Method for cultivating a bacterial strain and use thereof for preventing phage infection during fermentation

Abstract

The present disclosure provides a method for culture of bacterial strains, comprising the following steps: inoculating a bacterial strain in a first culture medium comprising 0.1 mg / L-0.8 mg / L of 1-bromohexane to obtain a first culture. The method for culture of bacterial strains provided by the present disclosure further comprises: inoculating the first culture into a second culture medium, wherein the second culture medium comprises 0.2 mg / L-0.8 mg / L of 1-bromohexane. The method can be used for fermentation of Gluconacetobacter melanogenum, Propionibacterium, Corynebacterium, Brevibacterium, Bacillus, Gluconobacter, Lactobacillus, Agromyces, Actinobacillus, Escherichia coli, Anaerobiospirillum, Trichoderma, Streptomyces, Aspergillus, Saccharomyces or Myceliopthora, effectively preventing infection of bacteriophages during fermentation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for cultivating a bacterial strain and a method for preventing bacteriophage infection in a fermentation process. BACKGROUND

[0002] Bacteriophage is a virus of prokaryotes. It is very small, with a diameter of 0.1 μm; does not have a complete cell structure; only contains a single nucleic acid. It has a very strong infection ability and is extremely easy to infect bacteria and actinomycetes used for fermentation. It spreads very quickly and is difficult to prevent and control, posing a great threat to fermentation. It can enter the fermentation system through environmental pollution, leakage or dead angles of equipment, air systems, medium sterilization processes, feeding, sampling and other operation processes. Bacteriophage widely exists in soil, sewage and air suitable for the survival of host bacteria, and almost all microorganisms used in fermentation production have the possibility of being infected by bacteriophage.

[0003] Bacteriophage contamination can easily cause the extension of the fermentation cycle, slow down the consumption of fermentation substrate and the formation of product, affect the yield and quality of the fermentation product, and even cause the termination of fermentation leading to fermentation failure, causing great losses to production. The existing measures for preventing bacteriophage contamination include controlling the source of infection, rotating strains, screening bacteriophage receptor mutant resistant strains, etc., but these measures still cannot effectively solve the technical problems of microbial strain infection and poor fermentation. Therefore, a new method for preventing bacteriophage infection in a fermentation process is needed. SUMMARY

[0004] The purpose of the present application is to provide a method for preventing bacteriophage infection in a fermentation process using 1-bromohexane.

[0005] In one aspect of the present disclosure, a method for cultivating a bacterial strain is provided, the method comprising the following steps: inoculating a bacterial strain in a first culture medium for cultivation to obtain a first culture product, wherein the first culture medium comprises 0.1 mg / L to 0.8 mg / L of 1-bromohexane.

[0006] In some embodiments, the first culture medium comprises 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 mg / L of 1-bromohexane. In some preferred embodiments, the first culture medium comprises 0.3, 0.4, 0.5 or 0.6 mg / L of 1-bromohexane.

[0007] In some embodiments, the bacterial strain is inoculated into the first culture medium at an inoculation amount of 0.05-0.5% (volume %).

[0008] In some embodiments, based on the total mass of the first culture medium, the first culture medium comprises 1-5 wt% sorbitol, 0.1-0.8 wt% corn steep liquor, 0.2-1 wt% peptone, 0.2-1 wt% urea, and the remainder being water. Preferably, based on the total mass of the first culture medium, the first culture medium comprises 1-2 wt% sorbitol, 0.2-0.4 wt% corn steep liquor, 0.4-0.6 wt% peptone, 0.3-0.5 wt% urea, and the remainder being water.

[0009] In some embodiments, the method further includes inoculating the first culture product into a second culture medium for culturing to obtain a second culture product, wherein the second culture medium contains 0.2 mg / L to 0.8 mg / L of 1-bromohexane.

[0010] In some embodiments, the second culture medium contains 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8 mg / L of 1-bromohexane. In some preferred embodiments, the second culture medium contains 0.5 or 0.6 mg / L of 1-bromohexane.

[0011] In some embodiments, the first culture product is inoculated into the second culture medium at an inoculation rate of 0.5-5% (volume %).

[0012] In some embodiments, based on the total mass of the second culture medium, the second culture medium comprises 2-10 wt% sorbitol, 0.5-1.5 wt% corn steep liquor, 0.8-1.5 wt% peptone, 0.5-1.5 wt% urea, and the remainder being water. Preferably, based on the total mass of the second culture medium, the first culture medium comprises 4-6 wt% sorbitol, 0.6-1.0 wt% corn steep liquor, 1-1.2 wt% peptone, 0.8-1.0 wt% urea, and the remainder being water.

[0013] In some embodiments, the bacterial strain is selected from Bacillus megaterium, Gluconobacterium oxysporum, Propionibacterium, Corynebacterium, Short bacillus, Bacillus, Staphylococcus, Lactobacillus, Agrobacterium, Actinobacterium, Escherichia coli, Anaerobic Spirulina, Trichoderma, Streptomyces, Aspergillus, yeast, or Trichoderma.

[0014] Another aspect of this disclosure provides the application of the above-described method in preventing bacteriophage infection during fermentation. Attached Figure Description

[0015] Figure 1 A microscopic photograph of bacteria infected by bacteriophage in one embodiment of this disclosure is shown.

[0016] Figure 2 A microscopic photograph of bacteria infected by bacteriophage in one embodiment of this disclosure is shown. Detailed Implementation

[0017] 1-Bromohexane can resist bacteriophage invasion, thereby preventing its infection and reproduction. The mechanism by which 1-bromohexane inhibits bacteriophages mainly includes the following three aspects: 1. Disruption of the bacteriophage's plasma membrane: Bacteriophages rely on their plasma membrane to adhere to and penetrate bacteria when infecting them. 1-Bromohexane can disrupt the bacteriophage's plasma membrane, thus preventing further bacterial infection; 2. Inhibition of bacteriophage DNA synthesis: Bacteriophages need to synthesize new DNA to replicate and transmit genetic information when infecting bacteria. 1-Bromohexane can inhibit bacteriophage DNA synthesis, thereby preventing its replication and reproduction; 3. Enhancement of host cell immunity: 1-Bromohexane can enhance the immunity of host cells and improve their antiviral capabilities, thereby preventing bacteriophage infection and reproduction. In summary, 1-Bromohexane inhibits bacteriophage infection and reproduction through multiple pathways, including disrupting the bacteriophage's plasma membrane, inhibiting its DNA synthesis, and enhancing the immunity of host cells.

[0018] The inventors discovered that 1-bromohexane is more suitable for the fermentation process described in this application compared to other compounds among various bromoalkanes. Furthermore, this application creatively discovers that adding 1-bromohexane during both inoculation and fermentation processes can more effectively interfere with phage infection during fermentation, and that the concentration of 1-bromohexane used in each process—in the inoculation and fermentation stages—effectively prevents phage infection during fermentation.

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0020] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0021] The culture medium and detection methods used in the following examples are as follows: Seed culture medium: based on the total mass of the seed culture medium, it contains 1.5 wt% sorbitol, 0.3 wt% corn steep liquor, 0.5 wt% peptone, 0.4 wt% urea, and the remainder is water.

[0022] Fermentation medium: Based on the total mass of the fermentation medium, it contains 4.5 wt% sorbitol, 0.8 wt% corn steep liquor, 1.1 wt% peptone, 0.9 wt% urea, and the remainder is water.

[0023] Yield = [Target product amount (g) / Substrate consumption (g)] × 100%.

[0024] Dissolved oxygen and pH were measured online using an online dissolved oxygen electrode, and OD was measured using a spectrophotometer at 562 nm.

[0025] The detection steps for the target product are as follows:

[0026] Preparation of sorbitol standard solution: Weigh 100 mg of sorbitol into a 100 mL volumetric flask and dilute to the mark with purified water. Dilute this solution serially to prepare a standard solution of the desired concentration.

[0027] Chromatographic column: Shim-Pack SCR-101C (7.9mm x 300mm); mobile phase: purified water; detector: Waters-2414 differential refractive index detector; flow rate: 1.0 mL / min; column temperature: 80°C; injection volume: 20 μL; the sample was filtered through a 0.22 μm filter membrane before chromatographic analysis. Sorbitol fermentation broth was transferred, filtered through a 0.22 μm organic filter membrane, and 1.0 mL of the filtrate was transferred to a 50 mL volumetric flask, diluted to the mark with water, filtered through a 0.22 μm organic filter membrane, and then analyzed by liquid chromatography.

[0028] Example 1: Detection of Fermentation Product Yield

[0029] The applicant's preserved Bacillus megaterium in the laboratory was inoculated into the seed culture medium at an inoculation rate of 0.1% (volume %) and cultured at a rotation speed of 150 rpm, a pressure of 0.05 MPa, and a ventilation rate of VVM=1:1 to obtain the bacterial solution.

[0030] The above bacterial solution was inoculated into the fermentation medium at an inoculation rate of 1% (volume%) and cultured at a rotation speed of 250 rpm, a tank pressure of 0.05 MPa, and an aeration rate of 1:1 vvm. The target product was then tested after the culture was removed from the tank.

[0031] The results showed that the sorbitol residue was 0.1 mg / L, the gulonic acid yield was 20.5 mg / L, and the gulonic acid yield was 20%.

[0032] Examples 2-6: Concentration Test of 1-Bromohexane in Seed Culture Medium

[0033] Examples 2-6 tested the effective concentration of 1-bromohexane in seed culture medium to prevent bacteriophage infection.

[0034] Example 2

[0035] Bacillus megaterium was inoculated into seed culture medium at a rate of 0.1% (v / v), and simultaneously inoculated with 0.1 mL of giant bacteriophage (selected from the infection vessel). The culture was carried out at 150 rpm, vessel pressure of 0.05 MPa, and aeration rate (VVM) of 1:1. After 7 hours, dissolved oxygen rose to 80%, pH rose to 7.8, and OD decreased to 15. Microscopic examination revealed bacterial lysis in the normal culture. Figure 1 As shown.

[0036] Example 3

[0037] Add 0.1 mg / L 1-bromohexane to the seed culture medium and sterilize. Inoculate Bacillus megaterium into the seed culture medium at an inoculum rate of 0.1% (v / v) and simultaneously inoculate 0.1 mL of bacteriophage. Culture under the conditions of 150 rpm rotation speed, 0.05 MPa pressure, and aeration rate (VVM) of 1:1. After 9 h, dissolved oxygen, pH, and OD showed signs of recovery. Microscopic examination revealed bacterial cell lysis in the normal culture.

[0038] Example 4

[0039] Add 0.2 mg / L 1-bromohexane to the seed culture medium and sterilize. Inoculate Bacillus megaterium into the seed culture medium at an inoculum rate of 0.1% (v / v) and simultaneously inoculate 0.1 mL of bacteriophage. Culture under the conditions of 150 rpm rotation speed, 0.05 MPa pressure, and aeration rate (VVM) of 1:1. After 13 h, dissolved oxygen, pH, and OD slowly decreased. Microscopic examination showed that the bacterial cells in the normal culture began to lyse.

[0040] Example 5

[0041] Add 0.3 mg / L 1-bromohexane to the seed culture medium and sterilize. Inoculate Bacillus megaterium into the seed culture medium at an inoculum rate of 0.1% (v / v), and simultaneously inoculate with 0.1 mL of bacteriophage. Culture under conditions of 150 rpm, 0.05 MPa, and aeration rate (VVM) = 1:1. After 15 hours, no increase in dissolved oxygen or pH was observed during transfer for fermentation. Microscopic examination showed that the bacterial cells in the normal culture were not lysed. Figure 2 As shown.

[0042] Example 6

[0043] Add 0.4 mg / L 1-bromohexane to the seed culture medium and sterilize. Inoculate Bacillus megaterium into the seed culture medium at an inoculum rate of 0.1% (v / v) and simultaneously inoculate with 0.1 mL of bacteriophage. Culture under the conditions of 150 rpm, 0.05 MPa, and VVM = 1:1. After 15 h, no increase in dissolved oxygen or pH was observed during the transfer fermentation. Microscopic examination showed that the cultured bacteria cells were not lysed.

[0044] Examples 7-12: Concentration test of 1-bromohexane in fermentation medium

[0045] Examples 7-12 tested the effective concentration of 1-bromohexane in fermentation medium to prevent bacteriophage infection.

[0046] Example 7

[0047] Add 0.3 mg / L 1-bromohexane to the seed culture medium and sterilize. Inoculate Bacillus megaterium into the seed culture medium at an inoculum rate of 0.1% (volume %), and simultaneously inoculate 0.1 mL of bacteriophage. Culture at 150 rpm, 0.05 MPa, and aeration rate (VVM) of 1:1 for 15 h. After 15 h, the bacterial culture was obtained. No increase in dissolved oxygen or pH was observed. Microscopic examination showed that the bacterial cells in the normal culture were not lysed.

[0048] The above bacterial solution was inoculated into the fermentation medium at an inoculation rate of 1% (volume%). After culturing for 8 hours, dissolved oxygen, pH and OD showed signs of recovery. Microscopic examination revealed that the bacterial cells in the normal culture were lysed.

[0049] Example 8

[0050] Add 0.3 mg / L 1-bromohexane to the seed culture medium and sterilize. Inoculate Bacillus megaterium into the seed culture medium at an inoculum rate of 0.1% (v / v) and simultaneously inoculate with 0.1 mL of bacteriophage. Culture at 150 rpm, 0.05 MPa, and aeration rate (VVM) of 1:1 for 15 h. After 15 h, the bacterial culture was obtained. No increase in dissolved oxygen or pH was observed. Microscopic examination showed that the cultured bacterial cells were not lysed.

[0051] The above bacterial culture was inoculated into the fermentation medium at an inoculum rate of 1% (volume%), and 0.2 mg / L 1-bromohexane was added at the same time. After sterilization and incubation for 10 h, dissolved oxygen, pH and OD were observed to rise and decrease significantly. Microscopic examination showed that the bacterial cells in the normal culture were lysed.

[0052] Example 9

[0053] Add 0.3 mg / L 1-bromohexane to the seed culture medium and sterilize. Inoculate Bacillus megaterium into the seed culture medium at an inoculum rate of 0.1% (v / v) and simultaneously inoculate with 0.1 mL of bacteriophage. Culture at 150 rpm, 0.05 MPa, and aeration rate (VVM) of 1:1 for 15 h. After 15 h, the bacterial culture was obtained. No increase in dissolved oxygen or pH was observed. Microscopic examination showed that the cultured bacterial cells were not lysed.

[0054] The above bacterial culture was inoculated into the fermentation medium at an inoculum rate of 1% (volume%), and 0.3 mg / L 1-bromohexane was added at the same time. After sterilization and incubation for 13 hours, dissolved oxygen, pH and OD showed signs of recovery and a significant decrease. Microscopic examination revealed that the bacterial cells in the normal culture were lysed.

[0055] Example 10

[0056] Add 0.3 mg / L 1-bromohexane to the seed culture medium and sterilize. Inoculate Bacillus megaterium into the seed culture medium at an inoculum rate of 0.1% (v / v) and simultaneously inoculate with 0.1 mL of bacteriophage. Culture at 150 rpm, 0.05 MPa, and aeration rate (VVM) of 1:1 for 15 h. After 15 h, the bacterial culture was obtained. No increase in dissolved oxygen or pH was observed. Microscopic examination showed that the cultured bacterial cells were not lysed.

[0057] The above bacterial culture was inoculated into the fermentation medium at an inoculum rate of 1% (volume%), and 0.4 mg / L 1-bromohexane was added at the same time. After sterilization and incubation for 18 hours, dissolved oxygen, pH and OD showed signs of recovery and significant decrease. Microscopic examination revealed that the bacterial cells in the normal culture were lysed.

[0058] Example 11

[0059] Add 0.3 mg / L 1-bromohexane to the seed culture medium and sterilize. Inoculate Bacillus megaterium into the seed culture medium at an inoculum rate of 0.1% (v / v) and simultaneously inoculate with 0.1 mL of bacteriophage. Culture at 150 rpm, 0.05 MPa, and aeration rate (VVM) of 1:1 for 15 h. After 15 h, the bacterial culture was obtained. No increase in dissolved oxygen or pH was observed. Microscopic examination showed that the cultured bacterial cells were not lysed.

[0060] The above bacterial culture was inoculated into the fermentation medium at an inoculum rate of 1% (volume%), and 0.5 mg / L 1-bromohexane was added at the same time. The culture was sterilized and cultured until fermentation was discharged from the tank. No phenomenon of dissolved oxygen recovery, pH recovery, or significant OD decrease was observed. Microscopic examination showed that the bacterial cells in the normal culture were not lysed.

[0061] The results of the detection of the target product showed that the residual amount of sorbitol was 0.1 mg / L and the yield of gulonic acid was 20.4975 mg / L.

[0062] Example 12

[0063] Add 0.3 mg / L 1-bromohexane to the seed culture medium and sterilize. Inoculate Bacillus megaterium into the seed culture medium at an inoculum rate of 0.1% (v / v) and simultaneously inoculate with 0.1 mL of bacteriophage. Culture at 150 rpm, 0.05 MPa, and aeration rate (VVM) of 1:1 for 15 h. After 15 h, the bacterial culture was obtained. No increase in dissolved oxygen or pH was observed. Microscopic examination showed that the cultured bacterial cells were not lysed.

[0064] The above bacterial culture was inoculated into the fermentation medium at an inoculum rate of 1% (volume%), and 0.6 mg / L 1-bromohexane was added at the same time. The culture was sterilized and cultured until fermentation was discharged from the tank. No phenomenon of dissolved oxygen recovery, pH recovery, or significant OD decrease was observed. Microscopic examination showed that the bacterial cells in the normal culture were not lysed.

[0065] The results of the detection of the target product showed that the residual amount of sorbitol was 0.1 mg / L and the yield of gulonic acid was 20.625 mg / L.

[0066] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for preventing bacteriophage infection during fermentation culture, the method comprising the following steps: Bacillus megaterium was inoculated into a seed culture medium and cultured to obtain a first culture product, wherein the seed culture medium contained 0.3 mg / L to 0.4 mg / L of 1-bromohexane.

2. The method according to claim 1, characterized in that, Based on the total mass of the seed culture medium, the seed culture medium comprises 1-5 wt% sorbitol, 0.1-0.8 wt% corn steep liquor, 0.2-1 wt% peptone, and 0.2-1 wt% urea.

3. The method according to claim 1, characterized in that, Based on the total mass of the seed culture medium, the seed culture medium comprises 1-2 wt% sorbitol, 0.2-0.4 wt% corn steep liquor, 0.4-0.6 wt% peptone, and 0.3-0.5 wt% urea.

4. The method according to claim 1, characterized in that, The method further includes: inoculating the first culture product into a fermentation medium for culturing to obtain a second culture product, wherein the fermentation medium contains 0.5 mg / L to 0.6 mg / L of 1-bromohexane.

5. The method according to claim 4, characterized in that, Based on the total mass of the fermentation medium, the fermentation medium comprises 2-10 wt% sorbitol, 0.5-1.5 wt% corn steep liquor, 0.8-1.5 wt% peptone, and 0.5-1.5 wt% urea.

6. The method according to claim 4, characterized in that, The fermentation medium comprises 4-6 wt% sorbitol, 0.6-1.0 wt% corn steep liquor, 1-1.2 wt% peptone, and 0.8-1.0 wt% urea.

Images