 20 days, anaerobic culture is carried out, the temperature is 37 ℃, and the volume ratio of mixed gas is H 2 ：CO 2 ：N 2 =10%: 10-20%: 70-80%, and then separating to obtain Novisyntrophophococcus fermenticelae and Clostridium fermenticelae.
The invention has the beneficial effects that:
the invention utilizes the bacteria Novisynophococci permmenticelle screened from the pit mud and the microorganisms Terrisporabacter petroleucus, clostridium sclerotiogenes, clostridium permmenticelle and Clostridium spongiones to establish a co-culture system, regulates and controls the abundance of each bacterium, and obviously improves the content of short-chain fatty acid and fatty acid ethyl ester. According to the invention, after the co-culture system is prepared into the pit mud culture strengthening microbial inoculum, the microbial inoculum can be used for biological maintenance of pit mud, the pit mud quality is obviously improved compared with new pit mud, the generation of flavor substances such as short-chain fatty acid, fatty acid ethyl ester and the like is further obviously promoted, and the method has an important significance for improving the white spirit quality.
Detailed Description
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention.
In the examples, the formulation of the enhanced liquid medium used was: caCl 2 ·2H 2 O 0.05g，NH 4 Cl 0.40g，MgSO 4 ·7H 2 O 0.40g，K 2 HPO 4 ·3H 2 O 0.50g，NaCl 0.40g，FeSO 4 ·7H 2 0.002g of O, 1.00g of tryptone, 15mL of yellow water, 1mL of microelement mother liquor, 20.0g of sodium acetate, 20.0g of glucose and the balance of water; wherein the formula (1L) of the microelement mother liquor is MnSO 4 ·H 2 O 1.0g，CoCl 2 ·6H 2 O 0.2g，ZnSO 4 ·7H 2 O 0.2mg，CuCl 2 ·2H 2 O20mg，NiCl 2 ·6H 2 O 20mg，Na 2 MoO 4 ·2H 2 O 20mg，Na 2 SeO 4 20mg，Na 2 WO 4 20mg, and the balance being water.
In the examples, novisterophorococcus fermenticelle, accession number CICC No.24502; clostridium fermenticelle, the preservation number is CGMCC No.17037; terrisporater petrolerius, accession number JCMNo.19845; clostridium scoriogenes with accession number DSM No.757; clostridium sporogenes, deposited under DSM No.795.
Example 1: research on microbial species in pit mud culture strengthening microbial inoculum
1.1, according to 1:1. Mixing Novitrophorococcus fermenticelle with Clostridium fermenticelle, terrisporabacter petrilerolerus, clostridium scoriogens and Clostridium sporogenes respectively according to a ratio of 1:10 (V/V) are respectively inoculated into an intensified liquid culture medium for pairwise co-culture under the culture condition of anaerobic culture at 37 ℃, and the volume ratio of mixed gas is H 2 ：CO 2 ：N 2 =1:1: and 8,96h later, measuring the content of short-chain fatty acid in the bacterial liquid.
The results show that co-culture of N.fermenticelle and C.fermenticelle, C.sponogenes improves the caproic acid yield of C.fermenticelle (4.56 g/L to 6.21 g/L) and C.sponogenes (6.64 g/L to 7.96 g/L); co-cultivation of N.fermenticelle with T.petrolerius, C.scoriogenes increased butyric acid production by T.petrolerius (1.33 g/L to 2.56/L) and C.scoriogenes (1.47 g/L to 2.65 g/L).
1.2, based on the characteristic that butyric acid can be used as a substrate for synthesizing caproic acid in a fatty acid metabolic pathway, mixing the five bacteria according to the proportion of 50% of N.fermenticelle and 12.5% of the rest four clostridiales bacteria respectively according to the proportion of 1:10 (V/V) inoculating into reinforced liquid culture medium for co-culture at 37 deg.C under anaerobic culture with mixed gas volume ratio of H 2 ：CO 2 ：N 2 =1:1: and 8,96h later, determining the content of butyric acid and caproic acid in the bacterial liquid.
1.3, comparing the short-chain fatty acid result of the co-culture of the five bacteria with the acid yield of the two-two co-culture and the single-bacteria culture, wherein the results are shown in the table 1, and the caproic acid content produced by the co-culture system of the five bacteria is obviously higher than the caproic acid yield of the two-bacteria co-culture and the single-bacteria culture. The evidence proves that under the conditions that acetic acid is provided by N.fermenticella and butyric acid is provided by T.petriarius and C.scorotogens, the caproic acid producing bacteria C.fermenticella and C.sponogenes in the system can obviously improve the yield of caproic acid, and is beneficial to biological maintenance of pit mud after the subsequent microbial inoculum is put into use.
TABLE 1 content of butyric acid and caproic acid in the bacterial solution after co-cultivation (unit: g/L bacterial solution)
Example 2: pit mud culture strengthening microbial inoculum strain ratio exploration
2.1, carrying out microorganism enrichment on the pit mud by using an intensified liquid culture medium. Inoculating 10g of pit mud of a 30-year pit-age pit of the strong aromatic Chinese spirits into 500mL of enhanced liquid culture medium, and carrying out enrichment culture for 15d in an anaerobic environment at 37 ℃, wherein the proportion of anaerobic mixed gas is H 2 ：CO 2 ：N 2 =1:1:8; novisterophorococcus fermenticelle and Clostridium fermenticelle are separated from pit mud and stored at-80 ℃.
2.2, respectively NoviSYNtrophococcus fermenticelle, clostridium fermenticelle, terrisporabacter petroleurus, clostridium scoriogenes and Clostridium sponogenes stored in a-80 ℃ refrigerator according to 1:10 (V/V) inoculating to reinforced liquid culture medium, activating, culturing at 37 deg.C under anaerobic condition, and mixing with mixed gas at volume ratio of H 2 ：CO 2 ：N 2 =1:1:8, growth of the microorganism in the culture Medium (OD) 600 ) Monitoring was performed until the bacteria in the medium grew to log phase, OD 600 Value of>1.0。
2.3, according to noviostrophophococcus cementiella: 60 percent; clostridium fermenticelle: 10 percent; clostridium sporogenes:10 percent; terrispobacter petrilerius: 10 percent; clostridium scoriogenes: mixing five bacterial solutions according to the relative abundance ratio of 10%, and then mixing the five bacterial solutions according to the ratio of 1:10 (V/V) inoculating the mixed bacteria liquid into an intensified liquid culture medium for anaerobic continuous culture at 37 ℃, wherein the volume ratio of anaerobic mixed gas is H 2 ：CO 2 ：N 2 =1:1:8, continuously carrying out passage for 6 times to obtain the stable compound microbial inoculum.
2.4, carrying out amplification culture on the composite microbial inoculum in an anaerobic fermentation tank. The intensified liquid culture medium is put into a 3L anaerobic fermentation tank, and the composite microbial inoculum is inoculated into the fermentation tank according to the proportion of 15% (V/V). The fermentation temperature is controlled at 37 ℃, the anaerobic environment is maintained by high-purity nitrogen with the flow of 0.8L/min, and the culture time is 3d, so that the seed solution of the compound microbial inoculum is obtained.
2.5, mixing the obtained seed liquid with new cellar mud prepared by a winery according to the weight ratio of 1:10, sealing the mixture in a cellar, and culturing the mixture for 60 days at the temperature of 30 ℃ to obtain the new cellar mud cultured by the composite microbial inoculum.
2.6, detecting the caproic acid content of the new cellar mud after the intensive culture of the composite microbial inoculum, and finding that the caproic acid content of the new cellar mud cultured according to equal proportion is 6.88g/kg (dry weight of the cellar mud), which is improved compared with the new cellar mud which is not cultured, but the improvement range is limited.
Example 3: construction and application of pit mud culture strengthening microbial inoculum
3.1, respectively, novisprophorococcus fermenticela, clostridium fermenticela, terrispora petrileroearius, clostridium scoriogenes and Clostridium spongiogenes stored in-80 ℃ refrigerator, according to 1:10 (V/V) inoculating to reinforced liquid culture medium, activating, culturing at 37 deg.C under anaerobic condition, and mixing with mixed gas at H ratio 2 ：CO 2 ：N 2 =1:1:8, growth of the microorganism in the culture Medium (OD) 600 ) Monitoring is performed until the bacteria in the medium grow to log phase, OD 600 Value of>1.0。
3.2 according to noviostrophophococcus cementiella: 40 percent; clostridium fermenticelle: 20 percent; clostridium sporogenes:20 percent; terrispobacter petrilerius: 5 percent; clostridium scoriogenes: five bacterial solutions were mixed at a relative abundance ratio of 5%, and then mixed according to a ratio of 1:10 (V/V) inoculating the mixed bacteria liquid into an intensified liquid culture medium for anaerobic continuous culture at 37 ℃, wherein the volume ratio of anaerobic mixed gas is H 2 ：CO 2 ：N 2 =1:1:8, continuously carrying out passage for 6 times to obtain the pit mud culture strengthening microbial inoculum.
3.3, carrying out amplification culture on the enhanced microbial inoculum in an anaerobic fermentation tank. The enhanced liquid culture medium is put into a 3L anaerobic fermentation tank, and the enhanced microbial inoculum is inoculated into the fermentation tank according to the proportion of 15 percent (V/V). The fermentation temperature is controlled at 37 ℃, the anaerobic environment is maintained by high-purity nitrogen with the flow of 0.8L/min, and the culture time is 3d, so that the enhanced microbial inoculum seed liquid is obtained.
3.4, mixing the obtained seed liquid with the new cellar mud prepared by a winery according to the weight ratio of 1:10, sealing the mixture in a cellar, and culturing the mixture for 60 days at the temperature of 30 ℃ to obtain the new cellar mud cultured by the enhanced microbial inoculum. Adding water into the control group of new cellar mud until the water content is similar to that of the enhanced culture new cellar mud, and then performing sealed culture at 30 ℃ for 60 days in the same cellar.
3.5, for the new cellar mud after the intensified culture (E) 1 Group) and control group new pit mud (W) 1 Panel) the results of the bacterial microbial community structure measurements and the four major acids measurements are shown in table 2. The relative abundance of caproic acid producing bacteria (caproliproducens) microorganisms in the new pit mud cultured with the enhanced microbial inoculum was increased from 15.4% to 20.2% compared to the new pit mud without the enhancement; the relative abundance of Clostridium (Clostridium) microorganisms increased from 2.5% to 16.3%; the content of lactic acid and acetic acid is reduced compared with that of unreinforced new pit mud; the content of the caproic acid is obviously increased, which shows that the enhanced microbial inoculum has good effects on the aging of the pit mud and the biological maintenance of the pit mud.
Example 4: construction and application of pit mud culture strengthening microbial inoculum
4.1, respectively, novisprophorococcus fermenticela, clostridium fermenticela, terrispora petrileroearius, clostridium scoriogenes and Clostridium spongiogenes stored in-80 ℃ refrigerator according to 1.2:10 (V/V) inoculating to reinforced liquid culture medium, activating, culturing at 36 deg.C under anaerobic condition, and mixing with mixed gas at a ratio of H 2 ：CO 2 ：N 2 =1:1.5:7.5 growth of the individual bacteria in the culture Medium (OD) during the cultivation 600 ) Monitoring was performed until the bacteria in the medium grew to log phase, OD 600 Value of>0.8。
4.2, according to noviostrophophococcus cementiella: 45 percent; clostridium fermenticelle: 17.5 percent; clostridium sporogenes:17.5 percent; terrispobacter petrilerius: 5 percent; clostridium pathogens: five bacterial solutions were mixed at a relative abundance ratio of 5%, and then mixed according to a ratio of 1.2:10 (V/V) is inoculated into an intensified liquid culture medium for anaerobic continuous culture at 37 ℃, and the proportion of mixed gas is H 2 ：CO 2 ：N 2 =1:1.5:7.5, continuously carrying out passage for 7 times to obtain the pit mud culture strengthening microbial inoculum.
4.3, carrying out amplification culture on the enhanced microbial inoculum in an anaerobic fermentation tank. The intensified liquid culture medium is put into a 3L anaerobic fermentation tank, and the intensified microbial inoculum is inoculated into the fermentation tank according to the proportion of 18% (V/V). The fermentation temperature is controlled to be 36 ℃, the anaerobic environment is maintained by high-purity nitrogen with the flow rate of 0.9L/min, and the culture time is 4d, so that the enhanced microbial inoculum seed liquid is obtained.
4.4, mixing the obtained seed liquid with new cellar mud prepared by a winery according to the weight ratio of 1.2:10, sealing the mixture in a cellar, and culturing the mixture for 55d at 31 ℃ to obtain the new cellar mud cultured by the enhanced microbial inoculum. Adding water into the control group of new cellar mud until the water content is similar to that of the enhanced culture new cellar mud, and then performing sealed culture at 31 ℃ for 55d in the cellar.
4.5, for the new cellar mud after the intensified culture (E) 2 Group) and control group new pit mud (W) 2 Panel) the results of the bacterial microbial community structure measurements and the four major acids measurements are shown in table 2. The results show that the relative abundance of caproic acid producing bacteria (caponicproducens) microorganisms in the new pit mud cultured with the enhanced microbial inoculum was increased from 15.2% to 18.7% compared to the new pit mud without the enhancement; the relative abundance of Clostridium (Clostridium) microorganisms increased from 2.5% to 17.2%; the content of lactic acid and acetic acid is reduced compared with that of unreinforced new pit mud; the content of the caproic acid is obviously increased, which shows that the enhanced microbial inoculum has good effects on the aging of the pit mud and the biological maintenance of the pit mud.
Example 5: construction and application of pit mud culture strengthening microbial inoculum
Respectively taking novistrophococcus fermenticela, clostridium fermenticella, terrispobacter pellerarius, clostridium scorotrogegens and Clostridium spongiogens stored in a refrigerator at the temperature of-80 ℃, and carrying out the following steps of 1.5:10 (V/V) inoculating to intensified liquid culture medium, activating, culturing at 38 deg.C under anaerobic condition, and anaerobic mixingIn a ratio of H 2 ：CO 2 ：N 2 =1:2:7, growth of the microorganism in the culture Medium (OD) 600 ) Monitoring was performed until the bacteria in the medium grew to log phase, OD 600 Value of>1.1。
5.2 according to Novisterophorococcus fermenticelle: 50 percent; clostridium fermenticelle: 15 percent; clostridium sporogenes:15 percent; terrispobacter petrilerius: 10 percent; clostridium scoriogenes: five bacterial solutions were mixed at a relative abundance ratio of 10%, and then mixed according to a ratio of 1.5:10 (V/V) is inoculated into an intensified liquid culture medium for anaerobic continuous culture at 38 ℃, and the proportion of anaerobic mixed gas is H 2 ：CO 2 ：N 2 =1:2:7, continuously carrying out passage for 8 times to obtain the pit mud culture strengthening microbial inoculum.
And 5.3, carrying out amplification culture on the enhanced microbial inoculum in an anaerobic fermentation tank. The enhanced liquid culture medium is put into a 3L anaerobic fermentation tank, and the enhanced microbial inoculum is inoculated into the fermentation tank according to the proportion of 20% (V/V). The fermentation temperature is controlled at 38 ℃, the anaerobic environment is maintained by high-purity nitrogen with the flow of 1.0L/min, and the culture time is 5d, so as to obtain the enhanced microbial inoculum seed liquid.
5.4, mixing the obtained seed liquid with new cellar mud prepared by a winery according to the weight ratio of 1.5:10, sealing the mixture in a cellar, and culturing the mixture at 29 ℃ for 65 days to obtain the new cellar mud cultured by the enhanced microbial inoculum. Adding water into the control group of new cellar mud until the water content is similar to that of the enhanced culture new cellar mud, and then performing sealed culture at 29 ℃ for 65d.
5.5, for the new cellar mud after the intensified culture (E) 3 Group) and control group new pit mud (W) 3 Panel) the results of the bacterial microbial community structure measurements and the four major acids measurements are shown in table 2. The results show that the relative abundance of caproic acid producing bacteria (Capriciduronucens) microorganisms in the new pit mud cultured with the enhanced microbial inoculum is increased from 16.8% to 22.4% compared with the new pit mud without the enhancement; the relative abundance of Clostridium (Clostridium) microorganisms increased from 3.9% to 15.8%; the content of lactic acid and acetic acid is reduced compared with that of unreinforced new pit mud; the content of the caproic acid is obviously increased, which indicates that the enhanced microbial inoculum has the effects of aging the pit mud and biologically curing the pit mudHas good effect.
TABLE 2 comparison of four major acid contents after inoculating pit mud with pit mud culture enhancing agent (unit: g/kg dry weight of pit mud)
| Index (I) | W 1 Group of | E 1 Group of | W 2 Group of | E 2 Group of | W 3 Group of | E 3 Group of |
| Lactic acid | 8.62 | 7.45 | 8.01 | 7.32 | 8.29 | 7.02 |
| Acetic Acid (AA) | 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 |
| Hexanoic acid | 5.69 | 8.61 | 5.48 | 7.34 | 5.30 | 7.58 |
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. The pit mud culture strengthening microbial inoculum is characterized in that: the microbial inoculum is prepared from the following microorganisms as raw materials:
novisyntropophococcus fermenticelle of the genus Novitrophophococci having a accession number of CICC No.24502; clostridium prefermenticelle of Clostridium, with a collection number of CGMCC No.17037; terrispobacter petrilerolearius of the genus terrispobacter, deposited under accession number JCM No.19845; clostridium scoriogens with the accession number DSM No.757; clostridium sporogenes of Clostridium genus, deposited under DSM No.795.
2. The pit mud culture enhanced bacterial agent according to claim 1, characterized in that: the relative abundance of the microorganisms is: novisyntropicoccus fermenticelle: 40 to 50 percent; terrispobacter petrilerius: 5 to 10 percent; clostridium scoriogenes: 5 to 10 percent; clostridium fermenticelle: 15 to 20 percent; clostridium sporogenes:15 to 20 percent.
3. The preparation method of the pit mud culture strengthening microbial inoculum, which is characterized by comprising the following steps: the method comprises the following steps:
A. inoculating Novitrophorococcus fermenticella, clostridium fermenticella, terrisporabacter petri, clostridium scoriogenes and Clostridium sporogenes into intensified liquid culture medium, culturing until the bacteria grow to logarithmic phase, OD 600 The value is 0.8-1.2, five kinds of bacteria liquid are obtained;
B. compounding the five bacterial liquids obtained in the step A according to the relative abundance of the microorganisms in the claim 2 to obtain a compound bacterial liquid;
C. and inoculating the compound bacterial liquid into an enhanced liquid culture medium for continuous culture, and obtaining the pit mud culture enhanced bacterial agent after the growth state of the compound bacteria is stable.
4. The method for preparing the pit mud culture strengthening microbial inoculum according to claim 3, which is characterized in that: the formula of the intensified liquid culture medium is as follows: caCl 2 ·2H 2 O 0.05～0.07g/L，NH 4 Cl 0.40～0.60g/L，MgSO 4 ·7H 2 O 0.40～0.60g/L，K 2 HPO 4 ·3H 2 O 0.50～0.70g/L，NaCl 0.40～0.60g/L，FeSO 4 ·7H 2 0.001-0.002 g/L of O, 0.80-1.00 g/L of tryptone, 15-20 mL/L of yellow water, 1mL/L of microelement mother liquor, 20.0-25.0 g/L of sodium acetate, 20.0-25.0 g/L of glucose and water as a solvent; the formula of the microelement mother liquor is MnSO 4 ·H 2 O 1.0g/L，CoCl 2 ·6H 2 O0.2g/L，ZnSO 4 ·7H 2 O 0.2mg/L，CuCl 2 ·2H 2 O 20mg/L，NiCl 2 ·6H 2 O 20mg/L，Na 2 MoO 4 ·2H 2 O20mg/L，Na 2 SeO 4 20mg/L，Na 2 WO 4 20mg/L and the solvent is water.
5. The method for preparing the pit mud culture strengthening microbial inoculum according to claim 3, which is characterized in that: in the step A, the culture conditions are as follows: culturing at 36-38 deg.C under anaerobic condition with mixed gas volume ratio of H 2 ：CO 2 ：N 2 ＝10％：10～20％：70～80％。
6. The method for preparing the pit mud culture enhancing microbial inoculum according to any one of claims 3 to 5, which is characterized in that: in step C, at least one of the following is satisfied:
during inoculation, the volume ratio of the composite bacterial liquid to the reinforced liquid culture medium is 1-1.5: 10;
the conditions for continuing the culture were: continuously passaging for 6-8 times at 36-38 ℃ under anaerobic condition, wherein the volume ratio of mixed gas is H 2 ：CO 2 ：N 2 ＝10％：10～20％：70～80％。
7. The method for using the pit mud culture enhancing microbial inoculum according to claim 1 or 2 or the pit mud culture enhancing microbial inoculum prepared by the method according to any one of claims 3 to 8 for biological maintenance of pit mud, which is characterized by comprising the following steps: the method comprises the following steps:
a. inoculating the pit mud culture strengthening bacterium agent into a strengthening liquid culture medium, and carrying out amplification culture to obtain a strengthening bacterium agent seed solution;
b. and mixing the enhanced microbial inoculum seed liquid with the new cellar mud, sealing and culturing in a cellar pool to obtain the new cellar mud cultured by the enhanced microbial inoculum.
8. The method of claim 7, wherein: in step a, at least one of the following is satisfied:
during inoculation, the volume ratio of pit mud culture strengthening bacterium agent to strengthening liquid culture medium is 15-20%;
the conditions of the amplification culture are as follows: the temperature is controlled to be 36-38 ℃, the anaerobic environment is maintained by high-purity nitrogen with the flow rate of 0.8-1.0L/min, and the culture time is 3-5 days.
9. The method of claim 7, wherein: in step b, at least one of the following is satisfied:
the mass ratio of the enhanced microbial inoculum seed liquid to the new pit mud is 1-1.5: 10;
the conditions of sealed culture are as follows: culturing at 29-31 deg.c for 55-65 days.
10. The use of the pit mud culture enhancing microbial inoculum according to claim 1 or 2 or the pit mud culture enhancing microbial inoculum prepared by the method according to any one of claims 3 to 8 in brewing white spirit, fermentation production of short-chain fatty acids, aging and maintenance of pit mud.
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