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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- 238000000855 fermentation Methods 0.000 title claims abstract description 158
- 230000004151 fermentation Effects 0.000 title claims abstract description 158
- MKJXYGKVIBWPFZ-UHFFFAOYSA-L calcium lactate Chemical compound [Ca+2].CC(O)C([O-])=O.CC(O)C([O-])=O MKJXYGKVIBWPFZ-UHFFFAOYSA-L 0.000 title claims abstract description 97
- 239000001527 calcium lactate Substances 0.000 title claims abstract description 97
- 229960002401 calcium lactate Drugs 0.000 title claims abstract description 97
- 235000011086 calcium lactate Nutrition 0.000 title claims abstract description 97
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 67
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 66
- 238000000034 method Methods 0.000 title claims abstract description 23
- 239000001963 growth medium Substances 0.000 title claims abstract description 16
- 108010081278 bacillomycin D Proteins 0.000 title claims abstract description 8
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- CLKOFPXJLQSYAH-ABRJDSQDSA-N bacitracin A Chemical compound C1SC([C@@H](N)[C@@H](C)CC)=N[C@@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]1C(=O)N[C@H](CCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2N=CNC=2)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCCCC1 CLKOFPXJLQSYAH-ABRJDSQDSA-N 0.000 claims abstract description 24
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- MKJXYGKVIBWPFZ-CEOVSRFSSA-L calcium;(2s)-2-hydroxypropanoate Chemical compound [Ca+2].C[C@H](O)C([O-])=O.C[C@H](O)C([O-])=O MKJXYGKVIBWPFZ-CEOVSRFSSA-L 0.000 claims description 15
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- 229910052791 calcium Inorganic materials 0.000 description 1
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- 229910001628 calcium chloride Inorganic materials 0.000 description 1
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- 238000012262 fermentative production Methods 0.000 description 1
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- 244000053095 fungal pathogen Species 0.000 description 1
- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(OC[C@]3(OC[C@]4(OC[C@]5(OC[C@]6(OC[C@]7(OC[C@]8(OC[C@]9(OC[C@]%10(OC[C@]%11(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 description 1
- 229940029339 inulin Drugs 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
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- 229940073490 sodium glutamate Drugs 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
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- 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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Abstract
Description
技术领域technical field
本发明属于微生物发酵技术领域，具体涉及乳酸钙在提高枯草芽孢杆菌合成杆菌霉素D的应用和乳酸钙发酵培养基及方法。The invention belongs to the technical field of microbial fermentation, and in particular relates to the application of calcium lactate in improving the synthesis of bacillus mycin D by Bacillus subtilis, a calcium lactate fermentation medium and a method.
背景技术Background technique
杆菌霉素D是由枯草芽孢杆菌中非核糖体肽合酶催化合成的一种属于iturin家族的抗菌脂肽。受潮的粮食很容易因霉变而产生真菌毒素(如黄曲霉毒素和赭曲霉毒素等)，而杆菌霉素D对农业病原真菌(如黄曲霉和赭曲霉等)具有很强的抑制作用。因此，杆菌霉素D在保障粮食和食品安全以及防治真菌污染等方面都具有重要应用前景。但是，枯草芽孢杆菌在常规培养基进行发酵培养时合成杆菌霉素D的能力有限，杆菌霉素D的产量低，限制了杆菌霉素D的大规模工业化应用。现有技术中多应用以酵母膏，L-谷氨酸和葡萄糖组成的培养基生产杆菌毒素D，但是杆菌霉素D的产量低。中国专利CN110016490A一种用于生产杆菌霉素D的发酵培养基和生产杆菌霉素D的方法公开了利用玉米秸秆纤维素酶酶解上清液、酵母提取物、L-谷氨酸钠和KH2PO4混合制备用于生产杆菌霉素D的发酵培养基，虽然提高了枯草芽孢杆菌发酵培养生产杆菌霉素D的产量，但是培养基的制备方法比较复杂；因此急需一种提高杆菌霉素D产量的、制备简单的发酵培养基。Bacitracin D is an antibacterial lipopeptide belonging to the iturin family synthesized by non-ribosomal peptide synthase in Bacillus subtilis. Damp grains are prone to mildew and produce mycotoxins (such as aflatoxin and ochratoxin, etc.), and bacitracin D has a strong inhibitory effect on agricultural pathogenic fungi (such as Aspergillus flavus and ochratoxin, etc.). Therefore, bacitracin D has important application prospects in ensuring food and food safety and preventing and controlling fungal contamination. However, the ability of Bacillus subtilis to synthesize bacillus mycin D is limited when it is fermented and cultured in a conventional medium, and the yield of bacillus mycin D is low, which limits the large-scale industrial application of bacillus mycin D. In the prior art, the culture medium composed of yeast extract, L-glutamic acid and glucose is mostly used to produce bacillus toxin D, but the yield of bacillus toxin D is low. Chinese patent CN110016490A, a fermentation medium for producing bacillus mycin D and a method for producing bacillus mycin D discloses the use of corn stalk cellulase enzymatic hydrolysis supernatant, yeast extract, L-sodium glutamate and KH 2 PO 4 mixed preparation for the production of bacinomycin D fermentation medium, although improved the production of bacillus subtilis fermented to produce bacinomycin D, but the preparation of the medium is more complicated; A simple, easy-to-prepare fermentation medium.
发明内容Contents of the invention
本发明的目的是提供了乳酸钙在提高枯草芽孢杆菌合成杆菌霉素D的应用，将乳酸钙添加在发酵培养基中对枯草芽孢杆菌进行发酵培养生产杆菌霉素D，能够提高杆菌霉素D的产量。The purpose of the present invention is to provide the application of calcium lactate in improving the synthesis of bacillus mycin D by Bacillus subtilis, adding calcium lactate in the fermentation medium to ferment and cultivate Bacillus subtilis to produce bacillus mycin D, which can improve the production of bacillus mycin D output.
为了解决上述技术问题，本发明提出以下技术方案：In order to solve the above technical problems, the present invention proposes the following technical solutions:
本发明提供了乳酸钙在提高枯草芽孢杆菌合成杆菌霉素D的应用。The invention provides the application of calcium lactate in improving the synthesis of bacillus mycin D by bacillus subtilis.
优选的，所述乳酸钙包括L-乳酸钙。Preferably, the calcium lactate includes L-calcium lactate.
本发明提供了一种乳酸钙发酵培养基，所述乳酸钙发酵培养基以水为溶剂，包含以下浓度的组分：酵母膏0.5～1.0g/L、L-谷氨酸2.0～5.0g/L、葡萄糖5.0～20.0g/L和乳酸钙3.5～9.5g/L。The invention provides a calcium lactate fermentation medium. The calcium lactate 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 L, glucose 5.0-20.0g/L and calcium lactate 3.5-9.5g/L.
本发明提供了一种提高杆菌霉素D产量的方法，包括以下步骤：采用上述技术方案所述乳酸钙发酵培养基对枯草芽孢杆菌种子液进行发酵培养获得杆菌霉素D；所述发酵培养过程分批补料乳酸钙发酵培养基。The invention provides a method for improving the output of bacillus mycin D, comprising the following steps: adopting the calcium lactate fermentation medium described in the above technical scheme to ferment and cultivate the Bacillus subtilis seed liquid to obtain bacillus mycin D; the fermentation and cultivation process Fed-batch calcium lactate fermentation medium.
优选的，所述发酵培养过程中分批补料乳酸钙发酵培养基的次数为3次；Preferably, the number of times of feeding calcium lactate fermentation medium in batches in the fermentation process is 3 times;
所述发酵培养的条件包括：所述枯草芽孢杆菌种子液接种于乳酸钙发酵培养基后培养48～84h后补加新的乳酸钙发酵培养基，随后每隔15～21h补加新的乳酸钙发酵培养基，最后一次补料发酵后获得发酵液。The conditions of the fermentation culture include: the Bacillus subtilis seed liquid is inoculated in the calcium lactate fermentation medium and cultured for 48 to 84 hours, and then a new calcium lactate fermentation medium is added, and then new calcium lactate is added every 15 to 21 hours. Fermentation medium, fermented liquid obtained after the last feed-fed fermentation.
优选的，每次补加的新的乳酸钙发酵培养基的体积为初始的乳酸钙发酵培养基体积的30％～55％。Preferably, the volume of the new calcium lactate fermentation medium added each time is 30%-55% of the volume of the initial calcium lactate fermentation medium.
优选的，所述发酵培养的温度为30～37℃，转速为150～200r/min；所述发酵培养的总时间为108～192h。Preferably, the temperature of the fermentation culture is 30-37° C., the rotation speed is 150-200 r/min; the total time of the fermentation culture is 108-192 hours.
优选的，所述枯草芽孢杆菌种子液的接种量为乳酸钙发酵培养基体积的3％～5％。Preferably, the inoculation amount of the Bacillus subtilis seed solution is 3%-5% of the volume of the calcium lactate fermentation medium.
优选的，所述枯草芽孢杆菌的种子液的制备包括：枯草芽孢杆菌接种于种子培养基，于33～37℃培养至种子培养基的OD600为0.8～1.0获得枯草芽孢杆菌种子液。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.
优选的，所述杆菌霉素D包括杆菌霉素D C14同系物、杆菌霉素D C15同系物、杆菌霉素D C16同系物和杆菌霉素D C17同系物中的一种或多种。Preferably, the bacitracin D includes one or more of bacitracin D C14 homologues, bacitracin D C15 homologues, bacitracin D C16 homologues and bacitracin D C17 homologues.
本发明的有益效果：本发明提供了乳酸钙在提高枯草芽孢杆菌合成杆菌霉素D的应用，利用乳酸钙配制发酵培养基对枯草芽孢杆菌进行发酵培养可以提高杆菌霉素D的产量。本发明乳酸钙能促进芽孢杆菌的杆菌霉素D合成酶基因表达进而有效提高杆菌霉素D的产量。实施例结果表明：应用本发明提供的乳酸钙发酵培养基对枯草芽孢杆菌进行发酵培养获得的杆菌霉素D的产量明显提高。Beneficial effects of the present invention: the present invention provides the application of calcium lactate in improving the synthesis of bacillus mycin D by bacillus subtilis, and the production of bacillus mycin D can be increased by using calcium lactate to prepare a fermentation medium for fermentation and cultivation of bacillus subtilis. The calcium lactate of the present invention can promote the expression of the bacillus mycin D synthetase gene of bacillus, thereby effectively increasing the output of the bacillus mycin D. The results of the examples show that the yield of bacillus mycin D obtained by fermenting and culturing Bacillus subtilis using the calcium lactate fermentation medium provided by the present invention is significantly improved.
具体实施方式detailed description
本发明提供了一种乳酸钙在提高枯草芽孢杆菌合成杆菌霉素D的应用。The invention provides an application of calcium lactate in improving the synthesis of bacillus mycin D by Bacillus subtilis.
在本发明中，所述乳酸钙优选包括L-乳酸钙。本发明所述乳酸钙能促进杆菌霉素D合成酶基因表达进而有效提高枯草芽孢杆菌合成杆菌霉素D的产量。本发明对所述L-乳酸钙的来源没有特殊限定，采用常规的市售产品即可。In the present invention, the calcium lactate preferably includes L-calcium lactate. The calcium lactate of the present invention can promote the expression of the bacillus mycin D synthetase gene, thereby effectively increasing the yield of the bacillus mycin D synthesized by the bacillus subtilis. In the present invention, the source of the L-calcium lactate is not particularly limited, and conventional commercially available products can be used.
本发明提供了一种乳酸钙发酵培养基，所述乳酸钙发酵培养基以水为溶剂，包含以下浓度的组分：酵母膏0.5～1.0g/L、L-谷氨酸2.0～5.0g/L、葡萄糖5.0～20.0g/L和乳酸钙3.5～9.5g/L。在本发明乳酸钙发酵培养基中，所述乳酸钙优选包括L-乳酸钙。The invention provides a calcium lactate fermentation medium. The calcium lactate 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 L, glucose 5.0-20.0g/L and calcium lactate 3.5-9.5g/L. In the calcium lactate fermentation medium of the present invention, the calcium lactate preferably includes L-calcium lactate.
在本发明中，所述乳酸钙发酵培养基包括酵母膏0.5～1.0g/L，优选为0.8～1.0g/L，更优选为1.0g/L。本发明酵母膏补充枯草芽孢杆菌杆菌生长繁殖中需要的蛋白质、微量元素等，促进杆菌霉素D合成酶基因表达提高枯草芽孢杆菌合成杆菌霉素D的产量。In the present invention, the calcium lactate fermentation medium includes 0.5-1.0 g/L of yeast extract, preferably 0.8-1.0 g/L, more preferably 1.0 g/L. The yeast paste of the present invention supplements the protein, trace elements and the like needed in the growth and reproduction of the bacillus subtilis bacillus, promotes the expression of the bacillus mycin D synthetase gene and improves the output of the bacillus mycin D synthesized by the bacillus subtilis.
在本发明中，所述乳酸钙发酵培养基包括葡萄糖5.0～20.0g/L，优选为15.0～20.0g/L，更优选为20.0g/L。本发明葡萄糖可以促进促进杆菌霉素D合成酶基因表达提高枯草芽孢杆菌合成杆菌霉素D的产量。In the present invention, the calcium lactate fermentation medium includes 5.0-20.0 g/L of glucose, preferably 15.0-20.0 g/L, more preferably 20.0 g/L. The glucose of the present invention can promote the expression of the bacillus mycin D synthetase gene and increase the yield of the bacillus mycin D synthesized by the bacillus subtilis.
在本发明中，所述乳酸钙发酵培养基包括L-谷氨酸2.0～5.0g/L，优选为4.0～5.0g/L，更优选为5.0g/L。本发明L-谷氨酸可以促进杆菌霉素D合成酶基因表达提高枯草芽孢杆菌合成杆菌霉素D的产量。In the present invention, the calcium lactate fermentation medium includes 2.0-5.0 g/L of L-glutamic acid, preferably 4.0-5.0 g/L, more preferably 5.0 g/L. The L-glutamic acid of the invention can promote the expression of the bacillus mycin D synthetase gene and increase the yield of the bacillus mycin D synthesized by the bacillus subtilis.
在本发明中，所述乳酸钙发酵培养基包括乳酸钙3.5～9.5g/L，优选为5～8g/L，更优选为6.5g/L。本发明所述乳酸钙能促进杆菌霉素D合成酶基因表达进而有效提高枯草芽孢杆菌合成杆菌霉素D。In the present invention, the calcium lactate fermentation medium contains 3.5-9.5 g/L of calcium lactate, preferably 5-8 g/L, more preferably 6.5 g/L. The calcium lactate of the present invention can promote the expression of the bacillus mycin D synthetase gene to effectively increase the bacillus subtilis synthesis of the bacillus mycin D.
生产杆菌毒素D的基础培养基组分为酵母膏，L-谷氨酸和葡萄糖，本发明以生产杆菌毒素D的基础培养基为基础，通过在基础培养中添加乳酸钙，得到乳酸钙发酵培养基，以乳酸钙发酵培养基为基础，并通过分批补料发酵生产杆菌霉素D，提高杆菌霉素D产量。The basal medium for producing bacillus toxin D consists of yeast extract, L-glutamic acid and glucose. The present invention is based on the basal medium for producing bacillus toxin D, by adding calcium lactate in the basal culture to obtain calcium lactate fermentation culture The base is based on calcium lactate fermentation medium, and bacillus mycin D is produced by fed-batch fermentation to increase the yield of bacillus mycin D.
本发明利用乳酸钙发酵培养基能高效发酵生产杆菌霉素D，成本低廉，操作简单，实用性高。本发明所述乳酸钙发酵培养基的灭菌条件优选为115℃，20min。The invention utilizes the calcium lactate fermentation medium to efficiently ferment and produce the bacillus mycin D, and has low cost, simple operation and high practicability. The sterilization condition of the calcium lactate fermentation medium of the present invention is preferably 115° C. for 20 minutes.
本发明提供了一种提高杆菌霉素D产量的方法，包括以下步骤：采用上述技术方案所述乳酸钙发酵培养基对枯草芽孢杆菌种子液进行发酵培养获得杆菌霉素D；所述发酵培养过程分批补料乳酸钙发酵培养基。The invention provides a method for improving the output of bacillus mycin D, comprising the following steps: adopting the calcium lactate fermentation medium described in the above technical scheme to ferment and cultivate the Bacillus subtilis seed liquid to obtain bacillus mycin D; the fermentation and cultivation process Fed-batch calcium lactate fermentation medium.
本发明优选直接应用斜面保藏的枯草芽孢杆菌制备枯草芽孢杆菌种子液。The present invention preferably directly uses the Bacillus subtilis preserved on the slant to prepare the Bacillus subtilis seed liquid.
本发明所述斜面保藏时应用的培养基组分优选包括3.0g/L牛肉浸膏，10g/L胰蛋白胨，5g/L氯化钠，15g/L琼脂和水1L。The culture medium components used during the slant preservation of the present invention 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.
本发明优选利用灭菌的竹签挑取斜面保藏的枯草芽孢杆菌接种于种子培养基中进行种子培养获得枯草芽孢杆菌种子液。在本发明中，所述种子培养优选为恒温培养，恒温培养温度优选为33～37℃，进一步优选为35～37℃，更优选为37℃；本发明优选培养至枯草芽孢杆菌种子液OD600为0.8～1.0，更优选为0.9即可。在本发明中，所述种子培养优选为液体培养，所述种子培养基的组成优选包括3.0g/L牛肉浸膏、10g/L胰蛋白胨、5g/L氯化钠和水1L。In the present invention, preferably, a sterilized bamboo stick is used to pick up the preserved Bacillus subtilis on a slant and inoculate it into a seed medium for seed cultivation to obtain a Bacillus subtilis seed liquid. In the present invention, the seed cultivation is preferably constant temperature cultivation, and the constant temperature cultivation temperature is preferably 33 to 37°C, more preferably 35 to 37°C, more preferably 37°C; in the present invention, it is preferred to cultivate until the OD 600 of the Bacillus subtilis seed liquid It may be 0.8 to 1.0, more preferably 0.9. In the present invention, the seed culture is preferably liquid culture, and the composition of 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.
本发明对所述固体培养基和种子培养基的灭菌条件无特殊限定，采用常规的灭菌条件即可。In the present invention, there is no special limitation on the sterilization conditions of the solid medium and the seed medium, and conventional sterilization conditions can be adopted.
获得枯草芽孢杆菌种子液后，本发明将所述枯草芽孢杆菌种子液接种于乳酸钙发酵培养基进行发酵培养获得发酵液。在本发明中，所述接种时枯草芽孢杆菌种子液的体积优选为乳酸钙发酵培养基体积的4～6％，进一步优选为4.5～6％，更优选为5％。After the Bacillus subtilis seed liquid is obtained, the present invention inoculates the Bacillus subtilis seed liquid into a calcium lactate fermentation medium for fermentation and cultivation to obtain a fermentation liquid. In the present invention, the volume of the Bacillus subtilis seed solution at the time of inoculation is preferably 4-6% of the volume of the calcium lactate fermentation medium, more preferably 4.5-6%, and more preferably 5%.
本发明所述乳酸钙发酵培养基在上文已经论述，在此不再赘述。The calcium lactate fermentation medium of the present invention has been discussed above and will not be repeated here.
在本发明中，所述发酵培养的温度优选为30～37℃，进一步优选为32～35℃，更优选为33℃。本发明所述发酵培养的转速优选为150～200r/min，进一步优选为170～190r/min，更优选为180r/min。In the present invention, the temperature of the fermentation culture is preferably 30-37°C, more preferably 32-35°C, more preferably 33°C. The rotational speed of the fermentation culture in the present invention is preferably 150-200 r/min, more preferably 170-190 r/min, more preferably 180 r/min.
在本发明中，所述发酵培养优选为补料分批发酵培养，本发明所述发酵培养过程分批补料乳酸钙发酵培养基。在本发明中，所述发酵培养的时间优选为108～192h，进一步优选为120～170h，更优选为162h。本发明所述发酵培养时间优选包括补加乳酸钙培养基之前的发酵培养时间和补加乳酸钙培养基之后的发酵培养总时间，即发酵培养的总时间；补加乳酸钙培养基之后的发酵培养时间包括第一次补加乳酸钙培养基之后的培养时间、第二次补加乳酸钙培养基之后的培养时间和第三次补加乳酸钙培养基之后的培养时间。本发明所述补加乳酸钙培养基之前的发酵培养时间优选为48～84h，进一步优选为56～72h，更优选为66h。In the present invention, the fermentation culture is preferably a fed-batch fermentation culture, and the fermentation culture process of the invention is a fed-batch calcium lactate fermentation medium. In the present invention, the fermentation culture time is preferably 108-192 h, more preferably 120-170 h, and more preferably 162 h. The fermentation culture time of the present invention preferably comprises the fermentation culture time before adding calcium lactate medium and the total time of fermentation culture after adding calcium lactate medium, i.e. the total time of fermentation culture; The culture time includes the culture time after the calcium lactate medium is supplemented for the first time, the culture time after the calcium lactate medium is supplemented for the second time and the culture time after the calcium lactate medium is supplemented for the third time. The fermentation culture time before adding the calcium lactate medium in the present invention is preferably 48-84 hours, more preferably 56-72 hours, more preferably 66 hours.
本发明所述发酵培养过程中分批补料乳酸钙发酵培养基的次数优选3次。The frequency of feeding calcium lactate fermentation medium in batches in the fermentation culture process of the present invention is preferably 3 times.
本发明所述补料分批发酵的条件优选包括：所述枯草芽孢杆菌种子液接种于乳酸钙发酵培养基培养48～84h后补加新的乳酸钙发酵培养基，随后每隔15～21h补加新的乳酸钙发酵培养基，最后一次补料发酵后获得发酵液。在本发明中，每次补加新的乳酸钙发酵培养基的时间间隔优选为15～21h，进一步优选为16～20h，更优选为18h。The conditions for fed-batch fermentation of the present invention preferably include: the Bacillus subtilis seed liquid is inoculated in a calcium lactate fermentation medium for 48 to 84 hours, and then a new calcium lactate fermentation medium is added, and then added every 15 to 21 hours. Add new calcium lactate fermentation medium, and obtain the fermentation broth after the last feed-fed fermentation. In the present invention, the time interval for adding new calcium lactate fermentation medium each time is preferably 15-21 h, more preferably 16-20 h, and more preferably 18 h.
在本发明中，每次补加的新的乳酸钙发酵培养基的体积优选为初始的乳酸钙发酵培养基体积的30％～55％，进一步优选为38％～53％，更优选为50％。本发明补加的新的乳酸钙发酵培养基及补加的新的乳酸钙发酵培养基的体积参数的选择可以提高枯草芽孢杆菌发酵生产杆菌霉素D的产量。In the present invention, the volume of the new calcium lactate fermentation medium added each time is preferably 30% to 55% of the initial calcium lactate fermentation medium volume, more preferably 38% to 53%, more preferably 50% . The new added calcium lactate fermentation medium and the selection of the volume parameter of the added new calcium lactate fermentation medium can increase the yield of bacillus subtilis fermented to produce bacillus mycin D.
在本发明中，当所述补料的次数为3次时，所述补料分批发酵的条件优选为：将枯草芽孢杆菌种子液接入初始乳酸钙发酵培养基中进行发酵培养，记为第1次发酵培养，向第1次发酵培养液中补加新鲜的乳酸钙发酵培养基，进行发酵培养，记为第2次发酵培养，向第2次发酵培养液内补加新鲜的发乳酸钙酵培养基，进行发酵培养，记为第3次发酵，向第3次发酵培养液内添加新鲜乳酸钙发酵培养基，进行发酵培养获得枯草芽孢杆菌发酵液。在本发明中，补料次数为3次时，枯草芽孢杆菌发酵生产的杆菌霉素D的效果较好，总杆菌霉素D的含量最高为1.01±0.02g/L，粗肽中总杆菌霉素D含量为38.06±0.72mg/g。In the present invention, when the number of times of the feeding is 3 times, the condition of the batch fermentation of the feeding is preferably: the Bacillus subtilis seed liquid is inserted into the initial calcium lactate fermentation medium for fermentation and cultivation, denoted as For the 1st fermentation culture, add fresh calcium lactate fermentation medium to the 1st fermentation culture liquid, carry out fermentation culture, record it as the 2nd fermentation culture, add fresh fermented lactic acid in the 2nd fermentation culture liquid Calcium fermented culture medium, carry out fermentation culture, record as the 3rd fermentation, add fresh calcium lactate fermentation medium in the 3rd fermentation culture liquid, carry out fermentation culture to obtain Bacillus subtilis fermentation liquid. In the present invention, when the feeding frequency is 3 times, the effect of the bacillus mycin D produced by Bacillus subtilis fermentation is better, the content of the total bacillus mycin D is the highest at 1.01±0.02g/L, and the total bacillus mycin D in the crude peptide The content of element D is 38.06±0.72mg/g.
获得枯草芽孢杆菌发酵液后，本发明优选对枯草芽孢杆菌发酵液进行离心、酸沉和萃取获得杆菌霉素D。本发明所述离心的转速优选为5800～6200r/min，更优选为6000r/min，离心时间优选为13～17min，更优选为15min。After obtaining the Bacillus subtilis fermentation broth, the present invention preferably performs centrifugation, acid precipitation and extraction on the Bacillus subtilis fermentation broth to obtain the bacillus mycin D. The rotational speed of the centrifuge in the present invention is preferably 5800-6200r/min, more preferably 6000r/min, and the centrifugation time is preferably 13-17min, more preferably 15min.
本发明将枯草芽孢杆菌发酵液离心，获得上清液。本发明优选对上清液进行酸沉浸提杆菌霉素D粗肽。在本发明中，所述酸沉的方式优选包括：利用HCl将上清液的pH值调至2.0以下，室温静置4h，室温静置4h后酸沉结束获得酸沉后的上清液；随后对酸沉后的上清液进行第二次离心，第二次离心的转速优选为5800～6200r/min，更优选为6000r/min，第二次离心时间优选为8～12min，更优选为10min；第二次离心后获得酸沉后的上清液粗肽沉淀，即为杆菌霉素D粗肽沉淀。The invention centrifuges the Bacillus subtilis fermented liquid to obtain supernatant liquid. In the present invention, the supernatant is preferably acid-immersed into the crude peptide of leviomycin D. In the present invention, the method of acid precipitation preferably includes: using HCl to adjust the pH value of the supernatant to below 2.0, standing at room temperature for 4 hours, and after standing at room temperature for 4 hours, the acid precipitation is completed to obtain the supernatant after acid precipitation; Then the supernatant after acid precipitation is centrifuged for the second time, the rotating speed of the second centrifugation is preferably 5800～6200r/min, more preferably 6000r/min, the second centrifugation time is preferably 8～12min, more preferably 10 min; after the second centrifugation, the crude peptide precipitate in the supernatant after acid precipitation was obtained, which was the crude peptide precipitate of bacillus mycin D.
酸沉后，本发明优选对获得的杆菌霉素D粗肽沉淀进行萃取和第三次离心，获得含有杆菌霉素D的上清液，本发明优选利用甲醇进行萃取，本发明所述甲醇优选为色谱纯甲醇。本发明所述萃取的时间为68～82min，进一步优选为70～75min，更优选为72min。本发明所述粗肽沉淀与甲醇的质量体积比优选为0.1～0.5g：0.5～2.0mL，进一步优选为0.3～0.5g：1.0～1.8mL，更优选为0.4g：1.5mL。本发明所述第三次离心的转速优选为9000～11000r/min，更优选为10000r/min，离心时间优选为8～12min，更优选为10min。After acid precipitation, the present invention preferably extracts and centrifuges the obtained bacillus mycin D crude peptide precipitate to obtain a supernatant containing bacillus mycin D. The present invention preferably utilizes methanol for extraction, and the methanol described in the present invention is preferably Chromatographically pure methanol. The extraction time of the present invention is 68-82 minutes, more preferably 70-75 minutes, more preferably 72 minutes. The mass volume ratio of the crude peptide precipitation to methanol in the present invention is preferably 0.1-0.5g: 0.5-2.0mL, more preferably 0.3-0.5g: 1.0-1.8mL, more preferably 0.4g: 1.5mL. The rotational speed of the third centrifugation in the present invention is preferably 9000-11000 r/min, more preferably 10000 r/min, and the centrifugation time is preferably 8-12 min, more preferably 10 min.
枯草芽孢杆菌发酵液进行三次离心、酸沉和萃取获得含有杆菌霉素D的上清液，本发明优选采用高效液相色谱(HPLC)法对含有杆菌霉素D上清液中的杆菌霉素D及其同系物的含量进行测定。本发明所述杆菌霉素D包括杆菌霉素DC14同系物、杆菌霉素DC15同系物、杆菌霉素DC16同系物和杆菌霉素DC17同系物中的一种或多种。在本发明中，所述杆菌霉素DC14同系物为十四碳β-氨基脂肪酸环七肽(C14-β-NH2COOH-Asn-Tyr-Asn-ProGlu-Ser-Thr)；所述杆菌霉素DC15同系物为十五碳β-氨基脂肪酸环七肽(C15-β-NH2COOH-Asn-Tyr-Asn-ProGlu-Ser-Thr)；所述杆菌霉素DC16同系物为十六碳β-氨基脂肪酸环七肽(C16-β-NH2COOH-Asn-Tyr-Asn-ProGlu-Ser-Thr)；所述杆菌霉素DC17同系物为十七碳β-氨基脂肪酸环七肽(C17-β-NH2COOH-Asn-Tyr-Asn-ProGlu-Ser-Thr)。利用本发明的技术方案枯草芽孢杆菌发酵生产杆菌霉素D的总产量、杆菌霉素DC14同系物产量、杆菌霉素DC15同系物产量、杆菌霉素DC16同系物产量和杆菌霉素DC17同系物产量均提高。Bacillus subtilis fermentation broth is centrifuged three times, acid precipitation and extraction to obtain the supernatant containing bacillus mycin D, the present invention preferably adopts high performance liquid chromatography (HPLC) method to contain the bacillus mycin in the bacillus D supernatant. The content of D and its homologues was determined. The bacillus mycin D in the present invention includes one or more of bacillus mycin DC14 homologs, bacillus mycin DC15 homologs, bacillus mycin DC16 homologs and bacillus mycin DC17 homologs. 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 bacitracin DC17 homologue is a seventeen carbon β-amino fatty acid cyclic heptapeptide (C 17 -β-NH 2 COOH-Asn-Tyr-Asn-ProGlu-Ser-Thr). Utilize technical scheme Bacillus subtilis fermentation of the present invention to produce the total output of bacillus mycin D, the output of bacillus mycin DC14 homologue, the output of bacillus mycin DC15 homologue, the output of bacillus mycin DC16 homologue and the output of bacillus mycin DC17 homologue Both increased.
本发明结合乳酸钙能促进杆菌霉素D合成酶基因表达进而有效提高枯草芽孢杆菌合成杆菌霉素D的特点，创制用于生产杆菌霉素D的乳酸钙发酵培养基，并采用补料分批发酵生产杆菌霉素D，显著提高了杆菌霉素D的产量，这为实现杆菌霉素D大规模的工业化应用提供了科学合理的技术基础。Combining with calcium lactate, the present invention can promote the expression of bacillus mycin D synthetase gene and then effectively improve the characteristics of bacillus subtilis to synthesize bacillus mycin D, create a calcium lactate fermentation medium for producing bacillus mycin D, and adopt fed batches Fermentative production of bacillus mycin D significantly increases the yield of bacillus mycin D, which provides a scientific and reasonable technical basis for the large-scale industrial application of bacillus mycin D.
为了进一步说明本发明，下面结合实施例对本发明提供的技术方案进行详细地描述，但不能将它们理解为对本发明保护范围的限定。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.
实施例1Example 1
(1)培养基(1) culture medium
乳酸钙发酵培养基组分：酵母膏1.0g/L、L-谷氨酸5.0g/L、葡萄糖20.0g/L、L-乳酸钙6.5g/L和1L水。乳酸钙发酵培养基115℃灭菌20min，冷却至室温后应用。Calcium lactate fermentation medium components: yeast extract 1.0g/L, L-glutamic acid 5.0g/L, glucose 20.0g/L, L-calcium lactate 6.5g/L and 1L water. Calcium lactate fermentation medium was sterilized at 115°C for 20 minutes, cooled to room temperature and used.
斜面保藏培养基组分：牛肉浸膏3.0g/L、胰蛋白胨10g/L、氯化钠5g/L和琼脂15g/L。Components of the slant preservation medium: beef extract 3.0g/L, tryptone 10g/L, sodium chloride 5g/L and agar 15g/L.
种子培养基组分：牛肉浸膏3.0g/L，胰蛋白胨10g/L和氯化钠5g/L。Seed medium components: beef extract 3.0g/L, tryptone 10g/L and sodium chloride 5g/L.
(2)补料分批发酵培养(2) Fed-batch fermentation culture
用灭菌竹签挑取斜面保藏的枯草芽孢杆菌菌落接入种子培养基，于37℃摇瓶培养至枯草芽孢杆菌种子液OD600为0.8，获得枯草芽孢杆菌种子液。The colony of Bacillus subtilis preserved on the slant was picked with a sterilized bamboo stick and inserted into the seed medium, and cultured in a shake flask at 37°C until the OD 600 of the Bacillus subtilis seed solution was 0.8 to obtain the Bacillus subtilis seed solution.
按体积比5％的接种量将枯草芽孢杆菌种子液接入体积为300mL的乳酸钙发酵培养基中，接种后在温度为33℃，180r/min的条件下进行发酵培养，记为第1次发酵。Put the Bacillus subtilis seed liquid into the calcium lactate fermentation medium with a volume of 300mL according to the inoculation amount of 5% by volume, and carry out fermentation culture under the conditions of temperature 33°C and 180r/min after inoculation, which is recorded as the first time fermentation.
从开始培养计时：第1次发酵至培养66h时，向第1次发酵液中添加新鲜的乳酸钙发酵培养基150mL，于温度为33℃，180r/min的条件下继续发酵培养，记为第2次发酵培养。第2次发酵培养至84h时，向第2次发酵培养液内添加新鲜的乳酸钙发酵培养基150mL，于温度为33℃，180r/min的条件下继续发酵，记为第3次发酵培养，第3次发酵培养至102h时，向第3次发酵培养液内添加新鲜发酵培养基150mL，于温度为33℃，180r/min的条件下继续发酵至162h获得枯草芽孢杆菌发酵液。即分批补料乳酸钙发酵培养基的次数为3次。补加乳酸钙发酵培养基前的发酵时间为66h，补加第1次培养基之后的发酵时间为18h，补加第二次发酵培养基之后发酵18h补加第3次发酵培养基，补加第3次发酵培养基之后继续发酵60h，获得枯草芽孢杆菌发酵液。总发酵时间为162h。Timing from the start of cultivation: From the first fermentation to 66 hours of cultivation, add 150mL of fresh calcium lactate fermentation medium to the first fermentation liquid, and continue the fermentation and cultivation at a temperature of 33°C and 180r/min, which is recorded as the second 2 fermentation cultures. When the second fermentation culture reaches 84 hours, add 150 mL of fresh calcium lactate fermentation medium to the second fermentation culture medium, and continue fermentation at a temperature of 33 ° C and 180 r/min, which is recorded as the third fermentation culture. When the third fermentation was cultivated to 102h, 150mL of fresh fermentation medium was added to the third fermentation culture medium, and the temperature was 33°C, and the fermentation was continued until 162h under the conditions of 180r/min to obtain a Bacillus subtilis fermentation broth. That is, the number of batch-fed calcium lactate fermentation medium was 3 times. The fermentation time before adding the calcium lactate fermentation medium was 66h, the fermentation time after adding the first medium was 18h, and after adding the second fermentation medium, fermented for 18h and added the third fermentation medium. Continue to ferment for 60 h after the third fermentation medium to obtain Bacillus subtilis fermentation broth. The total fermentation time is 162h.
取40.0mL枯草芽孢杆菌发酵液于6000r/min离心15min，弃菌体，取枯草芽孢杆菌发酵液的上清液，用HCl将上清液pH调至2.0以下浸提粗肽，室温静置4h，随后在6000r/min的条件下离心10min，得到杆菌霉素D粗肽沉淀。Take 40.0mL of Bacillus subtilis fermentation broth and centrifuge at 6000r/min for 15min, discard the bacteria, take the supernatant of Bacillus subtilis fermentation broth, adjust the pH of the supernatant to below 2.0 with HCl to extract the crude peptide, and let stand at room temperature for 4h , followed by centrifugation at 6000 r/min for 10 min to obtain a precipitate of crude bacitracin D peptide.
在0.4g杆菌霉素D粗肽沉淀中加入1.5mL甲醇萃取72min，将所得粗肽萃取液在10000r/min的条件下离心10min后取上清液即为含有杆菌霉素D的溶液。利于甲醇萃取的优势在于有效萃取杆菌霉素D。采用高效液相色谱(HPLC)法对含有杆菌霉素D的溶液中的杆菌霉素D及其同系物的含量进行测定。Add 1.5 mL of methanol to the 0.4 g bacitracin D crude peptide precipitate and extract for 72 min, centrifuge the obtained crude peptide extract at 10,000 r/min for 10 min, and then take the supernatant, which is the solution containing bacitracin D. The advantage of favoring methanol extraction is the efficient extraction of bacitracin D. The content of bacitracin D and its homologues in the solution containing bacitracin D was determined by high performance liquid chromatography (HPLC).
实施例1共5组平行实验。Embodiment 1 A total of 5 groups of parallel experiments.
实施例2Example 2
发酵培养基中添加L-乳酸钙，L-乳酸钙的添加量为3.5g/L，其余条件同实施例1。Add L-calcium lactate to the fermentation medium, the amount of L-calcium lactate added is 3.5g/L, and the rest of the conditions are the same as in Example 1.
实施例3Example 3
发酵培养基中添加L-乳酸钙，L-乳酸钙的添加量为9.5g/L，其余条件同实施例1。Add L-calcium lactate to the fermentation medium, the amount of L-calcium lactate added is 9.5g/L, and the rest of the conditions are the same as in Example 1.
对比例1Comparative example 1
不进行补料，其余条件同实施例1。Feeding is not carried out, and all the other conditions are the same as in Example 1.
对比例2Comparative example 2
发酵培养基中不添加L-乳酸钙，其余条件同实施例1。L-calcium lactate was not added to the fermentation medium, and the rest of the conditions were the same as in Example 1.
对比例3Comparative example 3
发酵培养基中添加氯化钙，其余条件同实施例1。Calcium chloride was added in the fermentation medium, and all the other conditions were the same as in Example 1.
对比例4Comparative example 4
发酵培养基中添加L-乳酸钙，L-乳酸钙的添加量为0.5g/L，其余条件同实施例1。Add L-calcium lactate in the fermentation medium, the addition amount of L-calcium lactate is 0.5g/L, other conditions are the same as embodiment 1.
对比例5Comparative example 5
发酵培养基中添加L-乳酸钙，L-乳酸钙的添加量为12.5g/L，其余条件同实施例1。Add L-calcium lactate to the fermentation medium, the amount of L-calcium lactate added is 12.5g/L, and the rest of the conditions are the same as in Example 1.
对实施例1～3和对比例1～5获得的枯草芽孢杆菌发酵液中的杆菌霉素D及其同系物的含量进行测定，测定方法采用高效液相色谱(HPLC)法。具体HPLC条件和测定方法参照现有技术(Qian Shiquan,Lu Hedong,Meng Panpan,Zhang Chong,Lv Fengxia,BieXiaomei,Lu Zhaoxin,Effect of inulin on efficient production and regulatorybiosynthesis ofbacillomycin D in Bacillus subtilis fmbJ,BioresourceTechnology,2015,179:260-267.)进行。对实施例1～3和对比例1～5的5次平行实验的测定值见表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 measured values of 5 parallel experiments of Examples 1-3 and Comparative Examples 1-5.
表1实施例1～3和对比例1～5获得的枯草芽孢杆菌发酵液中的杆菌霉素D及其同系物的含量(g/L)和粗肽中杆菌霉素D含量(mg/g)Table 1 The content (g/L) of bacitracin D and its homologs in the Bacillus subtilis fermentation broth obtained in Examples 1-3 and Comparative Examples 1-5 and the content (mg/g) of bacitracin D in the crude peptide )
根据表1可知，本发明以乳酸钙发酵培养基为基料进行三次补料发酵，有效提高了枯草芽孢芽孢杆菌的杆菌霉素D产量。According to Table 1, it can be seen that the present invention uses calcium lactate fermentation medium as the base material to carry out three fed-batch fermentations, effectively improving the bacillus mycin D output of Bacillus subtilis.
实施例4杆菌霉素D对黄曲霉的抑制效果The inhibitory effect of embodiment 4 bacitramycin D on Aspergillus flavus
PDA培养基：200g/L马铃薯、20g/L葡萄糖、18g/L琼脂和1L水。PDA medium: 200g/L potato, 20g/L glucose, 18g/L agar and 1L water.
验证实施例1制备得到的杆菌霉素D对黄曲霉(CICC2062)的抑制效果。使用PDA平板于温度为28℃培养黄曲霉4天后，取5mm菌丝块，将菌丝面朝下放置于含有不同浓度杆菌霉素D的PDA平板上于28℃静置培养5天作为试验组，试验组1～7的PDA平板中杆菌霉素D浓度依次为5μg/mL、10μg/mL、20μg/mL、40μg/mL、80μg/mL、160μg/mL、320μg/mL，以不含杆菌霉素D的PDA平板为对照组。The inhibitory effect of the bacitracin D prepared in Example 1 on Aspergillus flavus (CICC2062) was verified. After cultivating Aspergillus flavus on a PDA plate at 28°C for 4 days, take a 5mm piece of mycelium, place the mycelium face down on a PDA plate containing different concentrations of bacinomycin D, and culture it statically at 28°C for 5 days as the test group The concentrations of bacitracin D in the PDA plates of test groups 1 to 7 were 5 μg/mL, 10 μg/mL, 20 μg/mL, 40 μg/mL, 80 μg/mL, 160 μg/mL, and 320 μg/mL. The PDA plate of prime D was used as the control group.
培养5d后，采用十字交叉法测量菌落直径，计算菌丝抑制率。After culturing for 5 days, the colony diameter was measured by the cross method, and the mycelium inhibition rate was calculated.
培养5d后，在试验组1～7和对照组的PDA平板中加入无菌水5.0mL，用无菌水洗下孢子，用血球计数板计数，测定黄曲霉孢子数。结果见表2。After culturing for 5 days, add 5.0 mL of sterile water to the PDA plates of the test groups 1 to 7 and the control group, wash the spores with sterile water, and count them with a hemocytometer to determine the number of Aspergillus flavus spores. The results are shown in Table 2.
黄曲霉菌丝和孢子抑制率计算公式如下：菌丝抑制率(％)＝{(未经杆菌霉素D处理的黄曲霉菌落直径-经杆菌霉素D处理的黄曲霉菌落直径)/(未经杆菌霉素D处理的菌落直径)}×100％；The calculation formula of Aspergillus flavus hyphae and spore inhibition rate is as follows: Mycelia inhibition rate (%)={(the diameter of Aspergillus flavus colony without bacitramycin D treatment-the diameter of Aspergillus flavus colony treated with bacinomycin D)/(untreated Colony diameter treated with bacitracin D)}×100%;
孢子抑制率(％)＝{(未经杆菌霉素D处理的黄曲霉菌落孢子数-经杆菌霉素D处理的黄曲霉菌落孢子数)/(未经杆菌霉素D处理的菌落孢子数)}×100％。Spore inhibition rate (%)={(the number of colony spores of Aspergillus flavus treated without bacitracin D-the number of spores of Aspergillus flavus treated with bacitramycin D)/(the number of spores of colonies of Aspergillus flavus treated without bacinomycin D) } × 100%.
由表2可知，当添加320μg/mL杆菌霉素D时，能完全抑制黄曲霉的生长，可见本发明的技术方案制备得到的杆菌霉素D能够抑制黄曲霉的生长，说明本发明技术方案制备得到的杆菌霉素D的纯度较高。As can be seen from Table 2, when adding 320 μ g/mL bacitramycin D, the growth of Aspergillus flavus can be completely inhibited, it can be seen that the bacinomycin D prepared by the technical scheme of the present invention can inhibit the growth of Aspergillus flavus, which illustrates that the technical scheme of the present invention prepares The obtained bacinomycin D has higher purity.
表2杆菌霉素D对黄曲霉抑制作用Table 2 Inhibitory effect of bacitramycin D on Aspergillus flavus
综上，本发明利用乳酸钙配制发酵培养基对枯草芽孢杆菌进行发酵培养可以提高杆菌霉素D的产量，获得杆菌霉素D对黄曲霉有较好的抑制作用。In summary, the present invention uses calcium lactate to prepare a fermentation medium to ferment and cultivate Bacillus subtilis, which can increase the yield of bacillus mycin D, and the obtained bacillus mycin D has a better inhibitory effect on Aspergillus flavus.
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。Although the foregoing embodiment has described the present invention in detail, it is only a part of the embodiments of the present invention, rather than all embodiments, and 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.
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