A non-sterile production process for butyric acid
By employing a non-complete sterilization fermentation process using moist heat sterilization and a slightly positive pressure environment, the problems of high energy consumption, large carbon source loss, and contamination by miscellaneous bacteria caused by high-temperature and high-pressure sterilization have been solved. This process achieves high efficiency, stability, and high purity in butyric acid production, reduces equipment load, and improves production efficiency.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHUMADIAN HUAZHONG CHIA TAI CO LTD
- Filing Date
- 2026-04-30
- Publication Date
- 2026-06-23
AI Technical Summary
In existing industrial butyric acid fermentation production, high-temperature and high-pressure steam sterilization results in high energy consumption, significant carbon source loss, long production cycle, and easy equipment wear and tear. At the same time, under non-sterilization conditions, it is susceptible to contamination by other microorganisms, affecting product purity and yield.
A pure, highly active seed liquid was obtained by using a wet heat sterilized seed tank and seed culture medium. During the fermentation process, a slightly positive pressure environment was maintained, and combined with online pH adjustment, non-sterile fermentation was carried out to block the invasion of external bacteria.
Reduce production energy consumption, minimize carbon source loss, shorten production cycle, improve equipment turnover efficiency, ensure the purity and yield of butyric acid products, and inhibit the competitive growth of miscellaneous bacteria.
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Abstract
Description
Technical Field
[0001] This application relates to the technical field of organic acid production, and in particular to a non-sterilized production process for butyric acid. Background Technology
[0002] Butyric acid is an important organic acid with high application value in food, medicine, feed, flavorings, and chemical intermediates. Current industrial butyric acid fermentation production typically employs a complete sterilization process, where the seed tank, fermentation tank, and culture medium are sterilized by high-temperature, high-pressure steam before fermentation, followed by microbial propagation and fermentation to minimize the impact of contamination on the acid production process.
[0003] However, traditional fully sterilized fermentation processes still have some shortcomings in practical applications. First, the steam sterilization of fermenters and fermentation media requires processes such as heating, pressurizing, and cooling, resulting in significant consumption of steam, water, and electricity, leading to high overall energy consumption and production costs in the fermentation process. Second, for butyric acid fermentation systems using glucose as the main carbon source, the glucose is prone to caramelization, decomposition, or side reactions after being subjected to high temperature and high pressure, resulting in the loss of effective carbon source and adversely affecting subsequent microbial metabolism and butyric acid conversion. Third, the traditional fermenter sterilization process also prolongs the lead time for single-batch production, reduces equipment turnover efficiency, and repeated high-temperature and high-pressure operation can easily accelerate the aging and wear of tanks, pipelines, valves, and seals.
[0004] To reduce energy consumption, minimize carbon source heat loss, shorten production cycles, and alleviate equipment load, developing butyric acid production processes using non-sterilized or partially non-sterilized fermenters is of practical significance. However, butyric acid fermentation systems are highly sensitive to microbial contamination. Simply eliminating the sterilization steps of the fermenter and culture medium often leads to the invasion of exogenous microorganisms, competitive growth of other microorganisms, fermentation instability, and fluctuations in product purity and yield. Therefore, how to maintain the stable operation of the butyric acid fermentation process while ensuring product purity, yield, and production efficiency without high-temperature, high-pressure steam sterilization of the fermenter and fermentation medium has become a pressing technical problem to be solved in this field. Summary of the Invention
[0005] In order to improve product purity and yield without sterilizing the fermenter and fermentation medium with high temperature and high pressure steam, this application provides a butyric acid non-sterilized production process.
[0006] This application provides a non-sterile butyric acid production process, which adopts the following technical solution: A non-sterile production process for butyric acid includes the following steps: Seed culture: The seed tank and seed culture medium are sterilized by moist heat. After sterilization, butyric acid production bacteria are inoculated and cultured to obtain pure, highly active seed liquid. Fermentation medium preparation: Prepare glucose-based fermentation medium and transfer it to the fermenter; Non-sterile fermentation: Pure, highly active seed culture is introduced into the fermenter, and a slightly positive pressure environment is maintained inside the tank during the fermentation process to carry out butyric acid fermentation; Post-processing: After fermentation is complete, the fermentation products are extracted and purified to obtain butyric acid.
[0007] By adopting the above technical solution, the butyric acid production process is divided into a seed culture sterilization stage and a fermentation production non-sterilization stage. In the first stage, a pure, highly active seed culture is obtained through moist heat sterilization. In the second stage, butyric acid fermentation is carried out in a non-sterilized fermenter. During the fermentation process, a slightly positive pressure environment is maintained inside the fermenter. The pure, highly active seed culture establishes a growth advantage first, and the slightly positive pressure environment blocks the invasion of external bacteria, which helps maintain the stability of the fermentation system and ensures the purity and yield of butyric acid products. At the same time, it also reduces the steam consumption, cooling time, and equipment thermal shock caused by the sterilization of the fermenter and fermentation medium. This achieves the effects of reducing production energy consumption, reducing high-temperature glucose loss, shortening the production cycle, reducing equipment load, and improving equipment turnover efficiency.
[0008] In one specific implementation, during the seed culture step, the temperature for moist heat sterilization is 37°C, the sterilization pressure is 0.10-0.15 MPa, and the holding time is 20-40 min.
[0009] By adopting the above technical solution, the temperature, pressure and holding time of moist heat sterilization in the seed culture step are limited, so that the seed tank and seed culture medium can kill miscellaneous bacteria, spores and other contaminating microorganisms under relatively sufficient sterilization conditions, thereby providing a relatively clean culture environment for subsequent seed expansion culture, which is conducive to improving the feasibility and fermentation stability of the entire process.
[0010] In one specific implementation, the seed culture step comprises the following components per liter: 25-40g glucose, 8-15g yeast extract, 8-15g tryptone, 2-6g sodium acetate, 0.5-2.0g dipotassium hydrogen phosphate, 0.2-1.0g potassium dihydrogen phosphate, 0.1-0.5g magnesium sulfate, 0.005-0.05g manganese sulfate, 0.5-2.0g sodium chloride, 0.2-1.0g L-cysteine hydrochloride, and the remainder being water.
[0011] By adopting the above technical solution and setting components such as glucose, yeast extract, tryptone, sodium acetate, phosphate, magnesium salt, manganese salt, sodium chloride, and L-cysteine hydrochloride, a relatively complete carbon source, nitrogen source, inorganic salt, and growth cofactor can be provided for the expansion culture of butyric acid production strains. This helps to obtain seed liquid with high cell volume, strong activity, and good purity, thereby improving the inoculation quality of the subsequent non-sterilized fermentation stage.
[0012] In one specific implementation, the seed culture step includes the following steps: inoculating butyric acid producing bacteria into the cooled seed culture medium, culturing at 37°C for 12 hours, and controlling the stirring speed at 120 rpm to obtain a primary seed liquid; adding the primary seed liquid back into the seed culture medium, controlling the temperature at 37°C, and culturing for 8-16 hours to obtain a pure, highly active seed liquid.
[0013] By adopting the above technical solution, a primary seed culture is first formed under relatively stable conditions, and then the culture is further expanded to obtain a pure, highly active seed culture. This is beneficial to increase the cell concentration and metabolic activity when the culture is introduced into the fermenter, so that the target strain can form a quantitative and metabolic advantage more quickly in the subsequent non-sterilized fermentation system. This can more effectively inhibit the competitive growth of other bacteria and improve the stability and success rate of butyric acid fermentation under non-sterilized conditions.
[0014] In one specific implementation, the fermentation medium preparation step comprises the following components per liter: 60-120g glucose, 1-35g corn steep liquor, 3-12g yeast extract, 1-5g ammonium sulfate, 0.5-3g dipotassium hydrogen phosphate, 0.2-2g potassium dihydrogen phosphate, 0.1-1.0g magnesium sulfate, 0.005-0.05g manganese sulfate, 1-6g sodium acetate, 2-15g calcium carbonate, 0.05-0.5g defoamer, and the remainder being water.
[0015] By adopting the above technical solution and setting components such as glucose, corn steep liquor, yeast extract, ammonium sulfate, phosphate, magnesium sulfate, manganese sulfate, sodium acetate, calcium carbonate, and defoamer, sufficient carbon source, nitrogen source, inorganic salt, buffer components, and process auxiliary components can be provided for butyric acid fermentation. This is beneficial for maintaining cell growth and acid production metabolism under non-sterilized fermentation conditions, thereby improving butyric acid fermentation efficiency and process stability.
[0016] In one specific implementation, in the fermentation medium preparation step, the glucose-based fermentation medium is prepared at a temperature of 37°C and a stirring time of 20-60 minutes. After preparation, it is filtered through an 80-120 mesh filter and then transferred to a fermenter.
[0017] By adopting the above technical solution, the preparation temperature, stirring time and filtration conditions of the fermentation medium are limited, so that the medium is prepared under relatively mild conditions and enters the fermenter after being filtered through a certain mesh size. On the one hand, this is conducive to the full dispersion and uniform mixing of the components of the fermentation medium, avoiding local uneven concentration from affecting subsequent fermentation. On the other hand, filtration through 80-120 mesh can remove larger particulate impurities, reducing their impact on fermenter transportation, stirring and subsequent fermentation stability.
[0018] In one specific implementation, in the non-sterilized fermentation step, the inoculation amount of the pure, highly active seed culture is 10-15% of the volume of the glucose-based fermentation medium.
[0019] By adopting the above technical solution, the inoculation amount of pure high-activity seed liquid was limited, and the inoculation amount was controlled at 10-15% of the volume of glucose-based fermentation medium. This inoculation amount range is conducive to ensuring that the target strain can quickly establish a quantitative and metabolic advantage in the early stage of non-sterilized fermentation, thereby shortening the adaptation period and improving the target strain's ability to preferentially utilize nutrients in the culture medium, thus enhancing its competitive inhibition effect against exogenous bacteria.
[0020] In one specific feasible implementation, in the non-sterilized fermentation step, the fermentation temperature is 37°C, the fermentation pH is 6.0, the stirring speed is 60-250 rpm, and the fermentation time is 24-72 h.
[0021] By adopting the above technical solution, the temperature, pH, stirring speed and fermentation time in the non-sterilized fermentation process were limited, so that the fermentation conditions were more suitable for the growth and acid production metabolism needs of butyric acid producing strains.
[0022] In one specific implementation scheme, in the non-sterile fermentation step, an online pH adjustment is performed using a sodium hydroxide solution with a mass concentration of 20-40 wt% during the fermentation process.
[0023] By adopting the above technical solution and using a sodium hydroxide solution with a mass concentration of 20-40 wt% for online adjustment, it is beneficial to neutralize the acidic substances generated during fermentation in a timely manner, maintain the pH of the system within a suitable range, thereby ensuring cell activity, improving substrate conversion efficiency, and enhancing the stability of the fermentation process.
[0024] In one specific implementation, in the post-processing step, after fermentation, the fermentation broth is centrifuged at 6000 rpm for 20 min to remove cells and solids, and the supernatant is collected; the supernatant is acidified to pH=2.0 and then extracted and distilled to obtain butyric acid product.
[0025] By adopting the above technical solution, it is beneficial to separate the bacterial residue and solid impurities in the fermentation broth first, thereby reducing the impurity burden in the subsequent extraction and purification steps; and acidifying the supernatant to pH=2.0 is beneficial for butyric acid to exist in the form of free acid, thereby improving the efficiency of subsequent extraction and distillation; therefore, it is beneficial to improve the separation and purification effect and final purity of butyric acid products.
[0026] In summary, this application includes at least one of the following beneficial technical effects: The process described in this application divides the butyric acid production process into a seed culture sterilization stage and a fermentation production non-sterilization stage. In the first stage, a pure, highly active seed culture is obtained through moist heat sterilization. In the second stage, butyric acid fermentation is carried out in a non-sterilized fermenter. During the fermentation process, a slightly positive pressure environment is maintained inside the fermenter. The pure, highly active seed culture establishes a growth advantage first, and the slightly positive pressure environment blocks the invasion of external bacteria, which helps maintain the stability of the fermentation system and ensures the purity and yield of butyric acid products. At the same time, it also reduces the steam consumption, cooling time, and equipment thermal shock caused by the sterilization of the fermenter and fermentation medium. This results in reduced production energy consumption, reduced high-temperature glucose loss, shortened production cycle, reduced equipment load, and improved equipment turnover efficiency. The process described in this application limits the moist heat sterilization temperature, sterilization pressure, and holding time in the seed culture step, so that the seed tank and seed culture medium can be sterilized under relatively sufficient sterilization conditions to kill miscellaneous bacteria, spores and other contaminating microorganisms, thereby providing a relatively clean culture environment for subsequent seed expansion culture, which is conducive to improving the feasibility and fermentation stability of the entire process. The process described in this application uses a 20-40 wt% sodium hydroxide solution for online adjustment, which helps to neutralize acidic substances generated during fermentation in a timely manner, maintain the pH of the system within a suitable range, thereby ensuring cell activity, improving substrate conversion efficiency, and enhancing the stability of the fermentation process. Detailed Implementation
[0027] The present application will be further described in detail below with reference to the embodiments.
[0028] All raw materials used in the examples are commercially available. The defoamer is polypropylene glycol; the butyric acid producing strain is Clostridium butyricum. Example
[0029] Example 1 Example 1 provides a non-sterile production process for butyric acid, comprising the following steps: Seed culture: The seed tank and seed culture medium were sterilized by moist heat at 37℃ and 0.10 MPa for 40 min. Butyric acid production bacteria were inoculated into the cooled seed culture medium and cultured at 37℃ for 12 h with a stirring speed of 120 rpm to obtain the primary seed solution. The primary seed solution was then added back to the seed culture medium at a volume of 10% of the seed culture medium, maintaining the temperature at 37℃ and the pH at 6.5. The stirring speed was controlled at 140 rpm, and the culture was carried out for 16 hours to obtain a pure, highly active seed culture. The seed culture medium consisted of the following components per liter: glucose 32g, yeast extract 11g, tryptone 11g, sodium acetate 4g, dipotassium hydrogen phosphate 1.2g, potassium dihydrogen phosphate 0.6g, magnesium sulfate 0.3g, manganese sulfate 0.02g, sodium chloride 1.2g, L-cysteine hydrochloride 0.6g, and the remainder being water. The inoculum amount of butyric acid producing bacteria was 8% of the seed culture medium volume. Fermentation medium preparation: Prepare glucose-based fermentation medium at 37℃ with a stirring time of 60 minutes. After preparation, filter through a 100-mesh sieve and transfer to the fermenter. The glucose-based fermentation medium consists of the following components per liter: glucose 90g, corn steep liquor 18g, yeast extract 7g, ammonium sulfate 3g, dipotassium hydrogen phosphate 1.7g, potassium dihydrogen phosphate 1.1g, magnesium sulfate 0.5g, manganese sulfate 0.02g, sodium acetate 3g, calcium carbonate 8g, defoamer 0.2g, and the remainder is water. Non-sterile fermentation: Pure, highly active seed culture was inoculated into the fermenter at a volume of 10% of the glucose-based fermentation medium. During fermentation, a slightly positive pressure environment of 0.015 MPa was maintained inside the fermenter. The fermentation temperature was 37°C, the fermentation pH was 6.0, the stirring speed was 60 rpm, and the fermentation time was 72 h. Butyric acid fermentation was carried out, and the pH was adjusted online using a 30 wt% sodium hydroxide solution during the fermentation process. Post-processing: After fermentation is complete, the fermentation broth is centrifuged at 6000 rpm for 20 min to remove the cells and solids, and the supernatant is collected. The supernatant is acidified to pH=2.0 and then extracted and distilled to obtain butyric acid product.
[0030] Example 2 The difference between Example 2 and Example 1 lies in the seed culture: the seed tank and seed culture medium were subjected to moist heat sterilization at 37°C, a sterilization pressure of 0.12 MPa, and a holding time of 30 min. Butyric acid-producing bacteria were then inoculated into the cooled seed culture medium and cultured at 37°C for 12 h with a stirring speed of 120 rpm to obtain the primary seed solution. This primary seed solution was then added back to the seed culture medium at a volume of 10% of the seed culture medium, while maintaining the temperature at 37°C and the pH at 6.5. The stirring speed was controlled at 140 rpm, and the culture was carried out for 12 hours to obtain a pure, highly active seed culture. The seed culture medium contained the following components per liter: 32 g glucose, 11 g yeast extract, 11 g tryptone, 4 g sodium acetate, 1.2 g dipotassium hydrogen phosphate, 0.6 g potassium dihydrogen phosphate, 0.3 g magnesium sulfate, 0.02 g manganese sulfate, 1.2 g sodium chloride, 0.6 g L-cysteine hydrochloride, and the remainder was water. The inoculum amount of butyric acid producing bacteria was 8% of the seed culture medium volume. The remaining steps were the same as in Example 1.
[0031] Example 3 The difference between Example 3 and Example 1 lies in the seed culture: the seed tank and seed culture medium were subjected to moist heat sterilization at 37°C and 0.15 MPa for 20 minutes. Butyric acid-producing bacteria were then inoculated into the cooled seed culture medium and cultured at 37°C for 12 hours with a stirring speed of 120 rpm to obtain the primary seed solution. This primary seed solution was then added back to the seed culture medium at a volume of 10% of the seed culture medium, while maintaining the temperature at 37°C and the pH at 6.5. The stirring speed was controlled at 140 rpm, and the culture was carried out for 8 hours to obtain a pure, highly active seed culture. The seed culture medium contained the following components per liter: 32 g glucose, 11 g yeast extract, 11 g tryptone, 4 g sodium acetate, 1.2 g dipotassium hydrogen phosphate, 0.6 g potassium dihydrogen phosphate, 0.3 g magnesium sulfate, 0.02 g manganese sulfate, 1.2 g sodium chloride, 0.6 g L-cysteine hydrochloride, and the remainder was water. The inoculum amount of butyric acid producing bacteria was 8% of the seed culture medium volume. The remaining steps were the same as in Example 1.
[0032] Example 4 The difference between Example 4 and Example 2 lies in the preparation of the fermentation medium: a glucose-based fermentation medium was prepared at 37°C with a stirring time of 40 minutes. After preparation, the medium was filtered through a 100-mesh sieve and then transferred to a fermenter. The glucose-based fermentation medium, per liter, included the following components: 90g glucose, 18g corn steep liquor, 7g yeast extract, 3g ammonium sulfate, 1.7g dipotassium hydrogen phosphate, 1.1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.02g manganese sulfate, 3g sodium acetate, 8g calcium carbonate, 0.2g defoamer, and the remainder being water. The remaining steps were the same as in Example 2.
[0033] Example 5 The difference between Example 5 and Example 2 lies in the preparation of the fermentation medium: a glucose-based fermentation medium was prepared at 37°C with a stirring time of 20 minutes. After preparation, the medium was filtered through a 100-mesh sieve and then transferred to a fermenter. The glucose-based fermentation medium, per liter, comprised the following components: 90g glucose, 18g corn steep liquor, 7g yeast extract, 3g ammonium sulfate, 1.7g dipotassium hydrogen phosphate, 1.1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.02g manganese sulfate, 3g sodium acetate, 8g calcium carbonate, 0.2g defoamer, and the remainder being water. The remaining steps were consistent with those in Example 2.
[0034] Example 6 The difference between Example 6 and Example 4 is that the fermentation is not completely sterilized: a pure high-activity seed culture is introduced into the fermenter, and the inoculation amount of the pure high-activity seed culture is 12.5% of the volume of the glucose-based fermentation medium. During the fermentation process, a micro-positive pressure environment of 0.015 MPa is maintained in the fermenter, the fermentation temperature is 37°C, the fermentation pH is 6.0, the stirring speed is 150 rpm, and the fermentation time is 48 h for butyric acid fermentation. During the fermentation process, a 30 wt% sodium hydroxide solution is used for online pH adjustment; the remaining steps are the same as in Example 4.
[0035] Example 7 The difference between Example 7 and Example 4 is that the fermentation is not completely sterilized: a pure high-activity seed culture is introduced into the fermenter, and the inoculation amount of the pure high-activity seed culture is 15% of the volume of the glucose-based fermentation medium. During the fermentation process, a micro-positive pressure environment of 0.015 MPa is maintained in the fermenter, the fermentation temperature is 37°C, the fermentation pH is 6.0, the stirring speed is 250 rpm, and the fermentation time is 24 h for butyric acid fermentation. During the fermentation process, a 30 wt% sodium hydroxide solution is used for online pH adjustment; the remaining steps are the same as in Example 4. Comparative Example
[0036] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the fermentation was not completely sterilized: a pure, highly active seed culture was introduced into the fermenter, and the inoculation amount of the pure, highly active seed culture was 10% of the volume of the glucose-based fermentation medium. During the fermentation process, the pressure inside the tank was kept consistent with the external pressure. The fermentation temperature was 37°C, the fermentation pH was 6.0, the stirring speed was 60 rpm, and the fermentation time was 72 h for butyric acid fermentation. During the fermentation process, a 30 wt% sodium hydroxide solution was used for online pH adjustment. The remaining steps were the same as in Example 1.
[0037] Comparative Example 2 The difference between Comparative Example 2 and Example 1 lies in the seed culture: the moist heat sterilization of the seed tank and seed culture medium was omitted; butyric acid production bacteria were inoculated into the seed culture medium and cultured at 37°C for 12 hours with a stirring speed of 120 rpm to obtain a primary seed solution; the primary seed solution was then added back into the seed culture medium at a volume of 10% of the seed culture medium volume; the temperature was controlled at 37°C, the pH at 6.5, and the stirring speed at 140 rpm for 16 hours to obtain a pure, highly active seed solution; the seed culture medium, per liter, comprised the following components: 32 g glucose, 11 g yeast extract, 11 g tryptone, 4 g sodium acetate, 1.2 g dipotassium hydrogen phosphate, 0.6 g potassium dihydrogen phosphate, 0.3 g magnesium sulfate, 0.02 g manganese sulfate, 1.2 g sodium chloride, 0.6 g L-cysteine hydrochloride, with the remainder being water; the inoculation amount of butyric acid production bacteria was 8% of the seed culture medium volume; the remaining steps were consistent with Example 1. Performance testing experiment
[0038] Contamination rate: After fermentation, 10 mL of fermentation broth was taken under aseptic conditions and serially diluted 10-fold with sterile physiological saline. Appropriate dilutions were then plated onto RCM-enhanced Clostridium agar plates and anaerobically incubated at 37°C for 48 h. The total colony count was then recorded. Simultaneously, based on colony morphology, microscopic observation, and Gram staining results, typical non-caseinic Clostridium colonies were counted and recorded as contamination colonies. The contamination rate was calculated as: Contamination rate = (Contamination colony count / Total colony count) × 100%.
[0039] Butyric acid yield: After fermentation, the fermentation broth sample was taken, centrifuged at 6000 rpm for 10 min, and the supernatant was collected. After filtration through a 0.22 μm microporous membrane, the sample was detected by high performance liquid chromatography (HPLC). The chromatographic column was an organic acid analytical column; the mobile phase was 0.005 mol / L sulfuric acid aqueous solution; the flow rate was 0.6 mL / min; the column temperature was 55℃; the detection wavelength was 210 nm; and the injection volume was 10 μL. A standard curve was prepared using butyric acid standards, and the butyric acid concentration in the fermentation broth was calculated based on the peak area of the sample.
[0040] Butyric acid purity: Butyric acid products are tested using gas chromatography to determine the mass fraction of butyric acid in the products.
[0041] Table 1 Performance test results of butyric acid products
[0042] In conjunction with Examples 1-7 and Comparative Examples 1-2, the contamination rates in each example of this application are significantly lower than those in Comparative Examples 1 and 2. This indicates that the process of this application, through the combined control of "moist heat sterilization during seed culture, inoculation with pure, highly active seed liquid, and maintenance of a slightly positive pressure environment during the non-sterile fermentation stage," can effectively inhibit the invasion and competitive growth of exogenous contaminants, thereby maintaining the stability of the fermentation system. In particular, in Comparative Example 1, without maintaining a slightly positive pressure, the contamination rate significantly increased to 6.84%, indicating that under non-sterile fermentation conditions, a slightly positive pressure environment plays an important role in blocking the invasion of external contaminants. In Comparative Example 2, after eliminating moist heat sterilization before seed culture, the contamination rate further increased to 11.36%, indicating that the cleanliness of the initial seed system and the quality of the pure seed liquid also have a fundamental impact on the subsequent non-sterile fermentation.
[0043] In terms of butyric acid production, all examples showed higher yields than the comparative example, indicating that the process described in this application can not only control contamination but also ensure that the target strain can efficiently utilize glucose in the culture medium to produce acid.
[0044] Combined with Examples 1-3, Example 2 performed better than Examples 1 and 3, indicating that using moderate moist heat sterilization and expansion parameters during the seed culture stage is more conducive to obtaining seed liquid with higher activity and more stable state. Combining Examples 2, 4, and 5, Examples 4 and 5 further optimized the preparation conditions of the fermentation medium compared to Example 2. Among them, Example 4 performed best under the preparation conditions of 37°C and 40 min, indicating that a milder and more thorough preparation process is more conducive to the homogeneity of the medium and the stability of subsequent fermentation.
[0045] Combining Examples 4, 6, and 7, Example 6 showed the highest butyric acid yield, reaching 49.6 g / L. This indicates that the inoculum size, fermentation temperature, pH, stirring speed, and fermentation time were optimal in Example 6, which was more conducive to the target strain establishing a competitive advantage and continuously producing high-efficiency butyric acid. In contrast, although Example 7 maintained a low contamination rate, the higher fermentation temperature, pH, and stirring speed, coupled with a shortened fermentation time of 24 h, resulted in insufficient butyric acid accumulation, leading to a lower butyric acid yield than Example 6.
[0046] Regarding the purity of butyric acid, the butyric acid products obtained in all examples achieved a purity of over 98%, with Example 6 reaching the highest at 99.2%. In contrast, the butyric acid purity of Comparative Examples 1 and 2 decreased to 95.8% and 93.9%, respectively. This indicates that in the process of this application, a lower rate of contaminating microorganisms not only helps increase butyric acid yield but also reduces the accumulation of fermentation byproducts and impurities, thereby facilitating post-processing separation and purification, ultimately improving product purity. In particular, Comparative Examples 1 and 2, due to severe contamination by microorganisms, experienced disruptions to the target metabolic pathway and an increase in byproducts, ultimately resulting in a significant decrease in the purity of the obtained butyric acid products.
[0047] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.
Claims
1. A non-sterilized production process for butyric acid, characterized in that: Includes the following steps: Seed culture: The seed tank and seed culture medium are sterilized by moist heat. After sterilization, butyric acid production bacteria are inoculated and cultured to obtain pure, highly active seed liquid. Fermentation medium preparation: Prepare glucose-based fermentation medium and transfer it to the fermenter; Non-sterile fermentation: Pure, highly active seed culture is introduced into the fermenter, and a slightly positive pressure environment is maintained inside the tank during the fermentation process to carry out butyric acid fermentation; Post-processing: After fermentation is complete, the fermentation products are extracted and purified to obtain butyric acid.
2. The butyric acid non-sterilized production process according to claim 1, characterized in that: In the seed culture step, the temperature for moist heat sterilization is 37℃, the sterilization pressure is 0.10-0.15MPa, and the holding time is 20-40min.
3. The butyric acid non-sterilized production process according to claim 2, characterized in that: In the seed culture step, the seed culture medium comprises the following components per liter: 25-40g glucose, 8-15g yeast extract, 8-15g tryptone, 2-6g sodium acetate, 0.5-2.0g dipotassium hydrogen phosphate, 0.2-1.0g potassium dihydrogen phosphate, 0.1-0.5g magnesium sulfate, 0.005-0.05g manganese sulfate, 0.5-2.0g sodium chloride, 0.2-1.0g L-cysteine hydrochloride, and the remainder is water.
4. The butyric acid non-sterilized production process according to claim 3, characterized in that: The seed culture step is as follows: butyric acid producing bacteria are inoculated into the cooled seed culture medium and cultured at 37°C for 12 hours with the stirring speed controlled at 120 rpm to obtain a primary seed liquid; the primary seed liquid is added back into the seed culture medium and the temperature is controlled at 37°C for 8-16 hours to obtain a pure, highly active seed liquid.
5. The butyric acid non-sterilized production process according to claim 1, characterized in that: In the preparation step of the fermentation medium, the glucose-based fermentation medium comprises the following components per liter: 60-120g glucose, 1-35g corn steep liquor, 3-12g yeast extract, 1-5g ammonium sulfate, 0.5-3g dipotassium hydrogen phosphate, 0.2-2g potassium dihydrogen phosphate, 0.1-1.0g magnesium sulfate, 0.005-0.05g manganese sulfate, 1-6g sodium acetate, 2-15g calcium carbonate, 0.05-0.5g defoamer, and the remainder is water.
6. The butyric acid non-sterilized production process according to claim 5, characterized in that: In the fermentation medium preparation step, the glucose-based fermentation medium is prepared at a temperature of 37°C and a stirring time of 20-60 minutes. After preparation, it is filtered through an 80-120 mesh filter and then transferred to a fermenter.
7. The butyric acid non-sterilized production process according to claim 1, characterized in that: In the non-sterilized fermentation step, the inoculation amount of the pure, highly active seed culture is 10-15% of the volume of the glucose-based fermentation medium.
8. The butyric acid non-sterilized production process according to claim 7, characterized in that: In the non-sterilized fermentation step, the fermentation temperature is 37℃, the fermentation pH is 6.0, the stirring speed is 60-250 rpm, and the fermentation time is 24-72 h.
9. The butyric acid non-sterilized production process according to claim 8, characterized in that: In the non-sterilized fermentation step, a sodium hydroxide solution with a mass concentration of 20-40 wt% is used for online pH adjustment during the fermentation process.
10. The butyric acid non-sterilized production process according to claim 1, characterized in that: In the post-processing step, after fermentation, the fermentation broth is centrifuged at 6000 rpm for 20 min to remove the cells and solids, and the supernatant is collected. The supernatant is acidified to pH=2.0 and then extracted and distilled to obtain butyric acid product.