Method for improving fermentation level of antibacterial lipopeptide in bacillus amyloliquefaciens

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for improving the fermentation level of antibacterial lipopeptide in bacillus amyloliquefaciens, which comprises the following steps: preparing a fermentation culture medium containing alpha-linolenic acid, inoculating the bacillus amyloliquefaciens in the fermentation culture medium, and fermenting at 28-35 DEG C for 40-72 hours. According to the method, the expression condition of the bacitracin D synthetase gene in the fermentation process of the bacillus amyloliquefaciens can be regulated, and the yield of the bacitracin D is remarkably increased.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial fermentation, and particularly relates to a method for improving the fermentation level of antibacterial lipopeptide in Bacillus amyloliquefaciens. BACKGROUND

[0002] In recent years, food safety problems caused by the residues of chemical synthetic fungicides such as pesticides and antibiotics have attracted widespread attention. Long-term consumption of such substances may pose a threat to human health, such as inducing allergic reactions, carcinogenesis, etc. With the rapid development of China's economy and society, the public's awareness of food safety is constantly improving, and the use of chemical synthetic fungicides has been limited to some extent (Khakimov A A, Omonlikov A U, Utaganov S B U. Current status and prospects of the use of biofungicides against plant diseases [J]. GSC Biological and Pharmaceutical Sciences, 2020, 13(3): 119-126). Therefore, exploring and developing green and environmentally friendly antibacterial substances has become a hot spot in the current food industry, and more and more researches are focused on the research and application of natural fungicides and bacteriostatic agents.

[0003] Antibacterial peptides are natural defense components of most organisms against pathogen invasion. Because of its unique antibacterial mechanism, rapid bactericidal ability and the characteristics of not easily inducing drug resistance, it has become a natural drug with great development potential and is considered a safe alternative to antibiotics. It includes antibacterial substances, antibacterial proteins and lipopeptides, etc. Antibacterial lipopeptides are mainly produced by Bacillus through secondary metabolism, and different types of Bacillus can produce a variety of lipopeptides with broad-spectrum antibacterial activity. Among them, Bacillus amyloliquefaciens is widely studied because of its broad-spectrum antifungal and antibacterial ability and good biological control effect on plant diseases.

[0004] The main antibacterial lipopeptides found in Bacillus at present mainly include three families of Iturin, Surfactin and Fengycin. Bacillomycin D belongs to the Iturin family and is mainly composed of a heptapeptide and a β-amino fatty acid chain containing 14-17 carbon atoms. As an amphiphilic surfactant, bacillomycin D has excellent and stable physicochemical properties, strong inhibitory effect on conidia, and activity against many fungi such as Botrytis, and is a highly efficient cyclic antibacterial lipopeptide (Jin P, Wang H, Tan Z, et al. Antifungal mechanism of bacillomycin D from Bacillus velezensis HN-2 against Colletotrichum gloeosporioides Penz [J]. Pesticide Biochemistry and Physiology, 2020, 163: 102-107). Compared with traditional antibacterial agents, bacillomycin D has the characteristics of wide antibacterial spectrum, small molecular weight, strong thermal stability and biodegradability, and shows great application potential in the field of food storage and preservation. However, for a long time, the synthesis ability of bacillomycin D of wild-type Bacillus is generally weak. High cost and low yield seriously restrict its large-scale production and application (Wang Z, Liu C, Shi Y, et al. Classification, application, multifarious activities and production improvement of lipopeptides produced by Bacillus [J]. Critical Reviews in Food Science and Nutrition, 2024, 64(21):7451-7464). The existing research mainly improves the yield or changes the proportion of each component of the complex product by regulating the amino acid synthesis pathway and optimizing the fermentation conditions, which is complex and has many limitations. It is worth noting that the research on the regulation of bacillomycin D yield by unsaturated fatty acids is still in a blank state. SUMMARY

[0005] To solve the problems of high synthesis cost and low yield of bacillomycin D in the prior art, and the complex regulation process and many limitations of fermentation, the present application mainly provides a method for significantly improving the yield of bacillomycin D by using α-linolenic acid: A method for improving the fermentation level of antibacterial lipopeptide in Bacillus amyloliquefaciens comprises the following steps: preparing a fermentation medium containing alpha-linolenic acid, inoculating Bacillus amyloliquefaciens in the fermentation medium, and fermenting at 28-35 DEG C for 40-72 h.

[0006] Further, the inoculation amount of Bacillus amyloliquefaciens is 1-10%. The strain used is Bacillus amyloliquefaciens fmbJ (CGMCC 0943).

[0007] Further, the addition amount of alpha-linolenic acid is 0.45-0.75 mmol / L.

[0008] Further, the carbon-nitrogen ratio of the fermentation medium is 10-20:1.

[0009] Further, the fermentation is carried out at a speed of 150-250 rpm.

[0010] Further, the method comprises the following steps: inoculating Bacillus amyloliquefaciens in a seed culture medium after activation, and then inoculating in a fermentation medium.

[0011] Further, the bacterial liquid after activation is inoculated in a seed culture medium, and cultured to the logarithmic phase.

[0012] A method for preparing bacillomycin D by using the above method, after fermentation, the fermentation liquid is removed of solid impurities, then acid precipitation is carried out, and bacillomycin D is obtained after purification of the precipitate.

[0013] Further, the pH is adjusted to 1.5-2.

[0014] Further, the yield is not less than 450 mg / L.

[0015] Further, the synthesis enzyme related gene is up-regulated.

[0016] By using the above scheme, the method has the following advantages: 1. By adding alpha-linolenic acid to the fermentation medium, the fermentation yield of bacillomycin D can reach 781.45 mg / L, which is 1.4 times that of the control group.

[0017] 2. The method can up-regulate the expression of bacillomycin D synthesis enzyme gene of Bacillus amyloliquefaciens fmbJ during fermentation, so as to improve the yield of bacillomycin D.

[0018] 3. The formation of biofilm can promote the formation of bacillomycin D, and the method delays the formation of Bacillus amyloliquefaciens fmbJ biofilm, and the cell permeability of the bacteria is obviously increased during fermentation, solving the problem of feedback inhibition caused by too high concentration of metabolic products, and improving the yield of bacillomycin D.

[0019] 4、The method of the present application significantly delays the decay and cell body breakage of Bacillus amyloliquefaciens, and significantly increases the length of mycelium, the internal volume of the cell body, and provides more storage space for bacillomycin D synthesis.

[0020] 5、The method of the present application causes most of the cell walls to be damaged during fermentation, and the destruction of cell wall integrity is often regarded as an environmental threat signal, prompting it to eliminate competitors or inhibit the lysis of adjacent cells by secreting secondary metabolites, including bacillomycin D. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Effect of different concentrations of alpha-linolenic acid on bacillomycin D fermentation yield; Figure 2 Effect of different concentrations of gamma-linolenic acid, oleic acid, linoleic acid, arachidonic acid, EPA, and DHA on bacillomycin D fermentation yield; Figure 3 Effect of different fermentation times on bacillomycin D fermentation yield; Figure 4 Effect of alpha-linolenic acid on bacillomycin D synthesis gene expression; Figure 5 Effect of alpha-linolenic acid on Bacillus amyloliquefaciens fmbJ biofilm formation; Figure 6 Effect of alpha-linolenic acid on the permeability of Bacillus amyloliquefaciens fmbJ cell membrane; Figure 7 Effect of alpha-linolenic acid on the morphology of Bacillus amyloliquefaciens fmbJ cell body; Figure 8 Effect of alpha-linolenic acid on the microstructure of Bacillus amyloliquefaciens fmbJ cell wall. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0023] Example 1: (1) Strain activation: Bacillus amyloliquefaciens fmbJ taken out at -80℃ was cultured overnight at 37℃, 180 rpm in LB liquid medium, then streaked on LB plates and incubated at 37℃ for 12-16 h, and single colonies were picked to LB medium and incubated at 37℃, 180 rpm.

[0024] (2) Seed culture: the activated bacteria liquid was inoculated into NB seed culture medium, and cultured at 37°C and 180 rpm until the logarithmic phase.

[0025] (3) Concentration optimization: α-linolenic acid was added to Landy culture medium at 0.6 mmol / L, and the seed liquid was inoculated into each group of fermentation medium at an addition amount of 5%, and fermented at 33°C and 180 rpm for 72 h.

[0026] Example 2: The difference from Example 1 is that the addition amount of α-linolenic acid in step (3) is 0.8 mmol / L.

[0027] Example 3: The difference from Example 1 is that the addition amount of α-linolenic acid in step (3) is 0.4 mmol / L.

[0028] Example 4: The difference from Example 1 is that the fermentation time in step (3) is 48 h.

[0029] Example 5: The difference from Example 1 is that the fermentation time in step (3) is 72 h.

[0030] Comparative Example 1: The difference from Example 1 is that the addition amount of α-linolenic acid in step (3) is 0.

[0031] Comparative Example 2: The difference from Example 1 is that the addition amount of α-linolenic acid in step (3) is 1.0 mmol / L.

[0032] Comparative Example 3: The difference from Example 1 is that the addition amount of α-linolenic acid in step (3) is 0.2 mmol / L.

[0033] Comparative Example 4: The difference from Example 1 is that γ-linolenic acid is used instead of α-linolenic acid in step (3).

[0034] Comparative Example 5: The difference from Example 1 is that oleic acid is used instead of α-linolenic acid in step (3).

[0035] Comparative Example 6: The difference from Example 1 is that linoleic acid is used instead of α-linolenic acid in step (3).

[0036] Comparative Example 7: The difference from Example 1 is that arachidonic acid is used instead of α-linolenic acid in step (3).

[0037] Comparative Example 8: The difference from Example 1 is that eicosapentaenoic acid is used instead of α-linolenic acid in step (3).

[0038] Comparative Example 9: The difference from Example 1 is that docosahexaenoic acid is used instead of α-linolenic acid in step (3).

[0039] Comparative Example 10: The difference from Example 1 is that the fermentation time in step (3) is 24 h.

[0040] Comparative Example 11: The difference from Example 1 is that the fermentation time in step (3) is 36 h.

[0041] Example Sample Testing: Yield determination: The fermentation broths of each example and comparative example were transferred to centrifuge tubes, centrifuged at 10000 ×g for 10 min, and the supernatant was collected. The pH was adjusted to 2.0 with 6 mol HCl and placed at 4℃ overnight for acid precipitation. After centrifugation at 10000 ×g for 10 min, the supernatant was discarded and the precipitate was collected. The precipitate was concentrated in methanol at a ratio of 10:1. After complete dissolution, it was centrifuged at 10000 ×g for 10 min, and the supernatant was collected to obtain crude bacitracin D. The crude product was filtered through a 0.22 μm filter membrane and quantitatively analyzed by high performance liquid chromatography.

[0042] Depend on Figure 1 The comparison chart of the effects of different concentrations of α-linolenic acid shows that, according to HPLC analysis, compared with the culture medium without any added fatty acids, the yield of bacitracin D increased from 419.22 mg / L to 702.75 mg / L, an increase of 67.6%, when α-linolenic acid was added. However, with further increases in α-linolenic acid content, the yield of bacitracin D actually decreased, indicating that the relationship between α-linolenic acid content and bacitracin D yield is not a simple linear one; both need to be maintained within an appropriate range to maximize the yield of bacitracin D.

[0043] Depend on Figure 2 The comparison chart of the effects of different concentrations of γ-linolenic acid, oleic acid, linoleic acid, arachidonic acid, EPA, and DHA shows that the production of bacitracin D in treatment groups with any of the above fatty acids was lower than that in the blank culture medium without any polyunsaturated fatty acids. This indicates that the fatty acids with structures similar to α-linolenic acid all have an inhibitory effect on the production of bacitracin D. Therefore, α-linolenic acid has a unique promoting effect on the production of bacitracin D, which cannot be completely replaced by other polyunsaturated fatty acids; simple substitution may even produce negative effects.

[0044] The fermentation experiment was repeated according to the methods of Example 1 and Comparative Example 1. During the fermentation process, samples were taken every 12 hours from the 24th hour to the 72nd hour to determine the yield of bacitracin D. At the same time, total RNA of Bacillus amyloliquefaciens was extracted, and the RNA concentration and quality were detected by an ultra-micro spectrophotometer.

[0045] Production results as follows Figure 3The results are shown in Figure 2. As can be seen from the figure, compared with the blank culture medium, the bacillomycin D yield of the culture medium added with 0.6 mmol / L of α-linolenic acid is less than that of the control 1 at 24 h, the yield enters the plateau period at 36 h, exceeds the culture medium without any fatty acid at 48 h, reaches 1.4 times of the control group, and the subsequent yield is slowly increased, and finally reaches 781.45 mg / L at 72 h. Moreover, compared with the 72 h yield 702.75 mg / L of the control 2, it also shows that the method of the present application is not accidental, and the yield improvement effect can be stably reproduced by using the method of the present application. Figure 1

[0046] The expression amount of the synthetic enzyme gene (Sun J, Qian S, Lu J, et al. Knockout of fmbJ [J]. Journal of Agricultural and Food Chemistry, 2018, 66(17): 4422-4430) is shown in Figure 3. As can be seen from the figure, the expression amount of the synthetic enzyme gene presents an upward trend, and the expression of the synthetic related genes fmbA, fmbB, fmbC and fmbD is up-regulated. rapC Bacillus amyloliquefaciens The results are shown in Figure 3. As can be seen from the figure, the expression amount of the synthetic enzyme gene presents an upward trend, and the expression of the synthetic related genes fmbA, fmbB, fmbC and fmbD is up-regulated. Figure 4 bmyA, bmyB, bmyC bmyD At 48 h, the expression of fmbA is significantly up-regulated, and when the fermentation time reaches 60 h, it can be obviously found that the overall expression level of fmbD presents a downward trend, and the change is relatively significant, which can correspond to the change of the bacillomycin D yield of the control 2, and verifies the yield improvement effect of α-linolenic acid on bacillomycin D from the gene level. bmyA-D bmyA-D Figure 3

[0047] ​​​​​​​(1) Crystal violet staining assay of biofilm: Three groups of 180 μL LB medium with blank, DMSO and α-linolenic acid (0.6 mmol / L) were set in a 24-well plate, 20 μL of fmbJ seed liquid was inoculated into the three groups of medium, and the plate was incubated at 37°C for different time (12 h, 24 h, 36 h, 48 h, 60 h, 72 h) to monitor the formation of biofilm. At the end of each time, the medium was removed, washed with 200 μL distilled water for 3 times to remove non-adherent cells, and then fixed with 200 μL methanol for 15 min. Washed with distilled water for 3 times, dried the well at room temperature, and then stained with 1% crystal violet for 20 min. Excess dye was removed with distilled water. After drying at room temperature, 200 μL 33% glacial acetic acid was injected. The value change was measured at 570 nm by full-wavelength microplate reader.

[0048] (2) Fluorescent staining method for determining cell membrane permeability: The bacterial bodies at different time were collected by centrifugation, washed with 0.02 M PBS for 2 times and resuspended, and then fluorescent dye NPN was added to a final concentration of 10 μM. The multifunctional spectrophotometer (excitation wavelength 350 nm, emission wavelength 420 nm) was used to detect the fluorescence value.

[0049] (3) Scanning electron microscope observation: The bacterial body samples at three different time were collected, washed with 0.02 M PBS for 2 times, fixed with 2.5% glutaraldehyde at 4°C overnight, washed with 0.02 M PBS for 3 times, eluted with 10%, 30%, 50%, 70%, 90% ethanol gradient for 1 time, and 100% ethanol for 2 times, each for 15-20 min, and then dried, gold sprayed, and the surface roughness of the bacterial bodies under three conditions and the diameter of the bacterial bodies were observed by SEM.

[0050] (4) Transmission electron microscope observation: The method of fixation and dehydration was the same as above, then the bacterial bodies were embedded with epoxy resin, sliced by ultrathin freezing microtome, fixed on clean carbon-coated TEM grid, then stained with 100 μL of 0.5% uranyl acetate, and placed at room temperature for complete air drying, and the ultrastructure of the cell wall of the bacterial bodies was observed by TEM.

[0051] As Figure 5 As can be seen, the addition of α-linolenic acid fermentation delays the formation of Bacillus amyloliquefaciens fmbJ biofilm, and the formation of biofilm promotes the formation of bacillomycin D. From 12 h to 36 h, the biofilm formation of the experimental group was significantly lower than that of the blank group, and at 48 h, the biofilm began to form rapidly, which was consistent with the change of bacillomycin D production at the same time.

[0052] As Figure 6It can be seen that the addition of alpha-linolenic acid fermentation, 24 h, 36 h, 48 h, the cell permeability of bacteria significantly increased, the feedback inhibition caused by the high concentration of metabolic products was eliminated, thereby improving the yield.

[0053] As Figure 7 It can be seen that the addition of alpha-linolenic acid fermentation delays the decline of bacillus amyloliquefaciens and the damage of the cell, at 24 h and 36 h, the addition of alpha-linolenic acid significantly increases the length of the mycelium, and the internal volume of the cell is increased, thereby providing a larger storage space for bacillomycin D synthesis.

[0054] As Figure 8 It can be seen that the cell wall is mostly damaged when alpha-linolenic acid fermentation is added, and the destruction of cell wall integrity is often regarded as an environmental threat signal, which removes competitors or inhibits the lysis of adjacent cells by secreting secondary metabolites.

[0055] For those skilled in the art, various corresponding changes and modifications can be made to the technical solutions and concepts described above, and all these changes and modifications should belong to the protection scope of the present application.

Claims

1. A method for improving the fermentation level of antimicrobial lipopeptides in Bacillus amyloliquefaciens, characterized in that, Includes the following steps: In the preparation of fermentation medium containing α-linolenic acid, Bacillus amyloliquefaciens was inoculated into the fermentation medium and fermented at 28-35℃ for 40-72 h.

2. The method for improving the fermentation level of antimicrobial lipopeptides in Bacillus amyloliquefaciens according to claim 1, characterized in that, The inoculation amount of Bacillus amyloliquefaciens is 1-10%.

3. The method for improving the fermentation level of antimicrobial lipopeptides in Bacillus amyloliquefaciens according to claim 1, characterized in that, The amount of α-linolenic acid added is 0.45~0.75 mmol / L.

4. The method for improving the fermentation level of antimicrobial lipopeptides in Bacillus amyloliquefaciens according to claim 3, characterized in that, The carbon-to-nitrogen ratio of the fermentation medium is 10-20:

1.

5. The method for improving the fermentation level of antimicrobial lipopeptides in Bacillus amyloliquefaciens according to claim 1, characterized in that, Ferment at a speed of 150~250 rpm.

6. The method for improving the fermentation level of antimicrobial lipopeptides in Bacillus amyloliquefaciens according to claim 1, characterized in that, Includes the following steps: After activating Bacillus amyloliquefaciens, it was inoculated into seed culture medium and then into fermentation culture medium.

7. The method for improving the fermentation level of antimicrobial lipopeptides in Bacillus amyloliquefaciens according to claim 6, characterized in that, The activated bacterial culture was inoculated into the seed culture medium and cultured until the logarithmic growth phase.

8. A method for preparing bacitracin D using the method described in any one of claims 1 to 7, characterized in that, After fermentation, the fermentation broth was taken to remove solid impurities, and then acid precipitation was performed. The precipitate was purified to obtain bacitracin D.

9. The method for preparing bacitracin D according to claim 8, characterized in that, Adjust the pH to 1.5-2.

10. The method for preparing bacitracin D according to claim 8, characterized in that, It upregulates synthase-related genes.

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