CN110004093B - Bacillus subtilis culture medium raw material, preparation method and application thereof, and culture medium for increasing yield of bacillomycin D - Google Patents

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 bacteriocin D, and relates to the technical field of microbial fermentation. The Bacillus subtilis culture medium raw materials of the present invention are cellulase and wood Glycanase double-enzymatic hydrolysis supernatant of corn stover. Combining the enzymatic hydrolysis properties of cellulase and xylanase, the present invention utilizes cellulase and xylanase to double enzymatic hydrolysis of Bacillus zeae, and the enzymatic hydrolysis supernatant obtained by double enzymatic hydrolysis can be used as a culture medium raw material, which can effectively improve bacteriocin D yield and synthesis efficiency. This has laid a scientific and reasonable technical foundation for realizing the large-scale industrial application of bacteromycin D and developing bacteromycin D into a natural food preservative.

Description

A kind of Bacillus subtilis culture medium raw material and preparation method and application thereof, improving rod Medium for Bacteriocin D Production

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 bacillus subtilis D.

Background technique

China is a big agricultural country and produces a large amount of corn stover every year. Corn stover contains a large amount of macromolecular carbohydrates such as cellulose, hemicellulose and lignin. If corn stover is directly used for microbial fermentation, it is difficult to be utilized by microorganisms. Enzymatic hydrolysis of corn stalks to obtain smaller molecules of reducing sugars, which can be used in microbial fermentation to produce some valuable target metabolites, has become the consensus of researchers at home and abroad. However, due to the small number of substrates used by a single enzyme, the enzymatic hydrolysis efficiency is low, which restricts the application of enzymatic hydrolysis technology in the field of microbial fermentation. Enzymatic hydrolysis efficiency, obtaining more types of small molecules of reducing sugars, thus enriching the carbon source for microbial fermentation.

Bacillomyicin D is a secondary metabolite produced by Bacillus subtilis, which has physiological activities such as inhibiting the growth of pathogenic bacteria and anti-tumor, and has a very broad application prospect. Under natural conditions, the synthetic ability of bacteromycin D in Bacillus subtilis is very limited, which limits the industrial application of bacteromycin D.

SUMMARY OF THE INVENTION

In order to overcome the defect of insufficient production of Bacillus subtilis, the present invention provides a Bacillus subtilis culture medium raw material, a preparation method and application thereof, and a culture medium for improving the output of Bacteriocin D. The culture medium of the present invention is adopted. Constructing medium from raw materials and fermenting Bacillus subtilis can significantly increase the yield of bacillusomycin D, laying the foundation for the large-scale industrial application of bacillusmycin D.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides a Bacillus subtilis culture medium raw material, which is the enzymatic hydrolysis supernatant of the double enzymatic hydrolysis of corn stalks by cellulase and xylanase.

Preferably, the mass ratio of the cellulase and xylanase during enzymatic hydrolysis is 1-2:2-3.

Preferably, the specific activity of the cellulase is 10,000-120,000 U/g, and the specific activity of the xylanase is 10,000-120,000 U/g.

The present invention also provides a preparation method of the Bacillus subtilis culture medium material described in the above technical solution, comprising the following steps:

(1) Mixing corn stover, cellulase, xylanase and water, and enzymatically hydrolyzing at 50-60 °C for 60-80 hours to obtain an enzymatic hydrolysis complex;

(2) solid-liquid separation of the enzymolysis complex to obtain the enzymolysis supernatant.

Preferably, in the step (1), the ratio of the mass of the corn stover, the mass of the cellulase, the mass of the xylanase and the volume of the water is: 1-2 g: 1-2 g: 2-3 g: 54- 74ml.

Preferably, in the step (2), the method for solid-liquid separation is centrifugation.

The present invention also provides the application of the Bacillus subtilis medium raw material described in the foregoing technical solution in improving the production of bacillusmycin D.

The present invention also provides a Bacillus subtilis medium for improving the production of bacteriocin D, comprising the Bacillus subtilis medium raw materials, yeast extract, L-sodium glutamate and potassium dihydrogen phosphate described in the foregoing technical solution.

Preferably, in the medium, the ratio of the volume of the Bacillus subtilis medium raw material, the mass of the yeast extract, the mass of L-sodium glutamate and the mass of potassium dihydrogen phosphate is 100ml:1～4g: 5～15g: 0.5～2g.

Compared with the prior art, the beneficial effects of the present invention:

The invention provides a Bacillus subtilis culture medium raw material, which is the enzymatic hydrolysis supernatant of the double enzymatic hydrolysis of corn stalks by cellulase and xylanase. Combining the enzymatic hydrolysis properties of cellulase and xylanase, the present invention utilizes cellulase and xylanase to double enzymatic hydrolysis of Bacillus zeae, and the enzymatic hydrolysis supernatant obtained by double enzymatic hydrolysis can be used as a culture medium raw material, which can effectively improve bacteriocin D yield and synthesis efficiency. This has laid a scientific and reasonable technical foundation for realizing the large-scale industrial application of bacteromycin D and developing bacteromycin D into a natural food preservative.

The raw material of the Bacillus subtilis culture medium of the present invention has a wide range of raw material sources, low cost, and a simple and easy preparation method. The production cost of bacteriocin D can be further reduced, and the added value of corn stover can also be improved.

Detailed ways

The invention provides a Bacillus subtilis culture medium raw material, which is the enzymatic hydrolysis supernatant of the double enzymatic hydrolysis of corn stalks by cellulase and xylanase. The purpose of selecting cellulase and xylanase for double enzymatic hydrolysis in the present invention is to improve the enzymatic hydrolysis efficiency and the types of reducing sugars, facilitate the utilization of microorganisms, accumulate bacterial cells, and improve the synthesis ability of metabolites.

In the present invention, when the corn stover is hydrolyzed by double enzymes, the mass ratio of the cellulase and xylanase is preferably 1-2:2-3; more preferably 2:3.

In the present invention, the specific activity of the cellulase is preferably 10,000-120,000 U/g, more preferably 50,000-100,000 U/g; the specific activity of the xylanase is preferably 10,000-120,000 U/g, more preferably 50000～100000U/g.

The present invention also provides the preparation method of the Bacillus subtilis culture medium material described in the above technical solution, comprising the following steps:

(1) Mixing corn stover, cellulase, xylanase and water, and enzymatically hydrolyzing at 50-60 °C for 60-80 hours to obtain an enzymatic hydrolysis complex;

(2) solid-liquid separation of the enzymolysis complex to obtain the enzymolysis supernatant.

In the present invention, the corn stover is preferably pulverized to a size of 1-2 cm before enzymatic hydrolysis, and then dried to a constant weight and pulverized to a size of 60 mesh or more. In the present invention, the drying method is preferably hot air drying; more preferably, the temperature of the hot air drying is 60-70°C, further preferably 65°C. The purpose of drying the corn stalk to constant weight in the present invention is to sterilize and inactivate enzymes and to facilitate preservation.

In the present invention, the temperature of the enzymatic hydrolysis is preferably 50°C. In the present invention, the time of the enzymatic hydrolysis is preferably 70h. In the present invention, the ratio of the mass of the corn stover, the mass of cellulase, the mass of xylanase and the volume of water is preferably 1-2 g: 1-2 g: 2-3 g: 54-74 ml; more preferably It is 2g:2g:3g:54ml.

In the present invention, the method for solid-liquid separation is preferably centrifugation; the rotational speed of the centrifugation is preferably 7000-10000r/min, more preferably 8000r/min; the centrifugation time is preferably 8-15min, more preferably for 10min.

The present invention also provides the application of the Bacillus subtilis medium raw material described in the foregoing technical solution in improving the production of bacillusmycin D. Using the medium raw material of the present invention to ferment Bacillus subtilis can significantly increase the yield of bacteromycin D. As shown in the examples of the present invention, compared with the enzymatic hydrolysis supernatant obtained by single enzymatic hydrolysis as a medium raw material, the yield of bacteriocin D is significantly enhanced.

The present invention also provides a Bacillus subtilis medium for improving the production of bacteriocin D, comprising the Bacillus subtilis medium raw materials, yeast extract, L-sodium glutamate and potassium dihydrogen phosphate described in the foregoing technical solution. Preferably in the present invention, the ratio of the volume of the Bacillus subtilis medium raw material, the mass of the yeast extract, the mass of L-sodium glutamate and the mass of potassium dihydrogen phosphate is 100ml:1～4g:5～15g: 0.5 to 2g; more preferably 100ml:2g:10g:1g.

The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

Example 1

Select mature corn stalks without pests and diseases, remove dust, clean them, cut them to 1-2cm lengths with scissors, put them in an electric blast drying oven, control the temperature at 65°C, and dry them to constant weight. The dried corn stalks were taken out, placed at room temperature for 20 minutes, pulverized with a solid high-speed pulverizer, passed through a 60-mesh sieve, and prepared into powder for later use.

Accurately weigh 2.00 g of the corn stalk powder prepared above, place it in a high-pressure steam sterilizer, take it out after sterilizing at 121 ° C for 30 min, cool it to room temperature, and add 1 g (100,000 U/g of cellulase) to the sterilized corn stalk powder. ), 2g of xylanase (100000U/g) and 54ml of sterile water, shake well, and fully enzymolysis at a temperature of 50°C. The enzymolysis conditions are: the ratio of cellulase and xylanase added. 2:3 (mass ratio, g:g), the enzymatic hydrolysis time is 70h, and the liquid-to-material ratio is 27 mL/g (volume of enzymatic hydrolysis solution: mass of corn stalk powder, mL/g). After enzymolysis, the enzymolysis solution was taken out and centrifuged at 8000r/min for 10min to obtain the enzymolysis supernatant.

Comparative Example 1

Other conditions were the same as those in Example 1, except that no cellulase was added and the amount of xylanase was 0.1 g. The control enzymatic hydrolysis supernatant 1 was obtained.

Comparative Example 2

Other conditions were the same as those in Example 1, except that no xylanase was added and the amount of cellulase added was 0.1 g. The control enzymatic hydrolysis supernatant 2 was obtained.

Example 2

Take 100 ml of the enzymatic hydrolysis supernatant of Example 1, Comparative Example 1 and Comparative Example 2 respectively, and add 2.0 g of yeast extract, 10 g of L-glutamate (monosodium glutamate) and 41.0 g of KH ₂ PO to each of the enzymatic hydrolysis supernatants. , sterilized at 115° C. for 20 min, cooled to room temperature, and made into bacillusmycin D fermentation medium (Example 1), control bacillusmycin D fermentation medium 1 (comparative example 1), and contrast bacillusmycin D fermentation medium 2 (Comparative Example 2).

Inoculate the Bacillus subtilis stored in the laboratory on the slant medium, cultivate at a constant temperature of 37 °C for 24 hours, pick a single colony, insert it into the seed medium, and cultivate it in a shake flask at 37 °C until the OD ₆₀₀ is 0.8 ~ 1.0, press 6 % of the inoculum amount was respectively inserted into the above-mentioned three fermentation media, and fermented and cultured at 33° C., 180 r/min for 110 h.

After the fermentation, the fermentation broths obtained from the fermentation of three bacillimycin D fermentation media were respectively taken, and 10.0 mL of each fermented broth was centrifuged at 6000 r/min for 15 min, the bacterial cells were discarded, the supernatant was taken, and the pH of the supernatant was adjusted with HCl. to below 2.0, stand at room temperature for 4 h, centrifuge at 6000 r/min for 10 min, take the precipitate, and dry it at 60°C to constant weight to prepare bacillomycin D dry crude peptide solid.

Take a small amount of dry crude peptide solid of bacteriomycin D, grind it into powder, add 1.0 mL of methanol for extraction, centrifuge at 10,000 r/min for 10 min, take the supernatant, and use high performance liquid chromatography (HPLC) method to determine the content of bacteriocin D For the specific HPLC conditions and determination methods, please refer to the literature of Qian et al. 2015, 179:260-267). Repeat the above test 5 times and take the average value.

Table 1 Bacteriocin D content (n=5) obtained by different fermentation medium fermentation

The measurement results are shown in Table 1. It can be seen that the yield of bacteriocin D in the solid bacteriocin D crude peptide obtained by double enzymatic hydrolysis and fermentation with cellulase and xylanase has been significantly improved.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims (6)

1. a Bacillus subtilis culture medium raw material, is characterized in that, described culture medium raw material is the enzymolysis supernatant of cellulase and xylanase double enzymolysis corn stover; Described cellulase and xylanase enzyme The mass ratio during solution is 1:2; the specific activity of the cellulase is 100000U/g, the specific activity of the xylanase is 100000U/g; the quality of the corn stalk, the quality of the cellulase, The ratio of the mass of xylanase to the volume of water is: 2g:1g:2g:54ml. 2. the preparation method of the described Bacillus subtilis culture medium raw material of claim 1, comprises the following steps: (1) Mix corn stover, cellulase, xylanase and water, and enzymatically hydrolyze them at 50-60 °C for 60-80 hours to obtain an enzymatic-hydrolysis complex; (2) solid-liquid separation of the enzymolysis complex to obtain the enzymolysis supernatant. 3 . The preparation method according to claim 2 , wherein in the step (2), the method for solid-liquid separation is centrifugation, the rotational speed of the centrifugation is 7000-10000 r/min, and the centrifugation time is 8-15 min. 4 . 4. the application of the described Bacillus subtilis medium raw material of claim 1 in improving the output of bacillimycin D. 5. a Bacillus subtilis substratum that improves bacteromycin D output, is characterized in that, comprises the described Bacillus subtilis subtilis culture medium raw material of claim 1, yeast extract, L-sodium glutamate and potassium dihydrogen phosphate . 6. Bacillus subtilis culture medium according to claim 5, is characterized in that, in described culture medium, the volume of described Bacillus subtilis culture medium raw material, the quality of yeast extract, the quality of sodium L-glutamate The mass ratio of potassium dihydrogen phosphate is 100ml:1~4g:5~15g:0.5~2g.