A Chinese herbal medicine feed additive and its application in high-protein feed

Abstract

The present invention belongs to the field of feed additives, and specifically relates to a Chinese herbal feed additive and its application in high-protein feed, wherein the effective ingredients of the Chinese herbal feed additive are clove leaf extract and bitter herb extract, and the Chinese herbal feed additive is prepared by mixing clove leaf and bitter herb in a mass ratio of 1:3 to 3:1 and then extracting. The Chinese herbal feed additive prepared by the present invention has a significant improvement effect on nutritional diarrhea caused by high protein in pigs, with a total effective rate of up to 93.3%, and in terms of daily weight gain and daily feed intake, the growth-promoting effect of the Chinese herbal feed additive prepared by the present invention is also better than that of drugs such as chlortetracycline in the prior art.

Description

Technical Field

[0001] The invention belongs to the field of medicinal feed additives, and particularly relates to a Chinese herbal medicine feed additive and application thereof in high-protein feed. Background Art

[0002] As antibiotics are used more and more frequently in animal production, drug resistance caused by health-promoting growth-promoting antibiotics for feed is becoming more and more serious. Starting from July 2020, the Ministry of Agriculture and Rural Affairs strictly ordered to stop the production of growth-promoting drug feed additives (mainly feed antibiotics, but excluding traditional Chinese medicines) and stop the production of commercial feed containing growth-promoting drug feed additives (excluding traditional Chinese medicines). Therefore, the market has a strong demand for substitutes for feed antibiotics.

[0003] A large number of studies have confirmed that the crude protein (CP) level in feed is not only a key factor restricting the growth of piglets, but also has an important impact on piglet diarrhea. At present, the protein addition level in piglet feed is generally between 18% and 22%. When the protein level in the feed is insufficient (below 18%), it cannot meet the physiological and growth needs of piglets and will hinder the normal growth and development of piglets. When the protein level in the feed is too high (above 22%), it will cause excess amino acids in the body to be used for oxidation energy supply, resulting in feed waste. More importantly, when the protein level exceeds the body's energy and protein metabolism capacity, it will trigger a series of intestinal metabolic inflammation or chronic inflammation.

[0004] The internal mechanism of intestinal metabolic inflammation is: after the protein in the feed enters the intestine, it is hydrolyzed into amino acids under the action of trypsin, pancreatic chymotrypsin, and aminopeptidase and dipeptidase secreted by the pancreas, and the amino acids produce ammonia through joint deamination. The main bacterial species that degrade protein in the intestine are Bacteroides, Propionibacterium, Clostridium, Streptococcus, Lactobacillus, etc. Only 14% of the protein ingested by pigs is retained in the body as nutrients. The undigested and absorbed feed protein or its hydrolysis product amino acids produce a large amount of corruption products (amines, ammonia, phenols, indoles, hydrogen sulfide, etc.) under the corruption of intestinal bacteria. In particular, ammonia has a strong irritating odor, which can irritate the mucous membranes of animals and cause various inflammations. At the same time, ammonia in the body combines with hemoglobin to convert heme into iron hemoglobin, reducing the oxygen carrying capacity of hemoglobin, blood alkali storage and the oxidation capacity of heme, and then anemia and tissue hypoxia occur, and the body's resistance to disease decreases; in addition, ammonia will also affect the response of immune cells and reduce the immunity of animals. The detoxification of ammonia is a highly energy-intensive process, and just as metabolic inflammation consumes energy, the animal has less energy available for growth and production, thereby reducing the growth performance of pigs.

[0005] Yin Lanmei's research in 2019 confirmed that the continued occurrence of chronic inflammation under high-protein feed conditions leads to damage to the intestinal mucosa and increased susceptibility to pathogenic microorganisms such as harmful bacteria, clostridium, molds, coccidia, etc., which in turn affects the enterohepatic circulation, leading to repeated occurrences of enteritis and diarrhea, seriously affecting production performance. In July 2020, antibiotics were banned in feed, and plant essential oils, acidifiers, etc. have poor anti-diarrhea functions. Therefore, the market urgently needs a green feed additive that can both effectively resist high-protein nutritional diarrhea and promote growth. Summary of the invention

[0006] In view of the problems and shortcomings in the prior art, the purpose of the present invention is to provide a Chinese herbal medicine feed additive and its application in high-protein feed.

[0007] To achieve the purpose of the invention, the technical solution adopted by the present invention is as follows:

[0008] A first aspect of the present invention provides a Chinese herbal feed additive, wherein the active ingredients are clove leaf extract and euphorbia pulegiae extract. Calculated based on the tyrosol content in the clove leaf extract, the tyrosol content in the Chinese herbal feed additive is not less than 1.5 mg / g.

[0009] According to the above-mentioned Chinese herbal feed additive, preferably, the Chinese herbal feed additive is prepared by mixing clove leaves and Corydalis eryngii in a mass ratio of 1:3 to 3:1 and then extracting and preparing the mixture.

[0010] The second aspect of the present invention provides a use of the Chinese herbal medicine feed additive described in the first aspect in high-protein feed.

[0011] According to the above application, preferably, the content of the Chinese herbal feed additive in the high-protein feed is 5-10 ppm based on tyrosol content.

[0012] According to the above application, preferably, the protein content in the high-protein feed is greater than or equal to 22%.

[0013] The third aspect of the present invention provides a method for preparing the Chinese herbal feed additive described in the first aspect: clove leaves and bitter herb are mixed and added to an extraction solvent for extraction to obtain an extract, and the extract is concentrated and dried to obtain the Chinese herbal feed additive, wherein the extraction solvent is water or ethanol.

[0014] According to the above preparation method, preferably, during the extraction process, a pH adjuster is used to adjust the pH of the extraction solvent to 3.5-6.7, and the pH adjuster is a disodium hydrogen phosphate-citric acid solution.

[0015] According to the above preparation method, preferably, the extraction method is flash extraction, the time of the flash extraction is 5 to 10 minutes, the voltage is 130 to 220V, and the rotation speed is 6000 to 10000 r / min.

[0016] The fourth aspect of the present invention provides a high-protein feed, wherein the high-protein feed contains the Chinese herbal medicine feed additive described in the first aspect of the present invention.

[0017] According to the above-mentioned high-protein feed, the content of the Chinese herbal medicine feed additive in the high-protein feed is 5-10 ppm, and the protein content in the high-protein feed is greater than or equal to 22%.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The present invention uses clove leaves and bitter herb as medicinal raw materials, mixes and extracts to prepare a Chinese herbal feed additive, and applies the Chinese herbal feed additive to high-protein feed. The experimental results show that the Chinese herbal feed additive prepared by the present invention has a significant improvement effect on nutritional diarrhea of ​​pigs caused by high-protein feed, with a total effective rate of 93.3%, and in terms of daily weight gain and daily feed intake, the growth-promoting effect of the Chinese herbal feed additive prepared by the present invention is also better than that of drugs such as chlortetracycline in the prior art.

[0020] 2. The effective ingredients of the Chinese herbal feed additive prepared by the present invention are clove leaf extract and Corydalis syringae pumila extract. The clove leaf is bitter and cold in nature, and enters the stomach and large intestine meridians. The main components are tyrosol, syringoside and protocatechuic acid, and have strong intestinal anti-inflammatory and anti-dysentery effects; the main components of Corydalis syringae pumila are corydaline and acetyl corydaline, which mainly have anti-inflammatory and detumescent effects and promote intestinal mucosal repair. The Chinese herbal feed additive prepared by combining the two in the present invention has strong synergistic anti-inflammatory and growth-promoting effects.

[0021] 3. The medicinal materials of the feed additive prepared by the present invention are easy to obtain, low in cost, simple in preparation method, mild in conditions and short in time, and are very suitable for use by farmers. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a diagram showing the long-term toxicity of the Chinese herbal medicine feed additive prepared by the present invention on rats. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0024] (I) Preparation of Chinese herbal feed additives under different raw material ratios

[0025] Example 1

[0026] A method for preparing a Chinese herbal medicine feed additive comprises the following steps:

[0027] (1) Take 900 g of clove leaves and 450 g of bitter herb, mix and grind them, and pass them through a 20-mesh sieve to obtain a medicinal material powder;

[0028] (2) adding 10 times the weight of purified water to the medicinal material powder, and then adding disodium hydrogen phosphate-citric acid buffer to adjust the pH to 3.5, soaking for 30 minutes, flash extraction for 10 minutes, extraction voltage of 150 V, rotation speed of 7000 r / min, and filtering;

[0029] (3) adding purified water 8 times the weight of the medicinal material powder to the medicinal residue obtained after filtration, and then adding disodium hydrogen phosphate-citric acid buffer to adjust the pH to 6.7, flash extraction for 10 minutes, extraction voltage of 200 V, rotation speed of 9000 r / min, and filtering;

[0030] (4) The extracts filtered in steps (2) and (3) are combined and concentrated under reduced pressure to a relative density of 1.1 to obtain an extract, 600 g of lactose is added to the extract, the mixture is stirred evenly, and spray-dried to obtain a Chinese herbal feed additive. The tyrosol content in the clove leaf extract is not less than 1.5 mg / g.

[0031] Example 2

[0032] The contents of Example 2 are substantially the same as those of Example 1, except that in step (1), the mass ratio of clove leaves to bitter peony root is 1:1.

[0033] Example 3

[0034] The contents of Example 3 are substantially the same as those of Example 1, except that in step (1), the mass ratio of clove leaves to bitter peony root is 3:1.

[0035] Example 4

[0036] The contents of Example 4 are substantially the same as those of Example 1, except that in step (1), the mass ratio of clove leaves to bitter peony root is 1:2.

[0037] Example 5

[0038] The contents of Example 5 are substantially the same as those of Example 1, except that in step (1), the mass ratio of clove leaves to bitter peony root is 1:3.

[0039] Comparative Example 1: Preparation of clove leaf extract

[0040] The preparation method of the clove leaf extract in this example is basically the same as that in Example 1, except that only 900 g of clove leaves are taken in step (1).

[0041] Comparative Example 2: Preparation of Kuyede Extract

[0042] The preparation method of the Herba Corydalis extract in this example is substantially the same as that in Example 1, except that only 450 g of Herba Corydalis was taken in step (1).

[0043] (1) In vitro antibacterial activity test of the Chinese herbal medicine feed additive prepared by the present invention

[0044] The porcine Escherichia coli K88 and porcine Streptococcus type 2 used in the following experiments were isolated and preserved from the Preventive Veterinary Laboratory of Yangzhou University.

[0045] ① Experimental grouping:

[0046] Chinese herbal medicine feed additive group: a total of 5 groups, wherein the Chinese herbal medicine feed additives prepared in Examples 1 to 5 of the present invention were dissolved in sterile distilled water, and the concentration was 1024 mg / mL based on the crude drug;

[0047] Chlortetracycline control group: its specification is 1Kg / bag, calculated as chlortetracycline, 1000g: 200g (200 million units), production batch number is 211207, provided by Zhumadian Huazhong Zhengda Co., Ltd.;

[0048] Clove leaf extract group: the clove leaf extract prepared in Comparative Example 1 was dissolved in sterile distilled water to a concentration of 1024 mg / mL based on crude drug;

[0049] Kuyeding extract group: the Kuyeding extract prepared in Comparative Example 2 was dissolved in sterile distilled water to a concentration of 1024 mg / mL based on crude drug;

[0050] ②Test plan:

[0051] The tested strains were rejuvenated and strengthened according to the conventional method, and the bacterial solution was prepared. The MIC value of each group was determined by the micro-two-fold dilution method, and the contents of each test tube were cultured at 37℃ for 24 hours to observe the results.

[0052] ③Test results: as shown in Table 1.

[0053] Table 1 MIC (mg / ml) of Chinese herbal feed additives against Escherichia coli and Streptococcus suis

[0054]

[0055] As can be seen from Table 1, compared with the extracts of single ingredients of clove leaves and bitter herb, the Chinese herbal feed additive prepared when clove leaves and bitter herb are combined in a ratio of 2:1 (Example 1) and 3:1 (Example 3) has a better inhibitory effect on porcine Escherichia coli and Streptococcus, among which the result of 2:1 is the best, and the effect is better than that of the chlortetracycline group, which indicates that clove leaves and bitter herb have a better antibacterial effect after being combined.

[0056] (2) In vitro anti-inflammatory activity test of the Chinese herbal medicine feed additive prepared by the present invention

[0057] The mouse mononuclear macrophage cell line RAW264.7 used in the following experiments was donated by Professor Shi Dayong's laboratory at Zhengzhou University of Animal Husbandry and Economics. Hydrocortisone injection was produced by Shanxi Zhaoyi Biological Co., Ltd. with a specification of 20 ml:100 mg and batch number 202111232; lipopolysaccharide LPS was purchased from Solebao; mouse IL-6, TNF-α and other ELISA kits were purchased from Jiancheng Biological.

[0058] 1. Experimental plan

[0059] 1.1 Cell culture

[0060] Mouse mononuclear macrophage cell line RAW264.7 was cultured in a cell culture incubator using DMEM high-glucose medium (containing 10% inactivated FBS). The culture conditions were set at 37° C. and 5% CO 2 . Logarithmically growing cells were taken for subsequent experiments.

[0061] 1.2 Cell viability assay

[0062] The cells were divided into a blank control group and a group containing the Chinese herbal feed additives prepared in Examples 1 to 5 at doses of 1000, 750, 500, 250, 100, 50, and 10 μg / mL, respectively. Each group had three replicate wells. Cells in the logarithmic growth phase were taken at 1×10 6 The cells were inoculated with 100 μL of each well at a density of 100 μg / mL in a 96-well plate and cultured overnight. The next day, after rinsing with PBS twice, 100 μL of the corresponding concentration of drug-containing culture medium was added, and the blank control group was added with an equal volume of complete culture medium. After culturing for 24 hours, the culture medium was discarded, 10 μL of MTT solution (5 mg / mL) and 90 μL of fresh culture medium were added to each well, and the supernatant was removed after continuing to culture for 4 hours. DMSO 100 μL was added to each well, and the cells were placed on a shaker to fully dissolve the bottom precipitate. The absorbance at 490 nm was detected using a multifunctional microplate reader, and the cell survival rate was calculated. According to the cell survival rate of each group of 10-100 μg / mL, it was higher than 80%, indicating that this dose range had no significant inhibitory effect on the activity of RAW 264.7 macrophages, and the subsequent experimental drug administration concentration was set to 100 μg / mL.

[0063] 1.3 Detection of the effects of Chinese herbal feed additives on the secretion of nitric oxide (NO) and pro-inflammatory factors TNF-α and IL-6 by RAW264.7 macrophages

[0064] The cells in the logarithmic growth phase were divided into 10 groups (blank control group, model control group, drug control group, clove leaf extract group, bitter herb extract group and 5 groups of Chinese herbal feed additives prepared by the present invention), with 3 replicates in each group. 6The cells were inoculated with 500 μL of each well at a density of 100 μg / mL in a 24-well plate and cultured overnight. The next day, after rinsing with PBS twice, 500 μL of drug-containing culture medium with a concentration of 100 μg / mL was added to each group, and an equal volume of complete culture medium was added to the blank control group and the model control group. After incubation in a cell culture incubator for 30 minutes, lipopolysaccharide LPS (1 μg / mL) was added, and the cell supernatant was collected after 24 hours. The NO concentration in the cell supernatant was detected using a Griess kit and strictly followed the instructions of the kit, and the concentrations of inflammatory factors TNF-α and IL-6 in the cell supernatant were detected using the corresponding ELISA kit and strictly followed the instructions of the kit.

[0065] Trial group and medication:

[0066] ①Blank control group: healthy mouse mononuclear macrophage cell line RAW264.7 + complete culture medium;

[0067] ② Model control group: LPS-induced mouse mononuclear macrophage RAW264.7 + complete medium;

[0068] ③Drug control group: LPS-induced mouse mononuclear macrophage RAW264.7 + medium containing hydrocortisone;

[0069] ④ Clove leaf extract group: LPS-induced mouse mononuclear macrophage RAW264.7 + culture medium containing clove leaf extract prepared in Comparative Example 1;

[0070] ⑤ Corydalis erythrorhizon extract group: LPS-induced mouse mononuclear macrophage RAW264.7 + culture medium containing Corydalis erythrorhizon extract of comparative example 2;

[0071] ⑥-⑩ Chinese herbal medicine feed additive group: LPS-induced mouse mononuclear macrophage RAW264.7 + culture medium containing the Chinese herbal medicine feed additive prepared in Examples 1-5;

[0072] Test results: See Table 2 for the test results;

[0073] Table 2 Effects of Chinese herbal feed additives on the secretion of NO, TNF-α and IL-6 by RAW264.7 macrophages

[0074]

[0075] It can be seen from the results in Table 2 that, compared with the extracts of single components of clove leaves and bitter herbs, the Chinese herbal feed additive prepared by the present invention has better anti-inflammatory effect, among which the Chinese herbal feed additive prepared by combining clove leaves and bitter herbs in a mass ratio of 2:1 in Example 1 has the strongest anti-inflammatory activity.

[0076] (3) Experimental study on the efficacy of Chinese herbal feed additives on diarrhea in piglets induced by high-protein feed

[0077] 1. Experimental Animals

[0078] 650 weaned piglets (average weaning age 26 days) provided by Hebei Lvwang Animal Husbandry Co., Ltd. were selected, with an average weight of about 6.5 kg. Three days after weaning, the group had severe diarrhea, thin and gray feces, undigested curd in feces, decreased appetite, weight loss, red mouth and love to drink water. The on-site practicing veterinary laboratory test diagnosis ruled out viral and bacterial diarrhea. After inquiry, it was learned that the pig farm owner used a large amount of high-protein trough feed, with a crude protein content of about 21.5%. 15 typical diseased pigs were quickly sampled. The pig disease laboratory of the College of Animal Science and Technology of Henan Agricultural University found that the blood pro-inflammatory factors IL-2, IL-6, and TNF-α were significantly higher than normal, with average values ​​of 97.62pg / ml, 186.74pg / ml, and 310.42pg / ml, respectively. The on-site veterinarian comprehensively judged that the 15 cases were high-protein nutritional diarrhea in piglets, and after communication, they were willing to use Chinese medicine and antibiotic-free products for treatment. Therefore, 210 typical case pigs were selected from the large group, and then 150 pigs with similar weight (weight range 6.5kg±0.5kg) were selected from them. They were randomly divided into 5 pens according to their weight, with 30 pigs in each pen.

[0079] 2. Experimental plan

[0080] The specifications of chlortetracycline used in the following test are: 1Kg / bag, calculated as chlortetracycline, 1000g: 200g (200 million units), the production batch number is 220307, ​​provided by Zhumadian Huazhong Zhengda Co., Ltd.; the clove leaf extract prepared in Comparative Example 1, the bitter herb extract prepared in Comparative Example 2, and the Chinese herbal feed additive prepared in Example 1 are all 1g / g in terms of medicinal material equivalent;

[0081] The 150 selected case pigs were fed normal feed and had free access to food. The normal feed was still a high-protein creep feed used in the pig farm. The specific experimental groups and medication methods of each group are shown in Table 3.

[0082] Table 3 Medication methods for each group

[0083]

[0084] Each group was treated with the drug for 15 consecutive days. During the trial, the total feed intake, total daily weight gain and diarrhea score of each group of piglets were recorded every day. At the end of the trial, the diarrhea improvement index, average feed intake, average daily weight gain and average blood inflammatory factor content (IL-2, IL-6, TNF-α) of each group were statistically analyzed.

[0085] 3. Criteria for determining efficacy results

[0086] ① Cured: Diarrhea symptoms disappear, feces are formed and there is no feed overfeeding, feed intake and weight increase, and the appearance is lively and energetic;

[0087] ② Significant effect: Diarrhea and feed overload are alleviated, the color of feces is basically normal, but still not formed and porridge-like, the feed intake increases, the body weight remains unchanged, and the spirit improves;

[0088] ③ Ineffective: Diarrhea symptoms do not improve or even worsen after treatment, stool is thin and unformed, food intake and body weight remain unchanged or decrease, spirit and appetite deteriorate, and the patient becomes emaciated and tired.

[0089] Pigs that died during the experiment were counted as invalid results.

[0090] 4. Test results and analysis

[0091] ① Results and analysis of the improvement of high-protein diarrhea symptoms in piglets in each group

[0092] The results of improvement of high-protein diarrhea symptoms in piglets in each group are shown in Table 4, where cure rate (%) = number of cured heads / number of heads in group * 100%; effective rate (%) = number of significantly effective heads / number of heads in group * 100%; ineffective rate (%) = number of ineffective heads / number of heads in group * 100%; total effective rate (%) = cure rate + significantly effective rate.

[0093] Table 4 Results of each group on improvement of high protein diarrhea symptoms in piglets

[0094]

[0095] The results in Table 4 show that the cure rate of the Chinese herbal feed additive group for high-protein diarrhea symptoms in piglets is higher than that in other groups. The clove leaf extract and the bitter herb extract can also improve the nutritional diarrhea of ​​piglets caused by high protein to varying degrees. Among them, the improvement effects of the clove leaf extract and the Chinese herbal feed additive prepared in Example 1 of the present invention are better than those of the chlortetracycline group, and the improvement effect of the Chinese herbal feed additive prepared in Example 1 of the present invention is the most obvious, with a total effective rate of 93.3%.

[0096] ② Results and analysis of the effects of each group on feed intake and daily weight gain of piglets

[0097] The test results of the effects of each group on the feed intake and daily weight gain of piglets are shown in Table 5, where the calculation formula involved is:

[0098]

[0099]

[0100]

[0101] Table 5 Effects of each group on feed intake and daily weight gain of piglets caused by high-protein diarrhea

[0102]

[0103] The data in Table 5 show that in terms of daily weight gain and daily feed intake, the growth-promoting effect of the Chinese herbal feed additive prepared in Example 1 of the present invention is better than that of chlortetracycline. The reason may be that the synergistic anti-inflammatory effect of the total phenolic acids and flavonoids contained in clove leaves and the alkaloids corydaline and caffeic acid contained in bitter herb reduces the energy consumption caused by inflammation, which can be confirmed in the results of Table 6.

[0104] ③ Results and analysis of the effects of each group on inflammation caused by high protein in piglets

[0105] The results of the effects of each group on inflammation caused by high protein in piglets are shown in Table 6:

[0106] Table 6 Effects of each group on the content of inflammatory factors caused by high protein in piglets

[0107]

[0108] The data in Table 6 show that compared with the extracts of single ingredients of clove leaves and bitter herbs, the Chinese herbal feed additive group prepared by the present invention (clove leaves and bitter herbs in combination) has better anti-inflammatory effect, which is consistent with the results of daily weight gain and daily feed intake of piglets in Table 5.

[0109] The above results indicate that after the phenolic acid components in the clove leaf extract group, the bitter herb extract group and the Chinese herbal feed additive group were subjected to the same semi-bionic coupled flash extraction, the active ingredients were better released, and the Chinese herbal feed additive showed more excellent anti-inflammatory, anti-diarrhea and growth-promoting effects, indicating the synergistic advantage of combining clove leaves with bitter herb in the present invention, and the specific mechanism needs further study.

[0110] (4) Test on the long-term toxicity of the Chinese herbal medicine feed additive prepared by the present invention as a feed additive on rats

[0111] The Chinese herbal medicine feed additive prepared in Example 1 of the present invention was added to the basal feed of Wistar rats (6-7 weeks old, 13 males and 13 females, purchased from Hubei Experimental Animal Research Center, production license number: SCXK (E) 2020-2018) at a dosage of 1 wt%, and was added continuously for 60 days. The upper and lower limit test and the limit method acute toxicity method were used for the test, and the pathological sections of the heart, liver, spleen, lung, and kidney were observed by autopsy. Figure 1 It was found that the above-mentioned major organs had no obvious lesions, indicating that the Chinese herbal medicine feed additive prepared by the present invention is safe for long-term addition to feed.

[0112] In summary, according to the analysis of all test results, the present invention adds 0.5% feed additive to the feed and uses it continuously, which can significantly improve high-protein metabolic inflammatory diarrhea, reduce the diarrhea index, increase daily weight gain and feed intake, and is better than or equal to the commonly used representative feed antibiotic chlortetracycline, and is green and safe, and can be added as a substitute for pig feed antibiotics.

[0113] (II) Comparison of Chinese herbal feed additives prepared by different methods

[0114] Comparative Example 3: Preparation of Chinese herbal feed additives by water extraction

[0115] A Chinese herbal medicine feed additive is prepared by the following steps:

[0116] (1) Take 900 g of clove leaves and 450 g of bitter herb, mix and grind them, and pass them through a 20-mesh sieve to obtain a medicinal material powder;

[0117] (2) Add purified water to the medicinal material powder and boil and extract twice. The first amount of purified water added is 10 times the weight of the medicinal material, and the boiling and extraction is performed for 2 hours. The second amount of purified water added is 8 times the weight of the medicinal material, and the boiling and extraction is performed for 2 hours. Filter, combine the two decoctions, and concentrate the decoctions under reduced pressure to a relative density of 1.1 to obtain an extract. Add an appropriate amount of lactose to the extract, stir evenly, and spray dry to obtain a Chinese herbal feed additive.

[0118] Comparative Example 4: Preparation of Chinese herbal medicine feed additive by alcohol extraction

[0119] A Chinese herbal medicine feed additive is prepared by the following steps:

[0120] (1) Take 900 g of clove leaves and 450 g of bitter herb, mix and grind them, and pass them through a 20-mesh sieve to obtain a medicinal material powder;

[0121] (2) Add 70% ethanol to the medicinal material powder and heat under reflux for extraction twice. The first time, add 70% ethanol in an amount of 10 times the weight of the medicinal material and heat under reflux for extraction for 2 hours. The second time, add 70% ethanol in an amount of 8 times the weight of the medicinal material and heat under reflux for extraction for 2 hours. Filter and combine the two alcohol extracts. Reduce the pressure of the alcohol extract until there is no alcohol taste to obtain an extract. Add an appropriate amount of lactose to the extract, stir evenly, and spray dry to obtain a Chinese herbal feed additive.

[0122] Comparative Example 5: Preparation of Chinese herbal medicine feed additive by enzymatic hydrolysis

[0123] A Chinese herbal medicine feed additive is prepared by the following steps:

[0124] (1) Take 900 g of clove leaves and 450 g of bitter herb, mix and grind them, and pass them through a 20-mesh sieve to obtain a medicinal material powder;

[0125] (2) Add 10 times the weight of purified water to the medicinal material powder, heat to 50°C, adjust the pH to 4.5 with dilute hydrochloric acid, add a mixture of cellulase (enzyme activity 20,000 U / g) and pectinase (enzyme activity 40,000 U / g), the amount of cellulase is 1 g, the amount of pectinase is 1.5 g, stir evenly, enzymolyze at 45-50°C for 2.5 h, then heat to 80°C for enzyme inactivation treatment; after inactivation treatment, boil and extract in water for 2 h, and filter;

[0126] (3) Add 8 times the weight of the medicinal material to the filtered residue in step (2), decoct and extract for 2 hours, filter, combine the two decoctions and concentrate under reduced pressure to a relative density of 1.1 to obtain an extract, add an appropriate amount of lactose to the extract, stir evenly, and spray dry to obtain a Chinese herbal feed additive.

[0127] (1) Comparison of the active ingredient content of Chinese herbal feed additives prepared by different methods:

[0128] The components of clove leaves include tyrosol, chlorogenic acid, syringoside, syringin and protocatechuic acid. The main components of corydalis are corydalis and acetyl corydalis. The contents of the above components in the Chinese herbal feed additives obtained in Example 1 and Comparative Examples 3, 4 and 5 were tested, and the results are shown in Table 7:

[0129] Table 7 Results of the content of each index component by different extraction methods

[0130]

[0131] It can be seen from Table 7 that compared with water extraction, alcohol extraction and enzymatic hydrolysis, the Chinese herbal feed additive prepared by semi-bionic extraction coupled with flash extraction in the present invention has a higher content of effective ingredients. This is because semi-bionic extraction can fully release acid-soluble and alkali-soluble components, and flash extraction mixes the solute and solvent at a high speed, which can fully enrich the active ingredients such as tyrosol, protocatechuic acid, and corydaline.

[0132] (2) Comparison of the effects of Chinese herbal feed additives prepared by different methods on diarrhea and inflammation in piglets induced by high-protein feed

[0133] ①Prepare high-protein basic feed

[0134] Referring to the nutritional needs of piglets, the nutritional standards are strictly based on the NRC (2012) piglet feed formulation, and the feed protein content is 23% high-protein basic feed.

[0135] ②Construction of experimental animals

[0136] 135 weaned piglets without diarrhea (age: 30±2d) were selected from Wangji Agriculture and Animal Husbandry Co., Ltd. in Lankao County, Henan Province. The average weight was about 8 kg. The 135 weaned piglets without diarrhea were pre-fed with the above high-protein feed for 15 days. The experimental piglets began to have diarrhea and low feed intake from the 5th day of feeding. The cases with diarrhea for more than 3 days were selected, a total of 90 pigs, and divided into 6 groups as the experimental groups.

[0137] ③Test drugs

[0138] (a) the Chinese herbal medicine feed additive prepared in Example 1;

[0139] (b) the Chinese herbal medicine feed additives prepared in Comparative Examples 3 to 5;

[0140] (c) Chlortetracycline premix, specification: 1 kg / bag, calculated as chlortetracycline, 1000 g: 200 g (200 million units), production batch number 210702, provided by Zhumadian Huazhong Zhengda Co., Ltd.

[0141] ④ The experimental groups and medication methods of each group are shown in Table 8;

[0142] Table 8 Experimental groups and medication methods in each group

[0143]

[0144] Note: The dosage of Chinese medicine refers to the dosage in the Chinese Veterinary Pharmacopoeia, which is 0.5g / kg·bw.

[0145] ⑤ Diarrhea scoring criteria:

[0146] degree Appearance Initial water content in feces score normal Strip or granular <70 0 Mild Soft feces, can be formed 70～75 1 Moderate Thick, shapeless, no separation of feces and water 75～80 2 serious Liquid, no shape, no separation of feces and water >80 3

[0147] Diarrhea improvement rate (%) = (mean diarrhea score before the test - mean diarrhea score at the end of the test) / mean diarrhea score before the test × 100%

[0148] ⑥Test results and analysis

[0149] During the experiment, the total feed intake, total daily weight gain and diarrhea score of each group of piglets were recorded every day. At the end of the experiment, the diarrhea improvement index, average feed intake, average daily weight gain, and average blood inflammatory factor content (IL-2, IL-6, TNF-α) of each group were statistically analyzed.

[0150] The test results of each group on improving diarrhea symptoms in piglets are shown in Table 9:

[0151] Table 9 Results of the test on improvement index of diarrhea symptoms in piglets in each group

[0152]

[0153] As can be seen from Table 9, the compound medicinal feed additive prepared in Example 1 of the present invention has a significant effect on improving high-protein inflammatory diarrhea, with an improvement range of 70% to 80%, which is significantly higher than the antibiotic control group, enzymatic hydrolysis group, water extraction group and ethanol extraction group.

[0154] The effects of each group on the feed intake and daily weight gain of piglets are shown in Table 10:

[0155] Table 10 Effects of each group on feed intake and daily weight gain of piglets

[0156]

[0157] The effects of each group on the levels of inflammatory factors in piglets are shown in Table 11:

[0158] Table 11 Effects of each group on the level of inflammatory factors in piglets

[0159] Group TNF-α(pg / ml) IL-6 (pg / ml) IL-2 (pg / ml) Blank control group 288.97 203.44 98.89 Antibiotic control group (chlortetracycline group) 126.43 112.38 77.26 Chinese herbal medicine feed additive water extraction group (comparative example 1) 110.54 96.43 72.53 Chinese herbal medicine feed additive alcohol extraction group (comparative example 2) 128.49 113.25 79.05 Chinese herbal medicine feed additive enzymatic hydrolysis group (comparative example 3) 139.61 109.66 82.66 Chinese herbal medicine feed additive group (Example 1) 107.22 88.63 68.41

[0160] It can be seen from Table 10 that in terms of daily weight gain and daily feed intake, the Chinese herbal feed additive group prepared in Example 1 of the present invention is basically equivalent to chlortetracycline. The reason may be related to the synergistic anti-inflammatory effect of total phenolic acids contained in clove leaves and corydaline contained in bitter herb, which can be confirmed in the results of Table 11.

[0161] The above results indicate that the active ingredients of the phenolic acid components of the Chinese herbal feed additive prepared by the present invention are better released after semi-bionic flash extraction simulating the acid-base environment of the gastrointestinal tract. Compared with water extraction or alcohol extraction, it is more suitable for the acidic environment of the gastrointestinal tract and is beneficial to the release and absorption of phenolic acid components in the intestine. The specific mechanism needs further study.

[0162] The above embodiments are specific implementation methods of the present invention, but the implementation methods of the present invention are not limited to the above embodiments. Any other combination, change, modification, substitution and simplification that does not exceed the design concept of the present invention shall fall within the protection scope of the present invention.

Claims

1. Use of a Chinese herbal medicine feed additive in preparing feed for improving piglet diarrhea caused by high-protein feed, characterized in that: The content of the Chinese herbal feed additive in the feed is 5 to 10 ppm; the Chinese herbal feed additive is prepared by mixing clove leaves and bitter herb in a mass ratio of 2:1 to 3:1 and then extracting and preparing it. Calculated by the tyrosol content in the clove leaf extract, the tyrosol in the Chinese herbal feed additive is not less than 1.5 mg / g.

2. The use according to claim 1, characterized in that: The protein content in the feed is greater than or equal to 22%.

3. The use according to claim 1, characterized in that: The preparation method of the Chinese herbal feed additive comprises the following steps: mixing clove leaves and bitter herb, adding the mixture into an extraction solvent for extraction to obtain an extract, and concentrating and drying the extract to obtain the Chinese herbal feed additive, wherein the extraction solvent is water or ethanol.

4. The use according to claim 3, characterized in that: During the extraction process, a pH regulator is used to adjust the pH of the extraction solvent to 3.5-6.7, and the pH regulator is a disodium hydrogen phosphate-citric acid solution.

5. The use according to claim 4, characterized in that: The extraction method is flash extraction, the flash extraction time is 5 to 10 minutes, the voltage is 130 to 220 V, and the rotation speed is 6000 to 10000 r / min.

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