CN111357871A

CN111357871A - Preparation method of Chinese juniper fermentation enzymatic hydrolysis complex agent, preparation and application thereof

Info

Publication number: CN111357871A
Application number: CN202010178523.0A
Authority: CN
Inventors: 张世新; 李学刚; 陈波; 万宗敏; 贾付从
Original assignee: Ginten Biotechnology Beijing Co ltd
Current assignee: Ginten Biotechnology Beijing Co ltd
Priority date: 2020-03-14
Filing date: 2020-03-14
Publication date: 2020-07-03

Abstract

The invention discloses a preparation method of a Chinese medicinal composition, a preparation and an application thereof, wherein the preparation method comprises the following steps: preparing a composite microbial inoculum consisting of lactic acid bacteria, bacillus and aspergillus niger, fermenting the Chinese herbal medicines, adding a proper enzyme preparation after fermentation for fermentation and enzymolysis, and performing supercritical extraction; the invention also provides powder prepared from the compound agent prepared by the preparation method and application of the powder in preparing medicaments for improving anti-inflammatory reaction and improving animal immunity. The powder prepared by the invention has the advantages of being green, safe and efficient, and can replace antibiotics to be used for promoting the growth of animals, preventing and treating intestinal diseases, eliminating antibiotic residues, ensuring the safety and human health of animal products, promoting the upgrading of the industries of the feed industry and the breeding industry, and having important significance for improving the international competitiveness of domestic animal products.

Description

Preparation method of Chinese juniper fermentation enzymatic hydrolysis complex agent, preparation and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a preparation method of a Chinese juniper fermentation enzymatic hydrolysis complex agent, a preparation and application thereof.

Background

The Chinese herbal medicine rhizoma coptidis is one of the varieties of the traditional Chinese herbal medicine rhizoma coptidis in China, contains various bioactive substances, can resist bacteria, diminish inflammation, enhance the immunity of organisms, improve the anti-stress and anti-disease capabilities of animals, and improve the production performance of the animals; it also has the characteristics of difficult generation of bacterial drug resistance, low toxicity, no residue or low residue and the like.

At present, the medicinal materials of the Chinese medicinal material rhizoma coptidis are increasingly tense, more and more attention is paid to finding the substitute of the Chinese medicinal material, and if the fibrous root of the Chinese medicinal material rhizoma coptidis can be fully utilized, the existing resources can be effectively utilized, the production cost can be reduced, and economic benefit can be generated. The existing experiment proves that the residual fibrous root after the harvesting of the miscanthus edulis medicinal material contains berberine hydrochloride with higher effective component, and the medicinal material is used as veterinary drug at present, such as miscanthus edulis powder, which is used for treating white dysentery caused by escherichia coli infection.

The use of the common stamen of the odor is only limited to the compatibility with other medicinal materials or the direct use as veterinary drugs or feed additives, the preparation method is simple, the drug effect cannot be utilized to the maximum extent, and the further development of the common stamen of the odor has obvious social and economic benefits. Therefore, the development of a feed additive or veterinary drug with stable performance by using the flavor and the requirement of the continuous production of the flavor is a promising research direction by using the modern biotechnology.

Disclosure of Invention

Aiming at the defects in the prior art, the first purpose of the invention is to provide a preparation method of a flavor ligamentary fermentation enzymatic hydrolysis complex agent. The invention adopts modern biotechnology, and the prepared compound preparation not only has the function of preventing escherichia coli infection, but also has the effects of improving anti-inflammatory reaction and enhancing animal immunity through fermentation and enzymolysis of the traditional Chinese medicines.

The second purpose of the invention is to provide a taste desmodium fermentation enzyme hydrolysis composite powder. The powder prepared by the invention is convenient to use, can be used independently, and can also be mixed with other various feeds for use.

The third purpose of the invention is to provide the application of the taste-and-beard-fermentation enzymatic hydrolysis composite powder. The prepared compound powder has the function of enhancing the immunity of animals.

In order to realize the first invention purpose, the invention adopts the following technical scheme:

a preparation method of a flavor ligamentary fermentation enzymatic hydrolysis compound agent comprises the following specific steps:

s1: respectively carrying out amplification culture on lactobacillus, bacillus and aspergillus niger, and then, carrying out amplification culture on the bacterium liquid according to the volume ratio of the lactobacillus bacterium liquid: and (3) bacillus liquid: the aspergillus niger bacterial liquid is 1-4: 2-4: 1-3, and preparing a compound bacterial liquid;

s2: weighing the following Chinese herbal medicines in parts by mass: 2-6 parts of common corydalis, 2-6 parts of kudzuvine root and 2-4 parts of weeping forsythia, crushing, mixing and sterilizing at 110-;

s3: mixing the compound bacterial liquid and the sterilized Chinese herbal medicine mixture according to the mass ratio of liquid to solid of 15-30: 1, uniformly mixing, and carrying out anaerobic fermentation for 72-96h to obtain a fermentation mixture;

s4: continuously adding cellulose-decomposing enzyme 0.1-1.0% of the total mass of the mixture and lysostaphin 0.1-1.0% of the total mass of the mixture into the fermented mixture, mixing, continuously fermenting for 4-8h, and performing CO₂Performing supercritical extraction, and freeze drying the extract to obtain the finished product.

By adopting the scheme, the Chinese herbal medicine is sterilized at high temperature before fermentation, so that the problem of mixed bacteria in the fermentation process is avoidedInfluence. During fermentation, lactic acid, bacillus and aspergillus niger are selected as fermentation strains, almost complete sets of decomposition enzymes aiming at the botanical raw materials are contained, the decomposition enzymes are microorganisms with the strongest decomposition capability in the natural world, the toxicity of the Chinese herbal medicines is reduced through the powerful biotransformation function of the microorganisms, and natural plant cells are subjected to enzymolysis, so that the effective components in the Chinese herbal medicines are fully released freely and enriched. After fermentation, the added cellulase and lysostaphin can selectively degrade natural macromolecules, plant polypeptides and the like in the Chinese herbal medicines into micromolecular substances which are easily absorbed and utilized by organisms, such as various glycosides, alkaloids, polysaccharides, organic sugars, volatile oil and other immune active substances. CO 2₂The supercritical extraction process has low extraction temperature, and can minimize the damage to effective components and various enzymes of Chinese medicinal materials. The prepared finished product contains various immunocompetent substances, can regulate nerves, body fluid and cell molecules of an organism in an all-around way, activates cells and a humoral immune system of the organism and improves the disease resistance of the organism.

Further, in step S1, a specific culture method of each bacterial liquid is as follows:

culturing lactobacillus liquid:

inoculating lactobacillus strains into 50 mM MRRS liquid culture medium, fermenting for 16-24h at 35-40 deg.C by using a gas bath shaker 200rmp, inoculating the lactobacillus strains into the same liquid culture medium with 3% of inoculum size, and performing amplification culture under the same conditions to obtain lactobacillus strain liquid;

culturing bacillus liquid:

inoculating the bacillus strain into 50mL LB culture solution, culturing for 24-48h at 35-40 ℃ by using a gas bath shaker 200 rmp; then inoculating the bacillus subtilis into the same liquid culture medium by 3 percent of inoculation amount, and performing amplification culture under the same condition to obtain bacillus liquid;

culturing an Aspergillus niger bacterial liquid:

inoculating Aspergillus niger strains into 50mL liquid culture medium prepared by adding 8% of fermented soybean extract and 5% of defatted soybean powder into water solution, and continuously fermenting at 28 deg.C for 70-75 h; then inoculating the strain into the same liquid culture medium by 3 percent of inoculation amount, and carrying out amplification culture under the same conditions to obtain the Aspergillus niger liquid.

The three strains adopted by the invention are all conventional activated pure strains.

By adopting the technical scheme, as the enlarged culture process of the three strains is limited,

the lactobacillus and the bacillus adopt a conventional culture mode, but a special culture medium is provided for the aspergillus niger, so that the culture effect of the aspergillus niger is improved.

Further, the specific step of S1 is as follows:

culturing lactobacillus liquid:

inoculating lactobacillus strains into 50 mM MRS liquid culture medium for amplification culture, fermenting for 20h at 37 ℃ in a gas bath shaker 200rmp, then inoculating the lactobacillus strains into the same liquid culture medium by 3 percent of inoculum size, and performing amplification culture under the same condition to obtain lactobacillus bacteria liquid;

culturing bacillus liquid:

inoculating the bacillus strain into 50mL LB culture solution for amplification culture, and culturing for 36h at 37 ℃ in a gas bath shaker at 200 rmp; then inoculating the bacillus subtilis into the same liquid culture medium by 3 percent of inoculation amount, and performing amplification culture under the same condition to obtain bacillus liquid;

culturing an Aspergillus niger bacterial liquid:

inoculating Aspergillus niger strains into 50mL of liquid culture medium prepared by adding 8% by mass of fermented soybean extract and 5% by mass of defatted soybean flour into aqueous solution, continuously fermenting at 28 ℃ for 72h, inoculating the Aspergillus niger strains into the same liquid culture medium by 3% of inoculum size, and performing amplification culture under the same conditions to obtain Aspergillus niger liquid.

By adopting the preferable technical scheme, the bacterial liquid prepared by the method has higher colony count and good decomposition effect in subsequent Chinese herbal medicine fermentation.

Further, in step S2, the Chinese herbal medicine further includes the following medicinal materials in parts by mass: 2-4 parts of liquorice and 2-4 parts of Chinese pulsatilla root.

By adopting the scheme, the liquorice is sweet in taste and warm in nature, and has the effects of tonifying middle-jiao and Qi, and tonifying spleen and lung. Also has effects in clearing away heat and toxic materials, treating stranguria, and promoting urination. Pulsatilla chinensis has effects of clearing heat and detoxicating, cooling blood and stopping dysentery. Licorice root, radix Glycyrrhizae can relieve spasm and harmonize the herbs. The addition of the liquorice and the Chinese pulsatilla root can further improve the types of active ingredients in the compound preparation, thereby further improving the drug effect.

Further, in step S3, the anaerobic fermentation specifically comprises: firstly, controlling the temperature to be 32-35 ℃, and fermenting for 24-48 h; then controlling the temperature to be 28-30 ℃ until the fermentation is finished.

By adopting the technical scheme, the fermentation is controlled in two sections, the temperature of the front section is higher, the temperature reduction fermentation of the rear section is enzyme production fermentation, the enzyme yield is increased, and the Chinese herbal medicines are subjected to full enzymolysis.

Further, a synergist is added during anaerobic fermentation in step S3, and the synergist is Cu containing mineral elements²⁺、Co²⁺、Mg²⁺、Fe²⁺、Zn²⁺、Ca²⁺The water-soluble compound of (1).

By adding the synergist during fermentation, the mineral elements can provide various nutrient components for the growth of microorganisms, improve the number of viable bacteria in fermentation liquor, improve the stability and activity of the performance of the composite preparation, shorten the fermentation period and reduce the fermentation cost.

Further, containing Cu as a mineral element²⁺、Co²⁺、Mg²⁺、Fe²⁺、Zn²⁺、Ca²⁺Respectively is CuSO₄、Co(NO₃)₂、MgSO₄、FeSO₄、ZnSO₄、CaCl₂。

The compounds are conventional compounds, and have the advantages of readily available materials and low cost.

Further, the content of the water-soluble compound in the composite bacterial liquid is respectively as follows: CaCl₂2-4g/L，ZnSO₄0.5-1.5g/L，FeSO₄0.2-1.0g/L，CuSO₄0.01-0.08g/L，Co(NO₃)₂0.02-0.06g/L，MgSO₄0.1-0.5g/L。

By adopting the technical scheme, the dosage of each mineral compound is optimized, and more reasonable and sufficient nutrition is provided for the fermentation of the strains.

The invention also aims to provide the flavored desmodium fermentation enzymatic hydrolysis composite powder prepared by the preparation method of the flavored desmodium fermentation enzymatic hydrolysis composite agent.

The fermentation enzymolysis composite powder prepared by the technical scheme does not contain any additive, is convenient to use, can be fed independently, and can also be used together with other feeds or feed additives. Is a novel compound Chinese herbal medicine microecological preparation with high efficiency, greenness, no residue and no resistance.

The invention can also be added with proper auxiliary materials to prepare other formulations, such as granules and the like.

The invention also aims to provide the application of the Chinese medicinal herb flavored desmodium and fermented enzymolyzed composite powder in preparing medicaments for improving anti-inflammatory reaction and animal immunity.

The compound powder prepared by the invention has the functions of obviously improving anti-inflammatory reaction and improving animal immunity.

In conclusion, the invention has the following beneficial effects:

1. in the fermentation process, lactic acid bacteria, bacillus and aspergillus niger are selected, the synergistic effect of Chinese herbal medicines and probiotics is utilized, namely the Chinese herbal medicine active ingredients promote the proliferation efficiency of the probiotics, the probiotics carry out biotransformation on the Chinese herbal medicines to generate probiotic mycoprotein antibodies, oligosaccharides and derivatives thereof, and the probiotic mycoprotein antibodies, the oligosaccharides and the derivatives thereof have the effects of resisting inflammation and infection and promoting the propagation of intestinal beneficial flora and also have the effect of activating the immune function of organisms, so that the drug effect of the active ingredients in the Chinese herbal medicines is improved; the application of the medicine for treating animal diarrhea and various diarrhea and heat syndrome diseases. The antibiotic is replaced by the advantages of being green, safe and efficient, and the antibiotic is used for promoting the growth of animals, preventing and treating intestinal diseases, eliminating antibiotic residues, guaranteeing the safety and human health of animal products, promoting the upgrading of the feed industry and the breeding industry and improving the international competitiveness of domestic animal products.

2. The added cellulolytic enzyme and lysostaphin can selectively decompose natural macromolecules, plant polypeptides and the like in the Chinese herbal medicine to generate a plurality of immunocompetent substances such as glycosides, alkaloids, polysaccharides, organic sugars, volatile oil and the like, and the immunocompetent substances are matched with decomposed fermentation products to further improve the types and the activities of effective components in the Chinese herbal medicine and improve the utilization rate of the Chinese herbal medicine.

3. Selecting CO₂The supercritical extraction process has low extraction temperature, and can reduce the damage to the effective components of the Chinese herbal medicines as much as possible compared with the traditional Chinese herbal medicine decoction extraction mode.

4. The compound powder provided by the invention is not added with preservative, and is a novel compound Chinese herbal medicine microecological preparation which is efficient, green, residue-free, antibiotic-free and antibacterial.

5. The invention provides the Chinese medicinal powder for improving anti-inflammatory reaction and immunity of animals, which replaces antibiotics with the advantages of greenness, safety and high efficiency for promoting the growth of animals, preventing and treating intestinal diseases, starting a disease-resistant system, improving the immunity and disease resistance of organisms, reducing the death rate, and has the advantages of wide action range, quick curative effect and long-term addition. The method has the advantages of eliminating antibiotic residues, ensuring the safety and human health of animal products, promoting the upgrading of the feed industry and the breeding industry and improving the international competitiveness of domestic animal products.

Detailed Description

The present invention will be further described with reference to the following examples.

Example 1

A preparation method of a flavor ligamentary fermentation enzymatic hydrolysis complex agent comprises the following steps:

s1: inoculating lactobacillus strains into 50 mM MRS liquid culture medium for amplification culture, fermenting for 16h at 35 ℃ in a gas bath shaker 200rmp, then inoculating the lactobacillus strains into the same liquid culture medium by 3 percent of inoculum size, and performing amplification culture under the same condition to obtain lactobacillus bacteria liquid;

inoculating the bacillus strain into 50mL LB culture solution for amplification culture, and culturing for 24h at the temperature of 35 ℃ in a gas bath shaker at 200 rmp; then inoculating the bacillus subtilis into the same liquid culture medium by 3 percent of inoculation amount, and performing amplification culture under the same condition to obtain bacillus liquid;

inoculating Aspergillus niger strains into 50mL of liquid culture medium prepared by adding 8 mass percent of fermented soybean extract and 5 mass percent of defatted soybean flour into aqueous solution, continuously fermenting at 28 ℃ for 70h, then inoculating the Aspergillus niger strains into the same liquid culture medium according to the inoculation amount of 3 percent, and carrying out amplification culture under the same condition to obtain Aspergillus niger liquid;

and (3) preparing the bacterial liquid by volume ratio of lactic acid bacteria liquid: and (3) bacillus liquid: the ratio of the aspergillus niger liquid is 1: 3: 2, mixing to obtain a compound bacterial liquid;

s2: weighing the following components in parts by weight: 2 parts of common corydalis, 2 parts of kudzuvine root and 4 parts of weeping forsythia, crushing, uniformly mixing, and sterilizing at 110 ℃ for 20 min;

s3: mixing the compound bacterial liquid and the sterilized Chinese herbal medicine mixture according to the liquid-solid weight ratio of 15: 1, mixing, firstly controlling the temperature to be 32 ℃, carrying out anaerobic fermentation for 24 hours, then controlling the temperature to be 28 ℃, and then carrying out anaerobic fermentation for 48 hours;

s4: continuously mixing cellulose-decomposing enzyme with an amount of 0.1% of the total mass of the mixture and lysostaphin with an amount of 0.1% of the total mass of the mixture into the fermented mixture, mixing, fermenting for 4h, and performing CO₂Performing supercritical extraction, and freeze drying the extract to obtain the finished product.

Example 2

s1: inoculating lactobacillus strains into 50 mM MRS liquid culture medium for amplification culture, fermenting for 20h at 37 ℃ in a gas bath shaker 200rmp, then inoculating the lactobacillus strains into the same liquid culture medium by 3 percent of inoculum size, and performing amplification culture under the same condition to obtain lactobacillus bacteria liquid;

inoculating Aspergillus niger strains into 50mL of liquid culture medium prepared by adding 8 mass percent of fermented soybean extract and 5 mass percent of defatted soybean flour into aqueous solution, continuously fermenting at 28 ℃ for 72 hours, then inoculating the Aspergillus niger strains into the same liquid culture medium according to the inoculation amount of 3 percent, and carrying out amplification culture under the same condition to obtain Aspergillus niger liquid;

and (3) preparing the bacterial liquid by volume ratio of lactic acid bacteria liquid: and (3) bacillus liquid: the ratio of the aspergillus niger liquid is 4: 2: 3, mixing to obtain a compound bacterial liquid;

s2: weighing the following components in parts by weight: 4 parts of common corydalis, 4 parts of kudzuvine root and 3 parts of weeping forsythia, crushing, uniformly mixing, and sterilizing at 120 ℃ for 30 min;

s3: mixing the compound bacterial liquid and the sterilized Chinese herbal medicine mixture according to the weight ratio of liquid to solid of 20: 1, mixing, firstly controlling the temperature to be 34 ℃, carrying out anaerobic fermentation for 36 hours, then controlling the temperature to be 29 ℃, and then carrying out anaerobic fermentation for 46 hours;

s4: continuously mixing cellulose-decomposing enzyme with an amount of 0.4% of the total mass of the mixture and lysostaphin with an amount of 0.4% of the total mass of the mixture into the fermented mixture, mixing, fermenting for 6h, and performing CO₂Performing supercritical extraction, and freeze drying the extract to obtain the finished product.

Example 3

s1: inoculating lactobacillus strains into 50 mM MRS liquid culture medium for amplification culture, fermenting for 24h at 40 ℃ in a gas bath shaker 200rmp, then inoculating the lactobacillus strains into the same liquid culture medium by 3 percent of inoculum size, and performing amplification culture under the same condition to obtain lactobacillus bacteria liquid;

inoculating the bacillus strain into 50mL LB culture solution for amplification culture, and culturing for 48h at 40 ℃ in a gas bath shaker at 200 rmp; then inoculating the bacillus subtilis into the same liquid culture medium by 3 percent of inoculation amount, and performing amplification culture under the same condition to obtain bacillus liquid;

inoculating Aspergillus niger strains into 50mL of liquid culture medium prepared by adding 8 mass percent of fermented soybean extract and 5 mass percent of defatted soybean flour into aqueous solution, continuously fermenting at 28 ℃ for 75h, then inoculating the Aspergillus niger strains into the same liquid culture medium according to the inoculation amount of 3 percent, and carrying out amplification culture under the same condition to obtain Aspergillus niger liquid;

and (3) preparing the bacterial liquid by volume ratio of lactic acid bacteria liquid: and (3) bacillus liquid: the ratio of the aspergillus niger liquid is 3: 4: 1, mixing to obtain a compound bacterial liquid;

s2: weighing the following components in parts by weight: 6 parts of common corydalis, 6 parts of kudzuvine root and 2 parts of weeping forsythia, crushing, uniformly mixing, and sterilizing at 130 ℃ for 40 min;

s3: mixing the compound bacterial liquid and the sterilized Chinese herbal medicine mixture according to the liquid-solid weight ratio of 30: 1, mixing, firstly controlling the temperature to be 35 ℃, carrying out anaerobic fermentation for 48 hours, then controlling the temperature to be 30 ℃, and then carrying out anaerobic fermentation for 48 hours;

s4: continuously mixing the zymolytic enzyme with the dosage of 1.0% of the total mass of the mixture and the lysostaphin with the dosage of 1.0% of the total mass of the mixture into the fermentation mixture, uniformly mixing, fermenting for 8h, and then carrying out CO₂Performing supercritical extraction, and freeze drying the extract to obtain the finished product.

Example 4

This example was carried out as in example 2, with the following differences: the Chinese herbal medicines in the step S2 further comprise 2 parts of liquorice and 4 parts of Chinese pulsatilla root by weight.

Example 5

This example was carried out as in example 2, with the following differences: the Chinese herbal medicines in the step S2 further comprise 3 parts of liquorice and 3 parts of Chinese pulsatilla root according to parts by weight.

Example 6

This example was carried out as in example 2, with the following differences: the Chinese herbal medicines in the step S2 further comprise 4 parts of liquorice and 2 parts of Chinese pulsatilla root by weight.

Example 7

This example was carried out as in example 2, with the following differences: in the step S3, a synergist is added during closed fermentation, wherein the synergist is composed of a mineral element Cu²⁺、Co²⁺、Mg²⁺、Fe²⁺、Zn²⁺、Ca²⁺The specific compound and the amount of CaCl in the composite bacterial liquid are₂2g/L，ZnSO₄0.5g/L，FeSO₄0.2g/L，CuSO₄0.01g/L，Co(NO₃)₂0.02g/L，MgSO₄0.1g/L。

Example 8

This example was carried out as in example 2, with the following differences: step S3 middle cipherAdding synergist during fermentation, wherein the synergist is mineral element Cu²⁺、Co²⁺、Mg²⁺、Fe²⁺、Zn²⁺、Ca²⁺The specific compound and the amount of CaCl in the composite bacterial liquid are₂3g/L，ZnSO₄1g/L，FeSO₄0.5g/L，CuSO₄0.05g/L，Co(NO₃)₂0.04g/L，MgSO₄0.3g/L。

Example 9

This example was carried out as in example 2, with the following differences: in the step S3, a synergist is added during closed fermentation, wherein the synergist is composed of a mineral element Cu²⁺、Co²⁺、Mg²⁺、Fe²⁺、Zn²⁺、Ca²⁺The water-soluble compound of (2), the concrete compound and the content of CaCl in the composite bacterial liquid₂4g/L，ZnSO₄1.5g/L，Fe SO₄1.0g/L，CuSO₄0.08g/L，Co(NO₃)₂0.06g/L，MgSO₄0.5g/L。

Active ingredient detection

The data for the assay of the major ingredients of the finished products prepared in examples 1-9 are shown in Table 1.

TABLE 1 results of effective ingredient test data

。

As can be seen from examples 1 to 3 in Table 1, the setting of the preparation parameters has an influence on the amount of the effective ingredients in the finished product, and the preparation parameters in example 2 are preferred; the berberine hydrochloride and glycyrrhizic acid of the finished products prepared in the examples 4-6 are similar to those of the example 2, and in addition, the finished products prepared in the examples 4-6 also comprise various trace effective components such as sinigrin, adenosine, aucubin, pulsatilla saponin, various amino acids and the like; the berberine hydrochloride and glycyrrhizic acid contents of the finished products prepared in the examples 7-9 are higher than those of the finished product prepared in the example 2, because the addition of the synergist provides various nutrient components for the growth of microorganisms, improves the number of viable bacteria in fermentation liquor and further improves the fermentation decomposition effect.

Safety test for target animal pig

The sample prepared in example 2 was selected for safety tests to confirm the safety of the composite formulation prepared by the preparation method of the present invention, as follows.

Purpose of the test

According to the requirements of the technical Specification for experimental clinical tests (trial) of the Ministry of agriculture and the technical guide principle of clinical tests of traditional Chinese medicines and natural medicines for veterinary use of the Ministry of agriculture, the safety and the safe dose of the samples on the clinical application of the target animal pigs are evaluated.

2 materials and methods

2.1 materials

Test drugs: example 2 sample prepared.

Experimental animals: 40 weaned piglets of 50 days old are of unlimited varieties and are half female and half male.

2.2 Experimental grouping and feeding

Piglets were kept and observed in a standard animal house (certified by the institutional animal care committee in beijing) for 2 days after the purchase of piglets, and 40 piglets were randomly divided into 4 groups of 10 piglets each. 3 dose groups of the drug were set according to 1, 3, 5 times (i.e., 0.4g/kg, 1.2g/kg, 2.0 g/kg) of the maximum pharmacodynamic dose, while a blank control group without drug administration was set. During the test period, the feed and drinking water were fed according to the normal feeding schedule, and the temperature and humidity were maintained at 20 + -25 deg.C and 40-60% respectively. The administration mode adopts mixed feeding administration, 1 time per day and 15 days of continuous administration.

2.3 Effect evaluation index and method

The physical signs of the experimental piglets are as follows: the piglets were observed and recorded for feeding, mental and fecal (urine, feces) condition, and compared with the control group for sign difference.

Relative weight gain rate, weighing 1 time before the test starts and 1 time after the test ends, calculating the relative weight gain rate of the piglets, wherein the relative weight gain rate = the relative weight gain of the drug treatment group/the relative weight gain of the blank control group of × l 00%.

Blood routine and blood biochemical indices: after the test is finished, each group of piglets draws blood to carry out conventional blood examination and biochemical blood index examination.

And (4) pathological observation, namely, killing piglets after the experiment is finished, dissecting each group to take the heart, the liver, the spleen, the lung and the kidney, observing the condition of each organ, weighing the tissue and the organ of the piglets, and calculating the organ coefficient, wherein the organ coefficient = the weight of the organ/the total weight of × l 00%.

Pathological tissue section: corresponding lesion tissues are taken according to the lesion conditions of the piglets, and 3 piglets in each group are taken

Organ tissues are fixed by 10% formaldehyde solution, pathological sections are prepared, and the histopathological conditions are observed.

2.4 data analysis

Statistical software SPASS 18.0 is used for data processing, independent sample t test is adopted, test data are represented by mean values +/-standard deviation, and the difference is significant when P is less than or equal to 0.05.

3 results of the test

3.1 Observation of body signs

The piglets in each group in the test period have good overall condition, normal ingestion and good mental status, and do not die; on the 7 th day of the test, 2 pigs in the control group showed cough symptoms, and recovered to normal after 3 days; the physical signs of the pigs in the drug groups with 1 time, 3 times and 5 times of doses are normal.

3.2 relative rate of weight gain

Compared with the blank control group, the relative weight gain rate of the 1-time, 3-time and 5-time dose groups is increased, but the statistically significant difference (p >0.05) is not generated (Table 2).

Table 2 relative rate of weight gain of experimental piglets (n =10)

。

Note: the data in the same row without shoulder marks showed no significant difference (p >0.05), and data in the same row showed significant difference (p <0.05) compared to the control group.

3.3 blood routine test results

Compared with a blank control group, the difference between the detection indexes is not significant (p is greater than 0.05), and each detection index of each group is within a normal range (Table 3).

Table 3 piglet blood general indices (n =10) were tested.

。

3.4 Biochemical index test results of blood

Compared with a blank control group, the content of glutamic-oxaloacetic transaminase (GOT), urea nitrogen (BUN) and Total Protein (TP) in the serum of the 1-time dose group is obviously lower than that of the blank control group; the contents of glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT) and urea nitrogen (BUN) in the serum of the 3-time dose group are obviously lower than those of the blank control group; the contents of glutamic-pyruvic transaminase (GPT) and urea nitrogen (BUN) in the serum of the 5-time dose group are obviously lower than those of the blank control group; each group of data, although significantly different from the blank group, was still within the reference range (table 4).

Table 4 experimental piglet blood biochemical indices (n = 10).

。

Note: row data without shoulder marks indicated no significant difference (p >0.05) and row data indicated significant difference compared to control (p < 0.05).

3.5 organ coefficients

There was no significant difference in the organ coefficients compared to the blank control group (table 5).

Table 5 organ coefficients of experimental piglets (n = 10).

。

3.6 histopathological examination

After the test is finished, all piglets are sacrificed, no obvious lesion is found by the autopsy, and then the histological detection is carried out.

3.6.1 liver

The structure of each group of liver cells is clear and complete, the shapes and the sizes are consistent, and the cytoplasm is rich; the nucleus is round, centered and even with double or multiple cores; the liver cable structure of each group is clear.

3.6.2 spleen

Spleen tissues of each group are clear in color, and red marrow and white marrow have obvious boundaries; the number of lymphocytes is large, the shape is consistent, and the coloring is uniform.

3.6.3 Kidney

Each group of renal tubules has clear shape, uniform staining, clear renal tubule epithelial cell boundary and complete cell nucleus; the glomerulus structure is complete.

4 conclusion of the test

The general condition, the observation of visceral organs and the examination of blood physiological and biochemical indexes of the piglets are carried out in the experiment, so that the sample prepared in the embodiment 2 has no harmful effect on the piglets and has better safety.

Immunity enhancement test of compound preparation on target animal pig

The samples prepared in representative examples 2, 5 and 8 were selected to be subjected to an immunopotentiation test to confirm the effect of the complex formulation prepared by the preparation method of the present invention in enhancing the immunity of animals.

Purpose of the test

The clinical efficacy of the samples prepared in examples 2, 5 and 8 on enhancing the pig organism immunity was evaluated according to the requirements of the Ministry of agriculture, the technical Specification for clinical laboratory tests (trial) and the Ministry of agriculture, the technical guidelines for clinical tests of veterinary Chinese medicine and natural medicine.

2 materials and methods

2.1 materials

Test animals: for piglets, the number of male and female is 65 respectively, 15 +/-20 kg, and the porcine reproductive and respiratory syndrome antibody is negative, and the antibody is purchased from Dabei agriculture breeding technology Limited liability company in Tangshan; cyclophosphamide was purchased from Hubei Kangbao Tai Fine chemical Co., Ltd, lot number KBT 20170501; porcine reproductive and respiratory syndrome attenuated vaccine was purchased from bleegger hagaham, germany, lot no: 1245110-05; samples prepared in examples 2, 5, 8; lingqi kunjin vitamin was purchased from astragalis health products limited, shandong, and the batch number: 20170501.

2.2 groups of test animals

Referring to table 6, 130 piglets were randomly divided into 13 groups of 5 females/males each. Group 1 is blank, groups 2-3 are model, and groups 4-13 are drug groups. The blank group was not injected with cyclophosphamide, administered, and vaccinated; model group 1 cyclophosphamide injection, but no administration, no vaccination; model group 2 was vaccinated with cyclophosphamide injection, but no drug: the drug group 1 is injected with cyclophosphamide, inoculated with vaccine and administered with contrast drug; drug group 2-4 injections of cyclophosphamide, vaccination, different doses (0.1-0.4 g/kg-b.w.) of the samples prepared in example 2; drug groups 5-7 were injected with cyclophosphamide, vaccinated, and given different doses (0.1-0.4 g/kg-b.w.) of the samples prepared in example 5; drug groups 8-10 were injected with cyclophosphamide, vaccinated and given different doses (0.1-0.4 g/kg-b.w.) of the samples prepared in example 8. The molding administration procedure of cyclophosphamide is that 75 mg/kg.b.w. is intramuscular injected to cyclophosphamide injection on the 1 st to 4 th days of the experiment, and the same amount of normal saline is injected to a cyclophosphamide-free group; the drug administration program is that the drugs and the doses listed in the table 6 are mixed and fed for 1 time every day on the 1 st to 10 th days of the test; the procedure for vaccination was intramuscular injection of porcine reproductive and respiratory syndrome vaccine on day 11 of the experiment and the non-vaccinated group injected with an equivalent amount of normal saline. The test lasts for 50 days, the pigs are fed normally, and the condition of the pig herds is observed and recorded every day.

TABLE 6 test grouping

2.3 index evaluation

2.3.1 body weight determination

Pigs were weighed for each group at 0, 10, 25, 50 days of the trial, respectively, and the differences in weight of pigs for each group were compared.

2.3.2 blood routine, blood Biochemical assay and antibody level assay

Collecting peripheral blood of each group of pigs on 0, 20 and 40 days after immunization, and dividing the peripheral blood into two parts, wherein one part is used for carrying out conventional blood detection by using a full-automatic blood cell analyzer, and the other part is used for detecting GOT, GTP, ALP, BUN, TP and ALB by using a full-automatic biochemical analyzer.

And collecting peripheral blood of each group of pigs on 0, 7, 15, 25 and 40 days after immunization, separating serum, and detecting the porcine reproductive and respiratory syndrome antibody level of each group by using a porcine reproductive and respiratory syndrome antibody detection kit.

2.4 data analysis

The SPASS 18.0 statistical software is used for data processing, independent sample t test is adopted, test data are all expressed by mean values +/-standard deviation, and the difference P <0.05 is obvious.

2.5 test results

2.5.1 body weight

The daily weight gain was lower for the remaining test groups at 10 and 25 days compared to the blank group, and the daily weight gain for 50 days was essentially the same for each test group, but was not statistically significantly different. The daily weight gain of the first 10 days is obviously reduced compared with that of a blank group in a model group 1, a model group 2, a medicine group 1-3, a medicine group 5-6 and a medicine group 8-9; compared with the model group 2, the daily gain of the drug group 4, the drug group 7 and the drug group 10 was significantly increased, as shown in table 7.

Table 7 piglets were tested for daily gain (n =10) over different time periods.

。

Note that the difference between the two groups is significant compared with the blank group (P <0.05), and △ shows that the difference between the two groups is significant compared with the model group 2 (P < 0.05).

2.5.2 blood routine results

The blood routine detection is carried out on each group of piglets at 0, 20 and 40 days after immunization respectively, and the detection results are as follows:

day 0 after immunization, the level of White Blood Cells (WBC) was reduced and significantly different in each of the remaining test groups compared to the blank group, since the immunosuppressive effect of yellow phosphoramide was still affected; the Red Blood Cell (RBC) levels of model group 1, drug group 4, drug group 7, and drug group 10 were all significantly higher than the blank group. Compared with the model group 2, the Red Blood Cell (RBC) levels of the drug groups 2 to 3, 5 to 6 and 8 to 9 were significantly reduced, and the Red Blood Cell (RBC) levels of the drug group 1, 4, 7 and 10 were significantly increased, as shown in table 8.

Table 8 immunization day 0 standard index of piglet blood (n =10) was tested.

。

Note that the differences are significant compared to the blank group (P <0.05), and △ shows significant compared to the model group 2 (P < 0.05).

On day 20 post-immunization, the White Blood Cell (WBC) levels were significantly elevated in model group 2, drug groups 1-10, compared to the blank group. Model group 2 showed significantly higher White Blood Cell (WBC) levels compared to model group 1. The drug groups 1, 4, 7, 10 showed significantly higher Platelet (PLT) than the model group 2, as shown in table 9.

Table 9 standard indices of piglet blood (n =10) tested at day 20 post immunization.

。

Note that # indicates a significant difference compared to the blank group (P <0.05), # indicates a significant difference compared to model group 2 (P <0.05), and △ indicates a significant difference compared to model group 2 (P < 0.05).

On day 40 after immunization, the White Blood Cell (WBC) levels were significantly increased in each group except the model group 1, and the Red Blood Cell (RBC) and platelet (RLT) levels were significantly decreased in the drug group 1, as compared to the blank group. Model group 2 showed significantly higher White Blood Cell (WBC) levels compared to model group 1. The White Blood Cell (WBC) levels were significantly elevated for drug group 1, drug groups 3-4, drug groups 6-7, and drug groups 9-10 compared to model group 2. See table 10.

Table 10 standard indices of piglet blood (n =10) tested on day 40 after immunization.

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Note that: # indicates a significant difference compared to the blank group (<0.05), # indicates a significant difference compared to model group 2 (<0.05), and △ indicates a significant difference compared to model group 2 (< 0.05).

2.5.3 blood Biochemical index detection results

The blood biochemical detection is carried out on each group of piglets at 0, 20 and 40 days after immunization respectively, and the detection results are as follows.

Table 11 post immunization day 0 piglets were tested for blood biochemical markers (n = 10).

。

On day 0 after immunization, compared with the blank group, the levels of Total Protein (TP) of glutamic-oxaloacetic transaminase (GOT), Albumin (ALB) of model group 2, drug group 4, drug group 5, drug group 7, drug group 8 and drug group 10, drug group 2, drug group 5 and drug group 8 were all significantly reduced; alkaline phosphatase (ALP) in the group consisting of the model group 1, the model group 2, the drug group 1, the drug group 4, the drug group 7, and the drug group 10, glutamic-pyruvic transaminase (GPT) in the group consisting of the drug group 3, the drug group 6, and the drug group 9, and Albumin (ALB) level in the group consisting of the model group 2 were significantly increased. Urea Nitrogen (BUN), Albumin (ALB) levels were significantly reduced for model group 2 compared to model group 1; total Protein (TP) levels were significantly elevated. Compared with the model group 2, the glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) of the drug group 3, the drug group 6 and the drug group 9, the glutamic-oxaloacetic transaminase (GOT) level of the drug group 4, the drug group 7 and the drug group 10 were significantly increased, and the Total Protein (TP) and Creatinine (CRE) level of the drug group 2, the drug group 5 and the drug group 8 were significantly decreased, as shown in table 11.

Note that # indicates a significant difference compared to the blank group (P <0.05), # indicates a significant difference compared to model group 1 (P <0.05), and △ indicates a significant difference compared to model group 2 (P < 0.05).

On day 20 after immunization, the glutamic-pyruvic transaminase (GPT) levels of model group 1, glutamic-oxalacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) of model group 2, glutamic-pyruvic transaminase (GPT) of drug group 3, drug group 6 and drug group 9, and glutamic-pyruvic transaminase (GPT) levels were all significantly reduced compared to the blank group; alkaline phosphatase (ALP) in the model group 1, the model group 2, the drug group 4, the drug group 7, and the drug group 10, urea nitrogen (BUN) in the model group 1, the model group 2, the drug group 1, the drug group 2, the drug group 5, and the drug group 8, and Total Protein (TP) level in the model group 2 were significantly increased. The Albumin (ALB) was significantly reduced in model group 2 compared to model group 1. Compared with the model group 2, the Total Protein (TP) levels of the drug group 2, the drug group 5 and the drug group 8, the urea nitrogen (BUN) levels of the drug group 3, the drug group 6 and the drug group 9 and the Total Protein (TP) levels are obviously reduced; glutamic-oxaloacetic transaminase (GOT) of drug group 1, urea nitrogen (BUN) and glutamic-oxaloacetic transaminase (GOT) of drug group 2, drug group 5, and drug group 8, and glutamic-oxaloacetic transaminase (GOT) of drug group 4 were significantly increased, as shown in table 12.

Table 12 post immunization day 20 piglets were tested for blood biochemical markers (n = 10).

。

At day 40 after immunization, the Albumin (ALB) levels in model group 2 and glutamic-oxaloacetic transaminase (GOT) levels in drug group 2, drug group 5, and drug group 8 were significantly increased. Compared with the model 1, the glutamic-oxaloacetic transaminase (GOT) of the model 2 is significantly reduced, and the urea nitrogen (BUN) is significantly increased. Compared with the model group 2, the glutamic-oxalacetic transaminase (GOT) of the drug group 1, the drug group 2, the drug group 5, and the drug group 8 was significantly increased, and the level of glutamic-pyruvic transaminase (GPT) of the drug group 1 was significantly decreased. See table 13.

Table 13 piglets were tested for blood biochemical markers at day 40 after immunization (n = 10).

。

2.5.4 antibody detection results

And (3) collecting peripheral blood of the pigs on 0 th, 7 th, 15 th, 25 th and 40 th days after immunization, separating serum, detecting the porcine reproductive and respiratory syndrome antibodies of each group by using a porcine reproductive and respiratory syndrome antibody detection kit, and judging the porcine reproductive and respiratory syndrome antibodies to be positive if the detection result of the kit antibody level is more than 0.4 and judging the porcine reproductive and respiratory syndrome antibodies to be negative if the detection result is less than 0.4. Antibody levels were analyzed as follows:

before immunization, there was no significant difference between the test groups compared to the blank group. 7 days after immunization: compared with the blank group, the antibody levels of the model 2 group, the drug group 1, the drug group 4, the drug group 7 and the drug group 10 are obviously increased; compared with the model group 2, the antibody levels of the drug group 4, the drug group 7 and the drug group 10 were significantly increased. 15 days after immunization: compared with the blank group, the antibody levels of the model group 2 and the drug groups 1-10 are obviously increased; model group 2 antibody levels were significantly higher than model group 1; compared with the model group 2, the antibody levels of the drug groups have no significant difference. 25 days after immunization: compared with a blank group, the antibody levels of the model group 2 and the drug groups 1-10 are obviously increased; the antibody levels of the model group 2, the model group 5 and the model group 8 are obviously higher than that of the model group 1; compared with the model group 2, the antibody levels of the drug groups 3-4, 6-7 and 9-10 are obviously increased. 40 days after immunization: compared with the blank group, the antibody levels of the model group 2 and the drug groups 1-10 are obviously increased; model group 2 antibody levels were significantly higher than model group 1; compared with the model group 2, the antibody levels of the drug group 4, the drug group 7 and the drug group 10 were significantly increased, as shown in table 14.

Table 14 antibody level results.

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Note that # indicates a significant difference compared to the blank group (P < 0.05); # indicates a significant difference compared to model group 1 (P < 0.05; △ indicates a significant difference compared to model group 2 (P < 0.05).

2.6 conclusion

The analysis of the results shows that the compound preparation samples prepared in examples 2, 5 and 8 have the effect of improving the titer of the porcine reproductive and respiratory syndrome vaccine antibody, and the effect is better than that of a control medicament when the administration dosage is (0.2-0.4 g/kg-b.w.). The effect of the composite preparation samples prepared in examples 5 and 8 is better than that of example 2.

The above-mentioned embodiments are merely illustrative and not restrictive, and those skilled in the art can modify the embodiments without inventive contribution as required after reading this specification, but only fall within the scope of the claims of the present invention.

Claims

1. A preparation method of a flavor ligamentary fermentation enzymatic hydrolysis complex agent is characterized by comprising the following steps:

2. The method for preparing a fermented and enzymolyzed complex of claim 1, wherein in step S1, the specific culture method of each bacterial liquid is as follows:

culture of lactobacillus liquid

culturing bacillus liquid

culture of Aspergillus niger liquid

3. The method for preparing a fermented and enzymolyzed complex of claim 2, wherein in step S1, the specific culture method of each bacterial liquid is as follows:

culture of lactobacillus liquid

culturing bacillus liquid

culture of Aspergillus niger liquid

4. The method for preparing a fermented and enzymolyzed complex of Mandarin orange fibers as claimed in claim 1 or 3, wherein in step S2, the Chinese herbal medicines further comprise the following herbs in parts by mass: 2-4 parts of liquorice and 2-4 parts of Chinese pulsatilla root.

5. The method for preparing a fermented mixture of ingredients of claim 4, wherein in step S3, the anaerobic fermentation is specifically: firstly, controlling the temperature to be 32-35 ℃, and fermenting for 24-48 h; then controlling the temperature to be 28-30 ℃ until the fermentation is finished.

6. The method for preparing a fermented mixture of Leptospira spicata and Leptospira sinensis as claimed in claim 5, wherein step S3 is performed by adding synergist during anaerobic fermentation, wherein the synergist is Cu containing mineral element²⁺、Co²⁺、Mg²⁺、Fe²⁺、Zn²⁺、Ca²⁺The water-soluble compound of (1).

7. The method for preparing a fermented composition of claim 6, wherein the fermented composition contains Cu as mineral element²⁺、Co²⁺、Mg²⁺、Fe²⁺、Zn²⁺、Ca²⁺Respectively is CuSO₄、Co(NO₃)₂、MgSO₄、FeSO₄、ZnSO₄、CaCl₂。

8. The method for preparing a Leptospira Spectabilis fermentation enzyme complex agent according to claim 7, wherein the content of the water-soluble compound in the complex bacterial liquid is respectively: CaCl₂2-4g/L，ZnSO₄0.5-1.5g/L，FeSO₄0.2-1.0g/L，CuSO₄0.01-0.08g/L，Co(NO₃)₂0.02-0.06g/L，MgSO₄0.1-0.5g/L。

9. A fine powder of the slow-growing and long-growing enzyme complex prepared by the method for preparing the fine powder of the slow-growing and long-growing enzyme complex of any one of claims 1 to 8.

10. The use of the fine powder of claim 9 for the preparation of a medicament for enhancing anti-inflammatory response or enhancing immunity.