A fed-batch fermentation method for increasing the number of Bacillus licheniformis spores

Abstract

The present invention discloses a fed-batch fermentation method for increasing the number of Bacillus licheniformis spores, belonging to the field of microbial fermentation technology. The present invention uses a fed-batch technology to control the concentration of carbon and nitrogen sources to promote cell growth and spore formation, thereby increasing the number of Bacillus licheniformis BF-002 cells and spores. The number of Bacillus licheniformis cells can reach 2.88×10 at the end of fermentation. 10 cfu / mL, which is 1.07 times higher than the highest cell count achieved in the early stage. The number of spores reached 2.56×10 10 cfu / mL, which is 1.16 times higher than the optimal batch spore count in the previous period. The culture medium components used in the present invention are readily available and inexpensive, and the process parameters are simple, which significantly increases unit yield and reduces unit production cost, enabling large-scale industrial fermentation production.

Description

Technical Field

[0001] The invention relates to a fed-batch fermentation method for increasing the number of Bacillus licheniformis spores, and belongs to the technical field of microbial fermentation. Background Art

[0002] Bacillus licheniformis is a Gram-positive bacterium that is abundant in the soil. When encountering adverse environments during its growth process, such as nutrient deficiency, excessively high or low pH, and the accumulation of harmful metabolites, Bacillus licheniformis will produce endospores (spores) to resist the harsh environment. Probiotics, also known as live bacterial preparations or biocides, refer to preparations made through special processes using probiotics or probiotic growth-promoting substances that are beneficial and harmless to the host. Probiotics have been applied in various fields such as feed, agriculture, medical care, and food. Bacillus licheniformis is a new type of probiotic bacteria. It not only has many good uses in the aquaculture industry, but also plays an important role in plant disease prevention and control, environmental pollution control, etc. During use, it is green, efficient, and has low pollution. Due to its wide application in animal husbandry, environmental management, biological control, etc., Bacillus licheniformis has gradually attracted the attention of more and more researchers. The preparation of high-quality Bacillus licheniformis inoculants is one of the current key research directions. The number of viable cells and spores in the culture medium are important quality indicators for Bacillus licheniformis inoculants, with spore count being particularly crucial. During inoculant production and application, spores effectively withstand extreme environmental conditions and are resistant to inactivation during formulation, storage, and transportation, effectively maintaining the inoculant's activity. Once in the animal's intestines, spores rapidly germinate, forming new vegetative cells and exerting their physiological functions. Therefore, obtaining a culture medium containing a large number of spores is a crucial approach to improving inoculant quality. Summary of the Invention

[0003] According to the different effects of carbon source and nitrogen source concentrations on the spore formation process of Bacillus licheniformis, the present invention proposes a fed-batch method for increasing the cell and spore amounts by controlling the substrate concentration in a fermentation tank.

[0004] The present invention provides a fermentation method for increasing the number of Bacillus licheniformis spores. The method comprises the following steps: fermenting the Bacillus licheniformis at 35-40°C, adding a feed medium containing glucose starting from the 7th hour of the Bacillus licheniformis fermentation, and controlling the final concentration of glucose in the fermentation system to be 10-15 g / L.

[0005] In one embodiment, the culture medium for fermentation contains fish meal peptone, soybean meal, sodium chloride, calcium chloride, potassium chloride, magnesium sulfate, and manganese sulfate monohydrate.

[0006] In one embodiment, the fermentation is carried out in a fermenter, the dissolved oxygen is controlled within the range of 30% to 50%, the ventilation ratio is maintained at 2±0.2vvm, and the tank pressure is 0.02Mpa to 0.05Mpa.

[0007] In one embodiment, the carbon-nitrogen ratio in the feed medium is (10-20):1.

[0008] In one embodiment, the feed medium contains glucose and ammonium chloride.

[0009] In one embodiment, the feed medium is added in a continuous manner.

[0010] In one embodiment, the feed medium is fed from the 7th hour of fermentation until the 30th hour of fermentation.

[0011] In one embodiment, the method specifically comprises: controlling the initial glucose concentration of the fermentation system to 20 g / L, controlling the initial amino nitrogen concentration to 3.51 g / L, controlling the glucose concentration in the fermentation system to 10 g / L to 12 g / L from 7 h to 24 h, controlling the amino nitrogen concentration in the fermentation system to 1.4 g / L to 1.6 g / L from 7 h to 24 h, starting to reduce the glucose and amino nitrogen concentrations from 25 h, and controlling the glucose and amino nitrogen concentrations to 1.5 g / L to 2.5 g / L and 0.4 g / L to 0.6 g / L, respectively, from 28 h to 48 h. Fermentation is completed at 37° C. for 48 h.

[0012] In one embodiment, the Bacillus licheniformis is Bacillus licheniformis BF-002, which has been disclosed in the paper "Study on Nitrogen Source Fed-Batch Process for High Spore Production of Bacillus licheniformis BF-002".

[0013] In one embodiment, the Bacillus licheniformis is inoculated into the fermentation system in the form of seed liquid, and the OD after inoculation reaches above 3.5.

[0014] In one embodiment, the method for preparing the seed solution comprises:

[0015] (1) Primary seed preparation: Bacillus licheniformis BF-002 was cultured on solid LB medium at 35-37°C for 15-20 h;

[0016] (2) Preparation of secondary seeds: The single colony activated in step (1) was inoculated into liquid LB culture medium and cultured in a shaking incubator at 35-37°C and 160 rpm for 16-18 hours to obtain seed liquid.

[0017] The present invention also claims protection for the Bacillus licheniformis preparation prepared by the method.

[0018] Beneficial effects: The present invention controls the concentration of carbon and nitrogen sources through fed-batch technology to promote the cell growth and spore formation of Bacillus licheniformis, thereby increasing the number of cells and spores. The number of cells of Bacillus licheniformis BF-002 can reach 2.88×10 10 cfu / mL, which is 1.07 times higher than the highest cell count achieved in the early stage. The number of spores reached 2.56×10 10 cfu / mL, a 1.16-fold increase compared to the previous optimal batch spore count, achieving optimal cell and spore counts. The method of the present invention utilizes readily available and inexpensive culture medium, simple process parameters, and significantly increases unit yield while reducing unit production costs. This enables large-scale industrial fermentation production and provides relevant technical insights for optimizing fermentation processes for other Bacillus species. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results of Example 1 show the effects of different carbon source concentrations on cell growth and spore formation.

[0020] Figure 2 The results of Example 2 show the effects of different carbon-nitrogen ratios on cell growth and spore formation.

[0021] Figure 3 The results of Example 3 show the effect of intermittent carbon source addition on cell growth and spore formation.

[0022] Figure 4 The results of Example 4 showing the effect of controlling substrate feed on BF-002 fermentation;

[0023] Figure 5 This is the result of Example 5 on the effect of low carbon and nitrogen source concentration on bf-002 fermentation. DETAILED DESCRIPTION

[0024] The technical solution of the invention is described in detail below with reference to the accompanying drawings:

[0025] In the following examples, the Bacillus licheniformis used was sourced from Qingdao Genyuan Biotechnology Group Co., Ltd., numbered BF-002, and disclosed in the paper "Study on Nitrogen Source Fed-Batch Process for High Spore Production of Bacillus licheniformis BF-002".

[0026] The instruments used include: BIOTECH-5BG 5L fermenter; SBA-40C biosensor analyzer; Metso constant temperature water bath; UV spectrophotometer; LMK-2000A exhaust gas analyzer; BQ50-1J-A programmable peristaltic pump.

[0027] Detection of amino nitrogen concentration: After centrifugation of the fermentation broth, the supernatant was taken and the amino nitrogen concentration was determined by formaldehyde titration.

[0028] Determination of glucose concentration: After centrifugation of the fermentation broth, the supernatant was taken and diluted with distilled water to a glucose concentration in the range of 0.2 g / L to 0.8 g / L, and then detected using an SBA-40C biosensor analyzer.

[0029] Live bacteria count detection: Use dilution plate colony counting method. Dilute the fermentation liquid of Bacillus licheniformis BF-002 gradually to 10 -6 , 10 -7 , 10 -8 Three different concentration gradients were prepared. 100 μL of each dilution concentration was evenly spread on LB solid culture medium. Three plates were spread in each group and incubated upside down in a 37°C constant temperature incubator for 18 h. The number of colonies on the plates was counted, which was the number of viable Bacillus licheniformis cells and spores.

[0030] Spore quantity detection: After the live bacteria coating is completed, 10 -6 , 10 -7 , 10 -8 Three different concentrations of dilution were placed in a constant temperature water bath, treated at 80°C for 15 minutes, and then quickly cooled in ice. Then, 100 μL of each of the three concentration gradients was evenly spread on LB solid culture medium. Three plates were spread for each group, and the plates were inverted and cultured in a constant temperature incubator at 37°C for 18 hours. The number of colonies on the plates was counted, which was the number of Bacillus licheniformis spores.

[0031] Culture medium:

[0032] Primary seed culture medium (per L): peptone 10 g, sodium chloride 10 g, yeast powder 10 g, agar 20 g. Medium pH natural.

[0033] Secondary seed culture medium (per L): 10 g peptone, 10 g sodium chloride, 10 g yeast powder, and natural pH.

[0034] Fermentation medium (per L): 5g fish meal peptone, 15g soybean meal, 5g sodium chloride, 1g calcium chloride, 1g potassium chloride, 1g magnesium sulfate, 0.04g manganese sulfate monohydrate. The medium pH is 6.9-7.

[0035] Carbon source fed-batch medium (per L): 500 g glucose, 1 g calcium chloride, 1 g potassium chloride, 1 g magnesium sulfate, 0.8 g manganese sulfate monohydrate.

[0036] Nitrogen source feed medium (per L): 50 g ammonium chloride.

[0037] Example 1 Effects of different carbon source concentrations on cell growth and spore formation

[0038] To investigate the effects of different carbon source concentrations on BF-002 cell growth and spore formation, four batches of fermentation experiments were conducted in a 5L fermenter. The specific steps are as follows:

[0039] Preparation of seed solution: inoculate Bacillus licheniformis onto primary seed culture medium and culture at 37°C for 20 h; pick a single colony and inoculate onto secondary seed culture medium and culture at 37°C, 160 rpm for 18 h. The obtained fermentation liquid is used as seed solution.

[0040] Fermentation medium (per L): 20 g glucose, 5 g fish meal peptone, 15 g soybean meal, 5 g sodium chloride, 1 g calcium chloride, 1 g potassium chloride, 1 g magnesium sulfate, 0.04 g manganese sulfate monohydrate. The medium pH is 6.9-7.

[0041] Carbon source fed-batch medium (per L): 500 g glucose, 1 g calcium chloride, 1 g potassium chloride, 1 g magnesium sulfate, 0.8 g manganese sulfate monohydrate.

[0042] Fermentation conditions: 300 mL of seed liquid was inoculated into a 5 L fermentation system with 2.7 L of fermentation medium, and the OD after inoculation was 600 The dissolved oxygen concentration reached 3.75. A DO electrode was used to monitor dissolved oxygen online. The speed was manually adjusted to maintain the dissolved oxygen concentration between 30% and 50%. The ventilation ratio was maintained at 2 vvm. The tank pressure was maintained at 0.02 MPa to 0.05 MPa. The following four batches of fermentation experiments were set up:

[0043] Batch 1: Fermentation was carried out at 37°C for the first 6 h. The fermentation temperature was kept constant from 7h to 36h. The pH-Stat strategy was used to control glucose feeding. Specifically, the pH was set to 7.0, and the pH was adjusted with 4moL / L NAOH solution. When the pH was greater than 7, the peristaltic pump was automatically started to feed the carbon source and culture medium. When the pH was less than 7, the feeding of the carbon source and culture medium was automatically stopped to control the concentration of glucose in the fermentation system to ≤2g / L. The fermentation was continued for a total of 36h.

[0044] Batch 2: Fermentation was carried out at 37°C for the first 6 h, and the fermentation temperature was kept constant from 7h to 36h. The carbon source was added to the culture medium using an offline determination-manual control method to control the final concentration of glucose in the fermentation system at 5±1 g / L. The fermentation temperature was controlled at 37°C during the fermentation process, and the fermentation was terminated after 36h of reaction.

[0045] Batch 3: Fermentation was carried out at 37°C for the first 6 h, and the fermentation temperature was kept constant from 7h to 36h. The carbon source was added to the culture medium using an offline determination-manual control method to control the final concentration of glucose in the fermentation system at 10±1 g / L. The fermentation temperature was controlled at 37°C during the fermentation process, and the fermentation was terminated after 36h of reaction.

[0046] Batch 4: Fermentation was carried out at 37°C for the first 6 h. From the 7th to the 36th h of fermentation, the carbon source was added to the culture medium using an offline determination-manual control method to control the final concentration of glucose in the fermentation system at 15±1 g / L. The fermentation temperature was controlled at 37°C during the fermentation process, and the fermentation was terminated after 36 h of reaction.

[0047] like Figure 1 As shown in Figure 2, a final glucose concentration of 10 g / L is more conducive to cell growth, and the number of viable cells reached 2.05 × 10 after 15 h of culture. 10 cfu / mL, significantly higher than the levels observed in the other three fermentation batches at the same stage of culture. Simultaneously with nitrogen source depletion, a step-wise increase in spore count was observed. Spore formation began rapidly after nitrogen source depletion, with nitrogen source depletion times for the four batches reaching 21, 27, 15, and 24 hours, respectively. These results demonstrate a synergistic effect of carbon and nitrogen sources on cell growth.

[0048] Example 2 Effects of different carbon-nitrogen ratios on cell growth and spore formation

[0049] To investigate the effects of different carbon-nitrogen ratios on BF-002 cell growth and spore formation, three batches of fermentation experiments were conducted in a 5L fermenter. The specific steps are as follows:

[0050] The preparation of seed solution is the same as in Example 1.

[0051] Fermentation medium (per L): 20 g glucose, 1.5 g ammonium chloride, 5 g fish meal peptone, 15 g soybean meal, 5 g sodium chloride, 1 g calcium chloride, 1 g potassium chloride, 1 g magnesium sulfate, 0.04 g manganese sulfate monohydrate. The medium pH is 6.9-7.

[0052] Mixed medium of carbon source and nitrogen source (per L): 500 g glucose, 100 g, 33.3 g or 20 g ammonia chloride respectively.

[0053] Fermentation conditions: 300 mL of seed liquid was inoculated into a 5 L fermentation system with 2.7 L of fermentation medium, and the OD after inoculation was 600 The dissolved oxygen concentration reached 3.75. A DO electrode was used to monitor dissolved oxygen online. The speed was manually adjusted to maintain the dissolved oxygen concentration between 30% and 50%. The ventilation ratio was maintained at 2 vvm. The tank pressure was maintained at 0.02 MPa to 0.05 MPa. The following three batches of fermentation experiments were set up:

[0054] Batch 1: Control the initial glucose concentration to 20 g / L and the initial amino nitrogen concentration to 1.54 g / L (1.5 g / L ammonium chloride, 5 g / L fish meal peptone, and 15 g / L soybean meal in the initial culture medium). Ferment at 37°C for the first 6 h. At 6 h, the glucose concentration decreased to 9 g / L-11 g / L, and the amino nitrogen concentration decreased to 0.4 g / L-0.6 g / L. From h 7 to h 11, a mixed culture medium with a carbon and nitrogen source was fed at a glucose to ammonium chloride mass ratio of 5:1. Starting from h 12, only glucose was fed so that the final concentration of glucose in the fermentation system was controlled at 10 g / L. Fermentation was carried out at 37°C for 36 h.

[0055] Batch 2: The initial glucose concentration was controlled at 20 g / L, and the initial amino nitrogen concentration was 1.54 g / L (1.5 g / L ammonium chloride, 5 g / L fish meal peptone, and 15 g / L soybean meal in the initial culture medium). Fermentation was carried out at 37°C for the first 6 h. At 6 h, the glucose concentration decreased to 9 g / L-11 g / L, and the amino nitrogen concentration decreased to 0.4 g / L-0.6 g / L. From h 7 to h 11, a mixed culture medium with a carbon and nitrogen source was fed at a glucose to ammonium chloride mass ratio of 15:1. From h 12 onwards, only glucose was fed, so that the final glucose concentration in the fermentation system was controlled at 10 g / L. Fermentation was carried out at 37°C for 36 h.

[0056] Batch 3: The initial glucose concentration was controlled at 20 g / L, and the initial amino nitrogen concentration was 1.54 g / L (1.5 g / L ammonium chloride, 5 g / L fish meal peptone, and 15 g / L soybean meal in the initial culture medium). Fermentation was carried out at 37°C for the first 6 h. At 6 h, the glucose concentration decreased to 9 g / L-11 g / L, and the amino nitrogen concentration decreased to 0.4 g / L-0.6 g / L. From h 7 to h 11, a mixed culture medium with a carbon and nitrogen source was fed at a glucose to ammonium chloride mass ratio of 25:1. From h 12 onwards, only glucose was fed so that the final glucose concentration in the fermentation system was controlled at 10 g / L. Fermentation was carried out at 37°C for 36 h.

[0057] like Figure 2 As shown in Figure 2, when the carbon-nitrogen ratio was 15:1, the spore count was the highest after 36 h of fermentation, reaching 2.17×10 10 cfu / mL. When the carbon-nitrogen ratio was too high (5:1), the amino nitrogen concentration reached 2.67 g / L when the addition of NH4Cl was stopped at 12 h, which was much higher than the initial amino concentration. It was not completely depleted until 27 h of fermentation. During most of the fermentation process, the nitrogen source concentration was high and the cells grew rapidly. The cell count at the end of the fermentation reached 2.96×10 10 cfu / mL, which is 1.19 times and 1.66 times that of the batches with carbon-nitrogen ratios of 15:1 and 25:1. However, the high nitrogen concentration is not conducive to spore formation, and the number of spores at the end of fermentation is only 1.61×10 10cfu / mL, which is only 74% of that in the batch with a carbon-nitrogen ratio of 15:1. When the carbon-nitrogen ratio is too low (25:1), cell growth is restricted due to the low nitrogen source concentration. Although low nitrogen source concentration is conducive to spore formation, the number of viable cells in the fermentation broth is low. Even when the spore formation rate reaches 93.5% at the end of fermentation, the number of spores is only 1.7×10 10 cfu / mL, which is much lower than that of the batch with a carbon-nitrogen ratio of 15:1. Multiple experiments have shown that a carbon-nitrogen ratio of (10-20):1 is more conducive to spore formation.

[0058] Example 3 Effect of intermittent carbon source addition on cell growth and spore formation

[0059] In order to investigate the effect of different carbon source concentrations on the culture performance of the BF-002 strain, an intermittent feeding method was used to control the level of glucose in the fermentation broth. The specific steps are as follows:

[0060] Fermentation medium (per L): 30 g glucose, 5 g ammonium chloride, 5 g fish meal peptone, 15 g soybean meal, 5 g sodium chloride, 1 g calcium chloride, 1 g potassium chloride, 1 g magnesium sulfate, 0.04 g manganese sulfate monohydrate. The medium pH is 6.9-7.

[0061] Carbon source fed-batch culture medium (per L): 500 g glucose.

[0062] Fermentation conditions: DO was monitored online using a DO electrode. The speed was manually adjusted to maintain dissolved oxygen levels between 30% and 50%. The ventilation ratio was maintained at 2 vvm. The tank pressure was maintained between 0.02 MPa and 0.05 MPa.

[0063] Set up 2 batches of fermentation experiments as follows:

[0064] Batch 1: Control the initial concentration of glucose to 30 g / L, control the initial concentration of amino nitrogen to 3.51 g / L (5 g / L ammonium chloride, 5 g / L fish meal peptone and 15 g / L soybean meal in the initial culture medium), ferment at 37 ° C, when the amino nitrogen concentration dropped to 1.5 g / L, maintain the amino nitrogen concentration in the fermentation system at 1.4 g / L to 1.6 g / L for 12 h to 36 h, inoculate 300 mL of seed liquid into a 5 L fermentation system with a fermentation medium volume of 2.7 L, and make the bacterial OD in the fermentation system 600 The glucose concentration in the fermentation system was increased to 20 g / L by adding the carbon source feed medium. After that, no more glucose was added until the fermentation was completed after 36 h.

[0065] Batch 2: Control the initial concentration of glucose to 30 g / L, control the initial concentration of amino nitrogen to 3.53 g / L (5 g / L ammonium chloride, 5 g / L fish meal peptone and 15 g / L soybean meal in the initial culture medium). When the amino nitrogen concentration is reduced to 1.5 g / L, the amino nitrogen concentration in the fermentation system is maintained at 1.4 g / L to 1.6 g / L for 12 h to 36 h. Inoculate 300 mL of seed liquid into a 5 L fermentation system with a fermentation medium volume of 2.7 L. Make the bacterial OD in the fermentation system 600 The glucose concentration was 3.75, and the culture medium was added again at 37°C for 9 h until the glucose concentration dropped to 10 g / L, and the carbon source was increased to 40 g / L. After that, no more glucose was added until the fermentation was completed at 36 h.

[0066] The first batch fermentation performance is as follows Figure 3 As shown in a. It can be seen that under the conditions of sufficient carbon and nitrogen sources (0h-15h), only a very small amount of spores are formed. After 15h of fermentation, the nitrogen source is still sufficient, but the glucose concentration continues to decrease. At 18h, the glucose concentration has dropped to 2.6g / L, and spores begin to form. After 21h of fermentation, the glucose is almost completely consumed, and the number of spores reaches 0.52×10 10 cfu / mL, 2.36 times the level at 18 hours. After 21 hours of fermentation, the glucose concentration approaches 0 g / L. Even though nitrogen remains sufficient, the carbon source is insufficient, causing the number of viable cells and spores to gradually decline. Based on these experimental results, it is speculated that under conditions of sufficient nitrogen and carbon source, spore production is slow; however, a low carbon source concentration promotes rapid spore formation.

[0067] The fermentation performance of the second batch was as follows Figure 3 As shown in b. Because the amount of glucose added at 9 h was higher than that in the previous batch, the time of glucose depletion was postponed to 27 h. When the glucose concentration dropped to the critical value of 2.9 g / L (24 h), spores began to form rapidly. When glucose was completely depleted (27 h), the number of spores reached the highest level, which was 0.65×10 10 After 27 h of fermentation, glucose was completely consumed, but the number of cells and spores continued to decrease.

[0068] Example 4: Adjusting the substrate flow time to initiate spore formation

[0069] The preparation of seed solution is the same as in Example 1.

[0070] Fermentation medium (per L): 30 g glucose, 5 g ammonium chloride, 5 g fish meal peptone, 15 g soybean meal, 5 g sodium chloride, 1 g calcium chloride, 1 g potassium chloride, 1 g magnesium sulfate, 0.04 g manganese sulfate monohydrate. The medium pH is 6.9-7.

[0071] Carbon source fed-batch culture medium (per L): 500 g glucose.

[0072] Nitrogen source feed culture medium (per L): 50 g of ammonium chloride.

[0073] Fermentation conditions: 300 mL of seed liquid was inoculated into a 5 L fermentation system with 2.7 L of fermentation medium, and the OD after inoculation was 600 The dissolved oxygen level reached 3.75. A DO electrode was used to monitor dissolved oxygen online. The speed was manually adjusted to maintain the dissolved oxygen level between 30% and 50%. The ventilation ratio was maintained at 2 vvm. The tank pressure was maintained between 0.02 MPa and 0.05 MPa.

[0074] Set up the fermentation batches as follows:

[0075] Batch 1: Control the initial glucose concentration at 30 g / L, and the initial amino nitrogen concentration at 3.53 g / L (5 g / L ammonium chloride, 5 g / L fish meal peptone, and 15 g / L soybean meal in the initial culture medium). From 12h to 30h, the glucose concentration in the fermentation system was controlled to be between 10 g / L and 12 g / L by feeding the carbon source and feeding the culture medium. From 15h to 36h, the amino nitrogen concentration in the fermentation system was controlled to be between 1.4 g / L and 1.6 g / L by feeding the nitrogen source and feeding the culture medium. Stop feeding glucose at 30h, and no more glucose will be added thereafter. Fermentation is completed at 37°C for 36h.

[0076] Batch 2: Control the initial glucose concentration at 30 g / L, control the initial amino nitrogen concentration at 3.53 g / L (5 g / L ammonium chloride, 5 g / L fish meal peptone, and 15 g / L soybean meal in the initial culture medium), control the glucose concentration in the fermentation system at 10 g / L to 12 g / L from 12 h to 30 h, control the amino nitrogen concentration in the fermentation system at 1.4 g / L to 1.6 g / L from 15 h to 36 h by feeding the nitrogen source and culture medium, stop feeding glucose and ammonium chloride at the same time at 30 h, and ferment at 37 ° C for 36 h.

[0077] The first batch of fermentation was as follows Figure 4 As shown in a. 24 h before fermentation, the concentrations of carbon and nitrogen sources were sufficient, and the number of viable cells increased to 2.49×10 10 cfu / mL. The viable cell count remained stable thereafter and did not increase. After 30 hours of fermentation, glucose addition was stopped. By 33 hours, the glucose concentration had dropped to 0.88 g / L, and the spore count had tripled compared to 30 hours.

[0078] The second batch fermentation performance is as follows Figure 4As shown in b. After 30 h of fermentation, due to the synergistic effect of carbon source utilization and nitrogen source utilization, the nitrogen source concentration decreased, resulting in a slower glucose consumption rate. The glucose concentration at 33 h was higher than that of the previous batch. Due to the high glucose concentration, the spore initiation rate slowed down, and the number of spores at 33 h was 0.37×10 10 cfu / mL, which was 35% lower than that of the previous batch. As the carbon and nitrogen sources continued to be consumed, spores continued to form, and the number of spores reached 0.88×10 in 36 hours. 10 cfu / mL, a 15.4% increase over the highest level in the previous batch (reached at 33 h). This shows that initiating spore formation by simultaneously reducing carbon and nitrogen source concentrations can significantly increase spore counts.

[0079] Example 5: Adjusting substrate flow to extend the spore formation cycle

[0080] In order to prolong the time for transformation of vegetative cells into spores, the fermentation experiment was carried out under the following conditions:

[0081] The preparation of seed solution is the same as in Example 1.

[0082] Fermentation medium (per L): 20 g glucose, 5 g ammonium chloride, 5 g fish meal peptone, 15 g soybean meal, 5 g sodium chloride, 1 g calcium chloride, 1 g potassium chloride, 1 g magnesium sulfate, 0.04 g manganese sulfate monohydrate. The medium pH is 6.9-7.

[0083] Carbon source fed-batch medium (per L): 500 g glucose, 1 g calcium chloride, 1 g potassium chloride, 1 g magnesium sulfate, 0.8 g manganese sulfate monohydrate.

[0084] Nitrogen source feed culture medium (per L): 50 g of ammonium chloride.

[0085] Fermentation conditions: DO was monitored online using a DO electrode. The speed was manually adjusted to maintain dissolved oxygen levels between 30% and 50%. The ventilation ratio was maintained at 2 vvm. The tank pressure was maintained between 0.02 MPa and 0.05 MPa.

[0086] The initial glucose concentration was controlled at 20 g / L, the initial amino nitrogen concentration was controlled at 3.51 g / L (ammonium chloride 5 g / L, fish meal peptone 5 g / L and soybean meal 15 g / L in the initial culture medium), and 300 mL of seed liquid was inoculated into a 5 L fermentation system with a fermentation medium volume of 2.7 L. The bacterial OD in the fermentation system was 600The fermentation temperature was set to 3.75. From the 7th to 24th hour, the glucose concentration in the fermentation system was controlled at 10g / L-12g / L. From the 7th to 24th hour, the amino nitrogen concentration in the fermentation system was controlled at 1.4g / L-1.6g / L. From the 25th hour, the glucose and amino nitrogen concentrations began to decrease. From the 28th to 48th hour, the glucose and amino nitrogen concentrations were controlled at 1.5g / L-2.5g / L and 0.4g / L-0.6g / L, respectively. Fermentation was completed at 37°C for 48 hours.

[0087] It can be seen from the experimental results of Examples 1 to 4 that after 24 hours of culture, the number of BF-002 live cells reached a maximum value and no longer grew. Therefore, this batch of fermentation began to reduce the concentrations of glucose and amino nitrogen at 24 hours to initiate spore formation, and in the subsequent culture process, the concentrations of glucose and amino nitrogen were controlled at their respective critical values, which were 2g / L and 0.5g / L, respectively. After 3 hours of reducing the concentrations of glucose and amino nitrogen, spores began to form rapidly. After 30 hours of fermentation, the number of spores reached 2.78 times that of 24 hours. After 30 hours of fermentation, since the concentrations of glucose and amino nitrogen were at the critical level suitable for spore formation, the vegetative cells continued to transform into spores. After 48 hours of fermentation, the proportion of spores in the total number of live cells had reached 89.9%, and the number of spores in the fermentation broth reached 2.59×10 10 cfu / mL, and the total number of viable cells remained at the highest level. The results were as follows Figure 5 By adjusting the flow rate of glucose and NH4Cl, the number and rate of spores in the fermentation broth were effectively increased.

[0088] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A fermentation method for increasing the number of Bacillus licheniformis BF-002 spores, characterized in that: The initial glucose concentration in the culture medium was controlled at 20 g / L, the initial amino nitrogen concentration was controlled at 3.51 g / L, and 300 mL of seed liquid was inoculated into a 5 L fermentation system with a fermentation medium volume of 2.7 L. The OD of the bacteria in the fermentation system was 600 is 3.75, the glucose concentration in the fermentation system is controlled at 10 g / L-12 g / L from 7 h to 24 h, the amino nitrogen concentration in the fermentation system is controlled at 1.4 g / L-1.6 g / L from 7 h to 24 h, the glucose and amino nitrogen concentrations begin to decrease from 25 h, and the glucose and amino nitrogen concentrations are controlled at 1.5 g / L-2.5 g / L and 0.4 g / L-0.6 g / L from 28 h to 48 h, respectively; the fermentation is completed at a temperature of 37°C for 48 h; the culture medium contains 5 g / L of ammonium chloride, 5 g / L of fish meal peptone and 15 g / L of soybean meal; the fermentation is carried out in a fermentor, the dissolved oxygen is controlled within the range of 30%-50%, the ventilation ratio is maintained at 2 vvm, and the tank pressure is 0.02 MPa-0.05 MPa.

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