CN110812394A

CN110812394A - Formula of effective part of streptococcus agalactiae resistant traditional Chinese medicine extract and production process thereof

Info

Publication number: CN110812394A
Application number: CN201911297491.XA
Authority: CN
Inventors: 胡庭俊; 韦英益; 朱经国; 陈燕晴; 谢映红; 施君
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2019-12-17
Filing date: 2019-12-17
Publication date: 2020-02-21

Abstract

The invention discloses a formula of an effective part of an anti-streptococcus agalactiae traditional Chinese medicine extract, which is prepared from the following raw materials in parts by weight: 100-120 parts of rhubarb extract, 15-25 parts of berberine hydrochloride, 70-90 parts of scutellaria baicalensis extract and 75-95 parts of forsythia suspense extract. Because the berberine hydrochloride content in the prescription is low and the radix scutellariae extract has poor fluidity, on the basis of the prior experiment, a small amount of filling agent is premixed with the berberine hydrochloride and the radix scutellariae extract in the production process, and then the mixture is totally mixed, so as to increase the uniformity and ensure the stable drug effect. Experiments show that the minimum inhibitory concentration of the antibacterial agent to streptococcus agalactiae is 5.625 mug/mL; the minimum bactericidal concentration is 31.25 mug/mL, and good antibacterial effect is shown. Therefore, the streptococcus agalactiae resistant traditional Chinese medicine extract and the powder thereof which have the antibacterial effect, are non-toxic and residue-free and can replace antibiotics have important significance and broad prospects.

Description

Formula of effective part of streptococcus agalactiae resistant traditional Chinese medicine extract and production process thereof

Technical Field

The invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to a formula of an effective part of a streptococcus agalactiae resistant traditional Chinese medicine extract and a production process thereof.

Background

With the rapid development of the breeding industry, the application of antibiotics is more and more extensive, and the antibiotics play a certain positive role in the aspects of preventing and treating animal diseases and the like. However, in recent years, the problem of resistance caused by the abuse of antibiotics in the aquaculture industry has not been overlooked. Moreover, a large amount of unscientific antibiotics used for a long time can cause heavy metal residues in animal excrement, so that the environmental pollution is caused, and serious harm is brought to human health, and the rapid development of the breeding industry not only increases the supply of animal products, but also brings the problem of food safety. The application of the traditional Chinese medicine compound with broad spectrum, low toxicity and no residue in veterinary clinic is more and more extensive, and the characteristics of the traditional Chinese medicine compound of enhancing immunity, broad-spectrum antibiosis and promoting growth are more and more favored by the breeding industry.

Disclosure of Invention

The invention aims to solve the technical problem of providing a formula of effective parts of an anti-streptococcus agalactiae traditional Chinese medicine extract which has an antibacterial effect, is non-toxic and residue-free and can replace antibiotics and a production process thereof.

In order to solve the technical problems, the invention adopts the following technical scheme:

the formula of the effective parts of the streptococcus agalactiae resistant traditional Chinese medicine extract comprises the following raw materials in parts by weight: 100-120 parts of rhubarb extract, 15-25 parts of berberine hydrochloride, 70-90 parts of scutellaria baicalensis extract (calculated by baicalin) and 75-95 parts of forsythia suspense extract.

The powder of the traditional Chinese medicine extract for resisting streptococcus agalactiae is prepared by mixing the following raw materials and dextrin, wherein the raw materials comprise, by weight, 100-120 parts of rhubarb extract, 15-25 parts of berberine hydrochloride, 70-90 parts of scutellaria baicalensis extract (calculated by baicalin) and 75-95 parts of forsythia suspense extract.

The preparation method of the powder comprises mixing berberine hydrochloride and Scutellariae radix extract with part of dextrin, adding radix et rhizoma Rhei extract, fructus forsythiae extract and the rest amount of dextrin, and mixing completely.

The production process of the anti-streptococcus agalactiae traditional Chinese medicine extract powder comprises the following raw materials and dextrin, wherein the raw materials comprise 100-120 g of rheum officinale extract, 15-25 g of berberine hydrochloride, 70-90 g of scutellaria baicalensis extract (counted by baicalin) and 75-95 g of fructus forsythiae extract.

The production process of the powder of the traditional Chinese medicine extract for resisting streptococcus agalactiae comprises the steps of taking berberine hydrochloride and a scutellaria baicalensis extract, adding 100g of dextrin for premixing, then adding the rhubarb extract, the forsythia suspensa extract and the rest of the dextrin according to the prescription amount, and completely and uniformly mixing to obtain 1000g of powder.

The relative humidity of the production environment is controlled below 55 percent.

Aiming at the problem of antibiotics abuse in the breeding industry at present, the inventor develops a formula of an effective part of a streptococcus agalactiae resistant traditional Chinese medicine extract, and the formula consists of the following raw materials in parts by weight: 100-120 parts of rhubarb extract, 15-25 parts of berberine hydrochloride, 70-90 parts of scutellaria baicalensis extract (calculated by baicalin) and 75-95 parts of forsythia suspense extract. Accordingly, the related powder and the production process are also established. Because the berberine hydrochloride content in the prescription is low and the radix scutellariae extract has poor fluidity, on the basis of the prior experiment, a small amount of filling agent is premixed with the berberine hydrochloride and the radix scutellariae extract in the production process, and then the mixture is totally mixed, so as to increase the uniformity and ensure the stable drug effect. Experiments show that the minimum inhibitory concentration of the antibacterial agent to streptococcus agalactiae is 5.625 mug/mL; the minimum bactericidal concentration is 31.25 mug/mL, and good antibacterial effect is shown. Therefore, the streptococcus agalactiae resistant traditional Chinese medicine extract and the powder thereof which have the antibacterial effect, are non-toxic and residue-free and can replace antibiotics have important significance and broad prospects.

Drawings

FIG. 1 is a graph of the percent moisture absorption of the present invention at various relative humidities.

FIG. 2 is a gram-positive map of bacteria.

FIG. 3 is a second gram-positive pattern of bacteria.

FIG. 4 is an electrophoretogram of PCR detection of amplified products of Streptococcus agalactiae, in which: m is Marker 1000, lanes 1-4 in the figure are electrophoresis images of amplified products of Guangxi aquatic research institute samples, and lanes 5-7 are electrophoresis images of amplified products of standard strains.

Fig. 5 is a phylogenetic tree of test strains versus standard strains based on cfb sequences.

FIG. 6 is an electrophoretogram of PCR-detected S.agalactiae genotype amplification products, in which: s represents a test strain, B represents a standard strain; in a, lanes are Ia-S, Ia-B, II-S, II-B, III-S, III-B, V-S, V-B, Marker, VI-S, VI-B, VII-S and VII-B respectively; the lanes in B are Ib-S, Ib-B, IV-S, IV-B, VIII-S, VIII-B, Marker, d-S, d-B, respectively.

Figure 7 is a phylogenetic tree of test strain serotypes versus standard strain serotypes based on cfb sequence.

FIG. 8 is a growth curve of Streptococcus agalactiae.

Fig. 9 is a visible light absorption curve of the traditional Chinese medicine formulation of the invention.

FIG. 10 is a bactericidal curve of the Chinese medicinal formulation of the present invention against Streptococcus agalactiae.

Detailed Description

Research on streptococcus agalactiae resistant traditional Chinese medicine extract powder

First, the selection of dosage form

The formula is as follows: 100-120 g of rhubarb extract, 15-25 g of berberine hydrochloride, 70-90 g of scutellaria baicalensis extract (counted by baicalin) and 75-95 g of forsythia suspensa extract.

The product is finally determined to be powder, and the reason is as follows:

① in the composition, berberine hydrochloride and Scutellariae radix extract will generate serious precipitation reaction in water, so it is not suitable for liquid preparation.

② the aquatic food is divided into expanded feed or extruded feed according to molding process, and has spherical, granular, and columnar shape, etc. the recommended application is mixed feeding, and is not suitable for granulating (due to different shapes and densities, the mixed feeding is not uniform).

In conclusion, the preparation form is determined to be powder, and the production process of adding auxiliary materials and proper adhesive is researched according to the prescription and the using condition.

Secondly, research on process rationality

1.1 Process study protocol

The product is powder prepared by mixing the four extracts, and is administered in bait-mixing form, and the process factors influencing the product include ① mixing uniformity, ② influence of environmental humidity on mixing and subpackaging processes, and ③ influence on uniformity in bait-mixing.

1.2 selection of Filler

The filler serves primarily to fill the weight or volume of the formulation, thereby facilitating formulation. The fillers usually used include starches, sugars, celluloses, inorganic salts and the like.

① the starch mainly contains starch, dextrin, and compressible starch, and has the advantages of high quality, low cost, good fluidity, certain hygroscopicity, and capability of being used as adhesive when it is in contact with water.

② the saccharide mainly contains sucrose, lactose, glucose, and sucralose, and has the advantages of good quality, low cost, poor fluidity, high hygroscopicity, and flavoring effect.

③ cellulose such as microcrystalline cellulose (MCC), carboxymethyl cellulose, etc., has strong binding force, and can be used as "dry binder" for directly tabletting powder.

④ the inorganic salt is mainly inorganic calcium salt, which is usually calcium sulfate, calcium oxide, etc., but it generally has a certain acidity and alkalinity, which may affect the main component, and the divalent salt may complex some components.

Dextrin is finally selected as the filler of the product according to the properties of main components of the product (such as anthraquinone components contained in the rhubarb can generate color change reaction with metal ions), preparation technology (mixing technology, which requires good fluidity and does not absorb moisture), application method (bait mixing and feeding, which requires certain adhesive property), and cost factors.

1.3 study of mixing route and mixing homogeneity

The berberine hydrochloride accounts for 1-3% of the total amount of the prescription, the Scutellariae radix extract and fructus forsythiae extract account for 7-9% of the total amount, and the radix et rhizoma Rhei extract accounts for 11-13% of the total amount; wherein the content of berberine hydrochloride is low, and premixing is required; the Scutellariae radix extract has poor fluidity, and can be premixed to improve its uniformity. On the basis of the preliminary experiment, the mixing process route of the product is designed to take berberine hydrochloride and scutellaria baicalensis extract, add dextrin for premixing, and then add rhubarb extract, forsythia suspensa extract and the rest of the dextrin in the prescription amount for completely mixing.

1.4 research on critical relative humidity in mixing and subpackaging processes

The product is powder prepared by mixing the extract with adjuvants, has moisture absorption, and has improved stability, and Critical Relative Humidity (CRH) of the product is determined to determine relative humidity (RH%) of packaging environment.

1.4.1 Experimental methods

Taking 9 glass dryers, configuring a series of saturated salt solutions with different relative humidity (RH%), wherein the specific types and temperatures of the saturated salt solutions are set as shown in the attached table 1 (excessive salt exists at the bottom of the dryers to ensure the saturation of the salt solutions), sealing, and placing in a thermostat for 24 hours to obtain the corresponding constant-temperature and constant-humidity environment.

Placing about 1g of Chinese medicinal composition into a weighing bottle dried to constant weight, spreading at the bottom of the bottle, and having a thickness of about 2mm, weighing, drying to constant weight, precisely weighing, opening the weighing bottle cap, placing into the above different RH dryers, absorbing moisture at constant temperature to constant weight, precisely weighing to calculate moisture absorption rate, drawing corresponding moisture absorption equilibrium diagram, and determining Critical Relative Humidity (CRH).

Table 1 balance moisture absorption scale

The moisture absorption rate (%) is plotted as an ordinate and the relative humidity (RH%) is plotted as an abscissa, and the abscissa corresponding to the intersection of the tangents at both ends of the curve is the critical relative humidity (see fig. 1).

According to experimental results, the critical relative humidity of the traditional Chinese medicine formula is 55.3%, so that the relative humidity of the workshop environment is controlled to be below 55% in the mixing, subpackaging and encapsulating processes of the traditional Chinese medicine.

In summary, the production process of the streptococcus agalactiae resistant traditional Chinese medicine extract powder is determined as follows:

the formula is as follows: 1000g of the raw materials are mixed with dextrin, and the raw materials comprise 100-120 g of rhubarb extract, 15-25 g of berberine hydrochloride, 70-90 g of scutellaria baicalensis extract (counted by baicalin) and 75-95 g of forsythia suspensa extract. (Berberine hydrochloride, radix et rhizoma Rhei extract, Scutellariae radix extract, and fructus forsythiae extract are from Hengruitongda of Sichuan of Biotech limited)

The preparation method comprises the following steps: controlling relative humidity of production environment below 55%, taking berberine hydrochloride and Scutellariae radix extract, adding 100g dextrin, pre-mixing, adding radix et rhizoma Rhei extract, fructus forsythiae extract and the rest amount of dextrin, and mixing completely to obtain 1000g powder.

Example 1

As described above, this example was conducted according to the following formulation and preparation method.

The formula is as follows: 1000g of raw materials are mixed with dextrin, and the raw materials comprise 110g of rhubarb extract, 20g of berberine hydrochloride, 80g of scutellaria extract (counted by baicalin) and 85g of forsythia extract according to weight.

Thirdly, the traditional Chinese medicine formula of the invention is used for researching in-vitro antibacterial effect of tilapia streptococcus agalactiae

1 test Material

1.1 test strains

The streptococcus agalactiae strain is provided for fishery disease research laboratories of Guangxi aquatic research institute, and the standard strain is CMCC (B)32116 (2015-12-02).

1.2 test substances

Prepared as described in the above examples. Specification: the content of baicalin per gram is not less than 80 mg; packaging: 1000 g/bag.

1.3 Primary reagents

Streptococcus agalactiae CMCC (B)32116 was purchased from Guangdong province culture Collection; trace biochemical identification tube and brain-heart leachate broth culture medium (batch number: 171102) were purchased from Beijing road bridge technology, LLC; the ilex purpurea Hassk calf serum is purchased from Tian Hang Biotechnology Co., Ltd; penicillin sodium solution (100mg/mL) was given by Biotechnology (Shanghai) GmbH; phosphate buffered PBS, 2 × Taq PCR StarMix with Loading Dye from Guangzhou Kangrun Bio; DL1000 DNA Marker was purchased from Takara (Japan); the bacterial genome DNA extraction kit (batch No. 50241) and the agarose DNA recovery kit (batch No. 30314) are purchased from Beijing Kangwei century Biotech Co., Ltd.

1.4 Main Instrument

An ultra-clean bench (AIR TECH, sujing group altai), a DNP-9162 model electric heating constant temperature incubator (shanghai essence macro experimental facilities, ltd.), an AB104-L electronic analytical balance (mettler-toledo, switzerland); shaker incubators (U.S. shoe, Incushaker); s1000 TM Thermal cycle gradient PCR instrument and Gel Doc TMXR + type fully automatic Gel imaging system (Bio-Rad, USA); model DYY-8C electrophoresis apparatus (Beijing Liuyi instruments Co., Ltd.); UN-2100 ultraviolet spectrophotometer (Shanghai instruments, Unico., Umbelliferae).

2 test method

Preparation of 2.15% serum BHI broth

24.5g of brain-heart leachate broth culture medium, 1000mL of distilled water and 20g of nutrient agar, heating and stirring until the nutrient agar is completely dissolved, autoclaving at 121 ℃ for 15min, cooling to 56-60 ℃, adding fetal calf serum filtered by a 0.22-micron filter head, uniformly mixing to prepare a serum BHI culture medium with the final serum concentration of 5%, pouring into 9cm culture dishes, pouring 15mL of serum into each culture dish, after the culture medium is solidified, pouring into a 37 ℃ culture box for culturing for 18-24 h, checking no bacterial growth, storing at 4 ℃ and using within 7 days.

2.2 dissolution of the Chinese medicinal composition

Accurately weighing the medicine, adding appropriate liquid culture medium to dissolve the medicine, preparing to maximum concentration, filtering with 0.22 μm filter membrane, diluting to different concentrations, packaging, and storing at 4 deg.C.

2.3 Resuscitation of bacteria

Taking a preservation strain, thawing at the temperature of minus 20 ℃ and 4 ℃ in sequence, selecting a loop with an inoculating loop to be inoculated on a blood plate, culturing for 18-24 h in a constant-temperature incubator at the temperature of 37 ℃, selecting β hemolytic single bacterial colony, performing gram staining morphological examination to obtain a pollution-free pure culture, inoculating the pollution-free pure culture on a 5% serum BHI plate, and recovering for 24h at the temperature of 37 ℃.

2.4 identification of Streptococcus agalactiae

2.4.1 morphological and physiological Biochemical identification of bacteria

And (3) observing the morphology of bacteria and colonies: single colony is picked by an inoculating needle, and the shape of the thallus is observed under a microscope through smear, fixation and gram staining. And picking out a single colony by using an inoculating loop, culturing the single colony on a sheep blood agar culture medium at 37 ℃ overnight, and observing the hemolytic activity of the test bacteria.

The physiological and biochemical tests of 6.5% NaCl, salicin, lactose, etc. are carried out according to the conventional method.

2.4.2 PCR identification of bacteria and bacterial serotype specificity

(1) Inoculating the purified bacteria into a broth liquid culture medium containing 5% serum BHI, culturing at 37 ℃ for 24h, taking 1.5mL of bacterial liquid, extracting DNA according to the operation steps of a bacterial genome DNA extraction kit (Kangji Biotech Co., Ltd.), and detecting the quality of the DNA by electrophoresis of 1.0% agarose gel.

(2) Streptococcus agalactiae CAMP factors (cfb) are specific to tilapia-derived Streptococcus agalactiae, and the primer sequences are as follows: p1(5 '-AAGCGTGTATTCCAGATTTCCT-3') and P2(5 '-CAGTAATCAAGCCCAGCAA-3'), the expected amplified fragment was about 474bp, synthesized by Biotechnology engineering (Shanghai) GmbH. Reference is made to the 10 serotype-specific primer sequences synthesized by Biotechnology, Inc. (Shanghai), see Table 2 below.

TABLE 2 Streptococcus agalactiae serotype specific primer sequences and PCR amplification product fragments

(3) PCR amplification was performed using 40. mu.L PCR reaction (see tables 3 and 4).

(4) Recovering target fragment glue: the procedure was carried out according to the instructions of the agar gel DNA recovery kit of Bio-technology Co., Ltd.

(5) Sequencing: the recovered product was directly sent to the sequencing department of Biotechnology engineering (Shanghai) GmbH for sequencing. Sequence homology analysis was performed using the BLAST program and Editseq, Megalign software.

2.5 determination of growth Curve of Streptococcus agalactiae

Inoculating bacteria into 50mL sterile BHI liquid culture medium according to the inoculation amount of 2%, culturing at constant temperature of 37 ℃, and taking 2mL bacterial liquid for OD every 2h_600nmThe sampling was repeated 3 times at each time point and the detection was continued for 24 h.

Meanwhile, 2mL of the bacterial solution taken every 2h is added into an EP tube containing 0.9mL of sterile BHI liquid culture medium by sucking 100 μ L of the bacterial solution, mixed uniformly and diluted by 10 times in sequence (the tip of a pipette gun must be replaced for each dilution).

200 mu L of 3-dilution bacterial liquid obtained by a preliminary experiment is selected, added to the surface of a solid culture medium with a diameter of 9cm, the bacterial suspension is uniformly coated on the surface of the culture medium by an L rod, each gradient is repeated for 3 times, and the bacterial suspension is cultured at the constant temperature of 37 ℃ for 24 hours and then counted.

2.6 determination of minimum inhibitory concentration MIC and minimum bactericidal concentration MBC of Streptococcus agalactiae by traditional Chinese medicine formula

Preparing 13 BHI liquid culture media with concentration of 0.03-125 mg/mL for the traditional Chinese medicine formula by double dilution method, and adding 100 μ L bacterial solution (adjusting bacterial concentration to 6 × 10)⁶CFU/mL) was added to an EP tube containing BHI broth (1900. mu.L) containing the above Chinese medicinal composition at different concentrations, 2000. mu.L of the broth was added to the blank control group, 1900. mu.L of the BHI broth containing 100. mu.L of the bacteria without the Chinese medicinal composition was added to the bacteria control group, and 100. mu.L of penicillin (25. mu.g/mL and 50. mu.g/mL) was added to the drug control groupBHI medium (1900. mu.L) containing bacteria. Each concentration was repeated 3 times in parallel and incubated anaerobically at 37 ℃ for 24 h. And (3) taking the non-growing bacteria in the negative control group as quality control and the bacteria in the positive control group as reference, and observing whether the liquid in the medicine group becomes turbid or not by naked eyes to judge the Minimum Inhibitory Concentration (MIC).

Sucking out the bacterial suspension cultured by the traditional Chinese medicine formula of 1 × MIC, 2 × MIC and 4 × MIC, adding the bacterial suspension into a fresh 5% serum BHI broth culture medium without the traditional Chinese medicine formula, culturing at 37 ℃ for 24-48 h, and observing the formation of sterile colonies, wherein the corresponding drug concentration (the minimum dilution drug concentration of which the number of the bacterial colonies on a plate is less than 5) of the sterile colonies is the Minimum Bactericidal Concentration (MBC) of the drug concentration on the streptococcus agalactiae.

2.7 measurement of Sterilization Curve

(1) Scanning of absorption curve of visible region of Chinese medicinal composition

The traditional Chinese medicine formula is prepared into traditional Chinese medicine formula solutions of 1/2 XMIC, 1 XMIC and 2 XMIC by using 5% serum BHI broth culture solution, each concentration is repeated for 5 times, blank solution is used as reference, and a spectrophotometer is used for scanning at the wavelength of 230-650 nm.

(2) Determination of the Sterilization Curve

The traditional Chinese medicine formula is prepared into solutions with final concentrations of 1/2 × MIC, 1 × MIC and 2 × MIC respectively by using 5% serum BHI broth culture medium, and 3mL of each solution is taken as a reference tube. 1mL of test bacteria in the same growth period are respectively taken, and the concentration is 6 multiplied by 10⁶CFU/mL, added separately to the experimental groups containing 99mL of drug. 5% serum BHI broth without Chinese medicinal composition was used as blank control. Penicillin (25. mu.g/mL) was used as a positive drug control. Each group was incubated at 37 ℃ for 24 h. OD (optical Density) of the sample in an ultraviolet spectrophotometer every 1-2 h_600nmRead 3 times, repeat 3 counts per sample, average and make OD_600nm-t growth curve.

3 results

3.1 identification of bacteria

(1) Results of morphological and physiological biochemical identification of bacteria

The separated bacteria are inoculated on a sheep blood agar plate and cultured overnight at 37 ℃, bacterial colonies are milky white, round, protruding and neat in edge, β hemolysis, the separated and purified bacteria are gram-positive, and 2-5 thallus connected short chains can be seen under an oil lens, see attached figures 2 and 3.

TABLE 5 Biochemical identification of the test strains

(2) PCR and serotype identification results

1 streptococcus agalactiae is selected, 1 streptococcus agalactiae is cultured by standard bacteria beads, 3 repeats of each sample are taken, and PCR electrophoresis and PCR sequencing identification are carried out. The electrophoresis chart is shown in figure 4, the bacterial sequences are subjected to homology search in Genbank, the results show that the bacteria are streptococcus agalactiae, the homology and the login number of each bacterium are detailed in an attached table 6, and the genetic evolutionary tree is shown in figure 5.

TABLE 6 bacterial sequence homology alignment results

The results of serotype-specific PCR amplification of the standard and test strains are shown in FIG. 6. The sequencing result of the amplified target fragment is subjected to Blast comparison in Genbank, and the result shows that the homology of the sequencing result and the corresponding gene sequence of Streptococcus agalactiae in Genbank is 99 percent, see Table 7, and the genetic evolutionary tree is shown in FIG. 7.

TABLE 7 bacterial serotype sequence homology alignment

3.2 determination of growth Curve of Streptococcus agalactiae

Continuously culturing bacteria in 5% BHI liquid culture medium for 24 hr, taking 3mL bacterial suspension every 1 hr, adjusting to zero with culture solution, and performing OD₆₀₀And (4) measuring. By OD₆₀₀The ordinate represents the incubation time, and the abscissa represents the growth curve (FIG. 8). The result shows that the tilapia source streptococcus agalactiae is inoculated in vitro, the growth in 1h is relatively smooth, and the growth curve rapidly rises in the period from 1h to 5 h; the curve after 5h tends to be a straight line, which indicates that the growth state of the bacteria at the moment is relatively stable. It can be concluded from this that tilapia-derived streptococcus agalactiae were inoculated in vitro, with the 1h to 5h log phase of the bacteria.

3.3 determination results of Streptococcus agalactiae MIC and MBC by traditional Chinese medicine formula

As can be seen from Table 8, the Chinese medicinal formula of the invention has better bacteriostatic effect on Streptococcus agalactiae, the MIC is 15.625 mug/mL, and the MBC is 31.25 mug/mL.

TABLE 8 minimal inhibitory and bactericidal concentrations of traditional Chinese medicine formulations against Streptococcus agalactiae

Note: compared with a negative control group; "-" indicates clear liquid and "+" indicates turbid liquid.

3.4 Effect of Chinese medicinal formulation on growth Curve of Streptococcus agalactiae

3.4.1 absorption Curve of visible region of Chinese medicinal composition

The Chinese medicinal composition has no obvious absorption at 600nm (shown in figure 9), and has low and stable absorption value, so 600nm is selected as the detection wavelength.

3.4.2 influence of Chinese medicinal composition on growth of Streptococcus agalactiae

As shown in figure 10, the growth curve of the normal control group is the normal growth state of bacteria, and is consistent with the normal growth curve of tilapia-derived Streptococcus agalactiae detected in the early stage. After 1/2 XMIC, MIC and 2 XMIC Chinese medicine compositions are added, the growth curves of bacteria are changed differently. In the 1/2 × MIC and MIC concentration-treated groups, the bacteria entered the logarithmic growth phase at a slower rate than the normal control group. However, 1/2 × MIC concentration treated bacteria showed growth after 6h, while MIC concentration treated bacteria showed growth inhibition. 2 × MIC and positive drug treated bacteria, were unable to enter logarithmic growth phase.

4. Summary of the invention

The in-vitro MIC of the traditional Chinese medicine formula to tilapia-derived streptococcus agalactiae is 15.625 mug/mL, and the MBC is 31.25 mug/mL. The results of the sterilization curve show that: 1/2 XMIC and MIC, the traditional Chinese medicine formula mainly inhibits the division of bacteria in the logarithmic growth phase, but the bacteria treated at 1/2 XMIC concentration can grow after 6 hours, and the bacteria treated at MIC concentration can inhibit the growth. 2 × MIC and positive drug (penicillin 25. mu.g/ml) treated bacteria were unable to enter logarithmic growth phase. The formula of the traditional Chinese medicine has a good in-vitro antibacterial effect on tilapia streptococcus agalactiae.

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Claims

1. The formula of the effective parts of the traditional Chinese medicine extract for resisting streptococcus agalactiae is characterized by comprising the following raw materials in parts by weight: 100-120 parts of rhubarb extract, 15-25 parts of berberine hydrochloride, 70-90 parts of scutellaria baicalensis extract and 75-95 parts of forsythia suspense extract.

2. The powder is characterized by being prepared by mixing 100-120 parts of rhubarb extract, 15-25 parts of berberine hydrochloride, 70-90 parts of scutellaria baicalensis extract and 75-95 parts of fructus forsythiae extract.

3. The process for producing the powder of the Chinese medicinal extract for resisting streptococcus agalactiae of claim 2, wherein: taking berberine hydrochloride and Scutellariae radix extract, adding part of dextrin, premixing, adding radix et rhizoma Rhei extract, fructus forsythiae extract and the rest dextrin, and mixing completely.

4. The production process of the anti-streptococcus agalactiae traditional Chinese medicine extract powder according to claim 3, characterized in that 1000g of the anti-streptococcus agalactiae traditional Chinese medicine extract powder is prepared by mixing the following raw materials and dextrin, wherein the raw materials comprise 100-120 g of rhubarb extract, 15-25 g of berberine hydrochloride, 70-90 g of scutellaria baicalensis extract and 75-95 g of forsythia suspensa extract.

5. The process for producing the powder of the Chinese medicinal extract for resisting streptococcus agalactiae according to claim 4, wherein: taking berberine hydrochloride and Scutellariae radix extract, adding 100g dextrin, premixing, adding radix et rhizoma Rhei extract, fructus forsythiae extract and the rest dextrin, and mixing completely to obtain 1000g powder.

6. The process for preparing a powder of Chinese medicinal extract for treating Streptococcus agalactiae as claimed in claim 4, wherein the relative humidity of the production environment is controlled to 55% or less.