CN113913325A

CN113913325A - Bacillus subtilis, Ericin1a antibacterial peptide secreted by bacillus subtilis and application thereof

Info

Publication number: CN113913325A
Application number: CN202111068805.6A
Authority: CN
Inventors: 魏忠华; 杨波; 李杰峰; 李茜; 刘亚娟; 陈赛娟; 王珏; 贾国新; 王圆圆; 张绍兵; 高玉梅
Original assignee: Jinfusai Beijing Biotechnology Co ltd; HEBEI INSTITUTE OF ANIMAL HUSBANDRY AND VETERINARY
Current assignee: Jinfusai Beijing Biotechnology Co ltd; HEBEI INSTITUTE OF ANIMAL HUSBANDRY AND VETERINARY
Priority date: 2021-09-10
Filing date: 2021-09-13
Publication date: 2022-01-11
Anticipated expiration: 2041-09-13
Also published as: CN113913325B

Abstract

The invention discloses a bacillus subtilis with a preservation number of: CGMCC No. 23153. The bacillus subtilis is a strain with further enhanced antibacterial activity obtained by mutagenesis and screening, can continuously secrete and express antibacterial peptide in animal intestinal tracts, has the advantages of probiotics and small antibacterial activity peptides, has no toxic or side effect and no antibiotic residue, replaces the traditional antibiotics, and improves the technical level of life science. The Ericin1a antibacterial peptide provided by the invention can replace antibiotics to prepare bacteriostatic agents for preventing bacterial diseases, and plays an important role in reducing abuse of antibiotics. The invention discloses application of Ericin1a antibacterial peptide in preparation of a bacteriostatic agent for gram-negative bacteria. The invention discloses application of bacillus subtilis in preparing livestock feed for preventing or treating gram-negative bacterial infectious diseases. The invention discloses a livestock feed, which is added with bacillus subtilis.

Description

Bacillus subtilis, Ericin1a antibacterial peptide secreted by bacillus subtilis and application thereof

Technical Field

The invention relates to the technical field of bioengineering, and particularly relates to bacillus subtilis, Ericin1a antibacterial peptide secreted by the bacillus subtilis and application of the Ericin1a antibacterial peptide.

Background

The abuse of antibiotics is a very serious problem in the aquaculture industry. Not only do they cause antibiotic residues in meat, eggs and milk that affect human health, but also the rapid emergence of superbacteria that are resistant to multiple antibiotics has become a global concern over the past decade, prompting the search for alternative antimicrobial agents for livestock breeding. Antimicrobial peptides (AMPs) produced by bacteria, insects, amphibians and mammals, as well as by chemical synthesis, are promising candidates for new antimicrobial agents as alternatives to antibiotics due to their natural antimicrobial properties, lack of residues, and low tendency to develop resistance to microorganisms. According to the latest research progress abroad, England scientists such as Y.Kaznessis, D' Silva, L.Dicks and the like successfully apply the probiotics expressing the antibacterial active small peptide to experimental animals and breeding, obtain good antibacterial curative effect, and have no adverse reaction through long-term observation. A korean study showed that the addition of 60mg/kg of antimicrobial peptide P5(AMP-P5) instead of antibiotics to the daily feed improves the growth performance, nutrient absorption, intestinal health of broilers and reduces the intestinal flora in the intestines and feces of broilers.

The antibacterial peptide is a small molecular polypeptide with bacteriostatic activity generated by induction in an organism. Insects, mammals, microorganisms and the like of some species have the ability to produce small peptides having antibacterial activity, such as bombesin (Magainins) isolated from the skin of amphibian frogs and cecropin (AD) secreted from the larvae of the insect bombyx mori. The antibacterial active small peptide is a pure natural peptide, has broad-spectrum antibacterial activity, and can quickly search and kill a target, so that the antibacterial active small peptide can quickly become a potential therapeutic drug. The mechanism of action of antimicrobially active small peptides has not been fully elucidated to date. Current research shows that small peptides with antibacterial activity act by acting on the bacterial cell membrane. The cecropin antibacterial active small peptide acts on cell membrane, and is inserted into phospholipid membrane to form transmembrane channel, so as to destroy membrane integrity, cause intracellular substance leakage, and kill cells. However, the specific action process, the existence of specific membrane receptors, the existence of other factors, and the like are not clear, and different opinions exist. The mechanism of action of different antibacterial active small peptides may be different and still need further research. The antibacterial active small peptide has broad-spectrum antibacterial activity, and the action range comprises: gram negative bacteria, gram positive bacteria, fungi, parasites, tumor cells, and the like. The antibacterial active small peptide has strong killing effect on bacteria, and particularly has the killing effect on certain drug-resistant pathogenic bacteria, which draws more attention.

Live pigs, laying hens and broilers in livestock and poultry breeding in China have important proportions, various bacterial infectious diseases seriously threaten the breeding industry, so that the feed conversion rate is reduced, the survival rate is reduced, the weight is slowly increased, the growth and development are stopped and even die, and the healthy development of the breeding industry is seriously threatened. For example, piglet diarrhea is a typical multifactorial disease under intensive pig-raising production conditions, mainly caused by gram-negative bacteria and gram-negative bacteria treponema hyodysenteriae, has long epidemic period, high morbidity, low mortality and easy relapse; the chicken salmonellosis is a group of infectious diseases caused by salmonella members, mainly comprises pullorum disease, chicken typhoid and chicken paratyphoid, has high morbidity and mortality, reduces the production performance even if sick chicken recover, always carries bacteria for the whole life to become an infection source, and has great harm to a breeding farm; salmonellosis is a common disease of both human and animals, and food poisoning of people is often caused by bacteria-carrying products, so that the salmonellosis is a great harm to public health.

In order to prevent and treat bacterial diarrhea and other bacterial infectious diseases of pigs and salmonella diseases of chickens, various antibiotics and chemical drugs are used in farms in a large amount, so that the content of antibiotics and chemical drugs in meat exceeds the standard, the health of people is threatened, and the quality and the price of meat are difficult to improve.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.

The invention also aims to provide the bacillus subtilis, which is a strain obtained by mutagenesis and screening and further enhanced in antibacterial activity, can continuously secrete and express Ericin1a antibacterial peptide in animal intestinal tracts, has the advantages of probiotics and antibacterial activity small peptide, has no toxic or side effect and antibiotic residue, replaces the traditional antibiotics, and improves the technical level of life science.

Still another object of the present invention is to provide an Ericin1a antibacterial peptide, which can replace antibiotics to prepare bacteriostatic agents for preventing bacterial diseases, and plays an important role in reducing abuse of antibiotics.

To achieve these objects and other advantages in accordance with the present invention, there is provided a strain of Bacillus subtilis GF158, which has been deposited at the china general microbiological culture collection center on 8/17 th 2021, with the following deposition addresses: the Beijing West Lu No. 1 Hospital No. 3 of Chaoyang district, the preservation number is: CGMCC No. 23153.

An Ericin1a antibacterial peptide, wherein the Ericin1a antibacterial peptide is secreted from Bacillus subtilis.

Preferably, the amino acid sequence of the Ericin1a antibacterial peptide is shown as SEQ ID NO. 1.

Preferably, the nucleotide sequence of the Ericin1a antibacterial peptide is shown as SEQ ID NO. 2.

An application of Ericin1a antibacterial peptide in preparing a bacteriostatic agent for gram-negative bacteria.

Preferably, the gram-negative bacteria include Escherichia coli, Salmonella typhimurium, or Shigella dysenteriae.

An application of Bacillus subtilis in preparing the feed of fowls and animals for preventing and treating the infectious diseases caused by gram-negative bacteria is disclosed.

A livestock feed supplemented with the Bacillus subtilis of claim 1.

Preferably, the addition amount of the bacillus subtilis in the livestock feed is 150-600 g/T.

The invention at least comprises the following beneficial effects:

based on the idea of biological control, the invention widely screens probiotics capable of effectively inhibiting pathogenic bacteria from soil in various places, screens and obtains a strain with further enhanced antibacterial activity by a mutagenesis method, so that the strain can continuously secrete and express Ericin1a antibacterial peptide in animal intestinal tracts, has the advantages of probiotics and antibacterial activity small peptide, has no toxic or side effect and antibiotic residue, replaces the use of traditional antibiotics, and improves the technical level of life science.

The bacillus subtilis provided by the invention has a probiotic function, secretes and provides Ericin1a antibacterial peptide with a disease-resistant function, and is rarely seen in domestic markets at present. The Ericin1a antibacterial peptide provided by the invention is a natural high-efficiency antibacterial bioactive small peptide existing in nature, has the advantages of broad-spectrum antibacterial property, no toxic or side effect, no antibody generation, no drug resistance, no residue and no pollution, is a real green antibacterial agent, can greatly promote and solve the food safety problem of drug residue, enhances the development of green livestock breeding industry, and protects the food safety of Jingjin Ji people.

According to the invention, a proper amount of bacillus subtilis is added into the livestock feed, after the bacillus subtilis is ingested by animals, thalli can be damaged by strong acid gastric acid, so that probiotics can reach the digestive tract and propagate in the intestinal tract in a large quantity, and Ericin1a antibacterial peptide is continuously secreted into the intestinal tract of the animals, so that the bacillus subtilis can effectively replace antibiotics, and has an effective prevention effect on livestock infectious diseases caused by bacteria. The expression gene is positioned in the genome of the probiotics, and antibiotics are not needed to be added to maintain the existence of plasmids, and an inducer is not needed to be added to induce the expression. The bacillus subtilis strain does not contain any antibiotic resistance selective marker, and the biological safety is greatly improved.

The bacillus subtilis provided by the invention can be directly added into feed, not only has a continuous protection effect on livestock organisms, but also can greatly reduce the use of traditional antibiotics, is non-toxic and harmless, and is a green biological protective agent.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a three-dimensional structure of Ericin1a antimicrobial peptide of the present invention;

FIG. 2 is a bacteriostatic experiment of Bacillus subtilis GF158 (preservation number CGMCC number 23153) producing Ericin1a antibacterial peptide on Escherichia coli (left picture), Salmonella (middle picture) and Shigella dysenteriae (right picture);

FIG. 3 shows the organ weight ratio of the Bacillus subtilis GF158 in the mouse biosafety test;

FIG. 4 is a survival rate experiment of broiler chickens fed with poultry feed supplemented with Bacillus subtilis GF158 producing Ericin1a antimicrobial peptide, wherein 300 g of Bacillus subtilis GF158 is added to each ton of chicken feed;

FIG. 5 is a survival rate test of broiler chickens fed with poultry feed supplemented with Bacillus subtilis GF158 producing Ericin1a antimicrobial peptide, wherein 150g of Bacillus subtilis GF158 is added to each ton of chicken feed;

FIG. 6 is a survival rate test of broiler chickens fed with poultry feed supplemented with Bacillus subtilis GF158 producing Ericin1a antimicrobial peptide, wherein each ton of chicken feed is added with 600g of Bacillus subtilis GF 158;

FIG. 7 is a survival rate experiment of broiler chickens fed with poultry feed supplemented with Bacillus subtilis GF158 producing Ericin1a antimicrobial peptide, wherein 300 g of Bacillus subtilis GF158 is added per ton of pig feed.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

Example 1

1.1 separation, screening and identification of efficient antibacterial peptide probiotic strains

Separating and screening strains, sampling soil of a certain place of Beijing Pinggu, and separating the bacillus subtilis. Firstly weighing 1-10g of soil, pouring into a sterilized conical flask, adding 50-200ml of sterilized double distilled water, uniformly stirring by using a glass rod wiped by 70% alcohol cotton, and oscillating on a vortex mixer for 1-10 minutes to fully release microorganisms in the soil into the solution. Because the bacillus subtilis can generate spores with strong stress resistance and can tolerate high temperature, other soil microorganisms which are not heat-resistant are killed by adopting a high-temperature treatment method, so that the bacillus subtilis is selectively screened.

The conical flask is put into a water bath kettle with the temperature of 75-95 ℃ to be boiled for 12-20 minutes, and most of microorganisms which cannot resist high temperature can be killed in the process. After the reaction is finished, the conical flask is placed at room temperature for natural cooling. Then 1x10 with sterilized double distilled water²、1x10⁴And 1x10⁶The gradient dilution is carried out, 200-300ul of the dilution liquid is respectively taken and coated on an LB plate or other plates suitable for the growth of the bacillus subtilis, the mixture is cultured for 24-48 hours at the temperature of 28-37 ℃, and the grown colonies are analyzed.

And (3) carrying out 96-well plate analysis on the strains obtained by screening by using a Biolog strain identification system, and determining the screened strains as a strain of bacillus subtilis.

1.2 mutagenesis enhancement of strains

The screened bacillus subtilis probiotics have a certain inhibition effect on pathogenic bacteria, but the bacteriostasis is not high enough, so that the bacteriostasis is enhanced by adopting a mutation inducing method. The specific method comprises the following steps: single colonies were streaked on LB plates or other nutrient agar plates suitable for growth of Bacillus subtilis, and cultured overnight at 37 ℃. Single colonies with healthy morphology were picked, inoculated with 10 ml of LB liquid medium and cultured overnight.

The bacterial suspension was centrifuged at 1500-.

1500-.

The cells were resuspended in sterile water, diluted by an appropriate fold, and the absorbance at 600nm was measured using a visible spectrophotometer. The corresponding relationship between the absorbance and the number of the cells is as follows: when OD600 is 1, the number of bacteria per ml is about 8 × 10⁸And (4) respectively.

Based on the cell density, 1X10 was sampled⁸The cells were centrifuged to collect the cells, and the supernatant was discarded.

Preparation of EMS working solution: 50ul of liquid EMS (Ethyl metabisulphonate, also known as ethylmethanesulfonate, Sigma # M-0880) was added to a centrifuge tube containing 2ml of LB medium in a fume hood to make a 0.1M EMS solution. Gently stir until the viscous oily liquid completely dissolves. EMS working solutions were prepared in the same manner at concentrations of 25. mu.l/ml, 50. mu.l/ml, 75. mu.l/ml and 100. mu.l/ml.

EMS mutagenesis: the cells were gently resuspended in EMS working solution and left to stand at room temperature for 30,60,90,120 minutes, respectively. After the treatment, 1500-.

Diluting EMS-treated bacteria solution with bacteria PBS phosphate buffer solution, sterilized deionized water or other bacteria-free sterile solution to 1x10²-1x10⁷And coating the bacterial liquid with different dilutions on LB plate, and culturing at 37 deg.C for 24-48 h.

Inactivation of EMS solution: EMS solution was mixed with an equal volume of "inactivation solution" [0.1M NaOH, 20% w/v Na₂S₂O₃(sodium thiosulfate)]Mix for 24 hours to inactivate. All EMS-contaminated pipettes/tubes should be soaked in the inactivation solution for 24 hours prior to treatment.

Sequence comparison of mutagenized Ericin1a antimicrobial peptide to the original antimicrobial peptide:

sequencing shows that 4 mutations occur in the gene sequence of the Ericin antibacterial peptide screened by mutagenesis.

The method comprises the following steps: E4A, V15K, L16K and Q22F

Upper line:Ericin S,from 1to 32

Lower line:Modified Ericin that has enhanced antibiotic activity,from 1to 32

Ericin:Modified Ericin1a that has enhanced antibiotic activity identity＝87.5％(28/32)gap＝0.00％(0/32)

Compared with the original sequence, 4 of the 32 amino acids of the Ericin1a antibacterial peptide generate mutations, and the 3D structure of the Ericin1a antibacterial peptide is shown in FIG. 1.

Example 2

Detection of bacteriostatic activity

And respectively picking out the grown single colonies, and detecting the bacteriostatic activity of the single colonies on gram-negative pathogenic bacteria separated from chicken intestinal tracts and excrement after amplification culture.

The mutation strain with the bacteriostatic effect is detected by a classical bacteriostatic circle method, which comprises the following steps:

(1) inoculating experimental bacillus subtilis and control bacillus subtilis into 5mL LB liquid culture medium, and culturing at 37 ℃ and 200r/min for 12-16 h.

(2) Salmonella isolated from the feces of sick chickens were streaked on LB solid medium and cultured overnight at 37 ℃.

(3) And picking a single colony of the salmonella by using an inoculating needle, inoculating the single colony into an LB liquid culture medium, and culturing at 37 ℃ and 200r/min for 12-24 h.

(4) The cultured salmonella stock solution was diluted 10-fold with sterile water, and the bacterial concentration was measured spectrophotometrically at 560 nm.

(5) According to the measured concentration, the stock solution was diluted by a corresponding factor, and 100-250. mu.L of the diluted stock solution was pipetted and spread on an LB plate.

(6) A fermentation broth of Bacillus subtilis control, Bacillus foeniculi control, Bacillus subtilis GF118, which was commercially available, was centrifuged at 12000r/min for 1min, the supernatant was aspirated into a disposable syringe and sterile filtered through a sterilized needle filter (0.45 μm), and a kanamycin (1000 μ g/mL) (positive control) solution was prepared and sterile filtered.

(7) Four oxford cups are evenly placed on the LB solid culture medium coated with the E.coli pathogenic bacteria and marked, then the fermentation liquor of the commercial bacillus subtilis contrast, the bacillus fomentillis contrast and the bacillus subtilis GF118 is filled in the oxford cup, and 20 mu L kanamycin (positive contrast) is added in the rest oxford cup.

(8) Putting the finished plate into a constant-temperature incubator at 30-37 ℃ for culturing for 12-36 hours, and observing the size of the inhibition zone.

The result is shown in fig. 2, the fermentation supernatant of GF158 probiotics producing Ericin1a antibacterial peptide has obvious inhibiting effect on gram-negative pathogenic bacteria such as escherichia coli, salmonella, shigella dysenteriae and the like.

Example 3

Biological safety experiment

A mouse safety experiment is carried out, bacillus subtilis GF158 of the Ericin1a antibacterial peptide is added into mouse food in different bacterial amounts, BALB/c mice of SPF grade 4 weeks old are selected and randomly divided into 4 groups, 10 mice in each group are continuously fed for 2 weeks, and the groups are weighed. After the test, mice in the control group and the experimental group are dissected, and the liver, the kidney, the spleen and the lung are respectively weighed.

The experimental results show that the weight ratio of each organ of the test mice added with different dosages in the feed is not much different from that of the control group (figure 3), and the bacillus subtilis GF158 does not cause the side effect of organ swelling on the test mice. In the aspect of overall weight gain, the overall weight gain speed of the experimental group added with 1-time dose, 10-time dose and 50-time dose and the overall weight gain speed of the control group in 0-7 days and 0-14 days are not obviously reduced or even slightly improved (table 1), so that the bacillus subtilis GF158 is known to have safety.

TABLE 1 comparison of average daily gain for each group

Test group	Head with a rotatable shaft	Experiment period of 0-7 days	Experiment period of 0-14 days
				Control group	5	1.09±0.30	1.53±0.27
1X dosing	10	1.17±0.15	1.59±0.12
				10X dose addition	10	1.23±0.24	1.80±0.30
50X dose addition	10	1.16±0.09	1.62±0.10

Example 4

Experiment of survival rate of broiler chickens

In the same chicken farm, 2000 white feather broilers of the same chicken house, the same variety and the age of day are selected and randomly divided into 2 groups of 1000 broilers. 300 g of bacillus subtilis GF158 producing Ericin1a antibacterial peptide is added into each ton of chicken feed in the experimental group, and the bacillus subtilis GF158 is not added in the control group. The survival rate was calculated at the end of the experiment by continuous feeding for 3 weeks using the principle of double blind experiments. The calculation formula is adopted as follows:

the total survival rate is 3 weeks later, the residual broiler number is divided by the initial broiler number x 100 percent in the experiment

The experimental results are shown in fig. 4, the survival rate of the experimental group with 300 g bacillus subtilis GF158 producing Ericin1a antibacterial peptide added to each ton of chicken feed is 99.1% after 3 weeks, while the survival rate of the control group without bacillus subtilis GF158 is only 94.2%. Therefore, the survival rate of the broiler chicken can be remarkably improved by adding bacillus subtilis GF158 for producing Ericin1a antibacterial peptide into the feed.

Example 5

Experiment of survival rate of broiler chickens

In the same chicken farm, 2000 white feather broilers of the same chicken house, the same variety and the age of day are selected and randomly divided into 2 groups of 1000 broilers. 150g of bacillus subtilis GF158 producing Ericin1a antibacterial peptide is added into each ton of chicken feed in the experimental group, and the bacillus subtilis GF158 is not added in the control group. The survival rate was calculated at the end of the experiment by continuous feeding for 3 weeks using the principle of double blind experiments. The calculation formula is adopted as follows:

The experimental results are shown in fig. 5, the survival rate of the experimental group containing 150g of bacillus subtilis GF158 producing Ericin1a antimicrobial peptide per ton of chicken feed after 3 weeks was 98.6%, while the survival rate of the control group not containing bacillus subtilis GF158 was only 94.3%. Therefore, the survival rate of the broiler chicken can be remarkably improved by adding bacillus subtilis GF158 for producing Ericin1a antibacterial peptide into the feed.

Example 6

Experiment of survival rate of broiler chickens

In the same chicken farm, 2000 white feather broilers of the same chicken house, the same variety and the age of day are selected and randomly divided into 2 groups of 1000 broilers. 600g of bacillus subtilis GF158 producing Ericin1a antibacterial peptide is added into each ton of chicken feed in the experimental group, and the bacillus subtilis GF158 is not added in the control group. The survival rate was calculated at the end of the experiment by continuous feeding for 3 weeks using the principle of double blind experiments. The calculation formula is adopted as follows:

The experimental results are shown in fig. 6, the survival rate of the experimental group containing 600g of bacillus subtilis GF158 producing Ericin1a antimicrobial peptide per ton of chicken feed after 3 weeks was 99.8%, while the survival rate of the control group not containing bacillus subtilis GF158 was only 94.1%. Therefore, the survival rate of the broiler chicken can be remarkably improved by adding bacillus subtilis GF158 for producing Ericin1a antibacterial peptide into the feed.

Example 7

Experiment of livestock survival rate

300 piglets with the weight of about 12kg and about 42 days are selected and randomly divided into 6 groups, 3 experimental groups and 3 control groups, and each group comprises 50 piglets. 300 g of bacillus subtilis GF158 probiotics for producing Ericin1a antibacterial peptide is added into each ton of pig feed in the experimental group, and the control group is not added. The principle of double blind experiment is adopted, the feeding is continuously carried out for 90 days until the slaughtering, and the survival rate is calculated when the experiment is finished. The calculation formula is adopted as follows:

total survival rate is the number of live pigs after 90 days ÷ initial number of pigs tested x 100%

The experimental results are shown in fig. 7, the survival rate of the experimental group with 300 g bacillus subtilis GF158 probiotic bacteria producing Ericin1a antimicrobial peptide added to each ton of feed is 96.6% after 90 days, while the survival rate of the control group without bacillus subtilis GF158 added is only 85.1%. Therefore, the survival rate of pigs can be obviously improved by adding the bacillus subtilis GF158 probiotics for producing Ericin1a antibacterial peptide into the feed.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

<110> Jinfusai (Beijing) Biotechnology Ltd

<120> bacillus subtilis, Ericin1a antibacterial peptide secreted by bacillus subtilis and application thereof

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Claims

1. A bacillus subtilis strain is characterized in that the preservation number is as follows: CGMCC No. 23153.

2. An Ericin1a antimicrobial peptide, wherein the Ericin1a antimicrobial peptide is secreted from the Bacillus subtilis of claim 1.

3. The Ericin1a antimicrobial peptide of claim 2, wherein the Ericin1a antimicrobial peptide has the amino acid sequence shown in SEQ ID NO. 1.

4. The Ericin1a antimicrobial peptide of claim 2, wherein the nucleotide sequence of the Ericin1a antimicrobial peptide is set forth in SEQ ID NO. 2.

5. Use of the Ericin1a antimicrobial peptide of any one of claims 2-4 in the preparation of a bacteriostatic agent against gram-negative bacteria.

6. The use of claim 5, wherein the gram-negative bacteria comprise Escherichia coli, Salmonella typhimurium, or Shigella dysenteriae.

7. Use of the bacillus subtilis of claim 1 for the preparation of a livestock feed for the prevention or treatment of gram-negative bacterial infectious disease.

8. A livestock feed characterized by comprising the Bacillus subtilis of claim 1.

9. The livestock feed of claim 8, wherein Bacillus subtilis is added in an amount of 150-600 g/T.