A Chinese herbal medicine preparation for treating bee chalk disease and preparation method thereof

Abstract

The invention discloses a Chinese herbal medicine preparation for treating bee chalk disease and a preparation method thereof, 9 parts of Polygonum cuspidatum, 15 parts of White Peony Root, 9 parts of Radix Isatidis, 8 parts of Rhizoma Dioscoreae, 30 parts of Radix Astragali, soaked in 6 times the mass of deionized water, heated in a water bath at 95°C, filtered and centrifuged at high speed, filtered through a 0.22μm water system microporous filter membrane, inoculated with a composite probiotic according to an inoculation amount of 5% in the Chinese herbal medicine filtrate, 60% water content, 1% glucose, 0.5% brown sugar, fully mixed, and fermented in a 37°C incubator for 5 days. The present invention can maintain a slightly acidic environment in the intestine, inhibit the growth of harmful microorganisms, and probiotics can increase the types and quantity of intestinal flora, thereby maintaining the microecological balance in the animal body; during the fermentation process, probiotics can prompt Chinese herbal medicine to activate the body's nonspecific antibacterial factors, thereby effectively killing pathogens, and has no drug resistance, safety and high efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of insect disease prevention and treatment, and specifically relates to a Chinese herbal medicine preparation for treating bee chalkbrood and a preparation method thereof. Background Art

[0002] The chalk disease of bees is caused by a fungus called Ascocarpus mellifera, which only attacks young bees and has strong vitality. It is still active in nature for more than 15 years. The occurrence of chalk disease depends largely on the temperature and humidity at the time, and has obvious seasonality. Generally, this disease is prevalent in spring and early summer, especially in rainy and humid climates with frequent temperature changes. During this period, the bee colonies are mostly in the breeding period, the brood circle on the honeycomb is relatively large, and the chance of the honeycomb edge being cold is relatively large, so the incidence rate is high. In the bee colony, the corpses of sick larvae and contaminated feed and honeycombs are the main sources of disease transmission. The optimum temperature for the proliferation and formation of sporangia is about 30℃. When the hive temperature drops from 35℃ to 30℃, the larvae are most susceptible to infection. Therefore, when the bee colony reproduces in large numbers, it is most likely to occur when the larvae in the nest are cold due to poor insulation or insufficient nursing bees. It occurs from April to October every year, with a peak period from April to June. Humidity, excessive swarming, feeding stale and moldy pollen, excessive use of antibiotics that change the microbial flora in the bees' intestines, and weak bee colonies can all induce chalkbrood.

[0003] Chalk disease is often related to the inability of bees to adapt to sudden changes in climate. Prevention and control of this disease should start with the management of bee colony breeding. First, remove all diseased insect spleens and powder honey spleens in the diseased colony, and replace them with clean honeycombs for the queen bee to lay eggs. The replaced honeycombs must be carefully disinfected. Then strengthen the heat preservation of the bee colony, move the bee colony away from a relatively cold environment, and ensure the dryness inside and outside the beehive. For the diseased bee colony, on the basis of the above measures, certain drug treatments should be carried out. Generally, two tablets of mebendazole are ground into powder and mixed with pollen to feed the bee colony for 7 consecutive days. The bee colony should also be placed in a high and dry place to keep the nest clean and dry. Do not feed moldy and deteriorated feed, and do not use old and moldy spleens. After the disease occurs, first remove all diseased larval spleens and moldy powder honey spleens in the diseased colony, and replace them with clean and disease-free honeycombs for the queen bee to lay eggs. The replaced honeycombs are sterilized with closed sulfur dioxide fumigation for more than 4 hours, and the amount of sulfur is calculated as 3 to 5 grams per 10 frames of nests.

[0004] At present, the common method for treating the disease is to use antifungal drugs, but such drugs have strong toxic side effects and are not conducive to the development of bee colonies. In order to overcome these shortcomings, a new method for treating the disease needs to be developed. The existing technology for treating chalk disease in bees mostly adopts a spraying treatment method, which not only cannot ensure that every bee can be sprayed with drugs, but also spraying only treats the symptoms, and cannot fundamentally treat the disease, nor can it completely eradicate the residual chalk disease virus in the bees.

[0005] Therefore, in order to solve the above problems, this paper proposes a Chinese herbal medicine preparation for treating bee chalkbrood and a preparation method thereof. Summary of the invention

[0006] In order to solve the above technical problems, the present invention designs a Chinese herbal medicine preparation for treating bee chalkbrood and a preparation method thereof. The Chinese herbal medicine preparation is used with little side effects; the Chinese herbal medicine can maintain a slightly acidic environment in the intestine, inhibit the growth of harmful microorganisms, and also provide necessary nutrients for the reproduction of probiotics; probiotics can increase the types and quantity of intestinal flora, thereby maintaining the microecological balance in the animal body; during the fermentation process, probiotics can prompt the Chinese herbal medicine to activate the body's nonspecific antibacterial factors, thereby effectively killing pathogens, and has multiple advantages such as no drug resistance, safety and high efficiency; the two are used together, not only can the effective ingredients contained in the Chinese herbal medicine be fully utilized, but also its own performance can be enhanced, and the active ingredients can be better absorbed and utilized.

[0007] In order to achieve the above technical effects, the present invention is implemented by the following technical scheme: a Chinese herbal medicine preparation for treating bee chalk disease, characterized in that it comprises the following components by weight:

[0008] 7-11 parts of Polygonum cuspidatum, 12-18 parts of White Peony Root, 7-11 parts of Isatis Radix, 6-10 parts of Rhizoma Cynoglossi, and 25-35 parts of Radix Astragali.

[0009] Further, the following components are included in parts by mass:

[0010] 9 parts of Polygonum cuspidatum, 15 parts of the white paeonia lactiflora, 9 parts of Radix Isatidis, 8 parts of Rhizoma Cynoglossi, 30 parts of Radix Astragali.

[0011] Another object of the present invention is to provide a method for preparing a Chinese herbal medicine preparation for treating chalkbrood of bees, characterized in that the method comprises the following steps:

[0012] The component Chinese herbal medicines are mixed, soaked in 5 to 7 times the mass of deionized water for 0.5 to 1.5 hours, boiled in boiling water for 50 to 60 minutes, and the water bath temperature is 95°C to 100°C. The mixture is filtered through medical gauze, and the above steps are repeated for the residue. The two filtrates are combined, and the filtrate is subjected to high-speed centrifugation. The filtrate obtained by centrifugation is filtered through a 0.22 μm water system microporous filter membrane, and the composite probiotics are inoculated into the Chinese herbal medicine filtrate at an inoculation rate of 5%, 60% water content, 1% glucose, and 0.5% brown sugar. The mixture is fully mixed with the Chinese herbal medicines, and fermented in a 37°C incubator for 5 days. The fermentation is completed.

[0013] Furthermore, the high-speed centrifugation is 10000-12000 rpm / min, and the time is 15-20 min.

[0014] Furthermore, the composite probiotics are: five lactobacilli, namely, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus paracasei, Lactobacillus gasseri, Lactobacillus plantarum, and one bacillus, namely, Bacillus subtilis.

[0015] The beneficial effects of the present invention are:

[0016] (1) Chinese herbal medicine and probiotics interact with each other and can exert a synergistic effect. Chinese herbal medicine can maintain a slightly acidic environment in the intestine, inhibit the growth of harmful microorganisms, and also provide necessary nutrients for the reproduction of probiotics. This interaction can not only enhance immune response and stress resistance, but also improve feed utilization and promote their growth and development. The use of the two together can not only fully utilize the effective ingredients contained in Chinese herbal medicine, but also enhance its own performance and promote better absorption and utilization of the active ingredients.

[0017] (2) Enhance the utilization effect of Chinese herbal medicines and improve their efficacy. After fermentation, Chinese herbal medicines can fully release active substances and degrade macromolecular substances into small molecular active substances that are easy for the body to digest and absorb, thereby increasing the concentration of active ingredients and enhancing efficacy. During the fermentation process, extracellular enzymes can break down the cell walls, allowing active substances to pass through the cell walls more easily and be released, making them easier to be absorbed and utilized by the body. At the same time, the dosage can also be reduced, further enhancing the efficacy of the drug.

[0018] (3) Produce new active substances; in addition to secreting a variety of extracellular enzymes with biotransformation capabilities, probiotics can also produce a variety of secondary metabolites, which play an important role in the production of new compounds;

[0019] (4) Reduce the toxic side effects of drugs; fermented Chinese herbal medicines can decompose or transform the toxic substances in them, thereby eliminating their toxic effects and improving their efficacy;

[0020] (5) Regulate intestinal flora: probiotics can increase the types and quantity of intestinal flora, thereby maintaining the microecological balance in animals; during the fermentation process, probiotics can promote Chinese herbal medicines to activate the body's nonspecific antibacterial factors, thereby effectively killing pathogens. Moreover, probiotics have many advantages such as no drug resistance, safety and high efficiency, and are not prone to drug resistance;

[0021] (6) The combination of Chinese herbal medicine and microecological preparations can regulate the overall effects in animals and activate the body's nonspecific immune antibacterial factors to kill pathogens in the body in an all-round way; this antibacterial mechanism does not directly act on the bacteria, so it is not easy to produce drug resistance;

[0022] (7) Save medicine resources and protect the environment. By applying fermentation technology, the efficacy of Chinese herbal medicines can be improved, so the amount of medicine required is reduced, which indirectly protects Chinese herbal medicine resources. In addition, after the effective ingredients are extracted, traditional Chinese herbal medicines are often discarded, which will greatly waste precious Chinese herbal medicine resources and pollute the environment. However, the use of fermentation technology can convert Chinese herbal medicine waste into substances with high nutritional value, such as proteins and polysaccharides, which can effectively solve this problem. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0024] Figure 1 It is the standard curve diagram of rutin in Example 2;

[0025] Figure 2 This is the standard curve diagram of glucose in Example 2. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Example 1

[0028] A method for preparing a Chinese herbal medicine preparation for treating chalk disease in bees: 9g of Polygonum cuspidatum, 15g of White Peony Root, 9g of Isatis Indigotica Root, 8g of Rhizoma Cynoglossi, and 30g of Radix Astragali; the above Chinese herbal medicines are mixed together in a 1000ml conical flask, soaked in 500ml of deionized water for 1h, boiled in boiling water for 50min-60min, the water bath temperature is 95℃-100℃, filtered through medical gauze, the filter residue is repeated with the above steps, the two filtrates are combined, and the filtrate is divided into two parts;

[0029] A portion was subjected to high-speed centrifugation (10000rpm / min~12000rpm / min) for 15~20min, and the filtrate obtained by centrifugation was filtered through a 0.22μm water system microporous filter membrane. Six kinds of probiotics were inoculated into the Chinese herbal medicine filtrate at an inoculation rate of 5%, 60% water content, 1% glucose, and 0.5% brown sugar. They were fully mixed with the Chinese herbal medicine and fermented in an incubator at 37℃ for 5d. After fermentation, the changes of each component were measured.

[0030] Example 2

[0031] Another part of the water extract in Example 1 was filtered through petroleum ether to obtain extracts which were respectively spin-dried and concentrated to a paste, then dissolved in methanol, filtered through a 0.22 μm microporous filter membrane to remove impurities, fixed to 1 ml, and determined the various components therein.

[0032] Water-extracted petroleum ether phase: the stationary phase was Agilent ZORBAXSB~C18 (4.6mm*250mm, 5μm), the mobile phase was methanol~water (70:30, v / v), the flow rate was 0.8ml / min, the column temperature was 25℃, the detection wavelength was 254nm, the injection volume was 10μl, and the detection time was 15min.

[0033] Determination of total flavonoids by RP~HPLC

[0034] Preparation of flavonoid standard curve:

[0035] Take 10 mg of rutin (98%) + 100 ml of methanol (analytical grade), the mother liquor concentration is 0.98 mg / ml;

[0036] 0.20ml, 0.40ml, 1.0ml, 2.0ml and 3.0ml were drawn from the mother liquor into the measuring cylinder respectively, and then methanol was added to dissolve and the volume was fixed to 10ml, and the corresponding concentrations were 0.0196mg / ml, 0.0392mg / ml, 0.098mg / ml, 0.196mg / ml and 0.294mg / ml respectively, and filtered through a 0.45μm microporous filter membrane respectively, and injected into the chromatographic column according to the following chromatographic conditions: C18AgilentZORBAXSB~C18 (4.6mm×250mm, 5μm);

[0037] Mobile phase: methanol (chromatographic grade): water = 70:30; flow rate: 0.8 mL / min; wavelength 254 nm; column temperature: 25 °C; injection volume: 10 μL, peak time within 15 min, repeat injection 3 times at each concentration point of the configured concentration gradient, perform linear regression on injection concentration using peak area, and draw a standard curve using Origin software, such as Figure 1 .

[0038] Determination of total polysaccharides by phenol-sulfuric acid method

[0039] Preparation of standard curve: Weigh 20 mg of glucose accurately, place it in a 100 mL volumetric flask, add water to dissolve, dilute and shake well, and set aside. Accurately measure 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, 1.0 ml, and 1.2 ml of glucose solution, add water to 2 ml respectively, add 1 ml of 5% phenol solution, shake well to make it fully react, slowly add 5 ml of concentrated sulfuric acid, shake well, place in a boiling water bath for 20 min, cool to room temperature, measure the absorbance, OD = 490 nm, repeat 3 times for each concentration, record the values, and use Origin software to draw a standard curve for glucose, such as Figure 2 .

[0040] The prepared Chinese herbal medicine was analyzed by RP-HPLC method and UV spectrophotometer method before and after fermentation, and the content of flavonoids and glucose was calculated as shown in the following table:

[0041] Chinese herbal medicine water extract Flavonoid content (mg / ml) Glucose content (mg / ml) Fermentation 0.0632 0.0245 Unfermented 0.2531 0.0755

[0042] Example 3

[0043] Select a beehive with a relatively large and active colony from the apiary, pick out a honeycomb from it, put it in a special bee incubator that has been sterilized for a long enough time, and wait quietly for the young bees to break through the membrane by themselves (remember not to destroy the sealing film artificially); after successfully catching 120 young bees, feed the young bees with 1ml of chalkblight pathogen B5 + 9ml of sterile sugar water for 3-4 days until all three boxes of bees are infected with chalkblight, and then divide them into three groups, each with 40 bees:

[0044] Experimental group: 50% sugar water mixed with compound probiotic fermented Chinese herbal medicine was placed in EP tubes to feed young bees, and observations were recorded;

[0045] Control group 1: 50% sugar water was placed in EP tubes to feed sick larvae, and observations were recorded;

[0046] Control group 2: 50% sugar water mixed with unfermented Chinese herbal medicine was placed in EP tubes to feed young bees, and observations were recorded;

[0047] The sick bees are counted from the day they are fed sugar water etc. until they completely die;

[0048] Group Control group 1 Control group 2 Experimental Group Deaths 36 11 2 Number of cures 0 20 37 Number of people who survived but not cured 4 9 1

Claims

1. A Chinese herbal medicine preparation for treating bee chalkbrood, characterized in that: The invention is prepared from 7-11 parts of Polygonum cuspidatum, 12-18 parts of White Peony Root, 7-11 parts of Isatis Indigotica Root, 6-10 parts of Rhizoma Dioscoreae and 25-35 parts of Radix Astragali by weight.

2. A Chinese herbal medicine preparation for treating bee chalkbrood according to claim 1, characterized in that: The invention is prepared from 9 parts of Polygonum cuspidatum, 15 parts of White Peony Root, 9 parts of Isatis Indigotica, 8 parts of Rhizoma Dioscoreae and 30 parts of Radix Astragali by weight.

3. The method for preparing a Chinese herbal medicine preparation for treating bee chalkbrood according to claim 1, characterized in that: The following steps are involved: The component Chinese herbal medicines are mixed, soaked in 5-7 times the mass of deionized water for 0.5-1.5 hours, boiled in boiling water for 50-60 minutes, and the water bath temperature is 95°C-100°C. The mixture is filtered through medical gauze, and the above steps are repeated for the residue. The two filtrates are combined, and the filtrates are centrifuged at high speed. The filtrate obtained by centrifugation is filtered through a 0.22 μm water system microporous filter membrane, and the composite probiotics are inoculated into the Chinese herbal medicine filtrate according to an inoculation amount of 5%, 60% water content, 1% glucose, and 0.5% brown sugar. The mixture is fully mixed with the Chinese herbal medicines, and fermented in a 37°C incubator for 5 days. The fermentation is completed. The composite probiotics are five kinds of lactobacilli, namely, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus paracasei, Lactobacillus gasseri, and Lactobacillus plantarum, and one kind of bacillus, namely, Bacillus subtilis.

4. The method for preparing a Chinese herbal medicine preparation for treating bee chalkbrood according to claim 3, characterized in that: The high-speed centrifugation is 10000-12000 rpm / min, and the time is 15-20 min.

Images