Clostridium butyricum freeze-dried powder and preparation method thereof

Abstract

The invention relates to the technical field of fermentation strain preparation, and discloses clostridium butyricum freeze-dried powder and a preparation method thereof, and the freeze-dried powder comprises the following raw materials: clostridium butyricum liquid, a carrier, a freeze-drying protective agent and a dispersion stabilizer. The clostridium butyricum freeze-dried powder is arranged, the viable count of the clostridium butyricum freeze-dried powder is larger than 2 * 10 < 9 > cfu / g, the water content is smaller than 2%, and spores are in a dormant state, so that long-time preservation of the strain is facilitated, the strain cannot be degraded in the preservation process, the metabolic system cannot be affected, and the number of the strain in the inoculation process can be reduced. Due to decline caused by continuous passage and propagation and the clostridium butyricum in a spore dormant state, inoculation in fermentation production is facilitated, the inoculated dry powder strain can quickly germinate spores to form vegetative cells, inoculation steps are reduced, strain activity is maintained, and in industrial fermentation production, the clostridium butyricum has a good application prospect. The production cycle can be shortened by using the dry powder strain, the probability of spontaneous mutation in microbial passage is reduced, and the production efficiency is improved.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation strain preparation, in particular to a Clostridium butyricum freeze-dried powder and a preparation method thereof. Background Art

[0002] Clostridium butyricum, also known as butyric acid bacteria, belongs to the genus Clostridium of the family Bacillaceae of the phylum Firmicutes. It is a strictly anaerobic, spore-forming Gram-positive bacterium. Its metabolites, butyric acid, acetic acid, propionic acid and various digestive enzymes, can repair damaged intestinal mucosa and regulate the intestinal microecological environment, which is beneficial to animal health and growth and development. It is an ideal substitute for antibiotics in feed.

[0003] In order to facilitate the application of Clostridium butyricum in the production of nutritional health care, food, medicine and other products, Clostridium butyricum is usually proliferated by fermentation in liquid culture medium to obtain a high number of live bacteria, and then dried to obtain probiotic powder. Conventional hot air drying is not suitable for drying Clostridium butyricum due to its high mortality rate. Therefore, Clostridium butyricum is usually prepared by freeze drying. However, in the process of freeze drying, ice crystals produced by cell freezing will destroy the cell membrane and cause Clostridium butyricum to have a certain loss of live bacteria. In addition, during the shelf life of Clostridium butyricum, under room temperature storage conditions, Clostridium butyricum is kept in the commodity in the form of live bacteria, and it will also die due to its own growth and exhaustion of nutrients or due to excessively high ambient temperature, which will cause Clostridium butyricum to decay too fast during the shelf life. When the commodity is sold, the number of live bacteria of Clostridium butyricum cannot reach the content marked on the commodity label, resulting in quality problems. Therefore, how to provide a freeze-dried powder of Clostridium butyricum to achieve simple preservation of Clostridium butyricum and extend the preservation time while avoiding strain degradation is one of the technical problems that need to be solved urgently. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the shortcomings of the prior art, the present invention provides a lyophilized Clostridium butyricum powder and a preparation method thereof. By limiting the mass ratio of a carrier, a lyophilization protective agent and a dispersing stabilizer, the carrier is used to fully adsorb and carry Clostridium butyricum, so that the Clostridium butyricum in the lyophilized powder can be dormant in the form of spores; the lyophilization protective agent is used to reduce the damage of Clostridium butyricum cells caused by low-temperature freezing, and the overflow or freezing of free water in the Clostridium butyricum cells during the freezing process is reduced, so as to adjust the water activity of the Clostridium butyricum cells in the lyophilized powder; the dispersing stabilizer is used to make the Clostridium butyricum dispersed evenly and stably exist at the same time, so that the Clostridium butyricum can be stably exist on the carrier. At the same time, limiting the number of active Clostridium butyricum bacteria and further improving the survival rate of Clostridium butyricum during the storage of freeze-dried powder can allow enough strains to enter spore dormancy, which is not only beneficial to prolonging the storage time of the strains, but also can ensure that the strains do not degenerate during the storage stage and will not affect the metabolic system. The freeze-dried powder method has the advantage of simple preservation compared to test tube preservation, thereby realizing simple and long-term preservation of Clostridium butyricum, and at the same time avoiding strain degradation, thereby solving the above-mentioned technical problems.

[0006] (II) Technical solution

[0007] To achieve the above object, the present invention provides the following technical solution: a lyophilized powder of Clostridium butyricum, wherein the raw materials of the lyophilized powder include a Clostridium butyricum liquid, a carrier, a lyophilization protective agent and a dispersion stabilizer, wherein the mass ratio of the carrier, the lyophilization protective agent and the dispersion stabilizer is 5:5:1, and the number of viable bacteria of the Clostridium butyricum liquid is greater than 2×10 9 cfu / g.

[0008] Preferably, the carrier is soluble starch (50 g / L).

[0009] Through the above technical solution, the carrier limits the specific type and concentration, which can ensure that Clostridium butyricum can stably exist on the carrier.

[0010] Preferably, the lyoprotectant comprises sucrose (40 g / L), mannitol (8 g / L), cysteine ​​(1 g / L), and vitamin C (1 g / L).

[0011] Through the above technical solution, the specific type and concentration of the lyophilization protectant are limited, which can reduce the damage of low-temperature freezing to Clostridium butyricum cells.

[0012] Preferably, the dispersion stabilizer includes sodium carboxymethyl cellulose (10 g / L).

[0013] Through the above technical solution and the provision of a dispersion stabilizer, Clostridium butyricum can be dispersed uniformly and exist stably.

[0014] The present invention also provides a method for preparing lyophilized Clostridium butyricum powder, and the detection steps are as follows:

[0015] S1: Take out Clostridium butyricum strains stored at -80°C in the laboratory, inoculate them into Clostridium proliferation medium (10.0 g / L beef powder, 10.0 g / L peptone, 3.0 g / L yeast powder, 1.0 g / L soluble starch, 5.0 g / L glucose, 3.0 g / L sodium acetate, 5.0 g / L sodium chloride, 0.5 g / L L-cysteine ​​hydrochloride) at an inoculum volume of 1%, and culture them anaerobically at 37°C for 24 h to obtain a strain suspension;

[0016] S2: The bacterial suspension was inoculated into a primary Clostridium proliferation medium (10.0 g / L beef powder, 10.0 g / L peptone, 3.0 g / L yeast powder, 1.0 g / L soluble starch, 5.0 g / L glucose, 3.0 g / L sodium acetate, 5.0 g / L sodium chloride, 0.5 g / L L-cysteine ​​hydrochloride) at a volume ratio of 5%, and anaerobically cultured in an anaerobic incubator at 37°C for 24 hours to obtain a primary bacterial culture solution;

[0017] S3: Inoculate the primary bacterial culture solution at a volume ratio of 5% into the secondary Clostridium proliferation medium (10.0 g / L beef powder, 10.0 g / L peptone, 3.0 g / L yeast powder, 1.0 g / L soluble starch, 5.0 g / L glucose, 3.0 g / L sodium acetate, 5.0 g / L sodium chloride, 0.5 g / L L-cysteine ​​hydrochloride), and culture anaerobically in an anaerobic incubator at 37° C. for 24 h to obtain the secondary bacterial culture solution;

[0018] S4: Inoculate the secondary bacterial culture solution at a volume ratio of 5% into the tertiary Clostridium proliferation medium (10.0 g / L beef powder, 10.0 g / L peptone, 3.0 g / L yeast powder, 1.0 g / L soluble starch, 5.0 g / L glucose, 3.0 g / L sodium acetate, 5.0 g / L sodium chloride, 0.5 g / L L-cysteine ​​hydrochloride), and culture anaerobically in an anaerobic incubator at 37° C. for 24 hours to obtain the tertiary bacterial culture solution;

[0019] S5: Inoculate the third-grade Clostridium butyricum culture solution at a volume ratio of 5% into the Clostridium fermentation medium (beef extract 33.1g / L, peptone 32.7g / L, soluble starch 24.2g / L, calcium carbonate 24.7g / L, magnesium sulfate heptahydrate 0.25g / L, manganese sulfate monohydrate 0.25g / L, fermentation-specific defoaming agent 1.0g / L), stir at 37°C for 10min, and culture anaerobically in an anaerobic incubator at 37°C for 24h. During the culture process, stir for 5min every 2h, and take samples for Gram staining every 6h. The spores should be straight rod-shaped with blunt ends and Gram staining positive; at the same time, use a spectrophotometer to measure the OD value. When the number of viable bacteria is greater than 2×10 9cfu / g, it means that Clostridium butyricum has been cultured to the logarithmic phase, and Clostridium butyricum liquid can be obtained at this time;

[0020] S6: adding soluble starch (50 g / L) to the Clostridium butyricum liquid and mixing to obtain a mixture, then dissolving sucrose (40 g / L), mannitol (8 g / L), cysteine ​​(1 g / L), vitamin C (1 g / L), and sodium carboxymethyl cellulose (10 g / L) in water respectively, adding the mixture to mix, and performing refrigerated centrifugation (speed of 5500 rpm, time of 25 min) to obtain Clostridium butyricum slurry;

[0021] S7: The Clostridium butyricum sludge is balanced (temperature is 37°C, time is 50 min), then pre-frozen (temperature is -40°C, time is 12 h), and then vacuum freeze-dried (temperature ≤ -40°C, vacuum degree is 6 Pa, time is 22 h) to obtain Clostridium butyricum freeze-dried powder.

[0022] S8: The prepared Clostridium butyricum freeze-dried powder was activated by adding water in a gradient manner, and after half an hour of activation, it was shaken evenly, and 100 μL was pipetted onto the Clostridium selective medium and plated, and then placed at 37°C for anaerobically cultured for 16 hours to observe the growth of the colonies. In addition, a Clostridium butyricum stock solution control group was set up simultaneously in the experiment, and no additives were added to this group, and the colony growth ability was compared with the activated freeze-dried powder.

[0023] Compared with the prior art, the present invention provides a lyophilized Clostridium butyricum powder and a preparation method thereof, which have the following beneficial effects:

[0024] (1) A lyophilized powder of Clostridium butyricum provided in an embodiment of the present invention has a viable cell count of greater than 2×10 9 cfu / g, the moisture content is less than 2%, and the spores are in a dormant state, which is conducive to the long-term preservation of the strains. The strains will not degenerate during the preservation process, and will not affect the metabolic system, which can reduce the decline caused by continuous subculture and expansion during the inoculation process.

[0025] (2) The freeze-dried powder of Clostridium butyricum provided in the embodiment of the present invention is convenient for inoculation in fermentation production because Clostridium butyricum is in a spore dormant state. The inoculated dry powder strain can quickly germinate spores to form vegetative cells, thereby reducing the inoculation steps, maintaining the activity of the strain, and shortening the production cycle.

[0026] (3) A method for preparing a lyophilized powder of Clostridium butyricum provided in an embodiment of the present invention comprises activating and expanding the original bacterial strain, adding a carrier (soluble starch), a lyophilization protective agent (sucrose, mannitol, cysteine, vitamin C), and a dispersing stabilizer (sodium carboxymethyl cellulose) in proportion to a tertiary liquid culture medium, and collecting the bacterial mixture by centrifugation using a refrigerated centrifuge, and then subjecting the collected bacterial mixture to a vacuum freeze-drying treatment. The obtained lyophilized powder is subjected to a plate count method, and it can be seen that the number of live bacteria in the lyophilized powder is greater than 2×10 9 cfu / g.

[0027] (4) The method for preparing freeze-dried powder of Clostridium butyricum provided in an embodiment of the present invention has the advantages of long storage time, easy storage, less prone to degradation, large number of colonies, and convenient inoculation compared with test tube strains. In addition, in industrial fermentation production, the use of dry powder strains can shorten the production cycle, reduce the probability of spontaneous mutation during microbial culture, and improve production efficiency. DETAILED DESCRIPTION

[0028] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The present invention provides the following technical solution: a lyophilized powder of Clostridium butyricum, wherein the raw materials of the lyophilized powder include a Clostridium butyricum liquid, a carrier, a lyophilization protective agent and a dispersion stabilizer, wherein the mass ratio of the carrier, the lyophilization protective agent and the dispersion stabilizer is 5:5:1, and the number of viable bacteria of the Clostridium butyricum liquid is greater than 2×10 9 cfu / g.

[0030] Specifically, the carrier is soluble starch (50 g / L). The advantage is that the carrier limits the specific type and specific concentration, which can ensure that Clostridium butyricum can stably exist on the carrier. At the same time, there is sufficient carrier to carry Clostridium butyricum, ensuring that Clostridium butyricum in the freeze-dried powder can be dormant in the form of spores, ensuring that the number of live Clostridium butyricum meets expectations, and thus achieving simple and long-term preservation of Clostridium butyricum.

[0031] Specifically, the lyoprotectant includes sucrose (40 g / L), mannitol (8 g / L), cysteine ​​(1 g / L), and vitamin C (1 g / L). The advantage is that, through the above technical scheme, the specific type and specific concentration of the lyoprotectant are limited, which can reduce the damage of low-temperature freezing to Clostridium butyricum cells, reduce the overflow or freezing of free water in Clostridium butyricum cells during the freezing process, so as to adjust the water activity of Clostridium butyricum cells in the freeze-dried powder, put the live Clostridium butyricum in a dormant state, thereby further improving the survival rate of Clostridium butyricum during the storage of the freeze-dried powder.

[0032] Specifically, the dispersion stabilizer includes sodium carboxymethyl cellulose (10 g / L). The advantage is that the provision of the dispersion stabilizer can enable Clostridium butyricum to be dispersed uniformly and stably, thereby allowing Clostridium butyricum to stably exist on the carrier.

[0033] A method for preparing lyophilized Clostridium butyricum powder, the preparation steps of which are as follows:

[0034] S1: Take out Clostridium butyricum strains stored at -80°C in the laboratory, inoculate them into Clostridium proliferation medium (10.0 g / L beef powder, 10.0 g / L peptone, 3.0 g / L yeast powder, 1.0 g / L soluble starch, 5.0 g / L glucose, 3.0 g / L sodium acetate, 5.0 g / L sodium chloride, 0.5 g / L L-cysteine ​​hydrochloride) at an inoculum volume of 1%, and culture them anaerobically at 37°C for 24 h to obtain a strain suspension;

[0035] S2: The bacterial suspension was inoculated into a primary Clostridium proliferation medium (10.0 g / L beef powder, 10.0 g / L peptone, 3.0 g / L yeast powder, 1.0 g / L soluble starch, 5.0 g / L glucose, 3.0 g / L sodium acetate, 5.0 g / L sodium chloride, 0.5 g / L L-cysteine ​​hydrochloride) at a volume ratio of 5%, and anaerobically cultured in an anaerobic incubator at 37°C for 24 hours to obtain a primary bacterial culture solution;

[0036] S3: Inoculate the primary bacterial culture solution at a volume ratio of 5% into the secondary Clostridium proliferation medium (10.0 g / L beef powder, 10.0 g / L peptone, 3.0 g / L yeast powder, 1.0 g / L soluble starch, 5.0 g / L glucose, 3.0 g / L sodium acetate, 5.0 g / L sodium chloride, 0.5 g / L L-cysteine ​​hydrochloride), and culture anaerobically in an anaerobic incubator at 37° C. for 24 h to obtain the secondary bacterial culture solution;

[0037] S4: Inoculate the secondary bacterial culture solution at a volume ratio of 5% into the tertiary Clostridium proliferation medium (10.0 g / L beef powder, 10.0 g / L peptone, 3.0 g / L yeast powder, 1.0 g / L soluble starch, 5.0 g / L glucose, 3.0 g / L sodium acetate, 5.0 g / L sodium chloride, 0.5 g / L L-cysteine ​​hydrochloride), and culture anaerobically in an anaerobic incubator at 37° C. for 24 hours to obtain the tertiary bacterial culture solution;

[0038] S5: Inoculate the third-grade Clostridium butyricum culture solution at a volume ratio of 5% into the Clostridium fermentation medium (beef extract 33.1g / L, peptone 32.7g / L, soluble starch 24.2g / L, calcium carbonate 24.7g / L, magnesium sulfate heptahydrate 0.25g / L, manganese sulfate monohydrate 0.25g / L, fermentation-specific defoaming agent 1.0g / L), stir at 37°C for 10min, and culture anaerobically in an anaerobic incubator at 37°C for 24h. During the culture process, stir for 5min every 2h, and take samples for Gram staining every 6h. The spores should be straight rod-shaped with blunt ends and Gram staining positive; at the same time, use a spectrophotometer to measure the OD value. When the number of viable bacteria is greater than 2×10 9 cfu / g, it means that Clostridium butyricum has been cultured to the logarithmic phase, and Clostridium butyricum liquid can be obtained at this time;

[0039] S6: adding soluble starch (50 g / L) to the Clostridium butyricum liquid and mixing to obtain a mixture, then dissolving sucrose (40 g / L), mannitol (8 g / L), cysteine ​​(1 g / L), vitamin C (1 g / L), and sodium carboxymethyl cellulose (10 g / L) in water respectively, adding the mixture to mix, and performing refrigerated centrifugation (speed of 5500 rpm, time of 25 min) to obtain Clostridium butyricum slurry;

[0040] S7: The Clostridium butyricum sludge is balanced (temperature is 37°C, time is 50 min), then pre-frozen (temperature is -40°C, time is 12 h), and then vacuum freeze-dried (temperature ≤ -40°C, vacuum degree is 6 Pa, time is 22 h) to obtain Clostridium butyricum freeze-dried powder.

[0041] S8: The prepared Clostridium butyricum freeze-dried powder was activated by adding water in a gradient manner, and after half an hour of activation, it was shaken evenly, and 100 μL was pipetted onto the Clostridium selective medium and plated, and then placed at 37°C for anaerobically cultured for 16 hours to observe the growth of the colonies. In addition, a Clostridium butyricum stock solution control group was set up simultaneously in the experiment, and no additives were added to this group, and the colony growth ability was compared with the activated freeze-dried powder.

[0042] Working principle: The specific type and specific concentration of the carrier are limited. In the above invention, soluble starch (50g / L) is used as the carrier, so that Clostridium butyricum can be stably present on the carrier, and there is sufficient carrier to carry Clostridium butyricum, so that Clostridium butyricum in the freeze-dried powder can be dormant in the form of spores, and the number of live Clostridium butyricum is in line with expectations, thereby achieving simple preservation and long-term preservation of Clostridium butyricum. In the embodiment of the present invention, the specific type and specific concentration of the lyoprotectant are limited at the same time. The lyoprotectant includes sucrose (40g / L), mannitol (8g / L), cysteine ​​(1g / L), and vitamin C (1g / L). The setting of the lyoprotectant can reduce the damage of low-temperature freezing to Clostridium butyricum cells, reduce the overflow or freezing of free water in Clostridium butyricum cells during freezing, so as to adjust the water activity of Clostridium butyricum cells in the freeze-dried powder, so that Clostridium butyricum The live bacteria are in a dormant state, thereby further improving the survival rate of Clostridium butyricum during the storage of the freeze-dried powder, and at the same time limiting the specific type and specific concentration of the dispersing stabilizer. In the present invention, the dispersing stabilizer adopts sodium carboxymethyl cellulose (10g / L), which can ensure that the carrier can fully adsorb and carry Clostridium butyricum, and at the same time ensure that the number of live bacteria of Clostridium butyricum meets the requirements, thereby achieving simple preservation and long-term preservation of Clostridium butyricum. Finally, the mass ratio of the carrier, the freeze-drying protective agent and the dispersing stabilizer is 5:5:1, which is conducive to extending the preservation time of the strain, and can also ensure that the strain does not degenerate during the preservation stage, because in production, the enzyme system will be affected by the degradation of microorganisms, and the abnormality of the enzyme system will affect the strain's own anabolism system, and the metabolic products cannot be synthesized, so there is a risk of obtaining the target product. Therefore, by limiting the number of live bacteria, it can be effectively guaranteed that the strain does not degenerate during the preservation stage. Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A freeze-dried powder of Clostridium butyricum, characterized in that: The raw materials of the freeze-dried powder include Clostridium butyricum liquid, a carrier, a freeze-drying protective agent and a dispersion stabilizer, wherein the mass ratio of the carrier, the freeze-drying protective agent and the dispersion stabilizer is 5:5:1, and the number of viable bacteria of the Clostridium butyricum liquid is greater than 2×10 9 cfu / g.

2. The freeze-dried powder of Clostridium butyricum according to claim 1, characterized in that: The carrier is soluble starch (50 g / L).

3. The freeze-dried powder of Clostridium butyricum according to claim 1, characterized in that: The lyophilization protective agent includes sucrose (40 g / L), mannitol (8 g / L), cysteine ​​(1 g / L), and vitamin C (1 g / L).

4. The freeze-dried powder of Clostridium butyricum according to claim 1, characterized in that: The dispersion stabilizer includes sodium carboxymethyl cellulose (10 g / L).

5. A freeze-dried powder of Clostridium butyricum according to claims 1-4, characterized in that: The preparation method steps are as follows: S1: Take out Clostridium butyricum strains stored at -80°C in the laboratory, inoculate them into Clostridium proliferation medium (10.0 g / L beef powder, 10.0 g / L peptone, 3.0 g / L yeast powder, 1.0 g / L soluble starch, 5.0 g / L glucose, 3.0 g / L sodium acetate, 5.0 g / L sodium chloride, 0.5 g / L L-cysteine ​​hydrochloride) at an inoculum volume of 1%, and culture them anaerobically at 37°C for 24 h to obtain a strain suspension; S2: The bacterial suspension was inoculated into a primary Clostridium proliferation medium (10.0 g / L beef powder, 10.0 g / L peptone, 3.0 g / L yeast powder, 1.0 g / L soluble starch, 5.0 g / L glucose, 3.0 g / L sodium acetate, 5.0 g / L sodium chloride, 0.5 g / L L-cysteine ​​hydrochloride) at a volume ratio of 5%, and anaerobically cultured in an anaerobic incubator at 37°C for 24 hours to obtain a primary bacterial culture solution; S3: Inoculate the primary bacterial culture solution at a volume ratio of 5% into the secondary Clostridium proliferation medium (10.0 g / L beef powder, 10.0 g / L peptone, 3.0 g / L yeast powder, 1.0 g / L soluble starch, 5.0 g / L glucose, 3.0 g / L sodium acetate, 5.0 g / L sodium chloride, 0.5 g / L L-cysteine ​​hydrochloride), and culture anaerobically in an anaerobic incubator at 37° C. for 24 h to obtain the secondary bacterial culture solution; S4: Inoculate the secondary bacterial culture solution at a volume ratio of 5% into the tertiary Clostridium proliferation medium (10.0 g / L beef powder, 10.0 g / L peptone, 3.0 g / L yeast powder, 1.0 g / L soluble starch, 5.0 g / L glucose, 3.0 g / L sodium acetate, 5.0 g / L sodium chloride, 0.5 g / L L-cysteine ​​hydrochloride), and culture anaerobically in an anaerobic incubator at 37° C. for 24 hours to obtain the tertiary bacterial culture solution; S5: Inoculate the third-grade Clostridium butyricum culture solution at a volume ratio of 5% into the Clostridium fermentation medium (beef extract 33.1g / L, peptone 32.7g / L, soluble starch 24.2g / L, calcium carbonate 24.7g / L, magnesium sulfate heptahydrate 0.25g / L, manganese sulfate monohydrate 0.25g / L, fermentation-specific defoaming agent 1.0g / L), stir at 37°C for 10min, and culture anaerobically in an anaerobic incubator at 37°C for 24h. During the culture process, stir for 5min every 2h, and take samples for Gram staining every 6h. The spores should be straight rod-shaped with blunt ends and Gram staining positive; at the same time, use a spectrophotometer to measure the OD value. When the number of viable bacteria is greater than 2×10 9 cfu / g, it means that Clostridium butyricum has been cultured to the logarithmic phase, and Clostridium butyricum liquid can be obtained at this time; S6: adding soluble starch (50 g / L) to the Clostridium butyricum liquid and mixing to obtain a mixture, then dissolving sucrose (40 g / L), mannitol (8 g / L), cysteine ​​(1 g / L), vitamin C (1 g / L), and sodium carboxymethyl cellulose (10 g / L) in water respectively, adding the mixture to mix, and performing refrigerated centrifugation (speed of 5500 rpm, time of 25 min) to obtain Clostridium butyricum slurry; S7: The Clostridium butyricum sludge is balanced (temperature is 37°C, time is 50 min), then pre-frozen (temperature is -40°C, time is 12 h), and then vacuum freeze-dried (temperature ≤ -40°C, vacuum degree is 6 Pa, time is 22 h) to obtain Clostridium butyricum freeze-dried powder. S8: The prepared Clostridium butyricum freeze-dried powder was activated by adding water in a gradient manner, and after half an hour of activation, it was shaken evenly, and 100 μL was pipetted onto the Clostridium selective medium with a pipette to be coated, and placed in anaerobically cultured at 37°C for 16 hours to observe the growth of the colonies. In addition, a Clostridium butyricum stock solution control group was set up simultaneously in the experiment, and no additives were added to this group, and the colony growth ability was compared with the activated freeze-dried powder.