CN114107111A - Fermentation method of clostridium butyricum, microecological preparation and application thereof - Google Patents
Fermentation method of clostridium butyricum, microecological preparation and application thereof Download PDFInfo
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- CN114107111A CN114107111A CN202111435364.9A CN202111435364A CN114107111A CN 114107111 A CN114107111 A CN 114107111A CN 202111435364 A CN202111435364 A CN 202111435364A CN 114107111 A CN114107111 A CN 114107111A
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- clostridium butyricum
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- 241000193171 Clostridium butyricum Species 0.000 title claims abstract description 189
- 238000000855 fermentation Methods 0.000 title claims abstract description 132
- 230000004151 fermentation Effects 0.000 title claims abstract description 132
- 238000000034 method Methods 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 78
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims abstract description 78
- 239000007788 liquid Substances 0.000 claims abstract description 76
- 239000001963 growth medium Substances 0.000 claims abstract description 53
- 238000001694 spray drying Methods 0.000 claims abstract description 16
- 238000010438 heat treatment Methods 0.000 claims abstract description 8
- 230000001580 bacterial effect Effects 0.000 claims description 54
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 38
- 239000000843 powder Substances 0.000 claims description 37
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 19
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 claims description 18
- 238000011081 inoculation Methods 0.000 claims description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 238000005507 spraying Methods 0.000 claims description 13
- 238000012807 shake-flask culturing Methods 0.000 claims description 12
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- 239000004455 soybean meal Substances 0.000 claims description 10
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 9
- VLSOAXRVHARBEQ-UHFFFAOYSA-N [4-fluoro-2-(hydroxymethyl)phenyl]methanol Chemical compound OCC1=CC=C(F)C=C1CO VLSOAXRVHARBEQ-UHFFFAOYSA-N 0.000 claims description 9
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- 239000001888 Peptone Substances 0.000 claims description 8
- 108010080698 Peptones Proteins 0.000 claims description 8
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 8
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- 229910052760 oxygen Inorganic materials 0.000 abstract description 12
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- 230000000813 microbial effect Effects 0.000 abstract description 8
- 239000002068 microbial inoculum Substances 0.000 abstract description 8
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- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical group CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 2
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- 241000233866 Fungi Species 0.000 description 1
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- 229960003022 amoxicillin Drugs 0.000 description 1
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- 239000003613 bile acid Substances 0.000 description 1
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- PFRMQESZQDENEB-UHFFFAOYSA-L calcium;pyridine-2,3-dicarboxylate Chemical compound [Ca+2].[O-]C(=O)C1=CC=CN=C1C([O-])=O PFRMQESZQDENEB-UHFFFAOYSA-L 0.000 description 1
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- CYDMQBQPVICBEU-UHFFFAOYSA-N chlorotetracycline Natural products C1=CC(Cl)=C2C(O)(C)C3CC4C(N(C)C)C(O)=C(C(N)=O)C(=O)C4(O)C(O)=C3C(=O)C2=C1O CYDMQBQPVICBEU-UHFFFAOYSA-N 0.000 description 1
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- CYDMQBQPVICBEU-XRNKAMNCSA-N chlortetracycline Chemical compound C1=CC(Cl)=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O CYDMQBQPVICBEU-XRNKAMNCSA-N 0.000 description 1
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- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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Abstract
The invention discloses a fermentation method of clostridium butyricum as well as a microecological preparation and application thereof, belonging to the field of microbial fermentation and specifically comprising the following steps: firstly, clostridium butyricum is cultured to form dominant flora, then saccharomyces cerevisiae seed liquid is added through feeding, saccharomyces cerevisiae is inhibited or killed by heating when the clostridium butyricum starts to form spores after fermentation, so that the clostridium butyricum is promoted to form the spores, and then the solid powdery microbial inoculum is prepared through spray drying. Wherein, the high temperature in the spray drying process can inactivate the saccharomyces cerevisiae remained in the zymogen liquid. According to the method, the saccharomyces cerevisiae and the clostridium butyricum are fermented cooperatively, oxygen in the culture medium is consumed by aerobic respiration of the saccharomyces cerevisiae, the oxidation-reduction potential of the culture medium is reduced, the growth of the clostridium butyricum can be effectively promoted, and meanwhile, the utilization efficiency of the clostridium butyricum to raw materials can be promoted by extracellular enzyme generated during fermentation of the saccharomyces cerevisiae. It can be made into tolerant microbial inoculum and applied in feed to improve digestion and absorption capacity and immunity of animals.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a fermentation method of clostridium butyricum, a microecological preparation and application thereof.
Background
The clostridium butyricum is also called clostridium butyricum, is an obligate anaerobic gram-positive bacterium, can generate spores, is expanded into a spindle shape in the middle of the bacterium when the spores are generated, and is named clostridium butyricum because the main metabolite of the clostridium butyricum is butyric acid. Because the bacillus anaerobicus is anaerobic bacillus, the bacillus anaerobicus has stronger tolerance to gastric acid, bile acid and various feed antibiotics, and has wide application range.
The clostridium butyricum is used as an obligate anaerobic bacterium and is particularly sensitive to dissolved oxygen in a culture medium, the growth and the propagation of the clostridium butyricum can be greatly influenced by the dissolved oxygen and the oxidation-reduction potential, and at present, most manufacturers for producing the clostridium butyricum use complex equipment and process conditions to achieve an anaerobic environment, so that the current domestic clostridium butyricum products are low in yield, high in price and difficult to popularize. How to rapidly ferment and obtain clostridium butyricum by adopting a simpler method is always pursued in the field.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a fermentation method of clostridium butyricum as well as a microecological preparation and application thereof.
The invention is realized by the following steps:
the invention provides a fermentation method of clostridium butyricum, which comprises the following steps: adding yeast into a culture medium when clostridium butyricum is fermented and cultured to a logarithmic phase, heating to inhibit or kill the yeast when the clostridium butyricum starts to form spores so as to promote the clostridium butyricum to form the spores, and finishing fermentation when the spore rate of the clostridium butyricum is more than or equal to 96 percent to obtain clostridium butyricum liquid.
The invention also provides a microecological preparation containing the clostridium butyricum bacterial liquid or clostridium butyricum bacterial powder.
The invention also provides application of the clostridium butyricum microbial agent in preparation of probiotic microbial agents, feeds and feed additives.
The invention has the following beneficial effects:
the invention provides a fermentation method of clostridium butyricum, a microecological preparation and application thereof. The fermentation method of the clostridium butyricum comprises the following steps: firstly, clostridium butyricum is cultured to form dominant flora, then saccharomyces cerevisiae seed liquid is added through feeding, the saccharomyces cerevisiae seed liquid is gradually heated to inhibit or kill saccharomycetes to promote the clostridium butyricum to form spores when the clostridium butyricum starts to form the spores, and fermentation is finished when the spore rate of the clostridium butyricum is more than or equal to 96 percent, so that clostridium butyricum liquid is obtained. The invention provides a fermentation method of clostridium butyricum, which comprises the following steps: the fermentation process has the advantages that the saccharomyces cerevisiae and the clostridium butyricum are cooperatively fermented, oxygen in the culture medium is consumed by aerobic respiration of the saccharomyces cerevisiae, the oxidation-reduction potential of the culture medium is reduced, the growth of the clostridium butyricum can be effectively promoted, meanwhile, extracellular enzyme generated during fermentation of the saccharomyces cerevisiae can also promote the utilization efficiency of the clostridium butyricum to raw materials, the fermentation process is simple, the requirement on equipment is low, the popularization is easy, and the problems of complex fermentation process, low yield and the like of the clostridium butyricum are solved. After the clostridium butyricum bacterial liquid or clostridium butyricum bacterial powder is used as a microbial agent and added into livestock and poultry feed, the livestock and poultry feed is fed with the clostridium butyricum bacterial liquid or clostridium butyricum bacterial powder, the health condition of cultured animals can be effectively improved, the digestion and absorption capacity and the immunity capacity of the animals are improved, and the use of antibiotics is reduced.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The invention aims to provide a fermentation method of clostridium butyricum, which overcomes the defect that expensive and complicated anaerobic equipment is required for fermentation of the clostridium butyricum at present, skillfully utilizes saccharomyces cerevisiae, performs synergistic fermentation on the saccharomyces cerevisiae and consumes oxygen in a culture medium by utilizing aerobic respiration of the saccharomyces cerevisiae, reduces the oxidation-reduction potential of the culture medium, can effectively promote the growth of the clostridium butyricum, and simultaneously can promote the utilization efficiency of the clostridium butyricum to raw materials by using extracellular enzyme generated during fermentation of the saccharomyces cerevisiae.
To achieve the above object, the present invention is realized by:
in a first aspect, an embodiment of the present invention provides a fermentation method of clostridium butyricum, including the following steps:
1. preparation of seed liquid
And inoculating the clostridium butyricum and saccharomyces cerevisiae strains preserved on the inclined plane into a shake flask culture medium, and culturing for 24-30 hours to obtain a seed solution.
Wherein the shake flask culture condition of clostridium butyricum is 36-38 ℃, and the culture medium is RCM liquid culture medium;
wherein the shake flask culture condition of Saccharomyces cerevisiae is 36-38 deg.C, rotation speed of 200r/min, and culture medium is YPD broth;
2. clostridium butyricum propagation culture
Inoculating the clostridium butyricum seed liquid obtained in the step one into a fermentation tank, wherein the inoculation volume ratio is 0.8-1%, performing thallus propagation, and culturing for 4-6 hours to obtain clostridium butyricum fermentation liquid.
Wherein, the culture medium in the fermentation tank comprises: 8-10g/L of glucose, 6-8g/L of yeast powder, 12-16g/L of peptone, 10-12g/L of bran, 8-10g/L of soybean meal, 2-3g/L of sodium acetate, 5-6g/L of calcium carbonate, 1.8-2g/L of sodium sulfite, 0.4-0.5g/L of L-cysteine hydrochloride and 4-5g/L of sodium alginate.
Wherein the technological parameters in the fermentation process are as follows: regulating pH to 6.5 or higher with 10% sodium hydroxide solution, controlling temperature at 36-38 deg.C, rotating speed at 40-50rpm, and keeping aeration.
According to the fermentation method of clostridium butyricum provided by the embodiment of the invention, in the expanding culture process of clostridium butyricum, a culture medium for culturing clostridium butyricum is improved:
wherein, calcium carbonate and bran are used as solid matters and can be used as attachments of clostridium butyricum to promote growth and form spores, and simultaneously, the calcium carbonate is an important raw material for synthesizing an important component of calcium pyridine dicarboxylate in the spore structure of the clostridium butyricum;
wherein sodium sulfite and L-cysteine hydrochloride are used as reducing agents, so that the oxidation-reduction potential of the culture medium can be reduced, and the growth of clostridium butyricum serving as obligate anaerobe is promoted;
the sodium alginate is a natural thickening agent, and can reduce the bottom dissolved oxygen to a certain extent by increasing the viscosity of the culture medium, so that the growth of clostridium butyricum in a culture substrate layer is promoted.
3. Adding Saccharomyces cerevisiae seed solution
And (3) inoculating the saccharomyces cerevisiae seed liquid obtained in the step one into the clostridium butyricum fermentation liquid obtained in the step two, wherein the inoculation time is 0.8-1% of the inoculation volume ratio after the clostridium butyricum propagation for 4-5 hours, and the fermentation is continued for 12-14 hours after the inoculation.
Wherein the technological parameters in the fermentation process are the same as those in the second step.
Clostridium butyricum is an obligate anaerobe and is particularly sensitive to dissolved oxygen in a culture medium, and conventional fermentation equipment cannot reduce the dissolved oxygen by injecting nitrogen and other methods, so that the fermentation effect of the Clostridium butyricum is poor. The fermentation method of the clostridium butyricum provided by the embodiment of the invention aims to realize that the clostridium butyricum is rapidly fermented by a simpler method, overcomes the defect that the traditional fermentation method is carried out by using complicated anaerobic equipment, skillfully utilizes saccharomyces cerevisiae, carries out synergistic fermentation on the saccharomyces cerevisiae and the clostridium butyricum, consumes oxygen in a culture medium by utilizing aerobic respiration of the saccharomyces cerevisiae, reduces the oxidation-reduction potential of the culture medium, can effectively promote the growth of the clostridium butyricum, and simultaneously can promote the utilization efficiency of the clostridium butyricum to raw materials by using extracellular enzyme generated during fermentation of the saccharomyces cerevisiae. As facultative anaerobe, the saccharomyces cerevisiae can grow and move under the condition of low dissolved oxygen, and the effect can not be lost due to death caused by too low dissolved oxygen. The time for adding the saccharomyces cerevisiae is critical, and the clostridium butyricum is already formed into a dominant flora after the clostridium butyricum is subjected to propagation for 4-5 hours, so that the condition that the clostridium butyricum production is excessively influenced due to the large propagation of the saccharomyces cerevisiae is prevented.
4. Warming of the culture Medium
Increasing the temperature by 5 ℃ every half hour, continuing fermenting for 6-8 hours after the temperature of the culture medium is increased to 51-53 ℃, performing microscopic examination on the fermentation liquor, and completing fermentation when the spore rate reaches 96-98% to obtain the clostridium butyricum bacterial liquid.
The clostridium butyricum is used as bacillus, has stronger stress resistance after forming spores, gradually adjusts the temperature to 51-53 ℃ in a mode of gradually increasing the temperature when the clostridium butyricum is about to produce spores, and promotes the clostridium butyricum to produce spores by gradually deteriorating the environment on the one hand; on the other hand, due to the fact that metabolic stagnation is caused when the clostridium butyricum forms spores, the antagonistic capability is reduced, the temperature rise can inhibit or even kill saccharomyces cerevisiae, and the situation that the growth of the saccharomyces cerevisiae is greatly expanded due to the reduction of the antagonistic capability when the clostridium butyricum produces the spores is prevented, so that the formation of the spores of the clostridium butyricum is influenced.
The temperature rise adopts a gradual temperature rise mode because the clostridium butyricum has low stress resistance when spores are not formed yet, and the clostridium butyricum is prevented from dying before the spores are not formed yet due to the fact that the temperature rise is too fast.
5. Spray drying
Mixing the fermented clostridium butyricum bacterial liquid and light calcium carbonate according to a mass ratio of 20:1, and after the bacterial liquid and the protective agent are completely mixed, spraying the mixture in a spraying tower, wherein the air inlet temperature is set to be 170-180 ℃, and the air outlet temperature is controlled to be 80-85 ℃ by adjusting the feeding frequency.
The air inlet temperature and the air outlet temperature are respectively set to be 170-175 ℃ and 80-85 ℃ during spray drying, and only light calcium carbonate is added as a carrier during spray drying, mainly for killing yeast residual in fermentation liquor, ensuring the purity of bacterial powder, meanwhile, the clostridium butyricum after sporulation has strong stress resistance and does not die in spray drying.
In a second aspect, the embodiments of the present invention further provide a microecological preparation comprising a clostridium butyricum bacterial liquid or a clostridium butyricum bacterial powder.
In a third aspect, the embodiment of the invention also provides an application of the clostridium butyricum microbial agent in preparation of probiotic microbial agents, feeds and feed additives.
Preferably, the number of living bacteria in the piglet diet is not less than 1.0 multiplied by 108The addition amount of cfu/g of fermentation product is 10-15 kg/t.
Preferably, the microecological preparation is used for improving the production performance of weaned piglets, reducing the diarrhea rate and reducing the feed conversion ratio.
Preferably, the probiotic is used for increasing the daily gain and daily feed intake of piglets.
Preferably, the probiotics are used for improving the survival rate of piglets and reducing the diarrhea rate.
The features and properties of the present invention are described in further detail below with reference to examples.
In the following examples of the present invention, the raw material sources, components, preparation and experimental methods were the same as those of the comparative examples.
1. Preparation of seed liquid
And inoculating the clostridium butyricum and saccharomyces cerevisiae strains preserved on the inclined plane into a shake flask culture medium, and culturing for 24-30 hours to obtain a seed solution.
Wherein the shake flask culture condition of clostridium butyricum is 36-38 ℃, and the culture medium is RCM liquid culture medium;
wherein the shake flask culture condition of Saccharomyces cerevisiae is 36-38 deg.C, rotation speed of 200r/min, and the culture medium is YPD broth.
2. Clostridium butyricum propagation culture
Inoculating the clostridium butyricum seed liquid obtained in the step one into a fermentation tank, wherein the inoculation volume ratio is 0.8-1%, performing thallus propagation, and culturing for 4-6 hours to obtain clostridium butyricum fermentation liquid.
Wherein, the culture medium in the fermentation tank comprises: 8-10g/L of glucose, 6-8g/L of yeast powder, 12-16g/L of peptone, 10-12g/L of bran, 8-10g/L of soybean meal, 2-3g/L of sodium acetate, 5-6g/L of calcium carbonate, 1.8-2g/L of sodium sulfite, 0.4-0.5g/L of L-cysteine hydrochloride and 4-5g/L of sodium alginate.
Wherein the technological parameters in the fermentation process are as follows: regulating pH to 6.5 or higher with 10% sodium hydroxide solution, controlling temperature at 36-38 deg.C, rotating speed at 40-50rpm, and keeping aeration.
3. Adding Saccharomyces cerevisiae seed solution
And (3) inoculating the saccharomyces cerevisiae seed liquid obtained in the step one into the clostridium butyricum fermentation liquid obtained in the step two, wherein the inoculation volume ratio is 0.8-1%, and continuing to ferment for 12-14 hours.
Wherein the technological parameters in the fermentation process are the same as those in the second step.
4. Warming of the culture Medium
Increasing the temperature by 5 ℃ every half hour, continuing fermenting for 6-8 hours after the temperature of the culture medium is increased to 51-53 ℃, performing microscopic examination on the fermentation liquor, and completing fermentation when the spore rate reaches 96-98% to obtain the clostridium butyricum bacterial liquid.
5. Spray drying
Mixing the fermented clostridium butyricum bacterial liquid and light calcium carbonate according to a mass ratio of 20:1, and after the bacterial liquid and the protective agent are completely mixed, spraying the mixture in a spraying tower, wherein the air inlet temperature is set to be 170-180 ℃, and the air outlet temperature is controlled to be 80-85 ℃ by adjusting the feeding frequency.
Example 1
1. Preparation of seed liquid
And inoculating the clostridium butyricum and saccharomyces cerevisiae strains preserved on the inclined plane into a shake flask culture medium, and culturing for 24-30 hours to obtain a seed solution.
Wherein the shake flask culture condition of clostridium butyricum is 36-38 ℃, and the culture medium is RCM liquid culture medium;
wherein the shake flask culture condition of Saccharomyces cerevisiae is 36-38 deg.C, rotation speed of 200r/min, and the culture medium is YPD broth.
2. Clostridium butyricum propagation culture
Inoculating the clostridium butyricum seed liquid obtained in the step one into a fermentation tank, wherein the inoculation volume ratio is 0.8-1%, performing thallus propagation, and fermenting for 4 hours to obtain clostridium butyricum fermentation liquid.
Wherein, the culture medium in the fermentation tank comprises: 10g/L of glucose, 6g/L of yeast powder, 12g/L of peptone, 12g/L of bran, 10g/L of soybean meal, 2g/L of sodium acetate, 5g/L of calcium carbonate, 2g/L of sodium sulfite, 0.5g/L of L-cysteine hydrochloride and 5g/L of sodium alginate.
Wherein the technological parameters in the fermentation process are as follows: regulating pH to 6.5 or higher with 10% sodium hydroxide solution, controlling temperature at 36-38 deg.C, rotating speed at 40-50rpm, and keeping aeration.
3. Adding Saccharomyces cerevisiae seed solution
And (3) inoculating the saccharomyces cerevisiae seed liquid obtained in the step one into the clostridium butyricum fermentation liquid obtained in the step two, wherein the inoculation volume ratio is 1%, and continuing to ferment for 12 hours.
Wherein the technological parameters in the fermentation process are the same as those in the second step.
4. Warming of the culture Medium
Increasing the temperature by 5 ℃ every half hour, continuing fermenting for 6-8 hours after the temperature of the culture medium is increased to 51-53 ℃, performing microscopic examination on the fermentation liquor, and completing fermentation when the spore rate reaches 96-98% to obtain the clostridium butyricum bacterial liquid.
5. Spray drying
Mixing the fermented clostridium butyricum bacterial liquid and light calcium carbonate according to a mass ratio of 20:1, and after the bacterial liquid and the protective agent are completely mixed, spraying the mixture in a spraying tower, wherein the air inlet temperature is set to be 170-180 ℃, and the air outlet temperature is controlled to be 80-85 ℃ by adjusting the feeding frequency.
Example 2
The present embodiment is the same as embodiment 1 in other step parameters, and the difference is only that:
in the second step, the culture medium in the fermentation tank consists of: 8g/L of glucose, 8g/L of yeast powder, 16g/L of peptone, 10g/L of bran, 8g/L of soybean meal, 3g/L of sodium acetate, 6g/L of calcium carbonate, 1.8g/L of sodium sulfite, 0.4g/L of L-cysteine hydrochloride and 4g/L of sodium alginate.
Example 3
The present embodiment is the same as embodiment 1 in other step parameters, and the difference is only that:
the fermentation time in the second step is 6 hours.
Example 4
The present embodiment is the same as embodiment 1 in other step parameters, and the difference is only that:
the inoculation volume ratio of the seed liquid of the brewing mother fungus in the third step is 0.8 percent.
Example 5
The present embodiment is the same as embodiment 1 in other step parameters, and the difference is only that:
the fermentation was continued for 14 hours in step three.
Comparative example 1
This comparative example is identical to example 1 in other step parameters, except that:
in the second step, the culture medium in the fermentation tank consists of: 10g/L of glucose, 6g/L of yeast powder, 12g/L of peptone, 10g/L of soybean meal, 2g/L of sodium acetate, 2g/L of sodium sulfite, 0.5g/L of L-cysteine hydrochloride and 5g/L of sodium alginate.
Comparative example 2
This comparative example is identical to example 1 in other step parameters, except that:
in the second step, the culture medium in the fermentation tank consists of: 10g/L of glucose, 6g/L of yeast powder, 12g/L of peptone, 12g/L of bran, 10g/L of soybean meal, 2g/L of sodium acetate, 5g/L of calcium carbonate and 5g/L of sodium alginate.
Comparative example 3
This comparative example is identical to example 1 in other step parameters, except that:
in the second step, the culture medium in the fermentation tank consists of: 10g/L of glucose, 6g/L of yeast powder, 12g/L of peptone, 12g/L of bran, 10g/L of soybean meal, 2g/L of sodium acetate, 5g/L of calcium carbonate, 2g/L of sodium sulfite and 0.5g/L of L-cysteine hydrochloride.
Comparative example 4
The comparative example is identical to example 1 in other step parameters, except that step three is:
and (4) continuing fermentation for 12 hours without inoculating the saccharomyces cerevisiae seed liquid.
Wherein the technological parameters in the fermentation process are the same as those in the second step.
Comparative example 5
The comparative example is identical to example 1 in other step parameters, except that step three is:
and (3) inoculating the saccharomyces cerevisiae seed liquid obtained in the step one into the clostridium butyricum fermentation liquid obtained in the step two, wherein the inoculation volume ratio is 0.4%, and continuing to ferment for 12 hours.
Wherein the technological parameters in the fermentation process are the same as those in the second step.
Comparative example 6
The comparative example is identical to example 1 in other step parameters, except that step three is:
and (3) inoculating the saccharomyces cerevisiae seed liquid obtained in the step one into the clostridium butyricum fermentation liquid obtained in the step two, wherein the inoculation volume ratio is 2%, and continuing to ferment for 12 hours.
Wherein the technological parameters in the fermentation process are the same as those in the second step.
Comparative example 7
This comparative example is identical to example 1 in other step parameters, except that:
the fermentation time in the second step was 2 hours.
Comparative example 8
This comparative example is identical to example 1 in other step parameters, except that:
the fermentation time in the second step is 8 hours.
Comparative example 9
This comparative example is identical to example 1 in other step parameters, except that step four is:
and (3) continuously fermenting for 6-8 hours without heating the culture medium, performing microscopic examination on the fermentation liquor, and finishing fermentation when the spore rate reaches 96-98% to obtain the clostridium butyricum bacterial liquid.
Comparative example 10
This comparative example is identical to example 1 in other step parameters, except that:
and continuing fermenting for 10 hours in the third step.
Comparative example 11
This comparative example is identical to example 1 in other step parameters, except that:
the fermentation is continued for 16 hours in step three.
Comparative example 12
This comparative example is identical to example 1 in other step parameters, except that:
in step four, the temperature is increased by 10 ℃ every half hour.
Comparative example 13
This comparative example is identical to example 1 in other step parameters, except that:
in the fifth step, the air inlet temperature is set to be 180-185 ℃, and the air outlet temperature is set to be 97-92 ℃.
Experimental example 1
The amount of clostridium butyricum viable bacteria in the bacteria powder after the spraying is finished is detected by a plate dilution coating counting method, and the result is shown in table 1.
TABLE 1 viable cell count after spray drying in each example
As can be seen from the results in Table 1, the fermentation results were not greatly changed in the ranges of the recipes and the process variations as in examples 2 to 5.
As can be seen from the results of comparative examples 1 to 3 in Table 1, the amount of fungal powder is reduced when calcium carbonate, bran, sodium sulfite, L-cysteine hydrochloride, sodium alginate, etc. are not added to the fermentation medium.
From the results of comparative examples 4 to 5, it can be seen that the amount of the strain powder is greatly reduced when the saccharomyces cerevisiae seed solution is not added at the initial fermentation stage of clostridium butyricum or the amount of the saccharomyces cerevisiae seed solution is added is too small.
From the results of comparative examples 6-7, it can be seen that the addition of too much seed liquid of Saccharomyces cerevisiae or the addition of the seed liquid of Saccharomyces cerevisiae at a earlier time can result in the decrease of the amount of fungal powder, because Clostridium butyricum fails to form dominant bacterial flora compared with Saccharomyces cerevisiae, resulting in the extensive propagation of Saccharomyces cerevisiae and the consumption of more nutrients.
It can be seen from the results of comparative example 8 that delaying the addition of the saccharomyces cerevisiae seed solution also results in a decrease in the amount of fungal powder.
From the results of comparative examples 9-10, it can be seen that the absence of temperature rise or the delay of the start of temperature rise at the late stage of fermentation also leads to a large reduction in the bacterial powder bacterial load, because the large scale propagation of yeast in the spore formation process of clostridium butyricum affects the spore formation process of clostridium butyricum, and further leads to a low bacterial powder bacterial load.
From the results of comparative example 11, it can be seen that the temperature rise time is advanced, and the bacterial powder quantity is also reduced to some extent, because the clostridium butyricum does not use up the nutrients in the culture medium, and the temperature rise is advanced to the spore formation process.
From the results of comparative example 12, it can be seen that the culture medium is heated at an excessively high temperature, resulting in a decrease in the amount of fungal powder, because the spore formation rate of part of the Clostridium butyricum falls behind the temperature increase rate, and the activity is decreased in a high temperature state, resulting in the inability to form spores.
From the results of comparative example 13, it can be seen that the excessively high temperature of the inlet and outlet air of the spray drying leads to the reduction of the bacterial quantity of the clostridium butyricum bacterial powder, because part of the clostridium butyricum spores are incompletely formed or the heat resistance of the spores is poor, and the clostridium butyricum bacterial powder dies at an excessively high temperature, and in addition, the excessively low temperature of the inlet and outlet air of the spray drying leads to the high survival rate of the saccharomyces cerevisiae, and the high rate of mixed bacteria in the bacterial powder.
In conclusion, the invention greatly improves the effect of fermenting clostridium butyricum on common fermentation equipment and promotes production activities by improving the fermentation medium and the culture process.
Preparation of clostridium butyricum bacterial agent
Conventional fermentation group
Selecting clostridium butyricum bacterial liquid expanded and cultured in a fermentation tank, mixing the expanded and cultured bacterial liquid with light calcium carbonate according to the mass ratio of 20:1, after the bacterial liquid and the calcium carbonate are completely mixed, spraying in a spraying tower, diluting the clostridium butyricum bacterial powder prepared by spraying to the bacterial content of 1.0 multiplied by 10 by using a mixture of zeolite powder and broken bean pulp8And (3) CFU/g, uniformly mixing, subpackaging and sealing to obtain the clostridium butyricum microbial inoculum of the conventional fermentation group.
Wherein the fermentation medium is RCM medium.
Wherein the fermentation process parameters are as follows: the inoculation amount of the seed liquid is 0.3%, the temperature is controlled to be 36-38 ℃, the rotating speed is 50rpm, aeration is not needed, the pH value of the culture medium is regulated and controlled to be not lower than 6.5 by 10% of sodium hydroxide solution in the fermentation process, and the culture time is 24-28 hours.
Wherein, in the spraying process, the air inlet temperature is set to be 170-180 ℃, and the air outlet temperature is controlled to be 80-85 ℃ by adjusting the feeding frequency.
Wherein the mixture of zeolite powder and broken bean pulp comprises the following components: 50% of zeolite powder and 50% of broken bean pulp.
Experimental fermentation group
The Clostridium butyricum powder obtained in example 1 was diluted with a mixture of zeolite powder and crushed soybean meal to a bacterial count of 1.0X 108And (5) CFU/g, uniformly mixing, subpackaging and sealing to obtain the clostridium butyricum microbial inoculum of the experimental fermentation group.
Wherein the mixture of zeolite powder and crushed soybean meal is the same as that of conventional fermentation group.
Experiment of feeding
Mixing 0.2% of aureomycin and 0.025% of amoxicillin with conventional feed, and using as antibiotic group; mixing 1% of a clostridium butyricum bacterial agent of a conventional fermentation group with a conventional feed and using the mixture as a conventional bacterial agent group; the clostridium butyricum microbial inoculum of experimental fermentation groups is mixed with conventional feed according to 0.5 percent, 1 percent and 2 percent and then used as an experimental microbial inoculum group.
Feeding 30 weaned piglets according to the above 3 groups and blank group for 30 days.
Wherein, the diarrhea rate (%) is the number of diarrhea piglets in the test period/(number of test piglets multiplied by test days) × 100%;
wherein, the number of the diarrhea piglets in the experimental period is 1 day of diarrhea piglets plus 2 days of diarrhea piglets plus … … days of 30 diarrhea piglets.
TABLE 2 piglet production performance test results of each treatment group
As can be seen from table 2, compared with the blank group, the daily gain and daily feed intake of the conventional clostridium butyricum fermented inoculum group are improved, the feed conversion ratio, diarrhea rate and death rate are reduced, the effect is close to that of the antibiotic group and is slightly better than that of the antibiotic group, the daily gain and daily feed intake of the experimental clostridium butyricum fermented inoculum group are significantly higher than those of the antibiotic group and the conventional clostridium butyricum fermented inoculum group, and the feed conversion ratio and the diarrhea rate are also lower than those of the antibiotic group and the conventional clostridium butyricum fermented inoculum group. Meanwhile, in the experimental clostridium butyricum bacterial agent group, the effect of the addition amount of 0.5% is obviously inferior to that of 1% and 2%, and when the addition amount is increased from 1% to 2%, all indexes are not obviously changed, so that the addition amount of the clostridium butyricum bacterial agent is preferably 1% -2%.
The fermentation method of the clostridium butyricum provided by the embodiment of the invention greatly improves the effect of fermenting the clostridium butyricum on common fermentation equipment by improving the fermentation medium and the culture process, has low requirement on the equipment and is more beneficial to popularization, and meanwhile, the clostridium butyricum microbial inoculum prepared by the invention can obviously improve the growth speed and reduce the feed conversion ratio in the process of feeding piglets, can effectively improve the diarrhea and death conditions of the piglets and greatly promotes the related production activities.
To sum up, the embodiment of the present invention provides a fermentation method of clostridium butyricum, which specifically comprises: firstly, clostridium butyricum is cultured to form dominant flora, then saccharomyces cerevisiae seed liquid is added through feeding, saccharomyces cerevisiae is inhibited or killed by heating when clostridium butyricum begins to form spores after fermentation, clostridium butyricum is promoted to form spores, and then solid powdery microbial inoculum is prepared through spray drying. Wherein, the high temperature in the spray drying process can inactivate the saccharomyces cerevisiae remained in the zymogen liquid. Compared with the prior art that complicated anaerobic equipment is required for fermentation, the saccharomyces cerevisiae and clostridium butyricum are used for synergistic fermentation, oxygen in the culture medium is consumed by utilizing aerobic respiration of the saccharomyces cerevisiae, the redox potential of the culture medium is reduced, growth of clostridium butyricum can be effectively promoted, and meanwhile extracellular enzyme generated during fermentation of the saccharomyces cerevisiae can also promote utilization efficiency of clostridium butyricum to raw materials. The method has the advantages of simple fermentation process, low requirement on equipment, easy popularization and capability of solving the problems of complex fermentation process, low yield and the like of the clostridium butyricum. After the clostridium butyricum bacterial liquid or clostridium butyricum bacterial powder is added into livestock and poultry feed as a microbial agent, the livestock and poultry feed is fed with the clostridium butyricum bacterial liquid or clostridium butyricum bacterial powder, the health condition of cultured animals can be effectively improved, the digestion and absorption capacity and the immunity of the animals are improved, the daily gain and the daily feeding are increased, the feed conversion ratio, the diarrhea rate and the death rate are reduced, and the use of antibiotics is reduced.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A fermentation method of Clostridium butyricum, which comprises: adding yeast into a culture medium when clostridium butyricum is fermented and cultured to a logarithmic phase, heating to inhibit or kill the yeast when the clostridium butyricum starts to form spores so as to promote the clostridium butyricum to form the spores, and finishing fermentation when the spore rate of the clostridium butyricum is more than or equal to 96 percent to obtain clostridium butyricum liquid.
2. Fermentation process according to claim 1, characterized in that it comprises the following steps: inoculating the clostridium butyricum seed liquid into a fermentation tank for thallus propagation until a clostridium butyricum dominant flora is formed, obtaining clostridium butyricum fermentation liquid, then adding saccharomyces cerevisiae seed liquid through feeding, heating to inhibit or kill saccharomyces cerevisiae when the clostridium butyricum starts to form spores so as to promote the clostridium butyricum to form the spores, and finishing fermentation when the spore rate of the clostridium butyricum reaches 96-98%, thus obtaining the clostridium butyricum liquid.
3. The fermentation process of claim 2, wherein inoculating the clostridium butyricum fermentation broth with the saccharomyces cerevisiae seed solution comprises: and performing propagation culture on the clostridium butyricum seed liquid for 4-6h to form clostridium butyricum fermentation liquid, then inoculating the saccharomyces cerevisiae seed liquid according to the inoculation ratio of 0.8-1%, continuing fermenting for 12-14h until the clostridium butyricum begins to form spores, and preparing for heating.
4. The fermentation method according to claim 3, wherein the fermentation process of inoculating the seed solution of Saccharomyces cerevisiae to the Clostridium butyricum fermentation solution for fermentation comprises: regulating pH to be not less than 6.5, controlling temperature to be 36-38 deg.C, rotating speed to be 40-50rpm, and keeping aeration;
preferably, the culture medium for inoculating the saccharomyces cerevisiae seed liquid in the clostridium butyricum fermentation liquid is heated up in a gradual heating mode to inhibit or kill saccharomyces cerevisiae so as to promote clostridium butyricum to form spores;
more preferably, after the temperature of the culture medium is increased to 51-53 ℃ at a rate of increasing 5 ℃ every half hour, the fermentation is continued for 6-8 hours, and when the spore rate of clostridium butyricum reaches 96-98%, the clostridium butyricum is mature, and clostridium butyricum bacterial liquid is obtained.
5. The fermentation process of claim 4, further comprising: killing the residual yeast in the clostridium butyricum bacterial liquid through spray drying to obtain clostridium butyricum bacterial powder;
preferably, the fermented clostridium butyricum bacterial liquid and the light calcium carbonate are uniformly mixed according to the mass ratio of 20:1 and then are subjected to spray drying;
more preferably, the air inlet temperature of the spray drying equipment is 170-180 ℃, and the air outlet temperature is 80-85 ℃.
6. The fermentation process of claim 2, wherein the preparation of the clostridium butyricum fermentation broth comprises the steps of: inoculating the clostridium butyricum seed liquid into a fermentation tank, wherein the inoculation volume ratio is 0.8-1%, performing thallus propagation, and culturing for 4-6 hours to obtain clostridium butyricum fermentation liquid;
preferably, the composition of the medium in the clostridium butyricum fermenter is: 8-10g/L of glucose, 6-8g/L of yeast powder, 12-16g/L of peptone, 10-12g/L of bran, 8-10g/L of soybean meal, 2-3g/L of sodium acetate, 5-6g/L of calcium carbonate, 1.8-2g/L of sodium sulfite, 0.4-0.5g/L of L-cysteine hydrochloride and 4-5g/L of sodium alginate;
preferably, the fermentation process of the fermentation in the clostridium butyricum fermenter is: regulating pH to 6.5 or higher with 10% sodium hydroxide solution, controlling temperature at 36-38 deg.C, rotating speed at 40-50rpm, and keeping aeration.
7. The fermentation process of claim 2, wherein the preparation of the seed liquid comprises: inoculating clostridium butyricum and saccharomyces cerevisiae strains preserved on the inclined plane into a shake flask culture medium, and culturing for 24-30 hours to obtain seed liquid;
preferably, the shake flask culture condition of the clostridium butyricum is 36-38 ℃, and the culture medium is RCM liquid culture medium;
preferably, the shake flask culture condition of Saccharomyces cerevisiae is 36-38 deg.C, rotation speed of 200r/min, and the culture medium is YPD broth.
8. The fermentation process according to any one of claims 1 to 7, wherein the fermentation process of Clostridium butyricum comprises the steps of:
inoculating the clostridium butyricum seed liquid into a fermentation tank, wherein the inoculation volume ratio is 0.8-1%, performing thallus propagation, and culturing for 4-6 hours to obtain clostridium butyricum fermentation liquid;
then inoculating the saccharomyces cerevisiae seed liquid into the clostridium butyricum fermentation liquid, wherein the inoculation volume ratio is 0.8-1%, and continuing to ferment for 12-14 hours;
raising the temperature of the culture medium to 51-53 ℃ at a rate of raising the temperature by 5 ℃ every half hour, continuing fermenting for 6-8 hours, examining the fermentation liquor under a microscope, and completing fermentation when the spore rate reaches 96-98% to obtain clostridium butyricum bacterial liquid;
and mixing the fermented clostridium butyricum bacterial liquid and light calcium carbonate according to a mass ratio of 20:1, and after the bacterial liquid and the light calcium carbonate are completely mixed, spraying in a spraying tower, wherein the air inlet temperature is 170-180 ℃, the air outlet temperature is 80-85 ℃, and clostridium butyricum bacterial powder is obtained.
9. A microecological preparation comprising the Clostridium butyricum bacterial liquid of claim 1 or the Clostridium butyricum bacterial powder of claim 5.
10. Use of the probiotic according to claim 9 for the preparation of probiotic preparations, feed and feed additives.
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Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR100839874B1 (en) * | 2006-07-07 | 2008-06-19 | 자연과생명기술(주) | Powder-type fermeent material manufacturing method |
| CN101947295A (en) * | 2010-10-14 | 2011-01-19 | 上海创博食品技术发展有限公司 | Production method of microbial antioxidant |
| CN102319270B (en) * | 2011-09-26 | 2012-10-17 | 湖北绿雪生物产业有限公司 | Method for preparing high-density viable butyric acid bacterium preparation by adopting fed-batch fermentation method |
| CN110804564B (en) * | 2019-11-18 | 2020-10-09 | 江苏三仪生物工程有限公司 | Preparation of clostridium butyricum powder and detection culture medium thereof |
| CN111066947A (en) * | 2019-12-30 | 2020-04-28 | 江苏兴鼎生物工程有限公司 | Production method of clostridium butyricum feed additive |
| CN111034868A (en) * | 2019-12-31 | 2020-04-21 | 福建傲农生物科技集团股份有限公司 | Compound bacterium fermented feed and preparation method and application thereof |
| US20230201276A1 (en) * | 2020-05-21 | 2023-06-29 | Superbrewed Food, Inc. | Method of treating or preventing an infection |
| CN112481351B (en) * | 2020-12-16 | 2024-03-08 | 湖北华扬科技发展有限公司 | Counting method of clostridium butyricum spores in composite microecological product |
| CN116035146A (en) * | 2023-01-20 | 2023-05-02 | 江苏沃纳生物科技有限公司 | Production method of mulberry beverage with gastrointestinal tract health care function |
| CN116144544A (en) * | 2023-01-31 | 2023-05-23 | 福建益昕葆生物制药有限公司 | Clostridium butyricum pure microbial inoculum and preparation method thereof |
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| CN114672446A (en) * | 2022-05-31 | 2022-06-28 | 广东海洋大学 | Preparation method and application of clostridium butyricum preparation |
| CN116035146A (en) * | 2023-01-20 | 2023-05-02 | 江苏沃纳生物科技有限公司 | Production method of mulberry beverage with gastrointestinal tract health care function |
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