CN110016490A - A kind of fermentation medium for producing bacteriocin D and method for producing bacteriocin D - Google Patents

Abstract

The invention provides a fermentation medium for producing bacteriocin D and a method for producing bacteriocin D, belonging to the technical field of biological fermentation. The preparation method of the fermentation medium comprises: (1) drying the corn stalks and then pulverizing them to obtain corn stalk powder; (2) mixing the corn stalk powder, cellulase and sterile water, and the enzyme is heated at 40-60° C. Dissolve for 60-100h to obtain an enzymatic hydrolysis solution; (3) centrifuge the enzymatic hydrolysis solution to obtain an enzymatic hydrolysis supernatant; (4) separate the enzymatic hydrolysis supernatant, yeast extract, L-sodium glutamate and KH ₂ PO _4. Mixing to obtain a bacillimycin D fermentation medium; the mixing ratio of the enzymolysis supernatant, yeast extract, L-sodium glutamate and KH ₂ PO ₄ is: 80-120 mL: 1-3 g: 5-15 g: 0.5～1.5g. The fermentation medium of the present invention can be used to produce bacillomycin D, and the production cost is low; and the added value of the corn stover can also be improved.

Description

A kind of fermentation medium for producing bacteriocin D and production of bacteriocin D method

technical field

The invention belongs to the technical field of biological fermentation, and in particular relates to a fermentation medium for producing bacteriocin D and a method for producing bacomycin D.

Background technique

In recent years, the environmental problems caused by the unreasonable disposal of corn stalks are getting worse day by day. How to effectively utilize corn stalks has become the focus of research at home and abroad.

Bacillomyicin D is a secondary metabolite produced by Bacillus subtilis. It is a cyclic lipopeptide compound with physiological activities such as inhibiting the growth of pathogenic bacteria and anti-tumor. It is expected to be developed into a natural food preservative in the future. with broadly application foreground. At present, the most common method for producing Bacillus subtilis by fermentation is to use Landy medium as the fermentation medium. The composition of Landy medium is complex and expensive. The ability of the Landy medium to ferment bacomycin D is still limited, and the extraction process of bacteromycin D is complicated, so it is difficult to realize the large-scale industrial application of bacteromycin D. Therefore, it is very important to create a simple bacteriocin D fermentation medium, reduce costs, and simplify the preparation process of bacteromycin D, which is very important for green production of bacteromycin D and effectively improving the added value of corn stalks.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to provide a fermentation medium and a method for producing bacteromycin D; the production cost of bacteromycin D is low, and the added value of corn stover can be improved.

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

The present invention provides a fermentation medium for producing bacillin D, and the preparation method of the fermentation medium comprises the following steps:

(1) pulverizing the corn stalk after drying to obtain the corn stalk powder;

(2) mixing the corn stalk powder, cellulase and sterile water, and enzymatic hydrolysis at 40-60° C. for 60-100 hours to obtain an enzymatic hydrolysis solution;

(3) centrifuging the enzymolysis solution to obtain an enzymolysis supernatant;

(4) mixing the enzymatic hydrolysis supernatant, yeast extract, sodium L-glutamate and KH ₂ PO ₄ to obtain a fermentation medium for producing bacillin D;

The mixing ratio of the enzymatic hydrolysis supernatant, yeast extract, sodium L-glutamate and KH ₂ PO ₄ in step (4) is 80-120 mL: 1-3 g: 5-15 g: 0.5-1.5 g.

Preferably, the drying temperature in step (1) is 60-70° C., and the degree of pulverization is to pass through a 50-80 mesh sieve.

Preferably, the ratio of the corn stalk powder to the cellulase in step (2) is 1 g: 500-600 U; the solid-liquid ratio of the corn stalk powder during enzymatic hydrolysis is 1 g: 16-26 mL.

Preferably, the rotational speed of the centrifugation in step (3) is 6000-10000 r/min, and the centrifugation time is 5-15 min.

Preferably, after the corn stalk powder is obtained in step (1), it further includes a first sterilization; the sterilization temperature of the first sterilization is 118-124° C., and the sterilization time is 20-40 minutes; the step (4) described After mixing, a second sterilization is also included; the sterilization temperature of the second sterilization is 111-119° C., and the sterilization time is 15-25 minutes.

The present invention also provides a method for producing bacillin D, comprising the steps of:

1. Inoculate the Bacillus subtilis multiplication solution in the above fermentation medium, and ferment and culture at 28～38°C for 72～168h to obtain a fermentation broth;

II, the fermentation broth is centrifuged to obtain fermentation supernatant;

III, adjust the pH value of the fermentation supernatant to ≤3, let stand for 2-8 hours, collect the precipitate by centrifugation, and dry to obtain bacillomycin D dry crude peptide solid.

Preferably, the OD ₆₀₀ of the Bacillus subtilis multiplication solution in step I is 0.8-1.0; the amount of the inoculation is 4-8%.

Preferably, the rotational speed of the centrifugation in step II is 4000-8000 r/min, and the centrifugation time is 10-20 min.

Preferably, the rotational speed of the centrifugation in step III is 4000-8000 r/min, and the centrifugation time is 5-15 min.

Preferably, in step III, 2-3 mol/L HCl solution is used to adjust the pH value; the drying temperature in step III is 50-70°C.

Beneficial effects:

The present invention provides a fermentation medium for producing bacteromycin D and a method for producing bacteriocin D. The preparation method of the fermentation medium comprises: (1) drying the corn stalks and then pulverizing them to obtain corn stalk powder; (2) mixing the corn stalk powder, cellulase and sterile water, and the enzyme is heated at 40-60° C. hydrolyzed for 60-100 hours to obtain an enzymatic hydrolysis solution; (3) centrifuged the enzymatic hydrolysis solution to obtain an enzymatic hydrolysis supernatant; (4) mixed the enzymatic hydrolysis supernatant, yeast extract, L-glutamate sodium and KH ₂ PO ₄ Mixing to obtain a fermentation medium for the production of bacillomycin D; the mixing ratio of the enzymatic hydrolysis supernatant, yeast extract, L-sodium glutamate and KH ₂ PO ₄ is: 80-120 mL: 1-3 g: 5～15g: 0.5～1.5g. The invention utilizes cellulase to enzymolyze the corn stalks, and then prepares the fermentation medium with the corn stalk enzymatic hydrolysis solution, realizes the effective fermentation and production of bacillusmycin D, and simplifies the preparation process of bacillusmycin D. The present invention also provides a method for producing bacteromycin D, which utilizes the above fermentation medium to produce bacillusmycin D, reduces the production cost of bacteromycin D, and increases the added value of corn stalks.

Detailed ways

The present invention provides a fermentation medium for producing bacillin D, and the preparation method of the fermentation medium comprises the following steps:

(1) pulverizing the corn stalk after drying to obtain the corn stalk powder;

(2) mixing the corn stalk powder, cellulase and sterile water, and enzymatic hydrolysis at 40-60° C. for 60-100 hours to obtain an enzymatic hydrolysis solution;

(3) centrifuging the enzymolysis solution to obtain an enzymolysis supernatant;

(4) mixing the enzymatic hydrolysis supernatant, yeast extract, sodium L-glutamate and KH ₂ PO ₄ to obtain a fermentation medium for producing bacillin D;

The mixing ratio of the enzymatic hydrolysis supernatant, yeast extract, sodium L-glutamate and KH ₂ PO ₄ in step (4) is 80-120 mL: 1-3 g: 5-15 g: 0.5-1.5 g.

In the present invention, the corn stalk is first dried and then pulverized to obtain corn stalk powder. In the present invention, the corn stover is preferably mature, free from diseases and insect pests, dust-removed, and cleaned corn stover. Before drying, the corn stalk is preferably cut to 1-2 cm with scissors. The drying preferably uses an electric heating blast drying oven. The drying temperature is preferably 60-70°C, more preferably 65°C. In the present invention, the pulverization preferably uses a solid-state pulverizer. The degree of pulverization is preferably to pass through a 50-80 mesh sieve, more preferably through a 60 mesh sieve. After the corn stover powder is obtained, the present invention preferably performs the first sterilization. The sterilization temperature of the first sterilization is preferably 118-124°C, more preferably 121°C; the sterilization time is preferably 20-40 minutes, more preferably 30 minutes.

After the corn stalk powder is obtained, the present invention mixes the corn stalk powder, cellulase and sterile water, and uses the cellulase to enzymatically hydrolyze the corn stalk powder to obtain an enzymatic hydrolysis solution. In the present invention, the ratio of the corn stalk powder to the cellulase is preferably 1 g: 500-600 U, more preferably 1 g: 550 U; the solid-liquid ratio of the corn stalk powder during enzymatic hydrolysis is 1 g: 16-600 U 26 mL; more preferably 1 g: 18 to 24 mL, more preferably 1 g: 21 mL. The temperature of the enzymatic hydrolysis is preferably 40 to 60°C, more preferably 50°C. The pH of the enzymatic hydrolysis is preferably 5.0. The enzymatic hydrolysis time is preferably 60-100h, more preferably 70-90h, and more preferably 79.8h.

After enzymolysis, the present invention centrifuges the enzymolysis solution to obtain an enzymolysis supernatant. In this step, the rotational speed of the centrifugation is preferably 6000-10000 r/min, more preferably 8000 r/min. The time of the centrifugation is preferably 5-15 min, more preferably 10 min.

After the enzymatic hydrolysis supernatant is obtained, the present invention mixes the enzymatic hydrolysis supernatant, yeast extract, sodium L-glutamate and KH ₂ PO ₄ to obtain a bacillimycin D fermentation medium. In the present invention, the mixing ratio of the enzymatic hydrolysis supernatant, yeast extract, sodium L-glutamate and KH ₂ PO ₄ is 80-120 mL: 1-3 g: 5-15 g: 0.5-1.5 g, preferably 90 ~110 mL: 1.5 to 2.5 g: 8 to 12 g: 0.8 to 1.2 g, more preferably 100 mL: 2 g: 10 g: 1.0 g. In the present invention, the second sterilization is preferably performed after the mixing, and the sterilization temperature of the second sterilization is preferably 111-119°C, more preferably 115°C; the sterilization time is preferably 15-25min, more preferably for 15min.

The present invention also provides a method for producing bacillin D, comprising the steps of:

1. Inoculate the Bacillus subtilis multiplication solution in the above fermentation medium, and ferment and culture at 28～38°C for 72～168h to obtain a fermentation broth;

II, the fermentation broth is centrifuged to obtain fermentation supernatant;

III, adjusting the pH value of the fermentation supernatant to ≤ 3, standing for 2-8 hours, centrifuging, and drying to obtain bacillimycin D dry crude peptide solid.

In the present invention, the Bacillus subtilis propagation liquid is first inoculated into the fermentation medium for fermentation and culture to obtain a fermentation liquid. The present invention has no particular limitation on the specific species of Bacillus subtilis, and any type of Bacillus subtilis used in the art for producing bacillus subtilis D can be used. In order to illustrate the effect of the fermentation medium of the present invention, in a more specific embodiment of the present invention, the Bacillus subtilis propagation solution is preferably obtained by activation and propagation of Bacillus subtilis HSBS-177 stored in the laboratory. The OD ₆₀₀ of the Bacillus subtilis multiplication solution is preferably 0.8-1.0, more preferably 0.9; the inoculation amount is preferably 4-8%, more preferably 6%. In the present invention, the temperature of the fermentation culture is 28 to 38°C, preferably 30 to 35°C, and more preferably 33°C. The oscillation frequency of the fermentation culture is preferably 160-200 r/min, more preferably 180 r/min. The fermentation time is 72-168 hours, more preferably 96-144 hours, and more preferably 120 hours.

After the fermentation broth is obtained, the present invention centrifuges the fermentation broth to obtain a fermentation supernatant. In this step, the rotational speed of the centrifugation is preferably 4000-8000 r/min, more preferably 6000 r/min; the centrifugation time is preferably 10-20 min, more preferably 15 min.

After the fermentation supernatant is obtained, the present invention preferably uses 2-3 mol/L HCl solution, more preferably 2.5 mol/L HCl solution to adjust the pH value of the fermentation supernatant, and the pH value is less than or equal to 3, preferably less than or equal to 2. After adjusting the pH value, let it stand at room temperature, and the standing time is preferably 2 to 8 hours, more preferably 4 hours. After standing, the precipitate was collected by centrifugation and dried to obtain a dry crude peptide solid of bacillomycin D. In this step, the rotational speed of the centrifugation is preferably 4000-8000 r/min, more preferably 6000 r/min; the centrifugation time is preferably 5-15 min, more preferably 10 min. The drying temperature is preferably 50 to 70°C, and more preferably 60°C.

The method adopts cellulase to enzymolyze corn stalks, and the prepared bacillusomycin D fermentation medium has a simple formula, can effectively ferment and produce bacillusmycin D, and reduces the production cost of bacillusmycin D. The simple fermentation medium created by the invention is used to ferment and produce bacillusmycin D, and the cost is low, and the preparation method of bacillusmycin D is simple to operate and practical.

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.

Take out a small section of dried corn stalk, put it at room temperature, cool it appropriately (for 20 minutes), pulverize it with a solid high-speed pulverizer, pass it through a 60-mesh sieve, and prepare a powder for later use.

Accurately weigh 2.00 g of corn stalk powder, place it in a high-pressure steam sterilizer, sterilize it at 121°C for 30 minutes, take it out, and cool it to room temperature. Add cellulase (10000U/g) and sterile water to the sterilized corn stalk powder, shake it well, and carry out full enzymolysis at a temperature of 50 °C. The enzymatic hydrolysis conditions are: the amount of cellulase added 0.11 g, the enzymatic hydrolysis time is 79.8 h, and the liquid-material ratio is 21:1 (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. Add 2.0 g of yeast extract, 10 g of L-glutamate (monosodium glutamate) and 1.0 g of KH ₂ PO ₄ to 100 mL of the enzymatic hydrolysis supernatant, sterilize at 115° C. for 20 min, cool to room temperature, and prepare a bacillimycin D fermentation medium .

Example 2

Bacillus subtilis HSBS-177 stored in the laboratory was inoculated on the slant medium, incubated at 37°C for 24h, single colonies were picked, inserted into the seed medium, and cultured in a shaker flask at 37°C until the OD ₆₀₀ was 0.8-1.0 , insert the fermentation medium prepared in Example 1 according to the inoculum amount of 6%, and ferment and cultivate at 33° C., 180 r/min for 120 h.

Take 10.0 mL of fermentation broth and centrifuge at 6000 r/min for 15 min, discard the cells, take the supernatant, adjust the pH of the supernatant to below 2.0 with HCl, stand at room temperature for 4 h, centrifuge at 6000 r/min for 10 min, take the precipitate, and bake at 60 °C Dry to constant weight to prepare bacteriocin 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 , and the specific HPLC conditions and determination methods were carried out with reference to the literature of Qian et al. The content of bacteriocin D (bacteriocin D, mg; solid mass of bacteriocin D crude peptide, g.) was determined to be 9.42 mg/g in the bacteriocin D crude peptide solid. The experiment was repeated 5 times with a relative error of 1.82%.

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 (10)

1. a kind of for producing the fermentation medium of bacillomycin D, which is characterized in that the preparation method packet of the fermentation medium Include following steps:

(1) it is crushed after drying corn stover, obtains maize straw powder；

(2) maize straw powder, cellulase and sterile water are mixed, 40~60 DEG C of 60~100h of enzymatic hydrolysis are digested Liquid；

(3) enzymolysis liquid is centrifuged, obtains enzymatic hydrolysis supernatant；

(4) by the enzymatic hydrolysis supernatant, yeast extract, L-sodium and KH₂PO₄Mixing, obtains for producing bacillomycin The fermentation medium of D；

Step (4) the enzymatic hydrolysis supernatant, yeast extract, L-sodium and KH₂PO₄Mixed proportion be 80~120mL:1 ~3g:5~15g:0.5~1.5g.

2. fermentation medium according to claim 1, which is characterized in that the temperature of step (1) described drying is 60~70 DEG C, the degree of the crushing was 50~80 meshes.

3. fermentation medium according to claim 1, which is characterized in that step (2) maize straw powder with it is described The ratio of cellulase is 1g:500~600U；The solid-liquid ratio when maize straw powder digests is 1g:16~26mL.

4. fermentation medium according to claim 1, which is characterized in that the revolving speed of step (3) described centrifugation be 6000~ 10000r/min, the time of centrifugation are 5~15min.

5. fermentation medium according to any one of claims 1 to 4, which is characterized in that step (1) obtains corn stalk powder It is last, it further include the first sterilizing；The sterilising temp of first sterilizing is 118~124 DEG C, and sterilization time is 20~40min；Step It suddenly further include the second sterilizing after (4) described mixing；The sterilising temp of second sterilizing is 111~119 DEG C, and sterilization time is 15~25min.

6. a kind of method for producing bacillomycin D, which comprises the steps of:

Bacillus subtilis is expanded numerous liquid and is inoculated in any one of Claims 1 to 5 fermentation medium by I, 28~38 DEG C of hairs Ferment 72~168h of culture, obtains fermentation liquid；

The fermentation liquid is centrifuged, obtains fermented supernatant fluid by II；

III adjusts pH value≤3 of the fermented supernatant fluid, stands 2~8h, precipitating, drying is collected by centrifugation, obtains bacillomycin D Dry thick peptide solid.

7. according to the method described in claim 6, it is characterized in that, bacillus subtilis described in step I expands the OD of numerous liquid₆₀₀For 0.8~1.0；The amount of the inoculation is 4~8%.

8. according to the method described in claim 6, it is characterized in that, the revolving speed of centrifugation described in step II is 4000~8000r/ Min, the time of centrifugation are 10~20min.

9. according to the method described in claim 6, it is characterized in that, the revolving speed of centrifugation described in step III is 4000~8000r/ Min, the time of centrifugation are 5~15min.

10. according to the described in any item methods of claim 6~9, which is characterized in that step III is molten using 2~3mol/L HCl Liquid adjusts pH value；Dry temperature described in step III is 50~70 DEG C.