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WO2009068458A2 - Microparticules encapsulées présentant un noyau qui contient des virus, et procédé de préparation des microparticules - Google Patents

Microparticules encapsulées présentant un noyau qui contient des virus, et procédé de préparation des microparticules Download PDF

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Publication number
WO2009068458A2
WO2009068458A2 PCT/EP2008/065752 EP2008065752W WO2009068458A2 WO 2009068458 A2 WO2009068458 A2 WO 2009068458A2 EP 2008065752 W EP2008065752 W EP 2008065752W WO 2009068458 A2 WO2009068458 A2 WO 2009068458A2
Authority
WO
WIPO (PCT)
Prior art keywords
microparticles
microparticle
viruses
range
bar
Prior art date
Application number
PCT/EP2008/065752
Other languages
German (de)
English (en)
Other versions
WO2009068458A3 (fr
Inventor
Robert Schatz
Stefan Schwab
Eberhard Schlücker
Andreas Scheurer
Detlef Freitag
Original Assignee
Friedrich-Alexander-Universität Erlangen-Nürnberg
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Friedrich-Alexander-Universität Erlangen-Nürnberg filed Critical Friedrich-Alexander-Universität Erlangen-Nürnberg
Priority to EP08854538A priority Critical patent/EP2217362A2/fr
Publication of WO2009068458A2 publication Critical patent/WO2009068458A2/fr
Publication of WO2009068458A3 publication Critical patent/WO2009068458A3/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/04—Making microcapsules or microballoons by physical processes, e.g. drying, spraying
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/40—Viruses, e.g. bacteriophages

Definitions

  • the invention relates to encapsulated microparticles having a core containing viruses in free or carrier-bound form and a shell enclosing the core.
  • the invention further relates to an associated manufacturing method for the microparticles.
  • the encapsulation serves in particular to improve the UV stability and increase the rate of uptake of baculoviruses by a host organism used for pest control.
  • viruses for pest control is sought in organic and integrated agriculture.
  • the commercial use of viral particles necessitates the finding of formulations which permit a practicable handling of the viruses, that is to say the particular preparations to be used must have sufficient stability in the field of use, a sufficient dosage of viral particles and, if appropriate, satisfy existing safety regulations.
  • baculovirus-based biological insecticides combine positive ecological and economic aspects.
  • Baculoviruses against cotton bollworm, sponge moth, codling moth and apple peel winder In Germany, only baculoviruses of the genus granulovirus currently have a license. These are the apple peel worm granulovirus and codling granulovirus.
  • Granuloviruses generally have the property that they attack only the larval stages of their host (insects). In order to develop their pathogenicity, they must be actively taken up by the host and then endocytosed into the intestinal epithelial cells in the digestive tract, where they pass into their virulent form. As a result, the infected larvae are killed within 5 to 9 days and cause no damage to the apple. In practice, however, it has been found that the uptake rate of the virus is too low, with the consequence that the codling moth does not die shortly after eating and / or boring into the apple.
  • Arthurs et al. describe first the preparation and use of spray-dried, lignin-encapsulated formulations of the granulovirus CpGV for controlling Cydia pomonella, wherein the formulation TiC> 2 is used as UV stabilizer and sugar as Frausslockstoff (Evaluation of spray-dried lignin-based formulations and adjuvant as solar protectants for the granulovirus of the codling moth, Cydia pomonella (L) - Arthurs, SP; Lacey, LA; Behle, RW; Yakima Agricultural Research Laboratory, USA; Journal of Invertebrate Pathology (2006), 93 (2), 88-95 ).
  • the invention is based on encapsulated microparticles having a core which contains viruses in free or carrier-bound form and a shell which encloses the core.
  • the microparticles according to the invention are characterized in that the shell to at least 50% by weight of one or more triacylglycerides having a chain length of the fatty acids of C10 to C24.
  • the invention is based on the finding that saturated or unsaturated triacylglycerides having a chain length of the fatty acids with a length of 10 to 24 carbon atoms allow the encapsulation of viruses in free or carrier-bound form without their viral activity being inhibited.
  • the choice of material for the shell has, inter alia, the advantage that they allow rapid resorption of the virus after ingestion by the host, because it is precisely these fats happen easily epithelial cells in the digestive tract of the pests.
  • the triacylglyceride shell is particularly suitable for serving as a matrix for further additives, especially if they have a hydrophobic character. Kern within the meaning of the invention is understood to mean all components of the microparticle encompassed by the shell.
  • the core need not be compact as a singulated particle, but may contain multiple, separate fragments. It is therefore conceivable to encapsulate several carrier fragments for the viruses with a common shell.
  • the shell consists of at least 70% by weight of triacylglycerides.
  • Triacylglycerides are esters of the trivalent alcohol glycerol with three fatty acid molecules. Natural fats consist for the most part of triacylglycerides with three long-chain fatty acids, which usually consist of unbranched chains having 4 to 26, typically 12 to 22 carbon atoms. For the purposes of the invention are particularly suitable fatty acids having 10 to 24 carbon atoms.
  • the triacylglyceride preferably has a melting point in the range of 10 to 100 0 C, in particular in the range of 25 to 80 0 C, particularly preferably in the range of 25 to 65 ° C.
  • the triacylglyceride is tripalmitin or a tripalmitin-containing mixture of triacylglycerides, in particular palm fat.
  • Palm fat is characterized on the one hand by its low production costs and on the other contains carotene, which, as will be explained in more detail below, can serve as a UV stabilizer to protect the biological agent.
  • tripalmitin or tripalmitin-containing mixtures, such as palm oil are the main ingredients of apple cores and leaf adults and thus act as feed attractants for the codling moth, and are therefore particularly preferred in connection with the use of codling granulovirus.
  • the microparticles preferably have an average diameter in the range from 500 nm to 500 ⁇ m, in particular from 1 to 50 ⁇ m. Microparticles of this size can be discharged in particular with the already existing in agriculture conventional spray devices.
  • the number of viruses per microparticle should preferably be 1 to 10, more preferably 1 to 2 viruses per particle.
  • the shell contains an emulsifier as an additive.
  • the emulsifier is a mono- and diacylglyceride, lecithin, pectin, a sugar ester or mixtures thereof.
  • a proportion of the emulsifier in the total weight of the microparticle is preferably between 0.1 and 15% by weight.
  • the emulsifier serves primarily to facilitate the preparation of an aqueous suspension containing the microparticles.
  • a further preferred embodiment which can again be implemented in combination with all of the aforementioned embodiments, provides that the shell contains a UV stabilizer as an additive.
  • the UV stabilizer serves to improve the stability of the viruses to natural UV radiation.
  • the UV stabilizer used is preferably TiC> 2 , carotenoids, flavonoids, skimmed milk powder, mycosporine-like amino acids (MAA), scytonemin or mixtures thereof.
  • the use of UV stabilizers is particularly advantageous in the codling granulovirus.
  • the shell may contain a Frriblockstoff as an additive.
  • the Frriblockstoff is preferably a pheromone, in particular 2 (E), 4 (Z) - Ethyldecadienoat, glutamate or sugar, in particular sucrose, glucose or fructose.
  • Many pheromones are highly volatile, so that their biological effect no longer exists shortly after delivery. However, many of these pheromones are lipophilic, so incorporation into a lipophilic matrix delays the release behavior to the desired extent. This applies in particular to the aforementioned attractant of codling granulovirus.
  • the compound mentioned serves as a pheromone for adult females as well as Frriblockstoff for codling larvae.
  • baculoviruses in particular from the genus granulovirus.
  • the microparticles very particularly advantageously contain apple peel winder granuloviruses or codling granuloviruses.
  • a particularly preferred microparticle for the latter two viruses contains as triacylglyceride tripalmitin and / or a tripalmitin-containing mixture, in particular palm fat.
  • this microparticle contains 2 (E), 4 (Z) - Ethylenedecadienoate as Frausslockstoff and a UV stabilizer.
  • the viruses can be used in ring form or else bound to a carrier. Other, the stabilization of the virus serving additives are possible.
  • Another aspect of the invention is to provide a method of making an encapsulated microparticle.
  • the method according to the invention comprises the steps:
  • PGSS plant Providing a capsule material which consists of at least 50% by weight of one or more triacylglycerides having a chain length of the fatty acids of C10 to C24 in a pressure vessel of a high pressure particulate generation plant (PGSS plant);
  • Spraying the melt through a nozzle or capillary Spraying the melt through a nozzle or capillary.
  • the so-called PGSS process is a high-pressure method for producing particles.
  • any number of liquefiable solids can be mixed and processed into free-flowing powder.
  • supercritical fluids are used as auxiliaries. Melting is homogenized and pulverized by mixing the liquefied individual components with compressed carbon dioxide and subsequently depressurizing them, for example via a nozzle. The melt is thereby torn into tiny droplets. It forms the finest powder, which opens up new applications due to its specifically adjustable size (nanometer to micrometer), morphology and composition.
  • the PGSS process can be operated both continuously and batchwise.
  • the compressed gas is preferably selected from the group consisting of carbon dioxide, nitrous oxide, alkanes having a boiling point of less than 50 0 C at 1 bar, alkenes having a boiling point of less than 50 0 C at 1 bar, hydrofluorocarbons having a boiling point less than 50 ° C at 1 bar and mixtures the said gases.
  • Particularly preferred are carbon dioxide, ethane, propane or butane.
  • the compressed gas is carbon dioxide or propane.
  • Propane and carbon dioxide have a particularly favorable for the purposes of the invention Joule-Thompson effect and evaporate well from the shell, so that at most only small contaminants remain.
  • spray-drying may also be carried out, precipitation from suitable solvents or alternative high-pressure spraying processes may be used.
  • the method according to the invention has the advantage that no solvent is used which subsequently has to be separated off or contaminates the particles. Compared with spray drying, lower working temperatures result since, for example, the use of carbon dioxide lowers the melting temperature of the mixture and its viscosity.
  • Other high-pressure processes usually work with higher carbon dioxide and energy requirements, so they are less economical in this case.
  • the step of charging the melt with gas is preferably carried out at a pressure in the range of 100 to 200 bar. Further, the temperature in this step is preferably in the range of 25 to 80 0 C.
  • the nozzles or capillaries of the PGSS system preferably have a diameter in the range of 50 ⁇ m to 1500 ⁇ m.
  • the capsule material and the additives are melted and the viruses are dispersed in the melt.
  • the melt is charged with carbon dioxide, the pressure between 20 and 500 bar (preferably 100-200 bar) is selected and the temperature between 10 0 C and 100 0 C (preferably 25 ° C to 65 ° C) is set , but at least so high that the capsule material melts. Temperature and dwell time are chosen so that the viruses are not deactivated.
  • the capsule material is sprayed with the dissolved carbon dioxide through a nozzle or capillary (diameter between 50 microns and 1500 microns, preferably 200 - 500 microns). The resulting particles are collected.
  • 150 g capsule material (.. 80 wt% tripalmitin, 20% by weight of palmitic) are melted at 60 0 C and (4 ml of a glycerol-containing codling moth Granulovirus suspension available under the trade name Madex; virus concentration about 3 x 10 13 viruses per liter ).
  • the mixture is introduced in a pressure vessel with stirrer.
  • the temperature of the melt is maintained at 60 ° C and it is added via a compressor carbon dioxide, so that a pressure of 100 bar sets and stirred for 30 min.
  • a nozzle connected to the container 60 ° hollow cone nozzle, 400 microns
  • the mean size of the capsules obtained is 15 ⁇ m.
  • the calculated load is 1, 25 viruses per capsule.
  • Viruses heated to 65 ° C for 1 h have an efficiency of 95% compared to thermally untreated viruses. At 70 0 C heated viruses still have an efficiency of about 88%, at 80 0 C heated viruses kill only 30% of the larvae.
  • microparticles prepared by the above method showed in animal experiments an efficiency of over 88%, based on unencapsulated viruses.
  • the encapsulated viruses showed an improved efficiency compared to untreated viruses, indicating both improved UV stabilization and increased uptake rates.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Virology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Dispersion Chemistry (AREA)
  • Pest Control & Pesticides (AREA)
  • Dentistry (AREA)
  • Wood Science & Technology (AREA)
  • Environmental Sciences (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Agronomy & Crop Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Manufacturing Of Micro-Capsules (AREA)
  • Medicinal Preparation (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des microparticules encapsulées présentant un noyau qui contient des virus sous une forme libre ou liée à un support, et une enveloppe qui entoure le noyau. Les microparticules se caractérisent en ce que l'enveloppe se compose à au moins 50 % en poids d'un ou de plusieurs triacylglycérides dont la chaîne d'acides aminés mesure de C10 à C24.
PCT/EP2008/065752 2007-11-27 2008-11-18 Microparticules encapsulées présentant un noyau qui contient des virus, et procédé de préparation des microparticules WO2009068458A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP08854538A EP2217362A2 (fr) 2007-11-27 2008-11-18 Microparticules encapsulées présentant un noyau qui contient des virus, et procédé de préparation des microparticules

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102007057395.4 2007-11-27
DE102007057395A DE102007057395A1 (de) 2007-11-27 2007-11-27 Verkapselte Mikropartikel mit einem virenhaltigen Kern und Verfahren zur Herstellung der Mikropartikel

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WO2009068458A2 true WO2009068458A2 (fr) 2009-06-04
WO2009068458A3 WO2009068458A3 (fr) 2009-08-20

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DE (1) DE102007057395A1 (fr)
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012025533A1 (fr) * 2010-08-24 2012-03-01 Euroferm Gmbh Procédé de protection des cultures à l'aide de virus entomopathogènes et de sulfate de cellulose
WO2012025534A1 (fr) * 2010-08-24 2012-03-01 Euroferm Gmbh Produit phytosanitaire contenant des virus entomopathogènes, notamment des baculovirus et du sulfate de cellulose

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107232190B (zh) * 2017-05-31 2021-04-27 中国农业科学院植物保护研究所 噻虫胺控释包埋颗粒剂及其制备和应用

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IT1272943B (it) * 1994-02-01 1997-07-01 Bend Res Inc Composizioni insetticide labili
WO1996037106A1 (fr) * 1995-05-25 1996-11-28 The United States Of America, Represented By The Secretary Of Agriculture Formulation pulverisable a base de caseine destinee a la lutte antiparasitaire
WO1998015183A1 (fr) * 1996-10-07 1998-04-16 E.I. Du Pont De Nemours And Company Procede permettant d'enrober des pesticides biologiques et compositions contenant lesdits pesticides
US6506397B1 (en) * 1999-02-19 2003-01-14 Curt Thies Pest controlling
US6248363B1 (en) * 1999-11-23 2001-06-19 Lipocine, Inc. Solid carriers for improved delivery of active ingredients in pharmaceutical compositions
US6720001B2 (en) * 1999-10-18 2004-04-13 Lipocine, Inc. Emulsion compositions for polyfunctional active ingredients
US6365189B1 (en) * 1999-10-22 2002-04-02 3M Innovative Properties Company Method of delivering and releasing a pheromone
US20030180352A1 (en) * 1999-11-23 2003-09-25 Patel Mahesh V. Solid carriers for improved delivery of active ingredients in pharmaceutical compositions
WO2005005010A2 (fr) * 2003-02-21 2005-01-20 Ferro Corporation Procedes et appareil de production de particules composites utilisant un fluide supercritique comme agent plastifiant et d'extraction
US20050084513A1 (en) * 2003-10-21 2005-04-21 Board Of Regents Nanocoating for improving biocompatibility of medical implants
US20040224019A1 (en) * 2004-03-03 2004-11-11 Adi Shefer Oral controlled release system for targeted drug delivery into the cell and its nucleus for gene therapy, DNA vaccination, and administration of gene based drugs

Non-Patent Citations (1)

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Title
ARTHURS, S. P.; LACEY, L. A.; BEHLE, R. W.;, JOURNAL OF INVERTEBRATE PATHOLOGY, vol. 93, no. 2, 2006, YAKIMA AGRICULTURAL RESEARCH LABORATORY,, pages 88 - 95

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012025533A1 (fr) * 2010-08-24 2012-03-01 Euroferm Gmbh Procédé de protection des cultures à l'aide de virus entomopathogènes et de sulfate de cellulose
WO2012025534A1 (fr) * 2010-08-24 2012-03-01 Euroferm Gmbh Produit phytosanitaire contenant des virus entomopathogènes, notamment des baculovirus et du sulfate de cellulose

Also Published As

Publication number Publication date
EP2217362A2 (fr) 2010-08-18
WO2009068458A3 (fr) 2009-08-20
DE102007057395A1 (de) 2009-05-28

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