CN111991384A - Protocatechuic acid and application of compound protocatechuic acid thereof in improving performance of livestock and poultry - Google Patents
Protocatechuic acid and application of compound protocatechuic acid thereof in improving performance of livestock and poultry Download PDFInfo
- Publication number
- CN111991384A CN111991384A CN202010825749.5A CN202010825749A CN111991384A CN 111991384 A CN111991384 A CN 111991384A CN 202010825749 A CN202010825749 A CN 202010825749A CN 111991384 A CN111991384 A CN 111991384A
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- protocatechuic acid
- bacillus
- compound
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- acid
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- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/70—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in livestock or poultry
Abstract
The invention relates to protocatechuic acid and a new application of compound protocatechuic acid in feed industry, and the product can prevent and treat digestive tract diseases such as livestock and poultry diarrhea, gastric ulcer and the like caused by pathogenic bacillus (bacillus cereus, bacillus thuringiensis and bacillus pumilus) and immunosuppression. Wherein the protocatechuic acid compound comprises the following components in parts by weight: 70-90 parts of protocatechuic acid and 10-30 parts of fulvic acid. In vitro and in vivo experiments show that the compound protocatechuic acid can kill pathogenic bacillus cereus, bacillus pumilus and bacillus thuringiensis, and simultaneously can prevent and treat diarrhea, gastric ulcer and intestinal hemorrhagic diseases caused by the three bacilli. The additive amount is 12.5mg/kg body weight or 200 g/ton feed, and the compound protocatechuic acid kills pathogenic bacillus, thereby providing a new technical approach for healthy breeding of livestock and poultry.
Description
Technical Field
The invention relates to a new application of protocatechuic acid, in particular to an application of protocatechuic acid in preparing a medicament for preventing and treating diseases caused by pathogenic bacillus in livestock and poultry feed.
Background
Protocatechuic acid is present in leaves of red plum, Stenoloma chusana, and Aquifoliaceae. Protocatechuic acid has various biological activities aiming at different molecular targets, and has the effects of resisting bacteria, oxidation, inflammation, hyperglycemia and neuroprotection. In addition, protocatechuic acid has a potential chemoprotective effect, which can be anti-tumor and neuroprotective. At present, protocatechuic acid is mainly applied to antiviral drugs. However, their use for combating pathogenic Bacillus cereus and for antagonizing mycotoxin has not been found to date. Protocatechuic acid (PCA) with chemical name of 3, 4-dihydroxybenzoic acid (CAS registry number of 99-50-3, molecular formula of (HO)2C6H3 COOH) and molecular weight of 154.12.
The product is white to brown crystalline powder and discolors in air. Density 1.54g/cm3. Melting point about 200 deg.C (decomposition). Dissolving in 50 parts of water; dissolving in ethanol and diethyl ether.
At present, protocatechuic acid is researched, and found to have the effects of oxidation resistance, inflammation resistance, bacteria resistance and virus resistance, and the protocatechuic acid is widely applied to livestock and poultry breeding and disease prevention and treatment. Protocatechuic acid is used as an antioxidant, and can effectively relieve rat neurotoxicity induced by cadmium: the effects of oxidative stress, inflammation and apoptosis suggest that PCA can counteract cadmium-induced cortical toxicity by potentiating the antioxidant defense system and inhibiting inflammation and apoptosis. Protocatechuic acid can obviously inhibit the growth of pathogenic bacteria and fungi, and the bacteriostatic activity of protocatechuic acid still exists after being heated at 100 ℃. Protocatechuic acid has synergistic effect on antibiotics such as colistin, clinafloxacin, amikacin, and fosfomycin. In the SPF chicken model test inoculated with the Newcastle disease virus low virulent strain, compared with the oral administration of 125mg/kg astragalus polysaccharide group, the oral administration of 40mg/kg protocatechuic acid group obviously improves the NDV antibody level and the lymphocyte proliferation level compared with the control group, improves the immunity of the organism, and the survival rate (70%) of the chicken is higher than that (50%) of the astragalus polysaccharide group.
With the rapid development of livestock and poultry breeding industry in recent years, the harm of feed-derived diseases is increasingly prominent, and the serious threat to human health is also caused. Wherein: the feed sources are pathogenic bacillus cereus, bacillus thuringiensis and bacillus pumilus. The harm to livestock and poultry is serious, and the healthy development of livestock and poultry breeding is severely restricted.
1. The harm of bacillus cereus to livestock and poultry breeding industry: bacillus cereus, Bacillus anthracis and Bacillus thuringiensis belong to the same genus. Bacillus cereus is a gram-positive bacterium widely existing in nature, and a part of bacillus cereus can generate a large amount of nutrient substances in the growth and reproduction process, so that the bacillus cereus is widely applied to the field of animal feed additives as a microecology, but most of bacillus cereus can generate toxins, such as enterotoxins Nhe and Hbl, vomitoxin (cere mu Lide), cytotoxic protein Cytk and the like. If the toxins pollute food, the human body is harmed to different degrees such as diarrhea, vomiting and the like; if the bacillus cereus pollutes the livestock and poultry feed, the livestock and poultry feed can cause symptoms of diarrhea, vomiting and the like of different degrees, so that the utilization rate of the feed is greatly reduced, and immeasurable economic loss is brought to the livestock and poultry breeding industry. The source of bacillus cereus in the feed has 3 ways: pesticide pollution, feed pollution, microecologics and fermented traditional Chinese veterinary medicines.
2. Bacillus pumilus is commonly used to control cotton wilt, rice sheath blight, citrus sand blight, and also to reduce cadmium contamination. And secondly for the production of enzyme preparations, such as amylase, ferulic acid lipase and mannosidase B. The bacillus pumilus GBSW19 can not only accelerate the degradation speed of field crop straws, but also effectively improve the pH value of soil, increase the content of soil nutrients, improve the crop yield and has good field application value. After the bacillus pumilus is added into the feed, the white spot syndrome virus infection resistance of the litopenaeus vannamei can be enhanced, and the health condition of shrimp groups can be improved. The growth performance, the digestive enzyme activity and the immune function of the grouper can be obviously improved by adding the bacillus pumilus whole cell wall into the feed. However, the feed is added with excessive Bacillus pumilus to cause serious damage to the muscular stomach and duodenum of the chicken, which also directly indicates that pathogenic target organs of the strain of Bacillus pumilus are the muscular stomach and duodenum and belong to typical pathogenic bacteria of the digestive system. Compared with bacillus cereus separated from feed, the bacillus cereus has the advantages that the bacillus cereus is damaged to the digestive system of chickens, and particularly, the gastric ulcer symptom is remarkable. But the difference is that newly discovered bacillus pumilus does not cause obvious bleeding to duodenum, but the pathogenicity of the bacillus pumilus is the same as that of the bacillus cereus which is separated in the past in terms of morbidity and gastric ulcer degree, and the bacillus pumilus directly threatens the digestive tract health of livestock and poultry.
In conclusion, bacillus cereus, bacillus pumilus and bacillus thuringiensis are one of the important harm factors threatening the livestock and poultry breeding industry and the human food safety at present, so that how to solve the harm from the feed source and reduce the harm loss to the breeding industry and human is an urgent problem in the current breeding market.
Disclosure of Invention
The invention aims to provide a new application of protocatechuic acid in livestock and poultry breeding.
The research of the invention finds that protocatechuic acid has the function of inhibiting pathogenic bacillus in vitro. Further research shows that protocatechuic acid in vivo can prevent and treat diseases caused by pathogenic bacillus, and the protocatechuic acid has better effect and cost advantage when being matched with fulvic acid. Accordingly, the scheme of the invention is as follows:
application of protocatechuic acid in preparing medicine for inhibiting pathogenic bacillus is provided.
Application of protocatechuic acid in preparing medicine for preventing and treating diseases caused by livestock and poultry pathogenic bacillus.
Wherein the pathogenic bacillus is pathogenic bacillus cereus, bacillus pumilus or bacillus thuringiensis.
Wherein the disease is diarrhea, gastric ulcer or intestinal bleeding.
Further, the invention provides a medicine for preventing and treating diseases caused by livestock and poultry pathogenic bacillus, which comprises effective amount of protocatechuic acid.
Preferably, it also comprises fulvic acid. Protocatechuic acid and fulvic acid constitute the compound preparation of the invention.
Preferably, the medicament comprises the following components in parts by weight: 70-90 parts of protocatechuic acid and 10-30 parts of fulvic acid.
Preferably, the medicament comprises the following components in parts by weight: 80 parts of protocatechuic acid and 20 parts of fulvic acid.
The medicine can be powder, granules, oral liquid or tablets and the like.
The preparation method of the compound protocatechuic acid comprises the following steps:
weighing the components according to the parts by weight, crushing, uniformly mixing, and subpackaging into bags to obtain powder.
The compound protocatechuic acid can also be added with conventional auxiliary materials or carriers or can be prepared into oral preparations and the like according to conventional preparation methods in the field, for example, the compound protocatechuic acid can be powder, tablets, granules, oral liquid and the like, and the compound protocatechuic acid is preferably powder or granules. The compound protocatechuic acid medicine is administered orally according to 12.5mg/kg body weight or 200 g/ton feed additive.
Yellow humic acid: the fulvic acid is a short carbon chain molecular structure substance extracted from natural humic acid. It has high loading capacity and physiological activity. The method is applied to the agricultural and horticultural industries and has the following benefits: chelating macro and micro nutrients to make them better available to plants; preventing and treating plant diseases and enhancing waterlogging resistance; activating microscopic biological activity of the plant; the fertilizer is slowly released, and the utilization of chemical fertilizers and pesticides is improved; improving nutrient absorption and promoting plant germination and growth; accelerating the decomposition of the sediment and improving the soil structure.
The invention evaluates the effect of the compound protocatechuic acid on preventing and treating the pathogenic bacillus, finds that the compound protocatechuic acid can effectively prevent and treat diarrhea, gastric ulcer and immunosuppression caused by the pathogenic bacillus, and in vivo experiments show that: after the compound protocatechuic acid is continuously taken orally for 6 days according to the weight of 12.5mg/kg, the attack of pathogenic bacillus cereus on gastric mucosa and intestinal mucosa can be effectively resisted, and the incidence rate of gastric ulcer and intestinal bleeding can be reduced.
The invention has the following beneficial effects:
the compound protocatechuic acid can be widely applied to the poultry industry, the cattle industry and the pig industry, partially replaces mildew removal agent products and antibiotics, makes up for bacterial disease infection caused by the current antibiotic decrement plan of China, replaces drug-resistant antibiotics to prevent and treat bacterial diseases of livestock and poultry, controls the prevalence of infectious diseases, solves the residue of food antibiotics, ensures the food safety and improves the competitiveness of animal product export.
Detailed Description
The following examples further illustrate the present invention but should not be construed as limiting the invention and as imposing limitations upon the scope thereof, as well as modifications or alterations to the methods, procedures or conditions of the present invention.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Protocatechuic acid is purchased from Taishiai chemical industry development Limited company, the content is 99 percent, and the protocatechuic acid is analytically pure;
the fulvic acid is purchased from Ningxia Runtian biological science and technology limited company, and the content of the fulvic acid is 95%.
Example 1
The compound protocatechuic acid comprises the following components: 70 g of protocatechuic acid and 30 g of fulvic acid. The preparation process comprises the following steps:
weighing the components according to the weight, crushing, sieving with a 80-mesh sieve, uniformly mixing, and subpackaging to obtain the powder.
Example 2
The compound protocatechuic acid comprises the following components: 75 g of protocatechuic acid and 25 g of fulvic acid.
The preparation process comprises the following steps:
weighing the components according to the weight, crushing, sieving with a 80-mesh sieve, uniformly mixing, and subpackaging to obtain the powder.
Example 3
The compound protocatechuic acid comprises the following components: 80 g of protocatechuic acid and 20 g of fulvic acid.
The preparation process comprises the following steps:
weighing the components according to the weight, crushing, sieving with a 80-mesh sieve, uniformly mixing, and subpackaging to obtain the powder.
The compound protocatechuic acid used in the following experimental examples was the compound protocatechuic acid powder obtained in example 1.
Example 4
The compound citric acid comprises the following components: 85 g of protocatechuic acid and 25 g of fulvic acid.
The preparation process comprises the following steps:
weighing the components according to the weight, crushing, sieving with a 80-mesh sieve, uniformly mixing, and subpackaging to obtain the powder.
The compound citric acid used in the following experimental examples is the compound protocatechuic acid powder obtained in example 1.
Example 5
The compound protocatechuic acid comprises the following components: 90 g of protocatechuic acid and 10 g of fulvic acid.
The preparation process comprises the following steps:
weighing the components according to the weight, crushing, sieving with a 80-mesh sieve, uniformly mixing, and subpackaging into bottles to obtain the powder.
The compound citric acid used in the following experimental examples is the compound protocatechuic acid powder obtained in example 1.
Example 6
The compound protocatechuic acid comprises the following components: 80 g of protocatechuic acid and 20 g of fulvic acid.
The preparation process comprises the following steps:
dissolving a certain amount of the compound powder completely with distilled water to obtain a liquid with a concentration of 12.5mg/mL to obtain compound protocatechuic acid oral liquid (requiring light-blocking storage).
Unless otherwise specified, the compound protocatechuic acid mentioned in the following experimental examples was the compound protocatechuic acid powder obtained in example 1.
Experimental example 1 protocatechuic acid in vitro killing pathogenic Bacillus
1. Material
1.1 test strains of pathogenic Bacillus cereus DW strain, pathogenic Bacillus pumilus and Bacillus thuringiensis, which are separated, identified and stored in the subject group.
1.2 protocatechuic acid of the experimental drug is purchased from Taishiai chemical industry development Limited company, the content is 99 percent, and the protocatechuic acid is analytically pure; the detailed preparation method of the compound protocatechuic acid powder is shown in example 1.
2. Method of producing a composite material
2.1 amplification of pathogenic Bacillus cereus, Bacillus pumilus and Bacillus thuringiensis and preparation of bacterial liquid
Selecting and identifying the purified bacillus cereus, bacillus pumilus or bacillus thuringiensis, inoculating the bacillus cereus, bacillus pumilus or bacillus thuringiensis into 100mL of sterilized nutrient broth, and placing the broth in a constant-temperature oscillator at 37 ℃ for shake culture for 24 hours.
Counting of bacillus: taking 8 test tubes, respectively marking 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7 and 10-8, respectively adding 1800 common nutrient broth culture media into the 8 test tubes, respectively adding the proliferated bacterial suspension 200 into the 1 st test tube, mixing for 20S by using an electric mixer, sucking 200 liquid containers by using a micropipettor, adding into the 2 nd test tube, uniformly mixing, then, taking 200 samples by using the micropipettor, adding into the 3 rd test tube, and so on until the last tube, selecting a test tube with a proper dilution degree (15 cfu-300 cfu per plate according to the expected number of growing colonies), sucking 1.0ml of the uniformly mixed suspension, adding into a sterile plate, inoculating three plates per dilution degree, pouring a dissolved nutrient agar culture medium cooled to 40-45 ℃ into the plate with the added bacterial liquid, about 15-20ml per plate, cover the plate, shake gently immediately, mix well, lay flat on the laboratory bench. After the agar is solidified, turning over the plate to make the bottom face upward, placing the plate in a 37 ℃ incubator for culture, observing the growth condition of bacteria every day, and counting after culturing for 24 hours. Then, the average value is taken and converted into the concentration of the bacterial liquid according to the dilution. When the number of the counted bacterial colonies in the plate is not in the range of 15-300 cfu, the dilution degree needs to be adjusted according to the turbidity degree of the bacterial liquid, and the bacterial colonies are guaranteed to fall in the range.
2.2 minimum antimicrobial drug concentration (MIC) determination
Placing a 96-hole polystyrene U-shaped microporous plate on an ultra-clean workbench, opening a cover, opening an ultraviolet lamp for irradiating for 30min for sterilization, adding protocatechuic acid and compound protocatechuic acid diluent 100 into each hole by using a microsyringe, adding three holes in parallel at the same concentration by using a 100-hole sampler, sequentially adding 100 low-bacterial suspension into each hole according to the sequence from low concentration to high concentration, placing the microporous plate on a micro-oscillator for oscillation for 1min, uniformly mixing the solution in each hole, covering the microporous plate, sealing the microporous plate by using a sealing film to reduce evaporation in the incubation process, placing the microporous plate in a wet box, placing the box in a 37-DEG C incubator for culture for 24h, and observing an experimental result. Each tube was diluted to a concentration of about 5X 10, except for the broth control51mL of the suspension (Bacillus cereus, Bacillus pumilus or Bacillus thuringiensis) of (A). And (5) placing the test tube on a constant temperature shaking table at 37 ℃ to culture for 24h, and observing the result.
And observing an experimental result, wherein the lowest antibacterial drug concentration contained in the bacteria-free growth hole is MIC. 5 needs to be added into each hole to judge the bacteriostasis effect, and if the solution turns red, the existence of bacteria is indicated. The bacteria are indicated by adding 5 to each well in the order of the sample concentration from top to bottom. . To eliminate the effect of the drug itself, the incompletely defined test tube liquid was transferred to MYP agar plate medium and incubated at 37 ℃ for 24 h. The next day, the plates were observed for bacterial growth.
And (4) judging a result: and (3) comparing the growth characteristics of the detection bacteria and the standard bacteria in the growth control holes, such as turbid broth in the micropores, precipitate at the bottoms of the micropores and the like under a black background light source to judge, wherein the concentration of the lowest antibacterial drug contained in the bacteria-free growth holes is MIC. To make the result clear quickly, 5g/L of triphenyltetrazolium chloride (TTC)5 oxanil can be added into each well. After incubation for 1-3 h at 35 ℃, the holes with bacteria growth are red, which is helpful for result judgment.
3. Results
3.1 test results of bacterial species
Table 1 test results of the count of three Bacillus species
| Bacterial strain | Concentration (CFU) |
| Bacillus cereus | 2×108 |
| Bacillus pumilus | 2.0×109 |
| Bacillus thuringiensis | 1×109 |
From Table 1, it can be seen that the concentration of pathogenic Bacillus cereus is 2X 108The Bacillus pumilus concentration is 2.0 × 109The concentration of Bacillus thuringiensis is 1 × 109The three bacillus species are different in concentration.
3.2 measurement of minimum inhibitory concentration
Through a test tube dilution method, the MIC of protocatechuic acid to the bacillus cereus is 1.2mg/mL, the MIC of the bacillus pumilus is 0.98mg/mL, and the MIC of the bacillus thuringiensis is 0.85 mg/mL. The MICs of fulvic acid for the three species of Bacillus were 40.0, 20.0 and 35.0mg/mL, respectively. MICs of compound protocatechuic acid (90:10) to the above three kinds of bacilli are 1.25, 1.0 and 0.92 mg/mL, respectively. The MIC values for the three above mentioned bacilli gradually increased with the change of the concentrations of protocatechuic acid and fulvic acid. The results are shown in Table 2.
TABLE 2 minimum inhibitory concentrations of protocatechuic acid and Compound protocatechuic acid
4. Conclusion
From the three bacillus in vitro inhibition tests, the protocatechuic acid has the minimum inhibitory concentrations of 1.2, 0.98 and 0.85mg/ml in vitro on bacillus cereus, bacillus pumilus and bacillus thuringiensis respectively, and has a certain killing effect on the three bacillus. The MIC of the compound protocatechuic acid (80:20) and the compound protocatechuic acid (90:10) for inhibiting the three bacilli in vitro has no significant difference.
Experimental example 2 application of Compound protocatechuic acid in prevention and treatment of pathogenic Bacillus cereus
The test aims to evaluate the treatment effect of protocatechuic acid on the pathogenic bacillus cereus of the chicken by establishing an artificial infection morbidity model.
1. Material
1.1 test animals 24 days old SPF chickens, 80.
1.2 test strain pathogenic bacillus cereus, DW strain in lung of sick chicken is separated, identified and stored in the laboratory.
1.3 test drug 1 Compound protocatechuic acid (70:30) dose group, Compound protocatechuic acid (80:20) dose group, Compound protocatechuic acid (90:10) dose group, see Experimental examples 1, 3 and 5 preparation methods in detail.
1.4 control drug Ganfeishu, containing vitamin B premix, purchased from Shandong Xin De science and technology GmbH.
2. Method of producing a composite material
2.1 grouping: a compound protocatechuic acid (70:30) dose group, a compound protocatechuic acid (80:20) dose group, a compound protocatechuic acid (90:10) dose group, a glandular stomach easing drug group, a pathogenic bacillus cereus group (namely an infected group) and a blank control group, wherein each group comprises 10 SPF (specific pathogen free) chickens with the age of 24 days.
2.2 amplification of pathogenic Bacillus cereus and preparation of bacterial liquid
Selecting, identifying and purifying Bacillus cereus, inoculating into 100mL sterilized nutrient broth, placing in a constant temperature oscillator at 37 deg.C, and shake culturing to OD600The value is 0.5 to 0.6. Diluting the above bacterial liquid 1ml with nutrient broth 10-1、10-2、10-3、10-4And 10-5And (3) uniformly coating the five bacterial liquids with different concentrations on sterilized common nutrient agar after each bacterial liquid with the concentration of 100 mu 0 is respectively and aseptically absorbed, inversely placing the bacterial liquids in a constant-temperature incubator at 37 ℃ for 24 hours, then counting the bacterial colonies, and calculating the bacterial colonies of each concentration bacterial liquid.
Obtaining Bacillus cereus liquid at 5000rpm/min, centrifuging for 15min, discarding supernatant, remixing the precipitate with 20ml normal saline, centrifuging for 15min, discarding supernatant, repeating for 2 times, diluting the obtained Bacillus cereus precipitate with sterile normal saline to 108cfu/mL, and mixing well for use.
2.3 administration dosage and treatment mode:
the administration mode comprises the following steps: oral administration; oral administration was continued for 5 days according to the dose of table 3.
Control group for challenge: the pathogenic Bacillus cereus is administered at a dose of 1x108cfu/ml.
Test drug groups: the bacillus is continuously taken orally for 7 days, and then the compound protocatechuic acid is taken with drinking water.
Control drug group: the pathogenic waxy spores are given for 7 days first and then taken orally continuously.
Blank control group: the preparation is administered by infusing with normal saline.
The test duration is as follows: after 5 consecutive days of administration, 5 days were observed for 10 days.
2.4 methods of combating and administering:
counteracting toxic substances: except for the blank control group, all the groups orally take the pathogenic bacillus cereus, and each chicken is irrigated with 1ml of 10 ml8The diarrhea model was prepared by continuously drenching cfu/mL of Bacillus cereus solution once a day for 7 days. The control group was administered to each chicken with 1ml of sterile physiological saline daily for 7 days.
After 7 days in the artificial model, after confirming that 30% of chickens have yellow and red loose stools, the selective administration is started, and the administration is continuously carried out for 5 days, 1 time per day and continuously observed for 10 days.
TABLE 3 treatment trial groupings and treatment
2.5 test index
Overall observation indexes are as follows: recording the survival rate, the relative weight gain rate and the clinical diarrhea characteristics of the chickens;
relative weight gain (%) (end of experiment weight-initial experiment weight)/control group weight gain x control group gain;
pathological change indexes are as follows: on the day of the end of the test period, groups of SPF chickens were euthanized and observed for gastric ulcer, lung lesions and duodenal bleeding after dissection. Dissecting all SPF chickens, carefully picking the stomach, cutting with surgical scissors, cleaning the content of the stomach, observing whether the stratum corneum of the muscular stomach has a typical ulcer focus, scoring the pathological change degree and counting the incidence rate of gastric ulcer. The duodenum is pulled out by hand gently, the root is cut off by the surgical scissors, the whole intestine body is stretched, the intestinal canal is cut off by the surgical scissors, the content is clear, and whether the inner wall of the small intestine is bleeding or not is observed.
(1) Gastric ulcer scoring criteria:
0 minute: the stratum corneum of the muscular stomach has no ulcer spots visible to naked eyes;
1 minute: with minor bleeding or minor ulceration in one to two areas
And 2, dividing: in one to two areas there was significant bleeding and mild ulceration
And 3, dividing: has obvious bleeding point and ulcer focus in more than three areas
(2) Duodenum lesion scoring standard
0 minute: no visible bleeding point
1 minute: with small bleeding points in one to two regions
And 2, dividing: with apparent bleeding points in more than two areas
And 3, dividing: has obvious bleeding point or ulcer focus in more than three areas
3. Results
3.1 clinical diarrhea Rate and relative weight gain
After the bacillus cereus is orally taken, the chicken turns red and thin, which indicates that the model is successfully replicated. The clinical diarrhea rates of three dose groups of compound protocatechuic acid are respectively 10%, 20% and 30%, and the significance is lower than that of glandular stomach-soothing group (60%)) and bacillus cereus control group (90%). In the aspect of relative weight gain, the relative weight gain rates of three dose groups of compound protocatechuic acid are 131.0%, 120.1% and 103.1%, the relative weight gain rates of protocatechuic acid, fulvic acid, glandular stomach relaxing groups and bacillus cereus control groups are 123.5%, 90.2%, 89.7% and 51.0%, the difference between the dose groups of compound protocatechuic acid (70:30) and compound protocatechuic acid (80:20) and the glandular stomach relaxing groups is obvious (P is less than 0.05), and the difference between the dose groups of compound protocatechuic acid (70:30), compound protocatechuic acid (80:20) and the bacillus cereus groups is extremely obvious, which indicates that the bacillus cereus obviously reduces the weight development of the chicken. The results are shown in Table 4.
TABLE 4 diarrhea incidence and relative weight gain in SPF chickens from each group
| Grouping | Diarrhea Rate (%) | Average relative weight gain (%) |
| Compound protocatechuic acid (90:10) group | 10 | 131.0a |
| Compound protocatechuic acid (80:20) group | 20 | 120.1a |
| Compound protocatechuic acid (70:30) group | 30 | 103.1b |
| Protocatechuic acid group | 20 | 123.5a |
| Fulvic acid group | 70 | 90.2b |
| Glandular stomach relaxing group | 60 | 89.7b |
| Bacillus control group | 90 | 51.0c |
| Blank control group | 0 | 100.0 |
Note: in the same rowa-b,b-cP＜0.05；a-cP＜0.01
3.2 incidence and lesion index of gastric ulcer
After the medicine is orally taken for 5 days, the average incidence rates of gastric ulcer in three dose groups of compound protocatechuic acid are respectively found to be 5.0%, 10% and 30%, and the average incidence rates of the protocatechuic acid, the fulvic acid and the glandular stomach are respectively found to be 10.0%, 60.0% and 50%. The average gastric ulcer index is measured, and the compound protocatechuic acid (90:10) and the compound protocatechuic acid (80:20) are found to be remarkably lower than those of a protocatechuic acid group, a fulvic acid group and a glandular stomach easing control group and extremely remarkably lower than those of a bacillus control group, which indicates that the compound protocatechuic acid (90:10) and the compound protocatechuic acid (80:20) are better than those of the protocatechuic acid group and the fulvic acid group in preventing and treating gastric ulcer. The results are shown in Table 5.
TABLE 5 incidence of gastric ulcer and ulcer lesion index
| Grouping | Incidence of gastric ulcer (%) | Mean index of gastric ulcer lesion |
| Compound protocatechuic acid (90:10) group | 10.0a | 0.20a |
| Compound protocatechuic acid (80:20) group | 10.0a | 0.26a |
| Compound protocatechuic acid (70:30) group | 30.0b | 0.45b |
| Protocatechuic acid group | 10.0a | 0.40b |
| Fulvic acid group | 60.0b | 0.65c |
| Glandular stomach relaxing group | 50.0b | 0.73c |
| Bacillus control group | 80.0c | 1.5d |
| Blank control group | 0 | 0 |
Note: in the same rowa-b,b-c,c-dP＜0.05,a-c,a-dP＜0.01
3.3 duodenum bleeding incidence and disease index
After treatment, the intestinal hemorrhage rate of the three dose groups of the compound protocatechuic acid is 5.0 percent, 10.0 percent and 20.0 percent respectively. The incidence rate of the glandular stomach easing control group is 40 percent. Taking the intestinal bleeding lesion index as an index, and counting the average lesion index result as follows: compound protocatechuic acid (90:10) compound protocatechuic acid (80:20) has no significant difference with protocatechuic acid, but has lower significance than compound protocatechuic acid (70:30), fulvic acid and glandular stomach soothing groups. The results are shown in Table 6.
TABLE 6 duodenum incidence and lesion index
| Grouping | Incidence of disease (%) | Index of disease |
| Compound protocatechuic acid (90:10) group | 10.0a | 0.32a |
| Compound protocatechuic acid (80:20) group | 10.0a | 0.40a |
| Compound protocatechuic acid (70:30) group | 30.0b | 0.62b |
| Protocatechuic acid group | 10.0a | 0.45a |
| Fulvic acid group | 50.0b | 2.2c |
| Glandular stomach relaxing group | 40.0b | 2.9c |
| Bacillus control group | 80.0c | 3.2c |
| Blank control group | 10.0d | 0.28a |
Note: in the same rowa-b,b-cP＜0.05,a-cP＜0.01
4. Conclusion
The compound protocatechuic acid (80:20) dose group and the compound protocatechuic acid (90:10) dose group are 12.5mg/kg of body weight, 1 time per day and 5 days of continuous administration, so that the body is effectively improved, diarrhea and myogastric ulcer caused by artificial infection of bacillus cereus are reduced, and gastric ulcer lesion index and duodenal hemorrhage lesion index are effectively reduced, thereby improving production performance and reducing death and elutriation rate. Taking cost considerations into account, the recommended dose group of compound protocatechuic acid (80:20) is preferred.
Experimental example 3 application of Compound protocatechuic acid in prevention and treatment of pathogenic Bacillus pumilus
Test purpose evaluation of the treatment effect of the compound protocatechuic acid on diarrhea and gastric ulcer caused by bacillus pumilus after the broiler chicken artificially replicates the diarrhea and gastric ulcer model.
1. Material
1.1 broiler chickens 10 days old, 60, purchased from Beijing Aijia poultry breeding Limited.
1.2 broiler feeds were purchased from multi-food group ltd, makken, north Hebei.
1.3 test strain pathogenicity section small bacillus, which is separated and identified from the feed of a certain breeding hen in Hebei by the laboratory.
1.4 test drugs Compound protocatechuic acid (70:30) dose group, Compound protocatechuic acid (80:20) dose group, Compound protocatechuic acid (90:10) dose group, see Experimental example 1, Experimental example 3, and Experimental example 5 preparation method for details.
1.5 control drug Ganfeishu, containing vitamin B premix, purchased from Shandong Xin De science and technology GmbH.
2. Method of producing a composite material
2.1 grouping: compound protocatechuic acid (70:30) dose group, compound protocatechuic acid (80:20) dose group, compound protocatechuic acid (90:10) dose group, glandular stomach easing control drug group, bacillus pumilus group (namely, infection group) and blank control group, and 6 groups are counted, and 10 groups are used in each group.
2.2 amplification of Bacillus pumilus and preparation of bacterial solution
Selecting, identifying and purifying Bacillus pumilus, inoculating into 100mL sterilized nutrient broth, and shake culturing at 37 deg.C to OD600The value is 0.5 to 0.6. Preparation of bacterial liquid 10 by diluting 1ml with nutrient broth-1、10-2、10-3、10-4And 10-5And (3) uniformly coating the five bacterial liquids with different concentrations on sterilized common nutrient agar by respectively and aseptically sucking 100 bacterial liquids with the same concentration in each concentration, inversely placing the bacterial liquids in a constant-temperature incubator at 37 ℃ for 24 hours, then counting the bacterial colonies, and calculating the bacterial colonies number of the bacterial liquid with each concentration.
2.3 administration dosage and treatment mode:
the administration mode comprises the following steps: oral administration; oral administration was continued for 5 days according to the dose of table 7.
Control group for challenge: the bacillus pumilus is administered by drenching at a dose of 1x108cfu/ml.
Test drug groups: the compound protocatechuic acid is administered after 7 days of continuous oral administration of Bacillus pumilus, and administered with drinking water.
Control drug group: the Bacillus pumilus is administered for 7 days before the administration of the medicine for oral administration.
Blank control group: the preparation is administered by infusing with normal saline.
The test duration is as follows: after 5 consecutive days of administration, 5 days were observed for 10 days.
2.4 methods of combating and administering drugs
Except for the blank control group, the other groups orally take the bacillus pumilus, and each chicken is irrigated with 1ml of 10 ml8cfu/mL Bacillus pumilus solution was administered once daily for 7 consecutive days. The control group was administered to each chicken with 1ml of sterile physiological saline daily for 7 days.
TABLE 7 treatment trial groupings and treatment
| Grouping | Quantity (only) | Dosage of drug | Number of administrations |
| Compound protocatechuic acid (90:10) group | 10 | 12.5mg/kg body weight | 1 time per day |
| Compound protocatechuic acid (80:20) group | 10 | 12.5mg/kg body weight | 1 time per day |
| Compound protocatechuic acid (70:30) group | 10 | 12.5mg/kg body weight | 1 time per day |
| Glandular stomach relaxing group | 10 | 300mg/kg body weight | 1 time per day |
| Bacillus pumilus control group | 10 | 1 mL/piece (10)8cfu/mL) | 1 time/day |
| Blank control group | 10 | 1 mL/body of physiological saline | 1 time/day |
2.5 evaluation index
Overall observation indexes are as follows: recording the incidence rate of clinical diarrhea and the relative weight gain rate of the chickens;
relative weight gain (%) (end of experiment weight-initial experiment weight)/control group weight gain x control group gain;
the pathological change determination method comprises the following steps: on the day of the test period, the broilers were euthanized, and after dissection, gastric ulcer and duodenal bleeding were observed. The lesion assay method was as described in example 1.
3. Results
3.1 Effect of Compound protocatechuic acid on clinical diarrhea rate and relative weight gain rate of broiler chickens
After the broiler chickens are artificially orally taken with the bacillus pumilus, yellow loose stools of the chickens gradually appear red loose stools after 3 days, which proves that the orally taken bacillus pumilus can replicate a broiler chicken diarrhea model. The incidence rates of diarrhea of the compound protocatechuic acid group (90:10), the compound protocatechuic acid group (80:20), the compound protocatechuic acid group (70:30) and the control drug glandular stomach soothing group reach 10%, 20% and 40%, and the significance is lower than that of the bacillus pumilus group by 80%. In the aspect of relative weight gain, 125.7%, 108.4% and 95.2% of compound protocatechuic acid group (90:10), 80: 20% of compound protocatechuic acid group (70:30) and 92.1% of glandular stomach relaxing group are respectively adopted, wherein the difference between the compound protocatechuic acid group (70:30) and the glandular stomach relaxing group is not significant, but is higher than that of a bacillus pumilus control group, and the results are shown in table 8.
TABLE 8 incidence and relative weight gain of diarrhea in broilers
Note: in the same rowa-b,b-cP＜0.05；a-cP＜0.01
3.2 Effect of Compound protocatechuic acid on incidence of gastric ulcer and ulcer lesion index of broiler chickens
After the medicine is orally taken for 5 days, the incidence rates of the muscular gastric ulcer in the compound protocatechuic acid group (90:10), the compound protocatechuic acid group (80:20), the compound protocatechuic acid group (70:30) and the glandular stomach soothing control group are respectively 0, 10.0%, 20.0% and 30%, and the incidence rate is lower than that of the bacillus pumilus control group by 70%. In the aspect of measuring the indexes of the gastric ulcer, the average ulcer indexes of a compound protocatechuic acid group (90:10), a compound protocatechuic acid group (80:20), a compound protocatechuic acid group (70:30) and a glandular stomach easing group are respectively 0.21, 0.57, 0.68 and 1.24, and the three dose groups of the compound protocatechuic acid are all obviously lower than those of the glandular stomach easing group, thereby showing good curative effect of controlling the gastric ulcer. The results are shown in Table 9.
TABLE 9 incidence and lesion index of gastric ulcer in broilers
| Grouping | Incidence of gastric ulcer (%) | Mean index of gastric ulcer lesion |
| Compound protocatechuic acid (90:10) group | 0 | 0.21a |
| Compound protocatechuic acid (80:20) group | 10.0a | 0.57b |
| Compound protocatechuic acid (70:30) group | 20.0b | 0.68b |
| Glandular stomach relaxing group | 30.0b | 1.24c |
| Bacillus control group | 70.0c | 4.38d |
| Blank control group | 0 | 0 |
Note that in the same rowa-b,b-c,c-dP＜0.05；a-c.a-d,b-dP＜0.01
3.3 Effect of Compound protocatechuic acid on morbidity and pathological change index of duodenal bleeding of broiler chickens
The results are shown in Table 10. The incidence rates of intestinal hemorrhage of the compound protocatechuic acid group (90:10), the compound protocatechuic acid group (80:20), the compound protocatechuic acid group (70:30) and the glandular stomach comforting group are respectively 10%, 20%, 40% and 60%, and the incidence rate is remarkably lower than that of the bacillus pumilus group by 90%. The intestinal tract bleeding indexes of the three groups of the compound protocatechuic acid group and the glandular stomach relaxing group are respectively 0.51, 0.66, 0.76 and 1.26, and the intestinal tract lesion indexes of the three groups of the compound protocatechuic acid group (90:10), the compound protocatechuic acid group (80:20) and the compound protocatechuic acid group (70:30) are not obviously different. However, the significance is lower than that of glandular stomach relaxing group, which indicates that the compound protocatechuic acid can effectively reduce intestinal bleeding lesion and lesion index.
TABLE 10 incidence and disease index of duodenal bleeding in broilers
| Grouping | Incidence of disease (%) | Index of disease |
| Compound protocatechuic acid (90:10) group | 10.0a | 0.51a |
| Compound protocatechuic acid (80:20) group | 20.0a | 0.66a |
| Compound protocatechuic acid (70:30) group | 40.0b | 0.71a |
| Glandular stomach relaxing group | 60.0b | 1.26b |
| Bacillus pumilus control group | 90.0c | 3.80c |
| Blank control group | 10.0a | 0.41a |
Note: same as above
4. Conclusion
Considering the factors of comprehensive cost, diarrhea and gastric ulcer lesion indexes, the compound protocatechuic acid group (80:20) is recommended to be used for 12.5mg/kg of body weight, 1 time per day, and the broiler chickens are continuously administrated for 5 days, so that the lesions such as diarrhea, gastric ulcer, duodenal hemorrhage and the like caused by artificial infection of bacillus pumilus can be effectively reduced.
Claims (10)
1. Application of protocatechuic acid in preparing medicine for inhibiting pathogenic bacillus is provided.
2. The use according to claim 1, wherein the pathogenic bacillus is a pathogenic bacillus cereus, bacillus pumilus or bacillus thuringiensis.
3. Application of protocatechuic acid in preparing medicine for preventing and treating diseases caused by livestock and poultry pathogenic bacillus.
4. The use according to claim 3, wherein the pathogenic Bacillus is a pathogenic Bacillus cereus, Bacillus pumilus or Bacillus thuringiensis.
5. The use according to claim 3, wherein the disease is diarrhea, gastric ulcer or intestinal bleeding.
6. A medicine for preventing and treating diseases of livestock and poultry caused by pathogenic bacillus comprises effective amount of protocatechuic acid.
7. The medicament according to claim 6, characterized in that it further comprises fulvic acid.
8. The medicament of claim 6, which comprises the following components in parts by weight: 70-90 parts of protocatechuic acid and 10-30 parts of fulvic acid.
9. The medicament of claim 8, wherein the medicament comprises the following components in parts by weight: 80 parts of protocatechuic acid and 20 parts of fulvic acid.
10. The medicament according to any one of claims 6 to 9, wherein the medicament is a powder, a granule, an oral liquid or a tablet.
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