Preparation method of clostridium butyricum powder

Abstract

The invention discloses a preparation method of bacterial powder of clostridium butyricum, and belongs to the technical field of bacterial powder preparation. The preparation method of the bacterial powder provided by the invention comprises the following steps: adding a regulator for regulating culture, heating, and adding a protective agent for freeze drying. Experimental results show that the preparation method of the bacterial powder provided by the invention has high viable bacterium yield, and the loss of clostridium butyricum in the preparation process of the bacterial powder is reduced; the bacterial powder obtained according to the bacterial powder preparation method still has the retention rate of 90% or above after being stored for a long time for 180 days at the temperature of 35 DEG C, which indicates that the survival ability of clostridium butyricum is remarkably improved. The preparation method of the bacterial powder is simple in process, easy to operate and environmentally friendly, and the cost cannot be remarkably increased. The clostridium butyricum microecological product provides a new strategy for preparation and activity maintenance of clostridium butyricum microecological products, can be applied to the fields of animal husbandry industry and medical care, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of bacterial powder preparation, in particular to a method for preparing bacterial powder of Clostridium butyricum. Background Art

[0002] Clostridium butyricum, also known as Clostridium butyricum, belongs to the Bacillaceae family, genus Clostridium. It is an obligate anaerobic Gram-positive bacillus that can utilize a variety of carbon sources, including glucose, sucrose, fructose, maltose, starch, and cellulose, with butyrate, lactate, and acetate as its primary metabolites. Clostridium butyricum has a strong tolerance to gastric acid, bile acids, and various antibiotics in the digestive system. It has multiple functions, including regulating intestinal microecological balance, improving intestinal development, enhancing immunity, and producing prebiotics.

[0003] Based on these functions, Clostridium butyricum is often used clinically as a microecological agent for the treatment of antibiotic-associated diarrhea, ulcerative colitis, and as an adjunctive treatment for hand, foot, and mouth disease. It is also used as a feed additive in poultry, livestock, and aquaculture, improving daily weight gain and other production performance of livestock and poultry, enhancing animal resistance, and reducing morbidity and mortality.

[0004] Therefore, preparations of Clostridium butyricum can be widely used in production and life. Compared with liquid live bacteria preparations, Clostridium butyricum powder has good stability, is easy to transport and convenient to use; drying Clostridium butyricum to make dry bacterial powder is a common production method of enterprises. However, the currently commonly used spray drying process has an inlet and outlet air temperature that is higher than the extreme tolerance temperature of Clostridium butyricum, which seriously reduces the survival rate of Clostridium butyricum and is not conducive to maintaining bacterial activity during long-term storage. Therefore, there is an urgent need to provide a Clostridium butyricum powder preparation process with a high viable cell count retention rate, good stability, simplicity and economy. Summary of the Invention

[0005] The object of the present invention is to provide a method for preparing bacterial powder of Clostridium butyricum to solve the problems existing in the above-mentioned prior art. The bacterial powder preparation method provided by the present invention can obtain Clostridium butyricum bacterial powder from Clostridium butyricum fermentation broth with high yield, and the Clostridium butyricum in the bacterial powder can survive stably for a long time.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for preparing Clostridium butyricum powder, comprising the following steps:

[0008] mixing the Clostridium butyricum fermentation liquid with a regulator, regulating the culture, and heating to obtain a mixed liquid;

[0009] Centrifuging the mixed solution to prepare bacterial sludge, mixing the bacterial sludge with a protective agent, and freeze-drying to obtain the Clostridium butyricum powder;

[0010] Wherein, the regulator includes calcium lactate, sodium lactate, hydrogen peroxide and mannose;

[0011] The protective agents include sodium sulfide, glutathione, sodium pyruvate and licorice chalcone E.

[0012] Preferably, the regulator comprises the following components by mass: 25% calcium lactate, 20% sodium lactate, 0.3% hydrogen peroxide and 8% mannose, with the balance being water.

[0013] Preferably, the volume ratio of the Clostridium butyricum fermentation broth to the regulator is 100:(6-9).

[0014] Preferably, the initial pH of the regulated culture is 6.5, the temperature is 40° C., and the time is 1.5 h.

[0015] Preferably, the heating temperature is 70° C. and the heating time is 30 minutes.

[0016] Preferably, the protective agent comprises the following components by mass: 15% sodium sulfide, 24% glutathione, 20% sodium pyruvate and 0.5% licorice chalcone E, with the balance being water.

[0017] Preferably, the volume ratio of the fermentation broth to the protective agent is 100:(2-4).

[0018] The present invention also provides Clostridium butyricum powder obtained according to the above preparation method.

[0019] The present invention also provides a use of the Clostridium butyricum powder in preparing a probiotic preparation.

[0020] The present invention also provides a probiotic preparation, which contains the above-mentioned Clostridium butyricum powder as a main active ingredient.

[0021] The present invention discloses the following technical effects:

[0022] The present invention discloses a method for preparing bacterial powder of Clostridium butyricum, and belongs to the technical field of bacterial powder preparation. The bacterial powder preparation method provided by the present invention comprises the steps of adding a regulator for regulated culture, heating, and adding a protective agent for freeze-drying. Experimental results show that the bacterial powder preparation method provided by the present invention has a high live bacteria extraction rate, and reduces the loss of Clostridium butyricum in the bacterial powder preparation process; the bacterial powder obtained according to the bacterial powder preparation method still has a retention rate of more than 90% after long-term storage for 180 days at 35°C, indicating that the present invention significantly improves the survival ability of Clostridium butyricum. The bacterial powder preparation method of the present invention has a simple process, is easy to operate, is green and environmentally friendly, and does not significantly increase the cost. The present invention provides a new strategy for the preparation and vitality maintenance of microecological products of Clostridium butyricum, can be applied to the animal husbandry industry and the field of medical care, and has broad application prospects. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] Regarding the fermentation medium, the optional technical solutions in the prior art include: a fermentation medium for Clostridium butyricum, which includes, calculated by mass: 3 to 8 parts of a fast-acting carbon source, 22.5 to 34 parts of a slow-acting carbon source, 7 to 15 parts of yeast extract, 5 to 10 parts of fermented soybean meal, 5 to 10 parts of sodium acetate, 2 to 5 parts of disodium hydrogen phosphate, 0.2 to 1.0 parts of calcium chloride, 0.5 to 2.0 parts of magnesium sulfate, 0.1 to 0.3 parts of ferrous sulfate, and 0.2 to 0.3 parts of manganese sulfate. A "fast-acting carbon source" refers to a carbon-containing compound that can be directly utilized by Clostridium butyricum. A fast-acting carbon source can quickly participate in bacterial synthesis, energy production, and synthetic metabolites. For example, a fast-acting carbon source can be glucose. A "slow-acting carbon source" refers to a carbon-containing compound that cannot be directly absorbed and utilized by Clostridium butyricum. It is usually utilized by Clostridium butyricum in the absence of a fast-acting carbon source and relies on Clostridium butyricum to secrete extracellular enzymes to break it down into small molecules for utilization.

[0029] Regarding the preparation method of bacterial powder, the available technical solutions in the prior art include: a method of preparing bacterial powder containing Clostridium butyricum using pea protein process wastewater and Clostridium butyricum, specifically comprising the following steps: S1. Centrifugal removal: Take the pea protein process wastewater and filter it using a centrifuge to remove insoluble matter. S2. Evaporation concentration: Evaporate the pea protein process wastewater until the soluble solids therein are concentrated to the fermentation conditions; specifically, the soluble solids content in the pea protein process wastewater before evaporation is 1.5%-2.5% (mass fraction), and the soluble solids in the pea protein process wastewater after evaporation should be ensured to be concentrated to 15-25% (mass fraction). S3. Steam Sterilization: Add other nutrients, including sodium acetate, dipotassium phosphate, and magnesium sulfate, to the evaporatively concentrated pea protein process wastewater. For each liter of concentrated pea protein process wastewater, add 0.2g of sodium acetate, 0.1g of dipotassium phosphate, and 0.05g of magnesium sulfate. The pH is then adjusted to neutral, and after sterilization at 121-135°C, the pea protein wastewater is transferred to a fermentation tank. S4. Anaerobic Fermentation: Inoculate the fermentation tank with a pre-cultured strain of Clostridium butyricum, adjust the culture temperature to approximately 37°C, and conduct anaerobic fermentation. Sterile nitrogen is used to deoxygenate the fermentation process, and fermentation is terminated after the cells mature. S5. Cell Collection: After fermentation, the fermentation broth is subjected to solid-liquid separation. The filtered solid bacterial sludge is then transferred to an embedding tank for microencapsulation. S6. Spray drying: Using the bacteria as the core material and modified starch as the wall material, microencapsulation and embedding are carried out in a homogenization tank. After the reaction is completed, the homogenized material enters the spray drying tower through an atomizer and is spray dried.

[0030] Regarding the preparation method of bacterial powder, the technical solutions available in the prior art also include: S1. Concentrating the Clostridium butyricum fermentation broth to form a Clostridium butyricum slurry, adding an active protective agent to the Clostridium butyricum slurry, and mixing to obtain a mixed suspension; the active protective agent comprises the following components in percentage by weight: 4-7% peptone, 1-4.5% sodium chloride, 2-10% glucose, 2-6% beef extract powder, 2-4% glycerol, 1-3% corn starch, 0.3-3% sodium alginate, 0.1-1% cysteine ​​salt, and 0.5-1.5% dipotassium hydrogen phosphate, with the balance being water; the use of the active protective agent in the above ratio can stabilize the cell protein structure of Clostridium butyricum, increase the heat resistance and tolerance of Clostridium butyricum, and at the same time protect Clostridium butyricum during low-temperature electrostatic spray drying, so that the obtained Clostridium butyricum dry powder has a high number of effective live bacteria and good storage stability. S2. Low-temperature electrostatic spray drying the mixed suspension to obtain Clostridium butyricum dry powder. The inlet air temperature of low-temperature electrostatic spray drying is 80-90℃, and the outlet air temperature is 35-45℃; the electrostatic pressure of low-temperature electrostatic spray drying is 14-25kV, the liquid inlet pump speed is 25-35rpm, and the atomization pressure is 160-190kPa.

[0031] Regarding the preparation method of bacterial powder, the optional technical solutions in the existing technology also include: a preparation method of Clostridium butyricum powder, which comprises pre-treating the Clostridium butyricum fermentation broth by centrifugation, controlling the centrifugal separation parameters, centrifuging at a speed of 3000-5000 r / min, and centrifuging for 5-30 minutes; after centrifugation, discarding the supernatant, adding sterile liquid, and then adding a protective agent, which is preferably a composition of sodium alginate, maltodextrin, chitosan oligosaccharide, calcium lactate, sodium carboxymethyl cellulose and silicon dioxide, wherein the amount of maltodextrin added is 5%-30% of the mass of the fermentation broth, the amount of sodium alginate added is 5%-30% of the mass of the fermentation broth, the amount of chitosan oligosaccharide added is 1%-10% of the mass of the fermentation broth, the amount of calcium lactate added is 1%-10% of the mass of the fermentation broth, the amount of sodium carboxymethyl cellulose added is 0.05%-0.5% of the mass of the fermentation broth, and the amount of silicon dioxide added is 0.05%-0.5% of the mass of the fermentation broth. The spray drying is carried out, and the inlet temperature of the spray drying is 150-180°C.

[0032] The Clostridium butyricum of the present invention was purchased from the China Center for Type Culture Collection with a collection number of CCTCCAB 2017089. The public can purchase the strain from the above collection institution.

[0033] The Clostridium butyricum fermentation culture medium of the present invention is RCM culture medium, which is purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0034] The components of RCM solid culture medium are: tryptone 10g / L, beef powder 10g / L, yeast powder 3g / L, glucose 5g / L, soluble starch 1g / L, sodium chloride 5g / L, sodium acetate 3g / L, L-cysteine ​​hydrochloride 0.5g / L, agar powder 20g / L, pH = 6.8-7.0.

[0035] The components of RCM liquid culture medium are: tryptone 10g / L, beef powder 10g / L, yeast powder 3g / L, glucose 5g / L, soluble starch 1g / L, sodium chloride 5g / L, sodium acetate 3g / L, L-cysteine ​​hydrochloride 0.5g / L, pH = 6.8-7.0.

[0036] The present invention provides a method for preparing a Clostridium butyricum fermentation broth, comprising inoculating Clostridium butyricum into an RCM liquid culture medium, setting the initial pH to 7.0, and anaerobic culturing at 37°C. After culturing for 6 hours, samples are taken every 2 hours to measure the pH of the fermentation broth. When the pH drops below 5.20, or when the pH does not decrease within 2 hours, fermentation is stopped and the fermentation broth is collected.

[0037] Example 1

[0038] Take 25 g of calcium lactate, 20 g of sodium lactate, 1 g of 30% hydrogen peroxide solution and 8 g of mannose, mix them with 46 mL of water to prepare a regulator.

[0039] Take 15 g of sodium sulfide, 24 g of glutathione, 20 g of sodium pyruvate and 0.5 g of licorice chalcone E, mix them with 40.5 mL of water to prepare a protective agent.

[0040] Unless otherwise specified, the regulators and protective agents in the examples of the present invention are prepared according to the methods in these examples.

[0041] Example 2

[0042] Take 7 mL of the regulator and add it to 100 mL of Clostridium butyricum fermentation broth, mix well, add sodium bicarbonate to adjust the pH to 6.5, incubate anaerobically at 40°C for 1.5 h, heat at 70°C for 30 min, cool to room temperature, centrifuge at 4000 rpm for 15 min at 4°C, collect the precipitate, add 3 mL of the protective agent, precool at -80°C for 1 h, freeze-dry at -80°C for 36 h (vacuum degree is 25 Pa), and collect the dry bacterial powder.

[0043] Example 3

[0044] The only difference from Example 2 is that the amount of the regulator added is 9 mL and the amount of the protective agent added is 2 mL.

[0045] Example 4

[0046] The only difference from Example 2 is that the amount of the regulator added is 6 mL and the amount of the protective agent added is 4 mL.

[0047] Example 5

[0048] The only difference from Example 2 is that the amount of the regulator added is 8 mL, and the amount of the protective agent added is 3.5 mL.

[0049] Comparative Example 1

[0050] The only difference from Example 2 is that 7 mL of regulator is replaced by 3 mL of regulator.

[0051] Comparative Example 2

[0052] The only difference from Example 2 is that 3 mL of protective agent is replaced by 1 mL of protective agent.

[0053] Comparative Example 3

[0054] The only difference from Example 2 is that the freeze-drying process is replaced by a spray-drying process.

[0055] Spray drying conditions: inlet temperature 120 °C, flow rate 130 mL / h, rotation speed 50 r / min, pressure 1.0 × 10 4 Pa, outlet temperature 100℃.

[0056] Comparative Example 4

[0057] Take 7 mL of the regulator, add it to 100 mL of Clostridium butyricum fermentation broth, mix well, add sodium bicarbonate to adjust the pH to 6.5, incubate anaerobically at 40°C for 1.5 h, heat at 70°C for 30 min, cool to room temperature, centrifuge at 4000 rpm for 15 min at 4°C, collect the precipitate, add 7 g of maltodextrin, 0.6 g of sodium cysteine, 0.4 g of glycerol and 2 g of sodium alginate, precool at -80°C for 1 h, freeze-dry at -80°C for 36 h (vacuum degree is 25 Pa), and collect the dry bacterial powder.

[0058] Comparative Example 5

[0059] Take 7 mL of the regulator and add it to 100 mL of Clostridium butyricum fermentation broth, mix well, add sodium bicarbonate to adjust the pH to 6.5, incubate anaerobically at 40°C for 1.5 h, heat at 70°C for 30 min, cool to room temperature, centrifuge at 4000 rpm for 15 min at 4°C, collect the precipitate, add 7 g of peptone, 0.6 g of ferrous sulfate, 0.4 g of glucose and 2 g of corn flour, precool at -80°C for 1 h, freeze-dry at -80°C for 36 h (vacuum degree is 25 Pa), and collect the dry bacterial powder.

[0060] Experimental Example 1

[0061] 1. Experimental Methods

[0062] The viable bacteria content in the bacterial powders prepared in Examples 2-5 and Comparative Examples 1-5 was measured immediately after the bacterial powders were prepared (immediate detection), after storage at 35° C. for 30 days, and after storage at 35° C. for 180 days.

[0063] Determination method:

[0064] Accurately weigh 1.00 g of bacterial powder and dilute it 10-fold using RCM liquid medium. Transfer the appropriately diluted sample to RCM solid medium, spread on plates, and incubate anaerobically at 37°C for 24 h. Count the colonies on the plates and calculate the viable bacterial count (CFU / g of powder). Perform three biological replicates for each group of powder.

[0065] The retention rate is calculated according to the following formula:

[0066] Retention rate = 100 × viable bacteria content after 180 days of storage at 35°C / live bacteria content detected immediately.

[0067] 2. Experimental Results

[0068] The test results of the live bacteria content in the bacterial powder are shown in Table 1. When the bacterial powder was just prepared, the content of Clostridium butyricum in the bacterial powder prepared in Examples 2-5 was significantly greater than that in the bacterial powder prepared in Comparative Examples 1-5, indicating that the method for preparing Clostridium butyricum powder provided by the present invention has a better live bacteria yield and effectively reduces the loss of Clostridium butyricum caused by the drying process.

[0069] After short-term (30 days) and long-term (180 days) storage at 35°C, the retention rate of the live Clostridium butyricum content in the bacterial powder prepared in Example 2-5 remained above 90%, which was significantly greater than that in Comparative Example 2-5, indicating that the bacterial powder preparation method provided by the present invention significantly improves the survival ability of Clostridium butyricum in the bacterial powder state.

[0070] Table 1 Statistical results of viable bacteria content at different storage periods

[0071]

[0072] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing Clostridium butyricum powder, characterized in that: The following steps are involved: mixing the Clostridium butyricum fermentation liquid with a regulator, regulating the culture, and heating to obtain a mixed liquid; Centrifuging the mixed solution to prepare bacterial sludge, mixing the bacterial sludge with a protective agent, and freeze-drying to obtain the Clostridium butyricum powder; Wherein, the regulator includes calcium lactate, sodium lactate, hydrogen peroxide and mannose; The protective agents include sodium sulfide, glutathione, sodium pyruvate and licorice chalcone E.

2. The preparation method according to claim 1, wherein The regulator comprises the following components by mass: 25% calcium lactate, 20% sodium lactate, 0.3% hydrogen peroxide, 8% mannose and the balance water.

3. The preparation method according to claim 2, wherein The volume ratio of the Clostridium butyricum fermentation broth to the regulator is 100:(6-9).

4. The preparation method according to claim 1, wherein The initial pH of the regulated culture was 6.5, the temperature was 40° C., and the time was 1.5 h.

5. The preparation method according to claim 1, wherein The heating temperature is 70° C. and the heating time is 30 minutes.

6. The preparation method according to claim 1, wherein The protective agent comprises the following components by mass fraction: 15% sodium sulfide, 24% glutathione, 20% sodium pyruvate, 0.5% licorice chalcone E and the balance water.

7. The preparation method according to claim 6, wherein The volume ratio of the fermentation broth to the protective agent is 100:(2-4).

8. Clostridium butyricum powder obtained according to the preparation method according to any one of claims 1 to 7.

9. Use of the Clostridium butyricum powder according to claim 8 in the preparation of a probiotic preparation.

10. A probiotic preparation, characterized in that: The probiotic preparation contains the Clostridium butyricum powder described in claim 8 as a main active ingredient.

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