Preparation method of traditional Chinese medicine extract and application thereof

Abstract

This invention relates to the field of extracts, specifically to a method for preparing a traditional Chinese medicine extract and its application. The method includes pulverizing and sieving a traditional Chinese medicine formula, preparing a compound enzymatic hydrolysate using a compound enzyme, mixing the powdered medicine with the hydrolysate, performing enzymatic hydrolysis, ultrasonic extraction, fermentation, filtration, and freeze-drying to obtain the traditional Chinese medicine extract. The extract is used as an immune enhancer for animals. Existing methods for preparing traditional Chinese medicine extracts suffer from low extraction efficiency and poor drug efficacy. The method for preparing a traditional Chinese medicine extract and its application provided by this invention offers higher extraction efficiency and better drug efficacy.

Description

Technical Field

[0001] This invention relates to the field of extracts, and more specifically to a method for preparing extracts of traditional Chinese medicine and their applications. Background Technology

[0002] Animal husbandry is an important industry related to people's livelihood. In order to better ensure the quality and supply of meat, eggs and milk, the breeding model has changed from free-range to the establishment of large-scale, facility-based, intensive and standardized modern farms to improve the comprehensive production capacity of animal husbandry.

[0003] Modern farms concentrate animals in a relatively concentrated manner, which makes the spread of diseases faster and wider. Therefore, strict disease prevention and control measures must be implemented. Otherwise, all animals on the farm may be affected, resulting in significant economic losses.

[0004] Disease prevention and control in farms is mainly achieved through vaccination. If the corresponding vaccine is not administered, animals may still become infected. In addition, in order to further improve animal immunity, people have thought of using immune enhancers to enhance the immune response function of the animal's immune system. Traditional immune enhancers include aluminum salt adjuvants, calcium phosphate adjuvants, and water-in-oil emulsion adjuvants, but these immune enhancers are often accompanied by side effects caused by excessive immune activation.

[0005] Therefore, people began to study immune enhancers of natural origin. Natural immune enhancers are usually prepared from plant extracts, herbs or natural products, and usually have lower toxicity and better biocompatibility. In recent years, there have been cases of using Chinese herbal extracts as immune enhancers.

[0006] Currently, the most widely used method for preparing extracts of traditional Chinese medicine is fermentation. However, traditional Chinese medicine usually contains substances such as acids, phenols, or aromatic compounds that can inhibit bacterial growth, resulting in low fermentation efficiency or no fermentation at all. Furthermore, the drug effects of the fermented products are not ideal, which seriously restricts the development of immune enhancers derived from traditional Chinese medicine.

[0007] Therefore, existing methods for preparing Chinese herbal extracts suffer from low extraction efficiency and poor drug efficacy. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a method for preparing traditional Chinese medicine extracts with high extraction efficiency and good drug effect, and its application.

[0009] To solve the above-mentioned technical problems, the present invention provides a method for preparing a traditional Chinese medicine extract, comprising:

[0010] S1. After pulverizing the Chinese herbal formula, sieve it to obtain medicinal powder;

[0011] S2. Prepare a compound enzyme hydrolysate using a compound enzyme, with a volume of 5L to 1Kg of drug powder; add disodium hydrogen phosphate-citric acid buffer to the compound enzyme hydrolysate to adjust the pH to 4.8-5.2;

[0012] S3. Mix the powdered medicine with the compound enzymatic hydrolysate, heat it in a water bath to 45℃~50℃ and keep it at that temperature for 72 hours to carry out enzymatic hydrolysis, and obtain mixture A;

[0013] S4. Perform ultrasonic extraction on mixture A to obtain extract B;

[0014] S5. Ferment extract B to obtain fermentation broth C;

[0015] S6. Filter the fermentation broth C and freeze-dry it to obtain the Chinese herbal extract.

[0016] As a further improvement of the present invention: the traditional Chinese medicine formula in S1 includes 10g-16g of Atractylodes macrocephala, 10g-16g of Astragalus membranaceus, 3g-5g of Saposhnikovia divaricata, 3g-5g of Panax ginseng, 3g-5g of Bupleurum chinense, 1g-3g of Poria cocos, 0g-2g of Cimicifuga foetida, and 0g-2g of Angelica sinensis.

[0017] As a further improvement of the present invention: the mesh size of the sieve used for sieving in S1 is 50 to 100 mesh.

[0018] As a further improvement of the present invention: the complex enzyme in S2 includes acidic cellulase, protease and pectinase, and the enzyme activity of acidic cellulase: enzyme activity of protease: enzyme activity of pectinase in the complex enzyme is 2:1:1.

[0019] As a further improvement of the present invention: the concentration of the complex enzyme in the complex enzyme hydrolysate in S2 is 5 g / L.

[0020] As a further improvement of the present invention: the preparation of the complex enzyme hydrolysate using a complex enzyme as described in S2 includes:

[0021] S201. Weigh 3.659g of acidic cellulase powder, 0.732g of protease powder and 0.610g of pectinase powder to obtain a compound enzyme;

[0022] S202. Weigh out 6.3g of citric acid monohydrate and 17.6g of sodium citrate dihydrate;

[0023] S203. Dissolve citric acid monohydrate and sodium citrate dihydrate in 800ml of deionized water, add the compound enzyme, stir and mix for 10min to obtain 1L of compound enzyme hydrolysate.

[0024] As a further improvement of the present invention: the ultrasonic extraction described in S4 is performed by an ultrasonic extraction device, the ultrasonic power of which is 400w to 600w and the ultrasonic extraction time is 30min to 90min.

[0025] The present invention also provides a traditional Chinese medicine extract prepared by the method described above.

[0026] The present invention also provides an application of the above-mentioned Chinese herbal extract as an immune enhancer for animals.

[0027] The beneficial effects of the present invention are as follows: The method for preparing traditional Chinese medicine extract provided by the present invention uses enzymatic hydrolysis technology to enhance the efficacy of traditional Chinese medicine, thereby making the preparation method and its application have high extraction efficiency and good drug effect.

[0028] The preparation method shortens the extraction time and improves the extraction efficiency through enzymatic hydrolysis. Experiment (III) shows that the Chinese medicine extract prepared by this method has good drug effect. After being made into a vaccine, it can improve the antibody level induced by the vaccine, thereby achieving the antiviral effect and having a good immune enhancement effect. Experiment (I) shows that the animal mortality rate is significantly reduced, which indicates that the Chinese medicine extract can also play an immune enhancement role when directly used as a drinking water additive, making the epidemic prevention safety of the farm more stable.

[0029] In addition, the large amount of manure produced by animals in farms needs to be treated in a timely manner, otherwise it will breed bacteria, aggravate the risk of disease, and pollute the surrounding environment. Farms usually transport animal manure to organic fertilizer production areas for processing because direct use of animal manure will cause seedling burn, so it cannot be directly returned to the field and must be processed. However, as shown in Experiments (I) and (II), even a small amount of the herbal extract provided by this invention can improve feed conversion rate after animals use it. The improved feed conversion rate not only reduces feeding costs but also means that the nutrients in the feed are digested and absorbed more fully by the animals, leaving less nutrients in the manure. Therefore, the manure produced by animals that have taken the herbal extract provided by this invention can be directly returned to the field. With appropriate addition, seedling burn will not occur, and it can also promote crop root development, growth, yield increase, and nutrient content increase. In other words, this herbal extract can also help farms reduce a manure processing step and reduce workload. Attached Figure Description

[0030] Figure 1 Here is a photograph of the herbal extract obtained by this invention;

[0031] Figure 2 The diagram shows the drug use cycle for experimental groups 1 and 2 in Experiment (I).

[0032] Figure 3(A) is a photograph of experimental field 1;

[0033] Figure 3(B) is a photograph of experimental field 2;

[0034] Figure 3(C) is a photograph of experimental field 3;

[0035] Figure 3(D) is a photograph of the control field 1;

[0036] Figure 3(E) is a photograph of the control field 2;

[0037] Figure 4 A comparison chart of bok choy yields in different fields;

[0038] Figure 5(A) shows a photograph of the root system of the milk cabbage in experimental field 1;

[0039] Figure 5(B) shows a photograph of the root system of the milk cabbage in experimental field 2;

[0040] Figure 5(C) shows a photograph of the root system of the milk cabbage in experimental field 3;

[0041] Figure 5(D) shows a photograph of the root system of the milk cabbage in control field 1;

[0042] Figure 5(E) shows a photograph of the root system of the Chinese cabbage in control field 2;

[0043] Figure 6(a) shows a comparison of the vitamin C content of bok choy in different fields;

[0044] Figure 6(b) shows a comparison of β-carotene content in different fields of bok choy;

[0045] Figure 6(c) is a comparison of the carbohydrate content of bok choy in different fields;

[0046] Figure 6(d) is a comparison of ORAC (oxygen free radical absorption capacity) of bok choy in different fields;

[0047] Figure 6(e) is a comparison of the cellulose content of bok choy in different fields;

[0048] Figure 6(f) shows a comparison of the calcium content of bok choy in different fields;

[0049] Figure 6(g) is a comparison of the zinc content of bok choy in different fields;

[0050] Figure 6(h) is a comparison of the iron content of bok choy in different fields;

[0051] Figure 7 This is a comparison chart of the OD450 values ​​of each vaccine in Experiment (III). Detailed Implementation

[0052] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0053] The present invention provides a method for preparing a traditional Chinese medicine extract, comprising:

[0054] S1. The Chinese herbal medicine formula is pulverized using a toothed claw pulverizer according to the operating instructions, and then sieved through a sieve with a mesh size of 50 to 100 to obtain medicinal powder;

[0055] The traditional Chinese medicine formula includes Atractylodes macrocephala 10g~16g, Astragalus membranaceus 10g~16g, Saposhnikovia divaricata 3g~5g, Panax ginseng 3g~5g, Bupleurum chinense 3g~5g, Poria cocos 1g~3g, Cimicifuga foetida 0g~2g, and Angelica sinensis 0g~2g.

[0056] S2. A compound enzyme hydrolysate was prepared using a compound enzyme, with a volume of 5L to 1Kg of drug powder. To ensure the hydrolysis efficiency, disodium hydrogen phosphate-citric acid buffer was added to the compound enzyme hydrolysate to adjust the pH to 4.8-5.2, thus locking the pH of the compound enzyme hydrolysate within this range.

[0057] The concentration of the complex enzyme in the complex enzymatic hydrolysate is 5 g / L; the complex enzyme includes acidic cellulase, protease and pectinase, and the enzyme activity of acidic cellulase:protease:pectinase in the complex enzyme is 2:1:1; among them, the enzyme activity of acidic cellulase is 20000 u / g, the enzyme activity of protease is 50000 u / g, and the enzyme activity of pectinase is 60000 u / g.

[0058] The preparation of 1L of complex enzyme hydrolysate using a complex enzyme includes:

[0059] S201. Weigh 3.659g of acidic cellulase powder, 0.732g of protease powder and 0.610g of pectinase powder to obtain a compound enzyme;

[0060] S202. Weigh out 6.3g of citric acid monohydrate and 17.6g of sodium citrate dihydrate;

[0061] S203. Dissolve citric acid monohydrate and sodium citrate dihydrate in 800ml of deionized water, add the compound enzyme, stir and mix for 10min, and then bring the volume to 1L to obtain the compound enzyme hydrolysate.

[0062] S3. Mix the powdered medicine with the compound enzymatic hydrolysate, heat it in a water bath to 45℃~50℃ and keep it at that temperature for 72 hours to carry out enzymatic hydrolysis, and obtain mixture A;

[0063] The mixture was stirred with a glass rod in a beaker for 0.5 hours.

[0064] S4. Use an ultrasonic extraction device to perform ultrasonic extraction on mixture A to obtain extract B;

[0065] The ultrasonic power for ultrasonic extraction is 400w~600w, and the ultrasonic extraction time is 30min~90min.

[0066] S5. Place the extract B in a fermentation tank with a layer of far-infrared stones, and let it ferment for 5-7 days to obtain fermentation liquid C.

[0067] S6. The fermentation broth C is filtered through a plate filter press and freeze-dried to obtain the Chinese herbal extract. A pulverizing process can also be added after freeze-drying.

[0068] The present invention also provides a traditional Chinese medicine extract prepared by the method described above.

[0069] The present invention also provides the application of the Chinese Herbal Extract (CHE) as described above as an immune enhancer for animals.

[0070] The chemical reagents involved in the following examples are all commercially available products;

[0071] Example 1

[0072] S1. The Chinese herbal medicine formula is pulverized using a toothed claw pulverizer according to the operating instructions, and then sieved through a 50-mesh sieve to obtain medicinal powder;

[0073] The traditional Chinese medicine formula includes Atractylodes macrocephala 10g, Astragalus membranaceus 13g, Saposhnikovia divaricata 4g, Panax ginseng 5g, Bupleurum chinense 3g, Poria cocos 1g, and Angelica sinensis 2g.

[0074] S2. A compound enzyme hydrolysate was prepared using a compound enzyme, with a volume of 5 L of compound enzyme hydrolysate to 1 kg of drug powder. Disodium hydrogen phosphate-citric acid buffer was added to the compound enzyme hydrolysate to adjust the pH to 4.8.

[0075] S3. Mix the powdered medicine and the compound enzymatic hydrolysate in a beaker by stirring with a glass rod for 0.5 h, and heat in a water bath to 45°C and keep warm for 72 h to carry out enzymatic hydrolysis, to obtain mixture A;

[0076] S4. Use an ultrasonic extraction device to ultrasonically extract mixture A at an ultrasonic power of 400W for 90 minutes to obtain extract B;

[0077] S5. Place the extract B in a fermentation tank with a layer of far-infrared stones, let it ferment for 5 days, and obtain fermentation liquid C.

[0078] S6. Filter the fermentation broth C through a plate filter press and freeze-dry it to obtain CHE.

[0079]

Example 2

[0080] S1. The Chinese herbal medicine formula is pulverized using a toothed claw pulverizer according to the operating instructions, and then sieved through a 100-mesh sieve to obtain medicinal powder;

[0081] The traditional Chinese medicine formula includes 16g of Atractylodes macrocephala, 10g of Astragalus membranaceus, 3g of Saposhnikovia divaricata, 4g of Panax ginseng, 5g of Bupleurum chinense, 2g of Poria cocos, and 1g of Cimicifuga foetida.

[0082] S2. A compound enzyme hydrolysate was prepared using a compound enzyme, with a volume of 5 L of compound enzyme hydrolysate to 1 kg of drug powder. Disodium hydrogen phosphate-citric acid buffer was added to the compound enzyme hydrolysate to adjust the pH to 4.9.

[0083] S3. Mix the powdered medicine and the compound enzymatic hydrolysate in a beaker by stirring with a glass rod for 0.5 h, and heat in a water bath to 46 ℃ and keep warm for 72 h to carry out enzymatic hydrolysis, to obtain mixture A;

[0084] S4. Using an ultrasonic extraction device, the mixture A was ultrasonically extracted for 60 minutes at an ultrasonic power of 500W to obtain extract B;

[0085] S5. Place the extract B in a fermentation tank with a layer of far-infrared stones, let it ferment for 6 days, and obtain fermentation liquid C.

[0086] S6. Filter the fermentation broth C through a plate filter press and freeze-dry it to obtain CHE.

[0087]

Example 3

[0088] S1. The Chinese herbal medicine formula is pulverized according to the operating instructions using a toothed claw pulverizer and then sieved through a 75-mesh sieve to obtain medicinal powder;

[0089] The traditional Chinese medicine formula includes Atractylodes macrocephala 13g, Astragalus membranaceus 16g, Saposhnikovia divaricata 5g, Panax ginseng 3g, Bupleurum chinense 4g, Poria cocos 3g, Cimicifuga foetida 2g, and Angelica sinensis 1g.

[0090] S2. Prepare a compound enzyme hydrolysate using a compound enzyme, with a volume of 5 L of compound enzyme hydrolysate to 1 kg of drug powder; add disodium hydrogen phosphate-citric acid buffer to the compound enzyme hydrolysate to adjust the pH to 5.0;

[0091] S3. Mix the powdered medicine and the compound enzymatic hydrolysate in a beaker by stirring with a glass rod for 0.5 h, and heat in a water bath to 47.5 ℃ for 72 h to carry out enzymatic hydrolysis, to obtain mixture A;

[0092] S4. Use an ultrasonic extraction device to ultrasonically extract mixture A at an ultrasonic power of 600W for 30 minutes to obtain extract B;

[0093] S5. Place the extract B in a fermentation tank with a layer of far-infrared stones, let it ferment for 7 days, and obtain fermentation liquid C.

[0094] S6. Filter the fermentation broth C through a plate filter press and freeze-dry it to obtain CHE.

[0095]

Example 4

[0096] S1. The Chinese herbal medicine formula is pulverized using a toothed claw pulverizer according to the operating instructions, and then sieved through a 90-mesh sieve to obtain medicinal powder;

[0097] The traditional Chinese medicine formula includes 10g of Atractylodes macrocephala, 10g of Astragalus membranaceus, 3g of Saposhnikovia divaricata, 3g of Panax ginseng, 3g of Bupleurum chinense, 3g of Poria cocos, 2g of Cimicifuga foetida, and 1g of Angelica sinensis.

[0098] S2. A compound enzyme hydrolysate was prepared using a compound enzyme, with a volume of 5 L of compound enzyme hydrolysate to 1 kg of drug powder. Disodium hydrogen phosphate-citric acid buffer was added to the compound enzyme hydrolysate to adjust the pH to 5.1.

[0099] S3. Mix the powdered medicine and the compound enzymatic hydrolysate in a beaker by stirring with a glass rod for 0.5 h, and heat in a water bath to 49°C and keep warm for 72 h to carry out enzymatic hydrolysis, to obtain mixture A;

[0100] S4. Use an ultrasonic extraction device to ultrasonically extract mixture A at an ultrasonic power of 400W for 60 minutes to obtain extract B;

[0101] S5. Place the extract B in a fermentation tank with a layer of far-infrared stones, let it ferment for 5 days, and obtain fermentation liquid C.

[0102] S6. Filter the fermentation broth C through a plate filter press and freeze-dry it to obtain CHE.

[0103] Example 5

[0104] S1. The Chinese herbal medicine formula is pulverized according to the operating instructions using a toothed claw pulverizer and then sieved through a 60-mesh sieve to obtain medicinal powder;

[0105] The traditional Chinese medicine formula includes 16g of Atractylodes macrocephala, 16g of Astragalus membranaceus, 5g of Saposhnikovia divaricata, 5g of Panax ginseng, 5g of Bupleurum chinense, and 3g of Poria cocos.

[0106] S3. Mix the powdered medicine and the compound enzymatic hydrolysate in a beaker by stirring with a glass rod for 0.5 h, and heat in a water bath to 50 ℃ and keep warm for 72 h to carry out enzymatic hydrolysis, to obtain mixture A;

[0107] S4. Use an ultrasonic extraction device to ultrasonically extract mixture A at an ultrasonic power of 600W for 90 minutes to obtain extract B;

[0108] S5. Place the extract B in a fermentation tank with a layer of far-infrared stones, let it ferment for 7 days, and obtain fermentation liquid C.

[0109] S6. Filter the fermentation broth C through a plate filter press, freeze-dry, and pulverize to obtain CHE, as follows: Figure 1 As shown, CHE is a light yellow powder with strong hygroscopicity; the clumps within it crumble easily when pressed.

[0110]

experiment

[0111] Experiment (1): Feeding Experiment with Meat Ducks

[0112] The CHE prepared in Example 5 was used in a feeding experiment on meat ducks to verify its effects on growth performance and subgroup immunity of meat ducks.

[0113] The experiment used two independent experimental units, each of which included an experimental group and a control group;

[0114] All experimental groups received medication according to a cycle of drinking water with added CHE for 4 consecutive days each week, followed by drinking water without added CHE for 3 consecutive days each week. All control groups consistently drank water without added CHE. Apart from the water type, all other medication, immunization, and other management methods were the same for all groups. More detailed information for each group is shown in Table 1.

[0115] Table 1

[0116] Grouping Handling method CHE dosage per 1000kg of water Number of duck houses Shed No. Number of ducklings Experimental group 1 Drink water with added CHE 1g Building 1 H7 11000 Control group 1 Drink water without CHE 0 Building 1 H11 11000 Experimental group 2 Drink water with added CHE 1g Building 1 H12 28000 Control group 2 Drink water without CHE 0 Building 1 H13 28000

[0117] The medication cycles for experimental groups 1 and 2 in Table 1 are as follows: Figure 2 As shown;

[0118] The number of ducklings in Table 1 is the number of ducklings installed.

[0119] (1) Comparison of body weight among experimental group 1, control group 1, experimental group 2 and control group 2

[0120] The weight comparisons of experimental group 1, control group 1, experimental group 2, and control group 2 are shown in Table 2:

[0121] Table 2

[0122] Experimental group 1 Control group 1 Experimental group 2 Control group 2 weight difference Weight at 7 days old (g) 205 195 200 190 10 / 10 Weight at 14 days old (g) 575 550 565 540 25 / 25 The baby weighed only (g) at 21 days old. 1480 1440 1475 1430 40 / 45 Weight at 28 days old (g) 2220 2190 2240 2200 30 / 40 The weight at slaughter on the 36th day is (g). 2910 2860 2935 2875 50 / 60

[0123] Each shed has a representative column, and the weight in Table 2 is the weight of one meat duck in the representative column;

[0124] The slaughter weight is the average weight of all the meat ducks in the shed.

[0125] The formula for calculating weight difference is:

[0126] ,

[0127] In the formula, weight refers to the weight of each animal at different ages or the weight of each animal at slaughter.

[0128] As can be seen from Table 2, the weight of the ducks in each experimental group was significantly greater than that in each control group, which indicates that CHE has the effect of promoting animal growth and increasing weight.

[0129] (2) Comparison of mortality rates among experimental group 1, control group 1, experimental group 2 and control group 2

[0130] The mortality rates of experimental group 1, control group 1, experimental group 2, and control group 2 are compared in Table 3:

[0131] Table 3

[0132] Experimental group 1 Control group 1 Experimental group 2 Control group 2 Difference in the number of deaths Thousands of ratios Number of deaths in the first week 42 33 72 75 9 / -3 8.2 / -1.1 The number of deaths in the second week 10 32 16 56 -22 / -40 -20 / -14.3 Number of deaths in the third week 11 40 10 66 -29 / -56 -26.4 / -20 Number of deaths in the fourth week 9 10 4 17 -1 / -13 -0.9 / -4.6 Number of deaths in the fifth week + 36 days 19 61 27 118 -42 / -91 -38.2 / -2.5 Total number of deaths 91 176 129 332 -85 / -203 -77.3 / -2.9

[0133] The formula for calculating the difference in the number of deaths is:

[0134] ,

[0135] The formula for calculating the percentage in ten thousand parts is:

[0136] ;

[0137] As can be seen from Table 3, the number of duck deaths in each experimental group was less than that in each control group, which indicates that CHE can enhance animal immunity and thus improve animal survival rate.

[0138] (3) Comparison of survival rate and feed conversion ratio of each component;

[0139] Table 4 shows the comparison of survival rates and feed conversion ratios for each component: Table 4

[0140] The formula for calculating the survival rate is:

[0141] ;

[0142] As shown in Table 4, CHE increased the survival rate of ducks by 0.773% and 0.729% in the two independent experimental units, respectively, and reduced the number of deaths by 85 and 203, respectively. This indicates that CHE can improve animal survival rate and reduce animal mortality. Meanwhile, the average weight of each experimental group increased by 0.05 kg and 0.06 kg compared to the control group, respectively, and the Euler's index increased by 19 and 23, respectively, indicating that CHE can increase animal weight. The feed conversion ratio of each experimental group decreased compared to the control group, indicating that CHE can improve the utilization rate of feed and increase breeding efficiency.

[0143] Experiment (II): Experiment on returning manure to the field

[0144] To verify the effect of consuming the CHE provided by this invention on animal manure production, the manure produced by ducks in each experimental group and each control group in Experiment (I) was directly returned to the field.

[0145] The field is located in Feixian County, Linyi City, at an altitude of about 100 meters. The field is divided into five plots, including experimental plot 1, experimental plot 2, experimental plot 3, control plot 1, and control plot 2. Each plot is planted with bok choy. Except for the difference in the amount of fertilized plants, the planting and management methods are the same.

[0146] Table 5

[0147] Serial Number Field Applied Application amount Sources of feces A Experimental Field 1 feces <![CDATA[1m 3 / mu]]> The two experimental groups' sheds B Experimental Field 2 feces <![CDATA[2m 3 / mu]]> The two experimental groups' sheds C Experimental Field 3 feces <![CDATA[3m 3 / mu]]> The two experimental groups' sheds D Comparison Field 1 feces <![CDATA[4m 3 / mu]]> Two control group sheds E Comparison Field 2 fertilizer 30 kg / mu −

[0148] (1) Compare the growth of bok choy in each field

[0149] Photographs were taken of each field, resulting in Figures 3(A) to 3(E). It can be seen that in experimental field 1, no seedling burn was observed in the bok choy, and the growth was the best, with the greenest and thickest leaves, almost identical to the bok choy in the control field 2 where chemical fertilizer was applied. In experimental fields 2, control field 1, and experimental field 3, seedling burn was observed, and the severity of the burn gradually increased. This indicates that the amount of manure applied must be appropriate and not excessive.

[0150] (2) Compare the yield of bok choy in each field

[0151] The yield of bok choy in each field was statistically analyzed and plotted. Figure 4 , Figure 4 The horizontal axis represents the field number, and the vertical axis represents the weight of the bok choy (in kg). It can be seen that experimental field 1 had the highest bok choy yield, even higher than control field 2, indicating that the feces produced by animals consuming the CHE provided in this invention has a very good promoting effect on plant growth. Experimental field 3, which experienced severe seedling burn, had the lowest yield, suggesting that the amount of feces applied should not be excessive.

[0152] (3) Compare the root development of bok choy in each field.

[0153] Photographs of the root system of bok choy in each field were taken, resulting in Figures 5(A) to 5(B). It can be seen that the root system of bok choy in experimental field 1 is the best developed, long and lush, which is better than the root system development of bok choy in control field 2.

[0154] (4) Compare the nutritional components of milk cabbage in each field.

[0155] The content of various nutrients in bok choy in each field was analyzed and statistically analyzed, and Figures 6(a) to 6(h) were plotted. The nutrients included vitamin C, β-carotene, carbohydrates, ORAC (oxygen free radical scavenging capacity), cellulose, calcium, zinc, and iron. In Figures 6(a) to 6(h), the horizontal axis represents the field number, and the vertical axis represents the content of each nutrient in 100g of bok choy. It can be seen that the content of various nutrients in bok choy in experimental field 1 is generally in the lead. The bok choy in the field with applied manure has better content of cellulose, calcium, ORAC, iron, vitamin C, carbohydrates, and zinc than the two control fields. This indicates that manure can provide rich amounts of these nutrients and is more suitable for cultivating crops that prefer cellulose, calcium, ORAC, iron, vitamin C, carbohydrates, and zinc.

[0156] Experiment (3): Immune Enhancement Experiment

[0157] To verify the immune-enhancing effect of CHE, the following experiments were conducted using CHE prepared in Example 5:

[0158] 1. Evaluate the purity of CHE after removing impurities;

[0159] (1) Remove impurities from CHE, including proteins, fats, pigments, and small molecule components;

[0160] a) Dissolve CHE in pure water, add papain and hydrolyze overnight to obtain solution D;

[0161] b) Remove protein from solution D using the Sevag method: Mix Sevag reagent with solution D to obtain mixed solution E. Centrifuge to separate mixed solution E into three layers from top to bottom: protein-removed CHE solution, protein, and Sevag reagent. Collect the upper layer of protein-removed CHE solution. Repeat the operation until there is no intermediate layer.

[0162] In Sevag reagent, the volume of chloroform is 4 times the volume of n-butanol; the volume of CHE is 4 times that of Sevag reagent.

[0163] c) Add petroleum ether to the CHE solution that has been deproteinized, so that the solution separates into two layers from top to bottom: the petroleum ether layer and the CHE solution that has been defat removed. The petroleum ether can remove fat, and the lower layer of CHE solution that has been defat removed is collected.

[0164] d) Use macroporous resin AB-8 to remove pigments from the degreased CHE solution, and allow it to adsorb overnight to obtain a degreased CHE solution;

[0165] e) The small molecule components in the CHE solution with removed pigments are removed by dialysis through a 3000 Da (Dalton) dialysis bag. Small molecules with a molecular weight of less than 3000 Daltons can pass freely through the dialysis bag, while larger molecules will be trapped in the dialysis bag. After freeze-drying for 72 hours, CHE with impurities removed is obtained.

[0166] (2) The purity of CHE after impurities have been removed was evaluated using the sulfuric acid-phenol method. The purity was expressed as the total soluble sugar content.

[0167] The total soluble sugar content and purity of CHE after impurities have been removed were determined using the sulfuric acid-phenol method. The OD (optical density, absorbance) was measured at 490 nm to ensure that the difference in absorbance between pores in parallel experiments was less than 0.02.

[0168] 2. Vaccine preparation;

[0169] Four vaccines were prepared using CHE, Cap (catabolite gene activator protein), and ISA 15AVG (an adjuvant for preparing oil-in-water vaccines), including a Mock (simulated control group), PCV2 (Porcine circovirus type 2) Cap, PCV2 Cap + 1mg CHE, and PCV2 Cap + 4mg CHE. The components of each vaccine are shown in Table 6.

[0170] Table 6

[0171] Components name 1 ISA 15AVG Mock 2 Cap+ISA 15AVG PCV2 Cap 3 Cap+1mg CHE+ISA 15AVG PCV2 Cap+1mg CHE 4 Cap+4mg CHE+ISA 15AVG PCV2 Cap+4mg CHE

[0172] The mass of Cap is 10 μg;

[0173] 3. Verification of vaccine immunization efficacy;

[0174] (1) Each vaccine was injected into different mice via intramuscular injection.

[0175] The injection schedule is as follows: the second immunization is given 14 days after the first immunization (i.e., the first vaccination);

[0176] (2) Blood was collected from the cheeks of mice 14 and 28 days after the first immunization. The isolated serum was analyzed using an ELISA (enzyme-linked immunosorbent assay) kit according to the ELISA kit instructions to detect Anti-PCV2 Cap IgG (anti-porcine circovirus type 2 Cap protein IgG antibody) in the serum, and plotted as follows: Figure 7 ;

[0177] The ELISA kit reading is OD450 (absorbance at 450 nm). A higher OD450 value indicates a higher level of antibodies in the serum and a better immune enhancement effect. Therefore, from... Figure 7 It can be seen that the antibody levels of PCV2 Cap with added CHE (1 mg CHE) and PCV2 Cap with added CHE (4 mg CHE) were significantly higher than those of PCV2 Cap, and the antibody levels were directly proportional to the amount of CHE added, indicating that CHE has a good immune-enhancing effect.

Claims

1. A method for preparing a traditional Chinese medicine extract, characterized in that, include: S1. After pulverizing the Chinese herbal formula, sieve it to obtain medicinal powder; S2. Prepare a compound enzyme hydrolysate using a compound enzyme, with a volume of 5L to 1Kg of drug powder; add disodium hydrogen phosphate-citric acid buffer to the compound enzyme hydrolysate to adjust the pH to 4.8-5.

2. S3. Mix the powdered medicine with the compound enzymatic hydrolysate, heat the mixture in a water bath to 45℃~50℃ and keep it at that temperature for 72 hours to carry out enzymatic hydrolysis, and obtain mixture A; S4. Perform ultrasonic extraction on mixture A to obtain extract B; S5. Ferment extract B to obtain fermentation broth C; S6. Filter the fermentation broth C and freeze-dry it to obtain the Chinese herbal extract.

2. The method for preparing a traditional Chinese medicine extract according to claim 1, characterized in that, The traditional Chinese medicine formula in S1 includes 10g-16g of Atractylodes macrocephala, 10g-16g of Astragalus membranaceus, 3g-5g of Saposhnikovia divaricata, 3g-5g of Panax ginseng, 3g-5g of Bupleurum chinense, 1g-3g of Poria cocos, 0g-2g of Cimicifuga foetida, and 0g-2g of Angelica sinensis.

3. The method for preparing a traditional Chinese medicine extract according to claim 1, characterized in that, The mesh size of the sieve used for sieving in S1 is 50 to 100 mesh.

4. The method for preparing a traditional Chinese medicine extract according to claim 1, characterized in that, The complex enzyme in S2 includes acidic cellulase, protease, and pectinase, with the enzyme activity ratio of acidic cellulase to protease to pectinase being 2:1:

1.

5. The method for preparing a traditional Chinese medicine extract according to claim 1, characterized in that, The concentration of the complex enzyme in the complex enzymatic hydrolysate in S2 is 5 g / L.

6. The method for preparing a traditional Chinese medicine extract according to claim 1, characterized in that, The preparation of the complex enzyme hydrolysate using a complex enzyme in S2 includes: S201. Weigh 3.659g of acidic cellulase powder, 0.732g of protease powder and 0.610g of pectinase powder to obtain a compound enzyme; S202. Weigh out 6.3g of citric acid monohydrate and 17.6g of sodium citrate dihydrate; S203. Dissolve citric acid monohydrate and sodium citrate dihydrate in 800ml of deionized water, add the compound enzyme, stir and mix for 10min to obtain 1L of compound enzyme hydrolysate.

7. The method for preparing a traditional Chinese medicine extract according to claim 1, characterized in that, The ultrasonic extraction in S4 is performed using an ultrasonic extraction device with an ultrasonic power of 400W~600W and an ultrasonic extraction time of 30min~90min.

8. A traditional Chinese medicine extract prepared by a method according to any one of claims 1 to 7.

9. The application of the traditional Chinese medicine extract as described in claim 8 as an immune enhancer for animals.

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