CA2102707A1 - Microencapsulated agricuturally active agents and method of producing same - Google Patents

Abstract

2102707 9219102 PCTABS00017 A microencapsulated active agent, such as an insecticide, a hormone, or a fertilizer for application to vegetation and a method for its preparation is disclosed. An active agent is encapsulated in lignin, a naturally occurring polymer which is a structural component of the plant cell wall. Lignin serves not only as the capsulate material, but also as a sunscreen which protects the agent from ultraviolet radiation. There is also disclosed a process for the preparation of the microcapsules of the invention which does not use toxic materials requiring subsequent removal.

Description

2~0~0 ~ i 9 ~ / O ~ 7 ~ ~
~3 ,~ec'd PCt/P~ O 9 JUL 199:

~IC~O~NCAP~LA~ED AGRIC~LT~RALLY ACTIVB
AGENT8 ~ND ~ETHOD OF PROD~CING 8AM~
BACKGROUND OF THE INVENTION
1. Field of the Invention The preSent invention relates to agriculturally active preparations and, mor~ particularly, to microen~-apsulated bioloyical and chemical agricul~urally actiYe preparation3, such as pesticid~s and ~ertilizers having improved stability upon applic:a ion in a giv~n ~nvironment.
Plicroencapsulation o~ pesticide~ has been u~ed as a means for pxoviding controlle~-release preparation~, Ior reducing th~ toxic:ity to p~rson~ hand~ ing ths pesticide~, and ~or protecting th~ active in~redient from deactivation by exposur~
to environmental c:ondition~. Exposure of non-encapsulat~d in~ecticide to these conditions can lead to leaching and run-o~ by rainfall, 105s by volatilization, d~gradation by bact~riaI metabolites~ and unlight, especially ultraviolet inac:t$vation. Sel~ction of an appropriate carrier material f or us~ in an intera-:tiv~ delivery system can not only r~duc~
this de~ctivatiorl, but also producQ specif i.c de~ired ~ects when combined with the active insecticidal material.

~ In an effort to pro~,ride a delivery syst~m ~or an acti iri~edi~llt ~eting the criteria set forth above, th~ art ha~
propose~a a n~Db~r o~ ~ncapsulation materials.
~ ethod~ ~or pr~parillg microerlcap~ulatQd in ~cticidal p~khogerls haYe been d~cribed iI~ wh~ch a capsule mater~al its~ hi~la~ t~Q patho~en from sunlight-induaed i~acltiv~tion. US Pa~nt No. ~, 328, 20~ d~ ribes a micro3bial ins~eticide ~c~mpo~ition whic:h comprises a mi~robead of a ~i~2707 WO92/19102 . PCT/US92/03727 insecticide composition which comprises a microbead of a nucleic acid and a proteinaceous material which partially protects the insect pathogen from W inactivation. However, bscause these substances are not enYironmentally stable, the encapsulating material is easily broken down, and thus cannot efficiently protect the insecticide.
The u~e of sunscreens in controlled release preparations has also been described. Diisophorone derivatives are utilized in US Patent No. 3,839,56l to protect insacticidally acti~e cyclopropane carboxylic acid compounds from W -induced d~gradation. In US Patent No. 4,094,969, there is described the use of a sulfonated copolymer of catechin and leucocyanidin which stabili~es a pesticide by retarding degradation upon exposure to sunlight. However, these formulations do not maintain the sunscreen and insecti~idal preparation in close enou~h contact to be effectiv~ against W
irradiation. Ignoffo and Batzer, in l'Microencapsulation and Ultraviolet Protectants to Increase Sunlight Stability of an , Insect Virus," Journal of Econom~c Entomology, Vol G4, pp.
; ~ ~ 8s0-853 (1966) ~ attempt to solYe this problem through the use ~of~Buffalo Black, Car~o-Jet Black, car~on and aluminum oxide and powder with microencapsulatPd Heliothis nucleoPolYhedrosis viru~ to protect preparations from W irradiation. However, the microencapsulating materials used by Ignoffo, namely ~elatin and ethylcellulose, are not environmen~ally stable and are thus readily broken down by the environment. Similarly, US Pa~ent No. 2,090,lO9 discloses the use of chlorophyll green S~BSTITUTE~ SHEE~

'~092J191~2 2 1 0 2 7 0 7 PcT/us92/o3727 as a protectant of various insecticides in a gelatinous vehicle from UV light.
Fogle, et al. describe the use of W absorbing materials such as carbon b~ack in a polymer matrix composition containing an insect virus in US Patent No. 3,541,203. The microencapsulating process as well as the microcapsules obtained by Fogle et al. suffer from a number of disadvantages. The polymers forming the walls of the capsules are not always capable of retaining the sunscr~ening agent within the interior of the capsule. This diminishes the stability of the insecticidal preparation b cause ~he loss of sunscreening agent makes the pathogen more susceptible o the damaging ef~fects of ultraviole~ light. In addition, the microcapsules of Fogle,~et al. are prepare~ using hishly toxic materials, and cumbersome washing steps are required for their removal.
A~particularly preferred group of encapsulating polymers is disclosed in US Patents No. 4,844j896 and 4,948,586 by Bohm and Friend, of commo~ assignee. These polymers are synthesized ~rom acrylic and methacrylic acid esters with a low content of quaternary ammonium groups, and are known as Eudragit L, ~, RL and RS. They are useful because they can yield cap~ules with strong walls which can be made soluble at a pH~of 6 or 7.5, depending on the particular ~ormulation used~ The Eudragit capsules can be used effectively to retain sunscreening agents within the walls of the capsule until ingested by an insect.

SUBSTITUTE 5HE~

WO 92/1910~ 1 0 2 7 0 7 PCT/US92/03727 The use of lignin in certain types of controlled release formulations is also known in the art. US Patent No.

3,929,453 relates to controlled release preparations of lignin and biological materials which are produced via coprecipitation-inclusion from ~n aqueous alkaline lignin solu~ion, or via the elimination of a common solvent from a lignin-biologically active organic agent mixture. US Patent No. 4,244,72g describes a reversibly swellable carrier gel for the incorporation of pesticides. ~hese gels are formed by cro~slinking lignin with epichlorohydrin, leading to the formation of gels with differing surface chemical characteristics, which are able to su~tain controlled release of water-soluble and water-insoluble pesticides. The use of other cross-linking reagents such as formaldehyde, araformaldehyde, glyoxal a~d glutaric dialdehyde is described in related US Patent No. 4,244,728. Methods for microencapsulation and protection from W irradiation are not disclosed in any of these references.
: One of the disadvantages of prior art lignin-based pesticidal preparations and to microenrapsulated preparations using other known polymers, is that the insecticidal agent is subject to autocatalytic breakdown because of its partial exposure on the exterior of both the lignin gel matrices and che polymeric microGapsules. Thus, even in the presence of sunscreens added to certai~ polymeric microcapsules, insecticide which is present near the surface of th~
microcapsule which is not protected by sunscreen can undergo W degradation. As a result, this degradation produces an SlJBSTlTUTE SHE~

~092/19102 21~ 07 PCT/US92/03727 autocatalytic degradation of neighboring molecules of insecticide toward the interior of the microcapsule. These interior molecules might have otherwise been protected by sunscreen which was present within the microcapsule~
Therefore, prior art preparations, even those which seemed to effectively retain sunscreening agents within the microcapsule, were still not capable of protecting the insecticidal preparation from autocatalytic degradation which began at the surface of the microcapsule. In additi~n, :~ micr~encapsulated insecticidal formulations characterizing the prior art typically required use of toxic materials in their preparation.
; ;Another problem confronting the prior art was the inability to load;sufficiently large amounts of active agent ;into~a~capsule. ~Difficulties were also encountered in t~iloring~the~microcapsule sizes ~o the particular application. ~ ~
; SUMMARY~AND OBJECTS OF THE~INVENTION
In~v~iew of the aforementioned disadvantages attendant with;prior art formulations and processes as well as other disadvantages~not specifically mentioned above, it should be apparent that there still exists a neèd in the art for ~microencapsulated active agents which (l) include no toxic components~in eithèr~the~final product or in the materials use~ to~form such microcapsules, ~2~ provide protection for an activé agent against~environmental effects, such as sunlight, which could degrade an active agent and ( 3 ) break down to SUBSTITUTE SHEET' . ~

WO 92/~9l02 2 1 0 2 7 0~ PCT/US92/~3727 release the active agent on~e it has reached its desired target.
It is therefore a primary objective of the present inv~ntion to fulfill that need by providîng an active ag~nt, such as a biologi~al or chemi~al insecticide, which is microencapsulated in lignin.
Another obj~ct of the present invention is t~ provide an active agent, ~uc~ as a microeneapsulated insecticide, which provides controlled release of the active age~t wh~n contacted wit~ the desired target D
Y~t another object of the present invention is to load sufficien~ly large amounts of an ~ctive agent into a microcàp~ule.
Still an~ther ob~ect of the present in~ention is to provide a process for preparing microcapsules which enables ~he size ~f the microcapsule to be t-ailored to the particul~r : end use contemplated.
In particular, thes and other objects of the present invention are achieved by providing a controlled-release preparation of an active agent which us~s lignin not only as the capsule material, but also as a sunscreening agent which protects the agent from the harmful effeGts o~ light.
In a first aspect, the present invention relates to a microen apsulated active agent ~-omprising:
: ~i) an active agent such as an insecticidal agent, and (ii) a polymeric encapsulating agent comprising lignin or a lignin compound.

;~JBSTITUTE SHEET

WO92/19102 2 1 0 2 ~ 07 PCT/US92/037~7 In a second aspect, the present invention relates to a process for preparing a microencapsulated active agent comprising the steps of:
(i) mixing (l) an encapsulating agen~ comprising lignin or a lignin compound, (2) an active agent in a form compa~ible wi~h microcapsule formation and t3) an aqueous solvent or cosolvent system which i~ miscible with water and which dissolves lignin or the lignin compound to form a first mixture;
~ preparing an emulsifying solution comprising a surfactant and a liquid which is immiscible with said aqueous sol~ent or cosolvent system;
(iii) preparing a solution to harden the capsules;
~: (iv~ mixi~g the first mixture and the emulsifying solution to form an emulsion;
~ (v) mixing the resultant emulsion with the hardening `~ solution to harden emulsion droplets to form capsules; and (vi) concentrating and separating the capsules from the ~water insoluble liquid.
With the forsgoing and other objects, advantages and ~eatures of the invention that will become hereinafter apparent, the nature o~ the invention may be more clearly understood by reférence to the following detailed description of ~he prQferred Pmbodiments of the invention and th~ app~nded claimsO
BRIE~ DESCRIPTION.OF THE DRAWINGS
Figure l is a graph depicting the perce~t mortality of cabbage looper larvae as a ~ cff~ s of exposure to SllBSTlTOTE SHE~

2~ ~2 1 07 ~ .. . _ ~
8 ~ -^^ 9 2 / 0 3 7 ~ 7 ~3 Rec'd P~t/PTO O 9 JUL 1993 polymer which can be used in combination with the lignin to form the microcapsule. By Eudragit is meant a group of modi~ied acrylic acid polymers which can be made soluble at various pH
levels, depending on the modifying g~oups employed. Such polymers, as well as the techniques f~r modification thereo~ to obtain a desir~d pH solubility, are known in the art~
Any of the pathogens currently known to in~ect insect~, including viral ~ bact~rial and fungal pathogens or any chemical toxins known to act on insects, can b encapsulated by and protected from W light by 1 ignin. Such viruse~; include, but are not limited to th~ nuclear polyhedrosis viru~ (NPV) of th~
bullworm, Heliothis zea" of the gypsy moth Lymantria dis~ar, o~
th~ Douglas ~ir tosso¢k moth, Oraia pseudo~suqata" of the Europ~an pine saw fly ~Neodiprion sertifer or o~ Autoqra~ha californic~. B~c:teria lcnown to be pathogeTIic to targat insects, including but not limited to Bacillus thurincriensi~, Ba3::illus sphaeric:us, ~ yL ~ may also be encapsulated.
In8ecticidal ch~mical toxin~ including, but not limited to midin~ hydrazone~ ~uch a~ hydramethylnon (~mdro-) may alæo be u~ed. ~inally, it will b~ appreciated that a numb~r o~
recombinant acti~ agen~s which are now available may also b~
encapsulat~d ~n accordance with the present invention.
The insecticiaal agents are employ~d in combination with a lignin. ~h~ lign~n component is discuseed f irst . By " ~ignin" is m~ant any ligni~ or modifi~d lignin derived from th~ digestion o~
lignoc~Ilulo~ic mat~rial, ~nclud~ng sul~ated lignin~, hydrolysi~
lignin~t and lignin a~ine . mu~, any o~ the l~gnin~ may b~
~mployed to makel th~ carrier used in this inv~ntion. Most commonly, they ar~ obtained as byproduct~ ~rom SUBSTITIJTE SHEET

W~92/19102 2 1 ~ 2 q 0 7 PCT/U~g2/03727 ~ y of the pathogens currently known to infect insect~, incl~di ~ viral, bacterial and fungal pathogens or any chemical t~xins known to act on insects, can be encaps ~ ated by and prote ~ d from W light by lignin. Such viru ~ s include, but ar~\not limited to the nuclear polyhe~osis virus (NPV) of the bullw ~ , Heliothis zea, of the gyp~ moth LYmantria_dis~ar~ of ~ Douglas ~ir tossock ~ th, Orqia pseudotsuqata, of the Eur ~ an pine saw fl~ NeodiPrion se~t$~er or of Auto~ra~ha ça ~ ornica. ~ a~teria known to be pathogenic to target insects, i ~ udi ~ but not limited to Bacillus thurinqiensis, Bacillus S~ ericus, Bacillus Po~illiae may also be encapsulate~ ~nsecticidal chemical toxins including, but not limit~ to am~dino hydrazones su~h as hydramethylnon (Amdro) ma~ also be us ~ Finally, i~ will be appreciated that a numb ~ of recombinan ~active agents which~ are now available ~ y also be encapsul ~ed in accordance with the present inve ~ion.
The insectici ~ agents are employed in com ~natisn with a lignin. ~e lignin component is discussed f~rst, By "lignin~ nt any lignin or modified lignin d~ri~ d from the alkal/ digestion of lignocellulosic materi~l, in~luding sulfate~'lignins, hydrolysis lignins, and lignin amines.\
Thus ~ ny of the lignins may be employed to m~ke the carr us ~ in this invention.. Most commonly~ they are obtained the alkaline process of papermaking where sodium hydrvxide alone or in combination with sodium sulfide is employed. These lignins are generally referred to as soda or ~ulfate lignins after ~he pulping process used. In the S~BSTITUTE SH~

WO 92/lgl02 2 1 ~ 2 7 ~ 7 PCT/IS92/03727 preferred practice of this invention, the lignin employed is a kraft pine lignin. Likewise, lignins known as "hydrolysis lignins" obtained by enzymatic or acidic reactions with lignocellulosic materials may be used. Also, sulfite waste liquor lignins and sulfonated alkali lignins may be used if the degree of sulfonation is controlled. Thus, mixtures of alkali lignin and sulfite or sulfonated lignin may be usQd if an immediate release of pesticide is desired. Lignin amines may also be used to form capsules. Lignin amines ar~ soluble at a~pH ~3.5 or ~8.5, and are generally supplied at about pH
10. Lignin amine at pH 6 is not soluble in water, but will dissolve in ~o~utions of polyethylene glycol and waterJ for example, a solution of 70% PE~/30% water.
The present inv2ntors have discovered that lignin is effe~tive for use as a microencapsulating material as well as :a~sunscreening agent f~or agriculturally active agents such as pesticides. In some instances, the lignin and active agent : : :
can~be formed into a microcapsule which concentrates the active agent towards the center of the capsul~e. Depending on ~ , the~naturc~of the interaction between the lignin carrier and :the;:pesticide, the effects:demon~trated by the composite can ~ary from slmple dilution of th~ actîve ingredients to sustain~d release. The use of these types of microcapsules, w~ether interactive or ~ot, also allows for the reduction or elimination of some of the environmentally undesirable side effects of pe~ticide use such a~ contamination of non-target ar~as caused by toxicant run-off, leaching or ~vaporation. In addition, in~orporation of the pesticide into the de~ivery Sl3BSTlTUTE SHEEI-WO~2/19102 2 1 0 2 7 0 7 PCT/USg2/03727 system of the invention offers potential for decreasing chemical breakdown into biologically inactive compounds following exposure to light and extremes of pH. Thus, the active ingredient is shielded, its initial activity is reduced, and it5 release is sustained.
Lignin can be easily modified due to its phenolic hydroxyl, carb~xylate and aliphatic hydroxyl groups, as well as its high aromatic content. Matrices of varying pore structure, :polarity and solubility can be created by blocking certain groups on the :
lignin unit, and by crosslinking with reagents such as epiahlorohydrin, formaldehyde, ammonia and formaldehyde, and hexamethylenetetramine, among others. It will be appreciated hat the::ability to modify lignin enables the use thereo~
against different~targets. Thus, for example, when it is an insecticide which is encapsulated in the lignin, the lignin is designed to be insolubIe at approximately neutral or acidic pH
levels~:as encountered outdoors and soluble at alkaline pH
levels~as encountered within the gut of an insect~
The high aromatic content of lignin also gives it the property of excellent ultraviolet radiation absorption. This characteristic t along with its inert properties, makes lignin hiyhly suitable f c~r use with pesticides whirh ar~ sensitive to W-initiated or -catalyzed degradation~ Finally, the good ~dispe~sant qualities, the low cost and the environmentally benign nature of lignin compared to other carriers such as SVBSTITUTE SHEE~

high surface area, porous plastics, makes lignin highly desirable for use in virtually all pesticide formulations.
The microencapsulated preparations of the present invention, through the process of emulsif ication~ maintain their activity due to the physical-chemical protection and intrinsic sunscreening activity of the microencapsulating polymer, ligni~. The capsules formed are spherical and can be made to ha~e a large range of diameters depending on the final application. Thus, for example, wh~n insects are the target, they~can ba formed with average diameters of 10-50~ which allows the capsules to form free flowing suspensions ~hich can be sprayed and are small enou~h that they are easily ingested by the target insect.
Thus, lignin provides an ideal encapsulating agent f or an insecticide because it is insoluble in water at normal anvironmental pH values and thus pro~ects the pestici~al agent while present on vegetation, but will dissolve in the alkaline pH~:of the insect's: gut, releasing the pesticidal agent.
In a preferred embodiment, microcapsules are formulated with a mixture of lignin and the polymer Eudragit which, depending on the active agent, can prevent autocatalysis and degradatio~ until the ime of ingestion. In this regard, it will be apprecia~ed that Eudragits can be manufactured so a~
to have ei~her a positi~e or a negatiYe charge. Accordingly, ~depending on what char~e the acti~e agent has, an Eudragit having the opposite charge can be sele~ted. One particularly preferred combination is the chemical insecticide ~mdro~
which has a positive charge, and a negati~ely charged SIJE~STITUTE SHEE~

W~2/19102 2 1 Q 2 ~ 0 7 PC~/US92/03727 Eudragit. In fact, without being limited to theory9 it is believed that such combination has been found to result, during microcapsule formation with the lignin encapsulating agent, in a partial phase separation as the dropl~t forms and a pulling of the active agent into the core of the microcapsule where it is better protec~ed in the final product. This 9 in turn, increases the r~sistance of the microcapsulP to autocatalysisO
A water-immi~cible liquid such as vegetable oil, kerosene or liquid hydrocarbon containing a surfactant can be used to form an emulsion with the encapsulating polymer and insecticide. Solutions such as soybean oil and other vegetable oils, non-polar liquids such as kerosene and other liquid hydroc~rbons su~h as hexane, in combination with a surfactant can be used as the continuous phase for making the emulsion of the capsule material. The emu~.sifying agent can consist of a SPAN-80/Tween-80 mixture. For soybean oil, 20%
of a mixture of 30% Tween-80/70% SPAN-~0 has been found to be effective. For other cQntinuous phase liquids, the ratio of SPAN to Tween is adjusted to op~imize the formation of ~he emulsion. Sur~actants ~ther than SPAN and Tween may al~o be usedO Volume ratios between l.5 and 2.0 of oil to capsule material have been successfully used. Lower ratios are possible but ~i5c05ity increas~s at the lower ratios and the capsules which result are not as uniform in size and shape.
In addition, lower ratios may reverse the pha~e of the ~
emu1sion, resulting in oil droplets in the capsu1e material and fvrmation of distorted capsules or lack o~ capsules.

8VBSTITUTE SHE~

W092/19102 2 1 0 2 7 0 7 PC~/U~9~/03727 ~ ween-20 can be used as a surfactant in the hardening solution for hardening lignin capsules. Concentrations between 2 and lO% have been used, but too lit~le Tween results in poor separation of the capsules from the oil. 2.5% Tween is sufficient in most cases. Capsules made from alkaline solutions of lignin can be hardened using a water solution of an acid ~uch as acetic acid or an acid buffer. Capsules made with lignin dissolved in organic solvent/water can be hardened in water alone.
2. Preparatlon of Microcapsules A number of schemes ~or preparing microencapsulated ~active chemical toxins are now described. It will ~e ~:: :appreci~ted that such technigues are also applicable to the encapsulation of insecticidal viruses, bacteria and fungi as well a5 other actiYe agents.
The first step o* the process involves mixing the : encapsulating agent comprising lignin, an active agent in a :~ form compatible with microcapsule formation and a solvent or ` cos~l~ent system which is miscible with water and which ;~ : , : dissolves lignin. The lignin component, and the :
:;:: modi~i~ations which can be made thereto, were discussed previously. The active agent in a form which is compatible ~with microcapsu1e formation is one which does not interfere :~ with ~he solvents employed to prepare the microcapsules. In :
general, such actiYe agent is in the form of either a solid in an aqueous suspension or it is dissolved in a~ organic ~olvent which is not miscible with the microcapsule forming solvents~
: The solvent or cosolvent system includes water and is one SVlE3STITaJT~ SHEE~

W092/19102 2 1 ~ 2 ~ ~ 7 PCT/US92/037~7 The present method enables the preparation of microcapsules having a lignin content above lQ~ by weight, preferably between about 10 and 30% and most preferably betw~en about 20 and 30~. Typically, below about ~0%, it is possible to form microcapsules. However, at such low levels, an ef f ective sunscreening eff2ct is not obtained. At levels above about 30%, l ignin solutions can become quite ~iscous and di~fic~lt to handle. The present invention, by employing a solvent or cosolvent sy~tem which is miscible with water and whio~ di~solves lignin, enables handling o~ lignin ~olutions with such hi~h percentages of lignin therein.

21027~ 03 Rec'd P~T/~ 9 JUl l The apparatus for preparing microcapsules is one used conventionally in th~ art.
To prepare the microcapsules including Eudragit', Eudra~it-is dispersed in a solution containing PE& and water, th~ pH is adjusted with NaO~, and the solution mixed until dissolved.
Separately, lignin is dispersed in water to form a thick paste.
Because lignin is in~oluble in water, PEG is added to the solution, and the lignin i~ allowed to dissolYe. Alternatively, ammonium or sodium s~lts of lignin can ~e used which are soluble in water. Lignin has only limited solubility in acetone and isoprvpanol, but ~t i~ soluble in mixtures of such with water.
Lignin is soluble in PEGo400 solutions of greater than 50~ PEG, but if adjusted to pH 7 0 3, can become soluble in 35~ PEG.
Therefor~, capsules have been successfully made from lign~n dissolved in 50% P~G/water or 70% isopropanol/water, the ammonium salt o~ lignin dissolved in water, and from lignin ad~usted to p~
7:.3 dis~olv~d in 35% PEG/water.
Th~ Eudragit-PEG solution is added and mixea until the lignin is completely diesolvedO To this mixture is add~d the su pen~ion of tha in8ecticide. If it is desired to add an additional sunsereening agent, it can be added to th~ Eudragit-PEG solution~ After adding th~ suspendPd biopesticide to ths lignin-Eudraglt ~olut~on, SPAN-8S/Wesson oil ~ added and stirred at maximum speed for about one mlnu~ to form th~
~ulsion of cap ul~ material in oil. Thi~ is poured rapidly into ~ ardening ~olut~on of Twe~n-20 in ac~tic acid and s~irred ~or an additional ~inut~ to ~orm capsul2s. The mixtur~ ~8 c~tri~uged in a 50ml centrifug~ tube ~o separate the oil and th~
cap ule~. Th~ capsules are washed ~ev~ral times ~o remove a~

SUBSnTl)TE SHE~

2102707 PC~ S 92/0~7~ 7 - 16 ~ 03 Rec'd P~T/P~ o 9 JUL 19~3 much oil a~x pos~ible, and stored in water. The average diameter of the capsules is 25,um. For lignin amine capsules, the final pH
should be near neutralO 0.1M phosphate buffer pH 6.8 has been used to mainkain the pH in this range.
3. A~plication o~ Microcapsules to Veqetation The microcapsule~ prepared by any of the above-described method~ can generally be applied anywhere a conventional insecticide or other active ingredient such a~ a ~ertilizer could be applied. Generally, the concen~ration of the microcapsules and the rate o~ application depend on the nature of the pathog~n and on the nature of the vegetation being tr~ated. Such ar~
xeadily ascertainable by persons skilled in th~ art.
The followihg examples are presented in order to mor~ ~ully illustrate th~ preferred embodiments o~ the ~nventlon. Th~y should in no way b~ construed, however, as limiting the broad scop~ of the invention.
~: EXA~?L~, Microencapsulated Amdro- in Lignin-Eudragit was prepared by disæolving 14.4g Eudragit S-100 in 120g 40% PE~-~00, adding lg :NaOH and~m~xing until dissolved. In a separate beaker, 20g liqn~n was~added to lSg water and 15g of PEG ~nd mixed to ~orm a ~: th~ck pa~ta~ Ths mixtur~ wa~ stirred for 15 minutes on magneti~
s~irrer to im~rove ~ixing. 50g o~ the E~dragit~P~ solution wa~
adde~ and stirred 20-30 m~nute~ untiI all of ~he lig~in wa~
di3solvad,. 333,u1 micronized ~mdro~ su~pension wa~; added to lOg o~
th~ r~sulting ~ixture and stirred for about a ~inut~. 12.5g 50~6 SPA~8~t50% soyb~a~ oil waa added and ~tirx~d on~ minut~ to form the emulsion o~ capsule material in oil. 32ml o~ 7$ Tween-20 in aceta~ bu~f er was pour~d rapidly into the e~~ ion, and stirred SUBSTlllrrE SHEF~' 'VO92/19102 2 1 Q 2 7 0 7 PCT/US92103727 in water, and from lignin adjusted to pH 7.3 dissolved in 35%
PEG/water.
The Eudragit-PEG solution is added and mixed until the lignin is completely dissolved. To this mixture is added the suspension of the insecticide. ~f it is desired to add an additional sunscreening agent, it can be added to the Eudragit-PEG solution. After adding the suspended biopesticide to the lignin-Eudragit solution, SPAN-85/Wesson oil (1:1) is added a~d stirred at maximum speed for about sne minut'e to form the emulsion o~ capsule material in oil. This is poured rapidly into a hardening solutisn of Tween-20 in acetic acid and stirred for an additional minute to form capsules. The mixture is centrifuged in a 50ml cen~rifuge tube to separate the oil and the capsules. The capsules a~e washed several times to remove as much oil as possible, and stored in water. The average diameter of the capsules is 25~m. For lignin amine capsules, the final pH should be near neutral. O.lM phosphate buffer pH 6.8 has b~en used to maintain the p~ in this range.
3. AE$iLiaation of MicrocaPsules to Ve~e ation ~ The microcapsules prepared ~y any of the above-described methods can generally be applied anywhere a conven~ional insecticide or other active ingredient such a~ a fertilizer could be applied. Genera~ly, the concentration of the mic~ocapsul~s and th~ rate of application depend on the nature o~ the pathogen and on the naturP of th~ vegetation being treated. Such are readily ascertainable by persons skilled in the.art.

SUBSTITUTE SHEFI'' W092/19102 21~ 2 7 0 7 PCT/US92/03727 The following examples are presented in order to more fully illustrate the preferred embodiments of the invention.
They should in no way be construed, howev~r, as limiting the broad scope of the invention.
EX~MPLE 1 Microencapsulated Amdro in Lignin Eudragit was prepared by disso~ving 14.4g Eudragit S-lO0 in ~20g 40% PEG-400, adding lg NaOH and mixing until dissolved. In a separate beaker, 20g lignin was added to ~5g water and 15g of PEG and mixed to form a thlck pas~e. The mixture was stirred for 15 minutes on magnetic stirrer to improve mixing. 50g of the Eudragit-PEG
solution was added and stirred 20-30 minutes until all of the lignin was dissolved.: 333~1 micronized Amdro- was added to l0g of the resulting mixture and stirred for about a minute.
12,5~ 50~ SPAN-85/50% soybean oil was added and stirred one minute to form the emulsion of capsule material in oil. 32ml of 7%~Tween-20 in acetate buffer was poured rapidly into the emul~ion, and stirred for one minute to form capsules. The capsules~werz separated by.centrifugation at lOOOrpmO The capsules were then washed to remove the remaining oil.
otton leaves were treated with capsules with and without sunscreen, and with ~mdro- in a solution of acetone/water.
Leaves were excised and bioassayed as above. Amdro~

. . .
~;~ formulation5 were used at l00 ppm at (l ml per leaf.) Treated leaves were excised at various intervals and bioas ayed for control of first-instar larvae of _eliothis irescens. Plants received seven hours of direot sunlight in :

SUBSTITUTE SHlE~

~092/19102 2 1 ~ 2 7 0 7 PCTtUS92/037~7 the first 24 hours post-treatment, followed by a 14 hour photo period exposure to metal halide lamps in the greenhouse.
The above-described encapsulatPd produc~ was compared with nonencapsulated Amdro~. After days of exposure to the sun, the micrencapsulaked active agent of the in~ention retained most of its activity whereas the activity of the unprotected agent was mostly lost~
- , EXAMPLE_2 Bacillus thurinqiensis was encapsulated in lignin.
First~, the capsule material was prepared by mixing 135g of lignin and 135g of PEG-400 and ~ . lg of 25% NaOH in a 5 quart bowl thereby f orming a ~hLick paste . There wer~ then added 240g of Bacillus thurinc~iensis slurry in 4 aliquots, mixing thoroughly ~fter each addition. After the last addition, mixing was carried out f or an addi ~ ional 5 minutes to assure complete mixing and dissolution of the ligninO
An emulsifying solution was prepar d by mixing together 840g soy bean oil, 47g of SPAN-80 and 63g of TWEEN-80.
A hardening sol~tion was prepared by mixing together 138:$g water, 75g TWEEN-20 and 4~5ml acetic acid.
T~e emulsifying solution was then added to the capsule material in the mixing bowl and emulsified by mixing with a mixer. The mixing was continued for 15 minutes. The hardening solution was then poured in followed by mixing for another minute to harden and separate the capsules from the oil. The capsules were then separ2ted form th2 oil and concentrated followed by pouring off the oil and excess water.

SVBSTITOTE SHEE~

21~27~7 W092/~9102 PCT/US92/03727 Bioassays were done by first exposing a dilute water suspension of capsule~ or free Bacillus thurinqiensis to ultraviolet light for a given period of time followed by spreading aliquots of the sample on the surface of food in small cups~ Next several cabbage looper larvae were placed in each cup and allowed to feed for three days. The living and dead larvae were then counted and the results expressed as percent mortality, (dead larvae)/(~otal~. The capsules prepared above at dose~ of 2~g, l~g, and 0.5~g were compared ~o the free Bacillus at doses of 2~g and 4~g. As is shown in Figure 1, after only a few hours of exposure to ultraviolet radiation, the encapsulated products of the invention provided a much higher mortality against cabbage loopers as compared to the nonencap~ulated products.

Microencapsulated viruses were prepared. The lignin component was Llgnin AT from Westvaco. Polyethylene glycol having a molecular weight of 400 was employed~ As the emulsifying solution, there was used 10% of (60~6 Span-80/40%
Tween-80) by weight in soybean oil. A stock Eudragit S-l00 :
~as emp1Oyed including 14 . 4g Eudragit S-l00 and 120g PEG and lg NaOHO A working Eudragit S-l00 solution was prepared by adding 25g of the s~ock Eudragit S-l00 solution and 15 . og PEG
and lo. og water, to make a 596 Eudragit in 50~6 PEG/water~ A
2S~ NaOH w/w solution was prepared by adding 25g NaOH to 75y water. The virus used was A. cal from American Cyanamid o~
approximately 101 PIBs/g. A hardening solution was prepared SIJ~STITUTE SHE~

wo 92tl9la2 2 ~ O ~ 7 0 7 PCT/US92/03727 from 20g acetate buffer, 20g Tween-~0, 160g water and 100mg CaCl2 dihydrate.
To prepare the microcapsules, 2g of Lignin-AT were added to a 50ml beaker with 1. 5g o~ PEG~ 2g of 596 Eudragit S-100 in 50% PE~, and 180~1 of 25% NaOH. The liquids Were next mix~d into the lignin to form a.paste. Then, there was added lg of water f ollowed by mixing . The mixture was warmed slightly in order to speed the dissolution of the Lignin.
After all of the lignin was dissolved, the capsule solu~ion was allowed to cool. Then, there were added 1~0mg of virus dispersed in 0~3~ water followed by mixing. There were then added 10g of emulsifying solution. An ~mulsion was formed by stirring with a stir bar at maximum speed on a magnetic stir plate for- about 1 minute. Next, 30ml o~ the ~ : .
hardening soiution was poured in and stirring continued for ; another minute. The mixture was then poured into a 50cc , ~ centrifuge tube, shaken, and centrifuged for 3 minutes at 1000 :
rpm.: The oil and supernatant were removed and the capsules were retained and resuspended in water.
The encapsulated and nonencapsulated vîrus was again applied~against the cabbage looper larvae as described in Example 2. Th~ results, shown in Figure 2; demonstrate the dramatic dif~erence between the encapsulated and nonenc~psulated product in terms of the percent mortality after only a few hours of exposure to ultraviolet radiation.

~`:

While the invention has been described and illustrated herein by references to various specific mat~xialsl procedures SUB5TITUT~ $HE~

and examples it is understood that the invention is not restricted to the particular material combinations of material, and procedures selected for that purpose. Numerous variations of such details can be implied as will be.
appxeciated by those skilled in the art.

SUBSTITUTE SHEE~

Claims

- 2? -WHAT IS CLAIMED IS:
1. A microencapsulated active agent comprising:
(i) an active agent, and (ii) a polymeric encapsulating agent comprising lignin or a lignin compound which is soluble in an alkaline environment, said encapsulating agent being present in an amount effective to both form a microcapsule and provide a sunscreeing effect for said active agent.
2. The microencapsulated active agent of Claim 1 wherein said active agent is an insecticidal agent.
3. The microencapsulated active agent of Claim 2 wherein said insecticidal agent is an insecticidal pathogen selected from the group consisting of a virus, bacterium and fungi which infects insects.
4. The microencapsulated active agent of Claim 3 wherein said-insecticidal pathogen is a nuclear polyhedrosis virus.
5. The microencapsulated active agent of Claim 3 wherein said insecticidal agent is Bacillus thuringiensis., Bacillus sphaericus, Bacillus popilliae or a recombinantly produced pathogenic microorganism.
6. The microencapsulated active agent of Claim 4 wherein said nuclear polyhedrosis virus is a nuclear polyhedrosis virus of Heliothis zea, H. virescens, Lymantria dispar, Orgia pseudotsugata, Neodiprion sertifer, or Autographa californica.
7. The microencapsulated active agent of Claim 2 wherein said insecticidal agent is a chemical toxin.

- 2? -8. The microencapsulated active agent of Claim 1 wherein said polymeric encapsulating agent further comprises an adjuvant comprising an acrylic acid polymer which has been esterified to be alkaline or acid soluble, in an amount effective to prevent autocatalysis or futher enhance prevention of degradation of said active agent.
9. The microencapsulated active agent of Claim 8 wherein said acrylic acid polymer has a positive or negative charge and said active agent has a charge opposite to that of said acrylic acid polymer.
10. The microencapsulated active agent of Claim 9 wherein said active agent is hydramethylnon and wherein said polymer has a negative charge.
12. The microencapsulated active agent of Claim 1 comprising about 10-30% by weight of said lignin or lignin compound.
13. The microencapsulated active agent of Claim 12 comprising about 20-30% by weight of said lignin or lignin compound.
14. A process for preparing a microencapsulated active agent comprising the steps of:
(i) mixing (1) an encapsulating agent comprising lignin or a lignin compound, (2) an active agent in a form compatible with microcapsule formation and (3) an aqueous solvent or cosolvent system which is miscible with water and which dissolves lignin or the lignin compound to form a first mixture;
(ii) preparing an emulsifying solution comprising a - 2? -surfactant and a 1iquid which is immiscible with said aqueous solvent or cosolvent system;
(iii) preparing a solution to harden the capsules;
(iv) mixing the first mixture and the emulsifying solution to form an emulsion;
(v) mixing the resultant emulsion with the hardening solution the harden emulsion droplets to form capsules; and (vi) concentrating and separating the capsules from the water insoluble liquid.
15. The process of claim 14 wherein said aqueous solvent or cosolvent system which is miscible with water is selected from the group consisting of ethylene glycol, polyethylene glycol, isopropyl alcohol, acetone and mixtures thereof.
16. The process of Claim 14 wherein said active agent is an insecticide.
17. The process of claim 14 wherein said encapsulating agent is lignin amine or lignin amine sulfonate.
18. The process of claim 14 wherein said solvent is polyethylene glycol, isopropyl alcohol, or acetone.
19. The process of claim 14 wherein said emulsifying solution is prepared from an emulsifying agent selected from the group consisting of a liquid hydrocarbon and a vegetable oil.
20. The process of claim 19 wherein said liquid hydrocarbon is kerosene.
21. The process of Claim 14 wherein said encapsulating agent is lignin and said hardening solution comprises water and a surfactant.

- 2? -22. The microencapsulated agent of Claim 1 wherein said microcapsule is formed with an average diameter of 10-50 µ.
23. A microencapsulated active agent comprising:
(i) an active agent, and (ii) a polymeric encapsulating agent comprising a lignin or a lignin compound, said encapsulating agent being present in an amount effective to both form a microcapsule and provide a sunscreening effect for said active agent and said encapsulating agent being one which breaks down to release said active agent once it has reached a desired target.