CN115211501A

CN115211501A - Preparation method and application of compound fermented traditional Chinese medicine feed additive for preventing and treating common intestinal pathogenic bacteria

Info

Publication number: CN115211501A
Application number: CN202210755888.4A
Authority: CN
Inventors: 陈华友; 卢晨曦; 陈曦华; 蔡康涛; 姚丹; 陈华撑
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-06-29
Filing date: 2022-06-29
Publication date: 2022-10-21
Anticipated expiration: 2042-06-29
Also published as: CN115211501B

Abstract

The invention belongs to the field of biological feed, and particularly relates to a preparation method and application of a compound fermented traditional Chinese medicine feed additive for preventing and treating common intestinal pathogenic bacteria; the method comprises the following steps: activating the strains, and culturing to obtain bacillus, saccharomycetes and lactobacillus fermentation seed liquid; mixing the ground compound traditional Chinese medicinal materials with soybean meal and wheat middling, adding a complex enzyme to obtain a fermentation culture medium, inoculating a bacillus fermentation seed solution, a saccharomycete fermentation seed solution and a lactobacillus fermentation seed solution, and performing anaerobic fermentation under a closed condition to obtain a finished product; the compound traditional Chinese medicinal materials are adopted for bacterial enzyme synergistic fermentation, the fermentation is thorough, the compound traditional Chinese medicinal materials are rich in probiotics, organic acid and other probiotics, and the intestinal digestion level, the immunity and other aspects of the cultured animals can be effectively improved; has obvious prevention and treatment effect on bacterial infection caused by salmonella enteritidis, escherichia coli and shigella flexneri, is convenient to use, is beneficial to industrial production, and has high economic benefit.

Description

Preparation method and application of compound fermented traditional Chinese medicine feed additive for preventing and treating common intestinal pathogenic bacteria

Technical Field

The invention belongs to the field of biological feed, and particularly relates to a preparation method and application of a compound fermented traditional Chinese medicine feed additive for preventing and treating common intestinal pathogenic bacteria.

Background

Salmonella enteritidis (Salmonella enteritidis), escherichia coli (Escherichia coli), shigella flexneri (Shigella flexneri) are three of the more common enteropathogenic bacteria. For example, salmonella enteritidis is a zoonosis, has extremely wide animal hosts such as mammals, fishes, amphibians, birds, reptiles and the like, and often causes serious diseases after infecting animals, and is also a second most food-borne pathogen, namely, food-borne food eaten by people, which can cause food poisoning and cause diseases such as gastroenteritis, septicemia and the like. Escherichia coli, which is widely found in nature. Escherichia coli O157: h7 is a type of it, which causes zoonotic infectious diseases, infects breeding animals such as chicken, duck, pig, cow, fish, etc., and also infects humans, induces hemoenteritis and hemolytic uremia, and endangers human lives. Shigella flexneri is a gram-negative pathogen that causes bacillary dysentery, resulting in an inflammatory response in the intestine. Besides infecting human, studies show that young animals such as chicken, duck, rabbit, cow, mouse, dog, and macaque are also susceptible to the bacteria, and the bacteria can cause serious harm to young animals, such as diarrhea and intestinal hemorrhage. Researches prove that although the three intestinal pathogenic bacteria are conditional pathogenic bacteria, if the bacterial quantity of the three intestinal pathogenic bacteria is outbreak, the three intestinal pathogenic bacteria can cause huge influence in the breeding industry, antibiotics are often added blindly to farmers for preventing and treating diseases, and with the disablement of antibiotics, a new antibiotic substitute needs to be searched urgently.

At present, some researches on the use of fermented traditional Chinese medicines in animal breeding and disease prevention and treatment have been carried out, but none of the researches is designed by screening traditional Chinese medicines in a formula of a fermented traditional Chinese medicine feed additive aiming at specific pathogenic bacteria, such as patents CN113648393A and CN113521212A, which are used for poultry breeding to resist intestinal diseases and inflammation of other organs in the breeding process, but the selected traditional Chinese medicines are not screened, are simply added according to the characteristics of the traditional Chinese medicines, and have no specificity aiming at the pathogenic bacteria.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a preparation method of a compound fermentation traditional Chinese medicine feed additive for preventing and treating common intestinal pathogenic bacteria and application thereof in the breeding industry; the invention screens bacteriostatic traditional Chinese medicines of three main intestinal conditional pathogenic bacteria (salmonella enteritidis, escherichia coli and shigella flexneri), obtains a group of compound traditional Chinese medicines with strong inhibition capability on common intestinal pathogenic bacteria, and ferments the compound traditional Chinese medicines; by changing the addition ratio of different traditional Chinese medicines and auxiliary materials, the relationship between the compound traditional Chinese medicine and the fermentation strain is explored to obtain the optimal traditional Chinese medicine addition ratio. The obtained feed additive is rich in probiotics, can effectively improve the immunity of cultured animals, has sour flavor and certain bacteriostatic action, and is convenient to store.

Finally, the prepared compound traditional Chinese medicine feed additive is used for animal breeding (Chinese soft-shelled turtles); the experimental result shows that the feed can effectively improve the immunologic function, the intestinal digestion level and the like of the Chinese soft-shelled turtles when being fed for a long time, and can effectively improve the survival rate of young Chinese soft-shelled turtles in a short-term pathogenic bacterium challenge experiment. The fermented traditional Chinese medicine can be used as a high-quality feed additive for the breeding industry, and has unexpected control effect on common intestinal pathogenic bacteria such as salmonella enteritidis, escherichia coli and shigella flexneri.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

(1) Preparing a traditional Chinese medicine fermentation culture medium: grinding the compound traditional Chinese medicinal materials into fine powder, and fully mixing the fine powder with the soybean meal, the wheat middling and the compound enzyme to obtain a solid fermentation culture medium;

the fermentation medium comprises the following components in percentage by weight: 30 to 70 percent of compound Chinese medicinal material; 18-46% of soybean meal; 12 to 24 percent of wheat middling; the addition amount of the complex enzyme is 0.1 to 10 percent;

the compound traditional Chinese medicine is prepared by mixing coptis chinensis, schisandra chinensis, rhizoma anemarrhenae, gallnut, rheum officinale, scutellaria baicalensis, liquorice, myrobalan and catechu according to equal mass proportion;

the complex enzyme consists of cellulase, hemicellulase and tannase;

(2) Respectively inoculating bacillus, saccharomycetes and lactic acid bacteria to corresponding liquid culture media for activation culture to respectively obtain bacillus fermentation seed liquid, saccharomycetes fermentation seed liquid and lactic acid bacteria fermentation seed liquid;

(3) Inoculating the bacillus seed liquid, the saccharomycete seed liquid and the lactic acid bacteria seed liquid obtained in the step (2) into the fermentation culture medium obtained in the step (1), adjusting the fermentation temperature and the water content of the culture medium, uniformly mixing to obtain a fermentation mixture, and performing anaerobic fermentation under a closed condition to obtain the compound fermentation traditional Chinese medicine feed additive for preventing and treating the intestinal pathogenic bacteria.

Preferably, the soybean meal in the step (1) needs to be dried and ground into powder for use when in use; the powder is obtained by sieving with a 50-mesh sieve; the compound traditional Chinese medicine is ground into powder for use, and the powder is obtained by sieving with a 50-mesh sieve.

The mass ratio of the cellulase to the hemicellulase to the tannase is (2); in the complex enzyme, the activity of hemicellulase is 5 ten thousand u/g, and the activities of cellulase and tannase are both 1 ten thousand u/g.

Preferably, the fermentation medium comprises the following components in percentage by weight: 50% of compound Chinese medicinal materials; 30% of soybean meal; 20% of wheat middling; the addition amount of the complex enzyme is 1 percent.

Preferably, the bacillus in step (2) is bacillus amyloliquefaciens; the yeast is candida tropicalis; the lactobacillus is Lactobacillus plantarum, lactobacillus casei, or Lactobacillus rhamnosus.

Preferably, the inoculation amounts of the bacillus fermentation seed liquid and the yeast fermentation seed liquid in the step (3) are both 0.1-9% (V/W, mL/g) of the weight of the fermentation medium, namely the dosage relationship between the seed liquid and the culture medium is 0.1-9mL; the total number of viable bacteria of the bacillus and the microzyme is 1.0 multiplied by 10 ⁷ ～2.8×10 ⁹ cfu/mL；

The lactobacillus fermented seed liquid is lactobacillus plantarum fermented seed liquid, lactobacillus casei fermented seed liquid and lactobacillus rhamnosus fermented seed liquid, and the total number of viable bacteria is 1.5 × 10 ⁸ ～3.5×10 ⁹ cfu/mL, wherein the inoculation amount is 0.8-5.7% (V/W, mL/g; namely the dosage relationship between the seed solution and the culture medium is 0.8-5.7mL);

the volume ratio of the lactobacillus fermentation seed liquid to the bacillus fermentation seed liquid to the saccharomycetes fermentation seed liquid during inoculation is 2:2:3.

preferably, the inoculation amount of the bacillus fermentation seed liquid and the yeast fermentation seed liquid is 2% (V/W, mL/g) of the weight of the fermentation medium.

Preferably, the water content of the fermentation mixture in the step (3) is 40-60%; the fermentation time is 5-20 days, and the fermentation temperature is 24-38 ℃.

The additive prepared by the invention is used for inhibiting intestinal pathogenic bacteria when being added into aquaculture feed; the enteric pathogenic bacteria are salmonella enteritidis, escherichia coli or shigella flexneri; the specific operation is as follows: adding the fermented feed additive into aquatic feed to obtain mixed feed for feeding; the addition amount of the compound fermented traditional Chinese medicine feed additive accounts for 5-25% of the weight of the mixed feed.

The specific screening process of the technical scheme is as follows:

(1) Screening the traditional Chinese medicine raw materials:

from 45 bacteriostatic traditional Chinese medicines (dark plum, asparagus, dogwood, garlic, chinese magnoliavine fruit, coptis chinensis, heartleaf houttuynia herb, weeping forsythia, giant knotweed, purslane, green tangerine peel, common anemarrhena rhizome, ash bark, common selfheal fruit-spike, sappan wood, humifuse euphorbia herb, chinese pulsatilla root, myrobalan fruit, folium artemisiae argyi, baical skullcap root, astragalus mongholicus, amur corktree bark, chinese gall, wild chrysanthemum flower, glossy privet fruit, largehead atractylodes rhizome, indigowoad root, ginkgo leaf, dandelion, fineleaf schizonepeta herb, chinese angelica, eucommia bark, rhubarb, red paeony root, eucommia leaf, hawthorn fruit, catechu, rehmannia root, liquorice, szechuan lovage rhizome, honeysuckle flower, indigowoad leaf, siberian cocklebur fruit, pomegranate rind and common andrographis herb), the traditional Chinese medicines with strong bacteriostatic effects on salmonella enteritidis, escherichia coli and shigella flexnervosa before and after fermentation are screened through bacteriostatic experiments. The bacteriostasis experiment adopts an agar diffusion method, and the size of the bacteriostasis zone is shown in tables 1, 2 and 3.

TABLE 1 size of bacteriostatic circle of Chinese medicinal materials for Salmonella enteritidis before and after fermentation

Note: in each column of the diameter of the inhibition zone before and after fermentation, the representation difference marked by the same capital letters is not significant (P is more than 0.05), and the representation difference marked by different capital letters is significant (P is less than 0.05). In each line of the diameters of the inhibition zones before and after fermentation of different traditional Chinese medicines, the expression difference marked by the same lower case letters is not significant (P is more than 0.05), the expression difference marked by different lower case letters is significant (P is less than 0.05), and the letters are arranged from large to small in size from first to last to show the inhibition zones. "/" indicates no bacteriostatic effect.

TABLE 2 different fermented Chinese medicinal feeds have the size of the inhibition zone for escherichia coli

Note: in each column of the diameter of the inhibition zone before and after fermentation, the marked capital letters do not show significant difference (P is more than 0.05), and the marked capital letters show significant difference (P is less than 0.05). In each line of the diameters of the inhibition zones before and after fermentation of different traditional Chinese medicines, the expression difference marked by the same lower case letters is not significant (P is more than 0.05), the expression difference marked by different lower case letters is significant (P is less than 0.05), and the letters are arranged from large to small in size from first to last to show the inhibition zones. "/" indicates no bacteriostatic effect.

TABLE 3 size of inhibition zone of Chinese medicinal materials on Shigella flexneri before and after fermentation

According to the experimental result, in the bacteriostatic experiment aiming at salmonella enteritidis, the coptis chinensis, the schisandra chinensis, the rhizoma anemarrhenae and the gallnut have stronger bacteriostatic ability. In an experiment for inhibiting bacteria of escherichia coli, gallnut, rheum officinale, scutellaria baicalensis and liquorice have strong antibacterial effects. In an antibacterial experiment aiming at Shigella flexneri, the myrobalan, the catechu, the gallnut and the scutellaria baicalensis have better antibacterial ability. According to the comprehensive antibacterial result, coptis chinensis, schisandra chinensis, rhizoma anemarrhenae, gallnut, rheum officinale, scutellaria baicalensis, liquorice, myrobalan and catechu are selected as the fermentation raw materials.

(2) Identification of part of key traditional Chinese medicinal material active ingredients in compound fermented traditional Chinese medicinal feed additive

1. Change of effective components of Scutellariae radix before and after fermentation

A, chromatographic conditions: gradient elution (0-10min, 15A → 45A, 85% B → 55B 10-20min, 45A → 57% A,55% B → 43% B20-40min, 57% A → 85% A,43% B → 15% B) with methanol (A) -0.1% phosphoric acid solution (B) as the mobile phase, the detection wavelength was 335nm, the flow rate was 0.8mL/min, the sample intake: 10 μ L.

B, preparation of standard solution

Precisely weighing baicalin, baicalein and quercetin standard substances of each 2mg, dissolving with 60% methanol to constant volume of 10mL, and taking the solution as a mother solution of the standard substance. Sucking 1mL of each of baicalin, baicalein and quercetin standard substance mother liquor, mixing to obtain a mixed standard substance mother liquor, and placing in a refrigerator at 4 deg.C for use.

C, preparation of Chinese medicine extract

The preparation method of the scutellaria baicalensis fermented feed additive comprises the following steps: 50% (W/W) of scutellaria baicalensis, 30% (W/W) of soybean meal and 20% (W/W) of wheat middling. Taking yeast liquid, bacillus liquid and lactobacillus liquid of each single strain (cultured to logarithmic phase) to ferment the compound traditional Chinese medicine feed additive, adding the yeast liquid, the bacillus liquid and the lactobacillus liquid into a fermentation raw material with the addition of 10 percent (V/W, mL/g), adding distilled water to control the water content to be 50 percent (V/W, mL/g), uniformly stirring, placing the mixture into a fermentation bag with one-way air holes, and placing the fermentation bag in an incubator with 30 ℃ for anaerobic fermentation for 7 days.

The preparation method of the Chinese gall fermented feed additive comprises the following steps: 50 percent (W/W) of nutgall, 30 percent (W/W) of soybean meal and 20 percent (W/W) of wheat middling. Other fermentation conditions and methods are the same as those of the preparation of the scutellaria baicalensis fermentation feed additive.

Weighing 1g of the scutellaria baicalensis fermented feed additive and the gallnut fermented feed additive into a 50mL conical flask, and adding 10mL of 60% methanol solution for leaching for 30min; filtering to obtain supernatant, and storing in a refrigerator at 4 deg.C.

The chromatogram before and after fermentation of baicalein, baicalin, quercetin and Scutellariae radix is shown in figure 1. The baicalein content of the fermented scutellaria baicalensis is 9.99mg/g, which is 3.86 times of that of the baicalein content of the scutellaria baicalensis before fermentation, which is 2.59 mg/g. The content of quercetin is 4.99mg/g, which is 1.11 times of that of quercetin 4.49mg/g before fermentation. The main active ingredients of the scutellaria baicalensis are baicalin, baicalein and the like, and the scutellaria baicalensis has multiple functions of resisting bacteria, viruses, oxidation, tumors, liver and the like. The pharmacological research of the traditional Chinese medicine shows that the activity and cytotoxicity of the baicalein for inhibiting HIV-1 are both stronger than those of baicalin, and the effects of diuresis, antifungal and the like of the baicalin are also obviously better than those of the baicalin. Pharmacokinetic studies show that baicalin is not easy to be orally taken by organisms, and needs to be converted into baicalein after hydrolysis by beta-glucuronidase in intestinal tracts, and the baicalein is converted into baicalin by uridine diphosphate glucuronyl transferase in intestinal tracts in small intestinal mucosa and then absorbed into blood. The research and measurement result shows that the baicalin in the scutellaria baicalensis is reduced after fermentation, the baicalein content is increased, and the conclusion that a part of the baicalin in the scutellaria baicalensis can be converted into the baicalein in advance in the microbial fermentation process can be inferred, so that the baicalin is easier to absorb and utilize by the organism. This is proved by Wang research, and the fermentation strain contains the strain producing beta-glucuronidase, which converts baicalin in scutellaria into baicalein. According to bacteriostatic experiments, the bacteriostatic ability of the fermented feed against various pathogenic bacteria is enhanced, and the possible reasons of the fermented feed are that organic acid and antibacterial peptide in the fermented feed are increased, and the content of baicalein in the scutellaria baicalensis feed is increased.

2. Change of effective components before and after fermentation of Galla chinensis

A, gallic acid determination chromatographic conditions: asahi XB-C18 (250 mm. Times.4.6 mm,5 μm) column was used, and methanol (A) -0.1% phosphoric acid solution (B) was used as a mobile phase, and the mobile phase was eluted at the same ratio. Methanol (a) -0.1% phosphoric acid solution (B) =15:85. the detection wavelength is 270nm, the flow rate is 0.8mL/min, and the sample injection amount is as follows: 10 μ L.

B, preparation of standard solution

Accurately weighing gallic acid standard substance 20mg and 60% methanol, dissolving to constant volume of 10mL, using the solution as a standard substance mother solution, and placing the solution in a refrigerator at 4 ℃ for later use after the preparation is finished.

C, preparation of Chinese medicine extract

Weighing 1g of the scutellaria baicalensis fermented feed additive and the gallnut fermented feed additive into a 50mL conical flask, and adding 10mL of 60% methanol solution for leaching for 30min. Filtering to obtain supernatant, and storing in a refrigerator at 4 deg.C.

The standard curves of baicalein, baicalin, quercetin and gallic acid are shown in Table 4.

TABLE 4 Standard Curve of the ingredients of drug action

The chromatogram of gallic acid before and after fermentation is shown in figure 2; the gallic acid content before fermentation is 1.20mg/g, and the gallic acid content after fermentation is increased to 5.78mg/g, which is 4.82 times of that before fermentation. The main component of the gallnut is tannin substances, such as tannic acid, gallic acid and the like, and the gallnut has strong broad-spectrum antibacterial effect. It is proved by research that tannic acid has the efficacies of antibiosis, antivirus, antioxidation and the like, but has the defect of having an anti-nutritional effect, can react with protein, metal ions and the like, and is not easy to be utilized by organisms. The gallic acid also has the effects of antibiosis, antivirus, anti-inflammation and antioxidation, and in addition, researches show that the gallic acid also has the functions of protecting the liver, protecting heart and cerebral vessels, reducing blood sugar, reducing blood fat, resisting tumors and the like, but reports that the gallic acid has no anti-nutritional effect and is more beneficial to the utilization of organisms. The experimental result shows that the gallic acid content of the gallnut is greatly improved and the tannic acid content is reduced after the gallnut is fermented. The tannase produced by microbial fermentation can hydrolyze tannin to produce gallic acid.

(3) Screening of fermentation Strain

Screening of fermentation strains: different fermentation strains act on the compound traditional Chinese medicine to produce different effects on the compound traditional Chinese medicine. Therefore, it is necessary to screen the fermentation strains to effectively improve the effective components of the traditional Chinese medicine.

Taking compound traditional Chinese medicine, bean pulp and wheat middling as fermentation raw materials;

taking the culture solution of each single strain to ferment the compound traditional Chinese medicine feed additive, adding the culture solution into a fermentation raw material with the addition of 10% (V/W, mL/g), adding distilled water to control the water content to be 50% (V/W, mL/g), uniformly stirring, placing the mixture into a fermentation bag with one-way air holes, placing the mixture into an incubator at 30 ℃ for anaerobic fermentation, and fermenting for 7 days to obtain different single-strain compound fermented traditional Chinese medicine feed additives; detecting the change of the effective components of the medicine, and measuring the total polyphenol content of the effective components of the medicine by adopting a UV method.

Wherein the fermentation strain is Saccharomyces cerevisiae, candida utilis, candida tropicalis, bacillus subtilis, bacillus licheniformis, bacillus coagulans, bacillus amyloliquefaciens, bacillus natto, lactobacillus casei, lactobacillus paracasei, lactobacillus rhamnosus, lactobacillus fermentum, lactobacillus bifidus, and Lactobacillus plantarum

The fermentation raw materials comprise (by mass percent): 50% of compound traditional Chinese medicine (coptis chinensis, schisandra chinensis, rhizoma anemarrhenae, gallnut, rheum officinale, scutellaria baicalensis, liquorice, myrobalan and catechu which are mixed according to equal mass proportion), 30% of soybean meal and 20% of wheat middling.

The total phenol determination method comprises the following steps:

1. preparation of Total phenol Standard Curve

Accurately weighing 10mg (to 0.1 mg) of gallic acid standard, dissolving with distilled water to constant volume of 50mL, wherein the gallic acid content in the solution is 200mg/L, and placing in a refrigerator at 4 deg.C in dark place.

Accurately measuring 0.0,0.2,0.4,0.6,1.0 and 1.5mL of standard stock solution, respectively adding into a 10mL volumetric flask, and carrying out constant volume with 60% methanol solution to obtain gallic acid solutions with different concentrations. Then transferring 1.0mL of gallic acid with different concentrations into a 20mL test tube with a plug, and adding 2.5mL of 0.1mol/L Formica FuscaMixing with the linphenol reagent, adding 2.5mL of 20% Na ₂ CO ₃ Adding water to the solution until the volume is up to 10mL, and uniformly mixing. Water bath at 35 deg.C for 40min, taking out, cooling at room temperature, and standing in dark for 20min. Preparing standard concentration solution with concentration of 0, 4, 8, 12, 20, 30mg/L, measuring absorbance value at 765nm, and drawing standard curve with concentration as abscissa and absorbance as ordinate.

2. Determination of total phenols in samples

Sucking 1.0mL of leaching liquor of compound fermentation traditional Chinese medicine feed additive sample of different single bacteria in a 20mL colorimetric tube, shaking up 2.5mL of 0.1mol/L Folin phenol reagent, adding 2.5mL of 20% Na ₂ CO ₃ Adding water to the solution until the volume reaches 10mL, and shaking up. Water bath at 35 deg.C for 40min, taking out, cooling at room temperature, standing in dark place for 20min, and measuring its absorbance at 765 nm. And calculating the concentration of total polyphenol in the solution to be detected according to the standard curve.

The standard curve of total phenol is shown in figure 3, and the measurement results of total phenol after fermentation of the traditional Chinese medicine feed additive by single-bacterium compound fermentation are shown in figure 4; the determination result of the total phenol can observe that the content of the total phenol can be improved to a certain extent by single-strain fermentation of different strains. Among yeasts, candida tropicalis can increase the total phenol content in the traditional Chinese medicine to 142.25% before fermentation, and the total phenol content is the highest in the yeasts. In bacillus, bacillus amyloliquefaciens has the strongest capacity of improving total phenols, and can improve the total phenols in the traditional Chinese medicine to 139.29 percent before fermentation. Among lactobacillus, lactobacillus casei, lactobacillus plantarum and lactobacillus rhamnosus have good effects, and can increase the total phenols in the traditional Chinese medicine to be more than 135.82% before fermentation. Therefore, candida tropicalis, bacillus amyloliquefaciens, lactobacillus casei, lactobacillus plantarum and Lactobacillus rhamnosus are selected as fermentation strains.

(4) Determination of optimum Chinese medicine adding proportion

The results of bacteriostatic experiments show that the traditional Chinese medicine selected by the formula has broad-spectrum bacteriostatic effect and may have certain influence on the fermentation strain, so that a proper traditional Chinese medicine adding proportion needs to be determined, so that the traditional Chinese medicine can exert the bacteriostatic effect of the bacteriostatic traditional Chinese medicine and can also enable the fermentation strain to grow.

The invention changes the adding proportion of different compound traditional Chinese medicinal materials in fermentation. The PMA-qPCR detection method is used for measuring the number of viable bacteria of fermentation strains in different Chinese medicine addition ratio groups in the fermentation process, researching the relationship between the compound Chinese medicine and the fermentation strains, and searching for a group of Chinese medicine addition ratios which do not influence the growth of the fermentation strains and can play the antibacterial effect of the Chinese medicines.

Mixing Coptidis rhizoma, fructus Schisandrae, rhizoma anemarrhenae, galla chinensis, radix et rhizoma Rhei, scutellariae radix, glycyrrhrizae radix, fructus Chebulae, and Catechu at equal mass ratio to obtain compound Chinese medicinal premix. Mixing soybean meal secondary powder according to the mass ratio of 6 to serve as an auxiliary premix, designing seven groups of experiments, and taking the mixture of the compound traditional Chinese medicine and the auxiliary premix as a fermentation raw material, wherein the mass sum of the compound traditional Chinese medicine and the auxiliary premix is 100%, and the adding mass ratios of the compound traditional Chinese medicine premix are respectively 0%, 10%, 30%, 50%, 70%, 90% and 100%; the zymophyte is added in the form of bacterial liquid, the adding amount accounts for 2 percent (V/W, mL/g) of the fermentation raw material, the water content is 50 percent (V/W, mL/g), and the fermentation temperature is 30 ℃; the change of flora in the fermentation process is determined by a PMA-qPCR method.

Variation of bacterial quantity of bacillus amyloliquefaciens under different traditional Chinese medicine adding proportions

The bacterial load of Bacillus amyloliquefaciens in the fermentation process is shown in FIG. 5. The bacterial load of 0 percent and 10 percent of bacillus amyloliquefaciens in the traditional Chinese medicine adding group is observed to be at an extremely low level (only 10 percent) ² 、10 ³ Order of magnitude) and does not exhibit the phenomenon of increased reproduction. The bacterial amount is increased with the increase of Chinese medicinal materials, wherein 50%, 70%, 90%, 100% of the Chinese medicinal materials are added at 7 days of fermentation and still maintain a higher bacterial amount level of 1.53 × 10 ⁵ 、1.91*10 ⁵ 、1.46*10 ⁵ 、1.30*10 ⁵ copies/g. About 200 times of the amount of 0% Chinese medicinal materials.

The experimental results show that the compound traditional Chinese medicine of the formula is beneficial to the growth of the bacillus amyloliquefaciens to a certain extent. When the addition amount of the traditional Chinese medicine is less than 70%, the gain effect is more obvious when the addition amount of the traditional Chinese medicine is larger. However, if the amount exceeds 70%, the gain effect is reduced.

II, change of bacterial quantity of candida tropicalis under different traditional Chinese medicine adding proportions

The bacterial load change of Candida tropicalis in the fermentation process under different Chinese medicine addition ratios is shown in FIG. 6. The experimental result shows that the bacterial amount of the candida tropicalis increases along with the increase of the adding amount of the traditional Chinese medicine when the adding amount of the traditional Chinese medicine is 0-50% on the 1 st day of fermentation. On the 1 st day of fermentation, when the Chinese medicinal material is added in 50%, the yeast amount is 6.22 x 10 ⁸ The dosage of the copies/g is 3.68 times of the bacterial dosage of 0 percent of the traditional Chinese medicine. But the traditional Chinese medicine has certain inhibition effect on the growth of yeast when the adding amount of the traditional Chinese medicine is too high (90 percent and 100 percent), and the bacterial amount of the 100 percent traditional Chinese medicine adding group is 6.83 x 10 on the 1 st day of fermentation ⁷ The copes/g is only 10.98 percent of the traditional Chinese medicine addition group with 50 percent. But at the 7 th day of fermentation, the high concentration of the traditional Chinese medicine added with the Candida tropicalis still maintains a higher bacterial load, and the bacterial loads of 70 percent, 90 percent and 100 percent of the traditional Chinese medicine added with the Candida tropicalis are respectively 2.09 x 10 ⁸ 、4.32*10 ⁸ 、2.07*10 ⁸ copies/g。

The experimental result shows that the addition of the traditional Chinese medicine is beneficial to the growth of the candida tropicalis to a certain extent.

III, the change of the bacterial quantity of the lactobacillus casei under different traditional Chinese medicine adding proportions

The bacterial load of lactobacillus casei during fermentation varies with the ratio of Chinese medicinal materials, as shown in FIG. 7. On the 1 st day of fermentation, the addition of low-concentration traditional Chinese medicines can effectively increase the bacterial load of lactobacillus fermentum, but the addition of high-concentration traditional Chinese medicines can inhibit the growth of lactobacillus fermentum. For example, the lactobacillus casei amount of the group added with 10 percent of traditional Chinese medicines is 1.01 x 10 ⁹ The copies/g is 6.21 x 10 of the group bacteria amount of 100 percent of the traditional Chinese medicine adding amount ⁶ 162.64 times copies/g. On the 7 th day of fermentation, the lactobacillus casei bacterial amount of 0-50% of the traditional Chinese medicine added group can still be maintained at 10 ⁸ However, the group of 100% Chinese medicine addition is only 10 ⁴ An order of magnitude.

IV, the bacterial quantity of the lactobacillus plantarum is changed under different traditional Chinese medicine adding proportions

In the fermentation process of lactobacillus plantarum under different traditional Chinese medicine adding proportionsThe bacterial load was varied as shown in FIG. 8. The experimental result shows that the lactobacillus plantarum bacterial count of 0% traditional Chinese medicine addition group is the maximum and is 1.85 x 10 on the 1 st day of fermentation ⁹ The amount of copies/g, other groups of bacteria was reduced, but still 10 ⁸ A quantity level. It is demonstrated that the traditional Chinese medicine has weak inhibition effect on the growth of the lactobacillus plantarum during the initial fermentation stage. However, on the 7 th day of fermentation, the experimental results show that the addition of a certain amount of traditional Chinese medicine (10%, 30%, 50%, 70%) can maintain the bacterial amount at 10 ⁸ A quantity level. The bacterial count of the group without the traditional Chinese medicine is only 1.90 x 10 ⁷ . The addition of a certain amount of traditional Chinese medicine is beneficial to the growth of the lactobacillus plantarum. However, when the amount of the traditional Chinese medicine is too high, the growth of the lactobacillus plantarum is inhibited, and when the amount of the traditional Chinese medicine is 100%, the bacterial load of the lactobacillus plantarum is only 1.84 x 10 on the 1 st day of fermentation ⁶ The dosage of the drugs is only 0.10 percent of the group of 0 percent of the traditional Chinese medicine.

V. change of bacterial amount of lactobacillus rhamnosus under different traditional Chinese medicine adding proportion

The bacterial load of lactobacillus rhamnosus during fermentation varies according to the ratio of the traditional Chinese medicines, as shown in fig. 9. On the 1 st day of fermentation, when the addition amount of the traditional Chinese medicine is 30%, the quantity of the lactobacillus rhamnosus is the maximum and is 6.20 x 10 ⁶ The copies/g is 0% of the Chinese medicinal material added with the group bacteria amount of 4.01 x 10 ⁵ 15.46 times copies/g. When the addition amount of the traditional Chinese medicine is 100%, the bacterial amount is only 3.32 x 10 ³ The copies/g is only 0.05 percent of the group with 30 percent of traditional Chinese medicines. On the 7 th day of fermentation, the amount of 50% of the traditional Chinese medicines added with the lactobacillus rhamnosus of the group is the largest, namely 5.42 x 10 ⁴ The copies/g is 0% of the amount of the Chinese medicinal additive group bacteria 1.06 x 10 ⁴ 5.11 times of copies/g. The amount of the added group bacteria of 100 percent of the traditional Chinese medicine is only 2.84 x 10 ² The dosage of the copes/g is only 0.52 percent of the amount of the other bacteria in the 50 percent of the traditional Chinese medicine. The experimental result shows that the growth of the lactobacillus rhamnosus is beneficial to the addition of a small amount of traditional Chinese medicines, and the growth of the lactobacillus rhamnosus is unfavorable to the addition of a large amount of traditional Chinese medicines.

According to the bacterial quantity results of various bacteria in different traditional Chinese medicine adding proportions, the traditional Chinese medicine adding proportion of 50% is comprehensively considered and selected as the traditional Chinese medicine adding proportion for subsequent experiments. Under the condition of the addition proportion of the traditional Chinese medicine, each zymophyte has better growth, and the addition amount of the traditional Chinese medicine is not too low to influence the action effect of the drug effect components.

(5) Determination of the optimum enzyme addition

Because the compound traditional Chinese medicine is rich in lignocellulose components and tannase components, the lignocellulose and the tannase are selected and added as enzymes for fermentation. The change of total polyphenol before and after fermentation is measured by changing the addition amount of different enzymes, and the optimal addition amount of the enzymes is determined. Mixing Coptidis rhizoma, fructus Schisandrae, rhizoma anemarrhenae, galla chinensis, radix et rhizoma Rhei, scutellariae radix, glycyrrhrizae radix, fructus Chebulae, and Catechu at equal ratio to obtain compound Chinese medicinal premix. Mixing soybean meal secondary powder according to a certain proportion (60% of soybean meal and 40% of secondary powder) to obtain an auxiliary premix; then taking the mixture of the compound traditional Chinese medicine and the auxiliary premix as a fermentation raw material, wherein the mass sum of the two is 100 percent,

the compound enzyme consists of cellulase, hemicellulase and tannase in a mass ratio of 2;

the results of the experiment are shown in FIG. 10. The results show that the total phenol content in the compound fermentation traditional Chinese medicine feed additive is continuously increased along with the addition of the compound enzyme, and the increase of the total phenol content tends to be gentle when the total phenol content is more than 1 percent. The reason for this is that the addition of enzyme can help to hydrolyze the plant cell wall components, so that intracellular polyphenols are more easily released out of cells. When the enzyme addition amount is 8%, the total phenol tends to be slightly lower than those in the first addition amounts. The reason for this may be that the amount of substrate is constant and the amount of enzyme has reached saturation, thus giving less significant gain in total phenol increase. And the addition amount of the enzyme is too high, so that the proportion of the traditional Chinese medicine in each gram of the fermented traditional Chinese medicine additive is reduced, and the total phenol content in each gram of the fermented traditional Chinese medicine additive is reduced.

The total phenol content was 30.42mg/g, although the maximum was found when the amount of enzyme added was 6%. However, considering that the total phenol content of the 1% enzyme-added group was not much different from that of the 6% group, it was 29.33mg/g. The 1% addition was chosen for economic reasons.

The invention has the beneficial effects that:

(1) The invention screens three main bacteriostatic traditional Chinese medicines of intestinal conditional pathogenic bacteria (salmonella enteritidis, escherichia coli and shigella flexneri), obtains a group of compound traditional Chinese medicines with strong inhibition capacity on common intestinal pathogenic bacteria, and ferments the compound traditional Chinese medicines. And creative research is carried out on the addition proportion of the traditional Chinese medicine and the auxiliary materials and the growth relation of the fermentation strain aiming at the fermentation strain and the enzyme, so as to obtain the optimal addition proportion of the traditional Chinese medicine. Finally, the prepared compound fermented traditional Chinese medicine feed additive is used for animal breeding (such as Chinese soft-shelled turtles), and experimental results show that the compound fermented traditional Chinese medicine feed additive can effectively improve the immune function, the intestinal digestion level and the like of the Chinese soft-shelled turtles when being fed for a long time, and can effectively improve the survival rate of young Chinese soft-shelled turtles in short-term pathogenic bacterium challenge experiments; and has excellent prevention and treatment effects on common intestinal pathogenic bacteria such as salmonella enteritidis, escherichia coli and shigella flexneri.

(2) According to the invention, chinese medicinal materials with good bacteriostatic effects on common enteropathogenic bacteria such as salmonella enteritidis, escherichia coli and shigella flexneri are obtained by screening bacteriostatic experiments; the compound traditional Chinese medicinal materials are fermented, so that the active ingredients of the compound traditional Chinese medicinal materials are improved or converted into other easily-absorbed active ingredients, and the feed additive is rich in probiotics, can effectively improve the immunity of cultured animals, has sour and fragrant smell and a certain bacteriostatic action, and is convenient to store.

Drawings

FIG. 1 is a chromatogram before and after fermentation of baicalein, baicalin, quercetin and Scutellariae radix; wherein, the standard peak of baicalin (a), the standard peak of baicalein (b), the standard peak of quercetin (c), the chromatogram before the fermentation of scutellaria baicalensis (d) and the chromatogram after the fermentation of scutellaria baicalensis (e) are obtained;

FIG. 2 is a chromatogram of gallic acid and before and after fermentation; wherein, the gallic acid standard peak (a), the Chinese gall standard peak before fermentation and the Chinese gall standard peak after fermentation are respectively obtained;

FIG. 3 is a standard graph of total phenol;

FIG. 4 is a graph of total phenol content after fermentation of Chinese medicine by different strains;

FIG. 5 is a graph showing the variation of the bacterial load of Bacillus amyloliquefaciens at different ratios of Chinese medicinal materials during fermentation;

FIG. 6 is a graph showing the variation of Candida tropicalis bacteria amount at different Chinese medicinal material addition ratios during fermentation;

FIG. 7 is a graph showing the variation of the bacterial count of Lactobacillus casei at different ratios of Chinese medicinal materials during fermentation;

FIG. 8 is a graph showing the variation of the bacterial load of Lactobacillus plantarum in the case of different ratios of Chinese herbal materials added during fermentation;

FIG. 9 is a graph showing the variation of the amount of Lactobacillus rhamnosus bacteria at different ratios of the traditional Chinese medicine during fermentation;

FIG. 10 shows the effect of different complex enzyme additions on total phenols in a compound fermented traditional Chinese medicine feed additive;

FIG. 11 is a paraffin section of the intestinal tract of Chinese softshell turtle; wherein (a) is a fermented traditional Chinese medicine; (b) is a Chinese medicinal group; and (c) is a complete powder group.

Detailed description of the preferred embodiment

The invention is further described with reference to specific examples.

Wherein the laccase is obtained from Soochow Biotech limited of Shandong, and has enzyme activity of 10000U/g. The acid cellulase, the xylanase, the mannanase and the tannase are purchased from Xiasang (Beijing) Biotechnology development limited company, and the enzyme activities are 10000U/g. The beta-glucanase is purchased from Xiasang (Beijing) Biotechnology development Limited, and the enzyme activity is 50000U/g.

The strain source is as follows: i, bacillus: bacillus amyloliquefaciens (Bacillus amyloliquefaciens); II, yeasts: candida tropicalis (Candida tropicalis); III, lactic acid bacteria: lactobacillus casei (Lactobacillus casei) ATCC11578, lactobacillus rhamnosus (Lactobacillus rhamnosus) CGMCC1.577 and Lactobacillus plantarum (Lactobacillus plantarum).

Example 1:

(1) Activating and culturing bacillus, yeast and lactic acid bacteria:

A. firstly, preparing a culture medium of bacillus, saccharomycetes and lactic acid bacteria;

bacillus liquid and solid media: 10g of tryptone, 10g of sodium chloride, 5g of yeast powder and 1L of distilled water, wherein the pH is natural, the mixture is sterilized for 20min at 121 ℃, and the components of a bacillus solid culture medium are the same as those of a liquid culture medium; except that 15g of agar was added.

Yeast liquid and solid media: boiling 200g of potatoes, softening, filtering with 8 layers of gauze, filtering with 20g of glucose, 3g of monopotassium phosphate, 1.5g of magnesium sulfate, diluting with distilled water to a volume of 1L, keeping the pH natural, and sterilizing at 121 ℃ for 20min; the yeast solid culture medium has the same components as the liquid culture medium, and is characterized in that 15g of agar is added;

lactic acid bacteria liquid and solid medium: 10g of beef extract, 10g of tryptone, 20g of glucose, 5g of yeast powder, 2g of dipotassium phosphate, 2g of diammonium hydrogen citrate, 5g of anhydrous sodium acetate, 1mL of Tween 80, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 1L of distilled water, pH7.2, and sterilization at 121 ℃ for 20min; the lactobacillus solid culture medium has the same components as the liquid culture medium, and is characterized in that 15g of agar is added;

B. the activation culture method of bacillus, yeast and lactic acid bacteria comprises the following steps:

culturing bacillus liquid strain: inoculating glycerol bacteria stored at-80 ℃ to a bacillus solid culture medium under the aseptic condition, culturing for 18h at 30 ℃ to recover strains, selecting a single colony, inoculating the single colony to a shake flask (250 mL) filled with 100mL of bacillus liquid culture medium, and culturing for 12h at 30 ℃ at 180r/min to obtain bacillus fermentation seed liquid; inoculating the strain into a liquid culture medium according to the actual requirement, wherein the inoculation amount of the strain is 2% (v/v, namely 2mL of fermentation seed liquid is inoculated into every 100mL of liquid culture medium) for amplification culture; the bacillus is bacillus amyloliquefaciens.

Culturing a yeast liquid strain: inoculating glycerol strain stored at-80 deg.C to yeast solid culture medium under aseptic condition, culturing at 28 deg.C for 20 hr to recover strain, selecting single colony, inoculating to shake flask (250 mL) containing 100mL yeast liquid culture medium, culturing at 28 deg.C for 12 hr at 180r/min to obtain yeast fermentation seed solution; inoculating the strain into a liquid culture medium according to the actual requirement and the inoculation amount of 2 percent for amplification culture; the yeast is candida tropicalis.

Culturing a lactobacillus liquid strain: respectively inoculating glycerol bacteria stored at-80 ℃ to a lactic acid bacteria solid culture medium under aseptic conditions, culturing at 37 ℃ for 24h to recover the strains, selecting a single colony, inoculating to an anaerobic bottle (50 mL) filled with 50mL of lactic acid bacteria liquid culture medium, standing at 37 ℃ for 18h to obtain lactic acid bacteria fermentation seed liquid, and inoculating to the liquid culture medium according to actual needs in an inoculation amount of 2% for amplification culture; the lactobacillus is Lactobacillus plantarum CGMCC1.557, lactobacillus casei ATCC11578 and Lactobacillus rhamnosus CGMCC1.577.

(2) Preparing an anaerobic fermentation culture medium, wherein the anaerobic fermentation culture medium comprises the following components in percentage by weight: 50% of compound traditional Chinese medicine (coptis chinensis, schisandra chinensis, rhizoma anemarrhenae, gallnut, rheum officinale, scutellaria baicalensis, liquorice, myrobalan and catechu which are mixed according to equal mass proportion), 30% of soybean meal, 20% of wheat middling and 1% of complex enzyme are uniformly mixed to obtain the anaerobic fermentation culture medium.

(3) Inoculating the activated and cultured bacillus amyloliquefaciens fermented seed liquid, the candida tropicalis fermented seed liquid, the lactobacillus plantarum fermented seed liquid, the lactobacillus casei fermented seed liquid and the lactobacillus rhamnosus fermented seed liquid in the step (1) into a fermentation culture medium, wherein the total inoculation amount is 2% of the weight of the fermentation culture medium, the adding proportion of each seed liquid is (lactobacillus: bacillus: yeast =2 ⁸ ～3.5×10 ⁹ cfu/mL; the total number of viable bacteria of the bacillus and the microzyme is 1.0 multiplied by 10 ⁷ ～2.8×10 ⁹ cfu/mL; the yeast fermentation seed liquid is candida tropicalis fermentation seed liquid. The total number of viable bacteria in the seed liquid of lactobacillus fermentation is 1.5 × 10 ⁸ ～3.5×10 ⁹ cfu/mL. Uniformly mixing, and adjusting the water content to 45% to obtain an anaerobic fermentation mixture; placing in a fermentation bag, and performing anaerobic fermentation at 30 deg.C for 7 days to obtain the final product, which is compound fermented Chinese medicinal feed additive, and the detection results of each index of the final product are shown in Table 5.

Table 5 shows the results of the measurements of the respective indexes of the fermented product of example 1

Detecting items	Content (c) of
		Total phenol content	30.83mg/g
Total flavone content	10.32mg/g
		pH	4.51
Viable count of Bacillus amyloliquefaciens	1.53×10 ⁵ cfu/g
		Viable count of Candida tropicalis	6.19×10 ⁷ cfu/g
Viable count of Lactobacillus casei	1.91×10 ⁸ cfu/g
		Viable count of Lactobacillus plantarum	2.50×10 ⁸ cfu/g
Viable count of Lactobacillus rhamnosus	5.42×10 ⁴ cfu/g

Example 2:

(1) The specific operation of activated culture of bacillus, yeast and lactic acid bacteria is the same as that of example 1;

(2) Preparing an anaerobic fermentation culture medium, wherein the anaerobic fermentation culture medium comprises the following components in percentage by weight: 50% of compound traditional Chinese medicine (coptis chinensis, schisandra chinensis, rhizoma anemarrhenae, gallnut, rheum officinale, scutellaria baicalensis, liquorice, myrobalan and catechu which are mixed according to equal proportion), 30% of soybean meal, 20% of wheat middling and 0% of complex enzyme are uniformly mixed to obtain the anaerobic fermentation culture medium.

(3) The other fermentation conditions were the same as in example 1, and the final fermentation product was obtained, and the results of each index are shown in Table 6.

Table 6 shows the results of the measurements of the respective indexes of the fermentation product of example 2

Detecting items	Content (wt.)
		Total phenol content	28.55mg/g
Total flavone content	9.55mg/g
		pH	4.61
Viable count of Bacillus amyloliquefaciens	1.54×10 ⁵ cfu/g
		Viable count of Candida tropicalis	6.21×10 ⁷ cfu/g
Viable count of Lactobacillus casei	1.90×10 ⁸ cfu/g
		Viable count of Lactobacillus plantarum	2.47×10 ⁸ cfu/g
Viable count of Lactobacillus rhamnosus	5.46×10 ⁴ cfu/g

Example 3:

(2) Preparing an anaerobic fermentation culture medium, wherein the anaerobic fermentation culture medium comprises the following components in percentage by weight: 50% of compound traditional Chinese medicine (coptis chinensis, schisandra chinensis, rhizoma anemarrhenae, gallnut, rheum officinale, scutellaria baicalensis, liquorice, myrobalan and catechu which are mixed according to equal proportion), 30% of soybean meal, 20% of wheat middling and 6% of complex enzyme are uniformly mixed to obtain the anaerobic fermentation culture medium.

(3) The other fermentation conditions were the same as in example 1, and the results of measurement of each index of the fermented product are shown in Table 7.

Table 7 shows the results of each measurement index of the fermented product of example 3

Detecting items	Content (wt.)
		Total phenol content	33.45mg/g
Total flavone content	13.44mg/g
		pH	4.22
Viable count of Bacillus amyloliquefaciens	1.58×10 ⁵ cfu/g
		Viable count of Candida tropicalis	6.44×10 ⁷ cfu/g
Viable count of Lactobacillus casei	1.94×10 ⁸ cfu/g
		Viable count of Lactobacillus plantarum	2.54×10 ⁸ cfu/g
Viable count of Lactobacillus rhamnosus	5.66×10 ⁴ cfu/g

Example 4:

(2) Preparing an anaerobic fermentation culture medium, wherein the anaerobic fermentation culture medium comprises the following components in percentage by weight: 30% of compound traditional Chinese medicine (coptis chinensis, schisandra chinensis, rhizoma anemarrhenae, gallnut, rheum officinale, scutellaria baicalensis, liquorice, myrobalan and catechu which are mixed according to equal proportion), 46% of soybean meal, 24% of wheat middling and 1% of complex enzyme are uniformly mixed to obtain the anaerobic fermentation culture medium.

(3) The other fermentation conditions were the same as in example 1, and the results of measuring various indexes of the fermented product are shown in Table 8.

Table 8 shows the results of each measurement index of the fermented product of example 4

Example 5:

(2) Preparing an anaerobic fermentation culture medium, wherein the anaerobic fermentation culture medium comprises the following components in percentage by weight: 70% of compound traditional Chinese medicine (coptis chinensis, schisandra chinensis, rhizoma anemarrhenae, gallnut, rheum officinale, scutellaria baicalensis, liquorice, myrobalan and catechu are mixed according to equal proportion), 16% of soybean meal, 14% of wheat middling and 1% of complex enzyme are uniformly mixed to obtain the anaerobic fermentation medium.

(3) The other fermentation conditions were the same as in example 1, and the results of measurement of each index of the fermented product are shown in Table 9.

Table 9 shows the results of each measurement index of the fermented product of example 5

Detecting items	Content (c) of
		Total phenol content	42.33mg/g
Total flavone content	14.55mg/g
		pH	4.67
Viable count of Bacillus amyloliquefaciens	1.91×10 ⁵ cfu/g
		Viable count of Candida tropicalis	2.09×10 ⁸ cfu/g
Viable count of Lactobacillus casei	1.14×10 ⁷ cfu/g
		Viable count of Lactobacillus plantarum	2.23×10 ⁸ cfu/g
Viable count of Lactobacillus rhamnosus	3.39×10 ⁴ cfu/g

Example 6: the effect experiment of long-term feeding of the product is carried out;

the compound fermented traditional Chinese medicine feed additive prepared in the embodiment 1 of the invention is added into common aquatic feed to obtain mixed feed for feeding special aquatic animals, and the addition amount is 5-25% (by weight) of the obtained mixed feed.

The method comprises the steps of selecting a soft-shelled turtle breeding test field of a sparrezhendi fly ecological cycle agricultural base in Danyang city of Jiangsu province for experiment, wherein the length of a breeding pond is 1.57m, the width of the breeding pond is 1.22m, the height of the breeding pond is 0.72m, and the water depth is adjusted to be 0.5m. Three parallel groups are arranged in each group, and 20 Chinese soft-shelled turtles with similar sizes and weights are cultured in each parallel group for 10 months from 2021 to 1 month from 2022, the pre-experiment lasts for 15 days, and the formal experiment lasts for 60 days. The average body mass (200 +/-15 g of the average body mass) of healthy Chinese soft-shelled turtles which are half of the female body mass and half of the male body mass are used for the test, the Chinese soft-shelled turtles are randomly divided into three groups, corresponding feeds are respectively a fermented traditional Chinese medicine group, a traditional Chinese medicine group and a complete powder group, the formula of the Chinese soft-shelled turtle feed is shown in a table 11, and the components of the complete powder are shown in a table 10.

Wherein: fermenting the traditional Chinese medicine: 10% of fermented compound traditional Chinese medicine feed additive and 90% of complete powder;

the traditional Chinese medicine comprises: the compound traditional Chinese medicine feed additive is a mixture which is not fermented after the compound traditional Chinese medicine raw materials, the soybean meal and the wheat middling are mixed in the embodiment 1; 10% of unfermented mixture and 90% of full-value powder;

the complete powder group is 100 percent of complete powder;

the fermented traditional Chinese medicine, the traditional Chinese medicine and the complete powder group are respectively fed with soft-mass feed prepared according to the formula shown in the table 11 before the beginning of the test for domestication for 2 weeks until the feed of the group is completely ingested, and the daily feeding amount of the feed is 2 percent of the weight of the Chinese softshell turtle. Daily 11:00-14: 00. 19:00-22:00 feeding soft mass feed of each group, wherein the daily feeding amount of the feed is 2 percent of the weight of the Chinese soft-shelled turtle, and cleaning residual feed by adopting a siphon after feeding for 1 hour. During the culture period, the pH value of the water body is between 7 and 8.5, the dissolved oxygen is 3 to 5mg/L, the ammonia nitrogen is less than 2mg/L, and the water temperature of the water body is 30 ℃.

TABLE 10 complete powder compositions

TABLE 11 Chinese softshell turtle feed formula

The feed coefficient of the trionyx sinensis is shown in table 12. The experimental result shows that the feed coefficient of the trionyx sinensis in the fermented traditional Chinese medicine group and the feed coefficient of the complete powder group have no significant difference, and the feed coefficient of the trionyx sinensis in the traditional Chinese medicine group is higher than that of the other two groups. The feed coefficient of the traditional Chinese medicine group is highest probably because the traditional Chinese medicinal materials are mainly lignocellulose components, and the traditional Chinese medicinal materials which are not fermented by bacterial enzymes are difficult to be absorbed and utilized by the bodies of the soft-shelled turtle. After the compound traditional Chinese medicine is fermented, the effective components and the nutrient components in the compound traditional Chinese medicine are more beneficial to the absorption of the body of the turtle, for example, the lignocellulose components in the compound traditional Chinese medicine are degraded into carbohydrate substances by bacterial enzymes, and crude protein is degraded into small peptide and the like. Secondly, fermentation strains in the fermented traditional Chinese medicine can enter the intestinal tract of the organism for field planting along with the intake of the Chinese soft-shelled turtle so as to improve the digestion and absorption functions of the intestinal tract.

TABLE 12 feed coefficients of Chinese softshell turtles

Note 1: feed factor = feed consumption/weight gain × 100%

Note 2: the data in the same row are labeled with the same letter to indicate that the difference is not significant (P > 0.05), and no label to indicate that the difference is significant (P < 0.05).

The intestinal section of Trionyx sinensis Wiegmann is shown in figure 11. Villus height, crypt depth, villus height/crypt depth are shown in table 12.

The small intestine is an important part for absorbing nutrients, and the villus height, the crypt depth and the ratio of the villus height to the crypt depth of the small intestine are important indexes for measuring the digestion and absorption functions of organisms. The longer the villi of the small intestine, the larger the ratio of the height of the villi of the small intestine to the depth of the crypt, the stronger the digestive absorption function of the intestinal tract. The ratio of the length of small intestine villi, the height of small intestine villi and the depth of crypt of the Chinese soft shell turtle in the fermented traditional Chinese medicine group is larger than that of the other two groups, and the biological feed group is larger than the complete powder group.

TABLE 13 influence of compound fermented Chinese medicinal feed additive on intestinal tract of Chinese soft-shelled turtle

Note: the data in the same row are labeled with the same letter to indicate that the difference is not significant (P > 0.05), and no label to indicate that the difference is significant (P < 0.05).

The blood biochemical indexes of the fermented traditional Chinese medicine, the traditional Chinese medicine and the complete powder group are shown in a table 14. Alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) are two important indicators for assessing whether liver function is normal. When the liver is damaged, the permeability of cell membranes is increased and transaminase activity is increased. In the three groups, ALT and AST are fermented traditional Chinese medicines which are larger than full-price powder materials. Experiments show that the traditional Chinese medicine has a certain protection effect on the liver function of the Chinese softshell turtles, and the liver protection function of the Chinese softshell turtles is obviously enhanced after fermentation. The contents of UREA (UREA), creatinine (CREA) and Uric Acid (UA) are related to the protein metabolism of the kidney function of trionyx sinensis. Once kidney function is reduced or lost, various endogenous chemical components such as UREA and UA are accumulated in blood, and hyperuricemia is caused. The test result shows that the UREA and UA of the fermented traditional Chinese medicine group are smaller than those of the other two groups, the UREA in the traditional Chinese medicine group is smaller than that of the complete powder group, and the difference between the UA and the complete powder group is not great. It can be shown that the traditional Chinese medicine can effectively improve the kidney function of the turtle, and the turtle has a gain effect after being fermented. The contents of Triglyceride (TG), high Density Lipoprotein (HDL), and Low Density Lipoprotein (LDL) are correlated with lipid metabolism. The main functions of the triglyceride are storage and energy supply, and the experimental result shows that the content of the triglyceride in the serum of the experimental group and the control group is equivalent, but the HDL in the traditional Chinese medicine for fermentation is higher than that in the other two groups. The experiment group has high HDL content, which shows that the compound fermented traditional Chinese medicine feed has better gain effect in participating in cholesterol metabolism of the Chinese soft-shelled turtle.

TABLE 14 influence of Compound fermented feed on Biochemical index of Chinese Soft shelled turtle

Note: the data in the same row are marked with letters to indicate no significant difference (P > 0.05) and no mark to indicate significant difference (P < 0.05).

The serum antioxidant immunity indexes of the experimental group and the control group of the Chinese softshell turtles are shown in the table 15.

Lysozyme (LSM) in serum is an enzyme produced by monocytes, neutrophils and macrophages that lyses the bacterial cell wall. It is an important nonspecific defense factor and also one of the important marks of the nonspecific immune defense level of aquatic animals. The three group lysozyme enzyme activities are respectively a fermented traditional Chinese medicine group, a traditional Chinese medicine group and a complete powder group from high to low. To a certain extent, the compound traditional Chinese medicine can effectively improve the nonspecific immunity of the Chinese softshell turtles, and the nonspecific immunity function of the Chinese softshell turtles is stronger after fermentation.

The body of the trionyx sinensis contains a large amount of polyunsaturated fatty acids, which are easily oxidized, thereby causing diseases. Research shows that the antioxidant capacity is related to non-specific immunity, and the antioxidant capacity of the organism is improved, so that the immune function of the organism can be stimulated, and the immunity and the disease resistance of the organism are improved. Therefore, the immune function of the trionyx sinensis can be laterally observed by measuring the antioxidant capacity of the serum of the trionyx sinensis. The result of measuring the total antioxidant capacity (T-AOC) of the blood serum of the three groups of Chinese softshell turtles can be obtained, and the three groups of Chinese softshell turtles respectively comprise a fermentation traditional Chinese medicine group, a traditional Chinese medicine group and a complete powder group with high to low total antioxidant capacity. To a certain extent, the compound traditional Chinese medicine can effectively improve the oxidation resistance of the Chinese softshell turtle, and the improvement effect is stronger after fermentation.

Acid phosphatase (ACP) is a marker enzyme for lysosomal enzymes in macrophages, and studies have suggested that ACP activity is associated with the mechanism of body injury. The ACP enzyme activity difference among the three groups is not large, and the experimental result shows that the addition of the compound fermentation traditional Chinese medicine or the traditional Chinese medicine to the daily ration does not cause related damage to organisms.

Alkaline phosphatase (AKP) is a very important enzyme for Chinese softshell turtles. It is involved in bone dorsal concha formation and calcium and phosphorus metabolism. The three groups of AKP enzyme activities are respectively a complete powder group, a fermented traditional Chinese medicine group and a traditional Chinese medicine group from large to small. The possible reasons are that the fermented traditional Chinese medicine and 10 percent of the traditional Chinese medicine are fermented traditional Chinese medicine and traditional Chinese medicine, the main component of the compound traditional Chinese medicine is lignocellulose component, the supplement of protein is lacked, and 10 percent of fish protein is less than that of the complete powder group. Therefore, the calcium and phosphorus metabolism of the turtle can be influenced to a certain extent. However, compared with the AKP enzyme activity of fermented traditional Chinese medicines and traditional Chinese medicine groups, the AKP enzyme activity is improved after fermentation, so that the nutrition value of the compound traditional Chinese medicines can be effectively improved after fermentation, and the compound traditional Chinese medicines are beneficial to calcium and phosphorus metabolism.

TABLE 15 influence of compound fermented Chinese medicinal feed additive on antioxidant immunity index of Chinese soft-shelled turtle serum

The test results are combined, so that the intestinal function of the soft-shelled turtle can be effectively improved by adding the compound traditional Chinese medicinal materials during long-term feeding, the immunity, the oxidation resistance and the like of the soft-shelled turtle are improved, and the compound traditional Chinese medicinal materials have better effects on various functions of the soft-shelled turtle after being fermented.

Example 7: pathogenic bacteria challenge experiment 1

The compound fermented traditional Chinese medicine feed additive prepared by the invention is added into common aquatic feeds to obtain mixed feeds for feeding special aquatic animals, and the addition amount is 5-25% (by weight) of the obtained mixed feeds.

The formula of the compound fermented traditional Chinese medicine feed additive comprises: 50% of compound traditional Chinese medicine (coptis chinensis, schisandra chinensis, rhizoma anemarrhenae, gallnut, rheum officinale, scutellaria baicalensis, liquorice, myrobalan and catechu which are mixed according to equal proportion), 30% of soybean meal, 20% of wheat middling and 1% of complex enzyme.

The compound Chinese medicinal feed additive group has the same formula as the compound fermented Chinese medicinal feed additive, and is only different from the compound fermented Chinese medicinal feed additive in that no fermentation is carried out.

Selecting young Chinese soft-shelled turtles with half male and half female, healthy and active properties, wherein the average body weight is 10 +/-5 g. The feed is randomly divided into three groups, and the three groups of feed A, B and C with the same quantity are respectively fed, wherein A is 10% of compound fermented traditional Chinese medicine feed additive and 90% of complete powder group, B is 10% of compound traditional Chinese medicine feed additive group and 90% of complete powder group, and C is 100% of complete powder feed. Each group had 3 replicates of 30 trionyx sinensis each.

Salmonella enteritidis is added into daily ration at the beginning of the challenge test, and the average pathogenic bacteria intake of each young turtle is about 10 ⁸ CFU bacterial load, followed by normal feeding on different diets. The survival rates of the trionyx sinensis in each group are shown in table 16.

TABLE 16 Salmonella enteritidis challenge test

Note: the data in the same row are marked with letters to indicate that the difference is not significant (P > 0.05), and no mark indicates that the difference is significant (P < 0.05).

Example 8: pathogenic bacteria challenge experiment 2

Other conditions were the same as in example 7 except that the challenge species was changed to E.coli. Adding Escherichia coli into daily ration at the beginning of challenge test, wherein the average pathogenic bacteria intake of each young turtle is about 10 ⁸ CFU bacterial load, followed by normal feeding on different diets. The survival rates of the trionyx sinensis in each group are shown in table 17.

TABLE 17 Escherichia coli challenge test

Example 9: pathogenic bacteria challenge experiment 3

Other conditions were the same as example 7 except that the challenge strain was changed to Shigella flexneri. Shigella flexneri is added into daily ration at the beginning of the challenge experiment, and the average pathogenic bacteria intake of each young turtle is about 10 ⁸ CFU bacterial load, followed by normal feeding on different diets. The survival rates of the trionyx sinensis in each group are shown in table 18.

TABLE 18 toxin challenge test of Fushi bacteria

Example 10: pathogenic bacteria challenge experiment 4

Other conditions were the same as in example 7 except that the challenge species was changed. Adding salmonella enteritidis, escherichia coli and shigella flexneri mixed bacteria into daily ration at the beginning of the challenge test, wherein the average pathogenic bacteria intake of each young turtle is about 10 ⁸ CFU bacterial load, followed by normal feeding on different diets. The survival rates of the trionyx sinensis in each group are shown in table 19.

TABLE 19 Salmonella enteritidis, escherichia coli, shigella flexneri challenge test

Example 11: pathogenic bacteria challenge experiment 5

The other conditions are the same as the example 10, and the formula of the compound fermentation traditional Chinese medicine feed additive is changed as follows: 30% of compound traditional Chinese medicine (coptis chinensis, schisandra chinensis, rhizoma anemarrhenae, gallnut, rheum officinale, scutellaria baicalensis, liquorice, myrobalan and catechu are mixed according to equal proportion), 60% of soybean meal, 30% of wheat middling and 1% of complex enzyme. And (5) carrying out a toxicity attack experiment. The survival rates of the trionyx sinensis in each group are shown in table 20.

TABLE 20 Salmonella enteritidis, escherichia coli, shigella flexneri challenge test

Example 12: pathogenic bacteria challenge experiment 5

The other conditions are the same as the example 10, and the formula of the compound fermentation traditional Chinese medicine feed additive is changed as follows: 70% of compound traditional Chinese medicine (coptis chinensis, schisandra chinensis, rhizoma anemarrhenae, gallnut, rheum officinale, scutellaria baicalensis, liquorice, myrobalan and catechu which are mixed according to equal proportion), 16% of soybean meal, 14% of wheat middling and 1% of compound enzyme are subjected to toxicity counteracting experiment. The survival rates of the trionyx sinensis in each group are shown in table 21.

TABLE 21 Salmonella enteritidis, escherichia coli, shigella flexneri challenge test

Example 13: pathogenic bacteria challenge experiment 6

The other conditions are the same as the example 10, and the feeding mode of the toxicity attacking experiment is changed. Adding Salmonella enteritidis, escherichia coli and Shigella flexneri mixed bacteria into daily ration, wherein the average pathogenic bacteria intake of each young turtle is about 10 ⁸ The total intake of the bacteria of the CFU is 2.1 × 10 in average 7 days ⁹ CFU/only. The survival rates of the trionyx sinensis in each group are shown in table 22.

TABLE 22 Salmonella enteritidis, escherichia coli, shigella flexneri challenge test

Example 14: pathogenic bacteria attacking experiment 8

The other conditions are the same as those in the example 10, and only the enzyme addition amount in the preparation process of the compound fermented traditional Chinese medicine feed additive is changed, so that the enzyme addition amount is 0 percent. The survival rates of the trionyx sinensis in each group are shown in table 23.

TABLE 23 Salmonella enteritidis, escherichia coli, shigella flexneri challenge test

Example 15: pathogen attack experiment 9

The other conditions are the same as those in the example 10, and only the enzyme addition amount in the preparation process of the compound fermented traditional Chinese medicine feed additive is changed, so that the enzyme addition amount is 6 percent. The survival rates of the trionyx sinensis in each group are shown in table 24.

TABLE 24 Salmonella enteritidis, escherichia coli, shigella flexneri challenge experiment

Description of the invention: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; thus, while the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted; all such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and within the scope of the following claims.

Claims

1. A preparation method of a compound fermented traditional Chinese medicine feed additive for preventing and treating common intestinal pathogenic bacteria is characterized by comprising the following preparation steps:

the fermentation medium comprises the following components in percentage by weight:

30 to 70 percent of compound Chinese medicinal material;

18-46% of soybean meal;

12 to 24 percent of wheat middling; the addition amount of the complex enzyme is 0.1 to 10 percent;

the compound enzyme consists of cellulase, hemicellulase and tannase;

(2) Respectively inoculating bacillus, saccharomycetes and lactic acid bacteria to corresponding liquid culture media for activated culture to respectively obtain bacillus fermentation seed liquid, saccharomycetes fermentation seed liquid and lactic acid bacteria fermentation seed liquid;

(3) Inoculating the bacillus seed liquid, the saccharomycete seed liquid and the lactic acid bacteria seed liquid obtained in the step (2) into the fermentation culture medium obtained in the step (1), adjusting the fermentation temperature and the water content of the culture medium, uniformly mixing to obtain a fermentation mixture, and performing anaerobic fermentation under a closed condition to obtain the fermented traditional Chinese medicine feed additive for preventing and treating the intestinal pathogenic bacteria.

2. The preparation method of the compound fermented traditional Chinese medicine feed additive for preventing and treating the common intestinal pathogenic bacteria according to claim 1, wherein in the step (1), the soybean meal is dried and ground into powder for use when in use; the powder is obtained by sieving with a 50-80 mesh sieve; the compound traditional Chinese medicine is ground into powder for use, and the powder is obtained by sieving with a 50-mesh sieve.

The mass ratio of the cellulase to the hemicellulase to the tannase is 2; in the compound enzyme, the activity of hemicellulase is 5 ten thousand u/g, and the activities of cellulase and tannase are both 1 ten thousand u/g.

3. The preparation method of the compound fermented traditional Chinese medicine feed additive for preventing and treating the common intestinal pathogenic bacteria according to claim 1, wherein the fermentation medium comprises the following components in percentage by weight:

50% of compound traditional Chinese medicine;

30% of soybean meal;

20% of wheat middling;

the addition amount of the complex enzyme is 1 percent.

4. The preparation method of the compound fermented traditional Chinese medicine feed additive for preventing and treating common intestinal pathogenic bacteria according to claim 1, wherein the bacillus in the step (2) is bacillus amyloliquefaciens; the yeast is candida tropicalis; the lactobacillus is Lactobacillus plantarum, lactobacillus casei, or Lactobacillus rhamnosus.

5. The preparation method of the compound fermented traditional Chinese medicine feed additive for preventing and treating common intestinal pathogenic bacteria according to claim 1, wherein the relationship between the dosage of the bacillus fermented seed liquid and the yeast fermented seed liquid in the step (3) and the fermentation culture medium is 0.1-9 mL:100g of the total weight of the feed; the total number of viable bacteria of the bacillus and the microzyme is 1.0 multiplied by 10 ⁷ ～2.8×10 ⁹ cfu/mL；

The lactobacillus fermented seed liquid is lactobacillus plantarum fermented seed liquid, lactobacillus casei fermented seed liquid and lactobacillus rhamnosus fermented seed liquid, and the total number of viable bacteria is 1.5 × 10 ⁸ ～3.5×10 ⁹ cfu/mL, wherein the relation between the inoculation amount of the lactobacillus fermentation seed solution and the fermentation culture medium is 0.8-5.7 mL:100g of the total weight of the mixture; the volume ratio of the lactobacillus fermentation seed liquid to the bacillus fermentation seed liquid to the yeast fermentation seed liquid during inoculation is 2:2:3.

6. the preparation method of the compound fermented traditional Chinese medicine feed additive for preventing and treating common intestinal pathogenic bacteria according to claim 5, wherein the relationship between the dosage of the bacillus fermented seed liquid and the yeast fermented seed liquid during inoculation and the fermentation culture medium is 2mL:100g.

7. The preparation method of the compound fermented traditional Chinese medicine feed additive for preventing and treating the common intestinal pathogenic bacteria according to claim 1, wherein the water content of the fermented mixture in the step (3) is 40-60%; the fermentation time is 5-20 days, and the fermentation temperature is 24-38 ℃.

8. The compound fermented traditional Chinese medicine feed additive prepared according to the method of any one of claims 1 to 7.

9. The use of the compound fermented traditional Chinese medicine feed additive according to claim 8 for inhibiting enteropathogenic bacteria when added to aquaculture feed, wherein the enteropathogenic bacteria are specifically salmonella enteritidis, escherichia coli or shigella flexneri.

10. Use according to claim 9, characterized in that the specific operations are: adding the compound fermented traditional Chinese medicine feed additive into aquatic feed to obtain mixed feed for feeding; the addition amount of the compound fermented traditional Chinese medicine feed additive accounts for 5-25% of the weight of the mixed feed.