CN111450167A

CN111450167A - Composite traditional Chinese medicine micro-ecological composition and preparation method and application thereof

Info

Publication number: CN111450167A
Application number: CN202010498238.7A
Authority: CN
Inventors: 王春凤; 单宝龙; 郭杨丽; 宋翔; 刘乃芝; 亓秀晔; 宁扬; 侯玉凤; 李鹏; 陈晓雯; 程福亮; 陈雷; 谷巍; 徐海燕; 王红
Original assignee: Shandong Boly Lely Bioengineering Co ltd
Current assignee: Shandong Boly Lely Bioengineering Co ltd
Priority date: 2020-06-04
Filing date: 2020-06-04
Publication date: 2020-07-28

Abstract

The invention provides a composite traditional Chinese medicine microecological composition and a preparation method and application thereof, the composition takes a traditional Chinese medicine compound and probiotics as raw materials, wherein the probiotics are lactobacillus Plantarum and/or a fermentation product thereof, the traditional Chinese medicine compound comprises goldthread root, golden buckwheat rhizome and houttuynia cordata, the lactobacillus Plantarum is lactobacillus Plantarum (L actinobacillus Plantarum) B L CC2-0125, the lactobacillus Plantarum is stored in China center for type culture Collection in Wuhan City at 24 months 4 in 2020, and the preservation number is CCTCC NO: M2020078.

Description

Composite traditional Chinese medicine micro-ecological composition and preparation method and application thereof

Technical Field

The invention relates to the field of immune microecology, in particular to a composite traditional Chinese medicine microecology composition and a preparation method and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

The mycoplasma gallisepticum is one of respiratory diseases of chickens, and causes great economic loss to the breeding industry and the animal husbandry. At present, antibiotics and vaccines are mainly used for preventing and treating the disease, and both the antibiotics and the vaccines have disadvantages. The disadvantages associated with antibiotics include (1) problems with bacterial resistance; (2) the immunity of the organism is destroyed; (3) serious food and safety hazards. The use of conventional vaccines has revealed problems of (1) stress-induced development of other diseases; (2) variant strains are easy to appear; (3) the animal has virus return; (4) the immune effect is not ideal, etc.

The traditional Chinese medicine is widely used for preventing and treating various diseases of livestock and poultry, including respiratory diseases, due to the characteristics of good curative effect, no side effect, no residue, capability of enhancing the immunity of the organism and the like. The probiotics has the characteristics of safety, no pollution, no residue, disease prevention and the like, can obviously improve the feed conversion rate and the production performance of animals, is a preferred green feed additive for replacing chemical synthetic substances such as feed antibiotics and the like, and has relatively great progress in application research of the probiotics in respiratory medicine and critical diseases in recent years. Research has proved that probiotics and Chinese herbal medicines supplement each other in the aspects of improving the immunity of the organism and preventing and treating diseases. At present, most of traditional Chinese medicines are crushed and then directly added with a mixing material for preventing and treating animal diseases, active ingredients of the traditional Chinese medicines cannot be fully utilized, and the digestive tract ratio of chickens is short, so that the active ingredients of the chickens cannot be absorbed and utilized, great waste is caused, and the medication cost is increased.

Disclosure of Invention

Therefore, the invention aims to provide the composite traditional Chinese medicine micro-ecological composition as well as the preparation method and the application thereof, and the composite traditional Chinese medicine micro-ecological composition has a good effect of preventing and treating mycoplasma gallisepticum infection, and can improve the immunity of chickens, enhance the disease resistance and increase the economic benefit.

Specifically, the technical scheme of the invention is as follows:

in the first aspect of the invention, a composite traditional Chinese medicine microecological composition is provided, which takes a traditional Chinese medicine compound and probiotics as raw materials, wherein the probiotics are lactobacillus plantarum and/or a fermentation product thereof; the Chinese herbal compound comprises coptis chinensis, golden cypress, wild buckwheat rhizome and houttuynia cordata;

the Lactobacillus Plantarum is Lactobacillus Plantarum (L actobacillus Plantarum) B L CC2-0125, which is preserved in China center for type culture Collection (address: Wuhan university, Wuhan, China) in 24 months and 4 months in 2020, and the preservation number is CCTCCNO: M2020078.

The lactobacillus plantarum B L CC2-0125 can grow on an MRS culture medium, and has the advantages of cell surface hydrophobicity, good cell adhesion, self-aggregation (self-aggregation) capacity (the self-aggregation rate is close to 50% in 2 h), high-yield extracellular polysaccharide (the yield of the extracellular polysaccharide can reach 996.67 mg/L after fermentation culture is carried out for 24 hours), acid resistance (the survival rate can reach 45%, 90% and 100% respectively when the pH value is pH2.5, pH3.0 and pH6.0, and the extracellular polysaccharide can survive under the environment with the pH value being more than 2.0), cholate resistance (the survival rate can survive in 0.1% of poultry bile salt culture for 4h and 92.1%, the poultry bile salt concentration can still survive when the poultry bile salt is 0.3%), strong tolerance to trypsin and pepsin (the survival rates are respectively 92% and 76% after the poultry bile salt is treated in trypsin and pepsin liquid, continuous generation of 5, 10 generation, 15 generation and 20 generation culture gram-positive bacilli with different viable bacterium contents and large passage contents and large pH value difference of about 15.4).

The lactobacillus plantarum B L CC2-0125 has a good immune function, the weight of an animal using the lactobacillus plantarum is steadily increased after 5 days of gastric lavage compared with a blank group, a model group and a positive drug levamisole control group in an immunosuppression (an immunosuppression model is prepared by cyclophosphamide), the thymus index and the spleen index are obviously increased compared with the model group, and the spleen index is not obviously different from the blank group in an animal serum cytokine detection experiment, the I L-2 level, the IFN-gamma level and the IgG level in the animal serum using the lactobacillus plantarum are obviously improved compared with the model group (the immunosuppression model is prepared by cyclophosphamide) in an animal serum cytokine detection experiment, so that the oral administration of the lactobacillus plantarum disclosed by the invention can improve the levels of the serum I L-2, the IFN-gamma and the IgG of an immunosuppression animal, and the lactobacillus plantarum B L CC2-0125 can prevent (prevent and treat, particularly prevent) mycoplasma gallisepticum infection and has an effect on low avian influenza (H9N 2).

In addition, the lactobacillus plantarum B L CC2-0125 has better safety, and in an animal oral acute toxicity test, the weight of an animal using the lactobacillus plantarum can be normally increased after administration, which shows that the lactobacillus plantarum has safety when orally taken.

The medicinal properties of the traditional Chinese medicines comprise:

coptis chinensis: cool and cold in nature, clearing heat and drying dampness, purging fire and removing toxicity.

Phellodendron bark: is bitter and cold in nature, and has the effects of clearing heat and drying dampness, purging fire and removing steam, and detoxifying and treating sore.

Wild buckwheat rhizome: sour, bitter and cold in nature. It enters lung, stomach and liver meridians. Clearing away heat and toxic materials; promoting blood circulation and removing carbuncle; dispelling wind and eliminating dampness. Mainly treats pulmonary abscess; cough and asthma due to lung heat; swollen and sore throat; dysentery; syndrome of wind-dampness arthralgia; traumatic injury; abscess, swelling and sore.

Houttuynia cordata: pungent flavor, cold and cool in nature, enter lung meridian. It has effects of clearing away heat and toxic materials, relieving swelling, treating sore, inducing diuresis, removing dampness, clearing away heat, relieving dysentery, invigorating stomach, and resolving food stagnation, and can be used for treating lung abscess, pyocutaneous disease, hemorrhoid hematochezia, and heat accumulation of spleen and stomach due to excess heat, heat toxin, dampness, and disease heat. Has antibacterial, antiviral, immunity improving, and diuretic effects.

The traditional Chinese medicine disclosed by the invention has a certain effect of preventing and treating mycoplasma gallisepticum infection after being compounded, can reduce the death rate of chickens infected with mycoplasma gallisepticum, reduce the damage to air sacs, improve the immunity of the chickens to a certain extent, and improve the levels of IgG and IFN-gamma in serum. In the traditional Chinese medicine formula, the addition of wild buckwheat rhizome, particularly the simultaneous addition of wild buckwheat rhizome and houttuynia cordata can particularly promote the achievement of the above effects.

According to the embodiment of the invention, after the traditional Chinese medicine compound is compounded with the lactobacillus plantarum B L CC2-0125, the effect of preventing and treating mycoplasma gallisepticum infection can be greatly improved, and an excellent synergistic effect between the traditional Chinese medicine compound and the lactobacillus plantarum B L CC2-0125 is shown.

In an embodiment of the invention, the Lactobacillus plantarum B L CC2-0125 and/or a fermentation product thereof is obtainable by fermentative cultivation of Lactobacillus plantarum B L CC 2-0125.

The fermentation can be carried out by conventional fermentation methods or, as described in certain embodiments of the present invention, by inoculating Lactobacillus plantarum B L CC2-0125 into MRS liquid medium under aseptic conditions and culturing in 37 ℃ incubator for 16-18 h.

In some embodiments of the present invention, the number of viable bacteria of the probiotics in the composite traditional Chinese medicine microecological composition is greater than or equal to 1 × 10⁹cfu/m L with the increase of viable bacteria, the antibacterial effect and therapeutic effect of the composition will also increase, but when the viable bacteria concentration is 1 × 10⁹The ideal effect can be achieved when cfu/m L, in some more preferable embodiments of the invention, the viable count of the probiotics in the traditional Chinese medicine microecological composition is 1 × 10⁹cfu/mL～1×10¹⁰cfu/mL。

In some embodiments of the invention, the Chinese herbal compound is prepared from the following raw materials in parts by weight: 5-8 parts of coptis chinensis, 10-20 parts of golden cypress, 10-15 parts of wild buckwheat rhizome and 10-15 parts of houttuynia cordata.

In some embodiments of the present invention, the amount of the coptis root in the traditional Chinese medicine compound can be 5 parts, 6 parts, 7 parts or 8 parts, the amount of the phellodendron bark can be 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts or 20 parts, the amount of the cymose buckwheat can be 10 parts, 11 parts, 12 parts, 13 parts, 14 parts or 15 parts, and the amount of the houttuynia cordata can be 10 parts, 11 parts, 12 parts, 13 parts, 14 parts or 15 parts; of course, the above examples are all the whole weight parts, the weight parts of the raw materials can be any value between any two integer values, for example, the usage of the coptis root can be 5.5-8 parts, 6-8 parts or 7.5-8 parts, the usage of the phellodendron can be 10.5-20 parts, 10.5-15 parts or 13.5-20 parts, the usage of the wild buckwheat can be 10.5-15 parts, 10-12.5 parts or 12-14.5 parts, and the usage of the houttuynia can be 10.5-15 parts, 10-12.5 parts or 12-14.5 parts.

In some preferred embodiments, the Chinese herbal compound is prepared from the following raw materials in parts by weight: 6-8 parts of coptis chinensis, 15-20 parts of golden cypress, 10-12 parts of wild buckwheat rhizome and 10-12 parts of houttuynia cordata, and further preferably 6 parts of coptis chinensis, 15 parts of golden cypress, 12 parts of wild buckwheat rhizome and 12 parts of houttuynia cordata.

In a second aspect of the invention, a method for preparing the composite traditional Chinese medicine microecological composition described in the first aspect is provided, which comprises mixing a traditional Chinese medicine compound and probiotics, wherein the traditional Chinese medicine compound is traditional Chinese medicine extract, and the probiotics is lactobacillus plantarum B L CC2-0125 and/or a fermentation product thereof.

In some embodiments of the invention, the method comprises: weighing raw materials of a Chinese herbal compound according to parts by weight, mixing, adding 8-10 times of water by weight (namely the adding amount of the water is 8-10 times of the weight of the mixed medicinal materials), soaking, heating to boil, decocting with water for at least one time, and concentrating the filtrate to obtain a compound Chinese herbal concentrated filtrate, namely a Chinese herbal extract; adding probiotics into the traditional Chinese medicine extracting solution, and uniformly mixing to obtain a composite traditional Chinese medicine micro-ecological composition;

the probiotic can be probiotic bacteria liquid or probiotic powder, for example, the probiotic bacteria liquid can be obtained by fermenting and culturing lactobacillus plantarum B L CC2-0125, the fermentation liquid is further processed, such as vacuum freeze-drying operation, to obtain a solid fermentation product, or further, the lactobacillus plantarum B L CC2-0125 is fermented and then centrifuged to obtain thalli, the thalli are subjected to vacuum freeze-drying, and the probiotic powder can be obtained, wherein the freeze-drying protective agent can be optionally added in the vacuum freeze-drying process, and the addition mode can be that the fermentation liquid or the thalli is mixed with the freeze-drying protective agent before freeze-drying.

In some embodiments of the present invention, the lyoprotectant may be a lyoprotectant that is conventionally used in the art, such as skim milk, sucrose, glycerol, etc., and the added amount of the lyoprotectant may be a conventional amount in the art, or the added amount of the lyoprotectant is 2 to 90 wt%, preferably 5 to 80 wt%, and especially 40 to 60 wt% of the weight of the mixed raw materials, for example, in some embodiments of the present invention, the mass parts of the lyoprotectant and the bacterial sludge (or bacterial cells) in the microecological preparation are 120 parts and 100 parts, respectively.

In the embodiment of the present invention, although the conventional lyoprotectant can protect the bacterial cells of the present invention to some extent during the lyophilization process, it was found in the research process of the present invention that the technical effects of the present invention can be more effectively achieved when a lyoprotectant with a specific composition is selected. For example, in some embodiments of the invention, the protectant comprises or consists of: skim milk, trehalose, manganese sulfate, sucrose, sodium erythorbate and glucose; alternatively, the protective agent comprises or consists of: skim milk, trehalose, manganese sulfate, sucrose, Vc, glycerol and glucose.

In some embodiments of the invention, the viable count of lactobacillus plantarum B L CC2-0125 contained in the probiotic powder (lyophilized powder) in the compound traditional Chinese medicine microecological preparation or compound traditional Chinese medicine microecological composition is not less than 1.00 × 10¹⁰CFU/g。

In a more preferred embodiment of the present invention, when the probiotic is an oral probiotic, the protective agent comprises or consists of: skim milk, trehalose, manganese sulfate, sucrose, sodium erythorbate and glucose. In particular, in these embodiments, the content of each component in the protective agent is: 1-10 parts of skim milk, 1-3 parts of trehalose, 0.1-0.5 part of manganese sulfate, 0.1-0.5 part of sucrose, 0.1-0.5 part of sodium erythorbate and 0-0.5 part of glucose (preferably 0.1-0.5 part by weight), and the components are uniformly mixed and dissolved in sterile water to obtain the compound; in a preferred embodiment, the ingredients of the protectant formulation are preferably pre-packaged and radiation sterilized, dissolved in sterile water (e.g., 100 parts by weight) in a suitable ratio, and placed in dry heat sterilized glass bottles for use.

In some embodiments of the invention, the water decoction is performed 3 times, comprising: decocting for 30-60min, filtering, retaining filtrate, adding 8-10 weight times of water into the residue, decocting for 30-60min for the second time, filtering, retaining filtrate, adding 6-8 weight times of water into the residue, decocting for 30-50min for the third time, and retaining filtrate; after the water decoction is finished, the three filtrates are combined.

In a third aspect of the present invention, there is provided a composite Chinese medicinal microecological preparation comprising the composite Chinese medicinal microecological composition as described in the above first aspect.

The composite traditional Chinese medicine microecological preparation can be an oral liquid preparation or an oral solid preparation. Wherein, the solid preparation can be taken orally with water or directly after being dissolved in water.

The compound traditional Chinese medicine microecological preparation can be prepared according to the preparation method of the compound traditional Chinese medicine microecological composition; or, other acceptable auxiliary materials or additives are further added on the basis of the composite traditional Chinese medicine microecological composition, and the auxiliary materials and the additives only have the function of being further convenient to eat or being convenient to prepare into required dosage forms. One skilled in the art can select appropriate adjuvants or additives as required and prepare the desired dosage forms according to conventional methods in the art.

In a fourth aspect of the present invention, an application of the composite traditional Chinese medicine micro-ecological composition in the first aspect or the composite traditional Chinese medicine micro-ecological preparation in the third aspect in preparing a product for preventing and treating mycoplasma gallisepticum or related diseases caused by mycoplasma gallisepticum is provided.

Wherein the product is a medicine, a health product, a feed or a feed additive.

Compared with the prior art, the invention has the beneficial effects that:

(1) the traditional Chinese medicine compound disclosed by the invention can be well used for preventing (preventing and treating) infection of mycoplasma gallisepticum by compounding with lactobacillus plantarum B L CC2-0125, has a very obvious effect starting to play at the initial stage of mycoplasma gallisepticum infection, reduces the death rate of infected chickens, can reduce air sac injury to a great extent, can improve the immunity of an organism, enhances the disease resistance of the organism and the like.

(2) The lactobacillus plantarum has the function of regulating gastrointestinal microecological balance, so that the immunity index of an organism is improved; promoting proliferation of Lactobacillus caecum and Lactobacillus rectum, inhibiting proliferation of Escherichia coli, and regulating intestinal flora of chicken.

(3) The invention combines the probiotics and the traditional Chinese medicine, and the probiotics and the traditional Chinese medicine supplement each other in the aspects of disease prevention and growth promotion. The traditional Chinese medicine can promote the proliferation of probiotics and the exertion of the functions of the probiotics, and the probiotics can promote the absorption and utilization of the traditional Chinese medicine. The probiotics and traditional Chinese medicine compound preparation has the advantages of being natural, non-toxic, free of side effect, free of residue, safe and clean, and shows wide development prospect.

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 invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1: a shows that the mycoplasma gallisepticum liquid medium culture changes from a clear transparent rose-red color to a yellow semi-turbid state (from left to right); b shows mycoplasma gallisepticum solid medium cultured as a typical "omelette" colony.

FIG. 2: a shows mycoplasma gallisepticum swiss staining results; b shows mycoplasma gallisepticum giemsa staining results.

FIG. 3: a shows the results of PCR identification of isolated Mycoplasma gallisepticum; b shows the degree of matching between the base sequence of the amplified gene fragment in a and the base sequence of Mycoplasma gallisepticum 16S RNA.

FIG. 4: and (3) a purification process of mycoplasma gallisepticum and a PCR identification result of a purified product.

FIG. 5: and (3) determining the result of the mycoplasma gallisepticum CCU.

FIG. 6: a, dissecting the air sacs of blank animals, wherein the air sacs are transparent; and b, the anatomical result of the air sac of the animal in the model group shows that the air sac is turbid and thickened and has light yellow cheese-like exudates.

Detailed Description

The invention will be further illustrated with reference to the following 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 invention. 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 invention can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present invention can be used in a conventional manner 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 invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1Primary screen for lactic acid bacteria with immunological activity

1 materials and methods

1.1 materials

MRS culture medium: calculated by mass percent, 2.0 percent of glucose, 0.2 percent of sodium citrate, 0.5 percent of sodium acetate, 0.5 percent of dipotassium hydrogen phosphate, 0.02 percent of manganese sulfate, 0.05 percent of magnesium sulfate, 1.0 percent of peptone, 1.0 percent of beef extract, 0.5 percent of yeast extract, 800.1 percent of tween-tween and pH 6.0.

IEC-6 cells (small intestine epithelial cells) were purchased from the cell bank of the culture Collection of type, national academy of sciences.

1.2 methods

1.2.1 preparation of bacterial suspension

Respectively inoculating 20 strains of lactobacillus provided by the strain preservation center of biological engineering research institute of bioscience, Inc. of Shandong Baolalilai into MRS liquid culture medium, culturing at 37 deg.C for 16-18h, centrifuging at 4 deg.C and 3000rpm for 15min, collecting thallus, suspending the thallus with PBS solution, centrifuging, and repeating for 3 times.

1.2.2 screening of hydrophobic lactic acid bacteria

The hydrophobicity of the surface of lactic acid bacteria was measured by the microbial adhesion hydrocarbon method (BATH). Resuspending the washed cells in 0.1M KNO₃In the solution, the absorbance (OD 600nm) of the cell suspension was adjusted to 0.5. + -. 0.02(A₀) Mixing bacterial suspension 2m L with 200 μ L xylene, standing at room temperature for 10min, vortexing and shaking for 2min, standing for 10min to form a two-phase system, carefully absorbing water phase, and measuring absorbance (A) at 600nm₁)。

Cell surface hydrophobicity calculation formula:

1.2.3 lactic acid bacteria adhesion screening

After the IEC-6 cells were recovered, they were placed in a flask containing DMEM complete medium at 37 ℃ with 5% CO₂Incubating in an incubator, carrying out adhesion test after about 5 passages, adjusting the concentration of IEC-6 cells to 2 × 10⁴cells/m L, inoculating in 6-well culture plate (placing sterile cover glass in advance), allowing cells to grow on the cover glass, after the cells are cultured to compact monolayer, rinsing with PBS buffer solution for 2 times, adding 1m L DMEM complete culture solution and 1m L prepared bacterial suspension into each well, shaking, mixing well, and adding into CO₂Incubators continued incubation, repeating 3 wells per strain. After culturing for 60min, the 6-well culture plate was taken out, the cells were washed 5 times with PBS buffer (to remove non-adherent lactic acid bacteria), fixed for 20min with anhydrous methanol, stained with gram, and 20 fields were randomly selected under a microscope to count the number of bacteria adhered to 100 cells, and the average number of bacteria adhered to each cell was used to express the adhesion ability.

1.2.4 screening of lactic acid bacteria having self-coagulating ability

The cells were washed twice with PBS solution and resuspended in PBS solution so that the absorbance (OD 600nm) of the cell suspension became 0.5. + -. 0.02 (A)_0h) Taking PBS solution as blank control, placing 4m L adjusted bacterial suspension in test tube, standing at room temperature for 2 hr, absorbing 1m L supernatant solution, and measuring 600nm absorbance (A)_2h)。

Self-agglomeration capacity calculation formula:

2 results

2.1 screening of hydrophobic lactic acid bacteria

In table 1, the surface hydrophobicity of 20 lactic acid bacteria was greatly different, and 8 lactic acid bacteria were found to have a surface hydrophobicity exceeding 45%, i.e., No.1, 4, 9, 11, 14, 15, 16, and 20 lactic acid bacteria, and 4 lactic acid bacteria were found to have a surface hydrophobicity of 30% to 45%. Wherein the surface hydrophobicity of the lactobacillus No.1 is the highest and reaches 62%, and the lactobacillus No. 16 is the next strain and reaches 54%.

TABLE 1 measurement of the surface hydrophobicity of lactic acid bacteria

Note: the shoulder letters in the table represent significant difference analysis (p < 0.05).

2.2 lactic acid bacteria adhesion screening

The adhesion of different lactic acid bacteria to IEC-6 cells is shown in Table 2. The difference of different lactic acid bacteria to the number of cell adhesion bacteria is large, 8 lactic acid bacteria with the adhesion number larger than 10 are respectively 1, 4, 9, 11, 14, 15, 16 and 20 strains, wherein the adhesion number of the 16 lactic acid bacteria is up to 18.

Table 2 number of adhesion of lactic acid bacteria to IEC-6 cells (n-3,

)

2.3 screening of lactic acid bacteria having self-coagulating ability

The self-polymerizing ability of 20 lactic acid bacteria is shown in Table 3. The polymerization ability of different strains is greatly different, and more than 37 percent of 10 strains are respectively No.1, 4, 9, 11, 12, 14, 15, 16, 17 and 20 strains. The 16 bacteria have the strongest self-polymerization ability which reaches 48 percent, the second bacteria are the 1, 9 and 15 bacteria which are 46 percent, the lowest bacteria are the 5 strain and the 19 strain, and the self-aggregation rate is only 11 percent.

TABLE 3 determination of the self-aggregation Capacity of lactic acid bacteria

Example 2Rescreening of immunocompetent lactic acid bacteria

1 materials and methods

1.1 materials

MRS culture medium: calculated by mass percent, 2.0 percent of glucose, 0.2 percent of sodium citrate, 0.5 percent of sodium acetate, 0.5 percent of dipotassium hydrogen phosphate, 0.02 percent of manganese sulfate, 0.05 percent of magnesium sulfate, 1.0 percent of peptone, 1.0 percent of beef extract, 0.5 percent of yeast extract, 800.1 percent of tween-tween and pH 6.0. The solid culture medium needs to be added with 1.5 percent of agar powder.

Modified MRS medium: sucrose is used to replace glucose, and the other culture media are the same as MRS culture media.

1.2 methods

1.2.1 Primary screening for exopolysaccharide-producing lactic acid bacteria (colony wiredrawing method)

8 strains of lactic acid bacteria (see Table 4) preliminarily screened in example 1 were inoculated into an MRS medium for activation, and the activated lactic acid bacteria were inoculated into a modified MRS solid medium by a plate-streaking method, cultured at 37 ℃ for 24 hours, and the colony morphology was observed.

1.2.2 extracellular polysaccharide-producing lactic acid bacteria rescreening (phenol-concentrated sulfuric acid method)

The strain obtained in the primary screening of example 1.2.1 was inoculated into modified MRS liquid medium and left to ferment at 37 ℃ for 24 h. Taking fermentation liquor, carrying out the whole process at 4 ℃, centrifuging at 10000rpm for 15min, taking supernate, adding trichloroacetic acid into the supernate until the final mass fraction is 4%, reacting for 12h, centrifuging at 10000rpm for 15min, collecting supernate, adding 3 times of 95% (volume fraction) ethanol solution for leaching for 12h, centrifuging at 10000rpm for 15min, collecting precipitate, washing with absolute ethanol, dissolving with deionized water to constant volume, dialyzing the solution with deionized water (8000-14000 Da) for 3d, changing water every 8h, and carrying out vacuum concentration under reduced pressure until the original volume is the crude polysaccharide solution after the dialysis is finished.

2 results

2.1 Primary screening of extracellular polysaccharide-producing lactic acid bacteria

The screening results are shown in table 4, and the single colonies No.1, No. 4, No. 15 and No. 16 which have sticky surfaces and can be drawn are selected, stored on the inclined plane of an MRS culture medium and stored in a refrigerator at 4 ℃.

TABLE 4 preliminary screening results for exopolysaccharide-producing lactic acid bacteria

Note: "+": is, "-": and no.

2.2 extracellular polysaccharide-producing lactic acid bacteria rescreening

The results of the rescreening are shown in Table 5, and the results show that the extracellular polysaccharide yield of the strains No.1 and 16 is 700-800 mg/L, and the extracellular polysaccharide yield of the strain No. 15 can reach 996.67 mg/L, so that the strains No.1, 15 and 16 are selected for immune function detection.

TABLE 5 results of rescreening extracellular polysaccharide-producing lactic acid bacteria

Example 3Experiment for detecting immunologic function of lactic acid bacteria

1 materials and methods

1.1 materials

Healthy Kunming mice are purchased from Shandong Taibang biological products, Inc., and mouse interleukin 2(I L-2) E L ISA detection kit, mouse gamma interferon (IFN-gamma) E L ISA kit and mouse immunoglobulin G (IgG) E L ISA kit are purchased from Beijing equation biotechnology, Inc.

1.2 methods

1.2.1 lactic acid bacteria immune function test

210 healthy Kunming mice with half male and female parts and 18-20g weight are selected, are placed in a quiet, warm and light-proof environment to be fed and adapted for 3d, and are randomly divided into 6 groups, wherein each group comprises 35 mice, and the groups respectively comprise a blank group, a model group, a positive control group, a No.1 strain group, a No. 15 strain group and a No. 16 strain group. Before the experiment, the other groups except the blank group are subjected to a method of continuously injecting 80mg/kg cyclophosphamide into 3D (D-2-D0) abdominal cavity to establish an immune low mouse model, and the immunosuppression effect is measured by the weight of the mouse.

Continuously performing intragastric administration for 3 weeks after immunosuppression, administering sterilized normal saline to blank group and model group, administering levamisole (concentration is 50mg/kg) to positive control group, and performing intragastric administration at dosage of 0.1m L/10 g/d, wherein strain No.1, strain No. 15, and strain No. 16 are 10⁸And (3) irrigating the stomach with CFU/0.2m L/mouse/day, weighing every 4 days during the period of the irrigation, weighing 5 mice in a blank group and other groups respectively on the 2 nd day after the last irrigation, collecting whole blood for detecting immune indexes, weighing the weights of spleen and thymus, observing the activity and death of the mice, and recording in detail.

2 results

2.1 mouse body weight analysis

Mice were weighed 6 times throughout the experiment. As shown in Table 6, there was no significant difference in initial body weight 2 days (D-2) before the start of the gavage test. Subsequently, the remaining five groups of mice showed a dramatic decrease in body weight after cyclophosphamide injection compared to the blank group, indicating that cyclophosphamide injection caused immunosuppression in the mice. During the period of the gavage treatment, the body weights of the mice of the strain group No.1, the strain group No. 15 and the strain group No. 16 were increased to some extent and gradually increased steadily on the 5 th day after the start of the gavage compared with the model group.

TABLE 6 weight change over time in mice

Note: d-2 means two days before the beginning of the gavage test, and D1, D5, D10, D15 and D20 mean the 1 st, 5 th, 10 th, 15 th and 20 th days of the gavage respectively.

2.2 detection of mouse immune organ index

As shown in table 7, the effect on mouse thymus and spleen indices: compared with the model group mice, the thymus indexes of the mice of the blank group, the strain group No.1, the strain group No. 15 and the strain group No. 16 are all obviously improved (P < 0.05); compared with the model group, spleen indexes of mice of the strain No.1 group, the strain No. 15 group and the positive control group are all increased remarkably (P <0.05), and in addition, spleen indexes of mice of the strain No. 15 group and the blank group are not different remarkably (P > 0.05).

TABLE 73 Effect of lactic acid bacteria on mouse thymus index and spleen index

2.3 detection of mouse serum cytokines

The results are shown in table 8, and the data in the table show that the I L-2 levels in the serum of mice in the strain group No.1, the strain group No. 15 and the strain group No. 16 are all obviously improved (P is less than 0.05) compared with the model group, the difference among different lactic acid bacteria (1, 15 and 16) is obvious (P is less than 0.05), wherein the I L-2 level of the strain group No. 15 is the highest, and the 539 analysis result shows that the lactic acid bacteria have in vivo immunological activity and can improve the I2-2 level of the serum of the mice.

IFN-gamma: as can be seen from the data in the table, the IFN-gamma levels in the serum of mice in the strain group No.1, the strain group No. 15 and the strain group No. 16 are all significantly improved (P <0.05), wherein the IFN-gamma level of the strain group No. 15 is significantly higher than that of the lactobacillus group No. 1. Analysis results show that the 3 strains of lactic acid bacteria have in vivo immunological activity and can improve the serum IFN-gamma level of immunosuppressive mice.

IgG: IgG is the major immunoglobulin present in blood, lymph, peritoneal fluid, and cerebrospinal fluid, and accounts for over 75% of serum total immunoglobulins. As can be seen from the data in the table: compared with the model group of mice, the No.1 strain group, the No. 15 strain group and the No. 16 strain group can improve the IgG level in the serum of the immunosuppression mice, and have obvious difference (P < 0.05). Analysis results show that the 3 strains of lactic acid bacteria have in-vivo immunological activity and can improve the serum IgG level of immunosuppressive mice, wherein the No. 15 strain has the most obvious effect.

TABLE 83 Effect of lactic acid bacteria on mouse serum I L-2, IFN-. gamma.and IgG levels

In conclusion, strain 15 was most effective in immunizing, and therefore strain 15 was prepared for later testing.

Example 4Detection and identification of biological characteristics of No. 15 lactobacillus

1 materials and methods

1.1 materials

Healthy adult SPF-grade KM mice, purchased from santopram bio ltd.

1.2 methods

1.2.1 acid resistance test

Culturing lactobacillus No. 15 for 15h, inoculating to MRS culture medium with pH of 2.0, 2.5, and 3.0 according to 1% (V/V), sterilizing with high pressure steam at 121 deg.C for 20min with MRS culture medium with pH of 6.0 as control, and cooling. Culturing at 37 deg.C for 4 hr, sampling, counting colonies, and calculating survival rate of lactobacillus.

Survival rate (%) (viable count of bacterial suspension/viable count of initial bacterial suspension for PH to be measured) × 100

1.2.2 bile salt resistance test

Culturing No. 15 lactobacillus for 16h, inoculating to MRS culture medium with fowl bile salt concentration of 0.1%, 0.2%, and 0.3% according to 1% (V/V), sterilizing with high pressure steam at 121 deg.C for 20min with MRS culture medium without fowl bile salt as control, and cooling. The strain was inoculated into the treated medium at an inoculum size of 2% (v/v), cultured at 37 ℃ for 4 hours, sampled, counted for colonies, and assayed for viability.

Survival rate (%) (viable count of bacterial liquid to be tested for fowl bile salt concentration/viable count of initial bacterial liquid) × 100

1.2.3 digestive enzyme tolerance test

Culturing lactobacillus No. 15 on MRS solid plate at 37 deg.C overnight, centrifuging bacterial liquid, collecting bacterial mud, re-dissolving with equal amount of normal saline, diluting by 10 times, mixing 1m L bacterial suspension and 9m L pepsin liquid, standing at 37 deg.C for 4 hr, and counting at 0 hr, 2 hr, and 4 hr respectively.

Mixing 1m L bacterial suspension with 9m L trypsin solution, standing and culturing at 37 ℃ for 4h, and counting at 0h and 4h respectively.

1.2.4 animal oral acute toxicology test

Selecting 20 healthy adult SPF (specific pathogen free) KM (KM) mice of 18-22 g, wherein each mouse is half male and female, dividing the mice into two groups, namely a control group and a test group, performing intragastric administration according to the weight ratio of 20.0m L/kg, and performing intragastric administration on the test group, namely the number 15 lactobacillus bacterial liquid, wherein the viable count is 1 × 10¹⁰CFU/m L, gavage once on an empty stomach, and gavage with sterile normal saline on an empty stomach in a control group, and immediately observing the activity performance, the weight and the physical condition change after gavage, wherein the observation period is 7 days.

1.2.5 passage stability assay

After culturing the 15 # lactobacillus on an MRS agar plate at 37 ℃ for 48 hours, picking single colonies in an MRS liquid culture medium, culturing the single colonies in a 37 ℃ constant temperature incubator for 16 hours to serve as a 1 st generation bacterial liquid, inoculating the single colonies in the MRS liquid culture medium in a 1% (v/v) proportion, culturing the single colonies in the 37 ℃ constant temperature incubator for 16 hours to serve as a 2 nd generation bacterial liquid, and continuously inoculating the single colonies to a 20 th generation bacterial liquid. Bacterial liquid counts were performed on the bacterial cultures of 5, 10, 15 and 20 generations, and the pH was measured and the results were recorded.

1.2.6 identification of strains

The lactobacillus No. 15 is inoculated in MRS culture medium and cultured for 20h, and the bacterial DNA is extracted by adopting a kit of Tiangen company and subjected to 16S rDNA sequence amplification. The primers used were universal primers: r5'-ggttaccttgttacgactt-3' (SEQ ID NO.2), F5'-agagttgatcctggctcag-3' (SEQ ID NO.3), PCR amplification program of 94 ℃ pre-denaturation for 5min, 94 ℃ denaturation for 1min, 52 ℃ annealing for 1min, 72 ℃ extension for 2min, 30 cycles, 72 ℃ extension for 10 min. The PCR product was sent to Beijing Boshang Biotechnology Co., Ltd for sequence determination.

2 results

2.115 lactic acid bacteria acid resistance detection

As can be seen from Table 9, the survival rates of lactic acid bacterium No. 15 reached 45% and 90% at pH2.5 and pH3.0, indicating that the strain has strong acid resistance.

TABLE 915 lactic acid bacteria acid resistance assay

2.215 detection of bile salt resistance of lactobacillus

As can be seen from table 10, the survival rate of the No. 15 lactic acid bacteria cultured in 0.1% of the poultry bile salt for 4 hours was 92.1%, the survival rate was significantly decreased with the increase of the poultry bile salt concentration, and the survival rate was 4.1% at the 0.3% of the poultry bile salt concentration, from which it can be seen that the No. 15 lactic acid bacteria had a strong poultry bile salt tolerance.

TABLE 1015 lactic acid bacteria bile salt resistance assay

2.3 digestive enzyme tolerance test results

As can be seen from Table 11, the survival rates of the No. 15 lactic acid bacteria after the treatment in the pepsin solution for 2h and 4h were 84% and 46%, respectively. After the treatment in the trypsin solution for 2 hours and 4 hours, the survival rates of the No. 15 lactic acid bacteria are respectively 92% and 76%, so that the No. 15 lactic acid bacteria have stronger tolerance to trypsin and pepsin.

TABLE 1115 lactic acid bacteria digestive enzyme tolerance test results

Safety test of lactic acid bacterium No. 2.415

As shown in Table 12, after the mice were gavaged with the bacterial solution, the weight of the mice increased normally before and after administration, and in addition, the mice had normal appetite, normal drinking water, smooth and non-moist hair color, and no abnormality in spirit, which indicates that the lactic acid bacterium No. 15 was safe for the mice in the range of the concentration tested.

Table 1215 post-gavage lactic acid bacteria mouse weight changes

2.515 lactic acid bacterium passage stability determination

As can be seen from Table 13, the forms of the continuous 5 th, 10 th, 15 th and 20 th generations of culture of the No. 15 lactic acid bacteria are gram-positive bacilli, the difference between the viable bacteria contents of different generations is not large, the pH value of the fermentation broth is about 4.15, and the passage stability of the No. 15 lactic acid bacteria strain is good.

TABLE 1315 results of the subculture stability measurement of lactic acid bacterium

2.6 identification of the Strain

The colony shape of the lactobacillus No. 15 on the solid MRS culture medium is white and circular, the center is convex, the colony surface is fine and smooth, the sequencing result is compared with a database and analyzed, the 16S rDNA of the separated strain has high homology (100%) with the lactobacillus plantarum JY53, so the strain is confirmed to be the lactobacillus plantarum and is named as the lactobacillus plantarum (L actinobacillus plantarum) B L CC2-0125, and the lactobacillus plantarum is stored in China center for type culture Collection (address: Wuhan university of Wuhan, China) at 24 months 4 and 2020, and the preservation number is CCTCC NO: M2020078.

The 16S rDNA sequence (SEQ ID NO.1) is as follows:

CGGCTGGTTCCTAAAAGGTTACCCCACCGACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTA CAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTG CAGCCTACAATCCGAACTGAGAATGGCTTTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTACCATCCA TTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACC GGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCA ACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAATCTCTTA GATTTGCATAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGC GGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCA GCACTGAAGGGCGGAAACCCTCCAACACTTAGCATTCATCGTTTACGGTATGGACTACCAGGGTATCTAATCCTGTT TGCTACCCATACTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATAT CTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCT TCGGTTGAGCCGAAGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAA CGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAAATACCGTCAATACCT GAACAGTTACTCTCAGATATGTTCTTCTTTAACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCG TTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGT CCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCATGGTGAGCCGTTACCCCACCATCTAGCTAA TACGCCGCGGGACCATCCAAAAGTGATAGCCGAAGCCATCTTTCAAACTCGGACCATGCGGTCCAAGTTGTTATGC GGTATTAGCATCTGTTTCCAGGTGTTATCCCCCGCTTCTGGGCAGGTTTCCCACGTGTTACTCACCAGTTCGCCACT CACTCAAATGTAAATCATGATGCAAGCACCAATCAATACCAGAGTTCGTTCGACTTGCATTATA

example 5Preparation of lactobacillus plantarum B L CC2-0125 bacterial powder

1 preparation of bacterial sludge

Inoculating the screened Lactobacillus Plantarum (L actinobacillus Plantarum) B L CC2-0125 strain to an MRS liquid culture medium, standing and culturing at 30 ℃ for 20h, inoculating the strain to the MRS liquid culture medium according to the inoculation amount of 5% after microscopic examination of free bacteria, standing and culturing at 30 ℃ for 20h, performing amplification culture at 220rpm, centrifugally collecting thalli, adding a certain volume of sterile water, and adjusting the bacteria concentration to be not less than 1 × 10⁸CFU/mL。

2 preparation of the protective agent

The formula comprises 3.4g of skim milk, 1.8g of trehalose, 0.25g of manganese sulfate, 0.2g of sucrose, 0.1g of sodium erythorbate and 0.1g of glucose, the reagents in the formula are sealed by a transparent packaging bag in advance, packaged, radiated and sterilized for later use, dissolved in 100m L sterile water and placed in a glass bottle sterilized by dry heat for later use.

3 vacuum freeze drying

Mixing bacterial sludge and protective agent under aseptic condition, wherein the mass parts of freeze-drying protective agent and bacterial sludge are respectively 120 parts and 100 parts, placing a glass bottle containing bacterial liquid into a precooling vacuum freeze-drying machine, pre-freezing at-20 ℃ for 2 hours, starting a vacuum pump, and drying for 24 hours to obtain lactobacillus Plantarum powder, wherein the viable count of lactobacillus Plantarum (L actinobacillus Plantarum) B L CC2-0125 is not less than 1.00 × 10¹⁰CFU/g。

Example 6Lactobacillus plantarum B L CC2-0125 immune effect research

1 materials and methods

1.1 materials

The bacterial powder prepared in the embodiment 5 of the invention (the content of the lactobacillus plantarum B L CC2-0125 is more than or equal to 1.00 × 10)¹⁰CFU/g), H9N2 subtype avian influenza virus, chicken secretory immunoglobulin A (sIgA) E L ISA kit, chicken immunoglobulin G (IgG) E L ISA detection kit, chicken IFN-gamma E L ISA detection kit (purchased from Beijing equation biotechnology, Inc.), and sprayer (purchased from Shandong Polycheng Wulifog environmental science, Inc.).

150 AA broilers (purchased from Ding Gushan agriculture) are randomly and averagely divided into 5 groups which are respectively (1) blank groups; (2) control group for counteracting toxic substance; (3) a set of protective agents; (4) a fungus powder group; (5) and (4) a vaccine group.

1.2 methods

1.2.1 immunization protocol

Blank group, challenge control group: no treatment is carried out;

bacterial powder group for two consecutive days of 15 and 16 days old products (oral microecologics prepared in the invention example 5) are drunk, and the oral microecologics are diluted to 1.00 × 10 by using sterile water⁸CFU/m L, according to the amount of 5m L/unit, the water is drunk up in 2 hours;

protective agent group: 15. the protective agent is orally taken for two consecutive days at the age of 16 days; the difference between the components of the powder and the powder prepared in example 5 of the present invention is that no bacteria are added; when the protective agent is used, the drinking water amount and drinking water mode are the same as those of the fungus powder group.

The vaccine group comprises 120 eyedrops (1.5 feathers) of new branches at 8 days of age, 0.3m L per new branch by triple subcutaneous injection, 2 feathers of bursa of Fabricius drinking water (2 feathers) of 15 days of age, 2 feathers of ND four-line drinking water (22 days of age) and 2 feathers of 29 days of age of bursa of Fabricius drinking water (29 days of age).

1.2.2 immunization methods

Each group was performed according to immunization protocol; the fungus powder group and the protective agent group are directly added into drinking water for use.

1.2.3 attack of H9N2 subtype avian influenza virus

After virus propagation E L D50 ═ 10^-8.150.2m L, except for blank group, the broiler chickens of 23 days old are detoxified, and the virus stock solution is injected into the wings at 0.8m L.

1.2.4 sample Collection

Randomly selecting 5 wing veins for blood collection in 7-day-old, 19-day-old, 30-day-old and 42-day-old chicken groups respectively, randomly selecting 3 wings to be euthanized, and taking the trachea and the intestinal tracts of the chickens for storage and detection.

After 7 days of toxin attack, 10 cloaca cotton swabs are randomly selected from each group.

1.2.5 sample testing

Detecting IgG antibody and IFN-gamma content of collected serum by adopting an E L ISA kit, detecting H9 antibody content of serum by adopting a hemagglutination inhibition test, and detecting sIgA antibody content of trachea and intestinal mucus which are obtained by grouping different ages in days by adopting an E L ISA kit.

The collected cloaca cotton swab is used for detecting the titer of the excreted virus by adopting a hemagglutination test.

2 results

2.1 growth and elimination rule of sIgA in caecum mucous membrane

See table 14, sIgA antibodies consistently maintained the highest levels in cecal mucosa of the bacterial powder group. The 19-day-old and 30-day-old bacterial powder groups are significantly higher than the control group, and have no significant difference from the vaccine group, while the sample results at 42-day-old show that the bacterial powder groups are significantly higher than the rest groups (p < 0.05).

TABLE 14 measurement of sIgA antibody levels in cecal mucosa of broilers of different ages of day (ng/m L)

2.2 SIgA growth and elimination rules in tracheal mucosa

See table 15, the highest level of sIgA antibodies was consistently maintained in the tracheal mucosa of the bacterial powder group. The 19-day-old powder group was significantly higher than the rest (p < 0.05).

TABLE 15 measurement results of sIgA antibody levels in tracheas of broilers of different ages of days (ng/m L)

2.3 the growth-diminishing rule of IgG antibodies in serum

As shown in Table 16, IFN-. gamma.was always maintained at the highest level in the serum of the bacterial powder group. The bacterial powder groups are obviously higher than the rest groups in 19 days, 30 days and 42 days (p is less than 0.05).

TABLE 16 measurement of IgG antibody level in serum of broiler chickens at different days (μ g/m L)

2.4 IFN-gamma growth rule in serum

As shown in Table 17, IFN-. gamma.was always at the highest level in the serum of the fungal powder group. The bacterial powder group is significantly higher than the blank group and the protective agent group (p <0.05) at the age of 42 days, and has no significant difference with the vaccine group.

TABLE 17 determination of IFN-. gamma.levels in sera of broiler chickens at different ages of day (pg/m L)

2.5 in serum H9 antibody

See table 18, H9 antibody remained consistently high in the mycoplasma group sera. Although the H9 antibody levels gradually decreased as the maternal antibody disappeared, the powder group delayed the tendency for antibody decline.

TABLE 18 measurement results of H9 antibody levels in serum of broiler chickens of different ages in days

2.6 hemagglutination assay

In Table 19, the antigen titer of the cloacal secretion of the bacterial powder group is significantly lower than that of the challenge control group, which shows that the product can effectively reduce the virus content of the organism by oral administration.

TABLE 19 cloaca secretion H9N2 antigen titers from various groups after challenge

Example 7Determination of toxicity attack protection rate

1 method of experiment

In the 23-day-old broiler chickens subjected to virus attack in example 6, 8 broiler chickens are randomly selected from each group after 7 days of virus attack and are euthanized, the trachea is dissected and taken, the tracheal mucosa is scraped for virus RNA extraction, cDNA is used as a template, avian influenza universal primers are used for PCR, and after agarose gel electrophoresis, the infection rate and the protection rate of each group are calculated respectively.

2 results

As shown in Table 20, the infection rate was reduced to various degrees in each group after the product was used, and the bacterial powder group was the most effective.

TABLE 20 infection and protection rates for each group after challenge

Example 8Separation culture, identification, separation and purification of mycoplasma gallisepticum

1, isolation and culture of mycoplasma gallisepticum:

the mycoplasma gallisepticum strain for test is obtained by separating from the air sac of sick chicken of a certain chicken factory in Ningyang county of Taian, cutting the air sac into pieces under aseptic condition, placing into mycoplasma gallisepticum liquid culture medium for overnight at 4 deg.C, filtering with 0.22 μm filter, inoculating into liquid culture medium at 37 deg.C and 5% CO₂The culture was carried out and the medium was immediately passaged when the color changed from rose-red to yellow. The medium profile is shown in FIG. 1.

The culture medium is mycoplasma gallisepticum basic culture medium (purchased from Haibo biology), and 100m L sterile horse serum, 10.1 g coenzyme, 0.1g L-cysteine, 0.4g arginine and 80 ten thousand units penicillin are added into each 1L liquid culture medium.

2, identification of mycoplasma gallisepticum:

2.1, method: the PCR identification method is mainly characterized by Swiss dyeing, Jiemsa dyeing and a PCR method, and the specific PCR identification method comprises the following steps:

(1) preparation of DNA template:

the extraction of DNA was carried out according to the procedure of Mycoplasma paramagnetic Almond genomic DNA extraction kit (ex Ranunculus Scutescens Biotech Co., Ltd.).

(2) Designing a primer:

according to AB680686.1 in GenBank, 1 pair of specific primers is designed aiming at the conserved gene 16SrRNA by using Primer3Plus Primer design software, and the sequences of the primers are as follows:

F:5’-cggatttattgggcgtaaaa-3’(SEQ ID NO.4)

R:5’-tgttaactgcagcaccgaag-3’(SEQ ID NO.5)

the product size was 308bp by conventional PCR.

(3) The PCR reaction system is as follows:

(4) reaction conditions are as follows:

pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 45s, extension at 72 ℃ for 1min for 30 cycles in total, final extension at 72 ℃ for 10min, and termination at 4 ℃.

(5) Sequencing is carried out

(6) Sequencing results nucleic acid alignment in ncbi

2.2 results: the results of PCR identification in combination with Swiss staining and Giemsa staining (FIGS. 2 and 3a) showed that the base sequence of the PCR-amplified gene fragment matched up to 99% of the base sequence of Mycoplasma gallisepticum 16S RNA (FIG. 3 b).

3, separation and purification of mycoplasma gallisepticum:

3.1 method: the purification of mycoplasma gallisepticum is mainly achieved by culturing on a solid culture medium, then selecting a single colony for liquid culture, and repeating the culture for 3 times.

3.2 results: after repeating the purification 3 times, the purified mycoplasma gallisepticum liquid culture was obtained, and the target band was still obtained by PCR (FIG. 4).

4 determination of mycoplasma gallisepticum CCU:

4.1 assay method: adding culture medium 180. mu. L to each well of 96-well cell culture plate, adding sample 20. mu. L to the first well of each row, diluting 10 times to 12 th well, discarding excess 20. mu. L, wherein the two rows are not added with test substance as blank, sealing the periphery with sealing film after capping to prevent air leakage, culturing at 37 ℃, observing and recording pH change every day, determining the end point when color change wells are not increased, and considering the highest dilution of test substance causing color change as 1 CCU/m L.

4.2 measurement results: after 7 days of continuous observation, as shown in FIG. 5, the color of the first 9 rows of culture medium was changed, that is, the culture CCU of Mycoplasma gallisepticum was 10⁹/mL。

Example 9Traditional Chinese medicine for inhibiting proliferation of mycoplasma gallisepticum by in vitro screening

The traditional Chinese medicines to be screened: wild buckwheat rhizome, heartleaf houttuynia herb, indigowoad root, densefruit pittany root-bark, amur corktree bark, goldthread root, Chinese angelica and Chinese mugwort leaf; each traditional Chinese medicine is extracted by the following method respectively:

weighing 50g of traditional Chinese medicines, respectively adding 10 weight times of distilled water, soaking for 2h, heating to boil, decocting for 60min for the first time, filtering, adding 10 weight times of water into filter residue, decocting for 50min for the second time, filtering, adding 8 weight times of water into filter residue, decocting for 30min for the third time, mixing filtrates, and concentrating to obtain filtrate with concentration of 1g/m L.

(1) The minimum effective Mycoplasma Inhibition (MIC) concentration of 8 traditional Chinese medicines:

taking a sterile 96-well cell culture plate, adding 150 mu L of culture medium into the first well of each row, adding 100 mu L of culture medium into the second-11 well of each row, adding 200 mu L of culture medium into the 12 th well of each row, adding 50 mu L of liquid medicine into the 1 st well, diluting the liquid medicine to the 10 th well by using a microsyringe from the 1 st well in a multiple ratio, uniformly mixing, sucking 10 from the 10 th well0 μ L was discarded Mycoplasma culture was taken and diluted to 1X10 with the corresponding liquid medium⁴CCU/m L as working medium liquid, then 100 mu L of working medium liquid is added into the 1 st to 11 th wells, so that the total volume of the liquid in all the wells is 200 mu L, the 11 th well is a positive control well (namely a growth control well), the 12 th well is a negative control well, the final concentration of the medicines is (unit: mg crude drug/m L) 250.00, 125.00, 62.50, 31.25, 15.625, 7.81, 3.91, 1.95, 0.98 and 9 dilution gradients in sequence from the 1 st to 10 th wells, the final concentration of the tylosin tartrate liquid is 10mg/m L, the plate is covered, the plate is placed in a constant temperature incubator for incubation at 37C, when the positive control wells have color change (namely, the phenol red indicator in the culture liquid changes from red to yellow or orange), and when no turbidity exists, the first result is judged (when the minimum medicine concentration of the first tube before the positive control wells have color change is the minimum), the second result is judged after the bacteria change, the second result is the minimum.

(2) Test results

Through a minimum inhibitory concentration determination test, the judgment is carried out according to the judgment standard reported by similar experimental documents, namely that the sensitivity is higher when the MIC is less than or equal to 7.81mg crude drug/m L, the moderate sensitivity is realized when the MIC is 7.81-250mg crude drug/m L, and the insensitivity is realized when the MIC is more than or equal to 250mg crude drug/m L, so that the golden cypress and the goldthread root belong to the sensitive range, the wild buckwheat rhizome and the houttuynia cordata belong to the moderate sensitivity, and the insensitivity is realized in the angelica, the argy wormwood leaf, the isatis root and the.

TABLE 218 MIC values of crude extracts of Chinese herbs for inhibiting Mycoplasma gallisepticum

Example 10Establishment of mycoplasma gallisepticum animal model

1 establishing method

30 SPF chickens 12 days oldDividing the test sample into a normal control group (15) and a model group (15), and adopting a trachea toxicity counteracting method to counteract the toxicity except the normal control group, wherein the turbidity of the bacterial liquid is 1 × 10⁹And ccu/m L and 0.5m L per chicken, and simultaneously creating stress conditions for inducing morbidity, such as temperature reduction, humidity increase, material breakage for 2 days, poor sanitary conditions and poor ventilation conditions.

2 result of molding

After the detoxification, the chicken flock has typical clinical symptoms and pathological changes of the mycoplasma gallisepticum, mainly including head swing, cough, neck stretching respiration, lassitude, anorexia, slight respiratory tract rale and other clinical symptoms, the trachea has faint yellow secretion, the trachea mucosa is thickened, the trachea wall has congestion and bleeding in different degrees, the air sac is turbid and thickened, a large amount of turbid viscous liquid exudates or faint yellow cheese exudates exist, therefore, when the MG animal model is prepared, the detoxification mode adopts the intratracheal detoxification, and the turbidity of the bacterial liquid is 1 × 10⁹The drug effect test method has the advantages that the drug effect test method is simple, the drug effect test speed is high, the drug effect test speed is.

Example 11Preparation of composite traditional Chinese medicine microecological preparation

The formula comprises 6 parts of goldthread root, 15 parts of phellodendron, 12 parts of wild buckwheat rhizome, 12 parts of cordate houttuynia and probiotic powder, wherein the probiotic is lactobacillus plantarum B L CC2-0125, and the preparation method of the probiotic powder is the same as that in example 5, wherein the number of live bacteria in the probiotic powder B L CC2-0125 is not less than 1.00 × 10¹⁰CFU/g。

The preparation method of the composite traditional Chinese medicine microecological preparation comprises weighing rhizoma Coptidis, cortex Phellodendri, rhizoma Fagopyri Dibotryis and herba Houttuyniae according to weight parts, mixing, adding 10 weight times of water (the amount of water is 10 times of the weight of the mixed medicinal materials), soaking for 2h, heating to boil, decocting for 60min for the first time, filtering, adding 10 weight times of water into the residue, decocting for 50min for the second time, filtering, adding 8 weight times of water into the residue, decocting for 30min for the third time, mixing filtrates, and concentrating until the concentration of the filtrate is 1g/m L;adding probiotic bacteria powder into the concentrated filtrate, and mixing to obtain composite Chinese medicinal microecological preparation with viable count of Lactobacillus plantarum B L CC2-0125 no less than 1.0 × 10⁹cfu/mL。

Example 12Preparation of composite traditional Chinese medicine microecological preparation

The formula comprises 5 parts of coptis chinensis, 10 parts of golden cypress, 15 parts of wild buckwheat rhizome, 15 parts of houttuynia cordata and probiotic powder, wherein the probiotic is lactobacillus plantarum B L CC2-0125, and the preparation method of the probiotic powder is the same as that in example 5, wherein the viable count of B L CC2-0125 in the probiotic powder is not less than 1.00 × 10¹⁰CFU/g。

The preparation method is the same as that of example 11, and the viable count of the lactobacillus plantarum B L CC2-0125 in the prepared composite traditional Chinese medicine microecological preparation is not less than 1.0 × 10⁹cfu/mL。

Example 13Preparation of composite traditional Chinese medicine microecological preparation

The formula comprises 8 parts of coptis chinensis, 20 parts of golden cypress, 10 parts of wild buckwheat rhizome, 10 parts of houttuynia cordata and probiotic powder, wherein the probiotic is lactobacillus plantarum B L CC2-0125, and the preparation method of the probiotic powder is the same as that in example 5, wherein the viable count of B L CC2-0125 in the probiotic powder is not less than 1.00 × 10¹⁰CFU/g。

Example 14Effect test of composite traditional Chinese medicine microecological preparation for preventing mycoplasma gallisepticum

1 materials and methods

1.1 Experimental materials: healthy SPF chickens at 1 day of age were purchased from knanseisi poultry science and technology limited.

1.2 experimental design:

150 SPF chickens were randomly and evenly divided into 5 groups, each group containing 30 SPF chickens, a normal control group, an offensive control group, a high-dose group (the compound Chinese medicinal microecological preparation prepared in the invention example 11 was added with water at a ratio of 30% (V: V)), a medium-dose group (the compound Chinese medicinal microecological preparation prepared in the invention example 11 was added with water at a ratio of 20% (V: V)), and a low-dose groupGroup III (using the composite traditional Chinese medicine microecological preparation prepared in the invention example 11, drinking water is added according to the adding amount of 10% (V: V)), drinking water is concentrated for use at the age of 6 days, and the trachea toxicity counteracting method is adopted to counteract toxicity when the patient is continuously used for 5 days and 11 days, wherein the turbidity of the bacteria liquid is 1 × 10⁹ccu/m L, 0.5m L/mouse, 2 weeks after challenge.

1.3 Breeding management

Adopts free food intake and drinking water. The illumination, temperature and humidity in the house are strictly controlled according to the conventional feeding management requirements. Sterilizing the trough and the drinking bowl before changing food and water every day. The health status of each group of test chickens was recorded in detail during the test.

1.4 sample Collection and detection indexes

At the end of the test, namely when the SPF chickens are 25 days old, 10 SPF chickens are randomly subjected to autopsy in each group, pathological changes of air sacs, air ducts and lungs are observed, air sac damage degrees are graded and scored, and air sac damage rate and death rate are evaluated.

1.5 test methods

1.5.1 mortality

And observing and recording the survival state of the SPF chickens every day after infection, judging that the SPF chickens are infected and dead after the SPF chickens have the typical clinical symptoms and pathological changes of the mycoplasma gallisepticum and die, and calculating the death rate.

Mortality was × 100% of the number of birds in the test group/total number of birds in the test group.

1.5.2 balloon injury Rate

Grading and scoring the damage degree of the air sac, evaluating the damage rate of the air sac, and dividing pathological damage of the air sac into 5 grades according to the H.WYoder scoring standard:

0 grade, normal, thin and transparent air bag, 0 point is recorded;

grade 1, the air bag has only slightly thickened gray areas or yellow exudation spots, and the mark is 2;

2, part of the air bags are easy to see grey to yellow exudation, sometimes foams appear, the air bags are thickened, and 4 points are marked;

3, thickening most of the air bags, and filling a large amount of yellow and white cheese substances, and recording for 6 minutes;

4, almost the whole air bag is covered with yellow and white cheese-like exudates, the air bag is seriously thickened, and the score is 8;

average pathological damage degree of air sac (score) is the sum of damage degree of air sac of all chickens/number of chickens to be detected

Calculating the percentage reduction of balloon injury according to the balloon injury score:

air sac injury reduction rate (average damage of air sac of each chicken in challenge control group-average damage of air sac of each chicken in test group)/average damage of air sac of each chicken in challenge control group

2 data statistics and processing

Data were analyzed using GraphPad prism (GraphPad Software, inc. california, USA) Software, all data are expressed as Mean ± SD, and p <0.05 indicates that the data differed significantly.

3 results of the test

3.1 Effect on mortality of Chicken results

As can be seen from table 22, the use of different doses of the complex herbal microecologics reduced the mortality of SPF chickens compared to the challenge control group, wherein the mortality of the chickens after the use of the high dose group was at least 3.3%.

TABLE 22 Effect on mortality in model chickens

3.2 test results on the rate of reduction of balloon Damage

As can be seen from Table 23, the pathological damage degree of the air sac is reduced after the compound Chinese medicinal microecological preparation with different dosages is used. Wherein, the use effect of the high-dosage group is best, the reduction rate of the air sac injury is up to 70 percent, and the reduction rate of the air sac injury is 50 percent in the medium-dosage group.

TABLE 23 results on the rate of reduction of balloon damage

	Normal control	Control of offensive toxin	High dose group	Middle dose group	Low dose group
						Average pathological damage degree of air bag (score)	0	4.0	1.2	2.0	2.8
Air bag Damage reduction Rate (%)	--	--	70	50	30

4. And (4) test conclusion: the effect test of preventing the mycoplasma gallisepticum by using the composite traditional Chinese medicine microecologics with different doses is evaluated to obtain the optimal effect of preventing the mycoplasma gallisepticum by using the high-dose group of the composite traditional Chinese medicine microecologics.

Example 15The prevention effect of the composite traditional Chinese medicine microecological preparation

1 materials and methods

1.2 experimental design:

300 SPF chickens were randomly and equally divided into 10 groups, each group containing 30 SPF chickens, a normal control group (group I), a toxicity counteracting control group (group II), a composite traditional Chinese medicine microecological preparation (group III, the composite traditional Chinese medicine microecological preparation prepared in the present invention example 11), probiotics (group IV, the probiotic bacteria powder prepared in the present invention example 5), group V (the composite traditional Chinese medicine concentrated filtrate in the present invention example 11), group VI (the composite traditional Chinese medicine concentrated filtrate of Coptidis rhizoma and cortex Phellodendri prepared by the method of the present invention example 11), group VII (the composite traditional Chinese medicine concentrated filtrate of rhizoma Fagopyri Dibotryis and herba Houttuyniae prepared by the method of the present invention example 11), group VIII (the composite traditional Chinese medicine concentrated filtrate of Coptidis rhizoma, cortex Phellodendri and herba Fagopyri Dibotryis prepared by the method of the present invention example 11), group IX (the composite traditional Chinese medicine concentrated filtrate of rhizoma Coptidis, cortex Phellodendri and rhizoma Fagopyri Dibotryis prepared by the method of the present invention example 11), group X (tylosin) with 6 days of age of drinking water, group III-IX groups are all used in a 30% (V: V) adding amount, group X is used in 500 mg/855 mg of drinking water, and 355 d drinking water is used as a continuous drinking water counteracting bacterium liquid for 3510 days of 1 d⁹ccu/m L, 0.5m L/mouse, 2 weeks after challenge.

1.3 Breeding management

The same as in example 14.

1.4. Sample collection and detection index

And (3) after the test is finished, namely when the SPF chickens are 25 days old, randomly performing dissection on 10 SPF chickens in each group, observing pathological changes of air sacs, air ducts and lungs, grading and scoring the damage degree of the air sacs, and evaluating the damage rate of the air sacs, collecting blood separation serum when the SPF chickens are 6 days old, 11 days old, 18 days old and 25 days old respectively in the test process, and detecting the contents of mycoplasma gallisepticum antibodies, serum immunoglobulin IgG and cell factors IFN-gamma by using an E L ISA kit.

1.5 test methods

1.5.1 mortality: the same as in example 14.

1.5.2 balloon injury rate: the same as in example 14.

1.5.3 immunological index

The content of mycoplasma gallisepticum antibody, IgG and IFN-gamma is respectively detected by using 4 times of serum collected in the test process by using an E L ISA kit, the operation is carried out strictly according to the instruction of the kit, and the specific operation steps are as follows:

(1) and (3) balancing the detection kit at room temperature for 20min, and then detecting.

(2) Setting a negative and positive control hole and a sample hole, wherein the negative control hole and the positive control hole are respectively added with 50 mu L of negative control and positive control;

(3) firstly adding a sample to be detected 10 mu L into a sample hole to be detected, and then adding a sample diluent 40 mu L into the sample hole to be detected;

(4) subsequently, 100. mu. L of detection antibody labeled with horseradish peroxidase (HRP) was added to each of the negative and positive control wells and the sample wells, the reaction space was sealed with a sealing plate film, and the reaction space was incubated at 37 ℃ for 60 min.

(5) Discarding liquid, drying on absorbent paper, filling each hole with cleaning solution, standing for 1min, throwing off cleaning solution, drying on absorbent paper, and washing the plate for 5 times.

(6) Substrate A, B was added to each well at 50. mu. L, and incubated at 37 ℃ for 15min in the absence of light.

(7) The OD of each well was measured at a wavelength of 450nm within 15min after adding stop solution to each well at 50. mu. L.

2. Data statistics and processing: the same as in example 14.

3. Test results

3.1. Effect on mortality of chickens results

As can be seen from table 24, compared with group II (challenge control group), each of the herbal compositions can reduce the mortality of SPF chicken caused by mycoplasma gallisepticum, wherein the mortality of the composite herbal microecological formulation group (group III) is 6.7% at the lowest, and the mortality of the group IV (probiotic group) and the group V (4 herbal medicines) is also significantly reduced.

TABLE 24 preventive test-Effect on mortality in model chickens results

	Group I	Group II	Group III	Group IV	Group V	Group VI	Group VII	Group VIII	Group IX	Group X
											Number of test chickens	30	30	30	30	30	30	30	30	30	30
Number of deaths	0	14	2	6	8	10	12	11	9	15
											Mortality (%)	0	46.7	6.7	20	26.7	33.3	40	36.7	30	50

3.2 test results on the rate of reduction of balloon Damage

It can be seen from table 25 that, except for group X (tylosin), pathological damage of air sacs of other groups is reduced to different degrees, wherein group III (composite traditional Chinese medicine microecological preparation) has the best effect, and the reduction rate of air sac damage is as high as 65.2%.

TABLE 25 preventive test-results on the rate of reduction of balloon damage

3.3MG antibody detection results

The detection of mycoplasma gallisepticum antibodies (MG antibodies) (table 26) shows that only mycoplasma gallisepticum antibodies in sera of group I (normal control) and group III (complex traditional Chinese medicine microecological preparation) are negative after 7d (18 days old) and 14d (25 days old) of SPF chickens have been challenged, which indicates that the complex traditional Chinese medicine microecological preparation of the present invention starts to act at the initial stage of mycoplasma gallisepticum infection to resist the mycoplasma gallisepticum infection.

TABLE 26 prophylaxis test-detection of Mycoplasma gallisepticum antibodies in serum (OD value)

3.4 detection results of IgG and IFN-. gamma.

By detecting the content of IgG and IFN-gamma in serum, the results are shown in tables 27 and 28, and the results show that 14d (25 days old) after challenge, compared with a challenge control group, the content of IgG in the groups III, IV, V and IX of SPF chickens is obviously increased, the content of IFN-gamma in the groups III and IV is obviously increased, and the content of IFN-gamma in the groups III and IV is the highest, namely the composite traditional Chinese medicine microecological preparation can obviously improve the immunity index of the SPF chickens.

TABLE 27 prophylaxis test-results of IgG content in serum (pg/m L)

Note: p is less than 0.05 compared with control group

TABLE 28 prophylaxis test-measurement of IFN-. gamma.content in serum (pg/m L)

Note: p is less than 0.05 compared with control group

Example 16The treatment effect of the composite traditional Chinese medicine microecological preparation

1 materials and methods

1.1 Experimental materials: 1-day-old healthy SPF (specific pathogen free) chicken

1.2 experimental design:

300 SPF chickens followedThe machine is divided into 10 groups on average, each group comprises 30 normal control groups (group I), a toxicity counteracting control group (group II), a compound traditional Chinese medicine microecological preparation (group III, the compound traditional Chinese medicine microecological preparation prepared in the invention example 11), probiotics (group IV, probiotic bacteria powder prepared in the invention example 5), group V (compound traditional Chinese medicine concentrated filtrate in the invention example 11), group VI (compound traditional Chinese medicine concentrated filtrate of coptis chinensis and golden cypress prepared in the invention example 11), group VII (compound traditional Chinese medicine concentrated filtrate of wild buckwheat rhizome and heartleaf houttuynia herb prepared in the invention example 11), group VIII (compound traditional Chinese medicine concentrated filtrate of coptis chinensis, golden cypress and heartleaf houttuynia herb prepared in the invention example 11), group IX (compound traditional Chinese medicine concentrated filtrate of coptis chinensis, golden cypress and golden buckwheat rhizome prepared in the invention example 11), group X (tylosin), and when the day is 11, a trachea toxicity counteracting method is adopted, and the turbidity of the bacteria solution is 1 × 10⁹ccu/m L, 0.5m L/mouse, after 7 days of toxin counteracting, water is concentrated for administration, group III-group IX are used with 30% (V: V) of water, group X is used with 500 mg/L of water, and the administration is continued for 5 days, and the observation is continued for 2 weeks.

1.3, feeding management: same as example 14

1.4. Sample collection and detection index

And (3) after the test is finished, namely when the SPF chickens are 36 days old, randomly performing dissection on 10 SPF chickens in each group, observing pathological changes of air sacs, air ducts and lungs, grading and scoring the damage degree of the air sacs, and evaluating the damage rate of the air sacs, collecting blood separation serum when the SPF chickens are 11 days old, 18 days old, 29 days old and 36 days old respectively in the test process, and detecting the contents of mycoplasma gallisepticum antibodies, serum immunoglobulin IgG and cell factors IFN-gamma by using an E L ISA kit.

1.5 test methods

1.5.1 mortality: same as example 14

1.5.2 balloon injury rate: same as example 14

1.5.3 immune index: same as example 14

2. Data statistics and processing: same as example 14

3. Test results

3.1. Effect on mortality of chickens results

Through the experiments of the drug groups on the treatment of mycoplasma gallisepticum infection, it is shown in table 29 that the mortality rate of group III (the complex traditional Chinese medicine microecological preparation group) is 13.3% at the lowest, and the mortality rate of group IV (the probiotic group) and group X (the tylosin group) is 16.7% at the lowest compared with the challenge control group.

TABLE 29 therapeutic test-Effect on mortality of model chickens results

Group of	Group I	Group II	Group III	Group IV	Group V	Group VI	Group VII	Group VIII	Group IX	Group X
											Number of test chickens	30	30	30	30	30	30	30	30	30	30
Number of deaths	0	13	4	5	7	11	12	11	9	5
											Mortality (%)	0	43.3	13.3	16.7	23.3	36.7	40	36.7	30	16.7

3.2 test results on the rate of reduction of balloon Damage

As can be seen from table 30, the highest reduction rate of air sac injury in group III (compound herbal microecological formulation group) was 42.9%, and the reduction rates of air sac injury in group X (tylosin) and group IV (probiotic group) were 23.8%.

TABLE 30 therapeutic test-results on the rate of reduction of balloon damage

3.3MG antibody detection results

The results of detection by an E L ISA kit show that (Table 31), except that 7d and 14d of mycoplasma gallisepticum antibodies of a group III (compound traditional Chinese medicine microecological preparation group) and a group X (tylosin) are negative after the medicine is taken, and the antibodies of the rest test groups are positive, which indicates that the compound traditional Chinese medicine microecological preparation disclosed by the invention has the effect of treating mycoplasma gallisepticum infection.

TABLE 31 therapeutic test-detection of Mycoplasma antibodies in serum (OD value)

3.4 detection results of IgG and IFN-. gamma.

As can be seen from Table 32, after SPF chickens were administered 7 days (29 days old), the IgG content in blood of groups III, IV and V was significantly increased compared with the challenge control group; after 14d (36 days old), only group III had significantly higher IgG content than the challenge control.

After the SPF chickens are taken for 7 days (29 days old), compared with an offensive control group, the IFN-gamma content in blood of the group III, the group IV and the group V is obviously increased, and after the SPF chickens are taken for 14 days (36 days old), the IFN-gamma content of the group III is obviously higher than that of the offensive control group.

TABLE 32 therapeutic test-results of IgG content in serum (pg/m L)

Note: p is less than 0.05 compared with control group

TABLE 33 treatment trials-determination of IFN-. gamma.content in serum (pg/m L)

Note: p is less than 0.05 compared with control group

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Shandong Baolaili Biotechnology Ltd

<120> composite traditional Chinese medicine micro-ecological composition and preparation method and application thereof

<130>202021307

<160>5

<170>PatentIn version 3.5

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Claims

1. A compound traditional Chinese medicine microecological composition takes a traditional Chinese medicine compound and probiotics as raw materials, wherein the probiotics are lactobacillus plantarum and/or fermentation products thereof; the Chinese herbal compound comprises goldthread root, phellodendron, wild buckwheat rhizome and houttuynia cordata;

the Lactobacillus Plantarum is Lactobacillus Plantarum (L actinobacillus Plantarum) B L CC2-0125, which is preserved in China center for type culture Collection in Wuhan City at 24/4/2020 with the preservation number of CCTCC NO: M2020078.

2. The complex Chinese herbal microecological composition according to claim 1, wherein the Lactobacillus plantarum B L CC2-0125 and its fermented product is obtainable by fermentative culture of Lactobacillus plantarum B L CC 2-0125.

3. The composite traditional Chinese medicine microecological composition according to claim 1 or 2, wherein the viable count of the probiotics in the composition is not less than 1 × 10⁹cfu/mL。

4. The composite traditional Chinese medicine microecological composition according to claim 1, wherein the traditional Chinese medicine compound is prepared from the following raw materials in parts by weight: 5-8 parts of goldthread root, 10-20 parts of phellodendron, 10-15 parts of wild buckwheat rhizome and 10-15 parts of houttuynia cordata.

5. A method of preparing the complex herbal micro-ecological composition of any one of claims 1 to 4, comprising mixing herbal compound and probiotic.

6. The method of claim 5, wherein the herbal compound is herbal extract, and the probiotic is Lactobacillus plantarum B L CC2-0125 and/or fermented product thereof;

preferably, the method comprises: weighing raw materials of a compound traditional Chinese medicine according to parts by weight, mixing, adding 8-10 times of water by weight, soaking, heating to boil, decocting with water for at least one time, and concentrating the filtrate to obtain a compound traditional Chinese medicine concentrated filtrate, namely a traditional Chinese medicine extracting solution; adding probiotics into the traditional Chinese medicine extracting solution, and uniformly mixing to obtain a composite traditional Chinese medicine micro-ecological composition;

preferably, the probiotics is probiotic powder, and the preparation method of the probiotic powder comprises the steps of mixing lactobacillus plantarum B L CC2-0125 and/or a fermentation product thereof with a protective agent under an aseptic condition, and then carrying out vacuum freeze drying to obtain the probiotic powder.

7. The method of claim 5, wherein the water decoction is performed 3 times, comprising: decocting for 30-60min, filtering, retaining filtrate, adding 8-10 weight times of water into the residue, decocting for 30-60min for the second time, filtering, retaining filtrate, adding 6-8 weight times of water into the residue, decocting for 30-50min for the third time, and retaining filtrate; after the water decoction is finished, the three filtrates are combined.

8. A composite traditional Chinese medicine microecological preparation comprising the composite traditional Chinese medicine microecological composition of any one of claims 1 to 4.

9. The composite traditional Chinese medicine microecological preparation according to claim 8, wherein the preparation is an oral liquid preparation or an oral solid preparation.

10. Use of the complex traditional Chinese medicine micro-ecological composition of any one of claims 1 to 4 or the complex traditional Chinese medicine micro-ecological preparation of any one of claims 8 or 9 for preparing a product for preventing and treating mycoplasma gallisepticum or related diseases caused by mycoplasma gallisepticum;

preferably, the product is a pharmaceutical, nutraceutical, feed or feed additive.