CN113699054A - Clostridium butyricum solid microbial inoculum and preparation method thereof - Google Patents

Clostridium butyricum solid microbial inoculum and preparation method thereof Download PDF

Info

Publication number
CN113699054A
CN113699054A CN202010430087.1A CN202010430087A CN113699054A CN 113699054 A CN113699054 A CN 113699054A CN 202010430087 A CN202010430087 A CN 202010430087A CN 113699054 A CN113699054 A CN 113699054A
Authority
CN
China
Prior art keywords
clostridium butyricum
content
microbial inoculum
solid microbial
solid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202010430087.1A
Other languages
Chinese (zh)
Other versions
CN113699054B (en
Inventor
卢宗梅
刘利利
张德国
周勇
佟毅
邴狄祥
李义
郭世堂
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cofco Biotechnology Co Ltd
Original Assignee
Cofco Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cofco Biotechnology Co Ltd filed Critical Cofco Biotechnology Co Ltd
Priority to CN202010430087.1A priority Critical patent/CN113699054B/en
Publication of CN113699054A publication Critical patent/CN113699054A/en
Application granted granted Critical
Publication of CN113699054B publication Critical patent/CN113699054B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

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
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Inorganic Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention relates to the field of microorganisms, and discloses a clostridium butyricum solid microbial inoculum and a preparation method thereof, wherein the method comprises the following steps: the clostridium butyricum thallus is used for preparing a clostridium butyricum solid microbial inoculum by a fluidized bed under the condition that a carrier exists. In a preferred case, the parameters of the clostridium butyricum solid bacterial agent prepared by the method comprise: the water content is less than or equal to 10 percent, and the viable count is more than or equal to 1 x 1011The spore rate of the cfu/g clostridium butyricum solid microbial agent is more than or equal to 95 percent. The invention uses the fluidized bed drying method, not only reduces the energy consumption, but also reduces the damage of the temperature to the thalli and protects the activity of the thalli. In the preferred embodiment of the present inventionIn the formula, the clostridium butyricum solid microbial inoculum with good fluidity, high viable count and high spore rate can be obtained by adopting the optimized fluidized bed working condition, the optimized carrier and the optimized ratio of the bacterial sludge to the carrier, and the solid microbial inoculum also has the advantage of long preservation period.

Description

Clostridium butyricum solid microbial inoculum and preparation method thereof
Technical Field
The invention relates to the field of microorganisms, and particularly relates to a clostridium butyricum solid microbial inoculum and a preparation method thereof.
Background
Clostridium butyricum (Clostridium butyricum), a Clostridium butyricum, is an anaerobic probiotic existing in human and livestock intestinal tracts, can improve the balance of a host intestinal bacteria ecosystem, and is widely applied to the field of medicines and the feed industry as a microecological preparation. The discovery and report of doctor of womb nearly treating of Qianye medical university of Japan in 1933 shows that the preparation not only can inhibit the growth of pathogenic bacteria such as escherichia coli, salmonella and the like, but also can promote the growth of beneficial bacteria such as lactic acid bacteria in intestinal tracts. China is introduced from Japan in 1993, the research time is only 30 years, and the method for screening and culturing clostridium butyricum has much exploration space. Previous studies have shown that the acute oral toxicity test and Ames test of clostridium butyricum are safe and, due to their sporulating ability, resistant to the acidic environment of the gastrointestinal tract. Researches show that the clostridium butyricum has good gastrointestinal tract adhesion, can inhibit pathogenic bacteria such as vibrio anguillarum and the like through space occupation, and simultaneously generates various beneficial organisms so as to enhance the immune function of a host. The clostridium butyricum is mainly in the state of a live bacterial preparation during use.
With the development of the fermented feed industry, the market has short supply and demand for feed, a large amount of microbial inoculum is needed to meet the market demand, most of the solid microbial inoculum in the market at present is prepared in a spraying mode, and the method has the advantages of high spore rate loss rate of the microbial inoculum, long spraying time and high cost.
Disclosure of Invention
The invention aims to solve the problems of high loss rate, long time and high cost in the preparation of a clostridium butyricum solid microbial inoculum by a spray method in the prior art, and provides a clostridium butyricum solid microbial inoculum and a preparation method thereof.
In order to achieve the above object, the first aspect of the present invention provides a method for preparing a clostridium butyricum solid bacterial agent, comprising: the clostridium butyricum thallus is used for preparing a clostridium butyricum solid microbial inoculum by a fluidized bed under the condition that a carrier exists.
Preferably, the clostridium butyricum thallus is provided in the form of bacterial sludge.
More preferably, the weight ratio of the clostridium butyricum bacterial sludge to the carrier is 1: 1-10.
Preferably, the support comprises a biomass material and a clay.
Preferably, the weight ratio of biomass material to clay in the carrier is 1: 0.2-5.
Preferably, the working condition of the fluidized bed ensures that the water content in the obtained clostridium butyricum solid microbial inoculum is less than or equal to 10 percent.
Preferably, the operating conditions of the fluidized bed include: the inlet temperature is 70-100 deg.C, the outlet temperature is 30-50 deg.C, and the drying time is 10-40 min.
The second aspect of the invention provides a clostridium butyricum solid microbial inoculum prepared by the method.
The invention uses the fluidized bed drying method, not only reduces the energy consumption, but also reduces the damage of the temperature to the thalli and protects the activity of the thalli.
In a preferred embodiment of the invention, the bacteria are removed from the culture by using the preferred fluidized bed operating conditions, the preferred carrier and the preferred bacterial sludge: the clostridium butyricum solid microbial inoculum with good fluidity, high viable count and high spore rate can be obtained by the proportion of the carrier, and the solid microbial inoculum also has the advantage of long preservation period.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The invention provides a preparation method of a clostridium butyricum solid microbial inoculum, which comprises the following steps: the clostridium butyricum thallus is used for preparing a clostridium butyricum solid microbial inoculum by a fluidized bed under the condition that a carrier exists.
In the present invention, the clostridium butyricum may be clostridium butyricum conventionally used in the art, and the kind thereof is not particularly limited.
In the present invention, the form of the fungus used for preparing the clostridium butyricum solid microbial inoculum may not be particularly limited, and preferably, the clostridium butyricum fungus is provided in the form of fermentation broth or bacterial sludge.
In the present invention, the fermentation broth may be a clostridium butyricum-containing liquid obtained by activating, expanding or fermenting clostridium butyricum, and for convenience of industrial production, a clostridium butyricum-containing liquid (or corresponding bacterial sludge) obtained by fermentation is often used to prepare a clostridium butyricum solid microbial inoculum.
In a preferred embodiment of the present invention, the method for producing a clostridium butyricum bacterial cell comprises: and sequentially activating, expanding culture, fermenting and optionally carrying out solid-liquid separation on the clostridium butyricum strain to obtain the clostridium butyricum strain.
It will be appreciated that when subjected to solid-liquid separation, the resulting is a Clostridium butyricum thallus in the form of a bacterial sludge.
In the present invention, the activation method may be a method conventionally used in the art, for example, a clostridium butyricum oil tube stored in an ultra-low temperature refrigerator may be naturally thawed and then inoculated into a seed medium at an inoculum size of 0.1 to 1% by volume for activation to obtain a primary seed solution.
In the present invention, the seed medium may be a medium conventionally used in the art, and preferably, the seed medium comprises yeast extract, beef extract, tryptone, glucose, soluble starch, sodium chloride, sodium acetate trihydrate and L-cysteine hydrochloride; the pH of the seed culture medium is 6-7.
Preferably, based on the total weight of the seed culture medium, the content of the yeast extract powder is 0.2-0.4 wt%, the content of the beef extract is 0.5-1.5 wt%, the content of the tryptone is 0.5-1.5 wt%, the content of the glucose is 0.3-0.8 wt%, the content of the soluble starch is 0.02-0.2 wt%, the content of the sodium chloride is 0.3-0.8 wt%, the content of the sodium acetate trihydrate is 0.1-0.5 wt%, and the content of the L-cysteine hydrochloride is 0.01-0.02 wt%.
In the present invention, preferably, the activating conditions include: the temperature is 34-38 deg.C, and anaerobic culturing.
In the present invention, the activation time may not be particularly limited, and is preferably 12 to 16 hours.
Preferably, activation is terminated when the OD of the primary seed liquid reaches above 3.5 and the pH is in the range of 4.5-5.5.
In the present invention, the method of expanding culture may be a method conventionally used in the art, for example, the first-stage seed solution may be inoculated into an expanding culture medium in an inoculum size of 0.1-1 vol% and expanded to obtain a second-stage seed solution.
In the present invention, the expanding medium may be a medium conventionally used in the art, and preferably, the expanding medium is a seed medium as described above.
In the present invention, preferably, the condition of the propagation includes: the temperature is 34-38 deg.C, and anaerobic culturing.
In the present invention, the time of the expanding culture may not be particularly limited, and is preferably 14 to 20 hours.
Preferably, when the OD of the secondary seed liquid reaches above 3.5 and the pH is in the range of 4.5-5.5, the propagation is finished.
In the present invention, the fermentation method may be a method conventionally used in the art, for example, the secondary seed liquid may be inoculated into a fermentation medium in an amount of 0.1 to 0.5 vol% and fermented to obtain a fermentation liquid.
In the present invention, the fermentation medium may be a medium conventionally used in the art, and preferably, fermentation is performed in a fermentation medium comprising soluble starch, yeast extract, ammonium sulfate, sodium chloride, potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate, and calcium carbonate; the pH of the seed culture medium is 6-7.
Preferably, based on the total weight of the fermentation medium, the content of the soluble starch is 1-4 wt%, the content of the yeast extract powder is 0.5-1.5 wt%, the content of ammonium sulfate is 2-4 wt%, the content of sodium chloride is 1-3 wt%, the content of monopotassium phosphate is 1-2 wt%, the content of magnesium sulfate is 0.01-0.04 wt%, the content of manganese sulfate is 0.01-0.04 wt%, and the content of calcium carbonate is 0.2-0.4 wt%.
Preferably, the conditions of the fermentation include: anaerobic culture at 35-39 deg.C.
In the present invention, the fermentation can be carried out in a fermentation tank, and the stirring speed can be adjusted by those skilled in the art according to actual conditions.
In the invention, nitrogen can be introduced in the fermentation process, and the introduction of the nitrogen is stopped after the clostridium butyricum produces the gas.
In the present invention, the fermentation time may not be particularly limited, and is preferably 12 to 18 hours.
Preferably, the fermentation is ended when the OD of the fermentation reaches above 6 and the pH is in the range of 4-5.
The quality of the prepared solid microbial inoculum can be improved under the conditions of the preferred activation, propagation and fermentation conditions and the corresponding culture medium.
In the present invention, the obtained fermentation liquid can be subjected to solid-liquid separation to obtain bacterial sludge, wherein the solid-liquid separation method can be a method conventionally used in the art, such as centrifugation and/or filtration.
In the present invention, preferably, the clostridium butyricum thallus is provided in the form of bacterial sludge.
In the present invention, the weight ratio of the clostridium butyricum bacterial sludge to the carrier can be selected within a wide range, and preferably, the weight ratio of the clostridium butyricum bacterial sludge to the carrier is 1: 1-10, for example, can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, and any range consisting of any two values, more preferably 1: 2-5. Under the preferable conditions, the quality of the solid microbial inoculum can be improved.
In the present invention, the kind of the carrier may not be particularly limited, and preferably, the carrier contains a biomass material and clay.
Preferably, the biomass material is selected from at least one of starch, soybean meal and inulin.
The starch may be a starch conventionally used in the art, and may be, for example, corn starch.
Preferably, the clay is diatomaceous earth and/or montmorillonite.
In the present invention, the weight ratio of the biomass material and the clay in the carrier can be selected within a wide range, and preferably, the weight ratio of the biomass material and the clay in the carrier is 1:0.1 to 5, for example, may be 1:0.1, 1:0.2, 1:0.4, 1:0.6, 1:0.8, 1:1, 1:2, 1:3, 1:4, 1:5 and any range of compositions between any two values, and more preferably 1:0.2 to 0.8.
In the invention, the working conditions of the fluidized bed can be those conventionally used in the art, and preferably, the working conditions of the fluidized bed are such that the water content in the obtained clostridium butyricum solid microbial inoculum is less than or equal to 10%.
Preferably, the operating conditions of the fluidized bed include: the inlet temperature is 70-100 deg.C, the outlet temperature is 30-50 deg.C, and the drying time is 10-40 min. Further preferably, the operating conditions of the fluidized bed include: the inlet temperature is 70-90 deg.C, the outlet temperature is 30-45 deg.C, and the drying time is 10-40 min. Under the preferable conditions, the quality of the solid microbial inoculum can be improved.
In another aspect, the invention provides a clostridium butyricum solid bacterial agent prepared by the method.
Preferably, the parameters of the clostridium butyricum solid bacterial agent comprise: the water content is less than or equal to 10 percent, and the viable count is more than or equal to 1 x 1011The spore rate of the cfu/g clostridium butyricum solid microbial agent is more than or equal to 95 percent.
In the invention, the parameter evaluation of the clostridium butyricum solid bacterial agent is shown in T/CSWSL 006-2019.
The present invention will be described in detail below by way of examples.
In the following examples, clostridium butyricum used was purchased from china industrial microbial strain collection management center, strain collection number: CICC 23847.
In the following examples, the raw materials were commercially available unless otherwise specified.
The parameter evaluation of the clostridium butyricum solid microbial inoculum is shown in T/CSWSL 006-2019.
Preparation example 1
This preparation example is for explaining a method for preparing a Clostridium butyricum puree
And (3) activation: the clostridium butyricum oil tubes stored in the ultra-low temperature refrigerator are naturally thawed 0.5h in advance, and inoculated into a seed culture medium with the inoculation amount of 0.5 volume percent. Culturing at 36 + -2 deg.C, standing in anaerobic bottle for 14 hr to obtain a first-stage seed solution with OD value of about 4, pH of about 5, and activation to obtain a first-stage seed solution with viable count of 1.0 x 108cfu/mL。
Expanding culture: inoculating the first-stage seed solution into seed culture medium with inoculation amount of 0.4 vol%, culturing at 36 + -2 deg.C in anaerobic box for 18 hr until OD value is about 4 and pH is about 5, and finishing the amplification culture to obtain second-stage seed solution with viable count of 5.0 x 108cfu/mL。
Fermentation: inoculating the secondary seed liquid into fermentation medium with the inoculation amount of 0.2 vol%, culturing in 50L fermentation tank at 37 + -2 deg.C, controlling rotation speed to 100rppm, charging nitrogen gas for the first 4 hr, and thenStopping filling nitrogen and continuing culturing for about 10h, wherein OD value of the fermentation broth is 6-7, pH is about 4.5, and viable count in the obtained fermentation broth is 8.5 x 10 after the fermentation tank culture is finished9cfu/mL。
Preparation of bacterial sludge: and (4) centrifuging the fermentation liquor by using a high-speed centrifuge at the rotating speed of 8000rpm for 5min to obtain the bacterial sludge.
Wherein, in the seed culture medium, the yeast extract powder is 0.3 weight percent, the beef extract is 1 weight percent, the tryptone is 1 weight percent, the glucose is 0.5 weight percent, the soluble corn starch is 0.1 weight percent, the sodium chloride is 0.5 weight percent, the sodium acetate trihydrate is 0.3 weight percent, the L-cysteine hydrochloride is 0.015 weight percent, and the initial pH value is 6.5 +/-0.2.
Wherein, in the fermentation medium, 2 weight percent of soluble corn starch, 1 weight percent of yeast extract powder, 3 weight percent of ammonium sulfate, 2 weight percent of sodium chloride, 1.5 weight percent of monopotassium phosphate, 0.02 weight percent of magnesium sulfate, 0.02 weight percent of manganese sulfate, 0.3 weight percent of calcium carbonate, and the initial pH value is 6.5 +/-0.2.
Example 1
This example is used to illustrate the preparation method of the clostridium butyricum solid bacterial agent of the present invention
100g of bacterial sludge and 300g of carrier (the weight ratio of corn starch to diatomite is 2:1), drying by a fluidized bed: the inlet temperature is 80 ℃, the outlet temperature is 37 ℃, and the drying time is 20 min.
The obtained solid microbial inoculum is stored for 18 months at room temperature, the change of the viable count is measured and recorded, and the specific result is shown in table 1.
Example 2
This example is used to illustrate the preparation method of the clostridium butyricum solid bacterial agent of the present invention
100g of bacterial sludge and 300g of carrier (the weight ratio of corn starch to diatomite is 2:1), drying by a fluidized bed: the inlet temperature is 90 ℃, the outlet temperature is 44 ℃, and the drying time is 18 min.
The obtained solid microbial inoculum is stored for 18 months at room temperature, the change of the viable count is measured and recorded, and the specific result is shown in table 1.
Example 3
This example is used to illustrate the preparation method of the clostridium butyricum solid bacterial agent of the present invention
100g of bacterial sludge and 300g of carrier (the weight ratio of corn starch to diatomite is 2:1), drying by a fluidized bed: the inlet temperature is 100 ℃, the outlet temperature is 48 ℃, and the drying time is 16 min.
The obtained solid microbial inoculum is stored for 18 months at room temperature, the change of the viable count is measured and recorded, and the specific result is shown in table 1.
Example 4
This example is used to illustrate the preparation method of the clostridium butyricum solid bacterial agent of the present invention
100g of bacterial sludge and 300g of carrier (the weight ratio of corn starch to diatomite is 2:1), drying by a fluidized bed: the inlet temperature is 70 ℃, the outlet temperature is 33 ℃, and the drying time is 35 min.
The obtained solid microbial inoculum is stored for 18 months at room temperature, the change of the viable count is measured and recorded, and the specific result is shown in table 1.
Example 5
This example is used to illustrate the preparation method of the clostridium butyricum solid bacterial agent of the present invention
Example 5-1: the preparation was carried out as described in example 1, except that corn starch was replaced by equal masses of soybean meal.
Example 5-2: the preparation was carried out as described in example 1, except that inulin powder of equal mass was used instead of corn starch.
The obtained solid microbial inoculum is stored for 18 months at room temperature, the change of the viable count is measured and recorded, and the specific result is shown in table 1.
Example 6
This example is used to illustrate the preparation method of the clostridium butyricum solid bacterial agent of the present invention
The preparation was carried out as described in example 1, except that an equal mass of montmorillonite was used instead of diatomaceous earth.
The obtained solid microbial inoculum is stored for 18 months at room temperature, the change of the viable count is measured and recorded, and the specific result is shown in table 1.
Example 7
This example is used to illustrate the preparation method of the clostridium butyricum solid bacterial agent of the present invention
Example 7-1: the preparation was carried out as described in example 1, except that a weight ratio of corn starch to diatomaceous earth of 1:0.2 was used.
Example 7-2: the preparation was carried out as described in example 1, except that a weight ratio of corn starch to diatomaceous earth of 1:0.8 was used.
Examples 7 to 3: the preparation was carried out as described in example 1, except that a weight ratio of corn starch to diatomaceous earth of 10:1 was used.
Examples 7 to 4: the preparation was carried out as described in example 1, except that a weight ratio of corn starch to diatomaceous earth of 1:5 was used.
The obtained solid microbial inoculum is stored for 18 months at room temperature, the change of the viable count is measured and recorded, and the specific result is shown in table 1.
Example 8
This example is used to illustrate the preparation method of the clostridium butyricum solid bacterial agent of the present invention
Example 8-1: the preparation was carried out as described in example 1, except that the weight ratio of the clostridium butyricum puree to the carrier was 1: 2.
Example 8-2: the preparation was carried out as described in example 1, except that the weight ratio of the clostridium butyricum puree to the carrier was 1: 5.
Examples 8 to 3: the preparation was carried out as described in example 1, except that the weight ratio of the clostridium butyricum puree to the carrier was 1: 1.
Examples 8 to 4: the preparation was carried out as described in example 1, except that the weight ratio of the clostridium butyricum puree to the carrier was 1: 10.
The obtained solid microbial inoculum is stored for 18 months at room temperature, the change of the viable count is measured and recorded, and the specific result is shown in table 1.
Example 9
The preparation was carried out as described in example 1, except that different bacterial purees were used. Specifically, the seed culture medium and the fermentation culture medium used in the preparation process of the bacterial sludge respectively have the following compositions.
Seed culture medium: 0.3 weight percent of yeast extract powder, 1 weight percent of beef extract, 1 weight percent of tryptone, 0.5 weight percent of glucose, 0.1 weight percent of soluble corn starch, 0.5 weight percent of sodium chloride, 0.3 weight percent of sodium acetate trihydrate, 0.015 weight percent of L-cysteine hydrochloride and initial pH of 6.5 +/-0.2.
Fermentation medium: 2% by weight of glucose, 1% by weight of yeast powder, 1% by weight of peptone, 2% by weight of calcium chloride, 1.5% by weight of potassium dihydrogen phosphate, 0.02% by weight of magnesium sulfate, 0.02% by weight of manganese sulfate, and an initial pH of 6.5. + -. 0.2.
The obtained solid microbial inoculum is stored for 18 months at room temperature, the change of the viable count is measured and recorded, and the specific result is shown in table 1.
Comparative example 1
The preparation method of the clostridium butyricum solid microbial inoculum for reference in the comparative example
The preparation was carried out as described in example 1, except that the drying was carried out statically in an oven, the temperature being set at 60 ℃ and the time being 120 min.
The obtained solid microbial inoculum is stored for 18 months at room temperature, the change of the viable count is measured and recorded, and the specific result is shown in table 1.
Comparative example 2
The preparation method of the clostridium butyricum solid microbial inoculum for reference in the comparative example
The preparation was carried out as described in example 1, except that the preparation was carried out by spray drying at an inlet temperature of 120 ℃ and 140 ℃, an outlet temperature of 80-85 ℃ and a pump speed of 22.7 rpm.
The obtained solid microbial inoculum is stored for 18 months at room temperature, the change of the viable count is measured and recorded, and the specific result is shown in table 1.
TABLE 1
Figure BDA0002500213720000111
Figure BDA0002500213720000121
Note: the units in the table are cfu/g.
The results in table 1 show that the microbial inoculum prepared by the method of the invention has the advantages of high viable count and high spore rate, and the viable count after 18 months is still kept at a high level, which indicates that the quality of the microbial inoculum is stable.
Compared with the method of the invention, the method of the comparative example 1 has the problem of higher loss rate of the spore rate of the thalli, and the quality of the microbial inoculum is obviously reduced after long-term storage.
Compared with the method of the invention, the method of the comparative example 2 has the problems of high loss rate of the spore rate of the thalli, more loss of viable bacteria after long-term storage of the microbial inoculum and high cost.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims (10)

1. A preparation method of a clostridium butyricum solid microbial inoculum is characterized by comprising the following steps: the clostridium butyricum thallus is used for preparing a clostridium butyricum solid microbial inoculum by a fluidized bed under the condition that a carrier exists.
2. The method according to claim 1, wherein the Clostridium butyricum is provided in the form of a fermentation broth or a bacterial sludge.
3. The production method according to claim 1 or 2, wherein the Clostridium butyricum thallus is provided in the form of a bacterial sludge;
preferably, the weight ratio of the clostridium butyricum bacterial mud to the carrier is 1:1 to 10, more preferably 1: 2-5.
4. The production method according to any one of claims 1 to 3, wherein the carrier comprises a biomass material and clay;
preferably, the biomass material is selected from at least one of starch, soybean meal and inulin;
preferably, the clay is diatomaceous earth and/or montmorillonite.
5. The method of claim 4, wherein the weight ratio of biomass material to clay in the carrier is 1: 0.1-5, preferably 1: 0.2-0.8.
6. The method according to any one of claims 1 to 5, wherein the fluidized bed is operated under conditions such that the water content in the obtained Clostridium butyricum solid bacterial agent is less than or equal to 10%;
preferably, the operating conditions of the fluidized bed include: the inlet temperature is 70-100 deg.C, the outlet temperature is 30-50 deg.C, and the drying time is 10-40 min.
7. The method according to any one of claims 1 to 6, wherein the Clostridium butyricum thallus is prepared by a method comprising: sequentially activating, expanding culture, fermenting and optionally carrying out solid-liquid separation on clostridium butyricum strains to obtain clostridium butyricum strains;
preferably, the activating conditions include: anaerobic culture at 34-38 deg.C;
preferably, the condition of the propagation includes: anaerobic culture at 34-38 deg.C;
preferably, the conditions of the fermentation include: anaerobic culture at 35-39 deg.C.
8. The method of claim 7, wherein activation and expansion are each independently performed in a seed medium comprising yeast extract, beef extract, tryptone, glucose, soluble starch, sodium chloride, sodium acetate trihydrate and L-cysteine hydrochloride; the pH value of the seed culture medium is 6-7;
preferably, based on the total weight of the seed culture medium, the content of the yeast extract powder is 0.2-0.4 wt%, the content of the beef extract is 0.5-1.5 wt%, the content of the tryptone is 0.5-1.5 wt%, the content of the glucose is 0.3-0.8 wt%, the content of the soluble starch is 0.02-0.2 wt%, the content of the sodium chloride is 0.3-0.8 wt%, the content of the sodium acetate trihydrate is 0.1-0.5 wt%, and the content of the L-cysteine hydrochloride is 0.01-0.02 wt%.
9. The process of claim 7, wherein fermentation is carried out in a fermentation medium comprising soluble starch, yeast extract, ammonium sulfate, sodium chloride, monopotassium phosphate, magnesium sulfate, manganese sulfate, and calcium carbonate; the pH value of the seed culture medium is 6-7;
preferably, based on the total weight of the fermentation medium, the content of the soluble starch is 1-4 wt%, the content of the yeast extract powder is 0.5-1.5 wt%, the content of ammonium sulfate is 2-4 wt%, the content of sodium chloride is 1-3 wt%, the content of monopotassium phosphate is 1-2 wt%, the content of magnesium sulfate is 0.01-0.04 wt%, the content of manganese sulfate is 0.01-0.04 wt%, and the content of calcium carbonate is 0.2-0.4 wt%.
10. A clostridium butyricum solid bacterial agent prepared by the method of any one of claims 1 to 9;
preferably, the parameters of the clostridium butyricum solid bacterial agent comprise: the water content is less than or equal to 10 percent, and the viable count is more than or equal to 1 x 1011The spore rate of the cfu/g clostridium butyricum solid microbial agent is more than or equal to 95 percent.
CN202010430087.1A 2020-05-20 2020-05-20 Clostridium butyricum solid microbial inoculum and preparation method thereof Active CN113699054B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010430087.1A CN113699054B (en) 2020-05-20 2020-05-20 Clostridium butyricum solid microbial inoculum and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010430087.1A CN113699054B (en) 2020-05-20 2020-05-20 Clostridium butyricum solid microbial inoculum and preparation method thereof

Publications (2)

Publication Number Publication Date
CN113699054A true CN113699054A (en) 2021-11-26
CN113699054B CN113699054B (en) 2023-08-01

Family

ID=78645563

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010430087.1A Active CN113699054B (en) 2020-05-20 2020-05-20 Clostridium butyricum solid microbial inoculum and preparation method thereof

Country Status (1)

Country Link
CN (1) CN113699054B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114410524A (en) * 2022-01-20 2022-04-29 厦门昶科生物工程有限公司 Industrial production method of clostridium butyricum powder

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2481867A1 (en) * 2002-04-09 2003-10-23 Institut National De La Recherche Scientifique Methods and apparatus for treating animal manure
CN102057046A (en) * 2008-04-09 2011-05-11 钴技术有限公司 Immobilized product tolerant microorganisms
CN102363754A (en) * 2011-05-07 2012-02-29 华中农业大学 Clear liquid fermentation medium for clostridium butyricum and fermentation culture method thereof
CN103710286A (en) * 2013-12-30 2014-04-09 广东海纳川药业股份有限公司 Processing method of viable clostridium butyricum preparation and culture medium composition
CN106479924A (en) * 2016-10-31 2017-03-08 湖北绿雪生物科技有限公司 The preparation of a kind of Clostridium butyricum and clostridium butyricum active bacteria preparation and application
CN107080053A (en) * 2017-03-21 2017-08-22 四川新华扬山野生物有限公司 A kind of heat-resistance type coating bioactive substance and preparation method thereof
CN108130296A (en) * 2018-01-17 2018-06-08 厦门昶科生物工程有限公司 The method and the preparation method of clostridium butyricum probiotics that a kind of clostridium butyricum high density is continuously fermented
CN108531427A (en) * 2018-04-13 2018-09-14 安徽天邦生物技术有限公司 A kind of multistage fermentation method for producing of high yield gemma state clostridium butyricum
CN109566863A (en) * 2019-01-18 2019-04-05 湖北华扬科技发展有限公司 Feed addictive and its preparation method and application
CN110151795A (en) * 2019-05-14 2019-08-23 广东大泽农生物科技股份有限公司 A kind of clostridium butyricum active bacteria preparation and its production technology
CN110408563A (en) * 2019-07-17 2019-11-05 天津市圣世莱科技有限公司 A kind of preparation method of clostridium butyricum high density fermentation and its microecological microbial agent

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2481867A1 (en) * 2002-04-09 2003-10-23 Institut National De La Recherche Scientifique Methods and apparatus for treating animal manure
CN102057046A (en) * 2008-04-09 2011-05-11 钴技术有限公司 Immobilized product tolerant microorganisms
CN102363754A (en) * 2011-05-07 2012-02-29 华中农业大学 Clear liquid fermentation medium for clostridium butyricum and fermentation culture method thereof
CN103710286A (en) * 2013-12-30 2014-04-09 广东海纳川药业股份有限公司 Processing method of viable clostridium butyricum preparation and culture medium composition
CN106479924A (en) * 2016-10-31 2017-03-08 湖北绿雪生物科技有限公司 The preparation of a kind of Clostridium butyricum and clostridium butyricum active bacteria preparation and application
CN107080053A (en) * 2017-03-21 2017-08-22 四川新华扬山野生物有限公司 A kind of heat-resistance type coating bioactive substance and preparation method thereof
CN108130296A (en) * 2018-01-17 2018-06-08 厦门昶科生物工程有限公司 The method and the preparation method of clostridium butyricum probiotics that a kind of clostridium butyricum high density is continuously fermented
CN108531427A (en) * 2018-04-13 2018-09-14 安徽天邦生物技术有限公司 A kind of multistage fermentation method for producing of high yield gemma state clostridium butyricum
CN109566863A (en) * 2019-01-18 2019-04-05 湖北华扬科技发展有限公司 Feed addictive and its preparation method and application
CN110151795A (en) * 2019-05-14 2019-08-23 广东大泽农生物科技股份有限公司 A kind of clostridium butyricum active bacteria preparation and its production technology
CN110408563A (en) * 2019-07-17 2019-11-05 天津市圣世莱科技有限公司 A kind of preparation method of clostridium butyricum high density fermentation and its microecological microbial agent

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114410524A (en) * 2022-01-20 2022-04-29 厦门昶科生物工程有限公司 Industrial production method of clostridium butyricum powder

Also Published As

Publication number Publication date
CN113699054B (en) 2023-08-01

Similar Documents

Publication Publication Date Title
CN110408563B (en) High-density fermentation of clostridium butyricum and preparation method of microecological microbial agent thereof
EP4317415A1 (en) Bacillus natto for producing menaquinone-7 and use thereof
CN115074290B (en) Lactobacillus casei for co-production of phenyllactic acid and gamma-aminobutyric acid and application thereof
CN114032260A (en) Method for improving fermentation level of Hainanmycin
CN112280707A (en) Preparation method of probiotics for feed
CN103966271B (en) Fermenting and producing DHA method
CN113699054B (en) Clostridium butyricum solid microbial inoculum and preparation method thereof
CN109234215B (en) Lactobacillus rhamnosus low-salt culture medium and culture method
CN111424058B (en) Method for preparing erythritol by adopting continuous fermentation mode
CN111187744A (en) High-density industrial fermentation medium for stratospheric bacillus and fermentation method thereof
CN114181872B (en) Enterococcus faecium high-density fermentation culture method
CN109722408B (en) Method for promoting bacillus subtilis to produce spores
CN114437975B (en) Lactic acid-producing bacillus coagulans and application thereof
CN109329648B (en) Application of compound microecological preparation for antagonizing vibrio prawn
CN111349578B (en) Preparation method of lactobacillus solid microbial inoculum
CN113647518A (en) Special biological fermentation feed for fattening cattle in northern Xinjiang area and preparation method thereof
JP2001106608A (en) Plant growth promoter
CN111349581A (en) Breeding and application of acidophilic lactobacillus
CN111733202A (en) Mixed fermentation method of tylosin
CN110699281A (en) Special compound bacterium preparation for hybrid mutton sheep and preparation method thereof
CN114410529B (en) Culture medium and culture method for solid-state high-density fermentation of bacillus megatherium
CN114672437B (en) Bacillus subtilis G-1 for producing organic acid and application thereof
CN115466700B (en) Novel bacillus licheniformis fermentation medium formula and culture method thereof
CN112795487B (en) Fermentation medium and fermentation method for producing fusidic acid
CN112175855B (en) Lactic acid bacteria culture medium, application thereof and culture method of lactic acid bacteria

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
CB02 Change of applicant information
CB02 Change of applicant information

Address after: No. 99, Kaiyuan Avenue, Bengbu, Anhui 233010

Applicant after: COFCO Biotechnology Co.,Ltd.

Address before: No. 1, central grain Avenue, Bengbu, Anhui Province, Anhui

Applicant before: COFCO Biotechnology Co.,Ltd.

GR01 Patent grant
GR01 Patent grant