WO2022205557A1 - 假蒟提取物在制备防治禽类曲霉菌病的添加剂中的应用 - Google Patents
假蒟提取物在制备防治禽类曲霉菌病的添加剂中的应用 Download PDFInfo
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- WO2022205557A1 WO2022205557A1 PCT/CN2021/092030 CN2021092030W WO2022205557A1 WO 2022205557 A1 WO2022205557 A1 WO 2022205557A1 CN 2021092030 W CN2021092030 W CN 2021092030W WO 2022205557 A1 WO2022205557 A1 WO 2022205557A1
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- extract
- aspergillus
- konjac
- aspergillosis
- extraction
- Prior art date
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- 239000000284 extract Substances 0.000 title claims abstract description 59
- 201000002909 Aspergillosis Diseases 0.000 title claims abstract description 27
- 208000036641 Aspergillus infections Diseases 0.000 title claims abstract description 27
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 241000271566 Aves Species 0.000 title claims abstract description 13
- 239000000654 additive Substances 0.000 title claims abstract description 11
- 230000000996 additive effect Effects 0.000 title claims abstract description 9
- 240000001724 Piper sarmentosum Species 0.000 title abstract description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 26
- 241000228197 Aspergillus flavus Species 0.000 claims abstract description 8
- 241001225321 Aspergillus fumigatus Species 0.000 claims abstract description 8
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 8
- 229940091771 aspergillus fumigatus Drugs 0.000 claims abstract description 8
- 241001465318 Aspergillus terreus Species 0.000 claims abstract description 5
- 239000006286 aqueous extract Substances 0.000 claims abstract 2
- 235000001206 Amorphophallus rivieri Nutrition 0.000 claims description 38
- 229920002752 Konjac Polymers 0.000 claims description 38
- 235000010485 konjac Nutrition 0.000 claims description 38
- 239000000252 konjac Substances 0.000 claims description 38
- 241000272525 Anas platyrhynchos Species 0.000 claims description 23
- 238000000605 extraction Methods 0.000 claims description 23
- 241000589516 Pseudomonas Species 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 241000228212 Aspergillus Species 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 241000228143 Penicillium Species 0.000 claims description 3
- 244000144977 poultry Species 0.000 claims description 3
- 241001312219 Amorphophallus konjac Species 0.000 claims 5
- 208000028774 intestinal disease Diseases 0.000 abstract description 2
- 210000002345 respiratory system Anatomy 0.000 abstract description 2
- 241000132177 Aspergillus glaucus Species 0.000 abstract 1
- 208000018569 Respiratory Tract disease Diseases 0.000 abstract 1
- 244000247812 Amorphophallus rivieri Species 0.000 description 33
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- 238000002474 experimental method Methods 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 240000001439 Opuntia Species 0.000 description 9
- 235000013389 Opuntia humifusa var. humifusa Nutrition 0.000 description 9
- VNFPBHJOKIVQEB-UHFFFAOYSA-N clotrimazole Chemical compound ClC1=CC=CC=C1C(N1C=NC=C1)(C=1C=CC=CC=1)C1=CC=CC=C1 VNFPBHJOKIVQEB-UHFFFAOYSA-N 0.000 description 9
- 229960004022 clotrimazole Drugs 0.000 description 9
- 230000002265 prevention Effects 0.000 description 9
- 230000000694 effects Effects 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 239000003651 drinking water Substances 0.000 description 6
- 235000020188 drinking water Nutrition 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 239000003674 animal food additive Substances 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- 241000272522 Anas Species 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- 230000000844 anti-bacterial effect Effects 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000009395 breeding Methods 0.000 description 4
- 230000001488 breeding effect Effects 0.000 description 4
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000011161 development Methods 0.000 description 3
- 241000411851 herbal medicine Species 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 206010012735 Diarrhoea Diseases 0.000 description 2
- 206010013975 Dyspnoeas Diseases 0.000 description 2
- 206010017533 Fungal infection Diseases 0.000 description 2
- 230000036528 appetite Effects 0.000 description 2
- 235000019789 appetite Nutrition 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 208000024386 fungal infectious disease Diseases 0.000 description 2
- 238000002386 leaching Methods 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 240000001008 Dimocarpus longan Species 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 235000000235 Euphoria longan Nutrition 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 241000758706 Piperaceae Species 0.000 description 1
- 244000184734 Pyrus japonica Species 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000012675 alcoholic extract Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000004596 appetite loss Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 235000020639 clam Nutrition 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 238000005553 drilling Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 208000001848 dysentery Diseases 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 239000000469 ethanolic extract Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 235000021266 loss of appetite Nutrition 0.000 description 1
- 208000019017 loss of appetite Diseases 0.000 description 1
- 239000002398 materia medica Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 235000013622 meat product Nutrition 0.000 description 1
- 229960000988 nystatin Drugs 0.000 description 1
- VQOXZBDYSJBXMA-NQTDYLQESA-N nystatin A1 Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/CC/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 VQOXZBDYSJBXMA-NQTDYLQESA-N 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
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- 230000001737 promoting effect Effects 0.000 description 1
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- 239000002904 solvent Substances 0.000 description 1
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- 208000024891 symptom Diseases 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000008736 traumatic injury Effects 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/67—Piperaceae (Pepper family), e.g. Jamaican pepper or kava
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
Definitions
- the present invention relates to the technical field of poultry breeding, in particular to the application of a pseudocone extract in the preparation of an additive for preventing and treating avian aspergillosis.
- Avian aspergillosis is a common fungal disease in the duck industry, especially in young ducks, with a high mortality rate of up to 50%.
- the pathogenic bacteria that cause fowl aspergillosis are Aspergillus, including Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus and Aspergillus penicillium.
- the weather in Hainan is high temperature and high humidity, the feed and bedding are easy to be mildewed, and there are many mold spores in the breeding environment, especially in the summer of Hainan, the disease is a common disease in duck breeding.
- the clinical symptoms of the disease are dyspnea, gasping for breath, increased breathing rate, loss of appetite, like drinking water, like to sleep, loose green stools, often standing or lying in corners, and often viscous secretions from the mouth and nose.
- 1 ⁇ copper sulfate, 1 ⁇ potassium iodide, 0.05% clotrimazole, and 0.5 million IU nystatin have obvious curative effects.
- the technical problem to be solved by the present invention is to provide the application of the konjac extract in the preparation of feed additives.
- the present invention provides the application of the extract of konjac japonica in the preparation of an additive for preventing and treating avian aspergillosis.
- the avian aspergillosis is caused by Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus and Aspergillus penicillium.
- the avian aspergillosis is duck aspergillosis.
- the duck aspergillosis is red duck aspergillosis.
- the extract of konjac is a water extract of konjac or an alcohol extract of konjac.
- the preparation method of the water extract of the pseudocone is as follows: the pseudocone is extracted three times with water under reflux, and the combined extract is concentrated to obtain the pseudocone water extract; in the three extractions, the first extraction 3h, the second extraction 2h, the third extraction 1h.
- the mass-volume ratio of pseudocone to water was 1kg: 0.5L
- the mass-volume ratio of pseudocone to water was 1kg: 0.4L
- pseudocone The mass-volume ratio with water is 1kg:0.3L.
- the preparation method of the alcohol extract of Pseudomonas konjac is: leaching Pseudomonas konjac with ethanol for three times, and the combined extracts are concentrated to obtain the alcohol extract of Pseudomonas konjac; in the three extractions, the first leaching 7 days of extraction, 5 days of second extraction, and 3 days of third extraction.
- the mass-volume ratio of pseudocone to ethanol was 1kg: 2L
- the mass-volume ratio of pseudocone to ethanol was 1kg: 1.5L
- pseudocone and ethanol were The mass-volume ratio of ethanol is 1kg:1.5L.
- the ethanol is 95% ethanol. That is, a solution in which ethanol-water is mixed in a volume ratio of 95:5.
- the present invention also provides an additive comprising the konjac extract.
- the additive described in the present invention can be added to feed or drinking water, which is not limited in the present invention. Its addition in drinking water is 1%. That is, the mass fraction added to the extract of konjac in drinking water is 1%.
- the present invention also provides a method for raising poultry, which is to administer the additive of the present invention.
- the administration time is 7 days of age, and the administration is continued for one week.
- the water extract and alcoholic extract of Pseudo konjac have inhibitory effects on Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus and other Aspergillus fungi, and it is verified that the pseudokonjac extract can be used to prevent and treat the respiratory tract of birds due to Aspergillus infection. , intestinal diseases, the effective rate can reach 81% to 84%.
- the plant resources of Pseudomonas spp. are widely distributed, the biomass is large, and the price is low, especially in Hainan forests, roadside can be seen everywhere, the extract preparation process is simple, the cost is low, the yield is high, and it is easy to carry out large-scale production.
- the present invention provides the application of Konjac chinensis extract in the preparation of feed additives, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention.
- the method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications herein without departing from the content, spirit and scope of the present invention, so as to realize and apply the present invention. Invention technology.
- test materials used in the present invention are all common commercial products and can be purchased in the market.
- Described Chinese herbal medicine pseudo konjac is also known as clam clam, no prickly pear, prickly pear, clam clam, prickly pear, clam clam, wood handle prickly pear, longan prickly pear, pig prickly pear, drilling bone wind, stinky prickly pear, mountain prickly pear, horseshoe prickly pear is pepper Piper sarmentosum (Piper sarmentosum Roxb.) of the family (Piperaceae) has the effects of dispelling wind and dispelling cold, promoting qi and relieving pain, activating collaterals, and reducing swelling.
- the preparation of the extract of Konjac can use the whole plant of Konjac, and also can use some tissues and organs. Before extraction, it goes through the steps of drying and crushing. In the embodiment of the present invention, the whole plant of Konjac is used to prepare the extract of Konjac.
- Activated the target strain diluted to 1 ⁇ 10 6 CFU/mL of bacterial solution for later use; prepared a YPD sterile plate; took 0.1 mL of the prepared bacterial solution and evenly spread it on the YPD plate with a sterile cotton swab;
- the konjac extract of Examples 1-2 was dissolved in methanol to prepare a solution with a concentration of 10 mg/mL; the clotrimazole was prepared into a solution with a concentration of 10 mg/mL with acetone, and 10 ⁇ L of the medicinal solution was added dropwise to a diameter of 6 mm.
- the konjac extract was dissolved in DMSO to an initial concentration of 1 mg/mL, and clotrimazole was used as a positive control to dissolve in DMSO to an initial concentration of 1 mg/mL, and DMSO was used as a blank control.
- a 96-well plate use sterile medium to carry out gradient dilution according to the 2-fold method, add the bacteria to be tested after dilution, and observe after culturing for 12 hours. This experiment was set up with 3 replicates.
- the minimum inhibitory concentration of the Pseudomonas konjac extract of the present invention is less than 100 ⁇ g/mL on the three bacteria in the genus Aspergillus, indicating that the extract has a certain inhibitory effect on the above three kinds of Aspergillus It can be used as a feed additive for the prevention and control of duck aspergillosis.
- the experiment was carried out in a paddy duck farm in Danzhou.
- the experiment time was July, which is the season of high temperature and humidity in Hainan and high incidence of aspergillosis.
- the ducks tested were 7-day-old seedlings of red duck.
- a total of 120 7-day-old ducks with confirmed aspergillosis were selected and divided into 4 groups, 30 ducks in each group.
- the above test subjects were tested with 1% aqueous solution of Pseudo konjac extract, 0.05% aqueous solution of clotrimazole, and water (blank control), instead of drinking water, and were fed continuously for 1 week to evaluate the control effect.
- Cure The duck's appetite and activity spirit returned to normal, the color of the feces was normal, and the dryness was moderate.
- the duck's appetite and activity spirit are significantly improved, and the number of diarrhea is reduced.
- the effective number includes the number of cures.
- the Pseudomonas konjac extract of the present invention has a very good preventive effect on the duck mycosis caused by Aspergillus, and compared with the clotrimazole effect of the control group, the effect is slightly worse, but sufficient.
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Abstract
假蒟提取物在制备防治禽类曲霉菌病的添加剂中的应用,假蒟水提物和醇提物可抑制烟曲霉菌、黄曲霉菌、黑曲霉菌、土曲霉菌、青曲霉菌,防治禽类由于曲霉菌感染引起的呼吸道、肠道疾病。
Description
本申请要求于2021年03月30日提交中国专利局、申请号为202110341832.X、发明名称为“假蒟提取物在制备防治禽类曲霉菌病的添加剂中的应用”的中国专利申请的优先权,其全部内容通过引用结合在本申请中。
本发明涉及禽类养殖技术领域,尤其涉及假蒟提取物在制备防治禽类曲霉菌病的添加剂中的应用。
禽曲霉菌病是养鸭业中常见的一种真菌性疾病,尤其在幼鸭中发病较多,死亡率较高,可达50%。引起禽曲霉菌病的病原菌为曲霉菌,包括烟曲霉菌、黄曲霉菌、黑曲霉菌、土曲霉菌和青曲霉菌等。海南天气高温高湿,饲料、垫草易霉变,养殖环境中霉菌孢子较多,尤其在海南夏季该病为鸭养殖中的常见疾病。该病的临床症状为呼吸困难,张口喘气,呼吸次数增多,食欲不振,喜喝水,爱睡觉,拉淡绿色稀便,常呆立或卧在角落处,口腔和鼻腔常流出粘性分泌物。目前该病的治疗没有特效药,用1‰硫酸铜、1‰碘化钾、0.05%克霉唑、0.5万IU制霉菌素具有明显的疗效。
随着我国经济的发展,人民生活水平的日益提高,对肉制品的品质要求也不断提高。用中草药替代抗生素作为饲料添加剂是发展趋势之一,这对鸭养殖业绿色健康发展具有重要意义,并对为社会提供健康肉、蛋起到重要作用。但目前尚未见关于防治鸭曲霉菌病的中草药的报道。
发明内容
有鉴于此,本发明要解决的技术问题在于提供假蒟提取物在制备饲料添加剂中的应用。
本发明提供了假蒟提取物在制备防治禽类曲霉菌病的添加剂中的应用。
本发明中,所述禽类曲霉菌病由烟曲霉菌、黄曲霉菌、黑曲霉菌、土曲霉菌和青曲霉菌导致。
本发明中,所述禽类曲霉菌病为鸭曲霉菌病。
一些实施例中,所述鸭曲霉菌病为红毛鸭的曲霉菌病。
本发明中,所述假蒟提取物为假蒟的水提物或假蒟的醇提取物。
本发明实施例中,所述假蒟的水提取物的制备方法为,将假蒟以水回流提取三次,合并提取液经浓缩获得假蒟水提物;所述三次提取中,第一次提取3h,第二次提取2h,第三次提取1h。所述第一次提取中,假蒟与水的质量体积比为1kg∶0.5L;第二次提取中,假蒟与水的质量体积比为1kg∶0.4L;第三次提取中,假蒟与水的质量体积比为1kg∶0.3L。
本发明实施例中,所述假蒟的醇提取物的制备方法为,将假蒟以乙醇浸提三次,合并提取液经浓缩获得假蒟醇提物;所述三次提取中,第一次浸提7天,第二次浸提5天,第三次浸提3天。所述第一次提取中,假蒟与乙醇的质量体积比为1kg∶2L;第二次提取中,假蒟与乙醇的质量体积比为1kg∶1.5L;第三次提取中,假蒟与乙醇的质量体积比为1kg∶1.5L。所述乙醇为95%的乙醇。即乙醇-水以体积比95∶5混合的溶液。
本发明还提供了一种添加剂,其包含假蒟提取物。
本发明所述的添加剂可以添加在饲料中,也可以添加在饮用水中,本发明对此不做限定。其添加在饮用水中的添加量为1%。即添加至假蒟提取物在饮用水中的质量分数为1%。
本发明还提供了一种禽类的饲养方法,其为给予本发明所述的添加剂。
本发明实施例中,所述给予的时间为7日龄,连续给予一周。
本发明研究发现假蒟水提物和醇提物对烟曲霉、黄曲霉、黑曲霉、土曲霉等曲霉属真菌存在抑制作用,并验证假蒟提取物能够用于防治禽类由于曲霉感染引起的呼吸道、肠道疾病,有效率可达81%~84%。并且,假蒟的植物资源分布广泛、生物量大、价格低廉,尤其在海南林下、路边 随处可见,所述提取物制备工艺简单、成本低、得率高,易进行大规模生产。
本发明提供了假蒟提取物在制备饲料添加剂中的应用,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
本发明采用的试材皆为普通市售品,皆可于市场购得。所述的中草药假蒟别名蛤蒟、不拨子、假荖、蛤荖、假蒌、蛤蒌、木柄蒌、荜拨子、猪拨菜、钻骨风、臭蒌、山蒌、马蹄蒌为胡椒科(Piperaceae)胡椒属(Piper L.)假蒟(Piper sarmentosum Roxb.)具祛风散寒,行气止痛,活络,消肿的功效,用于风寒咳喘,风湿痹痛,脘腹胀满,泄泻痢疾,产后水肿,跌打损伤(《中华本草》)。所述假蒟提取物的制备可以采用假蒟全草,也可采用部分组织器官。在提取前,经过干燥、粉碎的步骤。本发明实施例中,采用假蒟全草制备假蒟提取物。
下面结合实施例,进一步阐述本发明:
实施例1
干燥的假蒟全草1kg,粉碎后放入5L三角瓶中,加入95%的乙醇2L,常温浸泡7天,回收提取液;加入1.5L 95%的乙醇继续浸泡5天,回收提取液;再次加入1.5L 95%的乙醇继续浸泡3天,回收提取液,合并3次浸提液,减压浓缩得假蒟乙醇提取物110g。
实施例2
干燥的假蒟全草1kg,粉碎后放入2L圆底烧瓶中,加入水0.5L,接好冷凝系统,加热煮沸3个小时,回收提取液;加入0.4L水继续加热煮沸2小时,回收提取液;再次加入0.3L水加热煮沸1小时,回收提取液,合并3次浸提液,减压浓缩得假蒟水提取物150g。
实施例3
实施例1和实施例2制得的假蒟提取物的抑菌实验
活化目标菌株,稀释到菌数1×10
6CFU/mL的菌液备用;制备好YPD无菌平板;取0.1mL制备好的菌液用无菌棉签均匀涂布于YPD平板上;分别将实施例1~2的假蒟提取物用甲醇溶解,配制成浓度为10mg/mL的溶液;将克霉唑用丙酮配制成浓度为10mg/mL的溶液,取药液10μL分别滴加到直径为6mm的无菌滤纸片上,至溶剂挥发完后将滤纸片置于涂布好目标菌液的平板上,每个平板放6张滤纸片,密封好平板倒置于28℃培养箱进行培养24小时,用游标卡尺测量抑菌斑直径(mm)。该抑菌实验所用目标菌为烟曲霉菌、黑曲霉菌、黄曲霉菌;每种菌抑制实验设置3个重复,每个重复包括10mg/mL假蒟提取物、10mg/mL克霉唑(阳性对照)、甲醇(空白对照)各2片滤纸片。测试效果见表1:
表1实施例1~2制得的假蒟提取物抑菌实验(单位mm)
烟曲霉菌 | 黑曲霉菌 | 黄曲霉菌 | |
实施例1提取物 | 21.41±1.71 | 27.35±1.62 | 18.14±1.22 |
实施例2提取物 | 22.15±1.31 | 26.72±1.47 | 20.36±1.43 |
克霉唑 | 24.36±1.17 | 28.43±1.75 | 21.78±1.64 |
甲醇 | 0±0 | 0±0 | 0±0 |
从表1数据体外抑菌实验可以看出,本发明所述的两种方法制备的假蒟提取物对曲霉菌属中的3个菌抑制效果都较好,这3种曲霉菌是导致鸭曲霉菌病最常见的病原菌。通过上述实验说明假蒟提取物可以用于防控鸭曲霉菌病。
实施例4
在实施例3的基础上对实施例1和实施例2制得的假蒟提取物的抑菌实验
把假蒟提取物用DMSO溶解到1mg/mL的初始浓度,以克霉唑为阳性对照用DMSO溶解到1mg/mL的初始浓度,DMSO为空白对照。在96孔板上用无菌培养基按照2倍法进行梯度稀释,稀释好后加入待测菌,培养12小时后观察。本实验设置3个重复。
表2实施例1~2制得的假蒟提取物MIC实验(单位μg/mL)
烟曲霉菌 | 黑曲霉菌 | 黄曲霉菌 | |
实施例1提取物 | 10~25 | 25~50 | 50~75 |
实施例2提取物 | 25~50 | 10~25 | 25~50 |
克霉唑 | 0.65 | 0.65 | 1.26 |
DMSO | >100 | >100 | >100 |
从表2 MIC实验数据可以看出,本发明所述的假蒟提取物对曲霉菌属中的3个菌最小抑制浓度均小于100μg/mL,说明该提取物对上述3种曲霉菌有一定抑制效果,可以用于防控鸭曲霉菌病的饲料添加剂。
实施例5
实施例1和实施例2制得的假蒟提取物的鸭曲霉菌病防治试验
在儋州某稻田鸭养殖场进行该试验,试验时间为7月份,正是海南高温高湿,曲霉菌病高发季节,所试验鸭为红毛鸭7日龄幼苗。选择120只确证患曲霉菌病的7日龄鸭分4组进行试验,每组30只。以上试验对象分别以假蒟提取物1%的水溶液,克霉唑0.05%的水溶液,水(空白对照),代替饮用水进行试验,连续饲养1周,评估防治效果,治疗效果判定标准为:
治愈:鸭食欲、活动精神恢复正常,粪便颜色正常,干稀适度。
死亡:防治期内鸭病情无明显改善而死亡。
有效:鸭食欲、活动精神有明显改善,下痢次数减少,有效数包括治愈数。
无效:在防治期间鸭病情无明显好转,甚至加重,但不包括死亡数。评价结果如表3。
表3实施例1~2制得的假蒟提取物的鸭曲霉菌病防治试验
死亡率% | 治愈率% | 有效率% | 无效率% | |
实施例1提取物 | 6.67 | 73.33 | 83.33 | 10.00 |
实施例2提取物 | 6.67 | 70.00 | 80.00 | 13.33 |
克霉唑 | 3.33 | 83.33 | 90.00 | 6.67 |
饮用水 | 53.33 | 6.67 | 13.33 | 33.33 |
由表3数据可以看出,本发明所述的假蒟提取物对曲霉菌导致的鸭霉菌病具有很好的防治作用,与对照组的克霉唑效果相比较,效果稍差,但足可以作为饲料添加剂对鸭曲霉菌病进行防控。
以上仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
- 假蒟提取物在制备防治禽类曲霉菌病的添加剂中的应用。
- 根据权利要求1所述的应用,其特征在于,所述禽类曲霉菌病由烟曲霉菌、黄曲霉菌、黑曲霉菌、土曲霉菌和青曲霉菌导致。
- 根据权利要求1所述的应用,其特征在于,所述禽类曲霉菌病为鸭曲霉菌病。
- 根据权利要求3所述的应用,其特征在于,所述鸭曲霉菌病为红毛鸭的曲霉菌病。
- 根据权利要求1~4任一项所述的应用,其特征在于,所述假蒟提取物为假蒟的水提物或假蒟的醇提取物。
- 根据权利要求5所述的应用,其特征在于,所述假蒟的水提取物的制备方法为,将假蒟以水回流提取三次,合并提取液经浓缩获得假蒟水提物;所述三次提取中,第一次提取3h,第二次提取2h,第三次提取1h。
- 根据权利要求5所述的应用,其特征在于,所述假蒟的醇提取物的制备方法为,将假蒟以乙醇浸提三次,合并提取液经浓缩获得假蒟醇提物;所述三次提取中,第一次浸提7天,第二次浸提5天,第三次浸提3天。
- 一种添加剂,其特征在于,包含假蒟提取物。
- 一种禽类的饲养方法,其特征在于,给予权利要求8所述的添加剂。
- 根据权利要求9所述的方法,其特征在于,所述给予的时间为7日龄,连续给予一周。
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