A compound type clostridium butyricum oral liquid fermentation method based on step-by-step inoculation and static maturation

Abstract

A kind of compound Clostridium butyricum oral liquid fermentation method based on step-by-step inoculation and static maturation, Clostridium butyricum oral liquid is combined fermentation by strict anaerobic Clostridium butyricum, facultative anaerobic Bacillus and heterotrophic anaerobic lactic acid bacteria.The probiotic compound culture of the present application not only solves the problem of oxygen in culture medium inhibiting normal growth of Clostridium butyricum, but also can mutualistic symbiosis between different strains to strengthen the gastrointestinal health function of oral liquid, esterification and alcoholization reaction can occur between metabolic products produced by Clostridium butyricum, facultative anaerobic Bacillus and heterotrophic anaerobic lactic acid bacteria, so that the odor produced by Clostridium butyricum can be eliminated, the smell and taste of oral liquid are better, organic acids produced by flora can reduce the pH of oral liquid, which is beneficial to prolong the shelf life of product and reduce storage cost, so that the product stability and functionality of oral liquid are superior to bifidobacterium oral liquid.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of probiotic fermentation, and specifically belongs to a compound Clostridium butyricum oral liquid fermentation method based on stepwise inoculation and static curing. BACKGROUND

[0002] Clostridium butyricum is a strict anaerobic probiotic that can form endogenous spores and has strong stress resistance. The bacteria also have the characteristics of acid and high temperature resistance, can colonize in the human intestinal tract and promote the growth of intestinal bifidobacteria, and have the functions of anti-bacterial and anti-inflammatory, repairing gastrointestinal mucosa and reducing gastrointestinal stress syndrome. Therefore, it is a very effective probiotic for regulating intestinal flora imbalance and treating diarrhea and constipation. However, if oxygen is present in the culture medium during the culture of Clostridium butyricum, it will seriously hinder its growth. In addition to using anaerobic equipment or filling nitrogen to isolate oxygen during fermentation, it is usually necessary to add inorganic or organic oxygen scavengers to the culture medium to eliminate the harm of oxygen. However, the addition of oxygen scavengers will increase the psychological hidden danger of consumers and increase the production cost. Therefore, it is necessary to find a method for producing Clostridium butyricum oral liquid without using oxygen scavengers. The influence of oxygen also exists in the sterilization process of the culture medium. After the culture medium is sterilized by high temperature and high pressure, in order to prevent the deformation and damage of the tank due to the pressure difference between the inside and outside of the tank during cooling, gas needs to be injected to maintain the pressure in the tank. If cheap air is injected, oxygen will be brought in, making the fermentation tank in an aerobic state. Therefore, the manufacturer can only choose to inject nitrogen into the tank, which will obviously increase the production cost.

[0003] In addition, Clostridium butyricum liquid fermentation usually has a smell, which not only makes it difficult for consumers to accept, but also increases the difficulty of market expansion. Therefore, whether the problem of odor of Clostridium butyricum oral liquid can be solved or not is related to the market prospect of the product.

[0004] In order to solve the problems of oxygen hazard in the culture medium of Clostridium butyricum and the odor of fermentation products, and make the taste and flavor of the oral liquid acceptable to consumers, it is necessary to improve the production process and fermentation technology of Clostridium butyricum oral liquid. SUMMARY

[0005] The technical problem solved: In view of the problems existing in the existing fermentation technology of Clostridium butyricum, the present application provides a compound Clostridium butyricum oral liquid fermentation method based on stepwise inoculation and static curing. This method can solve the problem of the influence of oxygen in the culture medium on the growth and reproduction of strict anaerobic Clostridium butyricum without using oxygen scavenging equipment, filling nitrogen and oxygen scavengers. At the same time, without adding oxygen scavengers, only by utilizing the characteristics of different strains to complement each other, the taste and efficacy of the product are better, the quality is more stable, and the shelf life of the tea beverage is prolonged, which is beneficial to reduce the storage and sales cost of the product.

[0006] Technical solution: A compound Clostridium butyricum oral liquid fermentation method based on step-by-step inoculation and static maturation, the steps are as follows: Clostridium butyricum oral liquid is fermented by strict anaerobic Clostridium butyricum, facultative anaerobic Bacillus and heterotrophic anaerobic lactic acid bacteria. In order to ensure the normal growth and reproduction of strict anaerobic Clostridium butyricum, facultative anaerobic Bacillus is used to consume oxygen in the culture medium, so that the culture medium is in an anaerobic state, to meet the requirements of Clostridium butyricum anaerobic culture. 10-80L Clostridium butyricum seed is cultured in 100L seed tank A, and 1-8L facultative anaerobic Bacillus and heterotrophic anaerobic lactic acid bacteria are cultured in 10L seed tank B and 10L seed tank C respectively. Then 10-80L Clostridium butyricum seed and 1-8L facultative anaerobic Bacillus seed are inoculated into 3000L fermentation tank at the same time, the inoculation amount is 4vol.% and 0.4vol.% respectively, the fermentation temperature is 37℃, and the culture is stirred at regular intervals. After 24-48 hours, 1-8L heterotrophic anaerobic lactic acid bacteria in seed tank C is inoculated into 3000L fermentation tank, the inoculation amount is not more than 0.4vol.%. At this time, the oral liquid is in a mixed fermentation state of different strains, the fermentation temperature is 37℃, and the culture is stirred at regular intervals. After 24 hours, 0.01wt.%-2wt.% of anhydrous ethanol is added for filling, and the product needs to be statically matured at 20℃-37℃ for 5-7 days after filling.

[0007] The culture method of the above-mentioned Clostridium butyricum seed is as follows: the seed culture medium for culturing Clostridium butyricum is added to 100L seed tank A, warmed to 121℃, pressure is 102.9kPa, sterilized for 20 minutes, then cooled. When the temperature of the culture medium in the seed tank drops to 37℃, inoculate Clostridium butyricum strain, the inoculation amount is 300mL. After 3 days of culture at 37℃+regular stirring, 50L of Clostridium butyricum seed liquid is obtained.

[0008] The culture method of the above-mentioned facultative anaerobic Bacillus and heterotrophic anaerobic lactic acid bacteria is as follows: the culture medium is warmed to 121℃, the pressure is 102.9kPa, and the sterilization treatment is carried out for 20 minutes, then cooled. When the temperature of the culture medium in the seed tank drops to 37℃, the facultative anaerobic Bacillus and the heterotrophic anaerobic lactic acid bacteria are inoculated into 10L seed tank B and 10L seed tank C respectively, the inoculation amount is 250mL. Stir at regular intervals during culture, and the seed quantity is 5L.

[0009] The mixed fermentation method is as follows: 2000 L of culture medium is added to a 3000 L fermenter, which is treated at 121°C and 102.9 kPa for 20 minutes. When the temperature drops to 37°C, 50 L of Clostridium butyricum seed from seed tank A and 5 L of facultative anaerobic Bacillus from seed tank B are simultaneously inoculated into the 3000 L fermenter, with inoculation amounts of 2.5 vol.% and 0.25 vol.% respectively. The fermentation temperature is 37°C, and the culture is stirred at regular intervals. After 48 hours of culture, 5 L of heterotrophic anaerobic lactic acid bacteria from seed tank C is inoculated into the 3000 L fermenter, with an inoculation amount of 0.25 vol.%. At this time, the oral liquid is in a mixed fermentation state of the three types of bacteria. The fermentation temperature is 37°C, and the culture is stirred at regular intervals. After 24 hours of further culture, the colony count of Clostridium butyricum reaches 1 billion CFU / mL, the colony counts of facultative anaerobic Bacillus and heterotrophic anaerobic lactic acid bacteria are 1.0 billion and 6.5 billion respectively, and the pH is 3.6. 0.05 wt.% of anhydrous ethanol is added, stirred, and then filled into bottles. After filling, the product needs to be statically matured at 20°C-37°C for 6 days. After static maturation, the colony count of heterotrophic anaerobic lactic acid bacteria rises to 8 billion, and the pH of the oral liquid drops to 3.4.

[0010] The above-mentioned regular stirring is that the stirring system operates according to the set program. During the culture, the culture is stirred every 120 minutes, with each stirring lasting 5 minutes.

[0011] The above-mentioned static maturation is that the compound microbial agent is in a sealed and static anaerobic state in the bottle (barrel) after filling. At this time, the heterotrophic anaerobic lactic acid bacteria can further proliferate under anaerobic conditions with the promotion of the metabolic products of Clostridium butyricum. After the pH drops to 3.3-3.6, the growth of the bacterial flora in the oral liquid tends to stop, and the proportion of each bacterial flora is more stable. The oral liquid can be stored at room temperature, and the shelf life is extended.

[0012] The technical key points of the present application are: 1. The respective characteristics of the strict anaerobic Clostridium butyricum, facultative anaerobic Bacillus and heterotrophic anaerobic lactic acid bacteria are combined for fermentation, which can solve the problem that the Clostridium butyricum must use oxygen removal equipment, fill in nitrogen or add oxygen remover to normally ferment during the fermentation process. 2. The Clostridium butyricum must grow in the condition of isolating oxygen, while the facultative anaerobic Bacillus can grow in the condition of having oxygen or no oxygen, and the facultative anaerobic Bacillus can consume the oxygen in the culture medium after mixed culture, which creates an anaerobic growth environment for the Clostridium butyricum, and when the oxygen in the culture medium is consumed by the facultative anaerobic Bacillus, the anaerobic characteristics of the facultative anaerobic Bacillus can still keep alive and slowly grow in the anoxic culture medium, which can prevent the dead bacteria from affecting the taste and flavor of the oral liquid. 3. The metabolic products of the Clostridium butyricum can promote the growth of the heterotrophic anaerobic lactic acid bacteria, and the metabolic products of the facultative anaerobic Bacillus and the heterotrophic anaerobic lactic acid bacteria can strengthen the gastrointestinal regulation function of the Clostridium butyricum, the heterotrophic anaerobic lactic acid bacteria grow faster after inoculation, and the delayed inoculation can control the biomass, and the lactic acid produced by the heterotrophic anaerobic lactic acid bacteria can reduce the pH of the culture medium and inhibit the growth of acid-intolerant bacteria, which is beneficial to stabilize the quality of the oral liquid and realize the normal temperature preservation, which can prolong the shelf life and reduce the preservation cost. 4. The facultative anaerobic Bacillus has strong organic matter decomposition ability, which can decompose the organic matter in the culture medium into small molecule nutrients, which is beneficial to the rapid growth of the Clostridium butyricum. 5. The metabolic products of the selected facultative anaerobic Bacillus can promote the growth of the Clostridium butyricum, the metabolic products of the Clostridium butyricum can promote the growth of the heterotrophic anaerobic lactic acid bacteria, and the flora can form a mutualistic symbiotic relationship and do not produce antagonism to each other. 6. The metabolic products of the Clostridium butyricum contain butyric acid and short-chain fatty acids, which can react with ethanol in the metabolic products of the heterotrophic anaerobic lactic acid bacteria and the added ethanol to generate butyl acetate with aromatic flavor, which can play a role in eliminating odor and improving the taste of the Clostridium butyricum oral liquid. 7. The static maturation process makes the pH of the oral liquid further decrease, the growth of the flora tends to stop, the proportion of the flora is more stable, the shelf life of the oral liquid is prolonged, and the oral liquid can be stored at room temperature.

[0013] Beneficial effects: 1. Clostridium butyricum is a strict anaerobic probiotic bacteria, which requires an anaerobic culture medium to grow normally, usually requires special equipment, nitrogen or deoxidizing agent to eliminate oxygen in the culture medium, which not only increases the production cost, but also the deoxidizing agent is a reducing agent, some kinds of which added to the oral liquid will have certain side effects on the human body, at the same time, the amount of deoxidizing agent added is related to the oxygen content in the culture medium, and the oxygen content is affected by many factors and changes greatly, so the amount of deoxidizing agent added is not easy to control in actual operation, if added too little, it will not work, if added too much, it will inhibit the growth of Clostridium butyricum. The present application mixes and cultures Clostridium butyricum and facultative anaerobic Bacillus which is also a probiotic bacteria, uses the oxygen consumption characteristics of Bacillus to consume the oxygen in the culture medium, provides protection for the normal growth of Clostridium butyricum, and avoids the use of deoxidizing agent, and the anaerobic characteristics of facultative anaerobic Bacillus enables it to symbiosis with Clostridium butyricum under anaerobic conditions, the metabolic products of which can promote the growth of Clostridium butyricum, which is beneficial to the improvement of Clostridium butyricum biomass and the reduction of production cost.

[0014] 2. First inoculate Clostridium butyricum and facultative anaerobic Bacillus, the inoculation ratio of the two is 1:0.1, then inoculate the heterotrophic anaerobic lactic acid bacteria which grows faster, the inoculation ratio is less than one tenth of Clostridium butyricum, so that Clostridium butyricum is always the dominant species in the oral liquid, avoiding the disadvantage of less total number of Clostridium butyricum colonies caused by simultaneous inoculation or inoculation of the same dose.

[0015] 3. The mixed culture of facultative anaerobic Bacillus and Clostridium butyricum can keep the culture medium in an anaerobic state all the time, even if the air in the tank contains oxygen, it will not affect the normal growth of Clostridium butyricum, which makes the culture medium in the fermenter can be injected with cheap air containing oxygen for pressure preservation after high temperature and high pressure sterilization, without the need of nitrogen pressure preservation, which is conducive to reducing production cost.

[0016] 4. After inoculating the heterotrophic anaerobic lactic acid bacteria, under the promotion of the metabolic products of Clostridium butyricum, the heterotrophic anaerobic lactic acid bacteria can make the pH of the culture medium decrease rapidly, which has a strong inhibitory effect on acid-intolerant bacteria, is conducive to stabilizing the quality of the oral liquid, especially after static maturation, the pH can be reduced to 3.3-3.6, at this time the growth of the bacterial community tends to stop, which is conducive to the stability of the bacterial community ratio of the oral liquid, the acidic oral liquid is not easy to deteriorate, the shelf life is more than one year under the condition of room temperature and sealed state, at the same time, the cost of seed preservation is also reduced.

[0017] 5. The metabolic products of Clostridium butyricum contain butyric acid, acetic acid and short-chain fatty acids, which can react with ethanol in the metabolic products of heterotrophic anaerobic lactic acid bacteria and added ethanol to generate butyl acetate with aromatic smell, which can eliminate odor, and the added modulating ethanol can improve the taste of Clostridium butyricum oral liquid and play a role in increasing aroma.

[0018] 6. Static maturation can further reduce the pH of the oral liquid, thereby inhibiting the growth of the bacterial flora and stabilizing the proportion of the bacterial flora, and the oral liquid can be kept at room temperature without deterioration when the pH is as low as 3.3-3.6, and the shelf life of the product is prolonged.

[0019] 7. The synergistic effect of mutualism among the probiotic bacterial flora enhances the antibacterial properties of the product, further improving the quality of the product and the effect of taking the product.

[0020] 8. The functional tea beverage with complex probiotics not only has enhanced efficacy, but also has stronger product competitiveness and better market prospects.

[0021] 9. The fermentation process does not produce wastewater, exhaust gas, and waste.

[0022] In summary, after the complex cultivation of probiotics, the problem of oxygen inhibition of the normal growth of Clostridium butyricum in the culture medium is solved, and the mutualism among different strains can strengthen the gastrointestinal health care function of the oral liquid. The metabolic products produced by Clostridium butyricum, facultative anaerobic Bacillus, and heterotrophic anaerobic lactic acid bacteria can undergo esterification and alcoholization reactions, thereby eliminating the odor produced by Clostridium butyricum, making the smell and taste of the oral liquid better, and the organic acids produced by the bacterial flora can reduce the pH of the oral liquid, which is beneficial to prolong the shelf life of the product and reduce the storage cost, making the oral liquid superior to bifidobacterium oral liquid in product stability and functionality. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The production process flow chart of Clostridium butyricum oral liquid in the prior art; DETAILED DESCRIPTION

[0024] The present application is further illustrated by the following implementation cases, but these implementation cases are only for illustration and are not a limitation.

[0025] A complex Clostridium butyricum oral liquid fermentation method based on stepwise inoculation and static maturation, the steps are as follows:

[0026] Reference Figure 1The oral liquid of Clostridium butyricum is fermented by Clostridium butyricum, Bacillus and Lactobacillus. The seed culture medium of Clostridium butyricum is added into a 100L seed tank A, heated to 121℃, sterilized at a pressure of 102.9kPa for 20 minutes, and then cooled. When the temperature of the seed tank is reduced to 37℃, the Clostridium butyricum strain is inoculated at a volume of 200-500mL. After 3 days of culture at 37℃ with regular stirring, 10-80L of Clostridium butyricum seed liquid is obtained. The Bacillus and Lactobacillus are inoculated into 10L seed tank B and 10L seed tank C respectively (the sterilization method of the culture medium is the same as that of seed tank A). The inoculation temperature is 37℃, and the inoculation volume is 200-250mL. The culture is stirred regularly, and the seed volume is 1-8L. The 2000L batch culture medium is added into a 3000L fermentation tank, sterilized at 121℃ and 102.9kPa for 20 minutes, and then cooled to 37℃. The 10-80L Clostridium butyricum seed from seed tank A and the 1-8L Bacillus from seed tank B are inoculated into the 3000L fermentation tank (first inoculation) at a volume of 4vol.% and 0.4vol.% respectively. The fermentation temperature is 37℃, and the culture is stirred regularly for 24-48 hours. Then, the 1-8L Lactobacillus from seed tank C is inoculated into the 3000L fermentation tank at a volume of not more than 0.4vol.%. At this time, the oral liquid is in a mixed fermentation state of different strains. The fermentation temperature is 37℃, and the culture is stirred regularly for another 24 hours. Then, 0.01wt.%-2wt.% of anhydrous ethanol is added for filling. After filling, the product needs to be statically matured at 20℃-37℃ for 5-7 days before being sold.

[0027] Example 1:

[0028] A compound Clostridium butyricum oral liquid fermentation method based on stepwise inoculation and static maturation, the steps are as follows:

[0029] Referring to Figure 1The seed culture of Clostridium butyricum is added to 100L seed tank A, heated to 121℃, sterilized at 102.9kPa for 20 minutes, and then cooled. When the temperature of the culture in the seed tank drops to 37℃, the Clostridium butyricum seed is inoculated at a volume of 500mL. After 3 days of culture at 37℃ with timed stirring, 80L of Clostridium butyricum seed solution is obtained. The facultative anaerobic Bacillus and the heterotrophic anaerobic lactic acid bacteria are inoculated into 10L seed tank B and 10L seed tank C, respectively (the sterilization method of the culture is the same as that of seed tank A). The inoculation temperature is 37℃, and the inoculation volume is 250mL. The culture is stirred at a constant speed, and the seed volume is 8L. The 2000L batch culture medium is added to a 3000L fermentation tank, sterilized at 121℃ and 102.9kPa for 20 minutes, and then cooled. When the temperature drops to 37℃, the 80L Clostridium butyricum seed solution from seed tank A and the 8L facultative anaerobic Bacillus from seed tank B are inoculated into the 3000L fermentation tank (inoculated first). The inoculation volume is 4vol.% and 0.4vol.%, respectively. The fermentation temperature is 37℃, and the culture is stirred at a constant speed. After 24 hours of culture, the 8L heterotrophic anaerobic lactic acid bacteria from seed tank C is inoculated into the 3000L fermentation tank (inoculated last). The inoculation volume is 0.4vol.%. At this time, the oral solution is in a mixed fermentation state of the three bacteria. The fermentation temperature is 37℃, and the culture is stirred at a constant speed. After 24 hours of continuous culture, the total number of colonies of Clostridium butyricum reaches 700 million CFU / mL, the total number of colonies of facultative anaerobic Bacillus and heterotrophic anaerobic lactic acid bacteria is 120 million and 700 million, respectively, and the pH is 3.6. 0.1wt.% of anhydrous ethanol is added, stirred, and then filled. The product needs to be statically matured at 20℃-37℃ for 5 days. After maturation, the total number of colonies of heterotrophic anaerobic lactic acid bacteria increases to 800 million, the pH of the oral solution decreases to 3.4, the product quality is more stable, and the shelf life exceeds one year.

[0030] Example 2:

[0031] A compound Clostridium butyricum oral solution fermentation method based on stepwise inoculation and static maturation, the steps are as follows:

[0032] Referring to Figure 1The seed culture of Clostridium butyricum is added to 100L seed tank A, heated to 121°C, and sterilized at a pressure of 102.9kPa for 20 minutes, then cooled. When the temperature of the culture in the seed tank drops to 37°C, Clostridium butyricum seed is inoculated at a volume of 500mL. After 3 days of culture at 37°C with timed stirring, 40L of Clostridium butyricum seed solution is obtained. Facultative anaerobic Bacillus and heterotrophic anaerobic lactic acid bacteria are inoculated into 10L seed tank B and 10L seed tank C, respectively (the sterilization method of the culture medium is the same as that of seed tank A). The inoculation temperature is 37°C, and the inoculation volume is 200mL. The culture is stirred at a constant speed, and the seed volume is 8L. The 2000L batch culture medium is added to a 3000L fermentation tank, sterilized at 121°C and 102.9kPa for 20 minutes, and then cooled to 37°C. The 40L Clostridium butyricum seed solution from seed tank A and the 8L facultative anaerobic Bacillus from seed tank B are simultaneously inoculated into the 3000L fermentation tank (first inoculation) at a volume of 2vol.% and 0.4vol.%, respectively. The fermentation temperature is 37°C, and the culture is stirred at a constant speed. After 48 hours of culture, the 8L heterotrophic anaerobic lactic acid bacteria from seed tank C are inoculated into the 3000L fermentation tank (second inoculation) at a volume of 0.4vol.%. At this time, the oral solution is in a mixed fermentation state of the three bacteria. The fermentation temperature is 37°C, and the culture is stirred at a constant speed. After 24 hours of continuous culture, the total number of colonies of Clostridium butyricum reaches 8.5 billion CFU / mL, the total number of colonies of facultative anaerobic Bacillus and heterotrophic anaerobic lactic acid bacteria is 1.0 billion and 7 billion, respectively, and the pH is 3.6. 0.5wt.% of anhydrous ethanol is added, stirred, and then filled. The product needs to be statically matured at 20°C-37°C for 7 days. After maturation, the total number of colonies of heterotrophic anaerobic lactic acid bacteria increases to 8.5 billion, the pH of the oral solution decreases to 3.4, the product quality is more stable, and the shelf life exceeds one year.

[0033] Example 3:

[0034] A compound Clostridium butyricum oral solution fermentation method based on stepwise inoculation and static maturation, the steps are as follows:

[0035] Reference Figure 1The seed culture of Clostridium butyricum is added to 100L seed tank A, and sterilized by heating to 121°C under 102.9kPa pressure for 20 minutes, and then cooled. When the temperature of the culture in the seed tank drops to 37°C, Clostridium butyricum seed is inoculated at a volume of 500mL. After 3 days of culture at 37°C with timed stirring, 60L of Clostridium butyricum seed solution is obtained. Facultative anaerobic Bacillus and heterotrophic anaerobic lactic acid bacteria are inoculated into 10L seed tank B and 10L seed tank C, respectively (the sterilization method of the culture is the same as that of seed tank A). The inoculation temperature is 37°C, and the inoculation volume is 200mL. The culture is stirred at timed intervals, and the seed volume is 6L. The 2000L batch culture medium is added to a 3000L fermentation tank, and sterilized by heating to 121°C under 102.9kPa pressure for 20 minutes. When the temperature drops to 37°C, 60L of Clostridium butyricum seed solution from seed tank A and 6L of facultative anaerobic Bacillus from seed tank B are simultaneously inoculated into the 3000L fermentation tank (first inoculation). The inoculation volume is 3vol.% and 0.3vol.%, respectively. The fermentation temperature is 37°C, and the culture is stirred at timed intervals. After 24 hours of culture, 6L of heterotrophic anaerobic lactic acid bacteria from seed tank C is inoculated into the 3000L fermentation tank (second inoculation). The inoculation volume is 0.3vol.%. At this time, the oral solution is in a mixed fermentation state of the three bacteria. The fermentation temperature is 37°C, and the culture is stirred at timed intervals. After 24 hours of continuous culture, the total number of Clostridium butyricum colonies reaches 700 million CFU / mL, the total number of facultative anaerobic Bacillus colonies is 110 million, the total number of heterotrophic anaerobic lactic acid bacteria colonies is 750 million, the pH is 3.6, and 0.01wt.% of anhydrous ethanol is added. After stirring, the product is filled, and the product needs to be statically matured at 20°C-37°C for 5 days. After maturation, the total number of lactic acid bacteria colonies increases to 800 million, the pH of the oral solution decreases to 3.4, the product quality is more stable, and the shelf life exceeds one year.

[0036] Example 4:

[0037] A compound Clostridium butyricum oral solution fermentation method based on stepwise inoculation and static maturation, the steps are as follows:

[0038] Referring to Figure 1The seed culture of Clostridium butyricum is added to 100L seed tank A, heated to 121°C, and sterilized at a pressure of 102.9kPa for 20 minutes, and then cooled. When the temperature of the culture in the seed tank drops to 37°C, the Clostridium butyricum seed is inoculated at a volume of 300mL. After 3 days of cultivation at 37°C with timed stirring, 50L of Clostridium butyricum seed solution is obtained. The facultative anaerobic Bacillus and the heterotrophic anaerobic lactic acid bacteria are inoculated into 10L seed tank B and 10L seed tank C, respectively (the sterilization method of the culture medium is the same as that of seed tank A). The inoculation temperature is 37°C, and the inoculation volume is 250mL. The seed volume is 5L after cultivation with timed stirring. The 2000L batch culture medium is added to a 3000L fermentation tank, sterilized at 121°C and 102.9kPa for 20 minutes, and then cooled. When the temperature drops to 37°C, the 50L Clostridium butyricum seed solution in seed tank A and the 5L facultative anaerobic Bacillus in seed tank B are simultaneously inoculated into the 3000L fermentation tank (first inoculation). The inoculation volume is 2.5vol.% and 0.25vol.%, respectively. The fermentation temperature is 37°C, and the culture is stirred at a constant speed. After 48 hours of cultivation, the 5L heterotrophic anaerobic lactic acid bacteria in seed tank C are inoculated into the 3000L fermentation tank (second inoculation). The inoculation volume is 0.25vol.%. At this time, the oral solution is in a mixed fermentation state of the three bacteria. The fermentation temperature is 37°C, and the culture is stirred at a constant speed. After 24 hours of continuous cultivation, the total number of colonies of Clostridium butyricum reaches 1 billion CFU / mL, the total number of colonies of facultative anaerobic Bacillus is 0.1 billion, and the total number of colonies of heterotrophic anaerobic lactic acid bacteria is 0.65 billion. The pH is 3.6. 0.05wt.% of anhydrous ethanol is added, stirred, and then filled. The product needs to be statically matured at 20°C-37°C for 6 days. After maturation, the total number of colonies of heterotrophic anaerobic lactic acid bacteria increases to 0.8 billion, the pH of the oral solution decreases to 3.4, the product quality is more stable, and the shelf life exceeds one year.

[0039] Example 5:

[0040] A compound Clostridium butyricum oral solution fermentation method based on stepwise inoculation and static maturation, the steps are as follows:

[0041] Reference Figure 1The seed culture of Clostridium butyricum is added to 100 L seed tank A, heated to 121 °C, and sterilized at a pressure of 102.9 kPa for 20 minutes, and then cooled. When the temperature of the culture in the seed tank drops to 37 °C, Clostridium butyricum seed is inoculated at a volume of 500 mL. After 3 days of culture at 37 °C with timed stirring, 80 L of Clostridium butyricum seed solution is obtained. Facultative anaerobic Bacillus and lactic acid bacteria are inoculated into 10 L seed tank B and 10 L seed tank C, respectively (the sterilization method of the culture medium is the same as that of seed tank A). The inoculation temperature is 37 °C, and the inoculation volume is 250 mL. The culture is stirred at a constant speed, and the seed volume is 5 L. The 2000 L batch culture medium is added to a 3000 L fermentation tank, sterilized at 121 °C and 102.9 kPa for 20 minutes, and then cooled to 37 °C. The 80 L Clostridium butyricum seed solution in seed tank A, the 5 L facultative anaerobic Bacillus in seed tank B, and the 5 L heterotrophic anaerobic lactic acid bacteria in seed tank C are simultaneously inoculated into the 3000 L fermentation tank at a volume of 4 vol.%, 0.25 vol.%, and 0.25 vol.%, respectively. At this time, the oral solution is in a mixed fermentation state of the three bacteria. The fermentation temperature is 37 °C, and the culture is stirred at a constant speed. After 72 hours of culture, the total number of colonies of Clostridium butyricum reaches 600 million CFU / mL, the total number of colonies of facultative anaerobic Bacillus and heterotrophic anaerobic lactic acid bacteria is 100 million and 1.2 billion, respectively, and the pH is 3.6. 0.8 wt.% of anhydrous ethanol is added, stirred, and then filled. The product needs to be statically matured at 20-37 °C for 7 days. After maturation, the total number of colonies of heterotrophic anaerobic lactic acid bacteria increases to 1.4 billion, the pH of the oral solution decreases to 3.3, the product quality is stable, and the shelf life is more than one year.

[0042] Example 6:

[0043] A compound Clostridium butyricum oral solution fermentation method based on stepwise inoculation and static maturation, the steps are as follows:

[0044] Referring to Figure 1The seed culture of Clostridium butyricum is added to 100 L seed tank A, heated to 121 °C, and sterilized at a pressure of 102.9 kPa for 20 minutes, then cooled. When the temperature of the culture in the seed tank drops to 37 °C, Clostridium butyricum seed is inoculated at a volume of 500 mL. After 3 days of cultivation at 37 °C with timed stirring, 40 L of Clostridium butyricum seed solution is obtained. The heterotrophic anaerobic lactic acid bacteria is inoculated into 10 L seed tank C (the sterilization method of the culture medium is the same as that of seed tank A), the inoculation temperature is 37 °C, and the inoculation volume is 250 mL. The culture is stirred at a constant speed, and the amount of seed obtained is 4 L. The 2000 L batch culture medium is added to a 3000 L fermentation tank, sterilized at 121 °C and 102.9 kPa for 20 minutes, and then cooled to 37 °C. The 40 L Clostridium butyricum seed solution in seed tank A is inoculated into the 3000 L fermentation tank (first inoculation) at a volume of 2 vol.%. The fermentation temperature is 37 °C, and the culture is stirred at a constant speed. After 48 hours of cultivation, the 4 L heterotrophic anaerobic lactic acid bacteria in seed tank C is inoculated into the 3000 L fermentation tank (second inoculation) at a volume of 0.2 vol.%. At this time, the oral solution is in a mixed fermentation state of Clostridium butyricum and heterotrophic anaerobic lactic acid bacteria. The fermentation temperature is 37 °C, and the culture is stirred at a constant speed. After 24 hours of continuous cultivation, the total number of Clostridium butyricum colonies reaches 50,000 CFU / mL, the total number of heterotrophic anaerobic lactic acid bacteria colonies reaches 600 million CFU / mL, and the pH is 3.6. At this time, 2 wt.% of anhydrous ethanol is added, stirred, and then filled. The product needs to be statically matured at 20-37 °C for 5 days. After maturation, the total number of heterotrophic anaerobic lactic acid bacteria colonies is 800 million CFU / mL, and the pH is 3.4. The product can be sold.

[0045] Example 7:

[0046] A compound Clostridium butyricum oral solution fermentation method based on stepwise inoculation and static maturation, the steps are as follows:

[0047] Referring to Figure 1The seed culture medium for culturing Clostridium butyricum was added to a 100L seed tank A, heated to 121℃ and pressure 102.9kPa, and then cooled after 20 minutes of sterilization. When the temperature of the culture medium in the seed tank dropped to 37℃, Clostridium butyricum inoculum was inoculated at a volume of 300mL. After culturing at 37℃ with timed stirring for 3 days, 80L of Clostridium butyricum seed solution was obtained. 2000L of culture medium for batch production was added to a 3000L fermenter and treated at 121℃ and 102.9kPa pressure for 20 minutes. When the temperature dropped to 37℃, 80L of Clostridium butyricum from seed tank A and 80L of heterotrophic anaerobic lactic acid bacteria from seed tank C were simultaneously inoculated into the 3000L fermenter at an inoculation rate of 4 vol.%. The fermentation temperature was 37℃, and the mixture was stirred regularly. After 72 hours of cultivation, the total colony count of Clostridium butyricum was only 0.2 billion CFU / mL, and the total colony count of heterotrophic anaerobic lactic acid bacteria was 1.2 billion CFU / mL. The pH was 3.4. 0.1% anhydrous ethanol was added, and the mixture was stirred well before bottling and sale without static fermentation.

[0048] Example 8:

[0049] A fermentation method for a compound Clostridium butyricum oral liquid based on stepwise inoculation and static maturation, comprising the following steps:

[0050] See Figure 1 The seed culture medium for culturing *Clostridium butyricum* was added to a 100L seed tank A, heated to 121℃ and pressure 102.9kPa, and sterilized for 20 minutes before cooling. When the temperature of the culture medium in the seed tank dropped to 37℃, *Clostridium butyricum* inoculum was inoculated at a volume of 200mL. After culturing at 37℃ with timed stirring for 3 days, 20L of *Clostridium butyricum* seed solution was obtained. Facultative anaerobic spore-forming bacteria were inoculated into a 10L seed tank B (the sterilization method of the culture medium was the same as that of seed tank A), at an inoculation temperature of 37℃, at a volume of 250mL, and stirred timed during cultivation. The seed quantity for both was 1L. 2000L of culture medium for batch production was added to a 3000L fermenter and treated at 121℃ and pressure 102.9kPa for 20 minutes. When the temperature dropped to 37℃, the 20L of *Clostridium butyricum* seed solution from seed tank A and the 1L of *Clostridium butyricum* seed solution from seed tank B were inoculated into the fermenter. Facultative anaerobic Bacillus was simultaneously inoculated into a 3000L fermenter at inoculation rates of 1 vol.% and 0.05 vol.%, respectively. The fermentation temperature was 37℃, with regular stirring. At this stage, the oral liquid was in a mixed fermentation state of Clostridium butyricum and facultative anaerobic Bacillus. After 72 hours of cultivation, the total colony count of Clostridium butyricum reached 700 million CFU / mL, and the total colony count of facultative anaerobic Bacillus reached 120 million. Anhydrous ethanol was added at a ratio of 1 wt.% for flavoring, and after stirring, it was ready for bottling and sale. Because no heterotrophic anaerobic lactic acid bacteria were added, the pH of the oral liquid was 4.2, resulting in a poor taste. The product is prone to bacterial growth after opening, and its shelf life is only 3-6 months.

[0051] Example 9:

[0052] A compound type Clostridium butyricum oral liquid fermentation method based on stepwise inoculation and static maturation, the steps are as follows:

[0053] Referring to Figure 1 The seed culture medium for culturing Clostridium butyricum is added to a 100L seed tank A, warmed to 121℃, the pressure is 102.9kPa, sterilized for 20 minutes, and then cooled. When the temperature of the medium in the seed tank drops to 37℃, Clostridium butyricum is inoculated, the inoculation amount is 300mL, and after 3 days of culture at 37℃+ timed stirring, 10L of Clostridium butyricum seed liquid is obtained. The culture medium for 2000L batch production is added to a 3000L fermentation tank, treated at 121℃, 102.9kPa pressure for 20 minutes, and then cooled. When the temperature drops to 37℃, 10L of Clostridium butyricum seed from seed tank A is inoculated into the 3000L fermentation tank, the inoculation amount is 3vol.%, the fermentation temperature is 37℃, and the culture is stirred for 72 hours. After 72 hours of culture, the total number of Clostridium butyricum colonies reaches 0.2 billion CFU / mL, the pH is 4.5, and 0.03wt.% of anhydrous ethanol is added, stirred, and then filled. Since this culture is only Clostridium butyricum, without the inoculation of facultative anaerobic Bacillus and heterotrophic anaerobic lactic acid bacteria, the fermentation effect is poor, the odor is heavy, the oral liquid is easily infected with bacteria after the bottle is opened, and the shelf life is only 1-3 months.

[0054] Example 10:

[0055] A compound type Clostridium butyricum oral liquid fermentation method based on stepwise inoculation and static maturation, the steps are as follows:

[0056] Referring to Figure 1The seed medium is added to 100 L seed tank A, heated to 121 °C, pressure is 102.9 kPa, sterilized by 20 minutes of disinfection and cooling, when the temperature of the medium in the seed tank is reduced to 37 °C, facultative anaerobic bacillus and lactic acid bacteria are inoculated, the inoculation amount is 2000 mL, after 3 days of culture at 37 °C, 60 L of facultative anaerobic bacillus and heterotrophic anaerobic lactic acid bacteria mixed seed liquid is obtained. The 2000 L batch production medium is added to a 3000 L fermentation tank, treated at 121 °C, 102.9 kPa pressure for 20 minutes, when the temperature is reduced to 37 °C, 60 L of facultative anaerobic bacillus and lactic acid bacteria in seed tank A is inoculated into the 3000 L fermentation tank, the inoculation amount is 2 vol.%, the fermentation temperature is 37 °C, and the culture is stirred at regular intervals, after 72 hours of culture, the total number of colonies of facultative anaerobic bacillus and lactic acid bacteria is 100 million CFU / mL and 1 billion CFU / mL respectively, pH is 3.6, after filling of the product, static aging is required at 20-37 °C for 7 days, then the total number of colonies of lactic acid bacteria is increased to 1.2 billion CFU / mL, and pH is decreased to 3.4. Since the oral liquid lacks clostridium butyricum, its functionality is obviously decreased.

[0057] The effect of the example is explained.

[0058] In this example, the fermentation effect of oral liquid is compared under the conditions of stepwise inoculation, simultaneous inoculation, facultative anaerobic bacillus and heterotrophic anaerobic lactic acid bacteria inoculation, to verify the advancement of the scheme.

[0059] Table 1. Effect of facultative anaerobic bacillus and heterotrophic anaerobic lactic acid bacteria on the total number of colonies of clostridium butyricum in oral liquid

[0060]

[0061]

[0062] From Table 1, it can be seen that the total number of Clostridium butyricum colonies in the oral solution of Example 6 and Example 7, which are not combined with facultative anaerobic Bacillus, is 0.5×104CFU / mL and 0.2×104CFU / mL respectively, and the growth and functionality are poor; Example 9 only adds Clostridium butyricum for fermentation, and does not combine facultative anaerobic Bacillus and heterotrophic anaerobic lactic acid bacteria, and the number of Clostridium butyricum in the oral solution (0.2×104CFU / mL) is much lower than that of other examples, and the pH of the oral solution is 4.5, and the product quality is poor. Example 8 and Example 9 do not inoculate heterotrophic anaerobic lactic acid bacteria, and the pH of the oral solution is 4.2 and 4.5 respectively, and the taste and smell of the product are poor, and the shelf life is short; Example 5 and Example 7 both use the method of inoculating lactic acid bacteria, Clostridium butyricum and facultative anaerobic Bacillus at the same time, and the total number of heterotrophic anaerobic lactic acid bacteria and colonies is much higher than that of Clostridium butyricum, which weakens the functionality of the oral solution. Example 10 does not add Clostridium butyricum, and only uses facultative anaerobic Bacillus and heterotrophic anaerobic lactic acid bacteria for fermentation, and the taste of the oral solution lacking Clostridium butyricum is poor, and its functionality is significantly reduced. Except that Example 8 and Example 9 do not inoculate heterotrophic anaerobic lactic acid bacteria, the microbial flora of the other groups all combine heterotrophic anaerobic lactic acid bacteria, and the pH of the oral solution after static fermentation is lower than that before static fermentation, and the total number of colonies is higher than that before static fermentation, that is, the heterotrophic anaerobic lactic acid bacteria have further growth during the static fermentation period, and at the same time, it also indicates that the heterotrophic anaerobic lactic acid bacteria have the phenomenon of post-acidification, which is beneficial to the stability of product quality.

[0063] The results of the examples show that the characteristics of mutual benefit and symbiosis of Clostridium butyricum, facultative anaerobic Bacillus and heterotrophic anaerobic lactic acid bacteria can be utilized, and the above-mentioned strains are reasonably combined for oral solution production, which not only enhances the functionality of the oral solution, but also has a very obvious effect on improving the product quality, and the microbial flora can complement each other, making up for the shortcomings of single microbial flora in the fermentation process. For example, facultative anaerobic Bacillus can consume the oxygen in the culture medium, creating a good growth environment for strict anaerobic Clostridium butyricum, so that the total number of Clostridium butyricum colonies is greatly improved, and heterotrophic anaerobic lactic acid bacteria can reduce the pH of the oral solution, inhibit the odor of Clostridium butyricum metabolites, improve the taste of the product and prolong the shelf life of the product. In addition, the static maturation process in the technical solution of the present application can further reduce the pH of the oral solution and stabilize the microbial flora ratio, and the produced oral solution can be stored at room temperature and has a shelf life of more than one year, which is beneficial to reducing the cost of product transportation, sales and storage, and also makes the functionality of the oral solution more stable and reliable.

[0064] The embodiments are only used for explaining the present application, and are not used for limiting the present application, and the person skilled in the art can make the modification of the embodiments without the creative contribution according to the need after reading the description, and as long as the modification is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A Clostridium butyricum oral liquid fermentation method based on a combination of stepwise inoculation and static maturation, characterized in that, The steps are as follows: first, 10-80 L of Clostridium butyricum seed is cultivated in a 100 L seed tank A, and 1-8 L of facultative anaerobic Bacillus and heterotrophic anaerobic lactic acid bacteria are cultivated in 10 L seed tank B and 10 L seed tank C respectively; 2000 L of culture medium is added to a 3000 L fermentation tank, and the culture medium is treated at 121℃ and 102.9 kPa for 20 minutes; when the temperature is reduced to 37℃, 50 L of Clostridium butyricum seed in the seed tank A and 5 L of facultative anaerobic Bacillus in the seed tank B are simultaneously inoculated into the 3000 L fermentation tank, and the inoculation amounts are 2.5 vol.% and 0.25 vol.% respectively; the fermentation temperature is 37℃, and the culture is stirred at a constant time; after 48 hours of culture, 5 L of heterotrophic anaerobic lactic acid bacteria in the seed tank C is inoculated into the 3000 L fermentation tank, and the inoculation amount is 0.25 vol.%; at this time, the oral liquid is in a mixed fermentation state of the three kinds of bacteria, the fermentation temperature is 37℃, and the culture is stirred at a constant time; after 24 hours of continuous culture, 0.05 wt.% of anhydrous ethanol is added, and the product is filled after stirring; and the product needs to be statically matured at 20℃-37℃ for 6 days after filling.

2. The Clostridium butyricum oral liquid fermentation method based on the combination of step-by-step inoculation and static maturation according to claim 1, characterized in that, The culture method of the facultative anaerobic Bacillus and the heterotrophic anaerobic lactic acid bacteria is as follows: the culture medium is heated to 121℃, and sterilized and disinfected at a pressure of 102.9 kPa for 20 minutes; when the temperature of the seed tank is reduced to 37℃, the facultative anaerobic Bacillus and the heterotrophic anaerobic lactic acid bacteria are inoculated into 10 L seed tank B and 10 L seed tank C respectively, and the inoculation amounts are both 250 mL; the culture is stirred at a constant time, and the cultivated seed amounts are both 5 L.

3. The Clostridium butyricum oral liquid fermentation method based on the combination of step-by-step inoculation and static maturation according to claim 1, characterized in that, The constant time stirring is that the stirring system operates according to a set program, and the culture is stirred once every 120 minutes, and each time for 5 minutes.

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