Preparation method of viable clostridium butyricum preparation

Abstract

The invention relates to a preparation method of a clostridium butyricum viable bacteria preparation, which comprises the following steps: sequentially adding sterile water, corn starch, sodium glutamate, glutamine and Arabic gum into bacterial sludge obtained after clostridium butyricum fermentation liquor is centrifuged, uniformly mixing, and spray-drying to obtain clostridium butyricum bacterial powder, glutamine is used as a nutrition enhancer; arabic gum is used as a wall material; the prepared viable bacterium preparation is good in stability and large in viable bacterium number, and the storage validity period and the survival rate of the viable bacterium preparation can be obviously improved.

Description

Technical Field

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

[0002] Probiotics are active microorganisms that have a beneficial effect on animals by improving the balance of intestinal microecology. Probiotic preparations have become an ideal alternative to antibiotics due to their many advantages such as low toxicity, no residue, no drug resistance, and significant effects.

[0003] Clostridium butyricum, also known as butyric acid bacteria or butyric acid bacteria, is an anaerobic Gram-positive bacterium widely found in the intestines of animals and humans and in the soil. Clostridium butyricum can improve the immune performance of animal bodies, and has probiotic properties such as regulating the balance of intestinal microecology, enhancing immunity, and producing prebiotics. The main metabolic product of Clostridium butyricum is butyric acid, which can help maintain the morphology and function of intestinal epithelial cells and enhance the intestinal barrier function. The butyric acid clostridium (antimicrobial peptide) produced by Clostridium butyricum can significantly inhibit some common intestinal pathogens; Clostridium butyricum can also produce amylase, cellulase and various vitamins to provide nutrients for animal bodies and other probiotics. Therefore, Clostridium butyricum can maintain the balance of intestinal microecology, thereby maintaining animal intestinal health and improving production performance.

[0004] Probiotics need to pass through the strong acidic environment of the stomach to work in the intestines. Ensuring that the number of live bacteria remains high after entering the intestines is the key to the efficacy of probiotics. Probiotic preparations are mainly available in two forms: liquid and solid. Solid forms can be divided into powders, granules, capsules, tablets, etc. Liquid preparations usually have disadvantages such as short shelf life, inconvenient transportation, and easy contamination, while solid preparations have a relatively long shelf life, high live bacteria count, easy transportation and carrying, and are convenient to use. Common methods for preparing probiotic powder in solid dosage forms include natural drying, low-temperature drying, vacuum freeze drying, and spray drying. Spray drying refers to a drying technology in which liquid raw materials are dispersed into fine droplets through an atomizer, and the solvent components of the droplets in the drying medium evaporate rapidly to form dry powder. The characteristics of spray drying technology are: short drying time, less damage to the active ingredients in the product, and a simple drying process.

[0005] At present, the way to apply Clostridium butyricum to feed in the market is to prepare it as a live bacteria preparation. The live bacteria preparation of Clostridium butyricum has low live bacteria content, low survival rate during drying, poor storage stability under normal conditions, and easy inactivation. The application is not ideal. For example, CN200610125586.X directly uses plant fiber powder to dilute the fermentation liquid. The live bacteria preparation prepared by this process has a low number of live bacteria; CN201610927003.9 uses a freeze-drying method to prepare a live bacteria preparation of Clostridium butyricum. This process consumes a lot of energy, is costly, and has limited production capacity, which is not conducive to industrial large-scale production. Compared with other methods, spray drying has its unique advantages. Although spray drying is a more commonly used process, for different effective ingredients, such as Clostridium butyricum liquid products with poor stability, how to provide a bacterial powder preparation process with high live bacteria count and good stability is still a problem that needs to be solved. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the present invention discloses a method for preparing a live Clostridium butyricum preparation with high live bacterial count and good stability.

[0007] The present invention discloses a method for preparing a live Clostridium butyricum preparation, which comprises: S1: inoculating a glycerol tube containing Clostridium butyricum into RCM medium, anaerobic culture to obtain a seed solution, inoculating the Clostridium butyricum seed solution into a Clostridium butyricum fermentation medium, and fermenting under anaerobic conditions to prepare a fermentation solution of Clostridium butyricum; S2: taking the fermentation broth of Clostridium butyricum and subjecting it to centrifugation, discarding the supernatant to obtain bacterial sludge; S3: Add sterile water to the bacterial sludge for concentration; S4: adding a carbohydrate protective agent, an amino acid protective agent, a nutrient enhancer and a capsule material in sequence, stirring for a period of time, and then spray drying to obtain a live Clostridium butyricum preparation; Wherein, the carbohydrate protective agent is at least one of corn starch, maltodextrin or trehalose; and the amino acid protective agent is sodium glutamate.

[0008] Furthermore, the Clostridium butyricum fermentation medium in S1 is 20-30 glucose, 10-15 yeast extract, 5-10 corn flour, 10-15 corn steep liquor, 2-5 sodium bicarbonate, 0.1-0.5 magnesium sulfate, 0.5-1 calcium chloride and 0.5-1 ferrous sulfate in parts by weight. The Clostridium butyricum fermentation medium is prepared by weighing the above components in proportion, adding them into deionized water and stirring to dissolve, adjusting the pH value to 7.0-7.2 with HCl or NaOH, and packaging the prepared Clostridium butyricum fermentation medium into a fermentation tank for high-pressure steam sterilization. The sterilized Clostridium butyricum fermentation medium is cooled to room temperature for use.

[0009] Furthermore, the temperature of high-pressure steam sterilization is 110-130° C., and the sterilization time is 15-25 min.

[0010] Further, the specific method of S1 is to inoculate the glycerol tube containing Clostridium butyricum into RCM liquid culture medium at 35-37°C, anaerobically culture for 16-20h to obtain seed liquid, inoculate the seeds into Clostridium butyricum fermentation medium, the inoculation amount is 1-5%, at a temperature of 37°C±1°C, the pH is maintained at 6.5±0.5 with liquid alkali, stir and mix at a stirring speed of 180-220r / min, and add sugar solution 7-9h after the start of fermentation, the amount of added sugar is 2%-3% of the total mass of Clostridium butyricum fermentation medium, the sugar supplement time is controlled within 4-6h, culture for 24-30h, and the fermentation is terminated when the microscopic spore rate reaches more than 90%, thereby obtaining Clostridium butyricum fermentation liquid; The centrifugal treatment method in S2 includes high-speed centrifugation, disc centrifugation or plate and frame filtration. The centrifugal treatment method is high-speed centrifugation with a centrifugal speed of 4000-6000r / min and a centrifugal treatment time of 5-30min. Add sterile liquid to S3 to concentrate it 3-10 times; The inlet temperature of the spray drying in S4 is 150-180°C, and the outlet temperature is 70-85°C; The stirring speed of the stirring process in S4 is 150-250 r / min, and the stirring time is 20-30 min.

[0011] Further, the nutrition enhancer is at least one of glutamine or vitamin C; The capsule material is at least one of gum arabic or gelatin.

[0012] Furthermore, the amount of the protective agent added is 10%-30% of the mass of the concentrated solution finally obtained in S3; The amount of the nutrient enhancer added is 0.2%-1.5% of the mass of the concentrated solution finally obtained in S3; The amount of capsule material added is 0.5%-7% of the mass of the concentrated solution finally obtained in S3.

[0013] Furthermore, the protective agent is selected from corn starch and sodium glutamate, the amount of corn starch added is 10%-20% of the mass of the concentrated solution finally obtained in S3, and the amount of sodium glutamate added is 5%-10% of the mass of the concentrated solution finally obtained in S3; The nutritional enhancer is glutamine, and the amount of glutamine added is 0.1%-1% of the mass of the concentrated solution finally obtained in S3; The capsule material is gum arabic, and the amount of gum arabic added is 1%-5% of the mass of the concentrated liquid finally obtained in S3.

[0014] Furthermore, the protective agent is selected from corn starch and sodium glutamate, the amount of corn starch added is 15% of the mass of the concentrated solution finally obtained in S3, and the amount of sodium glutamate added is 8% of the mass of the concentrated solution finally obtained in S3; Glutamine was selected as the nutritional enhancer, and the amount of glutamine added was 0.2% of the mass of the concentrated solution finally obtained in S3; The capsule material is gum arabic, and the amount of gum arabic added is 3% of the mass of the concentrated liquid finally obtained in S3.

[0015] By means of the above technical solution, the present invention has at least the following advantages: 1. The Clostridium butyricum preparation prepared by the method of the present invention has a high number of effective live bacteria, and the live bacteria retention rate is still greater than 90% after being stored for 6 months at room temperature. The method of the present invention is simple to operate, low in cost, and highly controllable. The obtained Clostridium butyricum powder product has a high live bacteria retention rate and good product storage stability, which plays an important role in the application and promotion of Clostridium butyricum.

[0016] 2. The protective agent in the method of the present invention is considered from various aspects. The composite protective agent including sugars and amino acids can comprehensively protect Clostridium butyricum from possible damage during the spraying process. Corn starch has a fine particle structure, good fluidity, good hygroscopicity and water retention, which helps to maintain the stability of the bacterial powder and extend the shelf life. Sodium glutamate can regulate the electrolyte balance inside and outside the cell membrane of Clostridium butyricum.

[0017] 3. The present invention uniquely adopts gum arabic as the raw material of the wall material, which has good emulsification and film-forming properties, can effectively wrap the bacteria, form an impermeable film, prevent the entry of air, has an antioxidant effect, and ensures the stability of the bacterial powder. At the same time, compared with gelatin, sodium alginate, xanthan gum, etc., it is easy to dissolve, can reduce the difficulty of operation, and simplify the process; the addition of gum arabic can reduce the heat stress of the probiotics during the drying process, and can reduce the oxidative stress of the probiotics during the storage process, greatly reduce the loss rate during the drying process, and improve the survival rate and stability during the storage process. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The present invention is a flow chart of a method for preparing a live Clostridium butyricum preparation in an embodiment of the present application.

[0019] Figure 2 This is a diagram of Clostridium butyricum powder in an embodiment of the present application. DETAILED DESCRIPTION

[0020] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the specific implementation mode of the present invention will be clearly and completely described below. Unless otherwise specified, the equipment and reagents used in each embodiment and comparative example can be obtained from commercial channels.

[0021] The present invention discloses a method for preparing a live Clostridium butyricum preparation, which comprises: S1: inoculating a glycerol tube containing Clostridium butyricum into RCM medium, anaerobic culture to obtain a seed solution, inoculating the Clostridium butyricum seed solution into a Clostridium butyricum fermentation medium, and fermenting under anaerobic conditions to prepare a fermentation solution of Clostridium butyricum; S2: taking the fermentation broth of Clostridium butyricum and subjecting it to centrifugation, discarding the supernatant to obtain bacterial sludge; S3: Add sterile water to the bacterial sludge for concentration; S4: adding a carbohydrate protective agent, an amino acid protective agent, a nutrient enhancer and a capsule material in sequence, stirring for a period of time, and then spray drying to obtain a live Clostridium butyricum preparation; Wherein, the carbohydrate protective agent is at least one of corn starch, maltodextrin or trehalose; and the amino acid protective agent is sodium glutamate.

[0022] Through the optimized preparation process, the present invention can ensure that the Clostridium butyricum in the final product maintains a high number of live bacteria, and after being stored for 6 months at room temperature, the live bacteria retention rate can still be maintained at more than 90%. The problem of low live bacteria content and poor storage stability in the prior art is solved. The method of the present invention is simple to operate, low in cost, strong in controllability, and suitable for large-scale industrial production. Compared with other methods such as freeze drying, the spray drying method not only reduces energy consumption and cost, but also increases production capacity, which is more conducive to market promotion and application. The preparation process of the present invention is simple and easy, does not require complex equipment or special technology, and reduces production costs. At the same time, by optimizing the process flow, the entire production process is made more economical and efficient, which is conducive to large-scale industrial production. A composite protective agent composed of sugars such as corn starch, maltodextrin or trehalose and amino acids such as glutamic acid is used, and these ingredients can provide comprehensive protection to Clostridium butyricum at different levels, reduce its damage during the spray drying process, and improve the survival rate. In particular, the fine granular structure, good fluidity and hygroscopicity of corn starch help maintain the stability of the bacterial powder and extend the shelf life, while sodium glutamate regulates the balance of electrolytes inside and outside the cell membrane, further enhancing the protective effect.

[0023] As an embodiment, the butyric clostridium fermentation medium in S1 is 20-30 glucose, 10-15 yeast extract, 5-10 corn flour, 10-15 corn steep liquor, 2-5 sodium bicarbonate, 0.1-0.5 magnesium sulfate, 0.5-1 calcium chloride and 0.5-1 ferrous sulfate, and the method for preparing the butyric clostridium fermentation medium is to weigh the above components in proportion, add them to deionized water and stir to dissolve, use HCl or NaOH to adjust the pH value to 7.0-7.2, and divide the prepared butyric clostridium fermentation medium into a fermentation tank, sterilize it with high pressure steam, and cool the sterilized butyric clostridium fermentation medium to room temperature for standby use. The fermentation medium contains glucose as the main carbon source, yeast extract provides a rich nitrogen source and vitamins, and corn flour and corn steep liquor provide additional carbohydrates and other growth factors for microorganisms. This combination can meet the various nutrients required for the growth and reproduction of butyric clostridium, and promote the rapid proliferation of butyric clostridium. Use HCl or NaOH to adjust the pH value to 7.0-7.2 to ensure the appropriate initial fermentation conditions for Clostridium butyricum, which helps maintain the efficient metabolism of Clostridium butyricum to produce butyric acid and other beneficial products. Add appropriate amounts of magnesium sulfate, calcium chloride and ferrous sulfate. These trace elements promote the normal physiological production of Clostridium butyricum. Magnesium ions participate in the formation of the active center of the enzyme; calcium ions play an important role in the stability of the cell wall structure and signal transduction; iron is a cofactor for many enzymes, ensuring the healthy growth and metabolic function of Clostridium butyricum. Ferrous sulfate, as a reducing agent, can reduce the oxygen content in the fermentation broth and provide a low-oxygen environment for the initial growth of Clostridium butyricum, which is more suitable for its growth. High-pressure steam sterilization completely removes the risk of contamination by other bacteria and ensures aseptic conditions during the fermentation process. It can improve the product quality of Clostridium butyricum and avoid the problem of reduced yield or unstable quality due to the presence of competitive microorganisms.

[0024] As an implementation method, the temperature of high-pressure steam sterilization is 110-130°C, and the sterilization time is 15-25 minutes. Within the temperature range of 110-130°C, high-pressure steam can quickly destroy the microbial cell structure, ensuring that any pathogens or competitive microorganisms that may exist in the culture medium are completely killed. This ensures that Clostridium butyricum will not be interfered with by other microorganisms during the subsequent fermentation process, thereby improving the fermentation efficiency and product quality.

[0025] As an embodiment, the specific method of S1 is to inoculate a glycerol tube containing Clostridium butyricum into an RCM liquid culture medium at 35-37°C, anaerobically culture for 16-20h to obtain a seed solution, inoculate the seeds into a Clostridium butyricum fermentation medium, the inoculation amount is 1-5%, at a temperature of 37°C±1°C, the pH is maintained at 6.5±0.5 with liquid alkali, stir and mix at a stirring speed of 180-220r / min, and add a sugar solution 7-9h after the start of fermentation, the amount of sugar added is 2%-3% of the total mass of the Clostridium butyricum fermentation medium, the sugar supplement time is controlled within 4-6h, and the culture is continued for 1-2 hours. After 24-30 hours of cultivation, the fermentation is terminated when the microscopic spore rate reaches more than 90%, and the fermentation liquid of Clostridium butyricum is obtained. The centrifugal treatment method in S2 includes high-speed centrifugation, disc centrifugation or plate-frame filtration. The centrifugal treatment method is high-speed centrifugation with a centrifugal speed of 4000-6000r / min and a centrifugal treatment time of 5-30min. Sterile liquid is added in S3 for concentration by 3-10 times. The inlet temperature of the spray drying in S4 is 150-180°C, the outlet temperature is 70-85°C, and the stirring speed of the stirring treatment in S4 is 150-250r / min, and the stirring time is 20-30min. The present invention is anaerobically cultured for 16-20 hours at 35-37°C to ensure that Clostridium butyricum proliferates rapidly at the optimal growth temperature to form a high-concentration seed liquid. The inoculation amount is 1-5%, which ensures sufficient starting cell density; at the same time, through stirring and mixing and pH maintenance, an ideal fermentation environment is provided, which promotes the effective growth of Clostridium butyricum and the generation of metabolites. Sugar solution is added 7-9 hours after the start of fermentation, and the amount of sugar supplement is 2%-3% of the total mass, and the sugar supplement is completed within 4-6 hours. It can avoid the inhibitory effect of the initial excessively high sugar concentration on Clostridium butyricum, and at the same time ensure sufficient carbon source supply during the entire fermentation process, thereby improving the final yield and product quality. Three options are provided: high-speed centrifugation, disc centrifugation or plate and frame filtration. The speed of high-speed centrifugation is 4000-6000r / min and the time is 5-30 minutes. To ensure efficient cell separation, the process parameters can be adjusted according to different needs to obtain the best bacterial sludge quality. Sterile water is added for 3-10 times of concentration to maintain the activity of the bacterial sludge. The inlet temperature is 150-180℃ and the outlet temperature is 70-85℃. Such temperature settings can not only effectively remove moisture, but also minimize the impact of high temperature on Clostridium butyricum and its metabolites, and protect their activity. The stirring speed is 150-250r / min, and the stirring time is 20-30 minutes, so that the protective agent and the capsule material are evenly dispersed in the bacterial mud to form a stable particle structure, thereby enhancing the stability and preservation performance of the product. Through the above-mentioned strict operating conditions and optimized process parameters, the present invention can prepare a live Clostridium butyricum preparation with high viable count, good stability and high spore rate. The whole process is reasonably designed, easy to operate, suitable for large-scale industrial production, reduces production costs, and improves economic benefits.

[0026] As an embodiment, the nutritional enhancer is at least one of glutamine or vitamin C, and the capsule material is at least one of gum arabic or gelatin. Glutamine is an important substrate for cell energy metabolism, which is essential for maintaining cell membrane stability and intracellular pH balance, and can promote the rapid proliferation of Clostridium butyricum. Under high temperature conditions, glutamine can help maintain cell structural integrity and functional stability, thereby increasing the survival rate of Clostridium butyricum during the drying process. Vitamin C is a powerful antioxidant that can effectively neutralize free radicals and reduce the damage of oxidative stress to Clostridium butyricum cells, especially during spray drying. Vitamin C not only helps to protect Clostridium butyricum itself, but may also indirectly support the function of probiotics by enhancing the immune system of the animal body. Gum arabic can form a thin film that is difficult to penetrate on the surface of Clostridium butyricum to prevent air from entering, play an antioxidant effect, and ensure the long-term stability of the bacterial powder. Gum arabic can reduce the effect of heat on probiotics during the drying process, reduce the risk of oxidation during storage, and greatly improve the survival rate and product shelf life. Compared with other materials such as gelatin, gum arabic is easier to dissolve, simplifies the production process, and reduces the difficulty of operation. Gelatin is also a commonly used capsule material that can provide a certain physical barrier to protect the Clostridium butyricum inside from the influence of the external environment. Gelatin has good biocompatibility and will not cause adverse reactions to animals. It is suitable for a variety of applications. By adding glutamine or vitamin C as a nutritional enhancer, the vitality and tolerance of Clostridium butyricum can be further improved to ensure its survival ability under adverse conditions. Choosing gum arabic or gelatin as a capsule material not only enhances the physical protection of the product, but also simplifies the production process and improves efficiency.

[0027] As an embodiment, the amount of protective agent added is 10%-30% of the mass of the final concentrated solution obtained in S3, the amount of nutritional enhancer added is 0.2%-1.5% of the mass of the final concentrated solution obtained in S3, and the amount of capsule material added is 0.5%-7% of the mass of the final concentrated solution obtained in S3. The amount of protective agent added is 10%-30% of the mass of the concentrated solution to ensure that there is enough protective agent to cover all Clostridium butyricum cells and reduce the damage caused by high temperature and mechanical stress during the spray drying process. Under the premise of ensuring the protective effect, excessive use of protective agent is avoided to control production costs. The ratio of 10%-30% can provide effective protection without increasing unnecessary expenses due to excessive protective agent. The amount of nutritional enhancer added is 0.2%-1.5% of the mass of the concentrated solution, which can effectively supplement the specific nutrients required by Clostridium butyricum and promote its growth and metabolic activities without causing waste or negative effects due to excessive addition. The addition of capsule material at 0.5%-7% of the concentrate mass can form a stable protective film on the surface of Clostridium butyricum, which not only prevents the influence of the external environment, but also provides additional protection in the gastric acid environment, ensuring that more live bacteria can safely reach the intestines and play a role. By precisely controlling the addition amount of protective agent, nutritional enhancer and capsule material, the high live bacteria count, good stability and efficient probiotic function of Clostridium butyricum live bacteria preparation are ensured. Reasonable ratio avoids material waste, reduces production costs, and at the same time ensures product quality, achieving the best balance between economic benefits and product performance.

[0028] As an implementation method, corn starch and sodium glutamate are selected as protective agents, and the amount of corn starch added is 10%-20% of the mass of the final concentrated solution obtained in S3, and the amount of sodium glutamate added is 5%-10% of the mass of the final concentrated solution obtained in S3. Glutamine is selected as a nutritional enhancer, and the amount of glutamine added is 0.1%-1% of the mass of the final concentrated solution obtained in S3. Gum arabic is selected as a capsule material, and the amount of gum arabic added is 1%-5% of the mass of the final concentrated solution obtained in S3. The amount of corn starch added is 110%-20% of the mass of the concentrated solution, which can form a physical barrier during the spray drying process, reduce the direct damage of high temperature and mechanical stress to Clostridium butyricum cells, and improve cell survival rate. Corn starch has good hygroscopicity and water retention, which helps to maintain the moisture balance of bacterial powder, prevent cell inactivation caused by excessive drying, and extend the shelf life of the product. Corn starch particles are fine and have good fluidity, which is easy to disperse evenly during the spray drying process to ensure that each bacterial cell can be effectively protected. Sodium glutamate can regulate the balance of electrolytes inside and outside cells, reduce the impact of osmotic pressure changes on cell membrane structure, and thus improve the stability of cells under extreme conditions. Glutamine is an important substrate for cell energy metabolism. The appropriate addition of 0.1%-1% can effectively support the growth and metabolic activities of Clostridium butyricum without affecting the overall formula, especially maintaining its activity during storage. Glutamine helps maintain the stability and functional integrity of the cell membrane, provides additional protection in high temperature and oxidative environments, and ensures that more live bacteria can safely reach the animal intestine and play a role. Gum arabic can form an impermeable film on the surface of Clostridium butyricum to prevent air from entering, play an antioxidant effect, and ensure the long-term stability of the bacterial powder. The ratio of 1%-5% provides sufficient protection without affecting the convenience of subsequent operations. During the spray drying process, gum arabic can effectively reduce the impact of heat on probiotics and reduce the risk of oxidation during storage, greatly improving the survival rate and shelf life of the product. Compared with other materials such as gelatin, gum arabic is easier to dissolve, simplifies the production process, reduces the difficulty of operation, and improves production efficiency. By precisely controlling the addition amount of corn starch, sodium glutamate, glutamine and gum arabic, the high viable count, good stability and efficient probiotic function of the Clostridium butyricum live bacteria preparation are ensured. The reasonable ratio avoids the waste of materials, reduces the production cost, ensures the product quality, and achieves the best balance between economic benefits and product performance.

[0029] As an implementation method, corn starch and sodium glutamate are selected as protective agents, and the amount of corn starch added is 15% of the mass of the final concentrated solution obtained in S3, and the amount of sodium glutamate added is 8% of the mass of the final concentrated solution obtained in S3. Glutamine is selected as a nutritional enhancer, and the amount of glutamine added is 0.2% of the mass of the final concentrated solution obtained in S3. Gum arabic is selected as a capsule material, and the amount of gum arabic added is 3% of the mass of the final concentrated solution obtained in S3. The amount of corn starch added is 15% of the mass of the concentrated solution, which can form a solid physical barrier during the spray drying process, effectively reduce the direct damage of high temperature and mechanical stress to Clostridium butyricum cells, and significantly improve the cell survival rate. Corn starch has good hygroscopicity and water retention, which helps to maintain the moisture balance of the bacterial powder, prevent excessive drying from causing cell inactivation, and thus extend the shelf life of the product. The ratio of 15% ensures sufficient moisture absorption capacity without making the product too wet. Corn starch particles are fine and have good fluidity, which is easy to disperse evenly during the spray drying process, ensuring that each bacterial cell can be effectively protected, while simplifying the production process. The addition of sodium glutamate is 8% of the concentrate mass, which can effectively regulate the electrolyte balance inside and outside the cell, reduce the impact of osmotic pressure changes on the cell membrane structure, and thus improve the stability of the cell under extreme conditions. As an amino acid, sodium glutamate has a certain antioxidant capacity, which can reduce the damage of free radicals to cells and further improve the stability of the product and the retention rate of live bacteria. The 8% ratio provides sufficient protection without affecting the functions of other ingredients due to excessive amounts. Glutamine is an important substrate for cellular energy metabolism. The addition of 0.2% can effectively support the growth and metabolic activities of Clostridium butyricum without affecting the overall formula, especially maintaining its activity during storage. Glutamine helps maintain the stability and functional integrity of the cell membrane, provides additional protection in high temperature and oxidative environments, and ensures that more live bacteria can safely reach the animal intestine and play a role. The right amount of glutamine not only provides the necessary nutritional support, but also avoids the risks of excessive addition. The addition of gum arabic is 3% of the concentrate mass, which can form an impermeable film on the surface of Clostridium butyricum to prevent air from entering, play an antioxidant effect, and ensure the long-term stability of the bacterial powder. This ratio can provide adequate protection without affecting the convenience of subsequent operations. During the spray drying process, gum arabic can effectively reduce the impact of heat on probiotics and reduce the risk of oxidation during storage, greatly improving the survival rate and shelf life of the product. The 3% ratio has been carefully calculated to achieve the best protection effect. By precisely controlling the specific addition amounts of corn starch, sodium glutamate, glutamine and gum arabic, the high viable count, good stability and efficient probiotic function of the live Clostridium butyricum preparation are ensured. Through the reasonable use of protective agents and capsule materials, the product can maintain a high viable count for a long time at room temperature, which is very important for transportation and storage in practical applications. Example

[0030] Preparation of Clostridium butyricum fermentation medium: The Clostridium butyricum fermentation medium is 20-30 glucose, 10-15 yeast extract, 5-10 corn flour, 10-15 corn steep liquor, 2-5 sodium bicarbonate, 0.1-0.5 magnesium sulfate, 0.5-1 calcium chloride and 0.5-1 ferrous sulfate in parts by weight. The Clostridium butyricum medium is prepared by weighing the above components in proportion, adding them into deionized water and stirring to dissolve, adjusting the pH value to 7.0-7.2 with HCl or NaOH, and filling the prepared Clostridium butyricum medium into a 5L fermentation tank with a liquid volume of 4L, sterilizing it with high-pressure steam at 121°C for 20 minutes, and cooling the sterilized Clostridium butyricum medium to room temperature for use.

[0031] The specific steps of S1 are: inoculating the glycerol tube containing Clostridium butyricum into RCM medium, anaerobic culture to obtain seed liquid, inoculating the seed liquid of Clostridium butyricum into Clostridium butyricum fermentation medium, fermenting under anaerobic conditions to prepare Clostridium butyricum fermentation liquid, and testing the number of live bacteria in the Clostridium butyricum fermentation liquid after the preparation. The number of live bacteria in the Clostridium butyricum fermentation liquid was 1.2×10 9 CFU / mL. Embodiment 1:

[0032] The preparation method of the live Clostridium butyricum preparation of this embodiment comprises the following steps: S1 prepares Clostridium butyricum fermentation broth, wherein the number of viable bacteria in the Clostridium butyricum fermentation broth is 1.2×10 9 CFU / mL; S2: taking the fermentation broth of Clostridium butyricum and centrifuging it at a speed of 5000 r / min for 10 min, and discarding the supernatant to obtain bacterial sludge; S3: adding sterile water to the bacterial sludge to concentrate it 5 times to obtain a concentrated solution; S4 then adds 15% corn starch by mass of the concentrated solution, 8% sodium glutamate by mass of the concentrated solution, 0.2% glutamine by mass of the concentrated solution and 3% gum arabic by mass of the concentrated solution in sequence, stirs at a speed of 200 r / min for 30 minutes, and then spray-dries. The inlet temperature of the spray-drying is 160°C, the outlet temperature of the spray-drying is 80°C, and the butyric acid Clostridium powder is obtained by spray drying.

[0033] Viable bacteria count test: refer to "NY / T 4347-2023 Feed Additive Clostridium butyricum" for viable bacteria count, directly prepare Clostridium butyricum powder for viable bacteria count test, and take a part of Clostridium butyricum powder and store it at room temperature for 6 months before viable bacteria count test. The results are shown in Table 1: Embodiment 2:

[0034] S1 prepares Clostridium butyricum fermentation broth, wherein the number of viable bacteria in the Clostridium butyricum fermentation broth is 1.2×10 9CFU / mL; S2: taking the fermentation broth of Clostridium butyricum and centrifuging it at a speed of 5000 r / min for 10 min, and discarding the supernatant to obtain bacterial sludge; S3: adding sterile water to the bacterial sludge to concentrate it 8 times to obtain a concentrated solution; S4 then adds 10% corn starch by mass of the concentrated solution, 10% sodium glutamate by mass of the concentrated solution, 0.2% glutamine by mass of the concentrated solution and 2% gum arabic by mass of the concentrated solution in sequence, stirs at a speed of 200 r / min for 20 minutes, and then spray-dries. The inlet temperature of the spray-drying is 170°C, the outlet temperature of the spray-drying is 85°C, and the butyric acid Clostridium powder is obtained by spray drying.

[0035] Viable bacteria count detection: The directly prepared Clostridium butyricum powder was tested for viable bacteria count, and a portion of the Clostridium butyricum powder was stored at room temperature for 6 months and then tested for viable bacteria count. The results are shown in Table 1.

[0036] Comparative Example 1: S1: Preparation of a Clostridium butyricum concentrate with a high viable count: the concentration method and multiple are the same as those in Example 1; S2 then sequentially added 10% corn starch by mass of the concentrate, 5% trehalose by mass of the concentrate, 8% sodium glutamate by mass of the concentrate, 0.2% glutamine by mass of the concentrate and 3% gum arabic by mass of the concentrate, stirred at a speed of 200 r / min for 20 min, and then spray-dried; S3 spray drying parameters are the same as those in Example 1, and spray drying is performed to obtain Clostridium butyricum powder.

[0037] Viable bacteria count detection: The directly prepared Clostridium butyricum powder was tested for viable bacteria count, and a portion of the Clostridium butyricum powder was stored at room temperature for 6 months and then tested for viable bacteria count. The results are shown in Table 1.

[0038] Comparative Example 2: S1: Preparation of a Clostridium butyricum concentrate with a high viable count: the concentration method and multiple are the same as those in Example 1; S2 then adds 15% corn starch by mass of the concentrate and 8% sodium glutamate by mass of the concentrate in sequence, stirs at a speed of 200 r / min for 20 min, and then spray-dries; S3 spray drying parameters are the same as those in Example 1, and spray drying is performed to obtain Clostridium butyricum powder.

[0039] Viable bacteria count detection: The directly prepared Clostridium butyricum powder was tested for viable bacteria count, and a portion of the Clostridium butyricum powder was stored at room temperature for 6 months and then tested for viable bacteria count. The results are shown in Table 1.

[0040] Comparative Example 3: S1: Preparation of a Clostridium butyricum concentrate with a high viable count: the concentration method and multiple are the same as those in Example 1; S2 then adds 15% corn starch by mass of the concentrate, 8% sodium glutamate by mass of the concentrate, 0.5% vitamin C by mass of the concentrate and 3% gum arabic by mass of the concentrate, stirs at a speed of 200 r / min for 20 minutes, and then spray-dries; S3 spray drying parameters are the same as those in Example 1, and spray drying is performed to obtain Clostridium butyricum powder.

[0041] Viable bacteria count detection: The directly prepared Clostridium butyricum powder was tested for viable bacteria count, and a portion of the Clostridium butyricum powder was stored at room temperature for 6 months and then tested for viable bacteria count. The results are shown in Table 1.

[0042] Comparative Example 4: S1: Preparation of a Clostridium butyricum concentrate with a high viable count: the concentration method and multiple are the same as those in Example 1; S2 then adds 15% corn starch by mass of the concentrate, 8% sodium glutamate by mass of the concentrate, 0.5% glutamine by mass of the concentrate and 3% gelatin by mass of the concentrate in sequence, stirs at a speed of 200 r / min for 20 min, and then spray-dries; S3 spray drying parameters are the same as those in Example 1, and spray drying is performed to obtain Clostridium butyricum powder.

[0043] Viable bacteria count test: The directly prepared Clostridium butyricum powder was tested for viable bacteria count, and a portion of the Clostridium butyricum powder was stored at room temperature for 6 months and then tested for viable bacteria count. The results are shown in Table 1: Comparative Example 5: S1: Preparation of a Clostridium butyricum concentrate with a high viable count: the concentration method and multiple are the same as those in Example 1; S2 then added 15% corn starch by mass of the concentrate, stirred at 200 r / min for 20 min, and then spray-dried; S3 spray drying parameters are the same as those in Example 1, and spray drying is performed to obtain Clostridium butyricum powder.

[0044] Viable bacteria count detection: The directly prepared Clostridium butyricum powder was tested for viable bacteria count, and a portion of the Clostridium butyricum powder was stored at room temperature for 6 months and then tested for viable bacteria count. The results are shown in Table 1.

[0045] Table 1 As can be seen from the results in Table 1, the formula and process variation range of Examples 1-2 do not change much for the results of spray drying and storage survival rate. As can be seen from the results of Example 1 and Comparative Example 1 in Table 1, the test results are not much different, but it was found in the experiment that after adding trehalose, the bacterial powder is more likely to absorb moisture and agglomerate after spray drying, which is not conducive to preservation. As can be seen from the results of Example 1 and Comparative Example 2, corn starch and sodium glutamate play a key role in the spray drying process, and glutamine and gum arabic play a key role in the storage stability of bacterial powder. As can be seen from the results of Comparative Example 3, the use of vitamin C instead of glutamine has little effect on the results, but the bacterial powder is easy to absorb moisture and agglomerate after being stored at room temperature, which affects the properties of the bacterial powder. As can be seen from the results of Comparative Example 4, after gelatin replaces gum arabic, the spray drying survival rate and bacterial powder stability are reduced to a certain extent, and gelatin needs a longer soaking time in actual applications, and the steps are more complicated, which is not conducive to large-scale production. As can be seen from the results of Comparative Example 5, the protection effect of a single protective agent, corn starch, is very poor, so a mixed protective agent is required.

[0046] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary rather than restrictive, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0047] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for preparing a live Clostridium butyricum preparation, characterized in that: include: S1: inoculating a glycerol tube containing Clostridium butyricum into RCM medium, anaerobic culture to obtain a seed solution, inoculating the Clostridium butyricum seed solution into a Clostridium butyricum fermentation medium, and fermenting under anaerobic conditions to prepare a fermentation solution of Clostridium butyricum; S2: taking the fermentation broth of Clostridium butyricum and subjecting it to centrifugation, discarding the supernatant to obtain bacterial sludge; S3: Add sterile water to the bacterial sludge for concentration; S4: adding a carbohydrate protective agent, an amino acid protective agent, a nutrient enhancer and a capsule material in sequence, stirring for a period of time, and then spray drying to obtain a live Clostridium butyricum preparation; Wherein, the carbohydrate protective agent is at least one of corn starch, maltodextrin or trehalose; and the amino acid protective agent is sodium glutamate.

2. The method for preparing a live Clostridium butyricum preparation according to claim 1, characterized in that: The Clostridium butyricum fermentation medium in S1 is composed of 20-30 parts by weight of glucose, 10-15 parts by weight of yeast extract, 5-10 parts by weight of corn flour, 10-15 parts by weight of corn steep liquor, 2-5 parts by weight of sodium bicarbonate, 0.1-0.5 parts by weight of magnesium sulfate, 0.5-1 parts by weight of calcium chloride and 0.5-1 parts by weight of ferrous sulfate. The Clostridium butyricum fermentation medium is prepared by weighing the above components in proportion, adding them into deionized water, stirring and dissolving them, adjusting the pH value to 7.0-7.2 using HCl or NaOH, and dispensing the prepared Clostridium butyricum fermentation medium into fermentation tanks for high-pressure steam sterilization. The sterilized Clostridium butyricum fermentation medium is cooled to room temperature for use.

3. The method for preparing a live Clostridium butyricum preparation according to claim 2, characterized in that: The temperature of high-pressure steam sterilization is 110-130℃ and the sterilization time is 15-25min.

4. The method for preparing a live Clostridium butyricum preparation according to claim 2, characterized in that: The specific method of S1 is to inoculate a glycerol tube containing Clostridium butyricum into an RCM liquid culture medium at 35-37°C, anaerobically culture for 16-20h to obtain a seed solution, inoculate the seeds into a Clostridium butyricum fermentation medium at an inoculation amount of 1-5%, maintain the pH at 6.5±0.5 with liquid alkali at a temperature of 37°C±1°C, stir and mix at a stirring speed of 180-220r / min, and add a sugar solution 7-9h after the start of fermentation, the amount of added sugar is 2%-3% of the total mass of the Clostridium butyricum fermentation medium, the sugar supplementation time is controlled within 4-6h, culture for 24-30h, and terminate the fermentation when the spore rate under microscopic examination reaches more than 90%, thereby obtaining Clostridium butyricum fermentation liquid; The centrifugal treatment method in S2 includes high-speed centrifugation, disc centrifugation or plate and frame filtration. The centrifugal treatment method is high-speed centrifugation with a centrifugal speed of 4000-6000r / min and a centrifugal treatment time of 5-30min. Add sterile liquid to S3 to concentrate it 3-10 times; The inlet temperature of the spray drying in S4 is 150-180°C, and the outlet temperature is 70-85°C; The stirring speed of the stirring process in S4 is 150-250 r / min, and the stirring time is 20-30 min.

5. The method for preparing a live Clostridium butyricum preparation according to claim 1, characterized in that: The nutrition enhancer is at least one of glutamine or vitamin C; The capsule material is at least one of gum arabic or gelatin.

6. The method for preparing a live Clostridium butyricum preparation according to claim 5, characterized in that: The amount of protective agent added is 10%-30% of the mass of the concentrated solution finally obtained in S3; The amount of the nutrient enhancer added is 0.2%-1.5% of the mass of the concentrated solution finally obtained in S3; The amount of capsule material added is 0.5%-7% of the mass of the concentrated solution finally obtained in S3.

7. The method for preparing a live Clostridium butyricum preparation according to claim 6, characterized in that: Corn starch and sodium glutamate are selected as protective agents. The amount of corn starch added is 10%-20% of the mass of the concentrated solution finally obtained in S3, and the amount of sodium glutamate added is 5%-10% of the mass of the concentrated solution finally obtained in S3. The nutritional enhancer is glutamine, and the amount of glutamine added is 0.1%-1% of the mass of the concentrated solution finally obtained in S3; The capsule material is gum arabic, and the amount of gum arabic added is 1%-5% of the mass of the concentrated liquid finally obtained in S3.

8. The method for preparing a live Clostridium butyricum preparation according to claim 6, characterized in that: Corn starch and sodium glutamate are selected as protective agents. The amount of corn starch added is 15% of the mass of the concentrated solution finally obtained in S3, and the amount of sodium glutamate added is 8% of the mass of the concentrated solution finally obtained in S3. Glutamine was selected as the nutritional enhancer, and the amount of glutamine added was 0.2% of the mass of the concentrated solution finally obtained in S3; The capsule material is gum arabic, and the amount of gum arabic added is 3% of the mass of the concentrated liquid finally obtained in S3.

9. The method for preparing a live Clostridium butyricum preparation according to claim 1, characterized in that Application of live Clostridium butyricum preparations in the feed field.

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