CN110016490B - Fermentation medium for producing bacilycin D and method for producing bacilycin D - Google Patents

Fermentation medium for producing bacilycin D and method for producing bacilycin D Download PDF

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CN110016490B
CN110016490B CN201910332986.5A CN201910332986A CN110016490B CN 110016490 B CN110016490 B CN 110016490B CN 201910332986 A CN201910332986 A CN 201910332986A CN 110016490 B CN110016490 B CN 110016490B
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enzymolysis
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钱时权
张璐瑶
任清逸
胡卫成
沈媛媛
姚斐
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Huaiyin Normal University
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Abstract

The invention provides a fermentation medium for producing bacillomycin D and a method for producing the bacillomycin D, and belongs to the biological fermentation technologyThe field of the technology. The preparation method of the fermentation medium comprises the following steps: (1) drying and crushing corn straws to obtain corn straw powder; (2) mixing the corn straw powder, cellulase and sterile water, and carrying out enzymolysis for 60-100 h at 40-60 ℃ to obtain an enzymolysis liquid; (3) centrifuging the enzymolysis liquid to obtain enzymolysis supernatant; (4) mixing the enzymatic supernatant, yeast extract, sodium L-glutamate and KH2PO4Mixing to obtain a bacillomycin D fermentation medium; the enzymolysis supernatant, yeast extract, L-sodium glutamate and KH2PO4The mixing ratio of (A) to (B) is as follows: 80-120 mL: 1-3 g: 5-15 g: 0.5 to 1.5 g. The fermentation medium can be used for producing the bacillomycin D, and the production cost is low; and the additional value of the corn straw can be improved.

Description

Fermentation medium for producing bacilycin D and method for producing bacilycin D
Technical Field
The invention belongs to the technical field of biological fermentation, and particularly relates to a fermentation medium for producing bacillomycin D and a method for producing the bacillomycin D.
Background
In recent years, environmental problems caused by unreasonable treatment modes of corn straws are worsened, and how to effectively utilize the corn straws has become the focus of domestic and foreign research.
The bacillomycin D (bacillus mycin D) is a secondary metabolite generated by bacillus subtilis, belongs to a cyclic lipopeptide compound, has physiological activities of inhibiting the growth of pathogenic bacteria, resisting tumors and the like, is expected to be developed into a natural food preservative in the future, and has wide application prospect. Currently, the most common method for producing the bacillomycin D by utilizing the fermentation of the bacillus subtilis adopts a Landy culture medium as a fermentation culture medium. Landy medium is complex in composition and expensive. The ability of producing the bacitracin D by fermentation of the Landy culture medium is still limited, and the extraction process of the bacitracin D is complex, so that the large-scale industrial application of the bacitracin D is difficult to realize. Therefore, the method for creating the simple bacillomycin D fermentation medium reduces the cost, simplifies the preparation process of the bacillomycin D, and is very important for green production of the bacillomycin D and effective improvement of the added value of the corn straw.
Disclosure of Invention
In view of the above, the present invention aims to provide a fermentation medium for producing bacillomycin D and a method for producing bacillomycin D; the production cost of the bacillomycin D is low, and the added value of the corn straw can be improved.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a fermentation medium for producing bacillomycin D, and a preparation method of the fermentation medium comprises the following steps:
(1) drying and crushing corn straws to obtain corn straw powder;
(2) mixing the corn straw powder, cellulase and sterile water, and carrying out enzymolysis for 60-100 h at 40-60 ℃ to obtain an enzymolysis liquid;
(3) centrifuging the enzymolysis liquid to obtain enzymolysis supernatant;
(4) mixing the enzymatic supernatant, yeast extract, sodium L-glutamate and KH2PO4Mixing to obtain a fermentation culture medium for producing the bacillomycin D;
the enzymolysis supernatant fluid, the yeast extract, the L-sodium glutamate and the KH in the step (4)2PO4The mixing ratio of (A) to (B) is 80-120 mL: 1-3 g: 5-15 g: 0.5 to 1.5 g.
Preferably, the drying temperature in the step (1) is 60-70 ℃, and the crushing degree is 50-80 meshes.
Preferably, the ratio of the corn straw powder to the cellulase in the step (2) is 1 g: 500-600U; the feed-liquid ratio of the corn straw powder during enzymolysis is 1 g: 16-26 mL.
Preferably, the rotation speed of the centrifugation in the step (3) is 6000 to 10000r/min, and the centrifugation time is 5 to 15 min.
Preferably, after the corn straw powder is obtained in the step (1), first sterilization is further included; the sterilization temperature of the first sterilization is 118-124 ℃, and the sterilization time is 20-40 min; after the mixing in the step (4), performing second sterilization; the sterilization temperature of the second sterilization is 111-119 ℃, and the sterilization time is 15-25 min.
The invention also provides a method for producing the bacillomycin D, which comprises the following steps:
inoculating a bacillus subtilis propagation solution into the fermentation culture medium, and performing fermentation culture at 28-38 ℃ for 72-168 hours to obtain a fermentation liquid;
II, centrifuging the fermentation liquor to obtain fermentation supernatant;
and III, adjusting the pH value of the fermentation supernatant to be less than or equal to 3, standing for 2-8 h, centrifuging, collecting precipitate, and drying to obtain the bacitracin D dry crude peptide solid.
Preferably, OD of the Bacillus subtilis propagation solution in the step I6000.8 to 1.0; the inoculation amount is 4-8%.
Preferably, the rotating speed of the centrifugation in the step II is 4000-8000 r/min, and the centrifugation time is 10-20 min.
Preferably, the rotating speed of the centrifugation in the step III is 4000-8000 r/min, and the centrifugation time is 5-15 min.
Preferably, in the step III, 2-3 mol/L HCl solution is used for adjusting the pH value; and III, drying at the temperature of 50-70 ℃.
Has the advantages that:
the invention provides a fermentation medium for producing bacillomycin D and a method for producing the bacillomycin D. The preparation method of the fermentation medium comprises the following steps: (1) drying and crushing corn straws to obtain corn straw powder; (2) mixing the corn straw powder, cellulase and sterile water, and carrying out enzymolysis for 60-100 h at 40-60 ℃ to obtain an enzymolysis liquid; (3) centrifuging the enzymolysis liquid to obtain enzymolysis supernatant; (4) mixing the enzymatic supernatant, yeast extract, sodium L-glutamate and KH2PO4Mixing to obtain a fermentation culture medium for producing the bacillomycin D; the enzymolysis supernatant, yeast extract, L-sodium glutamate and KH2PO4The mixing ratio of (A) to (B) is as follows: 80-120 mL: 1-3 g: 5-15 g: 0.5 to 1.5 g. The invention utilizes the cellulase to carry out enzymolysis on the corn straws, and then prepares the fermentation medium by the corn straw enzymolysis liquid, realizes the effective fermentation production of the bacitracin D, and advances the preparation process of the bacitracin DThe simplification is performed. The invention also provides a method for producing the bacillomycin D, and the method utilizes the fermentation culture medium to produce the bacillomycin D, so that the production cost of the bacillomycin D is reduced, and the additional value of the corn straw is improved.
Detailed Description
The invention provides a fermentation medium for producing bacillomycin D, and a preparation method of the fermentation medium comprises the following steps:
(1) drying and crushing corn straws to obtain corn straw powder;
(2) mixing the corn straw powder, cellulase and sterile water, and carrying out enzymolysis for 60-100 h at 40-60 ℃ to obtain an enzymolysis liquid;
(3) centrifuging the enzymolysis liquid to obtain enzymolysis supernatant;
(4) mixing the enzymatic supernatant, yeast extract, sodium L-glutamate and KH2PO4Mixing to obtain a fermentation culture medium for producing the bacillomycin D;
the enzymolysis supernatant fluid, the yeast extract, the L-sodium glutamate and the KH in the step (4)2PO4The mixing ratio of (A) to (B) is 80-120 mL: 1-3 g: 5-15 g: 0.5 to 1.5 g.
The invention firstly dries and then crushes the corn straws to obtain the corn straw powder. In the invention, the corn stalks are preferably mature, free of plant diseases and insect pests, dust-removed and cleaned corn stalks. Before the corn straws are dried, the corn straws are preferably cut into small sections of 1-2 cm by using scissors. The drying preferably uses an electric hot air drying oven. The drying temperature is preferably 60-70 ℃, and more preferably 65 ℃. In the present invention, the pulverization preferably uses a solid-state pulverizer. The grinding degree is preferably 50-80 meshes, and more preferably 60 meshes. After the corn straw powder is obtained, the first sterilization is preferably carried out in the invention. The sterilization temperature of the first sterilization is preferably 118-124 ℃, and more preferably 121 ℃; the sterilization time is preferably 20-40 min, and more preferably 30 min.
After the corn straw powder is obtained, the corn straw powder, cellulase and sterile water are mixed, and the corn straw powder is subjected to enzymolysis by the cellulase to obtain an enzymolysis liquid. In the present invention, the ratio of the corn stalk powder to the cellulase is preferably 1 g: 500-600U, more preferably 1 g: 550U; the feed-liquid ratio of the corn straw powder during enzymolysis is 1 g: 16-26 mL; more preferably 1 g: 18-24 mL, more preferably 1 g: 21 mL. The enzymolysis temperature is preferably 40-60 ℃, and more preferably 50 ℃. The pH of the enzymatic hydrolysis is preferably 5.0. The enzymolysis time is preferably 60-100 h, more preferably 70-90 h, and more preferably 79.8 h.
After enzymolysis, the enzymolysis liquid is centrifuged to obtain enzymolysis supernatant. In the step, the rotation speed of the centrifugation is preferably 6000 to 10000r/min, and more preferably 8000 r/min. The time for centrifugation is preferably 5-15 min, and more preferably 10 min.
After obtaining the enzymolysis supernatant, the invention mixes the enzymolysis supernatant, the yeast extract, the L-sodium glutamate and the KH2PO4And mixing to obtain the bacillomycin D fermentation medium. In the present invention, the enzymatic supernatant, yeast extract, sodium L-glutamate and KH2PO4The mixing ratio of (A) to (B) is 80-120 mL: 1-3 g: 5-15 g: 0.5-1.5 g, preferably 90-110 mL: 1.5-2.5 g: 8-12 g: 0.8-1.2 g, more preferably 100 mL: 2 g: 10 g: 1.0 g. In the invention, after the mixing, a second sterilization is preferably carried out, wherein the sterilization temperature of the second sterilization is preferably 111-119 ℃, and more preferably 115 ℃; the sterilization time is preferably 15-25 min, and more preferably 15 min.
The invention also provides a method for producing the bacillomycin D, which comprises the following steps:
inoculating a bacillus subtilis propagation solution into the fermentation culture medium, and performing fermentation culture at 28-38 ℃ for 72-168 hours to obtain a fermentation liquid;
II, centrifuging the fermentation liquor to obtain fermentation supernatant;
and III, adjusting the pH value of the fermentation supernatant to be less than or equal to 3, standing for 2-8 h, centrifuging and drying to obtain the bacitracin D dry crude peptide solid.
Inoculating the bacillus subtilis propagation solution into the fermentation culture medium for fermentation culture to obtain fermentation liquor. The present invention is not particularly limited with respect to the particular species of Bacillus subtilis, and any Bacillus subtilis used in the art for producing baccatin D may be used. In order to illustrate the function of the fermentation medium of the present invention, in a more specific embodiment of the present invention, the Bacillus subtilis propagation medium is preferably obtained by activating and propagating Bacillus subtilis HSBS-177 stored in a laboratory. OD of the bacillus subtilis propagation liquid600Preferably 0.8 to 1.0, more preferably 0.9; the inoculation amount is preferably 4-8%, and more preferably 6%. In the invention, the temperature of the fermentation culture is 28-38 ℃, preferably 30-35 ℃, and more preferably 33 ℃. The oscillation frequency of the fermentation culture is preferably 160-200 r/min, and more preferably 180 r/min. The fermentation culture time is 72-168 h, more preferably 96-144 h, and more preferably 120 h.
After the fermentation liquor is obtained, the fermentation liquor is centrifuged to obtain fermentation supernatant. In the step, the rotating speed of the centrifugation is preferably 4000-8000 r/min, and more preferably 6000 r/min; the time for centrifugation is preferably 10-20 min, and more preferably 15 min.
After the fermentation supernatant is obtained, the pH value of the fermentation supernatant is preferably adjusted by using 2-3 mol/L HCl solution, more preferably 2.5mol/L HCl solution, and the pH value is less than or equal to 3, preferably less than or equal to 2. And standing at room temperature after the pH value is adjusted, wherein the standing time is preferably 2-8 h, and more preferably 4 h. And standing, centrifuging, collecting precipitate, and drying to obtain dry crude peptide solid of the bacillomycin D. In the step, the rotating speed of the centrifugation is preferably 4000-8000 r/min, and more preferably 6000 r/min; the time for centrifugation is preferably 5-15 min, and more preferably 10 min. The drying temperature is preferably 50-70 ℃, and more preferably 60 ℃.
According to the invention, the corn straws are subjected to enzymolysis by adopting cellulase, the prepared bacitracin D fermentation medium is simple in formula, and can effectively produce bacitracin D by fermentation, so that the production cost of bacitracin D is reduced. The simple fermentation medium created by the invention is utilized to produce the bacillomycin D by fermentation, the cost is low, and the preparation method of the bacillomycin D is simple to operate and practical.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Selecting mature corn straws without diseases and insect pests, removing dust, cleaning, cutting to small sections of 1-2 cm by using scissors, putting into an electric heating blast drying oven, controlling the temperature at 65 ℃, and drying to constant weight.
Taking out the dried small sections of the corn straws, placing the small sections at room temperature, properly cooling (placing for 20 minutes), crushing by using a solid high-speed crusher, and sieving by using a 60-mesh sieve to prepare powder for later use.
Accurately weighing 2.00g of corn straw powder, placing in a high-pressure steam sterilization pot, sterilizing at 121 ℃ for 30min, taking out, and cooling to room temperature. Adding cellulase (10000U/g) and sterile water into the sterilized corn straw powder, fully shaking up, and carrying out full enzymolysis at the temperature of 50 ℃, wherein the enzymolysis conditions are as follows: the addition amount of cellulase is 0.11g, the enzymolysis time is 79.8h, and the liquid-material ratio is 21: 1 (volume of enzymolysis liquid: mass of corn stalk powder, mL/g).
After enzymolysis, taking out the enzymolysis liquid, and centrifuging at 8000r/min for 10min to obtain enzymolysis supernatant. Adding yeast extract 2.0g, L-sodium glutamate (monosodium glutamate) 10g and KH into 100mL of enzymolysis supernatant2PO41.0g, sterilizing at 115 deg.C for 20min, cooling to room temperature, and making into bacillomycin D fermentation medium.
Example 2
Inoculating Bacillus subtilis HSBS-177 stored in laboratory onto slant culture medium, culturing at 37 deg.C for 24 hr, selecting single colony, inoculating into seed culture medium, and shake-culturing at 37 deg.C to OD600The fermentation medium prepared in example 1 was inoculated at an inoculum size of 6% to 0.8-1.0, and fermentation-cultured at 33 ℃ and 180r/min for 120 hours.
Centrifuging 10.0mL fermentation liquid at 6000r/min for 15min, discarding thallus, collecting supernatant, adjusting pH of the supernatant to below 2.0 with HCl, standing at room temperature for 4h, centrifuging at 6000r/min for 10min, collecting precipitate, oven drying at 60 deg.C to constant weight, and making into bacitracin D dry crude peptide solid.
Taking a small amount of bacilycin D dry crude peptide solid, grinding into powder, adding 1.0mL of methanol for extraction, centrifuging at 10000r/min for 10min, taking supernate, and measuring the content of the bacilycin D by adopting a High Performance Liquid Chromatography (HPLC) method, wherein the specific HPLC conditions and measuring method refer to Qian and other documents. The content of bacitracin D (bacitracin D, mg; mass of bacitracin D crude peptide solid, g.) in the bacitracin D crude peptide solid was measured to be 9.42 mg/g. The experiment was repeated 5 times with a relative error of 1.82%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (5)

1. A method of producing bacitracin D, comprising the steps of:
inoculating the bacillus subtilis propagation solution into a fermentation culture medium, and performing fermentation culture at 28-38 ℃ for 72-168 hours to obtain a fermentation liquid;
II, centrifuging the fermentation liquor to obtain fermentation supernatant;
III, adjusting the pH value of the fermentation supernatant to be less than or equal to 3, standing for 2-8 h, centrifuging, collecting precipitate, and drying to obtain a bacitracin D dry crude peptide solid;
the preparation method of the fermentation medium comprises the following steps:
(1) drying and crushing corn straws to obtain corn straw powder;
(2) mixing the corn straw powder, cellulase and sterile water, and carrying out enzymolysis for 60-100 h at 40-60 ℃ to obtain an enzymolysis liquid;
(3) centrifuging the enzymolysis liquid to obtain enzymolysis supernatant;
(4) mixing the enzymatic supernatant, yeast extract, sodium L-glutamate and KH2PO4Mixing to obtain a fermentation culture medium for producing the bacillomycin D;
the enzymolysis supernatant fluid, the yeast extract, the L-sodium glutamate and the KH in the step (4)2PO4The mixing ratio of (A) to (B) is 80-120 mL: 1-3 g: 5-15 g: 0.5-1.5 g;
the drying temperature in the step (1) is 60-70 ℃, and the crushing degree is that the powder is sieved by a sieve of 50-80 meshes;
the proportion of the corn straw powder in the step (2) to the cellulase is 1 g: 500-600U; the feed-liquid ratio of the corn straw powder during enzymolysis is 1 g: 16-26 mL;
the rotating speed of the centrifugation in the step (3) is 6000-10000 r/min, and the centrifugation time is 5-15 min;
after the corn straw powder is obtained in the step (1), first sterilization is also included; the sterilization temperature of the first sterilization is 118-124 ℃, and the sterilization time is 20-40 min; after the mixing in the step (4), performing second sterilization; the sterilization temperature of the second sterilization is 111-119 ℃, and the sterilization time is 15-25 min.
2. The method of claim 1, wherein the OD of the Bacillus subtilis propagation solution of step I6000.8 to 1.0; the inoculation amount is 4-8%.
3. The method according to claim 1, wherein the rotation speed of the centrifugation in the step II is 4000-8000 r/min, and the centrifugation time is 10-20 min.
4. The method according to claim 1, wherein the rotation speed of the centrifugation in the step III is 4000-8000 r/min, and the centrifugation time is 5-15 min.
5. The method according to any one of claims 1 to 4, wherein the pH value is adjusted in step III by using a 2 to 3mol/L HCl solution; and III, drying at the temperature of 50-70 ℃.
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CN113528404A (en) * 2021-08-31 2021-10-22 淮阴师范学院 Method for improving yield of bacillomycin D based on segmented fermentation of corn straw enzymatic hydrolysate base material
CN115478088A (en) * 2022-09-27 2022-12-16 淮阴师范学院 Application of calcium lactate in improvement of bacillus subtilis synthetic bacillomycin D, calcium lactate fermentation culture medium and method
CN115466765A (en) * 2022-09-27 2022-12-13 淮阴师范学院 Application of magnesium sulfate in improvement of bacillus subtilis to synthesis of bacillomycin D, magnesium sulfate fermentation medium and method
CN115466764A (en) * 2022-09-27 2022-12-13 淮阴师范学院 Application of sodium chloride in improvement of bacillus subtilis to synthesis of bacitracin D, sodium chloride fermentation medium and method

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