Preparation method and application of compound for improving cold resistance of apis cerana

Abstract

The invention discloses a preparation method and application of a composition for improving cold resistance of bees. The composition is prepared by performing compound fermentation on Chinese herbal medicine liquid, lactobacillus buchneri, lactobacillus reuteri and bacillus Zhongmuran, the Chinese herbal medicine liquid is prepared from the following raw materials in parts by mass: 9 parts of rhizoma polygoni cuspidati, 30 parts of radix astragali, 15 parts of radix paeoniae alba, 9 parts of radix isatidis and 8 parts of rhizoma dioscoreae nipponicae; the selected probiotics have strong protease production capacity and good antibacterial effect, and are sensitive to various antibiotics. The composition disclosed by the invention can be used for enhancing the immune defense capability of bees in the wintering period, reducing the possibility of disease occurrence and improving the overall survival rate; and the composition has no drug residue while improving the cold resistance of the bees in winter, shows huge sustainable development potential when being applied to bee breeding, and brings economic benefits and social benefits to bee keepers.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, and in particular to a preparation method and application of a composite material for improving the cold resistance of Chinese honey bees. Background Art

[0002] Honey bees are the main pollinating insect group in nature and have important economic and ecological value. As a species of honey bees in temperate regions, Chinese honey bees are more sensitive to low temperature environments and their physiological activities will be inhibited during the winter, resulting in weakened flight ability, reduced honey collection efficiency, reduced reproductive capacity and increased feed consumption. In severe cases, bees may even die of diseases such as cysticercus virus. In order to cope with the cold resistance problem, many beekeepers have adopted various methods such as strengthening the insulation of beehives to help the bee colonies survive the cold and long winter.

[0003] Different wintering management measures have a direct impact on the weakening of bee colonies during the winter, and are also directly related to the reproduction and production of bee colonies in the following year. Measures such as strengthening insulation and providing sufficient feed require a lot of manpower and material resources, which increases the cost of breeding. In addition, breeding varieties with strong cold resistance requires long-term practice and accumulation, and it is difficult to achieve significant results in the short term. Although these cold-resistant measures have alleviated the cold-resistant problem to a certain extent, they have not fundamentally solved the problem of weak cold resistance of Chinese bees.

[0004] Therefore, finding an economical and effective method to improve the cold resistance of Chinese bees is of great significance to promoting the healthy development of the Chinese bee breeding industry. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a composite material for improving the cold resistance of Chinese honey bees, so that after feeding the composite material of the present invention to honey bees, the cold resistance physiological and biochemical indicators of the honey bees during the wintering period change significantly; at the same time, the problem of more or less death of bee colonies during the wintering is solved, the immune defense ability of the honey bees is enhanced, the possibility of disease is reduced and their overall survival rate is improved, the honey yield and quality are increased, and higher economic benefits are brought to beekeepers.

[0006] In order to achieve the above technical effects, the present invention is implemented by the following technical scheme: a method for preparing a composite material for improving the cold resistance of Chinese honey bees, characterized in that it comprises the following steps:

[0007] S1. Weighing Chinese herbal medicine raw materials

[0008] Weigh 8-10 parts of Polygonum cuspidatum, 29-31 parts of Astragalus, 14-16 parts of White Peony Root, 8-10 parts of Isatis indigotica and 7-9 parts of Rhizoma Dioscoreae by weight;

[0009] S2. Preparation of Chinese herbal medicine liquid

[0010] Grind the Chinese herbal medicine raw materials weighed in S1 into powder and pass through a 40-mesh sieve;

[0011] The powdered astragalus is first sonicated with 90-110 ml of RO water for 8-12 minutes, and then soaked for 18-22 minutes to obtain an initial astragalus solution;

[0012] Mix the remaining Chinese herbal medicines together, add them into 200-220 ml of RO water and soak for 1-1.5 hours to obtain a mixed initial solution;

[0013] The container containing the initial Astragalus solution and the container containing the mixed initial solution are placed in a water bath at 90 to 110°C for 1 to 1.5 hours of water bath heating, and the two containers are centrifuged twice to remove insoluble matter, and then filtered, and the collected supernatants are combined and fixed to 600 ml to obtain the Chinese herbal medicine solution;

[0014] S3. Preparation of probiotic fermentation seed liquid

[0015] Use an inoculating loop to take a small amount of sample from the thawed solution of probiotics and perform streak culture on the plate;

[0016] After streaking culture on the plate, a single colony was selected and inoculated into 100 mL of MRS and NB liquid culture medium, and cultured at 37°C with shaking for 24 h. The absorbance was measured under a spectrophotometer and the concentration was adjusted to OD600 = 1, and mixed at a ratio of 1:1:1 to obtain probiotic fermentation seed liquid.

[0017] S4. Inoculate the prepared probiotic fermentation seed liquid into the Chinese herbal medicine liquid at an inoculation rate of 1% to 3% by volume, and ferment at 34 to 38° C. for 3 to 7 days to obtain a composition that improves the wintering cold resistance of Chinese honey bees.

[0018] Furthermore, in S1, the Chinese herbal raw materials, measured by mass, include 9 parts of Polygonum cuspidatum, 30 parts of Astragalus, 15 parts of White Peony Root, 9 parts of Isatis Indigotica and 8 parts of Rhizoma Dioscoreae.

[0019] Furthermore, in S3, the probiotics are a mixture of Lactobacillus buchneri, Lactobacillus reuteri and Bacillus Nakamura.

[0020] Furthermore, in S4, the inoculation amount of the probiotic fermentation seed liquid inoculated into the Chinese herbal medicine liquid is 1%, the fermentation time is 3 days, and the fermentation temperature is 36°C.

[0021] Another object of the present invention is to provide an application of a composition for improving the cold resistance of bees, characterized in that the composition is used to make a product for improving the cold resistance of bees in winter.

[0022] The beneficial effects of the present invention are:

[0023] The composition of the present invention uses Chinese herbal medicine raw materials and is rich in antibacterial, antiviral and antioxidant active ingredients as main ingredients. The active ingredients of these main ingredients have unique pharmacological effects such as antioxidant activity and free radical scavenging, and have a certain promoting effect on cellular immunity and humoral immunity; and the composite probiotics in the composition have strong protease production ability and good antibacterial effect, are sensitive to multiple antibiotics, and have multiple functions such as regulating intestinal microecological balance, improving the utilization rate of these nutrients, and enhancing immunity;

[0024] After the composition of the present invention is fed to bees, the protein and glycerol content, antioxidant enzyme activity and cold-resistant gene expression in the bees are significantly changed, thereby enhancing the immune defense ability of the bees, reducing the possibility of disease and improving their overall survival rate. In addition, the composition has no drug residue after use and is highly safe.

[0025] The composition of the present invention is prepared by combining the traditional Chinese herbal medicine liquid preparation process with the modern probiotic preparation process. Compared with other preparation methods, the components used in the preparation of the herbal medicine liquid in this method are all common Chinese medicines, which have low cost, high safety, and no side effects. It not only reduces the economic losses of farmers, but also meets people's demand for green food, which is in line with the development trend. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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.

[0027] Figure 1 This is a physical picture of the composition liquid of the present invention;

[0028] Figure 2 Antagonism between the strains of the present invention;

[0029] Figure 3 The statistics of the changes of glycerol content in bees of the present invention;

[0030] Figure 4 Statistics of changes in protein content in bees of the present invention;

[0031] Figure 5 It is the statistics of the insect oxidase (POD) activity of the present invention;

[0032] Figure 6 It is the statistics of catalase CAT activity of the present invention;

[0033] Figure 7 This is the total antioxidant capacity T-AOC measurement statistics of the present invention. DETAILED DESCRIPTION

[0034] 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.

[0035] Example 1 Example 1

[0036] A method for preparing a composition for improving the cold resistance of bees comprises the following steps:

[0037] Step 1: Prepare Chinese herbal medicine solution.

[0038] Weigh the Chinese herbs according to the following formula: Polygonum cuspidatum (9g), Astragalus membranaceus (30g), White Peony Root (15g), Radix Isatidis (9g) and Rhizoma Dioscoreae (8g). Grind the Chinese herbs into powder and pass through a 40-mesh sieve. Ultrasonicate the Astragalus membranaceus with 100ml RO water for 10 minutes and soak it for 20min. Then mix the remaining Chinese herbs together and add 200ml RO water to soak for 1h. Then, bathe in 100℃ water for 1h, centrifuge twice to remove insoluble matter, filter, repeat the above steps 3 times, and combine the collected supernatants to make a fixed volume of 600ml to obtain the Chinese herbal medicine solution.

[0039] Step 2: Prepare probiotic seed fermentation liquid.

[0040] Use an inoculating loop to dip a small amount of sample from the culture storage liquid of Lactobacillus buchneri, Lactobacillus reuteri and Bacillus Nakamura, perform streak culture on the plate, and then select a single colony from it and inoculate it into 100 mL of MRS and NB liquid culture medium. Incubate at 37°C with shaking for 24 hours, measure the absorbance under a UV spectrophotometer and adjust the concentration to OD600=1. Mix the two in a ratio of 1:1:1 to obtain the fermentation seed liquid.

[0041] Step 3: preparing the composition.

[0042] The probiotic seed fermentation liquid is inoculated into the Chinese herbal medicine liquid at an inoculation rate of 1% by volume, and fermented at 34° C. for 3 days to obtain a composition, such as Figure 1 shown.

[0043] After the honey bees were fed with the composition, the total antioxidant capacity of the honey bees during the winter increased by 26%.

[0044] Example 2

[0045] A method for preparing a composition for improving the cold resistance of bees comprises the following steps:

[0046] Step 1: Prepare Chinese herbal medicine solution.

[0047] Weigh the Chinese herbs according to the following formula: Polygonum cuspidatum (9g), Astragalus membranaceus (30g), White Peony Root (15g), Radix Isatidis (9g) and Rhizoma Dioscoreae (8g). Grind the Chinese herbs into powder and pass through a 40-mesh sieve. Ultrasonicate the Astragalus membranaceus with 100ml RO water for 10 minutes and soak it for 20min. Then mix the remaining Chinese herbs together and add 200ml RO water to soak for 1h. Then, bathe in 100℃ water for 1h, centrifuge twice to remove insoluble matter, filter, repeat the above steps 3 times, and combine the collected supernatants to make a fixed volume of 600ml to obtain the Chinese herbal medicine solution.

[0048] Step 2: Prepare probiotic seed fermentation liquid.

[0049] Use an inoculating loop to dip a small amount of sample from the culture storage liquid of Lactobacillus buchneri, Lactobacillus reuteri and Bacillus Nakamura, perform streak culture on the plate, and then select a single colony from it and inoculate it into 100 mL of MRS and NB liquid culture medium. Incubate at 37°C with shaking for 24 hours, measure the absorbance under a UV spectrophotometer and adjust the concentration to OD600=1. Mix the two in a ratio of 1:1:1 to obtain the fermentation seed liquid.

[0050] Step 3: preparing the composition.

[0051] The probiotic seed fermentation liquid is inoculated into the Chinese herbal medicine liquid at an inoculation rate of 2% by volume, and fermented at 34° C. for 5 days to obtain a composition, such as Figure 1 shown.

[0052] After the honey bees were fed with the composition, the total antioxidant capacity of the honey bees during the winter increased by 10%.

[0053] Example 3

[0054] A method for preparing a composition for improving the cold resistance of bees comprises the following steps:

[0055] Step 1: Prepare Chinese herbal medicine solution.

[0056] Weigh the Chinese herbs according to the following formula: Polygonum cuspidatum (9g), Astragalus membranaceus (30g), White Peony Root (15g), Radix Isatidis (9g) and Rhizoma Dioscoreae (8g). Grind the Chinese herbs into powder and pass through a 40-mesh sieve. Ultrasonicate the Astragalus membranaceus with 100ml RO water for 10 minutes and soak it for 20min. Then mix the remaining Chinese herbs together and add 200ml RO water to soak for 1h. Then, bathe in 100℃ water for 1h, centrifuge twice to remove insoluble matter, filter, repeat the above steps 3 times, and combine the collected supernatants to make a fixed volume of 600ml to obtain the Chinese herbal medicine solution.

[0057] Step 2: Prepare probiotic seed fermentation liquid.

[0058] Use an inoculating loop to dip a small amount of sample from the culture storage liquid of Lactobacillus buchneri, Lactobacillus reuteri and Bacillus Nakamura, perform streak culture on the plate, and then select a single colony from it and inoculate it into 100 mL of MRS and NB liquid culture medium. Culture at 37°C with shaking for 24 hours, measure the absorbance under a spectrophotometer and adjust the concentration to OD600=1. Mix the two in a ratio of 1:1:1 to obtain the fermentation seed liquid.

[0059] Step 3: preparing the composition.

[0060] The probiotic seed fermentation liquid is inoculated into the Chinese herbal medicine liquid at an inoculation rate of 3% by volume, and the composition is obtained after natural fermentation at 34° C. for 7 days. Figure 1 shown.

[0061] After the honey bees were fed with the composition, the total antioxidant capacity of the honey bees during the winter increased by 34%.

[0062] Example 4

[0063] A method for preparing a composition for improving the cold resistance of bees comprises the following steps:

[0064] Step 1: Prepare Chinese herbal medicine solution.

[0065] Weigh the Chinese herbs according to the following formula: Polygonum cuspidatum (9g), Astragalus membranaceus (30g), White Peony Root (15g), Radix Isatidis (9g) and Rhizoma Dioscoreae (8g). Grind the Chinese herbs into powder and pass through a 40-mesh sieve. Ultrasonicate the Astragalus membranaceus with 100ml RO water for 10 minutes and soak it for 20min. Then mix the remaining Chinese herbs together and add 200ml RO water to soak for 1h. Then, bathe in 100℃ water for 1h, centrifuge twice to remove insoluble matter, filter, repeat the above steps 3 times, and combine the collected supernatants to make a fixed volume of 600ml to obtain the Chinese herbal medicine solution.

[0066] Step 2: Prepare probiotic seed fermentation liquid.

[0067] Use an inoculating loop to dip a small amount of sample from the culture storage liquid of Lactobacillus buchneri, Lactobacillus reuteri and Bacillus Nakamura, perform streak culture on the plate, and then select a single colony from it and inoculate it into 100 mL of MRS and NB liquid culture medium. Incubate at 37°C with shaking for 24 hours, measure the absorbance under a UV spectrophotometer and adjust the concentration to OD600=1. Mix the two in a ratio of 1:1:1 to obtain the fermentation seed liquid.

[0068] Step 3: preparing the composition.

[0069] The probiotic seed fermentation liquid is inoculated into the Chinese herbal medicine liquid at an inoculation rate of 1% by volume, and the composition is obtained after natural fermentation at 36° C. for 3 days. Figure 1 shown.

[0070] After the honey bees were fed with the composition, the total antioxidant capacity of the honey bees during the winter increased by 24%.

[0071] Example 5

[0072] A method for preparing a composition for improving the cold resistance of bees comprises the following steps:

[0073] Step 1: Prepare Chinese herbal medicine solution.

[0074] Weigh the Chinese herbs according to the following formula: Polygonum cuspidatum (9g), Astragalus membranaceus (30g), White Peony Root (15g), Radix Isatidis (9g) and Rhizoma Dioscoreae (8g). Grind the Chinese herbs into powder and pass through a 40-mesh sieve. Ultrasonicate the Astragalus membranaceus with 100ml RO water for 10 minutes and soak it for 20min. Then mix the remaining Chinese herbs together and add 200ml RO water to soak for 1h. Then, bathe in 100℃ water for 1h, centrifuge twice to remove insoluble matter, filter, repeat the above steps 3 times, and combine the collected supernatants to make a fixed volume of 600ml to obtain the Chinese herbal medicine solution.

[0075] Step 2: Prepare probiotic seed fermentation liquid.

[0076] Use an inoculating loop to dip a small amount of sample from the culture storage liquid of Lactobacillus buchneri, Lactobacillus reuteri and Bacillus Nakamura, perform streak culture on the plate, and then select a single colony from it and inoculate it into 100 mL of MRS and NB liquid culture medium. Incubate at 37°C with shaking for 24 hours, measure the absorbance under a UV spectrophotometer and adjust the concentration to OD600=1. Mix the two in a ratio of 1:1:1 to obtain the fermentation seed liquid.

[0077] Step 3: preparing the composition.

[0078] The probiotic seed fermentation liquid is inoculated into the Chinese herbal medicine liquid at an inoculation rate of 2% by volume, and the composition is obtained after natural fermentation at 36° C. for 5 days. Figure 1 shown.

[0079] After the honey bees were fed with the composition, their total antioxidant capacity during wintering increased by 25%.

[0080] Example 6

[0081] A method for preparing a composition for improving the cold resistance of bees comprises the following steps:

[0082] Step 1: Prepare Chinese herbal medicine solution.

[0083] Weigh the Chinese herbs according to the following formula: Polygonum cuspidatum (9g), Astragalus membranaceus (30g), White Peony Root (15g), Radix Isatidis (9g) and Rhizoma Dioscoreae (8g). Grind the Chinese herbs into powder and pass through a 40-mesh sieve. Ultrasonicate the Astragalus membranaceus with 100ml RO water for 10 minutes and soak it for 20min. Then mix the remaining Chinese herbs together and add 200ml RO water to soak for 1h. Then, bathe in 100℃ water for 1h, centrifuge twice to remove insoluble matter, filter, repeat the above steps 3 times, and combine the collected supernatants to make a fixed volume of 600ml to obtain the Chinese herbal medicine solution.

[0084] Step 2: Prepare probiotic seed fermentation liquid.

[0085] Use an inoculating loop to dip a small amount of sample from the culture storage liquid of Lactobacillus buchneri, Lactobacillus reuteri and Bacillus Nakamura, perform streak culture on the plate, and then select a single colony from it and inoculate it into 100 mL of MRS and NB liquid culture medium. Incubate at 37°C with shaking for 24 hours, measure the absorbance under a UV spectrophotometer and adjust the concentration to OD600=1. Mix the two in a ratio of 1:1:1 to obtain the fermentation seed liquid.

[0086] Step 3: preparing the composition.

[0087] The probiotic seed fermentation liquid is inoculated into the Chinese herbal medicine liquid at an inoculation rate of 3% by volume, and the composition is obtained after natural fermentation at 36° C. for 7 days. Figure 1 shown.

[0088] After the honey bees were fed with the composition, the total antioxidant capacity of the honey bees during the winter increased by 14%.

[0089] Example 7

[0090] A method for preparing a composition for improving the cold resistance of bees comprises the following steps:

[0091] Step 1: Prepare Chinese herbal medicine solution.

[0092] Weigh the Chinese herbs according to the following formula: Polygonum cuspidatum (9g), Astragalus membranaceus (30g), White Peony Root (15g), Radix Isatidis (9g) and Rhizoma Dioscoreae (8g). Grind the Chinese herbs into powder and pass through a 40-mesh sieve. Ultrasonicate the Astragalus membranaceus with 100ml RO water for 10 minutes and soak it for 20min. Then mix the remaining Chinese herbs together and add 200ml RO water to soak for 1h. Then, bathe in 100℃ water for 1h, centrifuge twice to remove insoluble matter, filter, repeat the above steps 3 times, and combine the collected supernatants to make a fixed volume of 600ml to obtain the Chinese herbal medicine solution.

[0093] Step 2: Prepare probiotic seed fermentation liquid.

[0094] Use an inoculating loop to dip a small amount of sample from the culture storage liquid of Lactobacillus buchneri, Lactobacillus reuteri and Bacillus Nakamura, perform streak culture on the plate, and then select a single colony from it and inoculate it into 100 mL of MRS and NB liquid culture medium. Incubate at 37°C with shaking for 24 hours, measure the absorbance under a UV spectrophotometer and adjust the concentration to OD600=1. Mix the two in a ratio of 1:1:1 to obtain the fermentation seed liquid.

[0095] Step 3: preparing the composition.

[0096] The probiotic seed fermentation liquid is inoculated into the Chinese herbal medicine liquid at an inoculation rate of 1% by volume, and the composition is obtained after natural fermentation at 38° C. for 3 days. Figure 1 shown.

[0097] After the honey bees were fed with the composition, their total antioxidant capacity during the winter increased by 32%.

[0098] Example 8

[0099] A method for preparing a composition for improving the cold resistance of bees comprises the following steps:

[0100] Step 1: Prepare Chinese herbal medicine solution.

[0101] Weigh the Chinese herbs according to the following formula: Polygonum cuspidatum (9g), Astragalus membranaceus (30g), White Peony Root (15g), Radix Isatidis (9g) and Rhizoma Dioscoreae (8g). Grind the Chinese herbs into powder and pass through a 40-mesh sieve. Ultrasonicate the Astragalus membranaceus with 100ml RO water for 10 minutes and soak it for 20min. Then mix the remaining Chinese herbs together and add 200ml RO water to soak for 1h. Then, bathe in 100℃ water for 1h, centrifuge twice to remove insoluble matter, filter, repeat the above steps 3 times, and combine the collected supernatants to make a fixed volume of 600ml to obtain the Chinese herbal medicine solution.

[0102] Step 2: Prepare probiotic seed fermentation liquid.

[0103] Use an inoculating loop to dip a small amount of sample from the culture storage liquid of Lactobacillus buchneri, Lactobacillus reuteri and Bacillus Nakamura, perform streak culture on the plate, and then select a single colony from it and inoculate it into 100 mL of MRS and NB liquid culture medium. Incubate at 37°C with shaking for 24 hours, measure the absorbance under a UV spectrophotometer and adjust the concentration to OD600=1. Mix the two in a ratio of 1:1:1 to obtain the fermentation seed liquid.

[0104] Step 3: preparing the composition.

[0105] The probiotic seed fermentation liquid is inoculated into the Chinese herbal medicine liquid at an inoculation rate of 2% by volume, and the composition is obtained after natural fermentation at 38° C. for 5 days. Figure 1 shown.

[0106] After the honey bees were fed with the composition, the total antioxidant capacity of the honey bees during the winter increased by 12%.

[0107] Example 9

[0108] A method for preparing a composition for improving the cold resistance of bees comprises the following steps:

[0109] Step 1: Prepare Chinese herbal medicine solution.

[0110] Weigh the Chinese herbs according to the following formula: Polygonum cuspidatum (9g), Astragalus membranaceus (30g), White Peony Root (15g), Radix Isatidis (9g) and Rhizoma Dioscoreae (8g). Grind the Chinese herbs into powder and pass through a 40-mesh sieve. Ultrasonicate the Astragalus membranaceus with 100ml RO water for 10 minutes and soak it for 20min. Then mix the remaining Chinese herbs together and add 200ml RO water to soak for 1h. Then, bathe in 100℃ water for 1h, centrifuge twice to remove insoluble matter, filter, repeat the above steps 3 times, and combine the collected supernatants to make a fixed volume of 600ml to obtain the Chinese herbal medicine solution.

[0111] Step 2: Prepare probiotic seed fermentation liquid.

[0112] Use an inoculating loop to dip a small amount of sample from the culture storage liquid of Lactobacillus buchneri, Lactobacillus reuteri and Bacillus Nakamura, perform streak culture on the plate, and then select a single colony from it and inoculate it into 100 mL of MRS and NB liquid culture medium. Incubate at 37°C with shaking for 24 hours, measure the absorbance under a UV spectrophotometer and adjust the concentration to OD600=1. Mix the two in a ratio of 1:1:1 to obtain the fermentation seed liquid.

[0113] Step 3: preparing the composition.

[0114] The probiotic seed fermentation liquid is inoculated into the Chinese herbal medicine liquid at an inoculation rate of 3% by volume, and the composition is obtained after natural fermentation at 38° C. for 7 days. Figure 1 shown.

[0115] After the honey bees were fed with the composition, their total antioxidant capacity during the winter increased by 42%.

[0116] Example 10

[0117] The 9 groups of Chinese herbal medicine liquids fermented by composite probiotics obtained in Examples 1 to 9 above were tested for polysaccharide, total flavonoids, amino acids, lactic acid content, DPPH free radical scavenging ability and T-AOC. It was shown that the lactic acid and flavonoid contents of experimental groups Z4 to Z6 were the highest among the 9 experimental groups, and the polysaccharide content of experimental group Z1 was the highest at (0.29±0.15) mg / mL, among which the highest T-AOC content was in experimental group Z6. In measuring the lactic acid, flavonoids and T-AOC contents, experimental groups Z4, Z5 and Z6 showed significant changes compared with other experimental groups (P<0.05). The lowest DPPH free radical scavenging concentrations were in experimental groups Z4, Z5 and Z8. The results are shown in Table 1.

[0118]

[0119] Table 1 Indicators of Chinese herbal medicine after fermentation

[0120] Embodiment 11

[0121] An application of a composition for improving the cold resistance of bees, specifically as follows:

[0122] The compositions prepared in the above Examples 1 to 9 were used in the following experiments. The bees in the control group were fed with a 50% by mass sucrose solution prepared from RO water and sucrose, and the bees in the experimental groups were fed with a 50% by mass sugar solution of Chinese herbal medicine fermented by compound probiotics prepared from Z1 to Z9 and sucrose.

[0123] Chinese bees were captured, and 50 bees were used as one test group in a box, and each group was repeated 3 times. According to the experimental groups 1 to 9, they were fed separately for 12 days, and then placed in a low-temperature (4°C) incubator for cold training for two hours and then sampled. 20 bees were sampled for each repetition each time and stored in a -80°C refrigerator. The remaining samples were tested for antioxidant indicators. The results of weight changes before and after 12 days of feeding Chinese bees are shown in Table 2. Except for experimental group Z2, the weight of Chinese bees in other experimental groups was higher than that of the control group, and there was a very significant difference compared with the control group, indicating that feeding Chinese herbal medicine fermented with probiotics during the winter is beneficial to overwintering Chinese bees.

[0124]

[0125] Table 2 Changes in body weight before and after 12 days of rearing of Chinese honey bees

[0126] After the composition was fed to bees for 12 days, three intestines of each bee were placed in an Eppendorf tube, and each group was repeated three times. A certain amount of bee intestinal tissue was taken and added with the extract according to the kit. The supernatant was centrifuged and the glycerol and protein contents were determined using an enzyme marker. The results were as follows: Figure 3 and Figure 4As shown, the glycerol and protein contents in the body of Chinese bees were significantly different between the experimental group and the control group (P < 0.0001), and the experimental group Z9 had an extremely significant difference (P = 0.0004). The protein concentration in the experimental group Z1 was the highest, which was more than three times that of the control group. This shows that feeding Chinese herbal medicine can enhance the immunity of Chinese bees, and the cold-resistant substance glycerol in the body has been significantly improved.

[0127] The antioxidant enzyme system is an important resistance mechanism for insects and other cold-blooded animals to resist temperature stress. Under normal circumstances, the production and removal of free radicals in cells rely on the coordinated action of antioxidant enzymes such as peroxidase POD and catalase CAT to participate in the removal of free radicals in the bee body, which has a direct impact on the lifespan and disease resistance of bees. Figure 5 , 6 and Figure 7 As shown, the activities of peroxidase and catalase in experimental groups Z1 to Z9 were higher than those in the control group, the peroxidase activity in experimental group Z6 was the highest, and there was a significant difference between experimental groups Z6 and Z9 (P < 0.0001).

[0128] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0129] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing a composite material for improving the cold resistance of Chinese honey bees, characterized in that: The following steps are involved: S1. Weighing Chinese herbal medicine raw materials Weigh 8-10 parts of Polygonum cuspidatum, 29-31 parts of Astragalus, 14-16 parts of White Peony Root, 8-10 parts of Isatis indigotica and 7-9 parts of Rhizoma Dioscoreae by weight; S2. Preparation of Chinese herbal medicine liquid Grind the Chinese herbal medicine raw materials weighed in S1 into powder and pass through a 40-mesh sieve; The powdered astragalus is first sonicated with 90-110 ml of RO water for 8-12 minutes, and then soaked for 18-22 minutes to obtain an initial astragalus solution; Mix the remaining Chinese herbal medicines together, add them into 200-220 ml of RO water and soak for 1-1.5 hours to obtain a mixed initial solution; The container containing the initial Astragalus solution and the container containing the mixed initial solution are placed in a water bath at 90 to 110°C for 1 to 1.5 hours of water bath heating, and the two containers are centrifuged twice to remove insoluble matter, and then filtered, and the collected supernatants are combined and fixed to 600 ml to obtain the Chinese herbal medicine solution; S3. Preparation of probiotic fermentation seed liquid Use an inoculating loop to take a small amount of sample from the thawed solution of probiotics and perform streak culture on the plate; After streaking culture on the plate, a single colony was selected and inoculated into 100 mL of MRS and NB liquid culture medium, and cultured at 37°C with shaking for 24 h. The absorbance was measured under a spectrophotometer and the concentration was adjusted to OD600 = 1, and mixed at a ratio of 1:1:1 to obtain probiotic fermentation seed liquid. S4. Inoculate the prepared probiotic fermentation seed liquid into the Chinese herbal medicine liquid at an inoculation rate of 1% to 3% by volume, and ferment at 34 to 38° C. for 3 to 7 days to obtain a composition that improves the wintering cold resistance of Chinese honey bees.

2. The method for preparing a composite material for improving the cold resistance of honey bees according to claim 1, characterized in that: In S1, the Chinese herbal medicine raw materials, calculated by weight, include 9 parts of Polygonum cuspidatum, 30 parts of Astragalus, 15 parts of White Peony Root, 9 parts of Isatis Indigotica and 8 parts of Rhizoma Dioscoreae.

3. The method for preparing a composite material for improving the cold resistance of Chinese honey bees according to claim 1, characterized in that: In S3, the probiotics are a mixture of Lactobacillus buchneri, Lactobacillus reuteri and Bacillus Nakamura.

4. The method for preparing a composite material for improving the cold resistance of honey bees according to claim 1, characterized in that: In S4, the inoculation amount of the probiotic fermentation seed liquid inoculated into the Chinese herbal medicine liquid was 1%, the fermentation time was 3 days, and the fermentation temperature was 36°C.

5. The use of a composition for improving the cold resistance of bees according to any one of claims 1 to 4, characterized in that: The composition is used to make a product for improving the cold resistance of bees in winter.

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