CN110004093B - Bacillus subtilis culture medium raw material, preparation method and application thereof, and culture medium for increasing yield of bacillomycin D - Google Patents
Bacillus subtilis culture medium raw material, preparation method and application thereof, and culture medium for increasing yield of bacillomycin D Download PDFInfo
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Abstract
The invention provides a bacillus subtilis culture medium raw material, a preparation method and application thereof, and a culture medium for improving the yield of bacillomycin D, and relates to the technical field of microbial fermentation. The invention combines the enzymolysis characteristics of the cellulase and the xylanase, utilizes the cellulase and the xylanase to carry out double enzymolysis on the corn bacillus, and takes the enzymolysis supernatant obtained by double enzymolysis as the raw material of the culture medium, thereby effectively improving the yield and the synthesis efficiency of the bacillomycin D. The method lays a scientific and reasonable technical foundation for realizing large-scale industrial application of the bacitracin D and developing the bacitracin D into a natural food preservative.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a bacillus subtilis culture medium raw material, a preparation method and application thereof, and a bacillus subtilis culture medium for improving the yield of bacillomycin D.
Background
China is a big agricultural country, and a large amount of corn straws are produced every year, and contain a large amount of macromolecular carbohydrates such as cellulose, hemicellulose, lignin and the like. If the corn stalks are directly used for microbial fermentation, the corn stalks are difficult to be utilized by microorganisms. The corn straws are subjected to enzymolysis to obtain smaller-molecule reducing sugars, and the reduced sugars are applied to microbial fermentation to produce some valuable target metabolites and become the consensus of researchers at home and abroad. However, the application of the enzymolysis technology in the field of microbial fermentation is limited due to the fact that the kinds of acting substrates of the single enzyme are few, and the enzymolysis efficiency is low, and the double enzymolysis method is adopted, and due to the fact that the acting substrates are many, the enzymolysis efficiency can be remarkably improved, more types of micromolecule reducing saccharides are obtained, and therefore the carbon source for microbial fermentation is enriched.
The bacillomycin D (bacillus micin D) is a secondary metabolite generated by the bacillus subtilis, has physiological activities of inhibiting the growth of pathogenic bacteria, resisting tumors and the like, and has very wide application prospect. Under natural conditions, the synthesis capacity of the bacillomycin D in the bacillus subtilis is very limited, which limits the industrial application of the bacillomycin D.
Disclosure of Invention
The invention provides a bacillus subtilis culture medium raw material, a preparation method and application thereof and a culture medium for improving the yield of the bacillomycin D, aiming at overcoming the defect of insufficient yield of the existing bacillomycin D.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a bacillus subtilis culture medium raw material, which is an enzymolysis supernatant of corn straws subjected to double enzymolysis by cellulase and xylanase.
Preferably, the mass ratio of the cellulase to the xylanase in enzymolysis is 1-2: 2-3.
Preferably, the specific activity of the cellulase is 10000-120000U/g, and the specific activity of the xylanase is 10000-120000U/g.
The invention also provides a preparation method of the bacillus subtilis culture medium raw material in the technical scheme, which comprises the following steps:
(1) mixing corn straws, cellulase, xylanase and water, and carrying out enzymolysis for 60-80 h at 50-60 ℃ to obtain an enzymolysis compound;
(2) and carrying out solid-liquid separation on the enzymolysis compound to obtain the enzymolysis supernatant.
Preferably, in the step (1), the ratio of the mass of the corn stalks, the mass of the cellulase, the mass of the xylanase and the volume of the water is as follows: 1-2 g, 2-3 g, 54-74 ml.
Preferably, in the step (2), the solid-liquid separation method is centrifugation, the rotation speed of the centrifugation is 7000-10000 r/min, and the centrifugation time is 8-15 min.
The invention also provides application of the bacillus subtilis culture medium raw material in the technical scheme in improving the yield of the bacillomycin D.
The invention also provides a bacillus subtilis culture medium for improving the yield of the bacitracin D, which comprises the raw materials of the bacillus subtilis culture medium, a yeast extract, L-sodium glutamate and potassium dihydrogen phosphate.
Preferably, in the culture medium, the ratio of the volume of the raw material of the bacillus subtilis culture medium, the mass of the yeast extract, the mass of the sodium L-glutamate and the mass of the potassium dihydrogen phosphate is 100ml: 1-4 g: 5-15 g: 0.5-2 g.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a bacillus subtilis culture medium raw material, which is an enzymolysis supernatant of corn straws subjected to double enzymolysis by cellulase and xylanase. The invention combines the enzymolysis characteristics of the cellulase and the xylanase, utilizes the cellulase and the xylanase to carry out double enzymolysis on the corn bacillus, and takes the enzymolysis supernatant obtained by double enzymolysis as the raw material of the culture medium, thereby effectively improving the yield and the synthesis efficiency of the bacillomycin D. The method lays a scientific and reasonable technical foundation for realizing large-scale industrial application of the bacitracin D and developing the bacitracin D into a natural food preservative.
The raw materials of the bacillus subtilis culture medium have wide sources and low cost, and the preparation method is simple and easy to implement. The production cost of the bacillomycin D can be further reduced, and the added value of the corn straw is improved.
Detailed Description
The invention provides a bacillus subtilis culture medium raw material, which is an enzymolysis supernatant of corn straws subjected to double enzymolysis by cellulase and xylanase. The invention selects cellulase and xylanase for double enzymolysis, aims to improve the enzymolysis efficiency and reducing sugar types, is beneficial to the utilization of microorganisms, accumulates thalli and improves the synthesis capacity of metabolites.
In the invention, when the corn straws are subjected to double enzymolysis, the mass ratio of the cellulase to the xylanase is preferably 1-2: 2-3; more preferably 2: 3.
In the invention, the specific activity of the cellulase is preferably 10000-120000U/g, more preferably 50000-100000U/g; the specific activity of the xylanase is preferably 10000-120000U/g, and more preferably 50000-100000U/g.
The invention also provides a preparation method of the bacillus subtilis culture medium raw material in the technical scheme, which comprises the following steps:
(1) mixing corn straws, cellulase, xylanase and water, and carrying out enzymolysis for 60-80 h at 50-60 ℃ to obtain an enzymolysis compound;
(2) and carrying out solid-liquid separation on the enzymolysis compound to obtain the enzymolysis supernatant.
In the invention, the corn straw is preferably crushed to 1-2 cm before enzymolysis, and is crushed to more than 60 meshes after being dried to constant weight. In the present invention, the drying method is preferably hot air drying; more preferably, the temperature of the hot air drying is 60 to 70 ℃, and further preferably 65 ℃. The invention dries the corn straw to constant weight, aims to sterilize and inactivate enzyme and is convenient to preserve.
In the present invention, the temperature of the enzymatic hydrolysis is preferably 50 ℃. In the present invention, the time for the enzymatic hydrolysis is preferably 70 hours. In the invention, the ratio of the mass of the corn straws, the mass of the cellulase, the mass of the xylanase and the volume of water is preferably 1-2 g: 1-2 g: 2-3 g: 54-74 ml; more preferably 2g:2g:3g:54 ml.
In the present invention, the method of solid-liquid separation is preferably centrifugal separation; the rotation speed of the centrifugation is preferably 7000-10000 r/min, and more preferably 8000 r/min; the time for centrifugation is preferably 8-15 min, and more preferably 10 min.
The invention also provides application of the bacillus subtilis culture medium raw material in the technical scheme in improving the yield of the bacillomycin D. The bacillus subtilis is fermented by adopting the culture medium raw material, so that the yield of the bacillomycin D can be obviously improved. As shown in the embodiment of the invention, compared with an enzymolysis supernatant obtained by single enzyme enzymolysis as a culture medium raw material, the yield of the bacillomycin D is obviously enhanced.
The invention also provides a bacillus subtilis culture medium for improving the yield of the bacitracin D, which comprises the raw materials of the bacillus subtilis culture medium, a yeast extract, L-sodium glutamate and potassium dihydrogen phosphate. Preferably, the ratio of the volume of the raw material of the bacillus subtilis culture medium, the mass of the yeast extract, the mass of the sodium L-glutamate and the mass of the monopotassium phosphate is 100ml: 1-4 g: 5-15 g: 0.5-2 g; more preferably 100ml:2g:10g:1 g.
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 corn straw segments, standing at room temperature for 20 minutes, crushing by a solid high-speed crusher, and sieving by a 60-mesh sieve to prepare powder for later use.
Accurately weighing 2.00g of the prepared corn straw powder, placing the corn straw powder in a high-pressure steam sterilization pot, sterilizing at 121 ℃ for 30min, taking out, cooling to room temperature, adding 1g (100000U/g) of cellulase, 2g (100000U/g) of xylanase and 54ml of sterile water into the sterilized corn straw powder, fully shaking up, and carrying out full enzymolysis at 50 ℃, wherein the enzymolysis conditions are as follows: the addition ratio of the cellulase to the xylanase is 2:3 (mass ratio, g: g), the enzymolysis time is 70h, and the liquid-material ratio is 27mL/g (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.
Comparative example 1
The conditions were the same as in example 1 except that no cellulase was added and the amount of xylanase added was 0.1 g. Control enzymatic supernatant 1 was obtained.
Comparative example 2
The conditions were the same as in example 1 except that xylanase was not added and the amount of cellulase added was 0.1 g. Control enzymatic supernatant 2 was obtained.
Example 2
Taking 100ml of each of the enzymatic supernatants of example 1, comparative example 1 and comparative example 2, respectively, adding yeast extract 2.0g, sodium L-glutamate (monosodium glutamate) 10g and KH into each of the enzymatic supernatants2PO41.0g, sterilized at 115 ℃ for 20min, cooled to room temperature, and made into bacitracin D fermentation medium (example 1), control bacitracin D fermentation medium 1 (comparative example 1), and control bacitracin D fermentation medium 2 (comparative example 2).
Inoculating Bacillus subtilis stored in laboratory to slant culture medium, culturing at 37 deg.C for 24 hr, selecting single colony, inoculating to seed culture medium, and shake-culturing at 37 deg.C to OD600The inoculation amount is 0.8-1.0, the 3 fermentation culture media are respectively inoculated according to the inoculation amount of 6%, and fermentation culture is carried out for 110h at 33 ℃ and 180 r/min.
After fermentation is finished, 10.0mL of fermentation liquor obtained by fermenting three bacillomycin D fermentation culture media is respectively taken, centrifuged for 15min at 6000r/min, thallus is discarded, supernatant fluid is taken, the pH of the supernatant fluid is adjusted to be below 2.0 by HCl, the supernatant fluid is kept stand for 4h at room temperature, centrifuged for 10min at 6000r/min, precipitate is taken and dried to constant weight at 60 ℃, and the bacillomycin D dry crude peptide solid is prepared.
A small amount of dry crude peptide of bacitracin D is taken, ground into powder, added with 1.0mL of methanol for extraction, centrifuged at 10000r/min for 10min, the supernatant is taken, and the content of bacitracin D is determined by a High Performance Liquid Chromatography (HPLC) method, wherein the specific HPLC conditions and determination method refer to Qian et al (Shiquan Qian, Hedong Lu, Panpan Meng, Chong Zhang, Fengxia Lv, Xiaomei Bie, Zhaoxin Lu. Effect of insulin effect production and regulation biology biochemical analysis of Bacillus D in Bacillus subtilis fmb. Bioresource Technology,2015,179: 260-267). The above experiment was repeated 5 times and the average was taken.
TABLE 1 bacitracin D content by fermentation in different fermentation media (n ═ 5)
The results are shown in Table 1, and it can be seen that the yield of bacitracin D in the crude bacitracin D solid obtained by double enzymolysis and fermentation using cellulase and xylanase is significantly improved.
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 (6)
1. The bacillus subtilis culture medium raw material is characterized in that the culture medium raw material is enzymolysis supernatant of corn straws subjected to double enzymolysis by cellulase and xylanase; the mass ratio of the cellulase to the xylanase in enzymolysis is 1: 2; the specific activity of the cellulase is 100000U/g, and the specific activity of the xylanase is 100000U/g; the ratio of the mass of the corn straws, the mass of the cellulase, the mass of the xylanase and the volume of water is as follows: 2g:1g:2g:54 ml.
2. The method for preparing a Bacillus subtilis culture medium material according to claim 1, comprising the steps of:
(1) mixing corn straws, cellulase, xylanase and water, and carrying out enzymolysis for 60-80 h at 50-60 ℃ to obtain an enzymolysis compound;
(2) and carrying out solid-liquid separation on the enzymolysis compound to obtain the enzymolysis supernatant.
3. The preparation method according to claim 2, wherein in the step (2), the solid-liquid separation method is centrifugation, the rotation speed of the centrifugation is 7000-10000 r/min, and the centrifugation time is 8-15 min.
4. Use of the Bacillus subtilis media feedstock of claim 1 for increasing the yield of bacitracin D.
5. A Bacillus subtilis culture medium for increasing the yield of bacillomycin D, which comprises the Bacillus subtilis culture medium raw material of claim 1, a yeast extract, sodium L-glutamate and potassium dihydrogen phosphate.
6. The Bacillus subtilis medium according to claim 5, wherein the ratio of the volume of the Bacillus subtilis medium raw material, the mass of the yeast extract, the mass of the L-sodium glutamate and the mass of the potassium dihydrogen phosphate in the medium is 100ml: 1-4 g: 5-15 g: 0.5-2 g.
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| CN112251374A (en) * | 2020-05-08 | 2021-01-22 | 重庆大学 | High-temperature-resistant high-yield cellulase bacillus subtilis and application thereof |
| 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 |
| CN114350558A (en) * | 2021-12-31 | 2022-04-15 | 黄河三角洲京博化工研究院有限公司 | Solid oil-removing microbial inoculum and preparation method and application thereof |
| CN114774498B (en) * | 2022-05-06 | 2023-10-03 | 南京财经大学 | Method for producing bacitracin D by fermenting bacillus immobilized by diatomite |
| 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 |
| 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 |
| 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 |
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