CN114107111B - 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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- CN114107111B CN114107111B CN202111435364.9A CN202111435364A CN114107111B CN 114107111 B CN114107111 B CN 114107111B CN 202111435364 A CN202111435364 A CN 202111435364A CN 114107111 B CN114107111 B CN 114107111B
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- 241000193171 Clostridium butyricum Species 0.000 title claims abstract description 186
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/60—Feeding-stuffs specially adapted for particular animals for weanlings
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/18—Baker's yeast; Brewer's yeast
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
Abstract
The invention discloses a clostridium butyricum fermentation method, a microecological preparation and application thereof, belonging to the field of microbial fermentation, and specifically comprising the following steps: culturing clostridium butyricum to form dominant bacterial colony, adding saccharomyces cerevisiae seed liquid through feed supplement, inhibiting or killing saccharomyces cerevisiae by heating when clostridium butyricum starts to form spores after fermentation, promoting clostridium butyricum to form spores, and preparing the solid powdery microbial inoculum through spray drying. Wherein, the high temperature in the spray drying process can inactivate the residual saccharomyces cerevisiae in the zymotic bacteria liquid. According to the invention, the oxygen in the culture medium is consumed by the aerobic respiration of the saccharomyces cerevisiae and the clostridium butyricum through the synergistic fermentation, so that the oxidation-reduction potential of the culture medium is reduced, the growth of the clostridium butyricum can be effectively promoted, and meanwhile, the extracellular enzyme generated during the fermentation of the saccharomyces cerevisiae can also promote the utilization efficiency of the clostridium butyricum on the raw materials. It can be made into tolerating bacteria and applied in feed to improve animal digestion and absorption ability and immunity.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a clostridium butyricum fermentation method, a microecological preparation and application thereof.
Background
Clostridium butyricum, also known as clostridium butyricum, is an obligate anaerobic gram-positive bacterium that produces spores, i.e., when spores are produced, the middle of the thallus expands to form a shuttle shape, and because the main metabolite is butyric acid, the bacterium is called clostridium butyricum. Because the bacillus anaerobis, the bacillus anaerobis has stronger tolerance to gastric acid, bile acid and various feed antibiotics, and has wide application range.
Clostridium butyricum is used as an obligate anaerobic bacterium, is particularly sensitive to dissolved oxygen in a culture medium, and the growth and propagation of clostridium butyricum can be greatly influenced by the dissolved oxygen and the oxidation-reduction potential, so that the existing manufacturers for producing clostridium butyricum mostly use complex equipment and process conditions to achieve an anaerobic environment, and the existing domestic clostridium butyricum products have low yield, high price and difficult popularization. It has been sought in the art how to obtain clostridium butyricum by rapid fermentation using a simpler process.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a clostridium butyricum fermentation method, a microecological preparation and application thereof.
The invention is realized in the following way:
the invention provides a fermentation method of clostridium butyricum, which comprises the following steps: adding saccharomycetes into a culture medium when clostridium butyricum is fermented and cultured to a logarithmic phase, heating to inhibit or kill saccharomycetes when clostridium butyricum starts to form spores so as to promote clostridium butyricum to form spores, and completing fermentation when the spore ratio of clostridium butyricum is more than or equal to 96%, so as to obtain clostridium butyricum bacterial liquid.
The invention also provides a microecological preparation containing clostridium butyricum bacterial liquid or clostridium butyricum bacterial powder.
The invention also provides application of the clostridium butyricum microbial agent in preparation of probiotics microbial agents, feeds and feed additives.
The invention has the following beneficial effects:
the invention provides a clostridium butyricum fermentation method, a microecological preparation and application thereof. The provided fermentation method of clostridium butyricum comprises the following steps: culturing clostridium butyricum to form dominant bacterial colony, adding saccharomyces cerevisiae seed liquid through feed supplement, inhibiting or killing saccharomycetes through gradual heating when clostridium butyricum starts to form spores after fermentation, and completing fermentation when the spore rate of clostridium butyricum is more than or equal to 96%, so as to obtain clostridium butyricum liquid. The invention provides a fermentation method of clostridium butyricum, which comprises the following steps: the oxygen in the culture medium is consumed by the aerobic respiration of the saccharomyces cerevisiae and the clostridium butyricum through the synergistic fermentation, the oxidation-reduction potential of the culture medium is reduced, the clostridium butyricum can be effectively promoted to grow, meanwhile, extracellular enzyme generated during the fermentation of the saccharomyces cerevisiae can also promote the utilization efficiency of the clostridium butyricum on raw materials, the fermentation process is simple, the requirements on equipment are low, the popularization is easy, and the problems of complex fermentation process, low yield and the like of the clostridium butyricum are solved. After clostridium butyricum liquid or clostridium butyricum powder is used as microbial agent to be added into livestock and poultry feed, the livestock and poultry feed is fed to animals, so that the health condition of the cultured animals can be effectively improved, the digestion and absorption capacity and the immunity 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 more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The invention aims to provide a clostridium butyricum fermentation method, which overcomes the defect that expensive and complex anaerobic equipment is required for clostridium butyricum fermentation at present, and the method skillfully utilizes saccharomyces cerevisiae to cooperatively ferment, consumes oxygen in a culture medium by aerobic respiration of the saccharomyces cerevisiae, reduces oxidation-reduction potential of the culture medium, can effectively promote clostridium butyricum growth, and simultaneously, extracellular enzyme generated during the saccharomyces cerevisiae fermentation can also promote the utilization efficiency of clostridium butyricum on raw materials.
To achieve the above object, the present invention is achieved by:
in a first aspect, an embodiment of the present invention provides a fermentation method of clostridium butyricum, comprising the steps of:
1. preparation of seed liquid
Inoculating clostridium butyricum and saccharomyces cerevisiae strains stored on the inclined plane into a shake flask culture medium, and culturing for 24-30 hours to obtain seed liquid.
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 36-38deg.C, the rotation speed is 200r/min, and the culture medium is YPD broth;
2. clostridium butyricum 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 expansion culture, and culturing for 4-6 hours to obtain clostridium butyricum fermentation liquid.
Wherein, the culture medium in the fermentation tank comprises the following components: 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: the pH value is regulated to be not lower than 6.5 by 10 percent of sodium hydroxide solution, the temperature is controlled to be 36-38 ℃, the rotating speed is 40-50rpm, and no ventilation is realized.
According to the clostridium butyricum fermentation method provided by the embodiment of the invention, in the clostridium butyricum expansion culture process, 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 meanwhile, the calcium carbonate is an important raw material for synthesizing an important component of pyridine dicarboxylic acid calcium salt in the spore structure of clostridium butyricum;
wherein sodium sulfite and L-cysteine hydrochloride are used as reducing agents, so that the oxidation-reduction potential of a culture medium can be reduced, and the growth of clostridium butyricum serving as obligate anaerobic bacteria can be promoted;
wherein sodium alginate is a natural thickener, and can reduce dissolved oxygen of the bottom layer to a certain extent by increasing the viscosity of the culture medium, so as to promote the growth of clostridium butyricum on the culture substrate layer.
3. Adding Saccharomyces cerevisiae seed solution
Inoculating the Saccharomyces cerevisiae seed liquid obtained in the first step into clostridium butyricum fermentation liquid in the second step, wherein the inoculation time is that clostridium butyricum is subjected to expanded culture for 4-5 hours, the inoculation volume ratio is 0.8-1%, and fermentation is continued for 12-14 hours after inoculation.
Wherein, the technological parameters in the fermentation process are the same as those in the second step.
Clostridium butyricum is an obligate anaerobic bacterium and is particularly sensitive to dissolved oxygen in a culture medium, and conventional fermentation equipment cannot reduce the dissolved oxygen by a nitrogen injection method and the like, so that the clostridium butyricum has poor fermentation effect. According to the clostridium butyricum fermentation method provided by the embodiment of the invention, in order to realize that clostridium butyricum can be obtained by rapid fermentation by a simpler method, the defect that the traditional clostridium butyricum is fermented by using complex anaerobic equipment is overcome, the saccharomyces cerevisiae is skillfully utilized to ferment together with clostridium butyricum, oxygen in a culture medium is consumed by aerobic respiration of the saccharomyces cerevisiae, the oxidation-reduction potential of the culture medium is reduced, the clostridium butyricum growth can be effectively promoted, and meanwhile, extracellular enzymes generated during the saccharomyces cerevisiae fermentation can also promote the utilization efficiency of clostridium butyricum on raw materials. As facultative anaerobes, saccharomyces cerevisiae also can grow and move under the condition of low dissolved oxygen, so that the effect is not lost due to death caused by the excessively low dissolved oxygen. However, the time for adding the saccharomyces cerevisiae is critical, the time for adding the saccharomyces cerevisiae seed liquid is 4-5 hours after the clostridium butyricum is amplified, and the clostridium butyricum has formed dominant bacterial flora at the moment, so that the situation that the production of clostridium butyricum is excessively influenced due to the large-scale amplification of the saccharomyces cerevisiae is prevented.
4. Heating up the culture medium
Raising the temperature every half hour by 5 ℃, continuing fermenting for 6-8 hours after the temperature of the culture medium is raised to 51-53 ℃, and performing microscopic examination on the fermentation liquid, and completing the fermentation when the spore rate reaches 96-98%, thus obtaining clostridium butyricum bacterial liquid.
The clostridium butyricum is used as bacillus, and has stronger stress resistance after spores are formed, and the temperature is gradually adjusted to 51-53 ℃ in a section-by-section temperature rising mode when the clostridium butyricum is about to produce spores, so that the clostridium butyricum is promoted to produce spores by gradually deteriorating the environment on one hand; on the other hand, as clostridium butyricum forms spores in the era Xie Tingzhi, antagonism is reduced, and the temperature rise can inhibit or even kill saccharomyces cerevisiae, so that the phenomenon that the saccharomyces cerevisiae is subjected to mass propagation due to the reduction of antagonism in the process of producing spores by clostridium butyricum is prevented, and then the clostridium butyricum forms spores is influenced.
The temperature rise adopts a gradual temperature rise mode, and the clostridium butyricum has weak stress resistance when spores are not formed, so that the clostridium butyricum is prevented from dying before spores are formed due to too fast temperature rise.
5. Spray drying
Mixing the fermented clostridium butyricum liquid and light calcium carbonate according to the mass ratio of 20:1, and after the clostridium butyricum liquid and the protective agent are completely mixed, spraying in a spraying tower, wherein the air inlet temperature is set to 170-180 ℃, and the air outlet temperature is controlled to 80-85 ℃ by adjusting the feeding frequency.
The air inlet temperature and the air outlet temperature are respectively set to 170 ℃ to 175 ℃ and 80 ℃ to 85 ℃ during spray drying, and only light calcium carbonate is added as a carrier during spray, so that the residual saccharomycetes in the fermentation broth are mainly killed, the purity of the bacterial powder is ensured, and meanwhile, the clostridium butyricum after spores are formed is strong in stress resistance and cannot die during spray drying.
In a second aspect, embodiments of the present invention also provide a probiotic comprising clostridium butyricum bacterial liquid or 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 probiotics microbial agents, feeds and feed additives.
Preferably, the number of viable bacteria in the daily ration of the piglets is not less than 1.0X10 8 The addition amount of the cfu/g fermentation product is 10-15kg/t.
Preferably, the microecological formulation is used to improve productivity, reduce diarrhea rate and reduce feed to meat ratio in weaned pigs.
Preferably, the microecological preparation is used for improving daily gain and daily feed intake of piglets.
Preferably, the microecological preparation is used for improving the survival rate of piglets and reducing the diarrhea rate.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
In the following examples of the present invention, the raw material sources, components, preparation and experimental methods are the same as those of the comparative examples.
1. Preparation of seed liquid
Inoculating clostridium butyricum and saccharomyces cerevisiae strains stored on the inclined plane into a shake flask culture medium, and culturing for 24-30 hours to obtain seed liquid.
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 36-38deg.C, 200r/min rotation speed, and the culture medium is YPD broth.
2. Clostridium butyricum 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 expansion culture, and culturing for 4-6 hours to obtain clostridium butyricum fermentation liquid.
Wherein, the culture medium in the fermentation tank comprises the following components: 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: the pH value is regulated to be not lower than 6.5 by 10 percent of sodium hydroxide solution, the temperature is controlled to be 36-38 ℃, the rotating speed is 40-50rpm, and no ventilation is realized.
3. Adding Saccharomyces cerevisiae seed solution
Inoculating the Saccharomyces cerevisiae seed liquid obtained in the first step into clostridium butyricum fermentation liquid in the second step, wherein the inoculation volume ratio is 0.8-1%, and continuing fermentation for 12-14 hours.
Wherein, the technological parameters in the fermentation process are the same as those in the second step.
4. Heating up the culture medium
Raising the temperature every half hour by 5 ℃, continuing fermenting for 6-8 hours after the temperature of the culture medium is raised to 51-53 ℃, and performing microscopic examination on the fermentation liquid, and completing the fermentation when the spore rate reaches 96-98%, thus obtaining clostridium butyricum bacterial liquid.
5. Spray drying
Mixing the fermented clostridium butyricum liquid and light calcium carbonate according to the mass ratio of 20:1, and after the clostridium butyricum liquid and the protective agent are completely mixed, spraying in a spraying tower, wherein the air inlet temperature is set to 170-180 ℃, and the air outlet temperature is controlled to 80-85 ℃ by adjusting the feeding frequency.
Example 1
1. Preparation of seed liquid
Inoculating clostridium butyricum and saccharomyces cerevisiae strains stored on the inclined plane into a shake flask culture medium, and culturing for 24-30 hours to obtain seed liquid.
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 36-38deg.C, 200r/min rotation speed, and the culture medium is YPD broth.
2. Clostridium butyricum 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 expansion culture, and fermenting for 4 hours to obtain clostridium butyricum fermentation liquid.
Wherein, the culture medium in the fermentation tank comprises the following components: 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: the pH value is regulated to be not lower than 6.5 by 10 percent of sodium hydroxide solution, the temperature is controlled to be 36-38 ℃, the rotating speed is 40-50rpm, and no ventilation is realized.
3. Adding Saccharomyces cerevisiae seed solution
Inoculating the saccharomyces cerevisiae seed liquid obtained in the first step into clostridium butyricum fermentation liquid in the second step, wherein the inoculation volume ratio is 1%, and continuing fermentation for 12 hours.
Wherein, the technological parameters in the fermentation process are the same as those in the second step.
4. Heating up the culture medium
Raising the temperature every half hour by 5 ℃, continuing fermenting for 6-8 hours after the temperature of the culture medium is raised to 51-53 ℃, and performing microscopic examination on the fermentation liquid, and completing the fermentation when the spore rate reaches 96-98%, thus obtaining clostridium butyricum bacterial liquid.
5. Spray drying
Mixing the fermented clostridium butyricum liquid and light calcium carbonate according to the mass ratio of 20:1, and after the clostridium butyricum liquid and the protective agent are completely mixed, spraying in a spraying tower, wherein the air inlet temperature is set to 170-180 ℃, and the air outlet temperature is controlled to 80-85 ℃ by adjusting the feeding frequency.
Example 2
This example is identical to the other step parameters of example 1, except that:
in the second step, the culture medium in the fermentation tank comprises the following components: glucose 8g/L, yeast powder 8g/L, peptone 16g/L, bran 10g/L, soybean meal 8g/L, sodium acetate 3g/L, calcium carbonate 6g/L, sodium sulfite 1.8g/L, L-cysteine hydrochloride 0.4g/L and sodium alginate 4g/L.
Example 3
This example is identical to the other step parameters of example 1, except that:
and in the second step, the fermentation time is 6 hours.
Example 4
This example is identical to the other step parameters of example 1, except that:
in the third step, the seed liquid inoculation volume ratio of the brewing mother bacteria is 0.8 percent.
Example 5
This example is identical to the other step parameters of example 1, except that:
and step three, fermenting for 14 hours continuously.
Comparative example 1
This comparative example is identical to the other step parameters of example 1, except that:
in the second step, the culture medium in the fermentation tank comprises the following components: 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 the other step parameters of example 1, except that:
in the second step, the culture medium in the fermentation tank comprises the following components: 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 the other step parameters of example 1, except that:
in the second step, the culture medium in the fermentation tank comprises the following components: 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 the other step parameters of example 1, except that step three is:
the Saccharomyces cerevisiae seed solution was not inoculated, and fermentation was continued for 12 hours.
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 the other step parameters of example 1, except that step three is:
inoculating the saccharomyces cerevisiae seed liquid obtained in the first step into clostridium butyricum fermentation liquid in the second step, wherein the inoculation volume ratio is 0.4%, and continuing fermentation 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 the other step parameters of example 1, except that step three is:
inoculating the saccharomyces cerevisiae seed liquid obtained in the first step into clostridium butyricum fermentation liquid in the second step, wherein the inoculation volume ratio is 2%, and continuing fermentation 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 the other step parameters of example 1, except that:
in the second step, the fermentation time is 2 hours.
Comparative example 8
This comparative example is identical to the other step parameters of example 1, except that:
in the second step, the fermentation time is 8 hours.
Comparative example 9
The comparative example is identical to the other step parameters of example 1, except that step four is:
and (3) heating the culture medium, continuing fermenting for 6-8 hours, and microscopic examination of the fermentation liquid, and finishing fermentation when the spore rate reaches 96-98%, so as to obtain clostridium butyricum bacterial liquid.
Comparative example 10
This comparative example is identical to the other step parameters of example 1, except that:
and step three, fermenting for 10 hours continuously.
Comparative example 11
This comparative example is identical to the other step parameters of example 1, except that:
and step three, fermenting for 16 hours continuously.
Comparative example 12
This comparative example is identical to the other step parameters of example 1, except that:
in the fourth step, the temperature is increased by 10 ℃ every half hour.
Comparative example 13
This comparative example is identical to the other step parameters of example 1, except that:
in the fifth step, the inlet air temperature is set to 180-185 ℃ and the outlet air temperature is set to 97-92 ℃.
Experimental example 1
The live bacterial load of clostridium butyricum in the bacterial powder after the spraying is detected by a plate dilution coating counting method, and the result is shown in table 1.
TABLE 1 viable count after spray drying for each example
As can be seen from the results in Table 1, the formulation and process variation ranges in examples 2-5 did not change much with respect to the fermentation results.
As can be seen from the results of comparative examples 1 to 3 in Table 1, the bacterial load of the bacterial powder was reduced when no substances such as calcium carbonate, bran, sodium sulfite, L-cysteine hydrochloride, sodium alginate, etc. were added to the fermentation medium.
As can be seen from the results of comparative examples 4 to 5, the addition of the seed liquid of Saccharomyces cerevisiae in the initial stage of fermentation of Clostridium butyricum or the addition of too little seed liquid of Saccharomyces cerevisiae greatly reduces the bacterial load of the bacterial powder.
As can be seen from the results of comparative examples 6 to 7, excessive liquid amount of the seed liquid of the Saccharomyces cerevisiae or early time of adding the seed liquid of the Saccharomyces cerevisiae can lead to the decrease of the bacterial powder amount, because clostridium butyricum cannot form dominant bacterial groups compared with the Saccharomyces cerevisiae, and a large amount of the Saccharomyces cerevisiae is spread and cultivated, so that more nutrition is consumed.
As can be seen from the results of comparative example 8, the delay of the addition of the seed liquid of Saccharomyces cerevisiae also resulted in a decrease in the amount of fungus powder.
As can be seen from the results of comparative examples 9 to 10, the temperature is not raised in the later fermentation stage, or the temperature raising start time is delayed, so that the bacterial powder amount is greatly reduced, and the bacterial powder amount is low because the bacterial powder amount is greatly reduced due to the influence of the large expansion of saccharomycetes in the process of forming spores by clostridium butyricum.
As can be seen from the results of comparative example 11, the temperature rise time is advanced, and the amount of fungus powder and fungus is also reduced to some extent, because clostridium butyricum has not utilized the nutrients in the culture medium, because the temperature rise advances to the spore formation process.
As can be seen from the results of comparative example 12, too fast a temperature rise rate of the medium results in a decrease in the amount of fungus powder, because part of clostridium butyricum forms spores at a later rate than the temperature rise rate, and the activity is decreased at a high temperature, and thus spores cannot be formed.
As can be seen from the results of comparative example 13, too high a temperature of the air inlet and outlet of the spray drying results in a decrease in the amount of clostridium butyricum powder, because part of clostridium butyricum spores are incompletely formed or the heat resistance of spores is poor, so that the clostridium butyricum dies at too high a temperature, and in addition, too low a temperature of the air inlet and outlet of the spray drying results in a higher survival rate of saccharomyces cerevisiae and a high rate of mixed bacteria in the powder.
In conclusion, the invention greatly improves the effect of fermenting clostridium butyricum on common fermentation equipment and promotes the production activity by improving the fermentation medium and the culture process.
Preparation of clostridium butyricum microbial inoculum
Conventional fermentation group
Selecting clostridium butyricum bacterial liquid expanded and cultivated by a fermentation tank, mixing the bacterial liquid expanded and cultivated with light calcium carbonate according to the mass ratio of 20:1, and after the bacterial liquid and the calcium carbonate are completely mixed, spraying the mixture in a spraying tower, wherein clostridium butyricum bacterial powder prepared by spraying is diluted to the bacterial amount of 1.0x10 by using a mixture of zeolite powder and crushed soybean meal 8 CFU/g, mixing uniformly, split charging and sealing to obtain the conventional clostridium butyricum bacterial agent.
Wherein the fermentation medium is RCM medium.
Wherein, the fermentation process parameters are as follows: the seed liquid inoculation amount is 0.3%, the temperature is controlled to be 36-38 ℃, the rotating speed is 50rpm, no ventilation is realized, the pH value of the culture medium is regulated and controlled to be not lower than 6.5 through 10% 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 170 ℃ to 180 ℃, and the air outlet temperature is controlled to 80 ℃ to 85 ℃ by adjusting the feeding frequency.
Wherein the zeolite powder and the crushed soybean meal mixture comprise the following components: 50% of zeolite powder and 50% of crushed soybean meal.
Experimental fermentation group
The clostridium butyricum powder prepared in example 1 was diluted with zeolite powder and crushed soybean meal to a bacterial load of 1.0X10 8 CFU/g, mixing uniformly, split charging and sealing to obtain the clostridium butyricum bacterial agent of the experimental fermentation group.
Wherein the zeolite powder and the crushed soybean meal mixture are the same as the conventional fermentation group.
Feeding experiment
The antibiotic group is prepared by mixing 0.2% of chlortetracycline and 0.025% of amoxicillin with conventional feed; the clostridium butyricum bacterial agent of 1% of conventional fermentation group is mixed with conventional feed and then used as a conventional bacterial agent group; the clostridium butyricum bacterial agent of the experimental fermentation groups of 0.5%, 1% and 2% is mixed with conventional feed and then used as an experimental bacterial agent group.
The 3 groups and blank groups were fed for 30 days for 30 weaned pigs.
Wherein, diarrhea rate (%) = test period diarrhea piglet head time/(test piglet head number x test days) ×100%;
wherein, the experimental period diarrhea piglet head times = the number of diarrhea piglets at day 1 + the number of diarrhea piglets at day 2 + the number of diarrhea piglets at day … ….
Table 2 results of the production performance experiments for piglets in each treatment group
As can be seen from table 2, compared with the blank group, the daily gain and daily intake of the conventional clostridium butyricum agent group are improved, the feed-meat ratio, diarrhea rate and death rate are all reduced, the effect is similar to that of the antibiotic group, and is slightly better than that of the antibiotic group, while the daily gain and daily intake of the experimental clostridium butyricum agent group are obviously higher than those of the antibiotic group and the conventional clostridium butyricum agent group, and the feed-meat ratio and diarrhea rate are also lower than those of the antibiotic group and the conventional clostridium butyricum agent 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%.
According to the clostridium butyricum fermentation method provided by the embodiment of the invention, the effect of clostridium butyricum fermentation on common fermentation equipment is greatly improved by improving the fermentation medium and the culture process, the equipment requirement is low, popularization is facilitated, meanwhile, the clostridium butyricum agent prepared by the method can obviously improve the growth speed, reduce the feed conversion ratio and effectively improve diarrhea and death of piglets, and related production activities are greatly promoted.
In summary, the embodiment of the invention provides a fermentation method of clostridium butyricum, which specifically comprises the following steps: culturing clostridium butyricum to form dominant bacterial colony, adding saccharomyces cerevisiae seed liquid through feed supplement, inhibiting or killing saccharomyces cerevisiae and promoting clostridium butyricum to form spores through heating when clostridium butyricum starts to form spores after fermentation, and preparing the solid powdery microbial inoculum through spray drying. Wherein, the high temperature in the spray drying process can inactivate the residual saccharomyces cerevisiae in the zymotic bacteria liquid. Compared with the prior art that complex anaerobic equipment is required to be used for fermentation, the invention utilizes aerobic respiration of the saccharomyces cerevisiae to consume oxygen in a culture medium by synergistic fermentation of the saccharomyces cerevisiae and the clostridium butyricum, reduces oxidation-reduction potential of the culture medium, can effectively promote clostridium butyricum to grow, and simultaneously, extracellular enzyme generated during the fermentation of the saccharomyces cerevisiae can also promote the utilization efficiency of clostridium butyricum on raw materials. The invention has simple fermentation process, low requirement on equipment, easy popularization and solves the problems of complex fermentation process, low yield and the like of clostridium butyricum. After clostridium butyricum liquid or clostridium butyricum powder is added into livestock and poultry feed as a microbial agent, the livestock and poultry feed is fed to animals, so that the health condition of the cultured animals can be effectively improved, the digestion and absorption capacity and the immunity of the animals are improved, daily gain and daily ingestion are increased, the feed conversion ratio, diarrhea rate and 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, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (18)
1. A method for fermenting clostridium butyricum, comprising: clostridium butyricum is preparedClostridium butyricum) Fermenting and culturing to logarithmic phaseAdding saccharomycetes, and heating to inhibit or kill saccharomycetes to promote clostridium butyricum to form spores when clostridium butyricum starts to form spores, wherein when the spore rate of clostridium butyricum is more than or equal to 96%, fermentation is completed, and clostridium butyricum bacterial liquid is obtained.
2. Fermentation process according to claim 1, characterized in that it comprises the following steps: inoculating clostridium butyricum seed liquid into a fermentation tank for thallus expansion culture until clostridium butyricum dominant flora is formed, obtaining clostridium butyricum fermentation liquid, and then adding saccharomyces cerevisiae through feed supplementSaccharomyces cerevisiae) And (3) fermenting the seed solution until clostridium butyricum starts to form spores, heating to inhibit or kill saccharomyces cerevisiae so as to promote clostridium butyricum to form spores, and completing fermentation when the spore rate of clostridium butyricum reaches 96-98%, so as to obtain clostridium butyricum liquid.
3. The fermentation process of claim 2, wherein inoculating a clostridium butyricum fermentation broth with a saccharomyces cerevisiae seed broth comprises: and (3) performing expansion culture on clostridium butyricum seed liquid for 4-6 hours to form clostridium butyricum fermentation liquid, then inoculating saccharomyces cerevisiae seed liquid according to an inoculation ratio of 0.8-1%, continuing fermentation for 12-14 hours until clostridium butyricum starts to form spores, and preparing to heat.
4. A fermentation process according to claim 3, wherein the fermentation process of inoculating a saccharomyces cerevisiae seed solution into clostridium butyricum fermentation broth for fermentation comprises: regulating pH to not lower than 6.5, controlling temperature to 36-38deg.C, rotating at 40-50rpm, and preventing ventilation.
5. The fermentation method according to claim 4, wherein the temperature of the medium inoculated with the seed liquid of Saccharomyces cerevisiae in the fermentation liquid of Clostridium butyricum is raised stepwise to inhibit or kill Saccharomyces cerevisiae to promote the formation of spores of Clostridium butyricum.
6. The fermentation method according to claim 5, wherein the temperature of the culture medium is raised to 51-53 ℃ at a rate of 5 ℃ per half hour, and fermentation is continued for 6-8 hours, and when the spore rate of clostridium butyricum reaches 96-98%, the clostridium butyricum liquid is obtained.
7. The fermentation process of claim 4, further comprising: and killing residual saccharomycetes in the clostridium butyricum bacterial liquid by spray drying to obtain clostridium butyricum bacterial powder.
8. The fermentation method according to claim 7, wherein the fermented clostridium butyricum liquid and light calcium carbonate are uniformly mixed according to a mass ratio of 20:1, and then spray-dried.
9. The fermentation process of claim 8, wherein the spray drying apparatus has an inlet air temperature of 170 ℃ to 180 ℃ and an outlet air temperature of 80 ℃ to 85 ℃.
10. The fermentation process according to claim 2, wherein the preparation of clostridium butyricum fermentation broth comprises the steps of: inoculating clostridium butyricum seed liquid into a fermentation tank, wherein the inoculation volume ratio is 0.8-1%, performing thallus expansion culture, and culturing for 4-6 hours to obtain clostridium butyricum fermentation liquid.
11. The fermentation process of claim 10, wherein 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.
12. The fermentation process according to claim 10, wherein the fermentation process of the fermentation in clostridium butyricum fermentor is: the pH value is regulated to be not lower than 6.5 by 10 percent of sodium hydroxide solution, the temperature is controlled to be 36-38 ℃, the rotating speed is 40-50rpm, and no ventilation is realized.
13. The fermentation process of claim 2, wherein the preparation of the seed liquid comprises: inoculating clostridium butyricum and saccharomyces cerevisiae strains stored on the inclined plane into a shake flask culture medium, and culturing for 24-30 hours to obtain seed liquid.
14. The fermentation process of claim 13, wherein clostridium butyricum is shake flask cultured at 36-38 ℃ and stationary cultured in RCM liquid medium.
15. The fermentation process of claim 13, wherein the Saccharomyces cerevisiae is shake flask cultured at 36-38deg.C at 200r/min and YPD broth.
16. Fermentation process according to any one of claims 1 to 15, characterized in that the fermentation process of clostridium butyricum comprises the following steps:
inoculating clostridium butyricum seed liquid into a fermentation tank, wherein the inoculation volume ratio is 0.8-1%, performing thallus expansion culture, and culturing for 4-6 hours to obtain clostridium butyricum fermentation liquid;
inoculating Saccharomyces cerevisiae seed liquid into the clostridium butyricum fermentation liquid with the inoculation volume ratio of 0.8-1%, and continuing fermentation for 12-14 hours;
raising the temperature of the culture medium to 51-53 ℃ at a temperature raising rate of 5 ℃ every half hour, continuing fermentation for 6-8 hours, and microscopic examination of the fermentation liquid, and finishing fermentation when the spore rate reaches 96-98%, so as to obtain clostridium butyricum bacterial liquid;
mixing the fermented clostridium butyricum liquid and light calcium carbonate according to the mass ratio of 20:1, and spraying the mixture in a spraying tower after the clostridium butyricum liquid and the light calcium carbonate are mixed, wherein the air inlet temperature is 170-180 ℃ and the air outlet temperature is 80-85 ℃ to obtain clostridium butyricum powder.
17. A microbial ecological agent comprising the clostridium butyricum bacterial liquid obtained by the fermentation method of claim 1 or the clostridium butyricum bacterial powder obtained by the fermentation method of claim 7.
18. Use of the microecological formulation according to claim 17 for the preparation of probiotics, feed and feed additives.
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