CN114107134B - Brevibacillus laterosporus and application thereof - Google Patents
Brevibacillus laterosporus and application thereof Download PDFInfo
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- CN114107134B CN114107134B CN202111582162.7A CN202111582162A CN114107134B CN 114107134 B CN114107134 B CN 114107134B CN 202111582162 A CN202111582162 A CN 202111582162A CN 114107134 B CN114107134 B CN 114107134B
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Abstract
The application provides a strain of Brevibacillus laterosporus named as Brevibacillus laterosporus BLCC1-0716, which has been deposited in China center for type culture Collection at 11.11.2021 with the deposit numbers: CCTCC NO: m20211397. The strain is obtained by screening and separating medicinal plant honeysuckle tissue, has the advantages of wide antibacterial spectrum, good antibacterial performance, good tolerance and safety, and has good functions of resisting inflammation, protecting intestinal tract and preventing and treating enteritis.
Description
Technical Field
The application relates to the technical field of biology, in particular to a Brevibacillus laterosporus strain and application thereof.
Background
The information disclosed in this background of the invention is intended to enhance an understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information has become known as prior art to a person skilled in the art.
With the development of high density and intensification of breeding industry, the breeding environment of livestock and poultry is more and more severe, and the production level and the health condition of animals are seriously influenced. Abuse of antibiotics causes drug-resistant bacteria, drug residues, environmental pollution and the like, and potential safety hazards are generated to human health. Therefore, research on the application of new green additives to replace antibiotics has become a hot topic of feed additive research.
The bacillus has the advantages of strong resistance, low nutritional requirement, high growth and propagation speed, strong adaptability to the external environment and the like. Can inhibit the colonization or proliferation of external pathogenic microorganisms in the gastrointestinal tract, regulate the disturbance of animal intestinal flora, reduce the occurrence of intestinal diseases and further improve the disease resistance of organisms. In addition, researches show that the bacillus can also regulate the balance of intestinal flora in the organism, enhance the immune function of the organism, secrete various enzymes and vitamins and effectively promote the digestion and absorption of nutrient substances. As a novel green microecological preparation, the bacillus has very wide application prospect and larger market demand. Brevibacillus laterosporus is widely distributed in nature, has pathogenicity on invertebrates and has an inhibiting effect on a plurality of harmful microorganisms, more than 30 microorganisms used for feed additives are published in 2014 of Ministry of agriculture (publication No. 2045 feed additive variety catalog (2013) of Ministry of agriculture), wherein Brevibacillus laterosporus (original name Bacillus laterosporus) is listed, can be used for breeding broilers, meat ducks, pigs and shrimps and is widely applied to agriculture for pest control.
Probiotics function as probiotics by being ingested directly by the animal body, and the safety of probiotics is a prerequisite for its possible use. Currently, probiotic evaluation is primarily initiated by safety and efficacy, including primarily in vivo and in vitro evaluations. In-vitro effectiveness evaluation is mainly used for evaluating and confirming the probiotic effect of the strain from the aspects of bacteriostatic activity, tolerance and the like of the strain. The in vivo efficacy evaluation of probiotics is based on in vitro efficacy evaluation, and no matter how good the in vitro efficacy of the strain is, if the in vivo efficacy is not, the strain cannot be stated to have the probiotic property.
The intestinal tract is closely related to the healthy growth of animals, so that the health of the intestinal tract is protected, and the intestinal tract has an important effect on the growth of the animals. The intestinal tract is the most important digestion and absorption place of higher animals, and the healthy intestinal tract is extremely important to the growth and development of the animals. The intestinal mucosa has rich vascular networks, and inflammation occurring in the intestinal tract is easy to cause multi-system inflammation of animal organisms. Meanwhile, the occurrence of intestinal inflammation can seriously affect the digestion and absorption functions of animal organisms, cause animal malnutrition, emaciation, growth obstruction and even death, and bring huge loss to the breeding production. When the organism is in an adverse state such as stress or the abnormal proliferation of pathogenic microorganisms, the barrier function of the intestinal tract is easily affected, so that unbalance is caused, and the injury exceeds the self-repair level of the organism, thereby causing the occurrence of diseases. Pathogenic bacteria act on epithelial cells of the intestinal mucosa to cause damage and apoptosis of the epithelial cells, so that the pathogens enter submucosa, activate immune related cells in the intestinal mucosa, promote the immune related cells to secrete inflammatory cytokines and chemokines, cause inflammation and cause intestinal damage. At present, the bacillus has more reports on the aspects of in-vitro bacteriostasis, safety evaluation and the like, but has less reports on the aspects of animal intestinal inflammation intervention and repair.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides the brevibacillus laterosporus which has a wide antibacterial spectrum, has good antibacterial effects on common livestock and poultry pathogenic bacteria such as escherichia coli, salmonella, streptococcus pyogenes, serratia marcescens, bacillus cereus, bacillus circulans, clostridium perfringens and the like, and also has good antibacterial effects on aquatic common pathogenic bacteria such as vibrio alginolyticus, vibrio parahaemolyticus, aeromonas hydrophila and the like; in addition, the bacterium has good safety and stability; and the composition shows good effects of resisting inflammation, preventing and treating enteritis (including acute enteritis, ulcerative colitis and the like) and improving intestinal disorder in animal experiments, can play good roles of preventing and treating colon atrophy, preventing intestinal injury and the like and protecting intestinal health, and shows good application prospects.
Specifically, the technical scheme of the invention is as follows:
in the first aspect of the invention, the invention provides a Brevibacillus laterosporus strain named Brevibacillus laterosporus BLCC1-0716 which has been preserved in China center for type culture Collection (CCTCC for short, with the address: Wuhan, university of Wuhan, China) at 11 months and 11 days 2021, with the preservation number: CCTCC NO: m20211397.
The brevibacillus laterosporus BLCC1-0716 is obtained by separating and screening honeysuckle stems from local town of Pingyi county in Linyi city, Shandong province, and is a medicinal plant endophyte. The Brevibacillus laterosporus BLCC1-0716 is gram-positive, and the thallus is rod-shaped and can form special canoe accompanying spores. The strain is cultured at 37 ℃ for 24h to form a light yellow colony which is moist and glossy, is tightly attached to a culture medium, does not bulge and has a neat edge. The BLCC1-0716 is aerobic bacteria, can utilize oxygen in intestinal tract to create anaerobic environment, and can promote proliferation of lactic acid bacteria of facultative anaerobe.
The Bacillus laterosporus BLCC1-0716 of the present invention was identified and found to have the biochemical characteristics shown in Table 4:
TABLE 4 physiological and biochemical identification results of Brevibacillus laterosporus BLCC1-0716
Note: +: positive; -: and (4) negativity.
In some embodiments of the present invention, the present invention provides a culture medium suitable for brevibacillus laterosporus BLCC1-0716, which comprises: 0.5 percent of glucose, 1.0 percent of peptone, 0.5 percent of beef extract and 0.5 percent of sodium chloride, adjusting the pH value to 6.5, and sterilizing for 30min at the temperature of 120 ℃, wherein the percentages are mass percentages.
Good safety is an important prerequisite for the use of microorganisms, and therefore, in the embodiment of the present invention, the inventors verified the safety of Brevibacillus laterosporus BLCC1-0716 and found that 10 or more 8 -10 10 The body state, internal organs and the like of the mice are not damaged and the body weight of the mice is not significantly different when the mice are treated by CFU/CFU dosage, and the bacteria is safe for the mice at the dosage of at least 100 hundred million by intragastric administration.
And, in some embodiments of the invention, the inventors have experimentally found that brevibacillus laterosporus BLCC1-0716 shows good tolerance to bile salts, as indicated by treating the bacteria with different concentrations of bile salts, and there is almost no loss of viable count during short treatment, until after 4 hours of action, as the concentration of bile salts increases, the survival rate of BLCC1-0716 begins to decrease, and the survival rate of 0.1% concentration of bile salts after 4 hours of action is 99.87%; the survival rate can reach 73.57 percent after the bile salt with the concentration of 0.3 percent acts for 4 hours; the survival rate of the bile salt with the concentration of 0.5 percent still can reach 45.00 percent after the bile salt with the concentration of 0.5 percent acts for 4 hours.
In some embodiments of the invention, the inventor experimentally found that Brevibacillus laterosporus BLCC1-0716 shows good acid resistance and can survive well in artificial gastric juice and artificial intestinal juice. Wherein the survival rate can reach 75.86% under the pH2.5 condition, 97.44% under the pH3.5 condition and 103.13% under the pH4.5 condition after the reaction is carried out for 4 hours at 37 ℃; the survival rate of 4h in the artificial intestinal juice is up to 289.38%, and the survival rate of 4h in the artificial gastric juice is up to 120.28%.
The probiotics as the feed additive has the functions of bacteriostasis and good stress resistance. The gastrointestinal tract is an important organ for digestion and absorption of animals, and the balance of microflora plays an important role in maintaining the health of the animal body. The probiotics enter animal bodies through oral administration and must pass through the stomach and then colonize the small intestine, so that the probiotics can resist stronger acid environment and bile salt with higher concentration is an important prerequisite for good survival and normal function in the intestinal tract. The test proves that the strain BLCC1-0716 has good acid resistance, cholate resistance and tolerance in artificial gastrointestinal fluid, can smoothly enter the small intestine through the stomach to play a role, and improves the animal organism performance.
And, in some embodiments of the present invention, the inventors also found that the strain BLCC1-0716 shows good heat resistance, particularly when the strain BLCC1-0716 is treated in a high temperature water bath at 85 ℃ for 15min, and the survival rate of the strain can still reach 98.1%. The good heat resistance is beneficial to the storage of strains and the later-stage preparation treatment, overcomes the defects that most probiotics are thermolabile, difficult to survive and store at normal temperature and difficult to prepare at present, and has good market application prospect.
And, in some embodiments of the present invention, the inventors found that the strain BLCC1-0716 has a good bacteriostatic effect, a broad bacteriostatic spectrum, and a good bacteriostatic activity against at least 14 tested pathogenic bacteria, including but not limited to escherichia coli, staphylococcus aureus, clostridium perfringens, salmonella, streptococcus pyogenes, serratia marcescens, bacillus cereus, bacillus circulans, etc., all of which have a bacteriostatic circle of more than 12mm, and especially have a stronger bacteriostatic ability against clostridium perfringens, vibrio parahaemolyticus, and bacillus circulans, and all of which have a bacteriostatic circle of more than 18mm, which indicates that BLCC1-0716 can be used for preventing and treating livestock and poultry from pathogenic microorganisms such as escherichia coli, salmonella, streptococcus pyogenes, serratia marcescens, bacillus cereus, and bacillus circulans in a breeding process, Necrotic enteritis caused by clostridium perfringens and the like; and can be used for preventing and treating hemorrhagic septicemia and ulcer of fish caused by pathogenic bacteria such as aquatic pathogenic microorganisms Vibrio alginolyticus, Vibrio parahaemolyticus and Aeromonas hydrophila.
In a second aspect of the present invention, the present invention provides a use of Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0716, specifically, at least one selected from the following 1) to 6):
1) the application of Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0716 or its fermentation product or its metabolite in preparing anti-inflammatory product;
2) the application of Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0716 or its fermentation product or its metabolite in preparing product for preventing and treating diarrhea;
3) the application of Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0716 or its fermentation product or its metabolite in preparing product for preventing and treating enteritis; wherein the enteritis comprises enteritis caused by pathogenic bacteria such as enteritis caused by pathogenic microorganisms such as escherichia coli, salmonella, streptococcus pyogenes, serratia marcescens, bacillus cereus, bacillus circulans, clostridium perfringens and the like common to livestock and poultry; enteritis caused by said non-pathogenic bacteria such as acute enteritis and ulcerative enteritis (ulcerative colitis);
4) application of Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0716 or its fermentation product or its metabolite in preparing product for regulating digestive tract flora; wherein, the regulation of the digestive tract flora especially means the prevention and treatment of the digestive tract flora disturbance in the state of enteritis, and the enteritis is preferably acute enteritis and ulcerative enteritis;
5) the application of Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0716 or its fermentation product or its metabolite in preparing product for preventing and treating colon atrophy; wherein the colon atrophy is colon atrophy caused by enteritis; and/or the presence of a gas in the atmosphere,
6) the application of Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0716 or its fermentation product or its metabolite in preparing product for protecting intestinal tract; wherein, the protection of the intestinal tract refers to the protection of the intestinal tract caused by inflammation, pathogenic enteritis and non-pathogenic enteritis such as acute enteritis and ulcerative enteritis, and the protection includes but is not limited to keeping the intestinal tissue structure intact, such as the intestinal wall tissue clear, the villus structure intact, the outline clear, the arrangement orderly, the gland structure intact, the crypt normal and the like. The intestine is especially the colon.
In the above-mentioned application of the present invention, the Brevibacillus laterosporus is used as a fermentation product or a cell or a metabolite thereof. The fermented product is used for referring to a fermented product. The corresponding fermentation product can be obtained from the process of fermenting and culturing Brevibacillus laterosporus BLCC1-0716, and can be a solid fermentation product or a liquid fermentation product based on the difference of the culture forms, and the liquid fermentation product can also be called as fermentation liquor or culture solution; the fermentation product of the invention comprises thalli and metabolites thereof. In an embodiment of the present invention, a fermentation liquid or culture liquid containing bacterial cells is subjected to centrifugation, filtration, sedimentation, or other means known in the art to separate bacterial cells growing in the fermentation liquid or culture liquid from the liquid, and the liquid remaining when the bacterial cells are removed is a supernatant, and in the present invention, the supernatant contains a metabolite of Brevibacillus laterosporus BLCC 1-0716.
In the above application of the present invention, the product may be a medicine, a food, a health product or a feed additive.
In some embodiments of the invention, animal experiments are carried out, an animal acute enteritis model and an animal ulcerative colitis model are respectively constructed, and the effect of the brevibacillus laterosporus BLCC1-0716 is verified. Among them, the results of experiments on LPS-induced acute enteritis model revealed that different concentrations (3X 10) of the lavage fluid were used 6 ～10 8 CFU/Brevibacillus laterosporus BLCC1-0716 can relieve the diarrhea of mice, reduce the levels of mouse serum cytokines TNF-alpha, IL-6 and IL-1 beta caused by LPS (lipopolysaccharide) injected into the abdominal cavity at a dose of 10mg/kg, and increase the level of inhibiting inflammatory factors IL-10, so that the BLCC1-0716 is supposed to inhibit the generation of inflammation and relieve the inflammation through the two routes; and Brevibacillus laterosporus BLCC1-0716 can improve upper digestive tract flora disorder caused by LPS, increase the number of facultative anaerobe lactic acid bacteria, and reduce the number of intestinal pathogenic bacteria such as Escherichia coli and Staphylococcus aureus. In experimental results of a DSS ulcerative enteritis model, the Brevibacillus laterosporus BLCC1-0716 can inhibit weight loss of mice caused by DSS, can inhibit DSS-induced colon atrophy of mice, improve disease activity indexes of colitis of the mice, prevent and improve DSS-induced colon structure damage, such as inhibition and improvement of DSS-induced colon epithelial necrosis and desquamation, intestinal wall thinning, severe intestinal gland structure damage, glandular disorder, intestinal villus rupture and severe deletion, only leave a mucosal muscular layer and the adverse conditions of interstitial edema of the mucosal muscular layer. The results show that the brevibacillus laterosporus BLCC1-0716 can be well planted and survived in the intestinal tract of animals, can effectively relieve the intestinal inflammation of the animals, relieve diarrhea and improve intestinal lesion, has the functions of preventing and treating enteritis caused by non-pathogenic bacteria, such as acute enteritis, ulcerative colitis and the like, and has good prevention and treatment effects.
In addition, the strain BLCC1-0716 has good bacteriostatic property, so that it can be used as bacteriostatic agent or used for preparing bacteriostatic product.
In a third aspect of the present invention, the present invention provides a microbial preparation or feed, which comprises Brevibacillus laterosporus BLCC1-0716 described in the first aspect above and/or a fermentation product thereof and/or a metabolite thereof. The microbial agent or the feed can contain Brevibacillus laterosporus BLCC1-0716, and can also contain a fermentation product or a fermentation liquor thereof, and can also contain a metabolite thereof.
In some embodiments of the invention, the invention provides a culture medium particularly suitable for Brevibacillus laterosporus BLCC1-0716, which comprises the following components: 0.5 percent of glucose, 1.0 percent of peptone, 0.5 percent of beef extract and 0.5 percent of sodium chloride, adjusting the pH value to 6.5, and sterilizing for 30min at the temperature of 120 ℃, wherein the percentages are mass percentages.
And, accordingly, in an embodiment of the invention, the invention provides a method of obtaining a brevibacillus laterosporus BLCC1-0716 ferment or fermentation broth comprising: the brevibacillus laterosporus BLCC1-0716 is inoculated to a suitable culture medium and cultured by standing. The culture medium can be a solid culture medium or a liquid culture medium. Suitable media and culture conditions are as described above. On the basis of this, the person skilled in the art can also make suitable adjustments.
In a fourth aspect of the invention, the invention provides a pharmaceutical composition or a pharmaceutical formulation comprising Bacillus laterosporus BLCC1-0716 and/or its fermentates and/or its metabolites as described in the first aspect above.
In some embodiments of the present invention, the pharmaceutical composition or pharmaceutical preparation of the present invention comprises brevibacillus laterosporus BLCC1-0716, and based on this, the pharmaceutical composition or pharmaceutical preparation may further comprise at least one pharmaceutical carrier or pharmaceutically acceptable adjuvant, or other therapeutically effective agent. Suitable pharmaceutical Excipients may be of a kind known in the art, such as solvents, buffers, diluents and the like, and may be, for example, those described in the Handbook of pharmaceutical Excipients (Handbook of pharmaceutical Excipients) by the authors Paul J Sheskey et al. The pharmaceutical composition or pharmaceutical formulation may be in solid, semi-solid or liquid form, which may be prepared according to any conventional method known in the art. The feed can further comprise grain substances, trace elements, proteins and the like.
In some embodiments of the present invention, the microbial agent may be prepared into a bacterial powder or a bacterial liquid, the bacterial powder may further comprise a freeze-drying protective agent, the addition amount of the freeze-drying protective agent is 40-60% of the weight of the bacterial body obtained after the fermentation broth is centrifuged, and the freeze-drying protective agent may be a freeze-drying protective agent conventional in the art, such as skimmed milk powder, sucrose, and the like.
In a fifth aspect of the invention, the invention provides a method for treating enteritis, which comprises administering an effective dose of brevibacillus laterosporus BLCC1-0716 or a microbial inoculum, a pharmaceutical composition, a pharmaceutical preparation or a feed or the like comprising the strain to a subject. Such enteritis includes pathogenic (as defined above) or non-pathogenic (such as acute and ulcerative colitis); the administration mode is oral administration or intragastric administration.
In some embodiments of the invention, the lavage is performed more easily in the form of a bacterial suspension, which may be a liquid containing the bacterial cells, such as an aqueous solution or other safe and easy to use solution or suspension. For oral administration, it can be administered in liquid form or non-liquid form.
The "subject" refers to an animal, preferably a mouse, human or livestock avian animal, such as a pig, cow, sheep, horse, donkey, camel, rabbit, chicken, duck, goose, pigeon, quail, etc., that has been the subject of treatment, observation or experiment.
The "effective dose" refers to an amount that can induce a beneficial effect or ameliorate a condition such as diarrhea, hematochezia, colonic atrophy, dysbacteriosis in an animal, and in some embodiments of the invention, the effective dose of Brevibacillus laterosporus BLCC1-0716 is 1X 10 6 ～10 10 CFU/mL, preferably 1X 10 6 ～10 8 CFU/mL。
Compared with the prior art, the invention has the advantages that:
the brevibacillus laterosporus BLCC1-0716 has a wide antibacterial spectrum, has good in-vitro antibacterial action on common pathogenic bacteria of livestock and poultry and aquatic products, and can treat diseases (such as enteritis) caused by the pathogenic bacteria; and the BLCC1-0716 has good tolerance, good acid resistance and cholate resistance, can survive well in artificial gastrointestinal fluid, shows that the artificial gastrointestinal fluid can be well planted in animal intestinal tracts, has good heat resistance, can survive well at normal temperature and in the environment of not higher than 85 ℃, and is easy to store and prepare for production. Meanwhile, the brevibacillus laterosporus BLCC1-0716 has good safety performance, can inhibit diseases caused by pathogenic bacteria, has good treatment and improvement effects on enteritis caused by non-pathogenic bacteria such as acute enteritis, ulcerative colitis and the like, can relieve intestinal inflammation of animals, protect and improve intestinal lesions, improve flora disorder, improve the disease resistance of animals and is safe to feed.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application. Embodiments of the present application are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1: in example 1, the isolation of the endophytes from the leaves (left), roots (middle) and stems (right) of honeysuckle is shown.
FIG. 2 is a schematic diagram: in example 1, the in vitro bacteriostatic effect of BLCC1-0716 and BLCC1-0155 on Clostridium perfringens is shown.
FIG. 3: colonies and microscopic images of the strain BLCC1-0716 in example 2.
FIG. 4: phylogenetic analysis of the strain BLCC1-0716 in example 2.
FIG. 5: example 5 line graph of the change in body weight of mice during the construction of the model of ulcerative enteritis in mice.
FIG. 6: example 5 photographs of the hematochezia of mouse ulcerative enteritis model mice.
FIG. 7: effect of different concentrations of DSS on colon morphology in mice in example 5.
FIG. 8: example 5 efficacy evaluation of ulcerative enteritis in mice the body weight of the mice in each group was changed after drinking DSS.
FIG. 9: example 5 efficacy evaluation of ulcerative colitis in mice colonic tissue sections from different treatment groups of mice were observed.
FIG. 10: example 6 efficacy comparison of ulcerative enteritis in mice the body weight change of the mice in each group after drinking DSS in the experiment was evaluated.
FIG. 11: example 6 efficacy of ulcerative colitis in mice colon length in different treatment groups of the experiment was compared and evaluated.
FIG. 12: example 6 efficacy of ulcerative colitis in mice colon tissue sections from different treatment groups were observed in comparative evaluation experiments.
Detailed Description
The present application is further illustrated with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or starting materials used in the present application can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present application can be used in a manner conventional in the art or in accordance with the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present application. The preferred embodiments and materials described herein are intended to be exemplary only.
Example 1Screening of the strains
1. Materials and methods
1.1 sample: the medicinal herbs include root, stem and leaf of flos Lonicerae.
1.2 pathogenic bacteria
The test strains are shown in Table 1.
TABLE 1 pathogen identification and Source
1.3 isolation Medium
Modified MRS medium: peptone 1%, beef extract 1%, yeast extract 0.5%, glucose 2%, dipotassium hydrogen phosphate 0.2%, sodium acetate 0.5%, ammonium citrate 0.2%, magnesium sulfate 0.02%, manganese sulfate 0.05%, tween-800.1% and pH 6.5; used for separating lactobacillus.
LB culture medium: 0.5% of glucose, 1.0% of peptone, 0.5% of beef extract and 0.5% of sodium chloride, and adjusting the pH value to 6.5; can be used for separating bacteria such as Bacillus.
PDA culture medium: potato 20% (cooking juice), glucose 20g, agar 15g, pH natural; used for separating fungi.
1.4 fermentation Medium
The lactobacillus fermentation medium is MRS liquid medium; the fermentation medium of bacillus and other bacteria is LB liquid medium; the fungus fermentation culture medium is potato glucose liquid culture medium (PDB); and (3) indicating bacterium fermentation medium: the same bacillus culture medium.
1.5 test methods
1.5.1 sample Collection and handling
Collecting honeysuckle branches, leaves, barks and the like, placing the honeysuckle branches, leaves, barks and the like in a sterile plastic bag, and carrying out surface disinfection treatment within 24 hours.
1.5.2 isolation and purification of endophytes
The method comprises the steps of adopting a tissue block method (for example, refer to the processing modes in strict casting cloud, Pont bud, Roche, Wangyang, old, Wandexing. gingko endophytic fungi strains separation and identification [ J ] Huaxi pharmaceutical journal, 2006(05): 425-. Taking a collected fresh sample, cleaning the surface of a plant with distilled water, slightly drying, cutting roots, stems and leaves of the plant into small sections with proper sizes, soaking the small sections in 75% ethanol for l min, rinsing the small sections with sterile water for 3 times, then disinfecting the small sections with 2.5% NaClO, disinfecting the stems and leaves for 2min, disinfecting skins for 3min, finally rinsing the small sections with the sterile water for 3-4 times, and sucking the small sections with sterile filter paper to be dry. Removing edges, cutting into small pieces of about 0.5cm multiplied by 0.5cm, pasting on a separation culture medium, placing 4-6 pieces in each dish, culturing in a constant-temperature incubator at 37 ℃ and 28 ℃ for 2d-7d, after bacteria grow out around plant tissues on the culture medium, picking and transferring to a new culture medium plate for streaking purification, and preserving purified single bacteria to the inclined plane at 4 ℃ or preserving glycerin tubes and freeze-dried powder. In the process of endophyte separation, two types of blank controls (which can be set according to the records in the literature, good Liu-laughing, Zhao-inspiring, old jun. research on the method for separating the plant endophyte [ J ] food science, 2009,30(15): 180-.
1.5.3 screening of strains with bacteriostatic effect
Preparation of the supernatant: respectively inoculating the purified strains in an LB slant culture medium, culturing and rejuvenating at 37 ℃, subculturing for 24h, then transferring to a 250mL triangular flask filled with 50mL liquid seed culture medium, carrying out constant temperature shaking culture (180r/min) at 37 ℃ for 24h, then inoculating to a 500mL triangular flask filled with 100mL fermentation culture medium, carrying out constant temperature shaking culture (180r/min) at 37 ℃, centrifugally separating thalli and supernatant at 5000r/min, wherein the supernatant is a metabolite; then, the antibacterial activity of the metabolite is measured by a tube-disc method.
Preparation of double dish: taking a flat bottom double dish with the diameter of 90mm, injecting 15mL of sterilized nutrient agar (1.5 percent), horizontally placing to solidify the nutrient agar to be used as a bottom layer, taking another nutrient agar (with the concentration of 0.8 percent and being cooled to about 50 ℃) and an appropriate amount of indicator bacterium liquid cultured at the temperature of 37 ℃ for 24 hours, uniformly mixing the nutrient agar and the indicator bacterium liquid, taking 6mL of the indicator bacterium liquid, paving the indicator bacterium liquid on a bottom layer culture medium, horizontally placing to solidify the indicator bacterium liquid to be used as a bacterium layer.
Adding a sample: the sterilized oxford cup was gripped with sterile forceps, the lid opened, and placed on the culture medium. The same amount of test sample supernatant (300. mu.L) was filled in an Oxford cup, and each sample was replicated three times. Carefully placing the double dishes with the samples in a constant temperature box at 37 ℃, culturing for 16h, and taking out to measure the diameter of the bacteriostatic zone.
2. As a result, the
2.1 isolation of endophytes from Lonicera japonica
12 strains of bacteria are separated from roots, stems and leaves of the honeysuckle, and the separation condition is shown in figure 1.
2.2 screening results of bacterial strains with bacteriostatic effect
TABLE 2 screening results of antibacterial effect of honeysuckle
Note: the diameter of the inhibition zone is the average value of 2 repeated tests; (-) no inhibitory effect; (+)4< Φ <10 mm; (++)10mm < phi <14 mm;
(+++)14mm≤Φ<24mm。
low activity: the bacteriostatic circle phi is less than 10 mm; moderate activity: the bacteriostatic zone (++)10mm is not more than phi <14 mm; high activity: the bacteriostatic circle phi is more than or equal to 14 mm.
Table 2 shows the results of the antibacterial test of honeysuckle endophytic bacteria. As can be seen from the table, 12 strains of endophytes have better bacteriostatic action on more than 1 indicator bacterium, accounting for 27.91% of the total number; 7 strains which have the bacteriostatic action on more than 3 indicator bacteria account for 16.28 percent of the total number; 2 endophytes have an inhibiting effect on 4 pathogenic bacteria, accounting for 4.65% of the total number, and the bacterial strains with the inhibiting effect have a better inhibiting effect on salmonella. From the aspect of bacteriostatic effect, the activity on gram-negative bacteria is greater than that on gram-positive bacteria. 5 strains which have an inhibiting effect on staphylococcus aureus and account for 11.63 percent of the total strains; 6 strains which have inhibition effect on escherichia coli account for 13.95 percent of the total number; the total amount of 12 strains with the inhibiting effect on the salmonella is 27.91 percent; 7 strains having an inhibitory effect on Aeromonas hydrophila were present, 16.28% of the total strains. By integrating the bacteriostatic activity and the bacteriostatic spectrum, the strain JYH-J4 has better in-vitro bacteriostatic performance, and is numbered BLCC1-0716 (therefore, in the embodiment of the invention, the strain JYH-J4 and the strain BLCC1-0716 refer to the same strain) for the convenience of preservation management.
2.3 determination of the bacterial inhibition Profile of the Strain BLCC1-0716
TABLE 3 bacteriostatic results of flos Lonicerae endophyte BLCC1-0716
As shown in Table 3, the diameters of inhibition zones of the strains BLCC1-0716 to Escherichia coli BLCC8-0102, Staphylococcus aureus BLCC8-0004 and Clostridium perfringens BLCC8-0044 are 15mm, 15mm and 22.5mm respectively, which are significantly larger than the inhibition diameters of Bacillus subtilis BLCC1-0155 to Escherichia coli (12.5mm), Staphylococcus aureus (9.0mm) and Clostridium perfringens (18.0mm) reported by China patent CN105039223A, the name of the invention is Bacillus subtilis with the function of inhibiting Clostridium perfringens and the application thereof, and the like, wherein the in vitro inhibition effects of the strains BLCC1-0716 and BLCC1-0155 to Clostridium perfringens are shown in FIG. 2. And the BLCC1-0716 has good in-vitro antibacterial performance on pathogenic microorganisms such as common escherichia coli and salmonella of livestock and poultry, has good in-vitro antibacterial performance on streptococcus pyogenes, serratia marcescens, bacillus cereus, bacillus circulans, clostridium perfringens and the like, and can effectively prevent and treat diseases such as necrotic enteritis and the like caused by the pathogenic microorganisms in the livestock and poultry breeding process. In addition, the BLCC1-0716 also has good antibacterial effect on common aquatic pathogenic bacteria such as vibrio alginolyticus, vibrio parahaemolyticus, aeromonas hydrophila and the like, and can be used for preventing and treating diseases such as fish hemorrhagic septicemia, ulcer and the like caused by pathogenic microorganisms.
Example 2Identification of bacterial species and safety test
1. Materials and methods
1.1 strain: the strain BLCC1-0716 screened in example 1 of the invention.
Experimental animals: 40 healthy female Kunming-breed mice (weight 20 +/-2 g) were purchased from Jinan Pengye laboratory animal Breeding Co., Ltd.
1.2 routine biochemical identification of bacteria: unless otherwise specified, general morphological and physiological and biochemical tests were carried out according to Bergey's Manual of bacteria identification (9 th edition) and microbiological Manual of experiments.
1.3 identification of 16S rDNA of Strain
1.3.116 amplification and sequence analysis of S rDNA
The target strain is inoculated in a fresh fermentation medium for culturing for 20h, and the DNA of the strain is extracted by adopting a kit of Tiangen company and is subjected to 16SrDNA sequence amplification. The primers used were bacterial 16S rDNA universal primers 27F/1492R, and the PCR reaction system (50. mu.L) was: mix 25. mu.L (containing Taq DNA polymerase and dNTP, purchased from Tiangen Biochemical technology Co., Ltd.), 1. mu.L of each of the upstream and downstream primers, 2. mu.L of the template DNA, and 21. mu.L of ultrapure water. The PCR amplification program comprises pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 1min, annealing at 52 deg.C for 1min, extension at 72 deg.C for 2min, 30 cycles, and extension at 72 deg.C for 10 min. The PCR product was sent to Beijing Boshang Biotechnology Co., Ltd for sequence determination.
1.3.2 phylogenetic analysis
Logging in GenBank (http:// www.ncbi.nlm.nih.gov), retrieving the sequencing result of the 16S rDNA of the strain by using Blast, downloading the 16S rDNA sequence of related genus species, performing homology analysis by using DNAMAN, DNAClub, MEGA3.1 and other software, and constructing a phylogenetic tree.
1.4 safety test
Preparing the gavage bacteria liquid: inoculating the pure BLCC1-0716 culture in LB medium, culturing at 37 deg.C and 180rpm for 24h, transferring the bacterial liquid into a sterile centrifuge tube, centrifuging at 4000rpm for 5min, collecting thallus, adding sterile normal saline and supernatant respectively, mixing, and adjusting to viable count concentration of 5.0 × 10 8 CFU/mL (Low dose group), 5.0X 10 9 CFU/mL (Medium dose group), 5.0X 10 10 CFU/mL (high dose group).
The test method comprises the following steps: after clean-grade female Kunming mice are adapted to the environment for 3 days, the female Kunming mice are randomly grouped into a control group and groups with different dosages, and each group comprises 10 mice. Marking when grouping, respectively intragastrically irrigating the bacterial suspensions with different viable counts by using normal saline for the control group and different dosages of the bacterial suspensions with different viable counts, wherein each one is 0.2mL, and intragastrically irrigating for 1 time every day. Various indexes of the mouse are observed in the test and recorded. After feeding for one week, the mice are killed, the weight change of the mice is calculated, and the liver, spleen and kidney are dissected and observed to see whether pathological changes exist.
2. As a result, the
2.1 bacterial colony of strain BLCC1-0716 and microscopic examination form
A pure culture of the strain BLCC1-0716 is selected and inoculated in an LB culture medium, and cultured for 24h at 37 ℃ to form a light yellow colony which is moist and glossy, is tightly attached to the culture medium, is not convex, slightly convex in the center and neat in edge. Gram staining is positive, the thallus is rod-shaped, and unique canoe accompanying spores can be formed. Wherein, the colony and microscopic picture of the strain BLCC1-0716 are shown in figure 3.
2.2 physiological and Biochemical identification of strains
The results of the physiological and biochemical properties of the strain BLCC1-0716 are shown in Table 4.
TABLE 4 physiological and biochemical identification results of Brevibacillus laterosporus BLCC1-0716
Note: +: positive; -: and (4) negativity.
As shown in Table 4, the identification result of the physiological and biochemical characteristics of the strain BLCC1-0716 conforms to the characteristics of Brevibacillus laterosporus. And (3) primarily judging the strain to be the brevibacillus laterosporus by integrating the colony, the thallus morphology and the physiological and biochemical identification results.
2.3 identification of 16S rDNA of Strain BLCC1-0716
The 16S rDNA PCR band of strain BLCC1-0716 was a single amplification product of about 1.5 kb. The sequence was aligned for homology with nucleic acid data from GenBank. The results show that the sequence has 100 percent of homology with known Brevibacillus laterosporus DQ122932.2, KP209409.1 and MN907467.116S rDNA sequences published in GenBank. The phylogenetic analysis diagram of strain BLCC1-0716 is shown in FIG. 4. In conjunction with 16S rDNA sequence analysis, strain BLCC1-0716 was identified as Brevibacillus laterosporus. Wherein, the nucleotide sequence of the strain BLCC1-0716 is shown in SEQ ID NO. 1.
2.4 Strain BLCC1-0716 safety test
Grouping by adopting a random non-difference principle, wherein each mouse is subjected to oral gavage at different concentrations: 5.0X 10 8 CFU/mL (Low dose group), 5.0X 10 9 CFU/mL (Medium dose group), 5.0X 10 10 CFU/mL (high dose group) suspension, and the placebo group was gavaged with equal amount of saline (CK saline group). The test period is 7 days, and the mice freely eat the feed; after the experimental period, the mice were sacrificed by removing cervical vertebrae, and the liver, spleen and kidney were observed by dissection, and the analysis results are shown in table 5.
TABLE 5 mouse body weight and organ coefficients after termination of the test
Note: data in the same column, when the same letter or no letter is marked, indicates that the difference is not significant (P > 0.05).
As seen from Table 5, there was no significant difference in body weight among the groups of mice after oral administration of different doses of Brevibacillus laterosporus BLCC1-0716 test substance for 7 d. The test group mice have normal body states, no macroscopic change on the liver, spleen and kidney and no obvious difference on organ coefficients, and the strain is safe to the mice in the dosage of 1 hundred million to 100 hundred million by gavage. The results show that the screened strain BLCC1-0716 has good safety and is suitable for preparing candidate strains for subsequent experiments.
Example 3Measurement of Strain tolerance
1. Materials and methods
1.1 strain: the screened brevibacillus laterosporus BLCC1-0716 in the embodiment 1 of the invention;
1.2 Medium LB Medium as in example 1
1.3 methods
1.3.1 bile salt tolerance assay
Taking 1mL of Brevibacillus laterosporus BLCC1-0716 seed liquid (with a spore rate of about 80%) cultured for 24h in a shaking table at 37 ℃ to 50mL of liquid LB culture medium, adding cholate mother liquor with different volumes to make the final concentration of cholate be 0.1%, 0.3% and 0.5%, culturing for 4h at 37 ℃, and sampling for 0h and 4h respectively to count viable bacteria.
1.3.2 determination of acid resistance
0.3mL of Brevibacillus laterosporus BLCC1-0716 seed liquid (with a spore rate of about 80%) cultured by a shaking table at 37 ℃ is taken to 15mL of liquid LB culture medium with different pH values (2.5, 3.5 and 4.5), cultured for 4h at 37 ℃, and sampled for 0h and 4h respectively for viable count.
1.3.3 Artificial gastrointestinal fluid tolerance assay
Inoculating Brevibacillus laterosporus BLCC1-0716 to artificial gastric juice and intestinal juice at the inoculation amount of 10%, culturing at 37 deg.C and 180rpm for 4h, sampling, and counting viable bacteria.
Preparing artificial gastric juice: 4.1mL of 9.5-10.5% hydrochloric acid, adding 250mL of water for dilution to make the pH value reach 2.0, adding 2.5g of pepsin, filtering and sterilizing by a 0.2 mu m filter membrane, and storing in a refrigerator at 4 ℃ for later use.
Preparing artificial intestinal juice: 3.4g of monopotassium phosphate is dissolved by adding 250mL of water, the pH value is adjusted to 6.8 by using 0.4% sodium hydroxide solution, 1g of trypsin is added into each 100mL of liquid, the mixture is evenly mixed, filtered and sterilized by a 0.2-micron filter membrane, and the mixture is stored in a refrigerator at 4 ℃ for standby.
1.3.4 measurement of Heat resistance
Inoculating the seed liquid of Brevibacillus laterosporus BLCC1-0716 into a liquid LB culture medium, culturing at 37 ℃ and 180rpm for 24h, sampling when the microscopic spore rate reaches 80%, treating the fermentation liquid at 85 ℃ for 15min, performing gradient dilution to count the viable bacteria, and calculating the heat treatment survival rate.
2. Results
2.1 determination of bile salt tolerance
TABLE 6 Strain BLCC1-0716 cholate resistance survival (%)
The 2% inoculation amount is used for detecting the tolerance of the strain to bile salts with different concentrations after acting for 4 hours at 37 ℃, and the bacillus is proliferated by 1-2 orders of magnitude under normal conditions. After 4 hours of action, the survival rate is in a descending trend along with the increase of the concentration of the bile salts. As can be seen from Table 6, the survival rate of 0.1% of bile salt after 4 hours of action is 99.87%; the survival rate of the bile salt with the concentration of 0.3 percent is 73.57 percent after the bile salt with the concentration of 0.3 percent acts for 4 hours; the survival rate of the bile salt with the concentration of 0.5 percent is 45.00 percent after the bile salt with the concentration of 0.5 percent acts for 4 hours.
2.2 determination of acid resistance
TABLE 7 Strain BLCC1-0716 acid resistance survival (%)
The 2% inoculation amount detection strain acts for 4 hours at 37 ℃ and has tolerance to different pH values, and the bacillus proliferates by 1-2 orders of magnitude under normal conditions (pH 6.5). Culturing for 4h under different pH conditions, and the survival rate is 75.86% under the condition of pH 2.5; the survival rate under the condition of pH3.5 is 97.44%; the survival rate under the condition of pH4.5 is 103.13%. (see Table 7)
2.3 Artificial gastrointestinal fluid tolerance assay
TABLE 8 bacterial strain BLCC1-0716 number of viable bacteria for artificial gastrointestinal fluid tolerance and survival rate
The 10% inoculation amount is used for detecting the tolerance of the strain to the artificial gastric juice and the intestinal juice after 4h of action at 37 ℃, and as can be seen from the table 8, the strain BLCC1-0716 has better tolerance to the artificial gastric juice, and the survival rate of the strain after 4h of action is 120.28%; the survival rate of the artificial intestinal juice after 4 hours of the action is 289.38 percent.
2.4 measurement of Heat resistance
TABLE 9 determination of Heat resistance of Strain BLCC1-0716
As can be seen from Table 9, the survival rate of strain BLCC1-0716 after treatment in a water bath at 85 ℃ for 15min was 98.10%. The feeding bacillus can be widely applied to the first line of cultivation only by being added into feed under most conditions, and the granulation temperature is generally 85 ℃ for instant cooling. The strain acts for 15min at 85 ℃, the number of the viable bacteria is not obviously changed, the strain can overcome the defect that most probiotics in the market are not heat-resistant, and the strain has good market application prospect.
Example 4Verification of effect of strain BLCC1-0716 on acute enteritis of mice
1 construction of mouse acute enteritis model
1.1 materials
Lipopolysaccharide LPS (Escherichia coli 055 type), available from Sigma; the ELISA kit is purchased from Nanjing to build a bioengineering institute; 40 female Kunming mice were purchased from Jinan Pengye laboratory animal Breeding, Inc.
1.2 method:
TABLE 10 model construction mouse grouping and processing
About 18g Kunming mice 40 are pre-fed for 3d, the weight is not different, the Kunming mice are randomly divided into 4 groups, each group is 10, the grouping and the treatment are shown in table 10, the day of the grouping is marked as the 1 st day of the test, different concentrations of LPS 0.1mL are injected into the abdominal cavity at the 6 th day of the test, the symptoms are observed, and the related indexes are measured by taking blood from the eyeball 1h and 4h after the LPS is injected respectively.
Detection indexes are as follows: general behavioral indicators (mouse phenotype), diarrhea rate, serum cytokines (IL-6, INF-alpha, IL-1 beta).
1.3 results
1.3.1 Change in diarrhea Rate in mice injected with LPS over different periods of time
TABLE 11 change in diarrhea rates after LPS treatment in mice of different groups
After different doses of LPS are injected, apparent symptoms of mice are obvious, the mice present diarrhea symptoms in each group 1h after the LPS is injected, the diarrhea rates of a low dose group (I group), a medium dose group (II group) and a high dose group (III group) are respectively 10%, 40% and 60%, the mobility is lazy, the abdominal contraction is obvious particularly in the high dose group (III group); mice in each group were diarrhea and ataxia 4h after LPS injection, and the body temperature of the high dose group (group III) was lowered and was dying.
1.3.2 serum cytokine changes at different time periods after LPS injection in mice
Blood is taken from eyeballs 1h and 4h after LPS injection, the eyeballs are centrifuged at 3000rpm for 10min, serum is carefully sucked, and an ELISA kit measures the level changes of interleukin IL-6, IL-1 beta and tumor necrosis factor TNF-alpha in the serum after different time of LPS injection, and the results are shown in the following table:
TABLE 12 serum cytokine level changes (pg/mL) at 1h, 4h after LPS treatment in different groups of mice
Note: the difference of the shoulder mark letters of the data in the same column indicates that the difference is obvious (P < 0.05); shoulder letters are identical or no letter designation indicates no significant difference (P > 0.05).
As shown in Table 12, the levels of proinflammatory factors IL-6, IL-1 beta and TNF-alpha in the serum of I, II group were all elevated 1h after LPS injection compared to the blank group. Compared with the blank group, the levels of IL-6, IL-1 beta and TNF-alpha in the group I are respectively 0.93 percent, 16.24 percent and 5.18 percent higher than those of the blank group, but the difference is not obvious (P is more than 0.05); the IL-1 beta and TNF-alpha levels of the group II are obviously higher than that of the blank group (P <0.05), and the IL-6 level is 13.52 percent higher than that of the blank group, and the difference is not significant (P > 0.05); TNF-alpha levels in group III were significantly higher than those in the blank (P <0.05) and increased with increasing LPS injection dose, but IL-6, IL-1. beta. levels in this group were slightly lower or not significantly different from those in the blank.
4h after LPS injection, compared with a blank group, the levels of proinflammatory factors IL-6, IL-1 beta and TNF-alpha in serum of LPS injection groups (I, II and III) are all increased, and the levels of IL-6, IL-1 beta and TNF-alpha in the group I are respectively 4.57%, 11.02% and 4.52% higher than those in the blank group, but the difference is not obvious (P is more than 0.05); the IL-6, IL-1 beta and TNF-alpha levels of the group II are respectively 3.32%, 19.31% and 3.67% higher than those of the blank group, wherein the IL-1 beta level is obviously higher than that of the blank group (P <0.05), and the IL-6 and TNF-alpha levels are not obviously different from that of the blank group (P > 0.05); the levels of IL-6, IL-1 beta and TNF-alpha which are related to the group III serum proinflammatory factors are all obviously higher than that of a blank group (P < 0.05).
And (3) knotting: after LPS with different concentrations (5-20 mg/kg) is injected into the abdominal cavity of a mouse, symptoms such as lying, listlessness and diarrhea can appear within the range of 1-4 h, general behavior observation is consistent with literature reports (Tanya, Ganmai neighborhood, normal source, WangJing Jing, Zhangxiong, Huangbo, Zhu Li, Shun, Shi Zhi, protection effect of golden buckwheat on lipopolysaccharide induced mouse intestinal inflammation [ J ] Chinese veterinarian, 2020,47(02): 597) and serum proinflammatory factor level has the trend of rising along with the increase of LPS injection dose, the LPS 10mg/kg injection dose is moderate in constructing mouse enteritis model phenotype, and the serum proinflammatory factor level is stable, and the research sets the LPS 10mg/kg injection dose of the mouse injected into the abdominal cavity to construct mouse enteritis model.
Evaluation of efficacy of 2 strain BLCC1-0716 on acute enteritis of mice
2.1 materials
The mouse type, purchase route and LPS, ELISA kit are the same as in section 1.1 of example 4.
2.2 methods
80 female Kunming mice of about 18g were pre-fed for 3 days, and randomly divided into 8 groups, blank group (CK group), LPS group (model group), strain BLCC1-0716 treatment group (dose of 3X 10 respectively) 6 CFU/only, 3X 10 7 CFU/only, 3X 10 8 CFU/body), the treatment time of each group was as shown in Table 13, water was freely drunk during the treatment, the weight was weighed once per 3d, after the gastric lavage treatment for 12d, each group was subjected to one-time intraperitoneal injection of 0.1mL of physiological saline in CK, Y11, Y12 and Y13, and each group was subjected to one-time intraperitoneal injection of 10mg/kg of LPS (0.1mL) in LPS, Y11P, Y12P and Y13P, and the following indices were sampled and measured after observation for 4 h.
TABLE 13 grouping and Individual group handling
Detection indexes are as follows: general behavioral indicators (diarrhea rate, weight loss, mental, piloerection), serum cytokines (IL-6, INF-alpha, IL-1 beta, IL-10) and intestinal flora (E.coli, lactic acid bacteria) changes.
2.3 results
2.3.1 weight changes in mice during treatment
TABLE 14 weight change of mice during treatment
As shown in table 14, there was no significant difference in the body weight change of the mice in each treatment group, and the body weight difference of the mice in each group was not significant by the end of the treatment.
2.3.2 diarrhea Rate profiles in mice
TABLE 15 diarrhea in mice of each treatment group
As shown in Table 15, the diarrhea did not occur in the saline-injected groups (CK, Y11, Y12, Y13), the diarrhea occurred in the LPS-injected groups (LPS, Y11P, Y12P, Y13P), and the diarrhea rates (70% to 80%) of the LPS-injected and B.ventriculi-inoculated groups Y11P, Y12P, Y13P were all lower than those of the model group (90%), indicating that B.ventriculi has the effect of alleviating the diarrhea in mice and the amount of B.ventriculi is 10% 7 CFU/dose was more effective.
2.3.3 groups of mice having altered levels of serum cytokines
After 4h of LPS injection, the eyeballs of each group of mice are centrifuged at 3000rpm for 10min, serum is carefully sucked, and an ELISA kit is used for measuring the level changes of proinflammatory factors, namely interleukin IL-6, IL-1 beta, tumor necrosis factor TNF-alpha and anti-inflammatory factor IL-10 in the blood, and the results are shown in Table 16:
TABLE 16 serum cytokine levels (pg/mL) in mice of different groups
Note: the data in the same column are marked with different letters to indicate significant difference (P < 0.05); shoulder marks with the same letter or no letter designation indicate no significant difference (P > 0.05).
As can be seen from Table 16, the water levels of the mouse serum cytokines IL-6, IL-1 beta, TNF-alpha and IL-10 in the model group were higher than those in the blank group, which indicates that the LPS injected into the abdominal cavity at a dose of 10mg/kg can cause the level of the proinflammatory factors and the anti-inflammatory factors in the mouse serum to rise, and the LPS model was successfully constructed.
IL-6 levels in Y11P, Y12P and Y13P groups were all lower than the model group but did not differ significantly (P >0.05), with the IL-6 level in Y11P group being the lowest, 82.30pg/mL, 5.89% lower than the model group but did not differ significantly (P > 0.05); the level of the serum cytokine IL-1 beta is not obviously different among the Y11P, Y12P and Y13P except for the model group, wherein the level of the IL-1 beta of the Y13P group is the lowest and is 93.51pg/mL, which is 6.49 percent lower than that of the model group, but the difference is not obvious (P is more than 0.05); serum cytokine TNF- α levels were lowest in each LPS treated group at Y11P, 661.56pg/mL, 9.88% lower than model group, with significant differences (P <0.05), and TNF- α levels in Y12P and Y13P groups were 6.78% and 6.35% lower than model group, respectively, but not significant differences (P > 0.05).
LPS is the major component of gram-negative bacteria and is the major promoter mediating the systemic inflammatory response syndrome. The brevibacillus laterosporus BLCC1-0716 can reduce the levels of serum cytokines TNF-alpha, IL-6 and IL-1 beta to some extent in different doses in intragastric administration, and the effect of inhibiting inflammation can be realized by reducing the levels of TNF-alpha, IL-6 and IL-1 beta to inhibit the generation of inflammation.
In each LPS treatment group, the serum cytokine IL-10 level of the Y12P group is the highest and is 282.17pg/mL, which is 1.43% higher than that of the model group and 4.60% higher than that of 269.76pg/mL of the blank group, and the difference is significant, which indicates that the gavage is 3X 10 7 CFU/Brevibacillus laterosporus can relieve the inflammation of mice caused by LPS injected into the abdominal cavity at a dose of 10mg/kg to a certain extent. IL-10 is a pluripotent stem cell factor, IL-10 is an important regulator for inhibiting the development of inflammation in inflammatory pathways, and the expression level of IL-10 can be detected to be higher than normal in patients with inflammatory bowel disease. In this study, the serum IL-10 level of each group of mice injected with LPS was higher than that of the blank group, indicating that each group of mice started the autoinhibiting inflammation system to perfuse the stomach with 3X 10 7 CFIL-10 levels were highest in the U/dose group mice, which also coincided with the lowest diarrhea rates in this group of mice previously measured.
As can be seen from the above, the different concentrations of Brevibacillus laterosporus BLCC1-0716 in the intragastric administration can reduce the levels of mouse serum cytokines TNF-alpha, IL-6 and IL-1 beta caused by the intraperitoneal injection of LPS with 10mg/kg dose, and increase the level of IL-10 which is an inflammatory factor, so that the BLCC1-0716 can be presumed to inhibit the generation of inflammation and relieve the inflammation through the two routes. Comprehensive appearance and serum index, and adopts intragastric perfusion fermentation liquor 3X 10 6 Or 3X 10 7 CFU/mouse as a prophylactic protective dose for i.p. LPS inflammation model.
2.3.4 changes in the intestinal content flora of groups of mice
TABLE 17 analysis of upper gastrointestinal content flora in different groups of mice
Note: data in the same column are marked with different letters to indicate significant difference (P < 0.05); shoulder marks with the same letter or no letter designation indicate no significant difference (P > 0.05).
Changes in the gut flora in each group of mice are shown in table 17. Of these, the Y11 group, which was the highest among lactic acid bacteria, was used for intragastric administration, and was comparable to the blank group and significantly different from the model group (P)<0.05); y12 and Y13 are both lower than Y12P and Y13P, which may be caused by LPS causing imbalance of intestinal flora in mice, and the residual bacillus in gastric lavage consumes oxygen in upper digestive tract to promote proliferation of facultative anaerobe lactobacillus; the number of intestinal lactobacillus in the Y11P, Y12P and Y13P groups is higher than that in the model group, which indicates that the bacillus gastrolavage can promote the proliferation of lactobacillus in the environment of intestinal dysbacteriosis of mice injected with LPS. Against E.coli, the numbers of E.coli in the gut contents Y11, Y11P, Y12, Y12P, Y13, Y13P were all significantly lower than the model group (LPS group), with Y12 being the lowest and significantly lower than the model group (P12)<0.05). Against Staphylococcus aureus, the number of Staphylococcus aureus was the lowest in the blank group, 2.8X 10 4 CFU/g, in model set 56.5X 10 4 CFU/g is highest; y12, Y1The 2P group had a low number of Staphylococcus aureus and the difference between the two groups was not significant (P)>0.05)。
In summary, brevibacillus laterosporus BLCC1-0716 fermentation liquid with different concentrations for intragastric administration can relieve upper gastrointestinal flora disorder caused by mice intraperitoneal injection of LPS, improve the number of facultative anaerobe lactic acid bacteria, reduce the number of intestinal pathogenic bacteria Escherichia coli and Staphylococcus aureus, and can be intragastric administered by 3 × 10 7 CFU/only has better effect.
The brevibacillus laterosporus BLCC1-0716 fermentation liquor with different concentrations for intragastric administration can relieve the inflammation of mice caused by LPS with 10mg/kg dosage by intraperitoneal injection to a certain extent, and integrates the apparent symptoms, intestinal flora and serum indexes, and the intragastric administration fermentation liquor is adopted in the subsequent experiments for 3 multiplied by 10 7 CFU/mouse as a prophylactic protective dose for i.p. LPS inflammation model.
Example 5Evaluation of efficacy of strain BLCC1-0716 on ulcerative enteritis in mice
1 mouse ulcerative enteritis (UC) model construction
1.1 test materials
The mouse class and the purchase route were the same as in section 1.1 of example 4.
Dextran Sulfate Sodium (DSS) was purchased from Hippon Biotech (Shanghai) Inc. of MW: 36000-50000
1.2 methods
30 female Kunming mice of about 18g are fed with basic feed and freely drunk, and are randomly divided into a blank group (CK group), a 3% DSS free drinking group and a 5% DSS free drinking group after being adaptively fed for 1 week. Mice in the blank group had free access to water; the 3% DSS group mice freely drunk the 3% DSS aqueous solution, and the 5% DSS group mice freely drunk the 5% DSS aqueous solution, and the grouping and treatment conditions are shown in Table 18. Continuously feeding for 9 days, and performing grain-breaking and water-breaking treatment on the mice at night of 9 days. The general condition, body weight, fecal characteristics and occult blood of the mice were observed daily during the molding period. On day 10, the cervical vertebrae were removed and the mice were sacrificed, the colon was isolated, and the lesion condition was visually observed to measure the length of the colon.
Table 18 different groups and treatments of mice
1.3 results
1.3.1 conditions of feces and blood in mice
The molding experiment was carried out for 9 days. Mice in the 3% DSS group and the 5% DSS group each consumed approximately 600mL of DSS aqueous solution per day. During the test period, the mice were normal in mental status and did not die. Of these, mice in the 3% DSS group and 5% DSS group developed individually with soft stools starting on day 4 (see table 19). At the end of the test, the test groups showed soft stools and bloody stools (fig. 6), indicating that the mice developed acute colitis symptoms.
TABLE 19 statistics of mouse feces and blood
1.3.2 mouse weight Condition
During the molding period, the blank group mice gained weight. The body weight of mice in the 3% DSS group showed a trend of increasing followed by decreasing, with a weight loss at day 9, but was not significantly different from the blank group (P >0.05) (see table 20). Mice in the 5% DSS group began to decline in body weight on day 7, and were significantly lower on days 8, 9 than the blank (P <0.05) (fig. 5).
TABLE 20 daily body weight indices for mice
Note: the different letters of the same row data shoulder mark indicate significant difference (P < 0.05); shoulder marks with the same letter or no letter designation indicate no significant difference (P > 0.05).
1.3.3 Colon characteristics of mice
Colonic atrophy is an important pathological feature of ulcerative colitis. In this study, it was found that the colon was atrophied to different degrees in both the 3% DSS group and the 5% DSS group, and was significantly shortened (P <0.05) compared to the blank group (FIG. 7). Wherein, the colon length of the blank group of mice is the longest and is 10.03 cm; the colon of the 5% DSS group is the shortest and is 7.70 cm; the colon was 8.62cm in the 3% DSS group of mice (Table 21).
TABLE 21 Colon Length statistics in mice
Note: the data in the same row are marked with different letters to indicate that the difference is significant (P < 0.05); shoulder marks with the same letter or no letter designation indicate no significant difference (P > 0.05).
By integrating the weight, the fecal characteristics and the colon characters of the mice, the mice in the 3% DSS group and the 5% DSS group all have ulcerative colitis symptoms, which indicates that the mouse UC model is successfully constructed, wherein the acute colon inflammation symptoms of the mice in the 5% DSS group are more obvious, and the 5% DSS is selected to carry out the in-vivo effect evaluation test of the strains in the later period.
Evaluation of efficacy of 2 strain BLCC1-0716 on ulcerative enteritis in mice
2.1 materials
The route of purchase of mouse products and DSS were the same as in section 1.1 of example 5.
2.2 methods
30 female Kunming mice of about 18g are fed with basal feed and adaptively fed for 3 days. The samples were randomly divided into CK group, UC model group, and BLCC1-0716 group. Gavage 200 μ L1.5X 10 daily for 7 days before the experiment, starting from the group 8 CFU/mL BLCC1-0716 bacterial fluid, CK group (blank group) and UC model group were gavaged with 200 μ L of saline daily, during which time each group of mice had free access to water. From day 8, the blank group was gavaged with 200. mu.L of physiological saline and water ad libitum, the model group was gavaged with 200. mu.L of physiological saline and water ad libitum, and the strain group of BLCC1-0716 was allowed to drink 5% DSS solution ad libitum while gavaged with 200. mu.L of the bacterial solution for 10 days (see Table 22). The general condition, body weight, fecal characteristics and occult blood of the mice were observed daily during the trial. After the test is finished, the mouse is killed by taking off the cervical vertebra, blood is taken from eyeballs, the colon is separated, the pathological change condition is observed by naked eyes, and the length of the colon is measured.
TABLE 22 test grouping and handling
TABLE 23 DAI scoring criteria
2.3 results
2.3.1 mouse weight changes
In the growth process, the continuous rising of the body weight is a normal trend, and the body weight is reduced and fluctuated, which is probably caused by the fact that the mice are stimulated by external substances to cause the immune response of the mice. In the test process, except the CK mice freely drinking water, the mice in the other test groups freely drink 5% DSS water solution from 8d to the end of the test, and the weight change of the mice is shown in figure 8.
The test mice were weighed once a day and the change in body weight was recorded. The CK group mice continued to gain body weight throughout the duration of the experiment due to the absence of induced stimulation by DSS. During the period of drinking DSS, the weight of mice in the UC model group and the BLCC1-0716 group tends to increase and then decrease, and the weight of mice in the UC model group and the BLCC1-0716 group tends to decrease after 5 days of drinking. At the end of the test, the weight of the mice in the UC model group is reduced most remarkably, compared with the blank group, the weight is reduced by 3.56g, the difference is remarkable (P <0.05), and the weight of the mice in the Brevibacillus laterosporus BLCC1-0716 group is reduced by 1.58g, compared with the blank group, the difference is not remarkable (P > 0.05). As can be seen from the test results, Brevibacillus laterosporus BLCC1-0716 has the efficacy of inhibiting the weight loss of mice caused by free-drinking DSS.
2.3.2 Colon Length variations in mice
After the test was completed, the dissected mice were sacrificed by removing the cervical vertebrae, the intact colon was taken out, the residual feces were removed, and the colon length was measured.
TABLE 24 Colon Length in mice
Note: different letters indicate significant differences (P < 0.05).
Except that the CK group had intact colon tissues and formed feces in the intestinal lumen, bleeding appeared in the BLCC1-0716 and UC model groups, the feces in the intestinal lumen was reduced, the longest colon in the CK group was 11.03cm, the length of the colon in the UC model group was 7.81cm, which was 3.22cm shorter than that in the blank group, the length of the colon in the BLCC1-0716 group was 9.58cm longer than that in the UC model group, and the difference was significant (P < 0.05). The colon length of normal mice is 10-11cm, while the UC model group obviously shortens the colon length of the mice to about 7.8cm, and possibly the induction of DSS causes the mice to generate inflammation. Compared with the UC model group, the colon length of the Brevibacillus laterosporus BLCC1-0716 is increased, and compared with the UC model group, the colon length of the mouse of the Brevibacillus laterosporus BLCC1-0716 group is increased by 22.66%. The test result shows that the brevibacillus laterosporus can regulate and control the colon length of mice induced by DSS.
2.3.3 mouse colitis Disease Activity Index (DAI)
The mouse feces were scored for DAI at the end of the trial. The results are shown in Table 25, and the scoring criteria are shown in Table 23.
TABLE 25 mouse DAI score
Note: the different letters of the same row data shoulder mark indicate significant difference (P < 0.05); shoulder marks with the same letter or no letter designation indicate no significant difference (P > 0.05).
As seen from Table 25, the DAI score of the mice in the blank group was maintained at about 0.25, with no change, and the other mice in each group exhibited loose stools, brownish red bloodstains on stools, tiredness, lying, and laziness due to the induction of Dextran Sodium Sulfate (DSS), and the weight of the mice continuously decreased with the extension of molding time, the stools were not shaped, adhered to the anus, and bloody stools were observed with naked eyes, and the DAI score was high. The DAI score of the feces BLCC1-0716 group was 26.95% lower than that of the UC model group, which indicates that the Brevibacillus laterosporus BLCC1-0716 has a certain treatment effect on the mouse colitis caused by self-drinking DSS.
2.3.4 serum cytokine level changes
Each group of mice was bled from the eyes with DSS 10d and the level of each inflammatory factor was measured with an ELISA kit, and the results are shown in table 26.
TABLE 26 serum cytokine level changes (pg/mL) after free administration of DSS to each group of mice
Note: the data in the same row are marked with different letters to indicate that the difference is significant (P < 0.05); shoulder marks with the same letter or no letter designation indicate no significant difference (P > 0.05).
After mice freely drink DSS for 10 days, blood is taken from eyeballs immediately, the level of serum cytokine IL-1 beta is 67.41pg/mL which is the highest in a UC model group and is 12.41% higher than that in a blank group, but the difference is not significant (P is more than 0.05); the group BLCC1-0716 was 3.04% lower than the UC model group, but the difference was not significant (P > 0.05); serum TNF-alpha level is highest in UC model group, is 400.41pg/mL, is significantly higher than blank group (P <0.05), and BLCC1-0716 group is 360.15pg/mL, is 10.05% lower than UC model group, but the difference is not significant (P > 0.05); serum IL-6 levels were lowest in the blank group and were significantly lower than 80.20pg/mL for the UC model group (p <0.05), 68.49pg/mL for the BLCC1-0716 group and significantly lower than the UC model group (p < 0.05); serum IL-10 levels were highest in the BLCC1-0716 group at 197.11pg/mL, which is 3.11% higher than the UC model group, but the differences were not significant (P > 0.05).
2.3.5 Colon tissue Observation in mice
The same part of the colon was taken from each group of mice and the observation results are shown in FIG. 9.
As shown in fig. 9, the blank mice had clear colon walls, intact villi structures, clear outlines, regular and sharp arrangement, intact glandular structures, regular arrangement, normal crypts, and no inflammatory cell infiltration. The UC model group mice have colocolic epithelial necrosis and desquamation, thinned intestinal walls, seriously damaged intestinal gland structures, disordered glands, seriously broken and lost intestinal villi, and only leave mucous membrane muscularis and interstitial edema of the mucous membrane muscularis. The BLCC1-0716 group was significantly improved over the model group in that the intestinal wall was clear, the villus structure was substantially intact, the contour was substantially clear, the arrangement was relatively regular, the gland structure was substantially intact, the arrangement was substantially regular, the crypt was normal, and inflammatory cell infiltration was less, but inferior to that of the blank control group.
Example 6Comparative evaluation of the efficacy of the Strain BLCC1-0716 on ulcerative colitis in mice
1 Material
The route of purchase of mouse products and DSS were the same as in section 1.1 of example 5.
The strain is as follows: BLCC1-0716 and strain JYH-Y3 screened in the same batch and having relatively good in-vitro antibacterial property.
2 method
40 female Kunming mice of about 18g are fed with basal feed and are fed adaptively for 3 days. The samples were randomly divided into CK group, UC model group, JYH-Y3 group and BLCC1-0716 group, and the grouping and treatment were shown in Table 27, and the test methods and treatments were the same as those in section 2.2 of example 5. The general condition, body weight, fecal characteristics and occult blood of the mice were observed daily during the trial. After the test is finished, the cervical vertebrae are taken off, the mouse is killed, blood is taken from eyeballs, the colon is separated, the pathological change condition is observed by naked eyes, and the length of the colon is measured.
Table 27 test grouping and processing
3 results
3.1 mouse weight changes
In the test process, except the CK group mice for freely drinking water, the rest of the test group mice freely drink 5% DSS aqueous solution from 8d, and the mice are treated for 10d after the test is finished, and the weight change of the mice is shown in figure 10.
The test mice were weighed once a day and the change in body weight was recorded. The blank group of mice continued to gain body weight throughout the duration of the experiment due to the absence of induced stimulation by DSS. During the period of drinking DSS, the weight of mice in UC model group, JYH-Y3 group and BLCC1-0716 group tended to increase and then decrease, and the weight of mice in UC model group, JYH-Y3 group and BLCC1-0716 group tended to decrease after drinking day 5. By the end of the experiment, the final body weights of mice in the blank group, UC model group, JYH-Y3 and BLCC1-0716 groups were 33.37g, 28.99g, 31.11g and 32.09g, respectively. The weight loss of mice in the UC model group is the most significant, compared with a blank group, the weight loss is 4.38g, and the difference is significant (P < 0.05). The weight of the mice in the JYH-Y3 group and the Brevibacillus laterosporus BLCC1-0716 group are respectively reduced by 2.26 g and 1.28g compared with the blank group, the difference is not significant (P is more than 0.05), but the weight of the BLCC1-0716 group is reduced by less than that of the JYH-Y3 group. The test results show that the strains JYH-Y3 and BLCC1-0716 both have the efficacy of inhibiting the weight loss of mice caused by free drinking DSS, and the effect of Brevibacillus laterosporus BLCC1-0716 is better than that of JYH-Y3.
3.2 Colon Length Change in mice
After the experiment was completed, the dissected mice were sacrificed by removing the cervical vertebrae, the intact colon was taken out, the residual feces were removed, and the length of the colon was measured, and the result is shown in fig. 11. The intestinal tract of the blank control CK group mice is in a pink meat color, has healthy luster and granular feces. Through DSS induction, the feces of the mice in the model group are not shaped, and bloody stools appear in part of the mice. Shortening of the colon length, engorgement, thickening of the colon wall, development of ulcer surfaces, etc., is considered to be inversely related to the severity of the inflammatory condition. The colon length of BLCC1-0716 group and JYH-Y3 group are both longer than that of UC model group, and the effect of BLCC1-0716 group is most obvious, the colon length of the mouse in the group is obviously better than that of JYH-Y3 group (P is less than 0.05), meanwhile, the red and swollen phenomenon of the colon of the mouse in BLCC1-0716 group is reduced, the colon congestion phenomenon is not obvious, the stool morphology is obviously improved, and the stool is granular. The result shows that the Brevibacillus laterosporus BLCC1-0716 can regulate and control the length of the colon of a mouse induced by DSS, and the effect is obviously superior to JYH-Y3.
3.3 Colon tissue Observation in mice
The same part of the colon was taken from each group of mice and the observation results are shown in FIG. 12.
As shown in FIG. 12, the colon and intestine villi of the mice in the blank group (CK group) are complete, the gland structure is complete, the mucosal epithelial cells are arranged regularly, the crypts are normal, the submucosa is not swollen, and inflammatory cell infiltration is not seen. The UC model group mice have broken colon villi, damaged gland structure, disordered glands and serious deletion, the colon of the mice has obvious inflammatory reaction, and inflammatory cells such as neutrophils, lymphocytes and the like are seriously infiltrated. Compared with a UC model group, inflammatory cell infiltration of the JYH-Y3 group and the BLCC1-0716 group is obviously reduced, which shows that JYH-Y3 group and the BLCC1-0716 group both have certain anti-inflammatory efficacy, and particularly, no obvious inflammatory cell infiltration is seen in the BLCC1-0716 group, no obvious structural damage is caused, the structure is complete, the outline is clear, and the test result shows consistency with the test result of the 2.2 part. The results show that honeysuckle endophytes BLCC1-0716 and JYH-Y3 have the effects of improving the intestinal mucosa injury caused by DSS-induced mouse ulcerative colitis and protecting the intestinal tract, but the effects of BLCC1-0716 are more obvious and are obviously superior to JYH-Y3.
The test results show that the honeysuckle endophyte brevibacillus laterosporus BLCC1-0716 is an excellent probiotic strain, has a wide antibacterial spectrum, stable antibacterial performance and good tolerance, and can relieve animal intestinal inflammation, protect intestinal tracts, improve animal disease resistance and ensure feeding safety after entering the animal intestinal tracts. The brevibacillus laterosporus with superior performance screened by the invention provides more choices for antibiotics banned antibiotic substitutes for breeding animals at present, thereby protecting the ecological environment and ensuring the safety of animal-derived food. The method provides technical support for realizing high quality, high yield and high efficiency in the livestock breeding industry, is beneficial to controlling antibiotic residues, realizes standardization, normalization and intensification of drug residue-free breeding, and can obtain higher economic benefit and social benefit.
Although the present application has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described in the foregoing embodiments, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Shandong Baolaili Bio-engineering Ltd
<120> Brevibacillus laterosporus strain and application thereof
<130> 202127939
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1443
<212> DNA
<213> Brevibacillus laterosporus (Brevibacillus laterosporus) BLCC1-0716
<400> 1
ggccgtggcg gctgctatac atgcaagtcg agcgagggtc ttcggaccct agcggcggac 60
gggtgagtaa cacgtaggca acctgcctgt gagactggga taacataggg aaacttatgc 120
taataccgga tagggttttg cttcgcctga agcgaaacgg aaagatggcg caagctatca 180
cttacagatg ggcctgcggc gcattagcta gttggtgagg taacggctca ccaaggcgac 240
gatgcgtagc cgacctgaga gggtgaccgg ccacactggg actgagacac ggcccagact 300
cctacgggag gcagcagtag ggaattttcc acaatggacg aaagtctgat ggagcaacgc 360
cgcgtgaacg atgaaggctt tcgggtcgta aagttctgtt gttagggaag aaacagtgct 420
atttaaataa ggtagcacct tgacggtacc taacgagaaa gccacggcta actacgtgcc 480
agcagccgcg gtaatacgta ggtggcaagc gttgtccgga attattgggc gtaaagcgcg 540
cgcaggtggc tatgtaagtc tgatgttaaa gcccgaggct caacctcggt tcgcattgga 600
aactgtgtag cttgagtgca ggagaggaaa gtggtattcc acgtgtagcg gtgaaatgcg 660
tagagatgtg gaggaacacc agtggcgaag gcgactttct ggcctgtaac tgacactgag 720
gcgcgaaagc gtggggagca aacaggatta gataccctgg tagtccacgc cgtaaacgat 780
gagtgctagg tgttaggggt ttcaataccc ttagtgccgc agctaacgca ataagcactc 840
cgcctgggga gtacgctcgc aagagtgaaa ctcaaaggaa ttgacggggg cccgcacaag 900
cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc 960
cactgaccgc tctagagata gagcttccct tcggggcagt ggtgacaggt ggtgcatggt 1020
tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttatc 1080
tttagttgcc agcattcagt tgggcactct agagagactg ccgtcgacaa gacggaggaa 1140
ggcggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac gtgctacaat 1200
ggttggtaca acgggatgct acttcgcgag aagatgctaa tctcttaaaa ccaatctcag 1260
ttcggattgt aggctgcaac tcgcctacat gaagtcggaa tcgctagtaa tcgcggatca 1320
gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccacgggagt 1380
ttgcaacacc cgaagtcggt gaggtaaccg taaggagcca gccgccgaag gtggttgttt 1440
gct 1443
Claims (11)
1. A strain of Brevibacillus laterosporus named Brevibacillus laterosporus (A)Brevibacillus laterosporus) BLCC1-0716, which was deposited in China center for type culture Collection on 11/2021 with the deposition number of CCTCC NO: m20211397.
2. Use of Brevibacillus laterosporus as claimed in claim 1 for the preparation of an anti-inflammatory medicament or feed additive.
3. Use of Brevibacillus laterosporus as claimed in claim 1 for the preparation of a medicament or feed additive for the prevention and treatment of diarrhea.
4. Use of Brevibacillus laterosporus according to claim 1 for the preparation of a medicament or feed additive for the prevention and treatment of enteritis.
5. The use of claim 4, wherein the enteritis is pathogenic or non-pathogenic.
6. Use according to claim 5, wherein the non-pathogenic enteritis is selected from acute enteritis and ulcerative enteritis.
7. Use of Brevibacillus laterosporus according to claim 1 for the preparation of a medicament or feed additive for modulating the gut flora.
8. Use of Brevibacillus laterosporus as claimed in claim 1 for the preparation of a medicament or feed additive for the prevention and treatment of colonic atrophy.
9. Use of Brevibacillus laterosporus according to claim 1 for the preparation of a pharmaceutical or feed additive for the protection of the intestinal tract.
10. A microbial preparation or feed comprising the brevibacillus laterosporus of claim 1.
11. A pharmaceutical composition or pharmaceutical preparation comprising the brevibacillus laterosporus of claim 1.
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| WO2014011233A1 (en) * | 2012-07-09 | 2014-01-16 | Trachtman Ira Milton | Compositions and method for treatment and prophylaxis of inflammatory bowel disease |
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