CN115478088A - Application of calcium lactate in improvement of bacillus subtilis synthetic bacillomycin D, calcium lactate fermentation culture medium and method - Google Patents
Application of calcium lactate in improvement of bacillus subtilis synthetic bacillomycin D, calcium lactate fermentation culture medium and method Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
- C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
- C07K7/58—Bacitracins; Related peptides
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
Abstract
The invention belongs to the technical field of microbial fermentation, and particularly relates to application of calcium lactate in improvement of bacillus subtilis to synthesis of bacillomycin D, a calcium lactate fermentation culture medium and a method. The invention provides application of calcium lactate in improving bacillus subtilis to synthesize bacitracin D, and the yield of bacitracin D can be improved by preparing a fermentation culture medium by utilizing calcium lactate during fermentation culture of bacillus subtilis. The calcium lactate can promote the expression of the bacillomycin D synthetase gene of the bacillus so as to effectively improve the yield of the 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 calcium lactate fermentation culture medium provided by the invention is obviously improved.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to application of calcium lactate in improving bacillus subtilis to synthesis of bacillomycin D, a calcium lactate fermentation culture medium and a method.
Background
Bacitracin D is an antibacterial lipopeptide belonging to the iturin family that is catalytically synthesized by the non-ribosomal peptide synthase in bacillus subtilis. The damped grains are easy to generate mycotoxins (such as aflatoxin, ochratoxin and the like) due to mildew, and the bacillomycin D has a strong inhibiting effect on agricultural pathogenic fungi (such as aspergillus flavus, ochratoxin and the like). Therefore, the bacillomycin D has important application prospects in the aspects of guaranteeing the safety of grains and foods, preventing and controlling fungal pollution and the like. However, the bacillus subtilis has limited ability to synthesize bacitracin D when fermentation culture is carried out on a conventional culture medium, and the yield of bacitracin D is low, so that large-scale industrial application of bacitracin D is limited. In the prior art, a culture medium consisting of yeast extract, L-glutamic acid and glucose is mostly used for producing the bacitracin D, but the yield of the bacitracin D is low. Chinese patent CN110016490A fermentation medium for producing bacillomycin D and method for producing bacillomycin D disclose that corn stalk cellulase is used for enzymolysis of supernatant, yeast extract, L-sodium glutamate and KH 2 PO 4 The mixed preparation of the fermentation medium for producing the bacillomycin D improves the yield of the bacillomycin D produced by the fermentation culture of the bacillus subtilis, but the preparation method of the medium is complex; therefore, a fermentation medium which can improve the yield of the bacillomycin D and is simple to prepare is urgently needed.
Disclosure of Invention
The invention aims to provide application of calcium lactate in improving bacillus subtilis to synthesize bacitracin D, and the bacillus subtilis is fermented and cultured to produce the bacitracin D by adding the calcium lactate into a fermentation culture medium, so that the yield of the bacitracin D can be improved.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides application of calcium lactate in improving bacillus subtilis synthetic bacillomycin D.
Preferably, the calcium lactate comprises L-calcium lactate.
The invention provides a calcium lactate fermentation medium, which takes water as a solvent and comprises the following components in concentration: 0.5-1.0 g/L of yeast extract, 2.0-5.0 g/L of L-glutamic acid, 5.0-20.0 g/L of glucose and 3.5-9.5 g/L of calcium lactate.
The invention provides a method for improving yield of bacillomycin D, which comprises the following steps: the calcium lactate fermentation culture medium in the technical scheme is adopted to carry out fermentation culture on the bacillus subtilis seed liquid to obtain the bacillomycin D; the fermentation culture process is fed with a calcium lactate fermentation culture medium in batches.
Preferably, the number of times of feeding the calcium lactate fermentation medium in batches in the fermentation culture process is 3;
the conditions of the fermentation culture comprise: inoculating the bacillus subtilis seed solution into a calcium lactate fermentation culture medium, culturing for 48-84 h, then supplementing a new calcium lactate fermentation culture medium, then supplementing the new calcium lactate fermentation culture medium every 15-21 h, and finally performing fed-batch fermentation to obtain a fermentation broth.
Preferably, the volume of the new calcium lactate fermentation medium supplemented each time is 30-55% of the volume of the initial calcium lactate fermentation medium.
Preferably, the temperature of the fermentation culture is 30-37 ℃, and the rotating speed is 150-200 r/min; the total time of the fermentation culture is 108-192 h.
Preferably, the inoculation amount of the bacillus subtilis seed solution is 3-5% of the volume of the calcium lactate fermentation medium.
Preferably, the preparation of the seed solution of bacillus subtilis comprises the following steps: inoculating Bacillus subtilis to seed culture medium, and culturing at 33-37 deg.C to OD of the seed culture medium 600 Obtaining the bacillus subtilis seed liquid with the concentration of 0.8-1.0.
Preferably, the bacillomycin D comprises one or more of a bacillomycin D C14 homolog, a bacillomycin D C15 homolog, a bacillomycin D C16 homolog, and a bacillomycin D C17 homolog.
The invention has the beneficial effects that: the invention provides application of calcium lactate in improving bacillus subtilis to synthesize bacitracin D, and the yield of bacitracin D can be improved by preparing a fermentation culture medium from calcium lactate to perform fermentation culture on bacillus subtilis. The calcium lactate can promote the expression of the bacillomycin D synthetase gene of the bacillus so as to effectively improve the yield of the 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 calcium lactate fermentation culture medium provided by the invention is obviously improved.
Detailed Description
The invention provides an application of calcium lactate in improving bacillus subtilis synthetic bacillomycin D.
In the present invention, the calcium lactate preferably comprises L-calcium lactate. The calcium lactate can promote the expression of the bacillomycin D synthetase gene so as to effectively improve the yield of the bacillomycin D synthesized by bacillus subtilis. The source of the L-calcium lactate is not particularly limited, and conventional commercial products can be adopted.
The invention provides a calcium lactate fermentation medium, which takes water as a solvent and comprises the following components in concentration: 0.5-1.0 g/L of yeast extract, 2.0-5.0 g/L of L-glutamic acid, 5.0-20.0 g/L of glucose and 3.5-9.5 g/L of calcium lactate. In the calcium lactate fermentation medium of the present invention, the calcium lactate preferably comprises L-calcium lactate.
In the invention, the calcium lactate fermentation medium comprises 0.5-1.0 g/L of yeast extract, preferably 0.8-1.0 g/L, and more preferably 1.0g/L. The yeast extract of the invention supplements proteins, trace elements and the like required in the growth and reproduction of bacillus subtilis, promotes the expression of the gene of the bacillus subtilis D synthetase, and improves the yield of the bacillus subtilis D synthetase.
In the invention, the calcium lactate fermentation medium comprises 5.0-20.0 g/L glucose, preferably 15.0-20.0 g/L glucose, and more preferably 20.0g/L glucose. The glucose can promote the expression of the bacillus subtilis D synthetase gene and improve the yield of bacillus subtilis D.
In the invention, the calcium lactate fermentation medium comprises 2.0-5.0 g/L of L-glutamic acid, preferably 4.0-5.0 g/L, and more preferably 5.0g/L. The L-glutamic acid can promote the expression of the bacillus subtilis D synthetase gene and improve the yield of bacillus subtilis D.
In the invention, the calcium lactate fermentation medium comprises 3.5-9.5 g/L of calcium lactate, preferably 5-8 g/L, and more preferably 6.5g/L. The calcium lactate can promote the expression of the bacillomycin D synthetase gene so as to effectively improve the bacillomycin D synthesized by the bacillus subtilis.
The basic culture medium for producing the bacitracin D comprises yeast extract, L-glutamic acid and glucose, the basic culture medium for producing the bacitracin D is used as a base, calcium lactate is added into the basic culture medium to obtain a calcium lactate fermentation culture medium, the calcium lactate fermentation culture medium is used as a base, and the batched fermentation is used for producing the bacitracin D, so that the yield of the bacitracin D is improved.
The invention utilizes the calcium lactate fermentation medium to efficiently ferment and produce the bacillomycin D, and has the advantages of low cost, simple operation and high practicability. The sterilization condition of the calcium lactate fermentation medium is preferably 115 ℃ and 20min.
The invention provides a method for improving the yield of bacillomycin D, which comprises the following steps: the calcium lactate fermentation medium is adopted to carry out fermentation culture on the bacillus subtilis seed solution to obtain the bacillomycin D; the fermentation culture process is fed with a calcium lactate fermentation culture medium in batches.
The invention preferably directly uses the bacillus subtilis preserved on the inclined plane to prepare the bacillus subtilis seed solution.
The components of the culture medium applied in the slant preservation of the invention preferably comprise 3.0g/L beef extract, 10g/L tryptone, 5g/L sodium chloride, 15g/L agar and 1L water.
The invention preferably utilizes the sterilized bamboo sticks to pick the bacillus subtilis preserved on the inclined plane to inoculate the seeds in the seed culture medium for seed cultureCulturing to obtain the bacillus subtilis seed liquid. In the invention, the seed culture is preferably constant-temperature culture, and the constant-temperature culture temperature is preferably 33-37 ℃, more preferably 35-37 ℃, and more preferably 37 ℃; the invention is preferably cultured to the bacillus subtilis seed solution OD 600 Preferably 0.8 to 1.0, more preferably 0.9. In the present invention, the seed culture is preferably a liquid culture, and the composition of the seed culture medium preferably includes 3.0g/L beef extract, 10g/L tryptone, 5g/L sodium chloride and 1L water.
The sterilization conditions of the solid culture medium and the seed culture medium are not particularly limited, and the conventional sterilization conditions can be adopted.
After the bacillus subtilis seed solution is obtained, the bacillus subtilis seed solution is inoculated in a calcium lactate fermentation culture medium for fermentation culture to obtain a fermentation liquid. In the present invention, the volume of the bacillus subtilis seed solution at the time of inoculation is preferably 4 to 6%, more preferably 4.5 to 6%, and still more preferably 5% of the volume of the calcium lactate fermentation medium.
The calcium lactate fermentation medium of the present invention is discussed above and will not be described herein.
In the present invention, the temperature of the fermentation culture is preferably 30 to 37 ℃, more preferably 32 to 35 ℃, and still more preferably 33 ℃. The rotation speed of the fermentation culture is preferably 150-200 r/min, more preferably 170-190 r/min, and even more preferably 180r/min.
In the invention, the fermentation culture is preferably fed-batch fermentation culture, and the fermentation culture process of the invention is fed-batch calcium lactate fermentation culture medium. In the present invention, the time for the fermentation culture is preferably 108 to 192 hours, more preferably 120 to 170 hours, and still more preferably 162 hours. The fermentation culture time preferably comprises the fermentation culture time before the calcium lactate culture medium is supplemented and the total fermentation culture time after the calcium lactate culture medium is supplemented, namely the total fermentation culture time; the fermentation culture time after the calcium lactate culture medium is supplemented comprises the culture time after the calcium lactate culture medium is supplemented for the first time, the culture time after the calcium lactate culture medium is supplemented for the second time and the culture time after the calcium lactate culture medium is supplemented for the third time. The fermentation culture time before the calcium lactate is supplemented is preferably 48 to 84 hours, more preferably 56 to 72 hours, and even more preferably 66 hours.
In the fermentation culture process, the frequency of feeding the calcium lactate fermentation culture medium in batches is preferably 3 times.
The conditions of the fed-batch fermentation according to the invention preferably comprise: inoculating the bacillus subtilis seed solution into a calcium lactate fermentation culture medium, culturing for 48-84 h, then supplementing a new calcium lactate fermentation culture medium, then supplementing the new calcium lactate fermentation culture medium every 15-21 h, and finally performing fed-batch fermentation to obtain a fermentation broth. In the present invention, the time interval for each new supplement of calcium lactate fermentation medium is preferably 15 to 21 hours, more preferably 16 to 20 hours, and even more preferably 18 hours.
In the present invention, the volume of the new calcium lactate fermentation medium to be supplemented each time is preferably 30% to 55%, more preferably 38% to 53%, and still more preferably 50% of the volume of the initial calcium lactate fermentation medium. The supplemented new calcium lactate fermentation medium and the selection of the volume parameters of the supplemented new calcium lactate fermentation medium can improve the yield of the bacillomycin D produced by fermenting the bacillus subtilis.
In the present invention, when the number of times of feeding is 3, the conditions of fed-batch fermentation are preferably: and (2) inoculating the bacillus subtilis seed solution into an initial calcium lactate fermentation culture medium for fermentation culture, recording as 1 st fermentation culture, supplementing a fresh calcium lactate fermentation culture medium into the 1 st fermentation culture medium, performing fermentation culture, recording as 2 nd fermentation culture, supplementing a fresh calcium lactate fermentation culture medium into the 2 nd fermentation culture medium, performing fermentation culture, recording as 3 rd fermentation, adding a fresh calcium lactate fermentation culture medium into the 3 rd fermentation culture medium, and performing fermentation culture to obtain the bacillus subtilis fermentation liquid. In the invention, when the feeding times are 3 times, the effect of the bacillomycin D produced by fermenting the bacillus subtilis is better, the content of the total bacillomycin D is 1.01 +/-0.02 g/L at most, and the content of the total bacillomycin D in the crude peptide is 38.06 +/-0.72 mg/g.
After the bacillus subtilis fermentation liquor is obtained, the bacillus subtilis fermentation liquor is preferably subjected to centrifugation, acid precipitation and extraction to obtain the bacitracin D. The rotating speed of the centrifugation is preferably 5800-6200 r/min, more preferably 6000r/min, and the centrifugation time is preferably 13-17 min, more preferably 15min.
The invention centrifuges the bacillus subtilis fermentation liquor to obtain supernatant. The invention preferably acid-precipitates the crude bacitracin D peptide from the supernatant. In the present invention, the acid precipitation method preferably includes: adjusting the pH value of the supernatant to be below 2.0 by using HCl, standing for 4 hours at room temperature, and standing for 4 hours at room temperature to obtain supernatant after acid precipitation; then, carrying out second centrifugation on the supernatant after acid precipitation, wherein the rotation speed of the second centrifugation is preferably 5800-6200 r/min, more preferably 6000r/min, and the second centrifugation time is preferably 8-12 min, more preferably 10min; and centrifuging for the second time to obtain supernatant crude peptide precipitate after acid precipitation, namely the bacitracin D crude peptide precipitate.
After acid precipitation, the crude peptide precipitate of the bacillomycin D is preferably extracted and centrifuged for the third time to obtain a supernatant containing the bacillomycin D, the extraction is preferably carried out by using methanol, and the methanol is preferably chromatographic pure methanol. The extraction time of the invention is 68-82 min, more preferably 70-75 min, and still more preferably 72min. The mass-to-volume ratio of the crude peptide precipitate to methanol is preferably 0.1-0.5 g:0.5 to 2.0mL, more preferably 0.3 to 0.5g:1.0 to 1.8mL, more preferably 0.4g:1.5mL. The rotating speed of the third centrifugation is 9000-11000 r/min, more preferably 10000r/min, and the centrifugation time is preferably 8-12 min, more preferably 10min.
The bacillus subtilis fermentation liquor is centrifuged, acid-precipitated and extracted for three times to obtain the supernatant containing the bacillomycin D, and the content of the bacillomycin D and the homologues thereof in the supernatant containing the bacillomycin D is preferably determined by a High Performance Liquid Chromatography (HPLC) method. The bacillomycin D provided by the invention comprises one or more of bacillomycin DC14 homolog, bacillomycin DC15 homolog, bacillomycin DC16 homolog and bacillomycin DC17 homolog. In the present invention, the bacitracin DC14 homologue is fourteenCarbon beta-amino fatty acid cyclic heptapeptide (C) 14 -β-NH 2 COOH-Asn-Tyr-Asn-ProGlu-Ser-Thr); the bacillomycin DC15 homolog is pentadecane beta-amino fatty acid cyclo-heptapeptide (C) 15 -β-NH 2 COOH-Asn-Tyr-Asn-ProGlu-Ser-Thr); the bacillomycin DC16 homologue is hexadecane beta-amino fatty acid cyclo-heptapeptide (C) 16 -β-NH 2 COOH-Asn-Tyr-Asn-ProGlu-Ser-Thr); the bacillomycin DC17 homolog is heptadecarbyl beta-amino fatty acid cyclo-heptapeptide (C) 17 -β-NH 2 COOH-Asn-Tyr-Asn-ProGlu-Ser-Thr). According to the technical scheme, the total yield of the bacillomycin D, the yield of the bacillomycin DC14 homologues, the yield of the bacillomycin DC15 homologues, the yield of the bacillomycin DC16 homologues and the yield of the bacillomycin DC17 homologues are all improved by fermenting the bacillus subtilis.
The invention combines the characteristics that calcium lactate can promote the expression of the bacillomycin D synthetase gene and further effectively improve the bacillomycin D synthesis of bacillus subtilis, creates a calcium lactate fermentation culture medium for producing the bacillomycin D, adopts fed-batch fermentation to produce the bacillomycin D, obviously improves the yield of the bacillomycin D, and provides scientific and reasonable technical basis for realizing large-scale industrial application of the bacillomycin D.
In order to further illustrate the present invention, the following embodiments are described in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
(1) Culture medium
Calcium lactate fermentation medium components: 1.0g/L of yeast extract, 5.0g/L of L-glutamic acid, 20.0g/L of glucose, 6.5g/L of L-calcium lactate and 1L of water. Sterilizing the calcium lactate fermentation medium at 115 deg.C for 20min, and cooling to room temperature.
Slant preservation medium component: 3.0g/L beef extract, 10g/L tryptone, 5g/L sodium chloride and 15g/L agar.
Seed culture medium components: 3.0g/L of beef extract, 10g/L of tryptone and 5g/L of sodium chloride.
(2) Fed-batch fermentation culture
By sterilizing bambooPicking the bacillus subtilis colony preserved on the inclined plane, inoculating the bacillus subtilis colony into a seed culture medium, and performing shake-flask culture at 37 ℃ until bacillus subtilis seed liquid OD 600 And the yield is 0.8, and the bacillus subtilis seed liquid is obtained.
Inoculating the bacillus subtilis seed solution into a calcium lactate fermentation culture medium with the volume of 300mL according to the inoculation amount of 5% of the volume ratio, and performing fermentation culture at the temperature of 33 ℃ and under the condition of 180r/min, wherein the 1 st fermentation is recorded.
From the start of incubation timing: and (3) when the fermentation is carried out for 66 hours from the 1 st time, adding 150mL of fresh calcium lactate fermentation medium into the 1 st fermentation liquid, and continuing the fermentation culture at the temperature of 33 ℃ and under the condition of 180r/min, and recording as the 2 nd fermentation culture. And when the 2 nd fermentation culture is carried out for 84h, adding 150mL of fresh calcium lactate fermentation culture medium into the 2 nd fermentation culture solution, continuing fermentation at the temperature of 33 ℃ and under the condition of 180r/min, recording as the 3 rd fermentation culture, when the 3 rd fermentation culture is carried out for 102h, adding 150mL of fresh fermentation culture medium into the 3 rd fermentation culture solution, and continuing fermentation for 162h under the condition of the temperature of 33 ℃ and 180r/min to obtain the bacillus subtilis fermentation solution. Namely, the frequency of the fed-batch calcium lactate fermentation culture medium is 3 times. The fermentation time before supplementing the calcium lactate fermentation medium is 66 hours, the fermentation time after supplementing the 1 st culture medium is 18 hours, the fermentation time after supplementing the second fermentation medium is 18 hours, the 3 rd fermentation medium is supplemented, and the fermentation is continued for 60 hours after supplementing the 3 rd fermentation medium, so that the bacillus subtilis fermentation liquid is obtained. The total fermentation time was 162h.
And (3) centrifuging 40.0mL of bacillus subtilis fermentation liquor at 6000r/min for 15min, discarding thalli, taking supernatant of the bacillus subtilis fermentation liquor, adjusting the pH of the supernatant to be below 2.0 by using HCl, leaching crude peptide, standing at room temperature for 4h, and centrifuging for 10min at 6000r/min to obtain the bacitracin D crude peptide precipitate.
Adding 1.5mL of methanol into 0.4g of the crude peptide precipitate of the bacillomycin D for extraction for 72min, centrifuging the obtained crude peptide extract for 10min under the condition of 10000r/min, and taking supernatant fluid, namely the solution containing the bacillomycin D. The advantage of facilitating methanol extraction is that bacitracin D is efficiently extracted. The content of bacillomycin D and homologues thereof in the bacillomycin D-containing solution is determined by High Performance Liquid Chromatography (HPLC).
Example 1 a total of 5 parallel experiments.
Example 2
L-calcium lactate was added to the fermentation medium in an amount of 3.5g/L, and the other conditions were the same as in example 1.
Example 3
The fermentation medium was supplemented with L-calcium lactate in an amount of 9.5g/L, and the other conditions were the same as in example 1.
Comparative example 1
No feed was added and the rest of the conditions were the same as in example 1.
Comparative example 2
The fermentation medium was supplemented with no L-calcium lactate under the same conditions as in example 1.
Comparative example 3
Calcium chloride was added to the fermentation medium under the same conditions as in example 1.
Comparative example 4
L-calcium lactate was added to the fermentation medium at a concentration of 0.5g/L, and the other conditions were the same as in example 1.
Comparative example 5
L-calcium lactate is added into the fermentation medium, the addition amount of the L-calcium lactate is 12.5g/L, and the other conditions are the same as the conditions in the example 1.
The content of bacitracin D and its homologues in the Bacillus subtilis fermentation broth obtained in examples 1 to 3 and comparative examples 1 to 5 was determined by High Performance Liquid Chromatography (HPLC). Specific HPLC conditions and assay methods were performed with reference to the prior art (Qian Shiquan, lu Hedong, meng Panban, zhang Chong, lv Fengxia, bie Xiaomei, lu Zhaoxin, effect of insulin on efficacy production and regulation of biosynthesis of Bacillus subtilis fmbJ, bioresource Technology,2015, 179. The measured values of 5 parallel experiments for examples 1 to 3 and comparative examples 1 to 5 are shown in Table 1.
TABLE 1 content (g/L) of bacitracin D and its homologues in Bacillus subtilis fermentation broth obtained in examples 1 to 3 and comparative examples 1 to 5 and content (mg/g) of bacitracin D in crude peptide
According to the table 1, the feed supplement fermentation is carried out for three times by using the calcium lactate fermentation medium as the base material, so that the yield of the bacillomycin D of the bacillus subtilis is effectively improved.
Example 4 inhibitory Effect of Bacillamycin D on Aspergillus flavus
PDA culture medium: 200g/L potato, 20g/L glucose, 18g/L agar and 1L water.
The effect of the bacitracin D prepared in example 1 on inhibition of Aspergillus flavus (CICC 2062) was verified. After culturing Aspergillus flavus at 28 ℃ for 4 days using a PDA plate, a 5mm hypha block was taken, the hypha was placed face down on PDA plates containing different concentrations of bacillomycin D and subjected to static culture at 28 ℃ for 5 days to serve as test groups, the concentrations of bacillomycin D in the PDA plates of test groups 1 to 7 were 5. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL, 80. Mu.g/mL, 160. Mu.g/mL and 320. Mu.g/mL in this order, and the PDA plate containing no bacillomycin D was used as a control group.
After 5d of culture, the colony diameter was measured by the cross method, and the hypha inhibition rate was calculated.
After 5 days of culture, 5.0mL of sterile water was added to the PDA plates of the test groups 1 to 7 and the control group, and spores were washed with sterile water and counted on a hemocytometer to determine the number of spores of Aspergillus flavus. The results are shown in Table 2.
The calculation formula of the inhibition rate of aspergillus flavus hyphae and spores is as follows: hypha inhibition (%) = { (diameter of aspergillus flavus colony treated without bacitracin D-diameter of aspergillus flavus colony treated with bacitracin D)/(diameter of colony treated without bacitracin D) } × 100%;
spore inhibition rate (%) = { (number of aspergillus flavus colonies spore without bacillomycin D treatment-number of aspergillus flavus colonies spore without bacillomycin D treatment)/(number of colonies spore without bacillomycin D treatment) } × 100%.
As can be seen from Table 2, when 320. Mu.g/mL of bacitracin D is added, the growth of Aspergillus flavus can be completely inhibited, and it can be seen that the bacitracin D prepared by the technical scheme of the present invention can inhibit the growth of Aspergillus flavus, which indicates that the purity of the bacitracin D prepared by the technical scheme of the present invention is high.
TABLE 2 inhibition of Aspergillus flavus by Bactericin D
In conclusion, the invention utilizes calcium lactate to prepare a fermentation medium for carrying out fermentation culture on the bacillus subtilis, so that the yield of the bacillomycin D can be improved, and the obtained bacillomycin D has a good inhibition effect on aspergillus flavus.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (10)
1. Application of calcium lactate in improving synthesis of bacillomycin D by bacillus subtilis is provided.
2. Use according to claim 1, wherein the calcium lactate comprises L-calcium lactate.
3. A calcium lactate fermentation medium, which takes water as a solvent and comprises the following components in concentration: 0.5-1.0 g/L of yeast extract, 2.0-5.0 g/L of L-glutamic acid, 5.0-20.0 g/L of glucose and 3.5-9.5 g/L of calcium lactate.
4. A method for improving the yield of bacitracin D, which comprises the following steps: fermenting and culturing bacillus subtilis seed liquid by using the calcium lactate fermentation medium as defined in claim 3 to obtain the bacillomycin D; the fermentation culture process is fed with a calcium lactate fermentation culture medium in batches.
5. The method according to claim 4, wherein the number of fed-batch calcium lactate fermentation medium times during the fermentation culture is 3;
the conditions of the fermentation culture comprise: inoculating the bacillus subtilis seed solution into a calcium lactate fermentation culture medium, culturing for 48-84 h, supplementing a new calcium lactate fermentation culture medium, then supplementing the new calcium lactate fermentation culture medium every 15-21 h, and finally performing fed-batch fermentation to obtain fermentation liquor.
6. The process according to claim 4, wherein the volume of the new calcium lactate fermentation medium fed each time is 30% to 55% of the volume of the initial calcium lactate fermentation medium.
7. The method according to claim 4, wherein the temperature of the fermentation culture is 30-37 ℃, and the rotating speed is 150-200 r/min; the total time of the fermentation culture is 108-192 h.
8. The method of claim 4, wherein the bacillus subtilis seed solution is inoculated in an amount of 4 to 6% by volume of the calcium lactate fermentation medium.
9. The method of claim 4 or 8, wherein the preparation of the Bacillus subtilis seed solution comprises: inoculating the Bacillus subtilis to a seed culture medium, and culturing at 33-37 deg.C to OD of the seed culture medium 600 Obtaining the bacillus subtilis seed liquid with the concentration of 0.8-1.0.
10. The method of claim 4, wherein the bacomycin D comprises one or more of a bacomycin D C14 homolog, a bacomycin D C15 homolog, a bacomycin D C16 homolog, and a bacomycin D C17 homolog.
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