CA1239637A - Nematocidally active chitin-protein complex - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A novel nematocidally-active chitin-protein complex is prepared from crustacean shell waste material. The complex is different from both chitin and chitisan, in respect of its Lowry protein content, its solubility properties, its IR spectrum, the acetyl content of its water-insoluble demineralized chitin component, and the amino acid composition of its water-insoluble protein component. The complex is in the form of dry particles having a diameter of less than 0.5 mm. It has useful nematostatic and nematocidal activity for agricultural and horticultural applications by admixing nematocidally-effective amounts thereof with a plant growth medium. The complex also provides a source of nitrogen in slow-release form, making it particularly suitable for combination with fertilizers, and soil conditioners.

Description

lZ39~;37 This invention relates to a process for converting shellfish wastes into useful products and for avoiding costly traditional methods for disposing of low economic value waste products of the shellfish processing industry. More particularly, the invention relates to methods for the isolation and recovery of a naturally occuring chitin-protein complex from the tough polymer matrix of crustacean exos~eletons and to methods for using these polymeric compositions for inhibiting the growth of plant-parasitic and other nematodes of interest in horticulture and agriculture.

Nematodes ~nema -- thread; oides -- resembling), or unsecJmented roundworms with elongated, fusiform, or sacli~e bodies covered with cuticle, which belong to the phylum Nematheln,inthes, are virtually ubiquitous in nature, inhabitating soil, water and plants, and are importantly involved in a wide range of animal and plant parasitic diseases.

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lZ39~3'7 While there are some significant problems related to nematodiasis in animals, it seems li~ely that major interest will continue to focus on nematodes which parasitize the roots, stems, leaves and seeds of plants and are major contributing factors to crop losses and to serious economic losses to agricultural productivity on a worldwide basis.
These include, bY way of example, root-~not (MPlni~ngYnP spp.), root-lesion (~h~ylPnch~s sp~.), spiral (~Pl in~ntvlon~uc 54p,) ~ and burrowing nematode (BaJnphnl-lc cimil ic) ~ which is highly destructive of citrus crops and more than 2~ other species o~ plants. Damaging levels of stunt ~IylehLh~y:Lh~l sp~.), reniform (~ntvlonrh~ c sp~ ~
and foliar (emphPlPnLhni~o~ sp~.) nematodes are also found in foliage ornamentals. Ring (~aL~npns~hn~a ~enopl3Y) and dagger ~m~biD~r~
sp~.) nematodes infect peach orchards, and soybeans are often seriously infested with both the soybean cyst nematode (~e~rrn~Pra glvLi~es) and root-~not (~el~i~ngYnP cpr)-In the United States alone, approximately two million acres ofagricultural land are treated each year by prophYlactic and quarantine measures, chemical control, soil fumigation, hot-water treatment, and resistance-based selection methods in order to control nematodes. This includes land used to grow nonfood crops (e.g., cotton and tobacco), field crops (e.g., corn and wheat), orchard crops (e.g., apples, citrus and nuts), vegetables (e.g., potatoes) and a wide range of ornamentals. ~lmonds, apples, asparagus, citrus ~including oranges, grapefruit, lemons and limes), cotton, cJrapes, melons, peaches, peanuts, pineapples, soybeans and strawberries, among other edible plant foods, and essentiallr all vegetables are susceptible to r,ematode infestation, as are home gardens and lawns, commercial turf, ornamentals and most other plants. Furthermore, large areas of otherwise arable agricultural lands (i.e., croplands, pasturelands, forest lands and lands in other agricultural use) may lie fallow or unused owing directly or indirectly to overwhelming or uncontrolled nematode infestations.
Plant-protection methods for nematode contr~l, including crop rotation, soil-treatment and fertili~ation practices, and green manuring- with sweet clover or mustard, as well as physical methods of soil treatment, e.g., steaming of soil and hot-water treatment of 1~239~37 planting stoc~s, have generally met with only limited success.
Chemical methods, on the other hand, emploYing a range of systemic pesticides, have been reasonablY successful, particularly in horticultural practice, despite the fact that nematodes tend to be resistant to many of the pesticidal agents which have been mar~eted for application either in a gaseous form ~fumigation) or dispersed in soil in liquid or solid forms; (see, for example, A.C. Tarjan and P.C. Cheo, ~The Nematode Screening Program of the University of Rhode ~sland,~
~ontribution 887, ~gricultural Experiment Stati~R~ Kingston, ~.1., March, 1956).
~ urrently, only son,e 25 nematocidal chemicals are registered with the U.S. Environmental Protection ~gency (EP~ for use on important food, feed and fi~er crops. Most nematocides now availaole in commercial mar~ets are, moreover, quite toxic to both man and animals, in large part being organic thiophosphate ~phosphorothioate and phosphorodithioate) compounds and cholinesterase inhibitors. Many of them are also phytotoxic. Because of their adverse effects on the environment, several nematocides which are currently mar~eted are subject to review which may result in cancellation of registration.
Thus, issues of safety and efficacy as well as of agricultural economics are critical considerations in the control of plant-parasitic nematodes. ~ clear and present co~mercial need exists for nematostatic or nematocidal materials, preferably biological control agents, which are non-toxic for plants, animals and man.
Despite the widespread presence in almost all soils, especially those with a high content of decomposing organic matter, protozoa, bacteria, predatory nematodes, and fungi which can in theory act as biological control agents against plant-parasitic nematodes, biological control programs have not been instituted on a large-scale anywhere in the world; see, for example, K. F. Ba~er and R. J. ~oo~, Binlngiral rnntrnl n~ Pl~nt P~t~fnc~ W. H. Freeman and ~ompany, San Francisco, California, 1974 and H. ~ec~er, Plant NPmatn~Pc ~n~ ThPir rnntrnl PhvtnnPmatnlnrJv~ published for the U.S. Department of ~griculture and the National Science Foundation, Washington, ~.C. by Amerind Publishing ~o., Ltd., New ~elhi, ]ndia, 1981.

1~39~37 Few effort- have been made to analyze in a systematic and orderly manner the biological effects of deliberate introduction into soils of relatively large amounts of the trpe of non-toxic organic wastes which n,ay accumulate and cause serious environmental and econon,ic consequences in speci4ic geographic regions, ~-g-, the Chesapea~e Bay ~e.g., R. A. A. Muzzarelli and E. R. Pariser, Eds., P~ocee~ings~ the Einst_ lntern~$i~n~ LonfenenLe___Dn__~hi~in~Lhitnsan, MIT Sea Grant Report, MITSG 7~-7, Gam~ridge, Mass., 197B; e. L. Averbach, 'Chitin/Chitosan Production for Utilization of Shellfish Wastes,~ pp.
285-3~, in W. S. Otwell, Ed. ~P~Do~__W~stP--~an~pmp~--in-~he-l9 LnnfenenLe _ ~n~Lee~in~s~ Florida Sea Grant Program, Report No. 4~, February, 19BI; T. P. Cathcart et al., ~Compcsting elue Crab Processing Plant Solid Waste~, Annual Repc.rt, Department of Agriculture Engineering, Uni~ersity of Maryland, College Par~, Maryland, December 31, Iq81; and T. M. Coo~, ~Development of a Fermentation Process to Use Wastes from the Chesapea~e Bay Industry~, Report F-lb-BI-~
Cepartment of Microoiology, UniversitY of Maryland, College Par~, Maryland, March 1~82).
Laboratory studies underta~en by Brown et al. have, however, shc~n that dried and powdered seafood wastes -- including commercially available chitin and chitosan, al~ali-treated (3.5~. NaOH for 24 hours) and non-treated shrimp shell wastes -- when utilized as a soil amendn,ent cause a statistically significant decrease in root-~not infestation and a statistically significant increase in the number of chitinolytic actinomycetes in cboth tomato and ornamental plants ~see, for example, L. R. Brown et al.: ~The Use of Chitinous Seafood Wastes for The Control of Parasitic Plant Nematodes~, MMRC Proiect No.
GR-76-0~4 and C0-76-~2~, Mississippi Marine Resources Chronical, Long Beach, Mississippi, Octo~er, Iq77 and The Use of Chitinous Seafood Wastes for the Control of Plant Parasitic NematodesL, BMR Project No.
GR-ST-78-~3 and GR-ST-78-0~4, Bureau of Marine Resources, Mississippi Department of Wildlife Conservation, Long Beach, Mississippi, Septem~er, 197~).
Problems associated with the accumulation of crabshell wastes in the Chesapea~e Bay region (or, by way of further example, shrimp shell wastes around the Gulf of Mexico) and often conflicting o~servations on 1~39~i37 the role and effect of chitin and chitc,san materials isolated frc,m these wastes prompted the laboratory investigations which led to the product and process ct c3~pects of the present invention- Relevant observ<ltionsinclude published reports that: ~a) the addition to soils of crop residues and other c~rbonaceous materials appears to suppress t,oth nematodes and certain fungi in soil populations (e.g., M. B. Linford et al., ~Reduction of Soil Populations of the Poot-~ot Nematode During Decomposition of Organic Matter', ~ Li_ 45: 127, 193~, and C. B.
Davey and G. C. Papavizas, ~Effect of Org~nic Soil~r,endments of the ~hi~ct~ia Disease of Snap Beans-, f~nt_.L~ ~l 493, 1959); ~
small additions of commerciallY availatlle chitin, but not of chitosan or N-acetylglucosamine, seem at,le to stimulate chitinase-prbducing microorganisms in the soil and to reduce the severity of root-rot of beans caused ~y El~c~ri~ ~e.s., F~. Mitchell and M. ~lexander, LThe Mycolytic Phenomenon and Biological Control of Ell~riLlm in Soil~, ~l~tllrP 1S~Q: 1961~ and R. Mitchell and M. ~lexander, ~Chitin and the Biological Control of Ells~i~ Diseases~ el~t_ l~iseaseQf ~nrtpr 45 4S7, July 15, 1961; ~c) chitosan, but not chitin, inhibits the growth of many fungi, including plant and animal pathogens, in culture media ~e.g., C. ~. ~llan and L. ~. Haclwiger, The Fungicidal Effect of Chitin on Fungi of ~arying Cell Wall Composition-, E~ MvfnlnfJy 3 2S5, ~1979); and ~d) commercial preparations of chitosan have little or no effect in reducing either the chemical or ~iolosica1 o~ygen demand of wastewater effluents from crab processing operations ~e.g., F. W.
Wheaton et al., Wastewater Characterization and Treatment System De~lelopment for a Blue Crab Processing Plant-, WRRC Technical Report No. 65, University of Maryland, Colle~e Par~, Maryland, ~pril 1981.) Several attempts at developinf,~ useful methods for dealing with chitin disposal have also been descri~ed in the patent literature, e.g., see f~.ustin, U.S. Patents 3~879~377; 3~B92~731 and 4,28~,0S7;
Balassa, U.S. Patents 3,9~3,268; 3,911,11S and 3,914,413; Dunn, U.S.
Patent 3,847,897; Casey, U.S. Patent 4,ûS9,a97; Muralidhara, U.S.
Patent 4,293,û9B; and Mu2~arelli. U.S. Patent 4,282,351 Various techniques are also known in the art for recovering chitosan from chitin, e.g., Rigby, ~39~37 U.S. Iatent 2 040 879 and Pennislon ~.S. Patents 3 862 122 4.195 175 and 4 199 496.
It is a general object- of ihis invention to provide improved and economically advantageous methods ~or disposing of otherwise ]ow economic value wastes remaining after commercial shellfish processing operations.
An object of another aspect of this invention is to provide a pro-cess for converting chitin-containing biomass waste materials into indus-trially useful compositions preEerably into forms and compositions of matter which have use in agriculture horticulture and animal husbandry.
An object of a further aspect of the invention is to provide an improved and inexpensive means for obtaining commercial quantities of m.ter-ials from nat:urally occurring chitin-containing biomass which can be demon-strated to induce nematostatic and nematocidal activity in culture media and in soil samples.
An object of a more particular aspect of the invention is to pro-vide a newly isolated chitin-protein complex which can be obtained from naturally occurring sources and has demonstrable nematocidal activity for prototypical nematode species without evidence of a direct toxic effect on nematodes.

9~i37 The present invention in~ol~es the discoverY that a nematocidally acti~e chitin-protein complex can be easily and economically prepared by mild acid hydrolysis of crustacean shell wastes, with or without recovery of carbon dioxide and other volatile gases produced during den,inerali2ation and partial protein degradation. The resulting chitin-protein complex induces nematocidal activity in nematode cultures i~ ~itrD, characteri2ed by microscopic evidence of premature senescence and gas vacuole formation accompanied by loss of motility and death. Dead and dying nematodes, in sharp contrast to viable and highly motile forms, ta~e up Brilliant Green and Brilliant CresYl Blue stains.

Because of the nematocidal activity that is induced by the chitin-protein complex described herein and the ease and low cost of its manufacture in commercial quantities, addition of this material to agricultural and horticultural soils for the purpose of control of plant-pathogenic nematodes provides an economically and environmentally attractive neans for the use of otherwise low value shellfish wastes and a means for reducing food, fiber and economic losses due to nematode infestations. Incorporation of these materials into animal feeds also offers a potential means for control of intestinal tract nematodiasis and a rich source of dietary protein.

By a broad aspect of this invention, a composition of matter is provided comprising a nematocidally-active, chitin-protein complex derived from crustacean shell waste material and consisting essentially of a water-insoluble demineralized chitin component complexed with a water-insoluble protein component, the complex being essentially free of low molecular weight peptides, amino acids, and calcium chlori.de brine formed by acid hydrolysis of such waste material, and being characterized by (a) a Lowry protein content of at least 50~ by weight, an ash content of not lOmore than 15% by weight, and a moisture content of less than 10~
by weight, based on the total composition; (b) having solubility properties similar to those of chitin in being insoluble in neutral dilute acid solutions but being solubilized with significant decomposition of the protein component in concentrated mineral salts; (c) having an IR spectrum similar to that of chitin but with a characteristic extra absorption band at 1738 cm~'; (d) the water-insoluble, demineralized chitin component having an acetyl content substantially identical to that of chitin as shown by a characteristic infrared spectrum 20absorption band at 1558 cm~l, but which is substantially free of carbonates and contains not more than 15~ of the ash content of chitin; (e) the water-insoluble protein component having an amino acid composition substantially identical to that of the untreated chitin-containing waste material, a molecular weight primarily in the range of 1Q-50 kdal as determined by sodium dodecyl sulfate lZ3~37 - 8a -gel electrophoresis, and being essentially insoluble in common protein solvents; and (f) being in the form of dry particles having a diameter of less than 0.5 mm. Preferably, thé
composition also has a protein content of at least 70~ by weight, an ash content of not more than 5% by weight, and a moisture content of less than 5~ by weight, based on the total composition. The composition above described is generally in the form of a B

~239~37 pulverulent solid.
The composition of another aspect of rhis invention may comprise a plant growth medium ~IdmixLure with d nematocidally-effective amount of the chitin-protein complex of the various aspec~s and embodiments cE this in-vention as described above. The plant growth medium in the above composition may be soil, or a plant potting soil suitable for growing stock or d par-ticuiate inorganic material, e.g. an expanded mica. The compositions describecl above preferably further include llarposporlum fungus in an amount effective to enhance the nematocidal activity of the chitin-protein complex.
The plant growth media described above may be planted with a liv-ing plant susceptible to nematode infection.
By another aspect of this invention, a nematocidal composition is provided comprising a nematocidally-effective amount of the chitin-protein complex of aspects of the invention described above in its various embodi-ments, in admixture with a horticulturally-acceptable carrier material. The composition may be in the form of a pulverulent solid composition, prefera-bly where the carrier material includes a soil conditioning agent.
By another aspect of this invention, a method is provided for in-hibiting the growth of saprophytic nematodes in a plant growth medium capable of supporting such growth, which method comprises admixing at least a nema-tostatically-effective amount of the chitin-protein complex of aspects of the present invention described above in its various embodiments, with the plant growth medium to inhibit the growth of the nematodes. In this method, the plant growth medium may be soil, or a particulate inorganic material, or may be one which is suitable for growing nursery stock.
By a variant thereof, the process includes admixing a nematocidally-effective amount of the chitin-protein complex, e.g. at least 5%, with the D lant growth medium.

123~S~37 In th~ annexcd drclwings, Figure 1 illustrates the process for producing the chitin-protein complex of an aspect of this invention and identiEies by-products which can be covered;
Figure 2 illustrat:es the subunit composition of the protein compo-nent ot crabshell waste treated according to the process of one aspect of this invention;
Figure 3 illustrates conventional processec used to treat shellfish wastes for ~he production ot commercial chitin and chitosan products as they compare with the process of an aspect of the invention disclosed herein, Fig-ure 3 also showing the chemical structures of chitin and chitosan;
Figure 4 i]lustrates the subunit composition of protein components of the chitin-protein complexes prepared according to aspects of this inven-tion, and of commercial chitin and chitosan preparations;
Figure 5 illustrates the subunit composition of the protein compo-nent of the chitin-protein complex obtained by acid treatment of dried fer-mentor cake from a commercial gibberellin fermentation process;
Figure 6 is a plot of the alternating current (ac) conductivity of chitin, chitosan and the crabshell chitin-protein complex as a function of applied voltage;

Figure 7 demonstrates the infrared spectra of (A) chitin, (B) the chitin-protein complex of an aspect of this invention, and (C) chitosan;
Figure 8 illustrates the infrared spectra of (A) untreated fungal fermentation cake and (B) an acid-treated fungal fermentation cake product of an aspect of this invention;

Figure 9 demonstrates the light microscopic appearance of Panagrellus spp. nematodes in control culture media (A and B) at 16-20 days after innoculation and the appearance of nematodes at 16-20 days after inno-,, _. 10 --1~23~;37 culation in test media contc3ining the chitin-protein complex (C and D) of an aspect of this invention; and Figure 10 shows living and dead nematodes stained with Brilliant Green.
The chitin-protein complex of an aspect of this invention can be prepared from any suitable chitin-containing biomass raw materiill. Such materials include bu~ are not limited to invercebrate marine org<lnisms hav-ing visible shells. Examples of such organisms are ~ucthropods~ including crustaceans, mollusks, marine benthic organisms and krill fish. Preferred shellfish waste is that obtained from crustaceans~ e.g. crabs, lobsters, crayfish, shrimp and prawns. Cell walls and filamentous masses of true fungi, including Phycomycetes and Ascomycetes species (which can be digested by one or more of the over 30 enzymes, including chitinase, glucanase and mannase, contained in the digestive juice of the snail Helix pomatia or produced by certain bacteria, such as some soil scavenging Pseudomonas species ~hich have been isolated from soils) contain chitin but do not ordinarily provide a suitable raw material or feedstock for a commercial process because of the amounts of these materials presently available. Because it is the presently preferred embodiment, the preparation of the chitin-protein polymer complex obtained from the shells of blue crabs (Callinectes sapidus) harvested from the Chesapeake Bay will be described in detail.
In conventional blue crab processing operations (see, for example, pages 206-207 in E. J. Middlebrooks, Industrial Pollution Control, -lZ3~37 ~nl~ Agr~=lnd~s~ s, John Wiley and Sons, New Yor~l 197~ crabs are dredged from the mud, caught in baited traps or lines or scraped from grassy shores during the molt. gaited pots are used to trap Dungeness, Tanner and King crabs which are then stored in circulating seawater in shipboard and/or in landt.ased tan~s prior to processing, usually by dry ~utchering. Blue crabs are transported live to the processing plant and are unloaded into trolleys for i~,ediate steam coo~ing at 121C for 1~-2~ minutes. The coo~ed cr~s are then stored overnight in a cooling loc~er after which the claws are removed and lo saved for later prc,cessincJ. After removal of the carapace and claws, the claws and sc~letimes the t.odies of the crabs are either run through a mechanical pic~er or pic~ed n,anually to separate residud1 meat from the shell. Crat. processing waste is generally discharged to a waterwaY
or a municipal sewer, hauled to a sanitary landfill or otherwise rendered, frequently by drying and shredding for eventual use as a feed meal, espeLially for chic~ens ~see, for example, P. R. Austin et al.
U.S. Patent 4,32~ ; W. P. Uri Yrains, Jr. and T. M. Miller, ^Prices ~ased on Nutritional Worth, Crab Meals and Crab Meal-Phosphoric Acid Supplements in the Diets of Monogastric Animals~, Final Report ~FIS-SI-005) to Department of Natural Resources, Maryland Tidewater Aclministration (March, 19S~).
The presently preferred starting or raw material for the embodiments of the herein disclosed invention (Figure 1) is such crab processing waste material which has been oven-dried and shredded to a small particle size. To reduce costs c,f raw materials, the drying step can be o~,itted. The exact particle size which is used affects the rate but not the nature of the process. Composition of the crabmeal raw material varies both with the season and with the thoroughness with which the meat is removed from the shells, but the raw n,aterial generally contains protein (40-50%), calcium carbonate and small amounts of other mineral salts ( 5~%), and chitin ( 10%).
Dried and shredded shell wastes are milled or ground to a desired particle size and either ued directly or washed with hot or cold water to remove contaminants which may have developed during transportation, if required, to a processing facility. Shell fragments are then lZ3~;37 demineralized in a stirred tan~ reactor using a dilute mineral acid, e.g. 1.3 N HCl, for a period of 30-6~ minutes, generallY under ambient temperature and pressure. Acids. e.~. suliuric and phosphoric are not suitable since they result in in~oluble calcium salts which interfere with recovery of the product. The deminerali~ation reaction, which is accompanied by significant modification of the protein component of the crab shells (Figure 2) and by the release of carbon dioxide gas containing detectable amounts of the ~fishy~ odors characteristic of al~yl amine~T-can be followed by titration or c,y observation of gas release.
The insoluble end-product of the reaction which is of specific interest to this disclosure is a chitin-protein con.plex which has distinctly different gel electrophoretic properties from the product resulting fron. deminerali2ation of crabshells by chelating agents e.g. ethylenediaminetetracetic acid (EDTA) ~Figure 4) and fr w chitin-protein complexes isolated from fungal residues (Figure 5).
The solid-state electrical properties of such chitin-protein material are also distinctly different from those of commercial preparations of chitin and chitosan which are produced by substantially more vigorous subsequent treatments (Figures 3 and 6).
After completion of deminerali~ation and protein modification (DM/PM) by mild acid hydrolysis5 usually at 6~ minutes after the start of the hydrolysis reaction, the resulting chitin-protein material is washed until neutral (pH 7.~) with water or wea~ soda ash (Na2C03) solutions. Effluents from the DM~PM and wash water tan~s can be recycled for recovery of low molecular weight peptides, amino acids and calcium chloride brine, or can be simply discharged to an approved waterway or wastewater treatment facility. The resulting chitin-protein ccmplex is then dried in a suitable drier and ground, if desired, to a particle size of lessthan ~.5mm. No further treatment, as is required in conventional chitin and chitosan processing operations ~Figure 3), is needed.
The resultant chitin-protein complex (Figures 4, 6 and 7) is: (i) insoluble in neutral and in dilute acid solutions but solubili~ed with significant decomposition of the protein component in concentrated mineral acids; (ii) low in ash content; (iii) high in _ 13 -1~39~37 bound nitrogen content owing to the presence of t:he protein moiety; and (iv) a naturally occurring, biodegradable materidl which, when added to namatode cultures in vitro, resu]ts in a significant reduction in the number of liv-ing organisms (Figures 9-10). The product can be produced commercially in better yield and at substantially less cost- than can either chitin or chito-san derived from crab, lobster, shrimp or other shellfish processing wastes or from the wall-s of chitin-contclining fungi, molds and yeasts. Morpholo-gical changes induced in namatode cultures in in vitro culture media are also distinctly different from those which are seen r~llowing exposure of phototypical namatode species to chit-in or chitosan (Figure 9).
Preferred rates for application of the chitin-protein complex of aspects of this invention to plant growth media range from I to 50 weight percent, generally in admixture with d plant growth medium containing the requisite nutrients. More preferred rates are in the range of 2 to 28 weight percent; the presently most preferred rates are in the range of 5 to 10 weight percent. The optimum amount within this range depends upon a number of variables which are well known to those skilled in the art of plant pro-tection. These variables include but are not limited to disease to be con-trolled, the type of crop, stage of development of the crop and rhe interval between applications. Applications within the range given may need to be repeated one or more times at intervals of 1 to 6 months.
The chitin-protein complex of aspects of this invention can be applied in a variety of formulations, preferably as granules, pelleLs, etc.
Powder and dust preparations can be made by blending the active ingredient, with or without surfactant, with finely divided solids, e.g.
talcs, natural clays, pyrophyllite, diatomaceous earth; flours, e.g. wheat, redwood, and soya bean; or inorganic substances, e.g. magnesium carbonate, calcium carbonate, calcium phosphate, sodium siliocoaluminate, sulfur and the like. The choice of a particular diluent is based on consideration of 1~3~ 7 the physical and chemical propertics rcquircd of t~he product~ the chemical and physical properties and concentration of the active ingredient, and the use for which the formualation is intended. The compositions arc made by thoroughly blending the active ingredient with the diluent and other additives.
Powdered compositions can be converted to granules by adding a liquid, treatillg mechanical]y, and usually drying. Mechanical devices, e.g.
granulating pans, mixcrs and extruders can be used. Compaction devices can be used even without a liquid in the mixture. Water soluble binders. e.g.
inorganic sdlts. urea, lignin sulfonates, methyl cellulose, ot-her watcr soluble polymers and the like, can be included in these particulate formu-lations in amounts up to 25% by weight of the finished granulc ~-r pellet.
Such materials also aid in disintegration of the pellet and release of the active ingredient under field conditions. Alternatively, a suspension of the active ingredicnt can be sprayed on the surface of preformed gr.~nules of clay, vermiculite, corn cob and the like. Surfactants may also be included in formulation of the latter type.
The compositions of aspects of the invention can contain, in addition, to the active ingredient of other aspects of this invention, conven-tional insecticides, miticides, bactericides, other nematocides, fungicides or other agricultural chemicals, e.g. fruit set agents, fruit thinning com-pounds, fertilizer ingredients and the like, so that the compositions can serve useful purposes in addition to its nematocidal activity.
Because of its protein, the chitin-protein complex of aspects of this invention contains 10% nitrogen in a slow-release form; it can advantage-ously be mixed with sources of metabolizable phosphorous and/or potassium to provide a balanced fertilizer. The nitrogen content can be enhanced by the further addition of other nitrogen fertilizer sources which are well -123~t;37 known in the Irt. The presently preferred embodiment oL aspec~s of this in-vention is for use in a po~:ting mixture with soil or a particulate inorgclnic material, e.g. vermiculite! e.g. for growing greenhouse plants or nursery stock. In one embodiment Harposporium fungus is added to enhance the nematocidal activity of the chitin-prottin complex.
Pathogenic plant namdtodes which mily be controlled in accordance with othe aspects of the present inven~ion include but are not- limited to those set forth in the following table.

T~BLE I
p~THnt~t3N~ pL~ FM~Tn~Es NFMATnrE ~LB~ DSI

t~4hPlPnLhDides_bPcceyi Strawberry ~ityle~Lh~s dicp~ri Root crops UPt~rnLe~a_~n~trrhiPncic Potato ~itYlPnrhus_L~s~ l4n Potato Pr~tvl~nLh~5-~2nptr~ns To~acco~ apple~ cherrY
~iphi _ Grasses, Citrus, Tot~ato MplnidnrJvnp-ha~3l~ Potato TYlPnrhlllllC semi4enPtr~nC Citrus ~titylPnrhllc myrPlinph;3rJllc Mushroc~
_ tnni Tobacco UPmirri~nnPmnidPc rhit~nn~i Cat~ellia HPmiryrlin4hnr Are~ia Citrus P~r.3trirhndnrllc rhri5~iei Celery ~a~a~vlPnrhuc prnjprtllc Grass Ola~4~ ch.s_zt~e Cor n knpl~imllc cnlllmhllc Soy~ean E~a~ylPn~hllc nPrJlPrtns Corn, straw~erries~ etc.

1,~,;3{~t;3~

Without ~urther ellbol-a~ion, i~ is believed Lhat one skilled in the art carl, using ~-he preceding dcscription, utilize the presen~ invention to its fullest extent. The fo]lowin~ preferred specific embodimcnts arc, therefore, to be cons~-rued as merely illustrative. All temperaturcs are set forth uncorrectd in degrees Celsius; unless otherwise indicated, all pressures are ambient and all parts and pcrcentages are by weight. The values obtained by elemental analysis are within the usual limits of experimen~al error; all new products gave t:he expected parent pe~lks in IR.

E~41P 1 PrPpAr~t irn_n~ RAhl MAtP~L1 Cra~meal processing waste o~tained fron. a cc~n.ercial supplier was water washed, dried in a hot-air o~en at 1~0C for approximately 16 hours and then shredded mechanically to a particle si~e such that all of the material passed through a No. 25 USA Standard Testing Sieve.
The resulting particles had a moisture content of 5-1~% and contained appro~imately 40-50~. protein and 30-50% CaC03 on a dry weight basis.
Elemental composition of three representati~e batches of otherwise untreated raw material feedstoc~ is illustrated in Table 11. Protein content was determined by the method of Lowry ~which measures tyrosine and peptide bond content). Nitrogen content was determined using combustion analysis based on the Pregl-Dumas method and an Elemental Analyzer known by the Trade Mark of PERKIN-ELMER MODEL 240B.

lZ39637 T~ble 11 ~p~siiiLln l~hell~ish_~rtin9 MAteei~ls Fl FMFNTL~ArTlnNATF~l) 51ZE~2)~nIN~3) Calcium (~g/g)92~00.0 ~49~.095~00.0 Sodium ~g~g)~470.0 12000.01050a.0 Magnesium ~g/g) 4880.05170.0 5030.0 Po t as5 i um ~g~g) 41b0.033~0.0 4420.0 Strontium ~g/g) 1970.01000.0 1170.0 lron ~g/g) 361.0 410.0716.0 ] ~luminum (~g/g~ 100.0103.0 206.0 8arium ~/g) 25.5 23.5 34.6 Boron (~g/g) 4.5 4.5 b.6 Cac~ium ~g/g)<0.5 ~0.5 <0.5 Chromium ~g/g)1.0 1.5 1.6 Co~alt ~g~g) ~1.0 ~1.0 <3.3 Copper ~gJg) 27.0 25.0 ~6.3 Lead ~g/g) <2.5 <2.5 <8.2 Manganese (~gJg) 150.0145.0 228.0 Moly~denum ~g/g) <1.0 <1.0 <3.3 Zinc ~g/Q) 69.0 57.5 74.2 Car~on ~%) 2S.84 34.7634.01 Hydrogen (%) 3.96 4.74 5.59 Nitrogen ~%) 5.46 6.2~ 7.1 Protein ~Lc~ry) (%) 32.4 43.0 41.8 ~sh ~%) 45.76 36.6537.22 Moisture (%) 7.41 10.478.11 ~1) Shredded shellwastes, as recei~ed (Batch 118)

(2) Shredded shellwastes, sized through ~STM 40 mesh screen (Batch 118)

(3) Shredded shellwastes, ground to <0.5mm size ~Batch 524) lZ3~37 E~mple_2 l~tlfl~tL~y-~LA~ at~ L~f r:~hshe~ hilin-er~in-~L~le~-hy ~Li~ ~inpr~ li2atlL~n The sutunit ccmposition was determined ty electrophoresis on a 10,' pol~acrylan,ide gel containing ~.1% sodium dodecyl sulfate ~SDS). Shown in Figure 2 are scans of the gels after staining of the proteine. with Coorr~assie erilliant elue R. The positions indicated ty the arrows are the positions rf standard mc.lecular weight ~given in daltcns in parentheses~ mar~er proteins: 'a) myosin - ~200,000), ~b~
teta-galactosidase (i~J~ C) phophorylase e ~7,40~ ine serum albumin S66,2Q0), ~e) o~altumin ~45,0Qa), ~f) car~onic anhydrase (81,000), ~g) c~oytean trypsin ihhititor ~21,500.), and (h) lyso~yme ~14,400). The samFles run were 5 n,g of ~Aj ctarting material, (B) starting material after 10 minutes in acid, (C) after 30 minutes, ~D) minutes, and ~E) the final product after acid treatment, washing, and drying.
Four hundred grams of the raw material descrit.ed in Example I were slowly added over a 30 minute period to 2 liters of 1.~ N HCl with continuous stirrincJ. The reaction caused rapid den,inerali~ation of the CaCO3 phase of the raw material feedstock as e~idenced t,y foaming of the reaction mixture ar,d the reiease of CO2 gas containing readily detectable amine odors. ~pproximately 40 mls. concentrated HCl were then added in small aliquots to the reaction mixture ~tc. maintain acidity at approximately pH 1.5) over a period of 60 minutes, after which no further foaming was otserved. The insclutle residue rerr~aining after the deminerali~ation and partial hydrolysis procedure was collected on a No. 270 U.S.~. Standard Testing Sieve and washed with water until t,oth the product and the washings were neutral (pH
7.8). The insoluble product was oven-dried at lOO C overnight yielding 128 grams of product (32% yield). The dried product was ground in a grinder known by the Trade Mark of WILEY LABORATORY MILL to a particle of less than O.5 mm. for use in all subsequent studies.

_ 19 --1239~37 F Y ~mp 1 ~_3 I ahnratnrY=S~ P~ $i~ n~ rrah~ hitin=ec:f~tf~in ~ e Il~ea tmPn ~ ~ ~ ~ ~ gen t Ten gra~s of the raw material feedstoc~ described in Exa~ple I were added to 1 liter Gf ~.IM ethylenediamine~etr~acetic acid ~EDT~)~ pH
7.5, and the mixture stirred continuously at 25C for 72 hours. The residual insoluble pr~duct was collected on a No. 27a U,S.A. Standard Testing Sieve and washed exh~usti~ely with water. The resulting product was oven-clried at 122C overnight with reco~ery of 2.75g of dry lo product (2~/. yield~.

Example 4 Pilot-Scale Preparation of Crabshell Chitin-ProLein Complex Fifteen kilograms of the raw material feedstock described in Example I were slowly added to 100 liters of 1.25 N ilCl in a 200 liter stainless steel stirred reacLor. The rate of addition of feedstock was regulated so as to minimize foaming over the course of a 60-minute demineralization and acid hydrolysis reaction. Insoluble produc~ remaining after demineraliza-tion and acid hydrolysis wai collected on cl separdtor known by the Trade Mark of SWEC0 VIBR0-ENERGY equipped with a 150-mesh self-cleaning stainless steel screen, washed with water and then 1% Na2C03 and~ finally, washed with water again to remove all soluble carbonates. The neutral (pH 7.0) product was oven-dried at 100 C overnight and then ground to a particle size of less than 0.5 mm. prior to use. The elemental compor~itions of the preparations from Examples 2, 3 and 4 as compared to chitin and chitosan are shown in Table III.
Figure 4 illustrates the subunit composition of protein components of tnc chitin-protein complexes prepared in Exdmples 2, 3 and 4 and of com-mercial chitin and chitosan preparations as determined by electrophoresis on a 10% polyacryldmide gel containing G.1% SDS. Shown are scans of Coomassie Brilliant Blue R- Stained gels. Samples are 5mg of (A) untreated crabshel~
wastes described in Example i, (B) chitin-protein complex obtained by mild acid hydrolysis described in Example 2, (C) chitin-protein con,plex preparea hy demineralization with ethylenediamlr:etetraacetic acid (EDTA) aescribed in lZ3~ 37 Examplc 3, (D) chi~in-protein complex prcparcd as in Examplc 4, (E) chitin obtained commercial]y, and (F) chitosan obtaincd commercially. Arrows indi-cate positions of migration of molecular wcight markers as in Figure 2.

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1239~37 Exam~ c 5 Laboratory-Scale lsolation of Chit;ll-Protein Complex Erom Dried Fungal 8iomass Dried fermentor cake obtained from a commercial gibberellin fer-mentation process was used as a raw material feedstock in place of thc Crab-shell raw material feedstock described in Examples I through 4. Two hunclred grams of dried fungal biomass were added to 1 000 ml. of 1.0 N HCl and the mixture stirred ccntinuously for a period of one hour. There wcls no appreci-able release of gas nor any significant neutralizati~n of the I~CI solution during the course of the reaction. Residual insoluble material WdS COI lected by centrifugation for 10 minutes at 10 0()0 rpm in a rotor known by the Trade Mark of SORVALL GSA at 40 C. The pellet was resuspended in water and centri-fuged again as described above. This procedure was repeated four times by which point the residual insoluble biomass material and the washings were neutral (pH 7.0). Insoluble material remaining after the fifth centrifuga-tion procedure was oven-dried overnight at 100 C with recovery of 88 grams of solid material ~44% yield). This material was ground in a grinder known by the Trade Mark of WILEY LABORATORY MILL to a particle size of less than 0.5 mm. prior to use. Its composition is shown in Table IV.
Figure 5 illustrates the subumit composition of the protein com-ponent of the chitin-protein complex obtained by acid treatment of dried fermentor cake from a commercial gibberellin fermentation process as described in Example 5. Scan (A) represents the acid-treated material and scan (B) represents untreated fungal fermentor cake. Arrows indicate the - positions of migration of molecular weight markers as in Figure 2.

T~ble 1~
LDm~DSi~inn n~_~ng~l Pre~ r ~ti~ns Acid-Treated Fl FMEt~[IFIID,9~1 FPrmPn~ PEllng~l_ e~nr~e Calcium (Pg/g) 76b.0 174.0 Sodium ~Pg/g) 102.0 160.0 Magnesium (~g/g)1~00.0 23.5 Potassium (Pg/g)827~.0 132.0 Strontium ~Pg/g) 3.2 - 1.9 IO Iron (~g/g) 106.0 237.0 ~luminum (Pg/g) 26.9 28.1 Barium tPg/g) 1.1 <0.9 Poron (~g/g) 2.1 3.7 Cadmium ~/9) <1.0 ~0 9 Chromium (Pg/g) 1.2 <1~9 Co~alt (Pg/g) <2.1 1.~
Copper (~g/g) 8.b 1.9 Lead (Pg/g) <5.4 <4.7 Manganese (Pg/g) 8.6 2.8 20 Moly~denun, (Pg/g)<2.1 <1.~
Zinc (Pg/g) 22.7 1.9 Carbon ~%~ 52.74 54.41 Hydrogen (%) 7.~7 7.61 Nitrogen t%) 6.73 6.20 Protein ~Lowry) (%)31.46 29.32 ~sh (~.) 4.96 3~.75 Moisture (%) 4.04 5.3 - 24 _ 1~39~37 Examplc 6 Characterizaticn of the Chicin/Protein Complexes Samples of crabsheJI raw material and each of the test materials (particle size less rhan 0.5 mm.) were analyzed for carbon~ hydrogen, nitrogen, ash and meta] contents; for total prot:cin and arnino acid content (Tables 11, llI, alld IV); for solid-state electric properties (Figure 6); and by infrared spectroscopy (Figures 7-8). Elemental composition WdS delermineC
using Industive]y Coupled P]asma Emission Spectroscopy (ICP) for metal analysis and an analyzer known by the Trade Mark PERKIN-ELMER 240B Elemental lo Analyzer for carbon, hydrogen and nitrogen ana]ysis. An Infrared Spectro-photometer known by the Trade Mark of PERKIN-ELMER MODEL 1320 WdS used to measure infrared spectra of clll materials. Total protein content was deter-mined by ex~:raction of each of the rnaterials with ].() N NaOH for 48 hours at 25 C, fol]owed by determination of the protein content in the solution by the Lowry method. ,For amino acid analysis, samples were hydrolyzed n vacuo in 6 N HCI for 24 hours at ]10 C and the amino acids were measured after after separation by high performance liquid chromatography (HPLC) according to standard methods, e.g. those recently reviewed by M.W. Dong and J.C.
DeCesare in Liq. Chrom. 1, 222-228 (1983). Solid-state electrical properties were measured by an electrical testing laboratory using standard techniques.
The amino acid compositions are shown in Table V.

1;Z3~37 E~ le_Z
L~r Lteri7~1i~n_~ he_eentein_~Qmp~nent_nf ~he ~hllin=~r ~ ~ ~ i n ~ple~
During the acid-demineralization of crab shell wastes, the amount of high molecular weight protein extractable by sodium dodecyl sulfate decreases ~Figure 2). The total amount of protein, howe~er, remains nearly constant (Table 111) and the protein can only be remoued by extended al~aline hydrolysis, indicating that in the acid treatment the protein is either made more detergent insoluble, is partially degraded, is co~alently lin~ed to the chitin matrix, or a combination of these possibilities. The amino acid analysis of the starting material and of the chitin-protein ccmplex ~Table V) demonstrates no significant change in amino acid compositi~n during mild ~cid hydr~lysis, indicatin~ that the relatiue amounts of the proteins present in the starting materials and in the final product are not very different. A5 seen in Figure 4, there is no significant amount of protein in commerclal preparations of chitin or chitosan. The chitin-protein cornplex prepared using EGTA to demineralize the material, on the other hand, contains a significant amount of protein which has not been modified.

- lZ3~ 37 I~BIF U
~miho_~Li~ Cnmp~siti r~n ~f rrah ~h P 1 1 ~A C t P C
ALid-dPminprAl i7~ rah ~hPll LlA<;tpc~ ~ d ~Li~n~=~emi~lilP~ C~~ h~l 1 LlA~t-eC

EDTA -deminerali~ed Lhitin/e~L~in ~rmpleY Grab Amin~ A~;~a ~t~rlin~ M~t~ial ~atLh_529 e~irh 11~ _Shell_J~s~es Aspb 10.8 g.3 1~ 5 13.3 Thr 3.6 8.6 4.5 13.6C
Ser 4.9 5.5 4.8 Glub ~.~ I3.4 12.~ 12.2 Gly 5.0 3.9 3.~ 4.2 Ala 5.6 5.3 5.4 5.5 ~al 3.6 5.2 5.~ 6.0 Met l.8 3.3 3.5 3.1 lle 4.4 5.4 5.5 5.1 Leu 10.5 8.5 ~.0 7.4 Tyr 4.9 5.~ 5.
Phe 10.8 ~.3 9.g 5.~
Lys 12.5 7.3 g.3 7.5 His 3.a 2.~ 3.2 3.0 Arg 8.8 7.l 7.0 7.2 a ~ysteine, proline, and tryptophan were not determined.
b Asparagine and glut~.ine are included with ~sp and Glu, re~pectively.
C Ser ~ Thr 123~>37 plP 8 il~rtpri7~ion nf thP rhitin l~nmpc~nent~f-~he rh i t i n -pr-kt P i n l~mnple~
The chitin-protein complex was analyzed by infrared spectroscopY
and compared with commercially available chitin and chitosan ~Figure 7). The chitin~protein complex gives a spectrum very similar to that of chitin with the exception of an extra absorption ~and at 1788 cm~l, possibly due to the protein component. The absorption band at 1550 cm~l in chitin and in the chitin-protein--~on,plex, which is shifted in chitosan, appears related to ace tyl at i on o~ the amino groups IO in chitin. The relative intensities of this band in chitin and the chitin-protein complex indicate that there is very little deacetYlation of chitin during preparation of the chitin-protein complex.
Infrared spectra of the fungal preparations were measured ~Figure 8). The spectra are very similar to that of chitin, and the spectra of both preparations (untreated, acid) are similar, indicating very little change in the form of the chitin in the materials.
The chitin-protein complex has solubility properties similar to those of chitin, i.e. it is insoluble in most ordinary sc,lvents. The protein portion can be partiallY solubilized ~y detergents and other protein solvents such as urea or guanidinium salts, or ~y treatment with alkali. Chitosan, on the other hand, is soluble in dilute organic acids ~1% acetic, lactic, propionic, and formic acids). All of the materials are soluble in concentrated mineral acids, but significant degradation occurs.

FY~n~rlP ~
E~U t i nn n~ t h P NPm~ t nr i ~1P T~L~iy~el:
Pan~rJrPll~s~ a saprophytic nematode obtained from Dr. Jul iU5 Feldmesser at the U.S. Department of ~griculture Plant Protection ]nstitute, Beltsville, Maryland, was cultured in a commercially available oatmeal cereal ~Gerber Products Co., Fremont, Michigan). The nematodes were cultured in 6~ x 22 n~ sterile plastic petri dishes containing 6 grams of autoclaved oatmeal cereal and 20 ml of sterile distilled water. ~he dishes were inoculated with approxi~ately 20~0 nematodes suspended in 2 ml of sterile distilled water. The cultures _ 28 -12~37 were then incuhated at 30C for 2I days. Control cultures contained only oatmeal cereal, distilled water, and nematodes in the ahove proportions. Materials to he tested for nematocidal activitY were autoclaved and added to the individual culture dishes at the level of 0.2 grams per dish. Both control and test cultures were set up in series of five samples.

F v ~ 1 R
t~ie~sllr Pn-P n rL~ NPma t nr i L~LL~LIy Cultures of ~na4~11~5 were prepared and observed according to the test system presented in Example ~. Ohservations were made beginning day S and continued through day ~1, or until the cultures died.
Microscopic observations were made using the wet mount slide technique on 0.~1 ml samples withdrawn from the active surface 20ne of the cultures where Pan~¢ell~s existed. Qn average of the numhers o~tained by counting the samples withdrawn from each of the culture dishes in the series was used to determine the relative population.
Counts were made beginning on the sixth and continuing through to the 2Ist day. ~veraged results are sh~wn in Table Vl, where a significar.t reduction in numhers of nematodes in cultures treated with chitin-protein complex of aspects of this invention can be seen.

T~hlP u~
E~Q~j~E~r P p a r a t i r n c nn t h P Nl Irb~P r n-f Liu ing NPm~tnrlPs i n_r"l tllrPC

Num~er_n~ I i~Cgan ism5 a Control 1,30~ - I,500 Chitin ~ ~0~ - 700 Chitosan h ~00 - 1,000 Chitin-Protein Complex b 0 - 400 a - Motile organisms counted in a 0.01 ml sample ta~en from the surface of the test plates ~ - Additions were present at ~/. (w/w) lZ3~37 The most- significant and reproduciblc reduction in the total number of organisms was seen at day 17 an(l latcr. While therc was a rcduction in population in ~:hc cultures treated with the chitin-protein complcx, chitin, and chitosan~ t:hc most significant reduction was observed with the chitin-protein complcx of aspects of this invention which also offers significant economic advalltages over the use of the more highly purified preparations of:
chitin and chitosan. The fungal fermentation cake is not pre-sently avai1able in quanrities sufficient to consider it as a practical raw material for this process.
Io Nematocidal activity in namatode test populations, as documcnted by photos shown in Figure 9 (A-D)~ includcd a variery of cvents evident in all stagcs of development. Loss of motilil-y~ a standard determination of death in namatodes~ was reinforced by avital staining with Brilliant Green, (C.l. 42040) and Brilliant Cresyl Blue (C.I. 51010). Staining was performed by adding a drop of a 0.05% aqueous solution to a preparation of the organisms on a microscope slide. Within three minutes, therc was a clear distinction between living and dead organisms (Figure 18). The living organisms wcre not stained, while the dye was taken up by the dead organisms. Cuticle dis-ruption as evident as shown in Figure 9. Unique to the cultures treated with the chitin-protein complex of aspects of this invention was the appearance of large vacuoles, indicating premature senescence, in namatodes of all stages as early as day 13. This appearance of vacuoles was not observed in cultures treated with any of the other materials.
Example 11 Nematostatic and Nematocidal Activity of Chitin-Protein Complex in Cultures with Soil Soil chosen randomly from agricultural fields was mixed with the chitin-protein complex of aspects of this invention at ratios of .05, .025 -lZ39~37 aTld .01 complex/s(!il (wt-/wt). Identical pcrtions o~ ~-hese mixtures were then spread on water agar plates and incubat-ed at room t-emperature. During this time the endogenous population of saprophyt-ic nematodes developed.
Several species were seen, predominantly Panagrellus sp. and Rhabditis sp.
The numbers of living and dead organisms in the cultures were counted. As shown in Table VII, the maximum killing was obtainecl witn 5/0 chitin-protein complex oE aspects of t~his invention. Control experiments with chitin and chitosan showed subs~antially less efficient killing f only 33/~ and 49%, rspectively, at day 33. Microscopic examination showed the nem~tophagous fungus Harpos~ to be present in the cultures where maximum kilLing occured. This may re1ect ttle mode of action OL the chitin-protein complex of aspects of this invention, which may stimul~te nematophagous fungi.
The following table shows the nematccidal activity observed in triplicate experiments, three plates per sample, of the chitin-protein com-plex of aspects of this invention on the growth of nematodes cultured in the presence of soil:

- 3~ -lZ39637 ~ BLE_l~ 1 1 ln_~'itrD ~nil Arti~it~

Percentages of dead organisms on test plates:

~_~5 ssliays ~a Gontrol 16% lk% 19/.

1% chitin-protein 52% 53% 91%
complex 2.S~ 9.7/. 5Z~. 77/.

5~ 93.6~. ~6% 8a.7J.

The preceding examples can ~e repeated with similar success ~y substituting the generically or speci f ically described reactants ancVor operating conditions of this invention for those specificallY used in the examples.

As can be seen from the preceding disclosure, the present invention in its various aspects is industrially useful in converting chitin-contain-ing biological waste material into product having nematostatic and nemato-cidal properties useful in hortiultural and agricultural applications.

Claims (20)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A composition of matter comprising a nematocidally-active, chitin-protein complex derived from crustacean shell waste material and consisting essentially of a water-insoluble, demineralized chitin component complexed with a water-insoluble protein component, said complex being essentially free of low molecular weight peptides, amino acids, and calcium chloride brine formed by acid hydrolysis of such waste material, said complex being characterized by:
(a) a Lowry protein content of at least 50% by weight, an ash content of not more than 15% by weight, and a moisture content of less than 10% by weight, based on the total composition;
(b) having solubility properties similar to those of chitin in being insoluble in neutral dilute acid solutions but solubilized with significant decomposition of the protein component in concentrated mineral salts;
(c) having an IR spectrum similar to that of chitin but with a characteristic extra adsorption band at 1738 cm-1;
(d) the water-insoluble, demineralized chitin component having an acetyl content substantially identical to that of chitin as shown by a characteristic infrared spectrum adsorption band at 1550 cm 1, but which is substantially free of carbonates and contains not more than 15% of the ash content of chitin;

(e) the water-insoluble protein component having an amino acid composition substantially identical to that of untreated crustacean shell waste material, a molecular weight primarily in the range of 10-50 kdal as determined by sodium dodecyl sulfate gel electrophoresis, and being essentially insoluble in common protein solvents; and (f) said complex being in the form of dry particles having a diameter of less than 0.5 mm.

2. A composition according to claim 1, having a Lowry protein content of at least 70% by weight, an ash content of not more than 5% by weight, and a moisture content of less than 5% by weight, based on the total composition.

3. A composition according to claim 1, in the form of a pulverulent solid.

4. A composition of matter comprising a plant growth medium in admixture with a nematocidally effective amount of the chitin-protein complex according to claim 1.

5. A composition according to claim 4, wherein said plant growth medium is soil.

6. A composition according to claim 5, wherein said plant growth is a plant potting soil suitable for growing nursery stock.

7. A composition according to claim 6, wherein said plant growth medium is planted with a living plant susceptible to nematode infection.

8. A composition according to claim 4, wherein said plant growth medium is a particulate inorganic material.

9. A composition according to claim 8, wherein said particulate inorganic material is an expanded mica.

10. A composition according to claim 8, wherein said plant growth medium is planted with a living plant susceptible to nematode infection.

11. A composition according to claim 8, and further including therein Harposporium fungus in an amount effective to enhance the nematocidal activity of said chitin-protein complex.

12. A nematocidal composition comprising a nematocidally effective amount of the chitin-protein complex according to claim 1 in admixture with a horticulturally- acceptable carrier material.

13. A composition according to claim 12 in the form of a pulveru-lent solid composition.

14. A composition according to claim 12 wherein said carrier material includes a soil conditioning agent.

15. A method for inhibiting the growth of saprophytic nematodes in a plant growth medium capable of supporting such growth, which method com-prises admixing at least a nematostatically-effective amount of the chitin-protein complex according to claim 1 with said plant growth medium to inhibit the growth of said nematodes.

16. A method according to claim 15, wherein said plant growth medium is soil.

17. A method according to claim 15, wherein said plant growth medium is a particulate inorganic material.

18. A method according to claim 15, wherein said plant growth medium is suitable for growing nursery stock.

19. A method according to claim 15, wherein a nematocidally effec-tive amount of the chitin-protein complex is admixed with said plant growth medium.

20. A method according to claim 19, wherein said nematocidally effective amount is at least 5% by weight of the plant growth medium.