阅读说明：本技术 一种产抗菌肽枯草芽孢杆菌的发酵工艺及其在畜禽抗病增产中的应用 (Fermentation process for producing antibacterial peptide bacillus subtilis and application of fermentation process in disease-resistant and yield-increasing of livestock and poultry ) 是由 汤法银 于 2020-05-12 设计创作，主要内容包括：本发明涉及一种产抗菌肽枯草芽孢杆菌的发酵工艺及其在畜禽抗病增产中的应用,包括以下步骤：1)产抗菌肽枯草芽孢杆菌种子液制备；2)发酵培养基配制；3)产抗菌肽枯草芽孢杆菌的发酵,以生产抗菌肽；发酵过程中对发酵液进行热冲击,以降低发酵液黏度；4)对发酵液进行纯化,得到抗菌肽。本发明采用高效抗菌肽生产菌株,使用优化发酵工艺,生产具有高效价的抗菌肽产品,用于预防和治疗畜禽腹泻、实现抗病增产。(The invention relates to a fermentation process for producing antibacterial peptide bacillus subtilis and application thereof in disease-resistant and yield-increasing of livestock and poultry, comprising the following steps: 1) preparing a bacillus subtilis seed solution for producing the antibacterial peptide; 2) preparing a fermentation medium; 3) fermenting bacillus subtilis for producing the antibacterial peptide to produce the antibacterial peptide; carrying out thermal shock on the fermentation liquor in the fermentation process so as to reduce the viscosity of the fermentation liquor; 4) and purifying the fermentation liquor to obtain the antibacterial peptide. The invention adopts high-efficiency antibacterial peptide production strains, uses an optimized fermentation process to produce antibacterial peptide products with high titer, and is used for preventing and treating livestock diarrhea and realizing disease resistance and yield increase.)

1. A fermentation process for producing antibacterial peptide bacillus subtilis is characterized by comprising the following steps:

1) preparing a bacillus subtilis seed solution for producing the antibacterial peptide;

2) preparing a fermentation medium;

3) fermenting bacillus subtilis for producing the antibacterial peptide to produce the antibacterial peptide; carrying out thermal shock on the fermentation liquor in the fermentation process so as to reduce the viscosity of the fermentation liquor;

4) and purifying the fermentation liquor to obtain the antibacterial peptide.

2. The fermentation process of the antibacterial peptide-producing bacillus subtilis according to claim 1, wherein the step 1) is specifically to prepare a seed culture medium, the seed culture medium comprises 3-10g/L of glucose, 15-25g/L of corn starch, 15-25g/L of soybean meal, 3-10g/L of corn steep liquor dry powder, 2-3g/L of magnesium sulfate and 3-10g/L of peptone, the pH value is adjusted to be 6.4-6.6, the antibacterial peptide-producing bacillus subtilis is inoculated into the seed culture medium and cultured for 9-10 hours at 37 ℃, the stirring speed is 130-150r/min in the culture process, air is continuously introduced to obtain a seed solution, and the bacteria number in the seed solution is adjusted to be 1.0-1.2 × 10 by using sterile physiological saline water⁸cfu/mL to obtain a seed solution for use.

3. The fermentation process of bacillus subtilis for producing the antibacterial peptide according to claim 1, wherein in the step 2), the fermentation medium consists of: 3-10g/L of glucose, 15-25g/L of corn starch, 15-25g/L of soybean meal, 3-10g/L of corn steep liquor dry powder, 2-3g/L of magnesium sulfate, 3-10g/L of peptone, 1-2g/L of urea, 0.1-0.2g/L of alpha-sodium naphthalene acetate and 0.2-0.3g/L of dimethyl phosphate, and the pH value is 6.4-6.6.

4. The fermentation process of bacillus subtilis for producing the antibacterial peptide according to claim 1, wherein the step 3) is specifically as follows: inoculating the spare seed liquid to a fermentation culture medium according to the inoculation proportion of 12-15%, culturing at 37 ℃ for 24-28h, wherein the stirring speed is 155-165r/min in the culture process, air is continuously introduced, and heat shock is performed every 6-8h in the culture process, and the heat shock step is as follows: heating the fermentation liquid within 8-12min to 5-10 deg.C, and cooling to 37 deg.C within 8-12 min.

5. The fermentation process of Bacillus subtilis for producing the antibacterial peptide according to claim 1, wherein the Bacillus subtilis is Bacillus subtilis BF 168.

6. An antimicrobial peptide produced by the fermentation process of any one of claims 1-5.

7. A composite veterinary drug product comprising an antibacterial peptide and Bacillus subtilis, wherein the antibacterial peptide is prepared by the fermentation process according to any one of claims 1 to 5, and the Bacillus subtilis is Bacillus subtilis bf 168.

8. The use of the antimicrobial peptide of claim 6, the fermentation process of any one of claims 1 to 5, and the compound veterinary drug of claim 7 in the preparation of a medicament for the disease resistance and yield increase of livestock and poultry.

Technical Field

The invention relates to a fermentation process for producing antibacterial peptide bacillus subtilis and application thereof in disease resistance and yield increase of livestock and poultry.

Background

In order to improve the trust of consumers on livestock products, the addition of antibiotics into animal feed is generally prohibited in 12 months and 31 days in 2020 and 31 days in China. The requirement of 194 th bulletin board of China rural agricultural department in 2019 is that all varieties of growth-promoting drug feed additives except traditional Chinese medicines are quitted from 1/2020, and commercial feed containing the growth-promoting drug feed additives (except traditional Chinese medicines) is stopped from 7/1/2020. Commercial feed which is produced before can be continuously circulated, sold and used for 12-31 days in 2020.

The antibacterial peptide has multiple effects, can replace antibiotics and antibacterial drugs which are harmful to human health to be added into the feed, is an ideal non-toxic, residue-free, efficient and broad-spectrum, antibacterial and growth-promoting green feed additive, and is considered as an ideal substitute of the antibiotics. Therefore, the invention aims to produce the antibacterial peptide product with high titer and the related compound medicament of the bacillus subtilis by adopting the high-efficiency antibacterial peptide production strain and using the optimized fermentation process.

Disclosure of Invention

In order to overcome the defects, the invention provides a fermentation process for producing antibacterial peptide bacillus subtilis and application thereof in disease resistance and yield increase of livestock and poultry, and the fermentation process has the functions of disease resistance and yield increase, diarrhea prevention and treatment and the like.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a fermentation process for producing antibacterial peptide bacillus subtilis is characterized by comprising the following steps:

1) preparing a bacillus subtilis seed solution for producing the antibacterial peptide;

2) preparing a fermentation medium;

3) fermenting bacillus subtilis for producing the antibacterial peptide to produce the antibacterial peptide; carrying out thermal shock on the fermentation liquor in the fermentation process so as to reduce the viscosity of the fermentation liquor;

4) and purifying the fermentation liquor to obtain the antibacterial peptide.

Preferably, step 1) is specifically: preparing a seed culture medium, wherein the seed culture medium comprises the following components: 3-10g/L of glucose, 15-25g/L of corn starch, 15-25g/L of soybean meal, 3-10g/L of corn steep liquor dry powder, 2-3g/L of magnesium sulfate and 3-10g/L of peptone, and the pH is adjusted to 6.4-6.6; inoculating bacillus subtilis for producing antibacterial peptide to seed cultureCulturing at 37 deg.C for 9-10h with stirring speed of 130-150r/min and continuously introducing air to obtain seed solution, and regulating the number of bacteria in the seed solution to 1.0-1.2 × 10 with sterile physiological saline⁸cfu/mL to obtain a seed solution for use.

In the step 2), the fermentation medium consists of: 3-10g/L of glucose, 15-25g/L of corn starch, 15-25g/L of soybean meal, 3-10g/L of corn steep liquor dry powder, 2-3g/L of magnesium sulfate, 3-10g/L of peptone, 1-2g/L of urea, 0.1-0.2g/L of alpha-sodium naphthalene acetate and 0.2-0.3g/L of dimethyl phosphate, and the pH value is 6.4-6.6.

The urea can generate ammoniation, the porosity of the soybean meal, the corn starch and the corn steep liquor dry powder is increased after ammoniation treatment, the contact area of the urea and the enzyme is increased, and the fermentation is facilitated; the alpha-sodium naphthalene acetate and the dimethyl phosphate in the special proportion have a synergistic effect, can promote the bacillus subtilis to enhance the secretion of acetyl coenzyme A, can greatly improve the tricarboxylic acid cycle speed of the bacillus subtilis, accelerate the growth, the propagation, the fermentation and the metabolism of the bacillus subtilis, promote the bacillus subtilis to metabolize more enzyme to act on a fermentation substrate, and further improve the yield and the output of the antibacterial peptide.

The step 3) is specifically as follows: inoculating the spare seed liquid to a fermentation culture medium according to an inoculation ratio (v/v) of 12-15%, culturing at 37 ℃ for 24-28h, wherein the stirring speed is 155-165r/min and air is continuously introduced in the culture process, and performing thermal shock every 6-8h in the culture process, wherein the thermal shock step is as follows: heating the fermentation liquid within 8-12min to 5-10 deg.C, and cooling to 37 deg.C within 8-12 min.

Preferably, the Bacillus subtilis is Bacillus subtilis BF 168. In addition, the bacillus subtilis can also be a strain disclosed by Xujian and the like, and the construction and characteristic research (2016 of Yanshan university) of the bacillus subtilis for expressing the porcine beta-defensin-2 and cecropin P1 antibacterial peptides, and can also be other bacillus subtilis strains capable of producing the antibacterial peptides.

Another object of the present invention is to provide the antibacterial peptide prepared by the fermentation process.

Still another object of the present invention is to provide a composite veterinary drug product comprising an antimicrobial peptide prepared by the aforementioned fermentation process and Bacillus subtilis, which may be Bacillus subtilis bf 168. By adopting the composite medicine of the antibacterial peptide and the bacillus subtilis, not only can the quick, accurate and powerful action of the antibacterial peptide be exerted, but also the effective components in the bacillus subtilis except the antibacterial peptide can be exerted (the digestion and absorption of animal intestinal tracts on nutrient substances are promoted, and the like), and the bacillus subtilis can also exert the intestinal tract constant value action. In the compound medicine, the mass ratio of the antibacterial peptide to the bacillus subtilis is 1: 1-2. The compound medicine can be added into feed for use, and the addition amount of the compound medicine is 0.2-0.8% (by mass) of the feed.

The invention also provides the antibacterial peptide, the fermentation process and the application of the composite veterinary drug in preparing the livestock and poultry disease-resistant yield-increasing drug.

The antibacterial peptide is combined through the physical action of anions and cations, after the antibacterial peptide contacts pathogenic bacteria, positive charges carried by the N end of the antibacterial peptide are electrostatically attracted with a large number of negative charges carried by the cell membrane of the pathogenic bacteria, and the hydrophobic C end is flexibly inserted into a hydrophobic area of the plasma membrane of the pathogenic bacteria to form an ion channel on the plasma membrane of the pathogenic bacteria, so that the cell contents of the pathogenic bacteria escape and die. The use effect of the feed can promote the growth of intestinal villi, increase the absorption area of nutrient substances, improve the feed reward and have obvious growth promoting effect. The non-specific immune response product can improve the immunity of the organism and enhance the disease resistance after being added for a long time, and the drug resistance is not easy to generate.

Compared with the prior art, the invention has the beneficial effects that: the bacillus subtilis is cultured in a seed culture medium which is weakly acidic but not neutral, and the seed culture medium with slightly high stirring speed and specific components and proportion is adopted, so that researches show that the culture time can be shortened, and the proliferation speed of strains can be improved. The inoculation proportion selected in the fermentation process is relatively high, the stirring speed is high, the concentration of the finally obtained fermentation liquor antibacterial peptide can be improved, and the fermentation time is shortened. The thermal shock operation is to prevent the fermentation efficiency from being reduced due to the increase of the viscosity of the fermentation broth as the fermentation is continued. The thermal shock operation can reduce the viscosity of the fermentation liquor, eliminate strains with poor or weak metabolic capability and improve the fermentation efficiency.

The antibacterial peptide, the bacillus subtilis and the compound medicine are used for preventing and treating animal diarrhea, promoting intestinal digestion and absorption of livestock and poultry, and realizing disease resistance and yield increase of the livestock and poultry.

Detailed Description

The following describes embodiments of the present invention with reference to examples.