CN115466764A - Application of sodium chloride in improvement of bacillus subtilis to synthesis of bacitracin D, sodium chloride fermentation medium and method - Google Patents

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a sodium chloride fermentation medium, a method for improving the yield of bacillomycin D and application of the method. The invention provides application of sodium chloride in improving bacillus subtilis to synthesize bacillomycin D, and calcium chloride can promote expression of bacillomycin D synthetase gene of bacillus so as to improve yield of bacillomycin D. The results of the examples show that: the yield of the bacillomycin D obtained by fermenting and culturing the bacillus subtilis by using the sodium chloride fermentation medium provided by the invention is obviously improved, and the content of the bacillomycin D in the fermentation liquid is 478.79 +/-13.41 mg/L. The content of the total bacillomycin D in the bacillus subtilis crude peptide is 17.34 +/-0.41 mg/g.

Description

The Application of Sodium Chloride and Sodium Chloride in Improving the Synthesis of Bacillus Mycin D by Bacillus subtilis Fermentation medium and method

technical field

The invention belongs to the technical field of microbial fermentation, and in particular relates to the application of sodium chloride in improving the synthesis of bacillus mycin D by Bacillus subtilis, a sodium chloride fermentation medium and a method.

Background technique

Bacitracin D is an antibacterial lipopeptide belonging to the iturin family synthesized by non-ribosomal peptide synthase in Bacillus subtilis. Bacitracin D can inhibit agricultural pathogenic fungi such as Aspergillus flavus and Ochra. Therefore, Bacitracin D is very important in ensuring food and food safety and preventing fungal contamination. However, in the prior art, when Bacillus subtilis is used for fermentation and culture in a conventional medium, the yield of bacinomycin D is low. Therefore, it is urgent to strengthen the research on increasing the production of bacitracin D.

Contents of the invention

In view of this, the object of the present invention is to provide a method that can effectively improve the synthesis of bacillus mycin D by Bacillus subtilis. The invention provides the application of sodium chloride in improving the synthesis of bacillus mycin D by Bacillus subtilis, adding sodium chloride to the basal medium to ferment and cultivate the bacillus subtilis to produce bacillus mycin D, which can improve the production rate of bacillus mycin D Yield.

In order to solve the problems of the technologies described above, the present invention proposes the following technical solutions:

The invention provides the application of sodium chloride in improving the synthesis of bacillus mycin D by bacillus subtilis.

The invention provides a sodium chloride fermentation medium. The sodium chloride fermentation medium uses water as a solvent and contains components at the following concentrations: 0.5-1.0 g/L of yeast extract, 2.0-5.0 g/L of L-glutamic acid g/L, glucose 5.0~20.0g/L and sodium chloride 1.0~6.0g/L.

Preferably, the sodium chloride fermentation medium uses water as a solvent and contains components of the following concentrations: yeast extract 1.0g/L, L-glutamic acid 5.0g/L, glucose 20.0g/L and sodium chloride 3.0 g/L.

The invention provides a method for increasing the output of bacillus mycin D, which is characterized in that it comprises the following steps: using the sodium chloride fermentation medium described in the above technical solution to ferment and cultivate the Bacillus subtilis seed liquid to obtain the bacillus mycin D; The fermentation culture process is a fed-batch sodium chloride fermentation medium.

Preferably, the fermentation culture is a fed-batch fermentation culture;

The conditions for the one-time fed-batch fermentation culture include: the Bacillus subtilis seed liquid is inoculated in the sodium chloride fermentation medium and cultured for 48-84 hours, and then a new sodium chloride fermentation medium is added once to continue the fermentation culture. Obtain a fermented broth.

Preferably, the volume of the added sodium chloride fermentation medium is 30% to 50% of the volume of the initial sodium chloride fermentation medium.

Preferably, the temperature of the fermentation culture is 30-37° C., the rotation speed is 150-200 r/min; the time of the fermentation culture is 139-171 h.

Preferably, the preparation of the Bacillus subtilis seed liquid comprises: inoculating the Bacillus subtilis on the seed medium, and culturing at 33-37° C. until the OD ₆₀₀ of the seed medium is 0.8-1.0 to obtain the Bacillus subtilis seed liquid.

Preferably, the inoculation amount of the Bacillus subtilis seed liquid is 3%-5% of the volume of the sodium chloride fermentation medium.

Preferably, the bacitracin D includes one or more of bacitracin DC14 homologues, bacitracin DC15 homologues and bacitracin DC16 homologues.

Beneficial effects of the present invention: the present invention provides the application of sodium chloride in improving the synthesis of bacillus mycin D by Bacillus subtilis. Production of mycin D. The results of the examples show that the output of bacillus mycin D obtained by fermenting and culturing Bacillus subtilis by using the sodium chloride fermentation medium provided by the present invention is significantly improved, and the content of bacillus mycin D in the fermentation broth is 478.79±13.41mg/L , the content of total bacitracin D in the crude peptide of Bacillus subtilis was 17.34±0.41mg/g.

detailed description

The invention provides the application of sodium chloride in improving the synthesis of bacillus mycin D by bacillus subtilis.

The invention provides a sodium chloride fermentation medium. The sodium chloride fermentation medium uses water as a solvent and contains components at the following concentrations: 0.5-1.0 g/L of yeast extract, 2.0-5.0 g/L of L-glutamic acid g/L, glucose 5.0~20.0g/L and sodium chloride 1.0~6.0g/L.

Unless otherwise specified, the present invention has no special limitation on the sources of yeast extract, L-glutamic acid, glucose and sodium chloride, and conventional commercially available products can be used.

In the present invention, the sodium chloride fermentation medium contains 5.0-20.0 g/L of glucose, preferably 13.0-18.0 g/L. In the embodiment of the present invention, the added amount of glucose is preferably 20 g/L or 15.0 g/L.

In the present invention, the sodium chloride fermentation medium includes 0.5-1.0 g/L of yeast extract, preferably 0.6-0.9 g/L. In the embodiment of the present invention, the amount of yeast extract added is specifically 1.0 g/L or 0.8 g/L. The yeast extract of the present invention can provide energy substances for the growth of the subtilis bacillus, and promote the subtilis bacillus to secrete more secondary metabolites.

In the present invention, the sodium chloride fermentation medium includes 2.0-5.0 g/L of L-glutamic acid, preferably 2.5-4.5 g/L, more preferably 4.0 g/L. In the embodiment of the present invention, the added amount of L-glutamic acid is preferably 5.0 g/L.

In the present invention, the sodium chloride fermentation medium contains 1.0-6.0 g/L of sodium chloride, preferably 2.0-5.0 g/L, more preferably 3.0 g/L.

In the present invention, the sodium chloride fermentation medium preferably uses water as a solvent, and comprises components of the following concentrations: yeast extract 1.0g/L, L-glutamic acid 5.0g/L, glucose 20.0g/L and sodium chloride 3.0 g/L.

The sodium chloride, glucose and L-glutamic acid in the sodium chloride fermentation medium of the present invention can all promote the expression of the bacillus mycin D synthetase gene so as to effectively increase the yield of the bacillus mycin D synthesized by the bacillus subtilis.

In the present invention, sodium chloride is added to the basal medium composed of yeast extract, L-glutamic acid and glucose, and the production of bacillus mycin D is fermented by feeding batches once, so that the output of bacillus mycin D is improved, and the method The method is simple and easy to operate, and solves the technical problem of low yield of bacillus subtilis bacillus mycin D in the prior art.

In the present invention, the sodium chloride fermentation medium is preferably sterilized, and the sterilization temperature is preferably 115° C., and the sterilization time is preferably 20 minutes.

The invention provides a method for increasing the output of bacillus mycin D, comprising the following steps: using the sodium chloride fermentation medium described in the above technical scheme to ferment and cultivate the Bacillus subtilis seed liquid to obtain bacillus mycin D; Process fed-batch sodium chloride fermentation medium.

In the present invention, the preparation of the Bacillus subtilis seed solution preferably includes: inoculating the Bacillus subtilis on the seed medium, culturing at 33-37° C. until the OD ₆₀₀ of the seed medium is 0.8-1.0 to obtain the Bacillus subtilis seed solution.

Before being inoculated in the seed medium, the present invention preferably inoculates the Bacillus subtilis preserved on the slant into the seed medium and cultivates it to obtain the Bacillus subtilis seed liquid. In the present invention, the temperature of the seed culture medium is preferably 33-37°C, more preferably 34-36°C, more preferably 35°C, and the temperature of the seed culture in the embodiment of the present invention is preferably 37°C; In the present invention, it is preferred to cultivate until the OD ₆₀₀ of the Bacillus subtilis seed solution is 0.8-1.0, more preferably 0.85-0.95, and more preferably 0.9. In the embodiment of the present invention, it is preferred to cultivate until the OD ₆₀₀ of the Bacillus subtilis seed solution is 0.8. The seed cultivation in the present invention is preferably constant temperature cultivation.

In the present invention, the bacillus subtilis seed liquid is fermented and cultured to obtain bacillus mycin D; the fermentation culture process is fed with sodium chloride fermentation medium in batches.

The present invention uses the sodium chloride fermentation medium to ferment and cultivate the Bacillus subtilis seed liquid to obtain the bacillus mycin D; the fermentation and cultivation process feeds the sodium chloride fermentation medium in batches.

In the present invention, it is preferred to inoculate the Bacillus subtilis seed solution in a sodium chloride fermentation medium for fermentation culture. In the present invention, the Bacillus subtilis seed liquid is preferably inoculated in the sodium chloride fermentation medium according to the volume ratio of 3%-5%, more preferably 3.5%-4.5%, more preferably 4%. In the embodiment of the present invention, the inoculation amount of the Bacillus subtilis seed solution inoculated into the sodium chloride fermentation medium is 5% by volume.

In the present invention, the rotational speed of the fermentation culture is preferably 150-200 r/min, more preferably 165-195 r/min, more preferably 180 r/min. The temperature of the fermentation culture in the present invention is 30-37°C, more preferably 31-34°C, more preferably 33°C. In the present invention, the fermentation culture time is preferably 139-171 h, more preferably 143-156 h, more preferably 147 h. In the present invention, described fermentation culture process feeds sodium chloride fermentation culture medium in batches; The time of fermentation culture described in the present invention is total culture time, comprises the total time of fermentation culture before adding sodium chloride culture medium and The total time of fermentation culture after supplementing the sodium chloride medium.

In the present invention, the fermentation culture is preferably a fed-batch fermentation culture. The conditions of the one-time fed-batch fermentation culture of the present invention preferably include: the Bacillus subtilis seed liquid is inoculated in a sodium chloride fermentation medium for 48-84 hours, and then a new sodium chloride fermentation medium is added to continue Fermentation broth is obtained after fermentation.

The fermentation culture time before adding the sodium chloride medium in the present invention is preferably 48-84 hours, more preferably 54-72 hours, more preferably 63 hours. In the embodiment of the present invention, the conditions of the fed-batch fermentation are preferably as follows: the Bacillus subtilis seed liquid is inserted into the initial sodium chloride fermentation medium for fermentation and cultivation for 63 hours, and new chlorine is added to the fermentation liquid. The sodium chloride fermentation medium was post-fermented for 84 hours, and fermented for 147 hours to obtain a Bacillus subtilis fermentation broth.

In the present invention, the volume of the new sodium chloride fermentation medium added is preferably 30% to 50% of the volume of the initial sodium chloride fermentation medium, more preferably 35% to 45%, more preferably 40% . In an embodiment of the present invention, the volume of the new sodium chloride fermentation medium added is preferably 50% of the initial sodium chloride fermentation medium volume, and the volume of the new sodium chloride fermentation medium added by the present invention 50% of the volume of the initial sodium chloride fermentation medium is to increase the yield of bacinomycin D produced by fermentation of Bacillus subtilis.

In the present invention, the components of the slant preservation medium preferably include 3.0 g/L beef extract, 10 g/L tryptone, 5 g/L sodium chloride, 15 g/L agar and 1 L of water.

The seed medium preferably includes 3.0 g/L beef extract, 10 g/L tryptone, 5 g/L sodium chloride and 1 L of water. The sodium chloride fermentation medium has been discussed above and will not be repeated here.

After the Bacillus subtilis fermentation liquid is obtained, the present invention preferably performs the first centrifugation, acid precipitation, second centrifugation, extraction and third centrifugation on the Bacillus subtilis fermentation liquid in order to obtain the bacillus mycin D. In the present invention, the Bacillus subtilis fermentation liquid is centrifuged for the first time to obtain the supernatant, and the supernatant is preferably subjected to acid immersion extraction and second centrifugation to obtain the crude bacillus mycin D peptide. The acid precipitation in the present invention preferably uses HCl to adjust the pH value of the supernatant to below 2.0, and leave it at room temperature for 4 hours to obtain the supernatant after acid precipitation for the second centrifugation. The rotating speed of the first centrifugation in the present invention is preferably 5500-6500r/min, more preferably 5900-6100r/min, and more preferably 6000r/min. The rotation speed of the second centrifugation in the present invention is the same as that of the first centrifugation. The time of the first centrifugation in the present invention is preferably 10-20 min, more preferably 13-18 min, more preferably 15 min, and the second centrifugation time is preferably 10 min.

After obtaining the bacitracin D crude peptide precipitate, the present invention preferably extracts and centrifuges the obtained bacitracin D crude peptide precipitate to obtain an extraction supernatant. In the present invention, the extraction time of the present invention is 70 to 80 minutes, more preferably 72 minutes; the extraction agent for the extraction is preferably methanol, and the ratio of the amount of the bacitracin D crude peptide precipitation to methanol is preferably 0.1 to 80 minutes. 0.5g: 0.5-2.0mL, more preferably 0.3-0.5g: 1.5mL, more preferably 0.4g: 1.5mL. In the present invention, the rotational speed of the third centrifugation is preferably 8000-12000r/min, more preferably 9500-10500r/min, more preferably 10000r/min, and the centrifugation time is preferably 7-11min, more preferably 9-10min. 10.5 min, more preferably 10 min. The methanol of the present invention can effectively extract the bacillus mycin D.

After the extraction supernatant is obtained, the present invention preferably uses a high performance liquid chromatography (HPLC) method to measure the content of the bacitracin D and its homologues in the extraction supernatant. The bacillus mycin D in the present invention includes one or more of the bacillus mycin DC14 homologue, the bacillus mycin DC15 homologue and the bacillus mycin DC16 homologue. In the present invention, the bacillus mycin DC14 homologue is fourteen carbon β-amino fatty acid ring heptapeptide (C ₁₄ -β-NH ₂ COOH-Asn-Tyr-Asn-ProGlu-Ser-Thr); the bacillus The DC15 homolog of bacitramycin is 15-carbon β-amino fatty acid ring heptapeptide (C ₁₅ -β-NH ₂ COOH-Asn-Tyr-Asn-ProGlu-Ser-Thr); the homolog of bacitramycin DC16 is 16 Carbon β-amino fatty acid cyclic heptapeptide (C ₁₆ -β-NH ₂ COOH-Asn-Tyr-Asn-ProGlu-Ser-Thr). The total yield of bacillus mycin D, the yield of bacillus mycin DC14 homologue, the yield of bacillus mycin DC15 homologue and the yield of bacillus mycin DC16 homologue are all increased by using the technical solution of the invention.

Metal salts can activate certain genes and enzymes in the biosynthetic pathway to improve the synthesis ability of target metabolites. The present invention innovatively adds sodium chloride into the basal medium to prepare a sodium chloride fermentation medium, and adopts fed-batch fermentation to produce bacillusmycin D, which solves the low yield of bacillusmycin D in the prior art technical problems, laying technical support for the large-scale industrial production of bacillus mycin D.

In order to further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with examples, but they should not be construed as limiting the protection scope of the present invention.

Example 1

1 medium

Sodium chloride fermentation medium components: yeast extract 1.0g/L, L-glutamic acid 5.0g/L, glucose 20.0g/L, sodium chloride 3g/L and 1L water.

Slant preservation medium components: beef extract 3.0g/L, tryptone 10g/L, sodium chloride 5g/L, agar 15g/L and 1L water.

Seed medium components: beef extract 3.0g/L, tryptone 10g/L, sodium chloride 5g/L and 1L water.

2-fed-batch fermentation culture

(1) Pick the Bacillus subtilis preserved on the slant and insert it into the seed medium, and culture it at a constant temperature at 37° C. until the OD ₆₀₀ of the Bacillus subtilis seed solution is 0.8 to obtain the Bacillus subtilis seed solution.

(2) inoculating the Bacillus subtilis seed solution in step (1) into a sodium chloride fermentation medium to carry out fed-batch fermentation culture. Start timing with the completion of inoculation: the volume of the sodium chloride fermentation medium is 300mL, the volume of the Bacillus subtilis seed solution is 15mL, and the inoculation volume of the Bacillus subtilis seed solution is 5% of the volume of the sodium chloride fermentation medium. The temperature is 33°C, and the rotation speed is 180r/min, and the fermentation is carried out for 63 hours, and 125mL of fresh sodium chloride fermentation medium is added to the fermentation liquid once, and the temperature is 33°C, and the fermentation is continued under the conditions of 180r/min After culturing for 147 hours, the Bacillus subtilis fed-batch fermentation culture broth was obtained. The total time of fed-batch fermentation was 147h.

Take 40.0mL of Bacillus subtilis fermentation broth and centrifuge at 6000r/min for 15min, discard the bacteria, take the supernatant, adjust the pH of the supernatant to below 2.0 with HCl, let it stand at room temperature for 4h, and again at 6000r/min Centrifuge for 10 min under the conditions to obtain the precipitate of crude bacitracin D peptide, add 1.5 mL of methanol to 0.4 g of the precipitate for extraction, the extraction time is 72 min, centrifuge at 10000 r/min for 10 min, take the supernatant, and use high-performance liquid chromatography (HPLC) The content of bacitracin D and its homologues in the supernatant was determined by this method.

A total of 5 parallel experiments were set up in this fed-batch fermentation experiment.

Example 2

Sodium chloride was added in the fermentation medium, and the addition amount of sodium chloride was 1.0g/L, and all the other conditions were the same as in Example 1.

Example 3

Sodium chloride was added in the fermentation medium, and the addition amount of sodium chloride was 6.0g/L, and all the other conditions were the same as in Example 1.

Comparative example 1

Feeding is not carried out, and all the other conditions are the same as in Example 1.

Comparative example 2

Sodium chloride is not added in the fermentation medium, and all the other conditions are the same as in Example 1.

Comparative example 3

Sodium chloride was replaced with sodium carbonate in the fermentation medium, and all the other conditions were the same as in Example 1.

Comparative example 4

Sodium chloride was added in the fermentation medium, and the addition amount of sodium chloride was 0.5g/L, and all the other conditions were the same as in Example 1.

Comparative example 5

Sodium chloride was added in the fermentation medium, and the addition amount of sodium chloride was 8g/L, and all the other conditions were the same as in Example 1.

The content of the bacillus mycin D and its homologues in the Bacillus subtilis fermentation liquid obtained in Examples 1-3 and Comparative Examples 1-5 were determined, and the determination method adopted a high performance liquid chromatography (HPLC) method. Specific HPLC conditions and assay methods refer to prior art (Qian Shiquan, Lu Hedong, Meng Panpan, Zhang Chong, Lv Fengxia, BieXiaomei, Lu Zhaoxin, Effect of inulin on efficient production and regulatory biosynthesis of bacillomycin D in Bacillus subtilis fmbJ, BioresourceTechnology, 2015, 179:260-267.) proceed. See Table 1 for the average values of the measurement results of 5 parallel experiments in Examples 1-3 and Comparative Examples 1-5.

Table 1 The content (mg/L) of bacitracin D and its homologs in the Bacillus subtilis fermentation broth obtained in Table 1 Examples 1-3 and Comparative Examples 1-5 and the content (mg/g) of bacitracin D in the crude peptide )

According to Table 1, it can be known that the present invention uses the sodium chloride fermentation medium as the base material to carry out 1 fed-batch fermentation, which effectively improves the bacillus mycin D output of Bacillus subtilis.

Although the above-mentioned embodiment has described the present invention in detail, it is only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments according to the present embodiment without inventive step. These embodiments All belong to the protection scope of the present invention.

Claims (10)

1. The application of sodium chloride in improving the synthesis of bacillus mycin D by Bacillus subtilis. 2. A sodium chloride fermentation medium, characterized in that, the sodium chloride fermentation medium uses water as a solvent, and comprises the components of the following concentrations: yeast extract 0.5～1.0g/L, L-glutamic acid 2.0 ～5.0g/L, glucose 5.0～20.0g/L and sodium chloride 1.0～6.0g/L. 3. the described sodium chloride fermentation medium of claim 2, is characterized in that, described sodium chloride fermentation medium is solvent with water, comprises the component of following concentration: yeast extract 1.0g/L, L-glutamine Acid 5.0g/L, glucose 20.0g/L and sodium chloride 3.0g/L. 4. A method for improving the output of bacillus mycin D, characterized in that it comprises the following steps: adopting the sodium chloride fermentation medium described in claim 2 or 3 to ferment and cultivate the bacillus subtilis seed liquid to obtain the bacillus mycin D; The fermentation culture process is a fed-batch sodium chloride fermentation medium. 5. The method according to claim 4, characterized in that, the fermentation culture is a fed-batch fermentation culture; The conditions for the one-time fed-batch fermentation culture include: the Bacillus subtilis seed liquid is inoculated in the sodium chloride fermentation medium and cultured for 48-84 hours, and then a new sodium chloride fermentation medium is added once to continue the fermentation culture. Obtain a fermented broth. 6. The method according to claim 5, characterized in that, the volume of the added sodium chloride fermentation medium is 30% to 50% of the initial sodium chloride fermentation medium volume. 7. The method according to claim 4, characterized in that, the temperature of the fermentation culture is 30-37°C, the rotation speed is 150-200r/min; the fermentation time is 139-171h. 8. The method according to claim 4 or 5, characterized in that, the preparation of the seed liquid of Bacillus subtilis comprises: inoculating Bacillus subtilis on the seed medium, cultivating it to the OD of the seed medium at 33～37°C ₆₀₀ is 0.8～1.0 to obtain Bacillus subtilis seed solution. 9. The method according to claim 4, characterized in that, the inoculation amount of the Bacillus subtilis seed solution is 3% to 5% of the volume of the sodium chloride fermentation medium. 10 . The method according to claim 4 , wherein the bacitracin D comprises one or more of bacitracin DC14 homologues, bacitracin DC15 homologues and bacitracin DC16 homologues. 11 .