WO1999057299A1

WO1999057299A1 - Cultured plant cell gums of aizoaceae: food, pharmaceutical, cosmetic and industrial applications of cultured plant cell gums of aizoaceae and other plant families

Info

Publication number: WO1999057299A1
Application number: PCT/AU1998/000318
Authority: WO
Inventors: Adrienne Elizabeth Clarke; Antony Bacic; Alan Gordon Lane
Original assignee: Albright & Wilson (Australia) Limited
Priority date: 1998-05-05
Filing date: 1998-05-05
Publication date: 1999-11-11
Also published as: AU7198998A

Abstract

Certain cultured plant cell gums, including those produced in suspension culture of plant cells of plants of the family Aizoaceae are described. Plant cell gums of plants of the genus Mesembryanthemum are specifically provided. Also described are the methods of using these cultured plant cell gums in the manufacture of food products, pharmaceuticals and veterinary products, cosmetics and other industrial products, such as paper, adhesive, ink, textiles, paint, ceramics, explosives, cleaning agents or detergents, products for firefighting, agricultural chemicals including pesticides and fungicides, for oil and gas production, and in photography, lithography, and other industries are described. Food, pharmaceutical, veterinary, industrial and cosmetic compositions containing certain cultured plant cell gums are also described.

Description

CULTURED PLANT CELL GUMS OF AIZOACEAE: FOOD, PHARMACEUTICAL, COSMETIC AND INDUSTRIAL APPLICATIONS OF CULTURED PLANT CELL GUMS OF AIZOACEAE AND OTHER PLANT FAMILIES

FIELD OF THE INVENTION

The subject invention relates generally to the use of cultured plant cell gums in food, pharmaceutical, cosmetic and other industrial applications, including their use in oil and gas well drilling and production and lithography, and in the manufacture of textiles, ink, adhesive, paper, paint, ceramics, agricultural chemical and cleaning or detergent agents.

BACKGROUND OF THE INVENTION A variety of natural and semisynthetic complex carbohydrates or polysaccharides have been commercially important in human and pet food manufacturing; in the cosmetic, paper, textile, paint, agricultural, explosives, hydrolube, adhesive, ceramic, cleaning polish, detergent, fire fighting, ink, photography, lithography, and deodorant gel industries; and in mining, and gas well drilling and production. Natural complex carbohydrates and polysaccharides include seaweed extracts, plant exudates, seed or root extracts, and microbial polysaccharides produced by fermentation. Semisynthetic complex carbohydrates and polysaccharides include cellulose derivatives, low-methoxyl pectin, propylene glycol alginate, triethanolamine alginate and guar gum derivatives. Sandford, P.

& Baird, J. (1983) "Industrial Utilization of Polysaccharides" in The Polysaccharides, Vol. 2, pp. 411-491.

The production of natural complex carbohydrates or polysaccharides is frequently problematic. For plant exudates and seed or root extracts, production is dependent on climate and harvest conditions. For example, gum arabic is an exudate from Acacia

Senegal trees. Gum production is stimulated by stripping the bark from the trees; the gum is collected by hand in the form of "dried tears." Production of gum arabic can vary each year as a function of weather conditions, labor strikes, natural disasters, etc. Meer et al. (1975) Food Technology 2£:22-30. The unreliable supply results in variable gum arabic cost. Seed gums, such as guar gums are expensive due to harvesting costs. Guar gum is derived from the seed of the guar plant Cyamopsis tetragonolobus. Processing involves removal of the seed coat, separation of the germ from the endosperm, and milling of the endosperm. Sandford, P. & Baird, J. (1983), supra. Further, gums obtained from such sources may have variable quality and exhibit variable functional properties.

The production of seaweed extracts can also be problematic. Agar production is labor intensive in that it involves the harvesting of red seaweed by hand: in some areas of the world, divers in full pressure suits collect individual plants in deep water; in other places, the seaweed can be collected at low tide without the use of diving equipment. Carrageenan or Irish Moss is produced from another red seaweed harvested by raking and hand gathering. Algin is produced from brown algae which can be harvested manually or with small mechanical harvesters. Sandford, P. & Baird, J. (1983), supra.

Further, hand harvesting can introduce a purity problem. For example, hand collected lots of gum arabic are seldom pure; samples are classified according to grade which depends on color, and contamination with foreign bodies such as wood or bark

(VanNostrand's Scientific Encyclopedia, 7th ed. (1989) D. Considine (ed.), Vol. I, p. 1389).

Microbial fermentation gums such as xanthan gum avoid many of the difficulties associated with harvesting of plant exudates or extraction of algae because production is carried out in fermentation facilities. However, xanthan gum production poses other problems. Xanthan gum is produced by Xanthamonas campestris, which presents a cell disposal problem because X. campestris is a plant pathogen (Scaad, N.W. (1982) Plant Disease 66(10):882-890). Xanthan gum has also been objected to as being too expensive for certain applications such as drilling mud. See, e.g., Kirk-Othmer Chemical Engineering Encyclopedia (3rd. ed. 1981) 17:153.

Thus, there is a clear need in a number of industries for a reliable, relatively inexpensive gum or class of gums that do not create a disposal problem. While a number of plant cells have been observed to produce polysaccharide and/or complex carbohydrates

2 when cultured (Aspinall, G. & Molloy, J. (1969) Canadian J. Biochem. 47: 1063-1070; Fincher, G. et al. (1983) Ann. Rev. Plant Physiol. 34:47-70; Clarke, A. et al. (1979) ϋ:521-540; McNeil, M. et al. (1984) Ann. Rev. Biochem. 53:625-663; Hale, A. et al. (1987) Plant Cell Reports 6:435-438; and Bacic, A. et al. (1987) Australian J. Plant Physiol. 14:633-641), it has not been suggested that such cultured plant cell gums might be suitable in the pharmaceutical, paper, textile, paint, agricultural, explosives, hydrolube, adhesive, ceramic, cleaning polish, detergent, fire fighting, ink, photography and lithography industries; or in mining, and oil and gas well drilling and production. Only Otsuji, K. et al. EP 0 285 829 (published October 12, 1988) have utilized cultured Polianthus gum in cosmetic applications.

Related work by the inventors hereof has been published in WO 8806627 (1988) and WO 9402113 (1994). WO 8806627 relates in general to the use of cultured plant cell gums in the manufacture of food products as emulsifiers, thickening agents, gelling agents and the like. Cultured plant cell gums of Pyrus, Prunus, and Rosa are specifically exemplified. US patents 5,133,979 (issued July 28, 1992) and US 5,296,245 (issued

March 22, 1994) are directed to similar subject matter. WO 9402113 relates to the general use of cultured plant cell gums as emulsifiers, viscosifiers, and the like for the manufacture of industrial, pharmaceutical or cosmetic products. Cultured plant cell gums from suspension cultures of Nicotiana, Pyrus, Phleum and Lolium are exemplified

SUMMARY OF THE INVENTION

The subject invention comprises the use of cultured plant cell gums produced from gum-secreting cells of vascular plants in a variety of industrial, pharmaceutical and cosmetic applications including, without limitation, textiles, paper, adhesives, inks, lithography, ceramics, cleaning detergents, firefighting, agricultural, explosives, oil and gas wells, and cosmetics. The cultured plant cell gums are useful in general as viscosifiers, as thickening, gelling, emulsifying, dispersing, suspending, stabilizing, encapsulating, flocculating, film-forming, sizing, adhesive, texture-modifying, enrobing, binding and/or coating agents, and/or as lubricants, water retention agents and coagulants. Any cultured plant cell gum can be useful in the subject industrial, pharmaceutical and cosmetic

3 applications described herein. This invention is more specifically directed to particular plant cell gums which have particularly useful rheological properties and the use of such gums in food, pharmaceutical, cosmetic and other industrial applications. The invention also relates to several specific applications for which cultured plant cell gums are particularly suitable.

In one embodiment the invention relates to cultured plant cell gum secreted in culture of plants of the family Aizoaceae. This family of succulents includes plants of the genera: Mesembryanthemum, Aptenia, Carpobrotus, Delosperma, Hereroa and Rushia, among others. Of more interest are plant cell gums of the Mesembryanthemum, Aptenia, or Carpobrotus. The plant cell gums of this family are particularly useful as emulsification agents and emulsion stabilizing agents. The plant cell gums of these plants are very active emulsifiers. Plant cell gums of species of Mesembryanthemum are particularly useful in the formation of low viscosity, low-droplet-size emulsions, e.g., cloud emulsions. Low-droplet-size emulsions find extensive use, for example in the food industry, for manufacture of soft drinks. Methods of use of these cultured plant cell gums are provided.

In another embodiment the invention relates to cultured plant cell gum secreted in culture of monocot plants, including plants of the family Poaceae including plants of the genera Phleum and Panicum. Cultured plant cell gums of Phleum (particularly those of timothy grass, P. pratense) exhibit good gelling ability and high viscosity. Phleum cultured plant cell gum can serve as a substitute for guar gum or hydroxymethylcellulose. Panicum gums exhibit high viscosity and visco-elastic properties and have a variety of applications in the food and other industries, particularly for the preparation of drilling muds. Panicum cultured plant cell gums are useful in the manufacture of chemical sprays, particularly for agricultural sprays to inhibit satellite droplet formation in such sprays. Methods of use of these cultured plant cell gums are provided.

Table 1A provides a preferred list of families, genera and species of plants that are useful in for the production of cultured plant cell gums. Table IB provides a list of more preferred families, genera and species of plants useful for production of cultured plant cell gums. Plant families of more interest for production of cultured plant cell gums include: Actinidaceae, Agavaceae, Aizoaceae, Asteraceae, Cucurbitaceae, Fabaceae, Malvaceae, Mimosaceae, Poaceae, Rosaceae, and Solanaceae. Plant genera of more interest for production of cultured plant cell gums include: Acacia, Actinidia, Chichorium, Cucumis, Glycine, Hibiscus, Hordeum, Letuca, Lycopersicon, Malus, Medicago,

Mesembryanthemum, Carpobrutus, Apentia, Nicotiana, Oryza, Panicum, Phalaris, Phleum, Polianthus, Pyrus, Rosa, Sida, Solanum, Trifolium, Trigonella, and Zea. Plant species of more interest for production of cultured plant cell gums include: Acacia Senegal, Actinidia deliciosa, Aptenia spp., Carpobrotus spp., Chichorium intybus, Cucumis sativus, Glycine max, domesticus, Medicago sativa, Mesembryanthemum spp., Oryza sativa, Panicum miliaceun, Hibiscus esculentus, Hordeum vulgare, Letuca sativa, Lycopersicon esculentum, Malus domesticus, Phalaris aquaticus, Phleum pratense, Polianthus tuberosa, Rosa glauca, Sida rhombifolia, Solanum, Trifolium repens, Trifolium pratense, Trigonella foenum-graceum, and Zea mays. Cultured plant cell gums produced by gum-secreting cells of plants of the foregoing families, genera and species are useful as emulsifying agents, viscosifying agents, gelling agents, thickening agents, dispersing or suspending agents, emulsion stabilizing agents, encapsulating agents, flocculating agents, film- forming agents, sizing agents, binding and/or coating agents, and/or as lubricants, water retention agents and coagulants or in adhesive compositions and the like in food, pharmaceutical, cosmetic and other industrial applications. Methods of use of these cultured plant cell gums are provided.

In general, plant cell lines that produce at least about 0.05% (w/v) gum in the final fermentor culture broth, are preferred to reduce production costs. Plant cell lines that produce at least about 0.5%, 2.0%, and 10.0% (w/v) gum in the final culture broth are increasingly preferred. In one embodiment, the cultured plant cell gums employed in such applications are cultured plant cell gums having arabinogalactan proteins (AGPs) of at least about 4.0% (w/w). As discussed herein, choice of explant and culture conditions for the plant cells can affect functional properties of the gum product.

Cultured plant cell gum products can be used as a substitute for prior art gums, such as gum arabic and guar gum. The cultured plant cell gums can also be used as a substitute for xanthan gum, alginic acid, agar, calcium alginate, carrageenan, guar gum,

SUBSTΓΓUTE SHEET (RULE 26) karaya gum, locust bean gum, potassium or sodium alginate, tragacanth gum and others. For example, the cultured plant cell gums can be used as thickening agents and/or emulsifying agents to replace gum arabic in adhesives, inks, textile printing and cosmetics. The cultured plant cell gums can be used to replace alginic acid as an emulsifier, thickening agent, suspending agent, waterproofing agent, etc. in toothpaste, cosmetics, pharmaceuticals, textile sizing, coatings, oil-well drilling muds, and concrete. The cultured plant cell gums can be used to replace agar as a gelling agent, protective colloid, in photographic emulsions or other applications. The cultured plant cell gums can be used to replace calcium alginate as a thickening agent, stabilizer, etc. in synthetic fibers. Carrageenan, which can be used as an emulsifier, protective colloid, stabilizing agent, etc. in toothpastes, cosmetics and pharmaceuticals, can be replaced by cultured plant cell gums. Cultured plant cell gums can substitute for guar gum, which functions as a thickening agent, emulsifier, etc. in paper, cosmetics, pharmaceuticals, textiles, printing, polishing, and as a fracture aid in oil wells. Cultured plant cell gums can also replace karaya gum as a protective colloid, stabilizer, thickener, emulsifier, etc. in pharmaceuticals, textile coatings and adhesives. Cultured plant cell gums can replace locust bean gum (carob-bean gum) as a stabilizer, thickener, emulsifier, etc. in packaging material, cosmetics, sizing and finishes for textiles, pharmaceuticals and paints. Potassium or sodium alginate, which can function as an emulsifier, thickening agent, stabilizer, etc. in pharmaceuticals, textile printing, cement compositions, paper coatings, and in some water-base paints, can be replaced by cultured plant cell gums. Cultured plant cell gums can replace tragacanth gum as an emulsifying agent, coating agent, thickening agent, stabilizer, etc. in pharmaceuticals, adhesives, leather dressings, textile printing and sizing, dyes, toothpastes, hairwave preparations, soap chips and powders. Xanthan gum, which is used as a thickening, suspending, emulsifying agent, stabilizing agent, etc. in oil and gas well drilling muds and other applications, can also be replaced by cultured plant cell gums. In replacing such prior art gums, the cultured plant cell gums can offer unexpectedly improved results. Often, cultured plant cell gums can surprisingly be used in smaller quantities than the prior art gums to achieve equivalent functional results. Further, production of the cultured plant cell gums do not present the cell disposal problem that xanthan gum production does. The cultured plant cell gums are not useful in applications where their utilities or properties are significantly compromised or destroyed. Organic solvents such as alcohol, acetone and ether and the like can disrupt function by causing precipitation of the cultured plant cell gums. To maintain the gums' emulsification, thickening or gelling properties, it is preferred that the temperature of the gum-containing solution or mixture be maintained between about 4° and 90°C and have a pH of neutral to slightly alkaline. As the pH increases, the thickening capacity of the gums decreases. However, even at elevated pH, viscosity can increase with increased ionic strength. Gum-containing solutions can gel in the presence of divalent cations such as calcium, and as temperature decreases, gel strength increases. Typically, stable gels are produced in the pH range of between about 3 to 10 and in the presence of calcium ions. Further, heating and cooling of gelled gum solutions between ambient and 80°C has not reduced gel strength, indicating that the gels can be thermo-reversible.

The cultured plant cell gums of this invention are useful in a wide variety of applications in part because they are stable over a wide range of temperatures. In an emulsion or solution, the gums are functional over a temperature range of about 0° to 100°C at neutral pH. The dried gum powder (neutral pH) is stable over a temperature range of about -70°C to about 10°C. If heated, the dried, powdered gum can caramelize. Furthermore, cultured plant cell gums of this invention can provide substantially non-toxic rheological agents (emulsifiers, etc.) of biological origin that can replace potentially harmful synthetic polymers and surface active agents.

The invention also provides isolated (i.e., substantially free of cell biomass) cultured plant cell gums which may be provided as aqueous solutions or suspension (more or less concentrated than in culture filtrate) or as dried powders. Isolated plant cell gums of this invention include those of plants of the genera: Acacia, Actinidia, Aptenia,

Carpobrotus, Chichorium, Cucumis, Glycine, Hibiscus, Hordeum, Letuca, Lycopersicon, Malus, Medicago, Mesembryanthemum, Oryza, Panicum, Phalaris, Phleum, Polianthus, Sida, Solanum, Trifolium, Trigonella, and Zea. Of particular interest are isolated cultured plant cell gums of plants of the family Aizoaceae (including those of the genera Mesembryanthemum, Carpobrotus and Aptenia).

7 DETAILED DESCRIPTION OF THE INVENTION The present work is an extension of the work disclosed in WO 8806627 (1988) and WO 9402113 (1994) which disclosed the general ability of cultured plant cell gums to function as emulsifying agents, thickeners, stabilizers, texture modifiers, gelling agents, binding or coating agents, suspending agents or the like. The present work specifically describes and exemplifies additional sources of and specialized applications of certain cultured plant cell gums.

The invention also provides isolated (i.e., substantially free of cell biomass) cultured plant cell gums which may be provided as aqueous solutions or suspension (more or less concentrated than in culture filtrate) or as dried powders. As will be appreciated in the art, isolated plant cell gums for use in food and veterinary applications should be substantially free of harmful levels of toxic plant components (oxalates, alkaloids and the like).

"Cultured plant cell gum" is defined as the substantially cell-free material recovered from cultured plant cells, and is used interchangeably herein with "gum product." The cultured plant cells are those which are capable of synthesizing components of the gum product and transporting the same extracellularly in culture. A variety of vascular plant cells, including those derived from gymnosperms and angiosperms, may be used in the subject method (see Table 1A and IB). Cells of plants of the Dicotyledonae class (e.g., the Rosidae and Asteridae subclasses) and Monocotyledonae class (e.g., the

Commelinidae subclass) can be used in the subject methods.

The cultured plant cell gum comprises complex carbohydrates and optionally glycoproteins, which are secreted into the medium by the cultured cells. The major classes of complex carbohydrate polymers are proteoglycans (e.g., arabinogalactan proteins (AGPs)), polysaccharides (e.g., neutral and acidic pectins), hetero- and homo-glucans, heteroxylans, and hetero- and homo-mannans (McNeil et al. (1984) Ann. Rev. Biochem. 53_:625-633). Complex carbohydrates and glycoproteins are known to be secreted by many cultured cell lines (Clarke, A. et al. (1979) Phytochemistry 18:521-540; Fincher et al. (1983) Ann. Rev. Plant Physiol. 34:47-70; Bacic, A. et al. (1987) Australian J. Plant Physiol. 14:633-641). Monocot gum-secreting plant cells have been found to secrete gums containing a root-slime-like material which contributes to the functionality of the gum as providing good gelling capacity and/or enhanced viscosity and/or visco-elastic properties compared in general to dicot gums.

The cells to be cultured can be initiated from a variety of explants, for example, a leaf, style, anther or stem of a plant, segments of which can be placed on solid plant culture medium. Callus cells may proliferate from any of the tissues of these organs and the callus cells can then be transferred to liquid suspension culture. Alternatively, seeds can be surface sterilized, and placed in a solid or liquid plant tissue culture medium to initiate germination. The germinating seedlings can then be maintained, for a time, in liquid suspension culture. The suspension culture medium can be any known suitable medium such as MS medium (Mirashige, T. & Skoog, F. (1962) Physiologia Plantarum 11:473-497; Wu, M. & Wallner, S. (1983) Plant Physiol. 72:817-820). or variants thereof. Suspension culture medium can be optimized for enhanced cell growth and/or enhanced gum production. Transfer to suspension culture is preferred because in general it increases gum production and because it is possible to scale up a liquid suspension culture. Air fermentors are preferred because they reduce shear stress on the cells. While cells can produce gum on a solid medium, mass culture on solid media poses a number of practical difficulties, including gum collection.

Usually, a plant cell hormone is employed to enhance cell growth and/or polysaccharide production. Plant hormones include, for example, the auxins such as 2,4- dichlorophenoxyacetic acid (2,4-D), naphthoxyacetic acid (NOA) and 2,4- dichlorophenoxybutyric acid (2,4-DB) or mixtures thereof. The use of given hormone may provide improved callus or cell growth for a given plant cell. For example, NOA gave improved results over 2,4-D for callus and cell cultures of Medicago sativa. Plant cell lines can be adapted using methods well-known in the art to exhibit good growth on lower levels (or no) of plant hormones. Using such adaptation methods, callus and suspension cultures of Pyrus and Mesembryanthemum that grow well on media containing no 2,4-D have been obtained. The use of lower hormone levels in suspension culture can decrease gum manufacture cost and alleviate the potentially detrimental effects of hormones on plant cells.

The methods described herein for generation of callus and the initiation and maintenance of suspension cell cultures are generally applicable or readily adaptable, employing art-known media, methods and expedients, to cells of any vascular plant including those listed in Table 1A and IB. The specific culture conditions for Nicotiana. plumb aginifolia, Pyrus communis and Phelum pratense are exemplified herein.

A variety of growth media suitable for the various plant cells of this invention are known in the art. A variety of carbon sources can be readily employed (or cells can be readily adapted for growth on a given carbon source) including sucrose, glucose, fructose and lactose. More complex carbon sources can be employed, e.g. double enzyme hydrolyzed glucose syrup or brewers' liquid maltose (BLM). Carbon source may affect the rheological properties of cultured plant cell gums. It has been observed that employing BLM as a carbon source increases cell growth and gum yield for certain types of cells. Growth of Nicotiana on BLM result in production of a plant cell gum having excellent film-forming properties. It has also been observed that an increase in osmotic pressure or in sucrose concentration in the medium can increase gum production by some cultured plant cells.

Addition of other additives to growth media may affect the viscosity or other properties of plant cell gums. For example, growth of a Nicotiana suspension on 5% glycerol, believed to function as an osmodicant in the medium, resulted in cultures having significantly higher viscosity.

The gum product can be recovered from the culture medium by methods well known in the art. See Johns, M. & Noor, E. (1991) Aust. J. Biotechnol. 5(2):73-77; Golueke, C. et al. (1965) U.S. Patent No. 3,195,271; Seviour, R. & Kristiansen, B. (1983)

Eur. J. Appl. Microbiol. Biotechnol. 17: 178-181; Mort, A. et al. (1991) Carbohydrate Res. 215:219-237; and Wu, M. & Wallner, S. (1983) Plant Physiol. 72:817-820. A specific recovery and purification method is exemplified herein. A "complexant" is a composition

10 or compound that sequesters calcium or other divalent metal ions from the gum product during the recovery procedure. For example, Na₂ -EDTA added during the recovery process chelates calcium. Other sequestering agents such as citrate, cyclohexane diamine tetraacetate (CDTA), imidazole, sodium hexametaphosphate may also be used. Sequestering of calcium is desirable to avoid the formation of insoluble complexes during drying of the recovered gum. Preservatives and antioxidants can be added to the culture filtrate to minimize gum degradation.

As noted above, plant cell gums are complex mixtures including components having a range of molecular weights. Molecular weight fractionation methods can be applied during plant cell gum isolation or to isolated plant cell gum to obtain gum fractions of different molecular weight range. These fractions can have distinct rheological properties, e.g., lower molecular weight fraction may exhibit generally lower viscosity.

In one embodiment this invention relates to cultured plant cell gums of plants of the family Aizoaceae including plants of the genera Mesembryanthemum, Aptenia and

Carpobrotus. These genera are closely related and species once classified in one of these genera may currently be classified in another of these genera, for example Mesembryanthemum chilense has been reclassified as Carpobrotus chilense. This invention includes plant cell gums of all plants classified into these genera, including varieties of each and plants that may result from a cross between two species of the genera. More specifically this invention includes cultured plant cell gums ( as well as uses thereof) of plants designated as Mesembryanthemum spp., including M. crystallinum, M. edulis, M. nodiflorum, M. barklyi, M. criniflorum, M. forsskalei Hochst, M. cordifolium and other species listed in Table IB, Carpobrotus spp., including Carpobrotus chilense, Carpobrotus acinaciformis, Carpobrotus edulis, Carpobrotus aequilaterus,

Carpobrotus modestus, Carpobrotus muirii and other species listed in Table IB, Apentia cordifolia, Aptenia cordii, varieties of each and plants resulting from crosses between two species. See: H. Jacobsen (1974) Lexicon of Succulent Plants Blandford Press Ltd. (London) and H. Jacobsen (1960) Handbook of Succulent Plants Blandford Press Ltd. (London) for descriptions of Aizoaceae including additional species of

Mesembryanthemum, Aptenia and Carpobrotus.

11 The skilled practitioner, using information available in the art and the teachings of the subject application, can identify cultured plant cell gums that are useful as thickening, gelling, emulsifying, dispersing, suspending, stabilizing, encapsulating, flocculating, film forming, sizing, adhesive, binding and coating agents, and as lubricants, water retention agents and coagulants, etc. in the aforementioned industries. The suitability of using a cultured plant cell gum for a particular application can be assessed by methods known to those of skill in the art.

The cultured plant cell gums can be used to establish and stabilize solid, liquid and gaseous dispersions. An emulsion is an intimate mixture of two immiscible liquids in which one phase is dispersed throughout the other as small, discrete droplets (Sandford, P.

& Baird, J., "Industrial Utilization of Polysaccharides" in The Polysaccharides (1983), Academic Press, Inc., Vol 2, pp. 411-491). The cultured plant cell gums can be used as emulsifying agents or stabilizing agents in emulsions. Suspensions are solid particles dispersed uniformly throughout a liquid phase (a suspension) mainly by increasing the viscosity of the suspension liquid phase with suspending agent. Foams are gas dispersed in a liquid or solid phase. When cultured plant cell gums are employed as foam stabilizers, they affect the surface properties (e.g., interfacial tension) of foams, thereby promoting a firm, stable foam.

Emulsification capacity can be assessed by, for example, measuring the reduction in aqueous surface tension or interfacial tension due to the gum product, measuring the critical micellar concentration (CMC), or measuring the hydrophile-lipophile balance (HLB; the ratio of polar to nonpolar portions of the composition). Additional methods of assessing emulsifying capacity include particle sizing and counting, and effect on viscosity and electrical properties of the emulsion due to the gum product. For a discussion of such methods, see Zajic, J. & Panchal, C. in CRC Critical Review in Microbiology (1976), pp.

39-66. The choice of a particular gum product for a desired application depends on additional factors such as solubility and compatibility with other chemicals in the emulsion mixture, and pH, ionic strength and temperature of the emulsion mixture. The specific method employed to measure the emulsification capacity for at least some of the gum

12 products described herein involves measurement of turbidity and droplet size and is described in the Examples.

Emulsion stabilizing capacity is the ability of a gum to maintain an emulsion over time. Emulsion stability can be tested by evaluating the turbidity of the emulsion (or industrial emulsion mixture) over time.

Thickening agents increase the viscosity of aqueous solutions or suspensions. They increase the resistance to flow of a liquid. Sandford, P. A. & Baird, J., supra. Viscosity imparted by cultured plant cell gums to mixtures or solutions can be measured with commercially available viscometers. Such viscometers commonly employ methods based on Stake's law, the capillary tube method, the rotating cylinder method or the oscillating disk method. The specific method employed to measure the viscosity of at least some of the gum products described herein is described in the Examples.

Assessment of gelling capacity of a gum product can be carried out by methods known in the art. The specific method employed to measure gelling capacity of at least some of the gum products described herein is set forth in the Examples. Gelling capacity can be assessed by measuring the rupture strength, shear modulus, back extrusion and melting and setting points of the gum product.

Lubricating capacity can be assessed by methods known in the art. For example, an adaptation of ASTM (American Society for Testing Materials) Method D4172 may be used.

Encapsulating capacity can be assessed by methods known in the art. A specific method is described in Example 3 of the U.S. Patent Application for "Plant Gum Material and Use Thereof in Food Products," filed on even date herewith.

It is desirable in certain emulsification applications, particularly in the manufacture of compositions of agricultural chemicals for spray applications, to inhibit satellite droplet formation and thereby to minimize undesired dispersion of potentially hazardous

13 materials. The ability of a plant cell gum to provide such inhibition can be tested by assessing splash inhibition in solutions/compositions containing the gum. Splashing behavior of droplets can be assessed by video monitoring of drop impact as is known and understood in the art.

In some cases, particular functional properties have been associated with particular gum components. It has been observed that AGP in the cultured plant cell gum product can enhance emulsification properties. For example, Pyrus communis and Nicotiana plumbaginifolia have higher levels (6-11 % (w/w)) AGPs, while Phleum pratense produces a gum with nondetectable AGP and poor gelling and emulsification capacity. Phleum pratense has comparable viscosity to Pyrus and Nicotiana gums without the gelling and emulsification properties. Phleum pratense is thus useful as a viscosity enhancer in applications where emulsification is not desired, e.g., in applications where guar gum and hydroxymethylcellulose have traditionally been used.

Those embodiments of the subject invention which use the gum products as emulsifiers preferably employ a gum product relatively rich in AGPs. In particular, cultured plant cell gums containing at least about 4% (w/w) AGP in the gum can be useful. Complex carbohydrates in the culture fluid can be determined by the method of Dubois et al. (1956) Anal. Chem. 28:350-356. AGP can be determined by the method of Van Hoist, G. & Clarke, A. (1985) Anal. Biochem. 148:446-450. AGP-containing gums have been found in higher plants (14 orders of angiosperms, 3 orders of gymnosperms), and in lower plants (e.g., Fontinalis anti-pyretica). Fincher, G. et al., supra.

It has been found that a gum product recovered from Pyrus communis cells suspension cultured in MS medium plus 2,4-D has complex carbohydrates at about 5.26 mg/ml of culture fluid as determined by the method of Dubois et al. (1956) Anal. Chem. 21:350-356; and 8.9% (w/w) AGP as determined by the method of Van Hoist et al. (1985)

Anal. Biochem. 148:446-450.

The cultured (MS medium) gum product of Lolium multiflorum and Nicotiana plumbaginifolia have been found to have an AGP % (w/w) of 11.0 and 4.5, respectively.

14

SUBSTTTUTE SHEET (RULE 26) In contrast, cultured cells (MS medium) of Phleum pratense have been found to have no detectable AGP (detection limit is about 0.25 μg by the method of Van Hoist et al. (1985)).

A description of particular applications in which the cultured plant cell gums can be employed follows. This discussion is not intended to be limiting.

Applications of cultured plant cell gums for food applications have been described in WO 8806627 and US patents 5,133,979 and 5,296,245. For example, plant cell gums of this invention are useful in the preparation of oil-free salad dressing and as whipping bases for mousses, desserts, yoghurts and fruit-based foams, as an encapsulating agent for flavors (e.g., flavors [garlic, onion, herbs, "smoky" flavors and "cheese" flavors] and flavor oils [orange and lemon oils]), film-forming agents for enrobing or coating foods, and thickening agents, for example for, sauces, toppings, spreads, fillings, dips, custards and gravies.

Gellan is currently employed as a film forming ingredient in bread crumb mixes and batters for coating various food items (e.g. meats, cheese, fish) or dough-enrobed foods (egg rolls) for frying to increase crispness and decrease oil absorption or to give items that are heated by microwave a "deep-fried" appearance. Cultured plant cell gums of this invention can be employed in such applications as a substitute for gellan. Film made from plant cell gums can exhibit excellent resistance to the application of dry heat (up to about 100°C) making them useful in various cooking applications.

Certain plant cell gums (e.g., that of Pyrus) can exhibit significant temperature stability such that gels prepared with the gum do not melt or soften up to temperatures of about 80°C. These gums are useful in the preparation of non-melting sauces, e.g., barbecue, cheese or butter sauces and other sauces for cooking,

Cultures plant cell gums can exhibit rapid cold gelling in cold water media. This property is useful in the production of instant dehydrated sauces, toppings, spreads, fillings, dips, custards, gravies and the like.

15 Gelatine is used extensively for preparing capsules for human and veterinary application, including drug, vitamin and food supplement applications. Gelatine is also used for the production of oil-filled capsules for nutrient oils including evening primrose oil, cod liver oil, vitamin E and the like. Films prepared from certain plant cell gums, including gums of Pyrus, are oil-resistant exhibiting little or no deterioration on exposure to oil. Cultured plant cell gums can be employed to prepare films that can be substituted for gelatine in capsule applications, particularly for use in oil-filled capsules. Oil-resistant gum film can also be employed to enrobe oily foods, e.g., gum films can be employed to enrobe nuts for subsequent coating with chocolate.

Plant cell gums of this invention can be employed as dietary fiber supplements.

Plant cell gums are useful in a variety of veterinary applications for preparation of food supplements and vitamins and in the preparation of slow release pellets that can be used to deliver vitamins or drugs (anthelminthic). Plant cell gums, particularly those that exhibit heat-stable gel formation, can be employed as gelling agents in the manufacture of canned pet food.

In the paper industry, prior art gums have been used in wet end beater aids, surface sizes (e.g., size press and calender), pigmented coatings (e.g., blade, roll airknife, and size press coatings), and in adhesives. Sandford, P. & Baird, J., supra. Cultured plant cell gums can be used as substitutes for such prior art gums as locust bean gum, karaya and guar gums as hydrophilic colloids employed in the wet end as beater aids to reduce flocculation of pulp suspensions and improve paper formation. The cultured plant cell gums can also replace prior art gums as a surface size which is typically applied after the formation of the sheet at calender rolls or at the size press. Sandford, P. & Baird, J., supra. As surface sizes, cultured plant cell gums can impart water resistance, oil and solvent resistance, glue holdout, scuff resistance, physical strength, curl control and gloss.

The cultured plant cell gums can also replace such prior art polysaccharides as sodium alginate, which is used as a thickener and dispersant in the pigment coating. The purpose of such an additive is to prevent agglomeration, and to produce adequate flow and leveling

16 of the coating, and to prevent pattern or orange peel in the coating. Sandford, P. & Baird, J., supra.

As exemplified herein, addition of the cultured plant cell gums as a beater aid at the wet end has been observed to result in superior tensile and burst strength, improved resistance to erasure, reduced lint on the paper surface and reduced rate of water penetration as compared paper manufactured without a beater aid. Without wishing to be bound by theory, it is believed that at least some of these improvements are due to a more uniform distribution of pulp fines.

In the adhesives industry, some prior art gums, waxes, tars, and natural resins have functioned as adhesives when dissolved or dispersed in water or organic solvents, applied between substrates and the solution/dispersal allowed to undergo solvent evaporation. Cultured plant cell gums have been found suitable for use in a water re-moistenable adhesive for paper or aluminum foil sheets. The cultured plant cell gum increases viscosity, thereby moderating the flow during application, and the finished film thickness and water retention. The gum product may also serve as a surface attaching agent. The cultured plant cell gum adhesive, when dried on the surface of paper or aluminum sheets, has good affinity for water and does not cause discoloration of the paper or become brittle on aging. The concentration range in the liquid adhesive concentration is between about 1.0 and 3.0% (w/v). The cultured plant cell gums can be used as an adhesive or cement in other applications.

Prior art gums have also been employed in oil and gas field applications including drilling, well completion (cementing and stimulation) and enhanced oil recovery. As used herein, "oil and gas well fluids" refers to all oil and gas well development or production fluids, including without limitation drilling fluids, cementing fluids, and enhanced oil recovery injection fluids. Drilling fluids or muds function to transport drill cuttings to the surface, control formation pressures, maintain bore hole stability, protect productive formations and cool and lubricate the bit and drill string. Prior art gums have been used to impart greater viscosity to the drilling fluid, to act as suspending agents for cuttings and weighting materials, and to reduce loss of water or fluid by preventing penetration into the

17 rock formation. The rheological requirements of the drilling fluid are that it have low viscosity at high shear rates (i.e., at the drill bit), but high pseudoplasticity to suspend solids in laminar flow. When mud circulation stops, the gel strength is preferably sufficient to suspend solids. Sandford, P. & Baird, J., supra; and Kirk-Othmer Chemical Engineering Encyclopedia (3rd. ed. 1981) 17:143-166. These rheology requirements have previously been addressed with combinations of bentonite, cellulose ethers, polyacrylamides and xanthan gum. Drilling mud additives for reduction of fluid loss have included carboxymethylcellulose, polyacrylates and xanthan gum. During well cementing, a cement lining is installed to isolate the productive zone from the remainder of formations. Fluid loss additives are also used during this stage to prevent cement dehydration and minimize water loss to the formation. Sandford, P. & Baird, J., supra. Following drilling and cementing, a completion may be used to remove undesirable formation particles and debris and prevent permeability damage to the producing zone. Completion fluids contain salts for density, and viscosifiers such as xanthan gum to provide suspension for the removal of debris. During well stimulation, hydraulic fracturing and/or acidizing fluids can be used to enhance hydrocarbon productivity. Hydraulic fracturing fluids require suspending agents such as guar or xanthan gums to carry propping solids. Acidizing fluids require a gelling agent effective in high acid concentrations (e.g., 15% HC1). In enhanced oil recovery, the injection fluids contain polymers to increase viscosity, resulting in better oil displacement. Xanthan gum has been a common component in enhanced oil recovery polymer flooding.

It has now been found that cultured plant cell gums can be employed in drilling fluids to increase viscosity, and as emulsifying, suspending, lubricating agents and fluid loss reduction agents. As an emulsifying agent in a drilling mud, the cultured plant cell gums can emulsify and stabilize oil-in-water or water-in-oil mixtures. As a suspending agent, the cultured plant cell gums disperse and suspend cuttings and weighting materials so as to provide a protective colloid for well equipment. As a lubricating agent, cultured plant cell gums can reduce frictional resistance between the drill string and the formation or casing or during string raising and lowering. The strong water affinity of the gum products can prevent water filtration into surrounding strata during drilling or cementing phases. The gum products can also be used as viscosifiers in completion fluids. In hydraulic fracturing fluids, the cultured plant cell gums can be used to impart viscosity, suspend propping solids and as gelling agents. In enhanced oil recovery, cultured plant cell gums can be used to increase viscosity of the injection fluid. The concentration of cultured plant cell gum in the drilling mud, completion, fracturing and enhanced oil recovery injection fluid is between about 0.1 and 3.0% (w/v). For P. communis gum, a soft gel begins to form at about 0.5% (w/v).

For each of the foregoing oil drilling applications, the whole fermentation mixture may be used, i.e., without removal of cells. This alternative has the advantage of simplifying the manufacture of oil and gas well fluids. The biodegradability and non- pathogenic nature of the cells makes such alternative possible.

An additional advantage of using cultured plant cell gums in oil and gas field fluids is that they have much less environmental impact than those using palm oil. This is particularly the case for drilling muds prepared for off-shore drilling where it is desirable that leakages from the well be easily dissipated. Aqueous-based drilling muds dissipate more effectively than oil- based muds.

In ink formulations, thickening, suspending and/or emulsifying agents are used to provide the proper viscosity for application and to increase the stability of the ink. Lithographic, letterpress and screen printing inks have higher viscosities and frequently contain thickeners. Flexographic (flexo) and rotogravure (gravure) printing inks have lower viscosities, but use emulsifying or suspending agents for uniform distribution of the pigment and to prevent the ink from separating. Flexographic inks can be alcohol or water based emulsions. Rotogravure inks also contain an emulsion and have the advantages of excellent press stability, printing qualities, the absence of fire hazard and the convenience and economy of water for reduction and cleanup. The ink distribution systems of flexo and gravure printing presses are simple and do not provide the means to distribute and level highly viscous inks; therefore, viscosity is typically 5-100 cP. Letterpress and litho inks can vary in viscosity from under 500cP for a letterpress-type news ink to over 500 P for special litho ink formulations. In lithography and letter press, uniform and adequate transfer of ink to the printing plate is ensured by a multitude of

19 rollers in the ink distribution unit. Rheology of the litho and letterpress inks is therefore important to roller-to-plate transfer, fidelity in printing, drying speed, holdout, and trapping properties obtained on the substrate. In general, higher press speeds require lower viscosity inks and slower press speeds employ more viscous inks. Low viscosity ink is used in fine-line flexography and shallow-cell gravure printing. Printing smooth, dense solids can best be achieved using higher viscosity ink. Rheology is also important as a color strength determinant. Over-pigmentation leads to a more thixotropic ink, thereby creating a balancing relationship between color intensity and rheology. Kirk-Othmer Chemical Engineering Encyclopedia, supra. Vol. 13, pp.374-376. Lasday, S. (ed.) Handbook for Graphic Communications: (1972) Ink, Paper, Binding, Vol 6., pp. 6-13.

It has been found that cultured plant cell gums can be used as emulsifying, suspending and/or thickening agents in a variety of printing inks, including litho, letterpress, screen printing, flexographic and gravure inks. The gum concentration in flexo inks can be between about 0.5 and 4.0% (w/v).

Additionally, in offset lithography, prior art gums have been used as emulsifying agents and viscosifiers in lithography solutions. Offset lithography is a planographic process where the image and non-image are in the same plane. The image area is oil receptive and the non-image area is water receptive so that following wetting of the plate with the fountain solution, the ink, when rolled across the plate will only be attracted to the oil receptive areas. As used herein, "lithography solution" refers to any non-ink solution used in lithography, including fountain solutions, sensitizing solutions and protecting solutions. The fountain solution is a desensitizing solution which prevents ink from adhering to the plate. Fountain solutions have contained gum arabic, typically at 0.2% (w/w). Lasday, S., supra. Vol. 6, pp. 93-95. The desensitizing use of gum arabic has taken advantage of the good wettability imparted to the fountain solution and also of the viscosity control that allows the wash solution to cling to the plate without running off or forming isolated droplets or pools on the plate. On metal plates, the desensitizing effect might be caused by the formation of an insoluble film of EG Aluminum or Zinc Arabate. A more plausible explanation is that the film of gum is absorbed by the plate. Studies have shown that such films occur on plates of zinc, aluminum, copper, silver, iron

20 tin, lead, glass and fused silica. These films are not mono-molecular but are composed of many molecular layers. LSC Printing Inks, Reinhold Publishing Corporation, New York (1940) pp. 230, 334, 346, 398-9 and 417. Measurement of the wettability of the desensitizing solutions can be evaluated by measurement and study of the contact angles. In this process a section of the plate is partially immersed in water or in a solution of the gum to be tested. The plate is then turned at an angle to the surface of the liquid until the meniscus appears to be eliminated. The resulting angle of the plate to the surface of the liquid is known as the contact angle and is the measure of the wettability of that particular plate with the solution being tested. Read REF Modern Lithography (1951) 47:62.

Cultured plant cell gums can be used as emulsifying agents in sensitizing or fountain solutions for the plates during operation and in protecting solutions during storage. The concentration range of the gum in fountain solutions is between about 0.01 and 2.0% (w/w).

In the textile industry, gums have been used as sizing and thickening agents. Sizing agents act during textile manufacture by binding the loose fibers of the warp, thereby imparting strength, flexibility and smoothness to the warp, allowing weaving to proceed efficiently. Thickeners control the viscosity of various formulations used in the textile industry including dyes, printing inks, coating and flocking solutions. Prior art gums, including guar, algin and xanthan gums have been used in printing and dyeing solutions. Sandford, P. & Baird, J., supra. Cultured plant cell gums can be useful as sizing or thickening agents in the textile industry. As exemplified herein, the gum product can function as a thickening agent for dyestuff used in wool and cotton fabric printing. The concentration range of the gum product in the dyestuff is between about 0.1 and 5.0% (w/v). Modified approaches can be used in the reactive dyestuff process and direct vat dyestuffs for silk and hydrophobic man-made fibers (nylon, acrylics, polyester and acetates).

In the paint industry, viscosifiers, thickeners, emulsifying agents, suspending agents, and dispersants are used to improve flow properties of the paint so that a smooth coat of desired thickness can be applied to a vertical surface without sagging, and to

21 stabilize the paint by preventing coagulation and pigment settling. Thixotropic character of the paint is important in providing good levelling, prevention of running, and avoidance of segregation or stratification of the paint during storage. Sandford, P. & Baird, J., supra: and Gamble, D. & Grady, D., U.S. Patent No. 2,135,936 (1938). As exemplified herein, cultured plant cell gums can be used as emulsifying agents in an acrylic resin paint or an oil emulsion paint. The concentration range of the gum product in acrylic or oil based paint is between about 0.2 and 0.3% (w/v).

In ceramics manufacturing, a glaze or a colored, opaque or transparent coating is applied to the ceramics before firing. The glaze forms a hard, nonporous surface. Glazes are usually made from powdered glass combined with colored oxides of such elements as cobalt, chrome, manganese or nickel. The mixture of powders is suspended in water and applied to the ceramic surface by spraying brushing or dipping. The glaze is then dried and fixed onto the ceramic surface by firing. Emulsifying agents, suspending agents or dispersants can be used to uniformly distribute the pigments in the glaze. The glaze causes the pigment to adhere to the surface during firing. As exemplified herein, cultured plant cell gums can be used as an emulsifying and suspending agent to produce a glaze of superior consistency, clarity and stability. Further, it has been found that if BLM (Brewers Liquid Maltose) is used as a carbon source during culturing of N. plumbaginifolia, the recovered gum product imparts excellent film-forming properties to the glaze. The gum product concentration range in the liquid glaze is between about 0.05 to 3.0% (w/v).

Cultured plant cell gums can also be useful in ceramics forming by plastic extrusion. Completely nonplastic materials can be extruded with the addition of suitable plasticizers such as gums, starches, polyvinylalcohol, waxes and wax emulsions. Grayson, M. (ed.) Kirk-Othmer Concise Encyclopedia of Chemical Technology (1985) p. 237.

Cultured plant cell gums can replace prior art gums in such processes. In ceramics forming by slip casting, cultured plant cell gums can be used in the suspension of raw materials to ensure uniform dispersion of the clay and other solid particles in the water.

22 In cleaning detersive systems, absorption of bath components to the substrate surface may be the most important and fundamental detergency effect. Adsorption is the mechanism whereby the interfacial free energy values between the bath and the solid components (substrate and soil thereon) of the system are lowered, thereby increasing the tendency of the bath to separate the solid components from one another. Surfactant adsorption reduces soil-substrate interactions and facilitates soil removal. Kirk-Othmer Chemical Engineering Encyclopedia, supra. Vol. 22, p. 408.

In cleaning detergent manufacturing, the addition of materials to increase viscosity and film-forming properties can enhance surfactant and substrate surface interactions, particularly for vertical surfaces. As exemplified herein, cultured plant cell gums have been found to be useful in improving the viscosity and film-forming properties of detergents. In particular, it has been found that use of BLM as a carbon source in the culturing of N. plumbaginifolia produces a gum product that can impart improved film- forming properties to the cleaning detergent. This is particularly useful for cleaning detergents used to clean vertical surfaces. Detergents can also contain soil antiredeposition or suspending agents, such as carboxymethylcellulose, polyvinylalcohol and polyvinylpyrollidone. These antiredeposition agents are believed to function by absorbing onto either the substrate or the soil particle, and imparting electrical charges that reduce the affinity between the soil and substrate. Sandford, P. & Baird, J., supra. It is believed that cultured plant cell gums can also function as an antiredeposition agent by coating the substrate and/or soil particles. The gum product concentration range in cleaning detergents is between about 1 and 10% (w/v).

Plant cell gums can function as encapsulating agents. As such , the plant cell gum can be used for encapsulation of lemon oil (or other suitable oils) in detergent powders and other dry or powdered cleaning agents.

Plant cell gums have been found useful in the manufacture of chemical compositions with decreased tendency to form fine mists or satellite droplets. Of particular interest are applications in agriculture to prevent unwanted dispersion of

23 potentially harmful agricultural chemicals (insecticides, fungicides, etc.) on spray application.

Cosmetic lotions and creams are water-in-oil or oil-in-water emulsions employing emulsifying and stabilizing agents. Emulsifiers, being surface active agents, lower surface and interfacial tensions and increase the tendency of the lotion or cream to spread. A purified acidic heteropolysaccharides obtained from cultured Polianthus has been used in cosmetic creams, lotions, shampoos and cleansing foams. Otsuji, K. et al. EP 0 285 829, published October 12, 1988. As exemplified herein, cultured plant cell gums can be used without prior purification of gum fractions in cosmetic lotions and creams. The gum product concentration range in the cosmetic lotions and creams is between about 0.5 and

4.0%> (w/w). Plant cell gums have been found useful for the formation of soft gels which spread well on the skin and feel smooth and supple to the touch. Humectant and perfume can optionally be added to these soft gels to provide moisturizing cosmetic gels. Plant cell gums have also be found useful in the preparation of deodorants, hair styling gels, and shampoos and conditioners.

Other applications for cultured plant cell gums include thickeners, emulsifiers or suspending agents for photographic preparations; thickeners for explosives; thickeners and suspending agents for foundry wash coats; thickeners, foam stabilizers and film formers for fire-fighting fluids; emulsifiers and suspending agents for flowable pesticides, suspension fertilizers and animal liquid feed supplements.

The advantages of the cultured plant cell gums over prior art gums include lower production costs, improved purity and improved production reliability. Because the production of cultured plant cell gums does not rely on labor-intensive harvesting of gum exudate from trees (e.g., as is required for gum arabic) or harvesting of seeds or plants for extraction (e.g., guar gum, agar algin, or carrageenan), and can instead be produced under automated conditions, labor costs associated with the production of cultured plant cell gums can be lower. As discussed hereinabove, in agar production, the harvesting of red seaweed is labor intensive in that it is carried out by hand; in some areas of the world, divers in full pressure suits collect individual plants in deep water; in other places, the

24 seaweed can be collected at low tide without the use of diving equipment. Carrageenan or Irish Moss is produced from another red seaweed harvested by raking and hand gathering. Algin is produced from brown algae which can be harvested manually or with small mechanical harvesters. Sandford, P. & Baird, J. (1983) in The Polysaccharides, Academic Press, Inc. Vol. 2, pp. 41 1-491. Additionally, since production of cultured plant cell gums is carried out in fermentation facilities, production does not rely on weather and is therefore more reliable than prior art gum production. See, for example, Meer et al. (1975) Food Technology 29:22-30. Further, because cultured plant cell gums are produced in fermentation facilities, they can be purer than prior art gums. As discussed hereinabove, because gum arabic is hand collected, it is seldom pure; samples are classified according to grade which depends on color, contamination with foreign bodies such as wood or bark (VanNostrand's Scientific Encyclopedia, supra at p. 1389).

An advantage of cultured plant cell gums over xanthan gum produced by cultured Xanthamonas campestris is that the cultured plant cells do not pose the same cell disposal problem presented by X. campestris, a plant pathogen (Scaad, N.W. (1982) Plant Disease

66(10):882-890). Further, cultured plant cell gums are less expensive than xanthan gum for a variety of applications, including drilling fluids (e.g., Kirk-Othmer Chemical Engineering Encyclopedia (3rd. ed. 1981) 17: 153).

A further advantage of the subject gum product is that it can often be used in smaller quantities than prior art gums to achieve comparable effectiveness as an emulsifying, stabilization, suspending, thickening, or gelling agent, as a film forming or coating agent, or as a protective colloid.

All references cited are incorporated herein by reference in their entirety.

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

25 EXAMPLES

Example 1 : Establishing suspension cultures A. - Phleum pratense

Seeds var. Kahu from Hodder & Tolley, seed merchants, 17 Binney Rd. Marayong, Australia, were sterilized by rinsing in ethanol and then soaking 5 minutes in hypochlorite

("chlorize" 1:4). The seeds were then rinsed three times with water and transferred to either liquid or solid medium of Hale et al., supra, containing 2 mg/1 2,4-D.

Suspension cultures were initiated from seeds germinating on either liquid culture or callus culture. In the liquid culture, most seeds germinated after five days. The seed and liquid were chopped in a small sterile blender and then returned to an Erlenmeyer flask and shaken for a further two weeks. The resulting culture was propagated by regular subculturing every 2-3 weeks into suspension culture.

The seeds germinating on agar medium began to form callus immediately. The small calli were dissected off and transferred to fresh agar medium. The calli were subcultured every 3-4 weeks. Initially, the calli are mucoid, but after a number of subcultures, they lose their mucoid appearance. Suspension cultures initiated from mucoid calli produced 2-5 g/1 of polysaccharide. Suspension cultures initiated from calli that lost their mucoid appearance and no longer produced polysaccharide.

The suspension medium and procedure were those employed in Hale, A. et al., supra.

Within three days of initiation into the suspension medium, culture filtrates are extremely viscous (i.e., filtrate runs from a 5 ml bulb pipette in about 70-80 seconds, as compared to 15 seconds for water and 16-20 seconds for Pyrus cell culture filtrate). Also, there is very little growth of cells, so the filtrate volume on harvesting is virtually the same as the culture volume (i.e., the packed cell volume is negligible). While polysaccharide production is lost from callus and suspension cultures on repeated subculture, this does not create a problem as it is easy to initiate a new cell line.

26 l .B. - N. plumbaginifolia

Callus was initiated from seeds cultured on 20-30 ml CSV (Gibson et al. (1976) Planta 128:233-239; and Schenk, R. & Hildebrandt, A. (1972) Can. J. Bot. 50:199-204)) medium (below) solidified with 0.5% (w/w) agar. The callus was maintained on the same solid medium, in the dark at 27°C. Maintenance subculturing occurred approximately every 3 weeks. If drying or discoloration of the culture was observed, it was immediately subcultured.

All stock solutions were made up with Milli-Q™ water in glass bottles. CS Macro salts

NH₄NO₃ 24.8 g

KNO₃ 50.1 g

(NH₄)H₂P0₄ 9.2 g

CaCl₂-2H₂O 4.0 g

MgSO₄-7H₂O 8.0 g The solution was made up to 1 liter with Milli-Q™ water and stored at 1°C in glass bottles.

CS organics

Thiamine-HC 1 100 mg

Nicotinic acid 1000 mg

Pyridoxine-HC 1 100 mg

The solution was made up to 200 ml with Milli-Q™ water and stored at -20°C in glass bottles.

CS micro salts

MnSO₄-4H₂O 6.5 g

H₂BO₃ 2.5 g

ZnSO₄-7H₂O 0.5 g

KI 0.5 g

CuSO₄-5H₂O 100 mg

NaMoO₄-2H₂O 50 mg

27 CoCl₂-6H₂O 50 mg

The solution was made up to 500 ml with Milli-Q™ water and stored at -20°C in glass bottles.

CS Iron solution Na₂EDTA-2H₂O 2.0 g

FeSo₄-7H₂O 1.5 g

The EDTA was dissolved in 60 ml Milli-Q water, while stirring and heating. It was then cooled to room temperature and the FeSO₄-7H₂O was slowly added while also adding NaOH (10 M = 400 g/liter) to keep pH at 5.9. The solution was made up to 100 ml with water and stored at -20°C in glass bottles.

To prepare one liter of CSV medium, the stock solutions and solids were mixed in the following quantities in approximately 800 ml of Milli-Q™ water:

CS Macro 50 ml

CS Micro 1 ml

CS Iron 1 ml

CS Organics 1 ml

Sucrose 30 g

myo Inositol 1 g

The pH was adjusted to 5.8 (20-30 drops of IM KOH). This medium can be modified in various ways without adverse effect, e.g., inositol can be reduced or deleted. The hormone stocks were added in the following quantities: 2.0 ml 2,4-D (stock 1.0 mg/ml) 0.5 ml of kinetin (stock 0.1 mg/ml). The solution was then made up to 1 liter with Milli-Q™ water and sterilized for 20 minutes at 10 psi (116°C).

Suspension cultures were passaged into fresh CSV medium at 7-day intervals using a 10% inoculum (i.e., 2 ml into 20 ml, 20 ml into 200 ml). Suspension cultures were maintained at a 27°C at a shaker speed of 100 rpm. The cultures were monitored visually

28 for departures from normal color and cell growth patterns. Cultures were also monitored for sterility (i.e., contaminating organisms) and healthy cell morphology (e.g., cell stress).

l.C. - Pyrus communis(Green Pear)

Callus was initiated from fruit cultured on 20-30 ml pear BAL (balanced) medium (below) solidified with 0.5% (w/w) agar. The callus was maintained on the same solid medium, in the dark at 27°C. Maintenance subculturing occurred approximately every 4 weeks. If drying or discoloration of the culture was observed, it was immediately subcultured.

All stock solutions for the pear BAL media were made up using Milli-Q™ water in glass bottles. Vitamins and hormone solutions were stored at -20°C; all other solutions were stored at 1°C.

Macro elements

NH₄NO₃ 165 g

KNO₃ 190 g

MgSO₄-7H₂O 37 g

The Macro solution was up to 1 liter with water.

Micro elements

H₃BO₃ 1 g

ZnSO4-7H₂O i g

MnSO₄ H₂O 1.44 g

NaMoO₄-2H₂O 0.029 g

CuSO₄-5H₂O 0.0025 g (*) CoCl₂-6H₂O 0.0025 g (*)

The Micro solution was made up to 100 ml with water. (*) To obtain 2.5 mg of these salts, 25 mg of each was weighed out in separate containers, and dissolved in 10 ml Milli-Q™; 1 ml of each solution was then used.

Vitamins

Ca pantothenate 0.1 g

29 myo-Inositol 10.0 g

Biotin 0.001 g (*)

Nicotinic acid 0.001 g (*)

Thiamine-HC 1 0.1 g

Pyridoxine-HC 1 0.05 g

The vitamin solution was made up to 100 ml with water.

(*) A stock solution containing 1 mg of Biotin + 1 mg of Nicotinic acid per 10 ml was prepared as follows: 10 mg of both vitamins was dissolved in 100 ml of Milli-Q; 10 ml of this solution was used to make up 100 ml of Stock Vitamins.

KH₂PO_₄ (potassium dihydrogen orthophosphate)

KH₂PO₄ 17 g

The solution was made up to 1 liter with water.

CaCl₂ 2H₂O (calcium chloride dihydrate) CaCl₂-2H₂O 6 g The solution was made up to 100 ml with water.

Fe-EDTA

FeSO₄-7H₂O 6.86 g

Na₂ EDTA-2H₂O 9.17 g

The EDTA was dissolved in 1 liter of Milli-Q™ (magnetic stirrer, room temperature). The ferrous sulphate was dissolved in the EDTA solution. The resulting solution was brought to a boil, cooled and stored in screw capped glass bottle at 1°C.

KI (potassium iodide) KI 0.03 g The KI was dissolved in 20 ml Milli-Q.

2.4-D (2,4-dichlorophenoxyacetic acid) 0.1 mg/ml 2,4-D 50 mg

30 The 2,4-D was dissolved in 5 ml of commercial grade ethyl alcohol (95%). The 2,4-D was injected slowly under the surface of 495 ml of Milli-Q™ water, using a Pasteur pipette and a magnetic stirrer.

To make up the pear BAL medium, the concentrated stock solutions and solids were mixed (magnetic stirrer) in the quantities indicated below and water added to approximate 900 ml.

Macro elements 10 ml

Micro elements 1 ml

Vitamins 1 ml KH₂PO₄ 10 ml

CaCl₂ 2.5 ml

Fe-EDTA 2.5 ml

KI 0.5 ml

2,4-D 10 ml L-Asparagine 180 mg

L-Ascorbic acid 50 mg

Thiourea 25 mg

Sucrose 40 grams

The pH was adjusted to 5.8 - 6.0 with KOH (0.1 or IM). The final volume was adjusted to 1 liter with water. For solid medium, 0.5% (5 g/liter) agar was added after adjusting pH and volume. The final medium was sterilized for 20 minutes at 10 psi (116°C).

Suspension cultures were passaged into fresh BAL medium at 14 day intervals using a 20% inoculum. The cultured were maintained at 27°C at a shaker speed of 100 rpm. Cultures were monitored for sterility, cell morphology, and departures from normal culture color and cell growth. After subculturing into fresh BAL medium, the packed cell volume (PCV) of the old culture is measured to assess whether the culture conditions are successfully maintaining the cell line in a stable growth pattern. If the PCV declined progressively over several subcultures, the cell line was revived with a single passage on double phosphate pear BAL medium.

31 1.D - Enhanced polysaccharide production using BLM

When BLM was used as a carbon source to enhance polysaccharide production by Nicotiana or Pyrus, it was typically used at a culture medium concentration of between about 80 to 200 g/liter of medium, or preferably at about 162 g (wet weight) per liter of medium.

Example 2: Recovery of Gum Product from Cultured N. plumbaginifolia

N. plumbaginifolia whole broth was harvested from a fermentor. The whole broth was filtered using a filter having a pore size of about 100 μm. The filtrate was then heated to 80°C for 30-60 minutes to denature enzymes in the filtrate. The filtrate was then cooled. Complexant (e.g., Na₂EDTA-2H₂0; 1 g/1) was added either prior to filtration, after filtration and prior to heating, or after cooling.

In some cases, the filtrate was stored prior to further processing. When storage time was longer than 18 hours, preservatives, e.g., 1.0 g/1 potassium sorbate and 0.34 g/1 sodium metabisulfate, were added. These preservatives allowed storage at ambient temperatures (15°-25°C) in sealed containers for prolonged periods.

The filtrate, warmed to 30-80°C to reduce viscosity, was next concentrated by ultrafiltration (10,000 MW membrane, Amicon Model DC10LA) to about 20-25% of its original volume or until viscosity made further significant concentration difficult. The concentrate was then diafiltered using the same membrane with five equal volumes of distilled H₂0, and concentrated again by ultrafiltration to the point at which viscosity or gelling inhibited further progress.

Where the gum product was intended to be used in industrial compositions such as in drilling mud, adhesives, cleaning detergents, dyestuffs, paper, acrylic resin and oil emulsion paints, or printing ink, the concentrate was directly spray dried (Niro Production Minor, Niro Atomizers, Denmark) using a 200°C inlet temperature and a 100°C outlet temperature.

32 Where the gum product was intended to be used in foods, pharmaceuticals, or cosmetics, the concentrate was further purified by an alcohol precipitation method comprising a precipitation and washing step. The concentrate was chilled to 1-4°C, and NaCl or KCl was added as a concentrated solution, followed by slow addition with stirring of 2-4 volumes cold (1-4°C) ethyl or isopropyl alcohol. The NaCl or KCl was added in an amount to give a concentration of 0.03-0.1% w/v in the alcohol-containing mixture. The mixture was allowed to stand at 1-4°C for 1-18 hours and then filtered using 2-4 layers of surgical gauze. The filtrate was washed in 67-80% alcohol at 1-4°C and the wash was removed by filtration using 2-4 layers of surgical gauze. The alcohol can be recovered and recycled by distillation.

Where further purification was desired, the alcohol purification procedure was repeated one or more times. A variation of the purification procedure comprises repeated precipitation and filtration steps without intervening washing steps.

The purified material was then directly drum dried (Blaw-Knox Co. Buffalo, NY). Alternative drying methods are fluidized bed, vacuum tumble drying and "flash-spin" drying. The purified material can also be spray-dried or freeze-dried if first rehydrated with 1-2 volumes distilled H₂0.

Example 3: Functional Assessment of Recovered Gum Products

3.A. - Emulsion Testing: Measurement of Droplet Size. Turbidity and Stability A comparison of the emulsifying properties of Pyrus gum and a prior art gum, gum arabic, was conducted to determine whether the claimed gum has emulsifying qualities comparable or improved relative to the prior art gum. Aqueous solutions of Pyrus gum and gum arabic were mixed with D-limonene oil to produce emulsions, which were then tested for droplet size, turbidity and shelf life stability.

In order to clarify or reduce complexing of the pectic fraction of the Pyrus gum prior to use, 5 grams of Pyrus gum were dissolved in 500 ml of distilled water and boiled for 5 minutes. Concentrated EDTA solution was added until the insoluble pectic material was dissolved. The solution was filtered through two layers of Whatman glass fiber filter

33 paper GF/F under vacuum and dialyzed (MW cutoff 14,000-16,000) against distilled water at 4°C for 24 hours. The volume of the solution was then reduced under vacuum by rotary evaporation and freeze dried. Gum arabic was obtained from Sigma, No. G-9752. D-Limonene (p-mentha-l,8-diene) was obtained from Bush Boake and Allen.

Stock solutions of gum arabic (250 mg/ml) and Pyrus gum (62.5 mg/ml or 12.5 mg/ml) were pipetted in duplicate to give final concentrations of 0, 0.2, 0.5, 1, 5, 10 and 20%) (w/v). The Pyrus solution could not be prepared at concentrations greater than 5% (w/v) due to its viscosity and gelling properties. Twenty percent D-limonene oil in water emulsions were prepared by injecting the oil into the aqueous solutions under the surface of the solutions while being mixed in an Ultraturrax (Ystal T1500, 25-240V, West

Germany) at setting 4 for 15 seconds. The speed of the Ultraturrax was increased to setting 7 for 45 seconds to produce the cloud emulsion. The emulsions were allowed to stand for 0.5 hours to allow bubble dispersal.

To determine emulsion capacity, droplet size, turbidity and shelf-life were measured for each emulsion. Emulsion capacity increases with decreased droplet size, increased turbidity and increased shelf-life stability. The droplet size of the cloud emulsion was examined microscopically by placing 2 drops of the emulsion on a slide and diluting with 2 drops of water and estimating droplet size using a calibrated eye piece graticule. Cloud turbidity was measured by diluting duplicate 5 μl aliquots of cloud emulsion into 5 ml of 0.1% (w/v) sodium dodecylsulphate and measuring absorbance at

500 nm. Cloud emulsions were tested for shelf-life stability by centrifuging at 2,500 rpm for 10 minutes and observation of the resulting separation of oil and water phases. The results are set forth in Table 2.

From these results, it is seen that when emulsifying 26% D-limonene in water, Pyrus gum on a weight for weight basis produces smaller droplets at a lower concentration than gum arabic. For example, at 0.2% (w/v) of Pyrus gum, the emulsion mixture has a film of free oil, a cream layer stable to centrifugation, oil droplets of 1-20 μm and a cloud turbidity at 500 nm of 0.127. In contrast, 0.2% (w/v) gum arabic in an emulsion mixture has an unstable cream which separates completely to oil on centrifugation, has a larger

34 droplet size (10-20 μm) and an average cloud turbidity reading of 0.023 at 500 nm. These results indicate that the Pyrus gum has improved emulsifying qualities relative to those of gum arabic at the same concentration.

Emulsion stability can also be assessed by the following method. An oil-in-water emulsion was produced with a range of gum product concentrations (e.g., 0.2, 0.5 and 0.7

% (w/v)):

gum (g) 0.1 0.25 0.35 oil (ml) 10.00 10.00 10.00 water (ml) 40.00 40.00 40.00

Total 50.00 50.00 40.00

The gum product was dissolved in water using the ultraturrax (John Morris Scientific Equipment) at a setting of 4. Oil (Crisco, polyunsaturated blend) was then added while mixing and held at setting 4 for 45 seconds. The solution was further mixed at setting 8 for 45 seconds. The emulsion obtained was poured into 50 ml measuring cylinders (21 mm internal diameter), sealed with aluminum foil and stored at 27°C. It was then observed for up to a week. Creaming or separation was expressed in percentage volume.

The volume of oil can be varied to provide an HLB in the emulsion that is typical for the intended application.

For measuring the stability of a water in oil emulsion comprising a cultured plant cell gum, ASTM method 3707 or an adaptation thereof can be used.

3.B. - Viscosity Testing

The flow behavior of the gum product in aqueous solutions or mixtures was assessed over a range of gum concentrations, temperatures and shear rates. The gum product was dissolved in water using the ultraturrax at setting number 4. The solution was then stirred and heated to 60°C. Viscosity was measured at decreasing temperatures from

35 60° to 10°C using an Epprecht Rheometer (Contraves AG Zurich) at various shear rates. Results were plotted as viscosity versus temperature for different shear rates.

Viscosity plotted as a function of shear rate indicates the thixotropic nature of the gum. Thixotropic profiles indicate whether a gum is suited for particular applications where shear thinning is required (e.g., in drilling muds).

Viscosity plotted as a function of temperature indicates the suitability of the gum as a viscosifier or thickener over the operating temperature range of the intended application.

3.C. - Gel Strength Testing Gel strength is assessed by measuring rupture strength, shear modulus and back extrusion. Back extrusion is of particular interest because it can distinguish between and characterize soft gels and viscous fluids.

3.C.1 - Rupture Strength

Rupture strength is the force required to compress and rupture a gel sample. For rupture strength, the force is proportional to the sample weight.

The gel samples were prepared by mixing P. communis gum (0.2-0.5 % (w/v)) or N. plumbaginofolia (0.5-l.o% (w/v)) in water in 50 mm plastic petri dishes and storing them at 15°C overnight. Rupture strength was measured by compression on the Instron 1122, using a probe of 150 mm in diameter at a cross-head speed of 50 mm/min.

3.C.2 - Shear Modulus

Shear modulus is a measure of the force required to shear/cut the gel. Shear modulus is expressed as stress divided by strain. For shear modulus, the force is proportional to the sample weight.

The gel samples were prepared as in C. l in a 24 mm diameter glass vial and stored at 15°C overnight. Shear force was measured on a modified puncture strength meter

36 (Oakenfull, D.G. et al. (1987) "A method for determining the absolute shear modulus of a gel from a compression test" in Gums and Stabilizers for the Food Industry, Vol 4, Phillips, G.O. et al. (eds.) IRL Press, Oxford) with a probe of 3 mm in diameter at the cross head speed of 5 mm/min for 20 seconds. Shear modulus was then calculated using a mathematical model set forth in Oakenfull, D.G. et al. (1987)..

3.C.3 - Back-extrusion

Back-extrusion force is the force required to compress and shear a gel sample. In back-extrusion, force is independent of sample weight.

The gel samples were prepared as in C. l in 200 ml beakers of 64 mm and stored at 15°C overnight. Back-extrusion was performed on the Instron 1122 by plunging a probe of 60 mm in diameter at a speed of 100 mm/min to a depth of 50% into the gel.

3.C.4. - Effective Temperature Range of Gel: Determination of melting and setting points The melting point is determined by observing the temperature at which a 10 ml gel begins to melt in a 11 mm diameter spectrophotometric tube. The determination was aided by observing small glass beads (0.08 g) sinking into the melting gel. As there can be temperature gradients within the gel, a melting range can be observed. The experiment was carried out in a Thermoline waterbath in 5°C steps.

The setting point was determined by observation of gelling in spectrophotometer tubes. The gel samples in the tubes were stored overnight (18 hours) at a range of temperatures, and the tubes were then inverted to observe if setting had occurred. The temperatures tested were 6°, 10°, 15°, 20°, 25°, 27°, 30°, 37.2° and 45°C.

3.P. - Encapsulating capacity

Encapsulating capacity can be assessed by evaluating a gum-containing spray dried emulsion in terms of flow characteristics and stability, as described in Example 3 of a

U.S. Patent application entitled "Plant Gum Material and Use Thereof in Food Products," and filed on even date herewith.

37 3.E. - Adhesive capacity

Adhesive capacity can be measured by using standard methods such as ASTM (American Society for Testing Materials) method D1713 ("Bonding Permanancy of Water- or Solvent- Soluble Liquid Adhesives for Automatic Machine Sealing Top Flaps of Fiberboard Specimens") and D1581 ("Bonding Permanancy of Water- or Solvent- Soluble

Liquid Adhesives for Labelling Glass Bottles"), or adaptations thereof.

Example 4: Papermaking - Preparation of Paper Hand Sheets

A superior strength paper can be produced using the procedure described in Australian Standard 1301 APPITA P203s/80 by adding N plumbaginifolia gum product at the wet end to improve the physical properties of the dry sheet. The observed improvements include increased paper strength (both burst and tensile), greater resistance to erasure, reduced "fuzz" or lint on the paper surface and reduced rate of water penetration as compared to hand sheets prepared without a gum beater aid. The gum product allows for a retention of wet strength and improved yield by providing a more uniform distribution of fines.

The following method (Australian Standard 1301 APPITA P203s/80) was used for the preparation of hand sheets:

Commencing with wood fibre pulp (sourced as chemically treated pulp, semi- chemical pulp, or mechanical pulp or recycled pulp), the gum product was dissolved in a quantity of water sufficient to produce a 2% solids solution. One liter of the dissolved solution was added to 4 liters of pulp placed in a container. Adequate mixing was ensured by sparging for at least 15 minutes. A sample of 500 ml was then place into a larger tapering 15 liter vessel with a 60 mesh screen at the base 100 mm in diameter. A further 10 liters of processed water was added and the mixture was sparged from the base of the vessel for at least 15 seconds to ensure thorough mixing. The base valve was then opened, allowing processed water to drain away, retaining all of the fibers on the wire mesh screen. The base screen was removed from the unit base and covered with a blotter, allowing the wet fibrous mat to be retained by the blotter. Successive cycles produce a number of samples which are then stacked and pressed in a stack to remove excess water.

38 They were then placed in a drying cabinet and maintained at a standard 23 °C, 50% relative humidity until testing.

Testing revealed that the subject gum-containing paper has superior tensile strength, stretch, work to rupture and extensional stiffness on an Alweitron Universal Testing machine. Methods for testing paper are known in the art and include, e.g., Australian

Standard 1301.403s-89 for "Bursting Strength of Paper;" Australian Standard Appita P404s-81 for "Tensile Strength of Paper and Paperboard;" Australian Standard 1301.419s- 89 for "Water Vapour Transmission Rate of Paper;" Australian Standard 1301.41 ls-89 for "Water Absorptiveness of Paper and Paperboard (Cobb Test);" and Australian Standard Appita P406m-86 for "Bending Quality of Paperboard."

Example 5: Adhesives - Preparation of re-moistenable adhesive

A satisfactory adhesive for envelopes, labels, stamps and aluminum foil sheets, which is of the water re-moistenable type, was prepared as follows:

1. N. plumbaginifolia (BLM carbon source) 1000 gm

2. Sodium Chloride 20.5 gm

3. Glycerol 20.5 gm

4. Potato starch 20 gm

5. Water 1300 ml

6. Preservative 1 gm

The water was placed in a high speed mixer and mixing was begun at a slow speed. The gum product was slowly added, allowing it to fully dissolve in the mixing process. After 4 minutes, the sodium chloride, glycerol, starch and preservative were added. After thorough mixing, the mixture was left to stand for VΛ hours.

This produced an adhesive which was applied to the surface of paper and dried. It remained inactive until moisture was reapplied. It was found to be a superior gum for use in these applications as it has good affinity for water and does not cause discoloration of the paper or become brittle on aging. It was found that the adhesive glued pieces of

39 aluminum foil to paper very firmly and also glued pieces of paper together in a manner similar to commercial adhesive pastes.

Example 6: Oil and Gas Well Applications - Preparation of Drilling Mud

A satisfactory drilling mud or fluid can be prepared in stirred tanks as follows:

A large 1 ,000 liter tank was filled with water. About 6% by weight bentonite

(montmorillonite) was added while stirring slowly and continuously until dissolved. In a second 1,000 liter tank filled with water, about 3% by weight N. plumbaginifolia gum product was added while stirring slowly until dissolved. In a third holding tank, equal quantities of gum product mixture and bentonite mixture were mixed. This produced a basic drilling fluid to which was added up to 30% solids of barium sulphate or 30% chalk as weighting agents depending upon the nature of the surrounding rock structure. If desired, a biocide can be added to prevent fermentation during storage or down-hole.

The resulting drilling fluid has increased viscosity, and can provide an improved flow of material from the bit to the surface and a uniform dispersion of the solids, thereby acting as a protective colloid. It can also lubricate and reduce fluid loss into porous rock.

The resulting drilling fluid is particularly efficacious in providing a uniform suspension and maintaining a consistent fluid in drilling through shale layers, broken rock that has been stabilized, or magnesium or calcium containing rock. During cementing, a stabilizing fluid containing the subject gum product will also have reduced fluid loss.

The N. plumbaginifolia gum product, when in an aqueous dispersion with calcium, possesses gelling properties. Such gelling properties can enhance suspension of solids in a drilling mud even when flow has stopped.

Example 7: Printing Applications

7.A. Preparation of Printing Ink A satisfactory emulsion or suspension water-based flexo ink for printing was prepared using the N. plumbaginifolia gum product as a suspension agent to provide

40 uniform dispersion of the pigment elements and prevent the ink from separating. To a typical ink formulation of:

1. Carbon Black

2. Mineral Oil 3. Sodium silicate

4. Sodium carbonate

5. Water was added about 2%> by weight of gum product to produce a fine uniform stable suspension of the solid ingredients. Using a high speed mixer running at low speeds the gum product was added to the mixture until thoroughly dispersed. The emulsion mixture was left to stand for 11 hours prior to use.

7.B. Preparation of a Lithography Fountain solution

The N plumbaginifolia gum product provides a satisfactory substitute for gum arabic in several lithography solutions or mixtures including the plate sensitizer solution, the fountain solution and the protecting solution (used during plate storage). The gum product imparts good wettability particularly to the fountain solution. It also supplies the viscosity required to allow the fountain solution to cling to the plate without running off or forming isolated droplets or pools on the plate.

A fountain solution was prepared as follows: 1. Water 700 ml

2. Propylene Glycol 50 ml

3. Biocide Parabens (methyl/ethyl- hydroxy parabenzoic acid at 0.5-2.0% (w/v) in water adjusted to pH 7.0 with phosphate buffer 1 ml

4. gum product solution 3% (w/w) 200 ml

5. pH buffer 40 ml

41 All ingredients other than the gum product solution were added to a mixture, and stirred until dispersed (10 mins). The gum product solution was then added, and stirring was continued. The mixture was then allowed to stand for 30 minutes before use.

7.C. Comparison of N. plumbaginifolia gum to gum arabic in fountain solutions The following formulae were made up by Cetec Pty. Ltd., a consultant. All values (except pH) are w/w percent.

FI F2 F3 F4

Water 70 90 70 90

Propylene Glycol 5 - 5 5

3% w/w gum arabic soln. 15-20 - - - biocide 0.1 0.1 0.1 0.1 pH buffer (phosphate) 5-7 5 5-7 5

Phosphoric acid - 2 - 2 gum arabic EDTA - 2 - 2

EDTA - 0.5 - 0.5

N. plumbaginifolia gum - - 0.3-0.4 -

3% (w/v)

N. plumbaginofolia gum - - - 0.3-0.4

FI and F2 are standard fountain solutions that employ gum arabic. F3 and F4 are identical to FI and F2, respectively, except that N. plumbaginifolia gum product has been substituted for the gum arabic in a weight that is 1/50 of the gum arabic weight.

When these fountain solutions were employed in an offset litho printing, it was found that the N. plumbaginifolia gum product performed comparably to the gum arabic fountain solutions in terms of ink-plate roll up and in degree of plate background desensitization. The plate wetting characteristics of the two products were also very similar. The N. plumbaginifolia gum was found to be less soluble in isopropyl alcohol than gum arabic; since isopropanol is very widely used as part of the dampening system of modern, fast lithographic offset presses, this may be a negative feature.

42 Example 8: Fabric Printing - Preparation and use of reactive dyestuff for wool or cotton Satisfactory dyeing of wool and cotton was accomplished as follows: First, a thickening was prepared:

1. N. plumbaginifolia gum product 150 gm 2. Cold water 2800 ml

3. Sodium metaphosphate (Calgon™) 30 gm

The water was agitated with a high speed mixer during gradual addition of thesodium metaphosphate. The gum product was then added slowly, but fast enough so that all the powder was added before the viscosity has risen appreciably. Stirring was continued for 5-10 minutes until all particles were swollen and had formed a thick suspension. The mixture was allowed to stand for 1-1/2 hours.

Then the following were added:

4. Diphasol™ solution 115 ml

5. Hot water 975 ml

6. White spirit 3750 ml

7. Resist salt L™ 150 gm

The thickening mixture was then stirred in the high speed mixer for 20 minutes.

The screen printing paste was prepared by mixing the following: 1. Dyestuff 3 gm 2. Urea 10 gm

3. Hot to boiling water 30 ml

4. Thickening (as above) 50 gm

5. Sodium bicarbonate 4 gm

Using a high speed mixer, the dyestuff and urea were thoroughly dry mixed. Then the hot water and thickening were added and mixed.

43 The printing paste was used in a standard fabric screen printing method. The printed cotton and wool were then dried followed by steaming for 8 minutes. They were then rinsed thoroughly in cold water followed by a soaping at or near the boiling point with a detergent solution of Lissapol ND (2% w/w solution) and finally rinsed in cold water. The printing on the wool and cotton material appeared stable.

Example 9: Paints

9.A. Preparation of Acrylic Resin Paint

A stable water emulsion was prepared using the following formulations: Premix in a ball mill: 1. Tap water 125 ml

2. Daxad 30™ Dispersant 8 gm

3. Tergitol NPX™ Surfactant 4 ml

4. Victawet 35B™ Wetting Agent 2.5 ml

Then the mill speed was increased and the following was added slowly: 5. Chemacoil TA-1001™ Resin 74 gm

The speed was adjusted to disperse the following pigments and additives:

6. Zinc oxide AZO-ZZZ-33™ 75 gm

7. Titanox RANC™ Rutile Titanium Dioxide 175 gm

8. Titanox A168L0™ Anatase Titanium Dioxide 25 gm 9. Asbestine 3X™ Talc 100 gm

10. Ethylene Glycol 18.5 gm

11. Nuodex PMA-18 Mildewoide™ 3 gm

12. Nopco NDW™ Defoamer 4 gm

The mill was then slowed to mixing speed. 13. N. plumbaginifolia gum product emulsion 165 ml

(2% w/w aqueous solution)

14. Rhoplex AC-34™ Acrylic Emulsion 372 gm

15. Super Cobalt™ Drier 1 gm

44 Mixing continued for at least 1/2 hour at mixing speed. Other pigments, such as carbon black or red oxide of iron, may be added to this to replace part of the titanium dioxide ingredients in items 7 & 8 and provide a differing color balance.

The above formulation was derived from Ernest Flick "Water-Based Paint Formulations" Noyes Publications, Parkridge, New Jersey.

9B. Oil Emulsion Paint

A satisfactory thixotropic paint was prepared as follows. Premix in a high speed stone mill:

1. Water 205 ml 2. Victawet 35B™ Wetting agent 4 ml

3. Potassium polyphosphate 5 gm

Adjust speed of the mill to disperse the following additives and pigments:

4. Ethylene glycol 10 gm

5. Titanox RANC™ Rutile TiO₂ 175 gm

6. Titanox A168L0™ Anatase TiO₂ 50 gm

7. Asbestine 3X™ Talc 50 gm

8. Zinc oxide AZO-ZZZ-33™ 125 gm

9. Nuodex PMA-18 Mildewoide™ 2 ml

10. Nopco NDW™ Defoamer 2 ml

11. Victawet 35B™ Wetting agent 16 gm

The mill was then slowed to mixing speed and the following were added:

12. N. plumbaginifolia emulsion

(2.5% w/w) 130 ml

13. Emulsified linseed oil (60% solids) 340 ml

14. Super Cobalt™ drier 11 gm

Milling was continued for approximately 1/2 hour.

45 This procedure resulted in a paint that tends to set to relatively stiff or "buttery" consistency upon standing, but thins down to relatively mobile liquid when mechanically agitated. This thixotropic character is such that the shearing action of the brush used to apply the paint to a surface is sufficient to render the paint adequately mobile. The paint leaving the brush remains fluid for a sufficient time to bring about good levelling (i.e., the brush marks disappear while the paint again sets to a stiff consistency before it has time to run appreciably on the surface painted).

This thixotropic property in paints is valuable in flat paints meant to be applied to interiors with a brush because it prevents the running of the paint and at the same time eliminates brush marks. Thixotropic paints possess a further advantage quite apart from their intended use for the reason above, in that the paint acquires a buttery or solid consistency upon standing in containers. Segregation or stratification of the paint during long periods of storage is thus prevented.

The above formulation and methods were derived from U.S. Patent No. 2,135,936, November 8, 1938, for "Use of gum arabic in paint" and from "Emulsion and Water

Soluble Paints and Coatings."

Example 10: Ceramic Glazes

The suspension of the glaze ingredients in a glaze slip for several hours or even days has been achieved using N plumbaginifolia gum product as an emulsifying and/or suspending agent. Further, the resulting glaze has superior clarity and stability.

A stable glaze slip was prepared as follows:

To prepare an emulsifying/stabilizing mixture, the following were combined: 1. N. plumbaginifolia dry gum product 10 gm

(grown on BLM as carbon source) 2. Cold water 500 cc

Example 1 1 : Clear Thixotropic Detergent or Cleaning Preparation

46 A satisfactory thixotropic cleaning detergent with superior grip and film forming properties was prepared as follows:

1. Water 812 ml

2. N. plumbaginifolia (BLM carbon source) 40 gm 3. Sodium chloride 20 gm

The gum product was added to the water in a high speed mixer running at a slow speed and was mixed for 15 minutes. The mixture was then left for 1-1/2 hours and the sodium chloride was added, mixing slowly for 3-5 minutes. Sodium ethylsulphate was then added while mixing continued:

4. Sodium ethylsu Iphate

(C12-14 2E.0.) (100% basis) 125 gm

5. Perfume <.5 gm

6. Dye <.5 gm

7. Preservative <.5 gm

The perfume, dye and preservative were then added, and mixing continued for another 10 minutes.

Because the foregoing formulation does not contain either ethyl alcohol or propylene glycol (which can be used in cleaning detergents), the possibility of precipitation of the gum due to a high alcohol concentration is averted.

The resulting product is a clear cleaning agent which tends to be relatively stiff and provides an adequate detergent which clings to the surfaces. This is a desirable characteristic particularly in the cleaning of vertical surfaces. It was found that the BLM carbon source enhanced the film forming properties of the detergent.

Example 12: Cosmetic Creams and Lotions 12.A. The concentrated gum from Nicotiana Batch 3-1000 (1.7% total solids) was found to have pleasant soft feeling on the skin and to dry without stickiness. When mixed with water, it makes a satisfying, i.e., moisturizing, skin treatment without any further

47 additions. Another product was prepared by perfuming the biopolymer solution with 0.1% v/v rose oil.

12.B. A cosmetic lotion was prepared with the ingredients indicated below. The vegetable oil, perfuming oil and glycerol were added to the biopolymer solution while mixing with a high speed stirrer such as an Ultraturrax at a setting of about 6.

Nicotiana gum #3-1000 2.4% (w/w in H₂0)

Orange oil 1.0% (w/w)

Olive oil 2.3% (w/w)

Glycerol 5.3% (w/w)

The Nicotiana gum was mixed in the water in a high speed stirrer such as an

Ultraturrax at a setting of about 6. The olive oil, orange oil and glycerol were then added. The result was a soft gel with a pleasant fresh aroma which can be spread on the hands or face, leaving skin feeling fresh and soft.

12. C. A cosmetic lotion was prepared using the following: Nicotiana gum #3-1000 1.7% (w/w in H₂0)

Sunflower oil 1.3%

Glycerol 4.0%

The ingredients were combined as in l l.B. The resulting product was soft enough to be used in a pump-action dispenser. A perfuming oil can be added if desired.

12.D. Other batches of gum from other cell lines were used to prepare products with different properties. For example, a cream was prepared from gum produced by Nicotiana cells growing in a medium containing Brewers Liquid Maltose ("BLM") 162 g/liter, as the source of sugar. The resulting gum produced a viscous solution and was used to prepare a lotion with the following formulation: Nicotiana gum 2.3% (w/w in H₂0)

Peanut oil 3.3%

Rose oil 0.1%

Glycerol 5.0%

48 Example 13: Compositions and Selected Rheological Properties of Plant Cell Gums

Table 4 lists relative weight proportions of protein and ash and types of polysaccharide in plant cell gums obtained from suspension cultures of a variety of vascular plants including both dicots and monocots. Table 5 lists explant sources and culturing conditions forplant cell cultures of Tables 4 and Table 6 lists the maximum polysaccharide concentration obtained in cultures during growth cycle measurements.

There is significant variation in the amount of protein + ash, from a low of about 12%) for Timothy Grass to a high of 58% for white clover, in the representative plant cell gums exemplified. Xyloglucan is relatively low in the gums of cells of Fabaceae and Poacea but relatively high in Aizoaceae and Malvaceae plant cell gums. Arabinogalactan which will be attached to protein also varies significantly from a high of 28% to a low of 10%. Heteroxylan is only significant in cells of monocots. Glucuronomannan is high in the monocot Poacea, but also in Nicotiana plumbaginifolia plant cell gum. Rhamnogalacturonan is also variable among the gums assessed.

The percentages by weight of the components measured add up approximately to

100%) for each cell line. Discrepancies in total weight percent may occur due to the inherent errors in analytical techniques or if the assumptions made with respect to the structures of polysaccharides present are inaccurate. For example, the presence of minor functional groups such as methyl or acetyl groups may not be taken into account. The relative amounts of individual polysaccharides is inferred from detailed methylation data from isolated gum.

Methylation analysis was performed using analytical methods well-known in the art. Protein and ash were also measured using standard methods well-known in the art. Briefly, total nitrogen (N) was determined using the Kjeldahl method and expressed as protein (N x 6.25). Inorganic material (ash) was determined by ash content.

Monosaccharide compositions were determined by GC/MS following carboxyl reduction and methylation analysis. Isolated gums were carboxyl reduced and methylated and the partially methylated alditol acetates were analyzed by GC-MS.

4 9 The proportions of individual polysaccharides present in the gum were determined based on characteristic linkage structures of purified polymers from dicotyledonous and monocotyledonous species as described by Shea et al. (1989) Planta 179:293. Xyloglucan was the sum of 4,6-Glc and terminal Xyl equal to 4,6-Glc, as well as 2-Xyl, 2-Gal, terminal Fuc and 4-Glc equal to one third that of 4,6-Glc, except for Solanum tuberosum and Lycopersicon esculentum in which the structure of xyloglucan from S. tubersum was used as a model (Ring and Selvendran (1981)). Galactoglucomannan was the sum of 4- Man and 4,6-Man, 4-Glc equal to the sum of these Man linkages and terminal Gal equal to 4,6-Man. Glucomannan was the sum of 4-Man and any 4-Glc not assigned to xyloglucan. 3,6-Arabinogalactans (type II) were the sum of 3-Gal, 6-Gal, and 3,6-Gal and terminal Ara equal to 3,6-Gal. Heteroxylans were the sum of 4-Xyl, 2,4-Xyl,and 3,4-Xyl and terminal GlcA and terminal Ara equal to 2,4-Xyl and 3,4-Xyl. 4-Galactan was the sum of 4-Gal and 2,4-Gal and terminal Gal equal to 2,4-Gal, Arabinan was the sum of 2-Ara, 3-Ara, 5-Ara, 2,5-Ara and terminal Ara equal to 2,5-Ara. Glucuronomannan was the sum of 2-Man, 2,3-Man and 4-GlcA, 3,4-GlcA and terminal Ara and terminal Gal equal to the sum of 2,3-Man and 3,4-GlcA. Galacturonan was the sum of 4-GalA, 4-GalA(6-O-Me) and 3,4-GalA, and rhamnogalacturonan was the sum of these linkages and 2,4-Rha.

Suspension cultures were initiated from explants indicated in Table 6 generally following the methods described in Example 2 except that different media, hormone balance (as indicated in Tables 5 and 6) and fermentation times were used. Suspension cultures were grown in 2L shake flasks shaken at 100 rpm at 27°C in the dark.

Table 6 lists gum yield produced by suspension cultures. Gum production in these cell lines has not as yet been optimized by variation of fermentation conditions and media components. Increased yields of gum can be achieved by such optimization methods.

Table 4 emulsification, gelling and the viscosity of various dicot and monocot plant cell gums. Gums were isolated essentially as described in example 2. Emulsification was tested by determining the droplet size of a limonene emulsion formed using 1% (w/v) of an aqueous gum solution essentially as described in example 3. Mesembryanthemum gum gave an excellent emulsion with low droplet size of 1.5 μm which is three to five times

50 smaller than emulsion droplets formed using Nicotiana and Pyrus gum, respectively. Gums which can generate an emulsion with droplet size less than about 5.0 μm are particularly useful in the drink industry. Low-droplet-size emulsions are those in which droplet size is 5.0μm or less; preferred low-droplet-size emulsions are those having droplet size less than about 2.0μm.

Viscosity was measured essentially as described in example 3B. Some of the gums including timothy grass and millet appear to be viscoelastic, i.e., displaying both solid-like (elastic) and liquid-like (viscous) properties. Viscoelastic properties are time dependent. A viscoelastic material can exhibit either linear or non-linear viscoelastic behavior. Viscoelastic behavior can be observed, for example, by rapidly twisting a bottle of gum and watching recoil. Linear viscoelastic behavior is observed at low strain (nondestructive testing). Measurements of storage modulus(G') and loss modulus (G"), both well-known measurements, give an indication of the amount of solid-like behavior (G') and liquid behavior (G").

Mesembryantheum gum has a relatively low viscosity combined with the capability to form low droplet size emulsions. Mesembryantheum gum is thus useful in emulsification applications and particularly well-suited for use in cloud emulsions. Cloud emulsions have applications in the food industry, for example for manufacture of soft drinks, and in the chemical industry and agriculture for preparation of chemical emulsions including emulsions of agricultural chemicals (pesticides, etc.).

Timothy grass gum displays good gelling ability and high viscosity. This gum is useful in applications where there is a need to suspend or stabilize or where water-holding properties are needed. This gum has application, among many others, in the food industry for ice cream and dessert items and in the preparation of spray emulsions, for example, for agricultural chemicals.

Millet gum has high viscosity associated with water thickening properties. In addition to a variety of uses in the food industry in sauces, bakery glaces and other foods, millet gum has application in the preparation of drilling muds.

51 Derek Morton "Determination of Primary Structure of Microbial Polysaccharides", Ohio Food HyrocoUoids Conference, September 1994, provides an exemplary listing of applications and specific industrial uses for gum having a given rheological property or characteristic.

Example 14: Additional Media for Plant Cell Cultures

Linsmaier and Skoog Powder (LS) Medium for Chicory

LS Powder 4.16g

Sucrose 30. Og

Organics stock 1.0ml 2,4-D (O.lmg/ml) 2.2ml

NAA (0.2mg/ml) 0.186ml

Dissolve all ingredients, except agar, in 800 ml Milli-Q water. Adjust pH to 6.0 with KOH and make up to 1 L. Add agar 5.0 g if making solid medium. Autoclave for 20 m at 121°C.

Organic Stock

Thiamine Hydrochloride 40mg

Myo-inositol 10g

Dissolve both ingredients and make up to 100ml with Milli-Q water and use stock at lml/L. Aliquot into 10 ml portions and freeze.

Tomato BAL Medium

Add concentrated stock solutions and sucrose to approximately 900ml of Milli-Q water with constant agitation using a magnetic stirrer: Macro elements 10ml

Micro elements 1ml Vitamins 1ml

CaCl₂ 2.6ml

KH₂PO₄ 10ml

52 Fe.EDTA 1ml

2iP 1ml

2,4-D 20ml

KI 0.5ml Sucrose 30g

Macro and micro elements and vitamins are the same as used in pear BAL. Adjust pH to 5.0-6.0 with KOH (0.1 or IM) and adjust final volume to 1 litre. For solid medium add 0.5% agar (5 g/L) after adjusting Ph and volume. Sterilize for 20 m at 10 psi (116°C).

TB1 Medium for Tuberose (PETAL)

LS powder 4.60g

Sucrose 30. Og

2,4-D 20.0ml (Stock Solution O.lmg/L)

Dissolve all ingredients in approximately 800 ml Milli-Q water and then make up to IL. Adjust pH to 5.8-6.0 with 0.1M KOH. To prepare solid medium, add 5.0g of agar to a 2L flask, add dissolved consitutents and steam until dissolved. Sterilize at 10 psi for 20 m.

A related medium TB2 (Tuberose 2) also used for petal culture is prepared in a similar manner but contains 1/lOth the amount of 2,4-D (i.e., add 2.0 ml of the Stock Solution of 2,4-D rather than 20 ml).

Example 15 - Production of Plant Cell Gum of Mesembryanthemum "pigface"

A commerically available succulent plant with orange flowers labelled Mesembryanthemum purchased in 1992 from Sherringham's Nursery (North Ryde, NSW, Australia) as supplied by Milingimbi Nursery (Jillibi, NSW, Australia) was employed to establish a Mesembryanthemum sp. suspension cell culture. This plant is commonly

53 called pigface and is a very common landscaping plant in the Central Coast region of New South Wales. It is possible that this plant is a cross between Mesembryanthemum and/or Carpobrotus or that it is more correctly designated Carpobrotus. The labelling employed by the Nursery is used herein.

Callus was initiated from leaf tissue cultured on Murashige and Skoog medium containing 2.0 mg/L 2,4-D ( MS2, see Murashige.T and Skoog, P. (1962) Physiol. Plant 15 473-97) medium solidified with 0.5%> (w/w) agar. Subculturing was performed substantially as described in Example 1 wherein the number of subcultures of callus is indicated in Table 5. Suspension cultures are established employing small portions of callus tissue wherein the number of subcultures are indicated in Table 5.

MS2 can be used for suspension cultures of Mesembryanthemum. Suspension cell media are typically provided with 2.0 mg/L of the plant hormome 2,4-D. However, Mesembryanthemum callus and cells can be adapted to grow well in the absence of 2,4-D. Plant cells are typically cultured at temperatures ranging from about 25-28°C with dissolved oxygen maintained at 20-70%> air saturation. Culture pH is typically not controlled externally.

Plant cell gum was isolated from Mesembryanthemum suspension cultures substantially as described in Example 2. Briefly, cells (biomass) are separated from culture filtrate, and Na₂EDTA-2H₂O (1 g/L) (as a sequestering agent) is added to the filtrate and the pH is adjusted to about 8 to complex calcium. Preservatives and antioxidants (ascorbic acid, potassium sorbate, sodium benzoatesodium metabisulfate, etc.) can be added to the culture filtrate to prevent deterioration of gum components. Treated filtrate is concentrated using ultrafiltration or in a dialysis sack covered with polyethylene glycol flakes to 1/3 or 1/4 its original volume. Concentrated filtrate is washed (by diafiltration or by dialysis). The washed filtrate is dried, for example, by spray drying or freeze drying.

Dried gum can be assessed for emulsification capacity (as described above) and inert fillers (such as maltodextrin) can be added to dried gum to provide a standardized sample having a selected emulsification power. Mesembryanthemum plant cell gum for

54 use in food or veterinary applications can be further purified by alcohol precipitation if desired or necessary.

Example 16 - Splash Inhibition Studies of Millet Plant Cell Gum

The formation of fine mists and satellite droplets is undesirable in certain agricultural chemical compositions, such as those containing pesticides, herbicides and fungicides. Formation of fine mists on chemical application to fields or gardens can result in undesired wide dispersion of hazardous materials. The use of components such as polyethylene oxide in agricultural chemical compositions can inhibit formation fine mists and satellite droplets. Satellite droplet inhibition is also important in ink applications, for example in application to ink-jet printing. Monocot plant cell gums, particularly those of millet and timothy grass are found to have viscoelastic properties which are associated with fine mist inhibition and satellite droplet inhibition. Splash inhibition assays can be used to assess the potential of a plant cell gum for use in applications needing fine mist and satellite droplet inhibition.

Splash inhibition in solutions containing millet plant cell gum (as in Table 4) was examined in a drop impact study in which millet solutions were compared to solutions containing polyethylene oxide (PEO). A 35% aqueous glycerol solution containing 0.15% millet plant cell gum was found to have a shear viscosity of about 5.5 cP at 25 °A. Aqueous glycerol (35%) solutions containing PEO (either 8,000 Mwt, 300,000 Mwt, 600,000 Mwt, or 1,000,000 Mwt PEO with concentration [ranging from 0.1% - 0.17%] dependent upon molecular weight of the PEO ) having the same shear viscosity (5.5 cP at 25 °C) were compared to the millet solution. A 50%> aqueous glycerol solution was also tested. Glycerol was included in solutions to enhance viscosity for measurement purposes. The Trouton ratio of the millet solution at 25°C is about 120 compared to that of a 1,000,000 Mwt. PEO solution which is 5.5. The millet gum solution is about 20-fold more elastic than PEO.

All of the solutions examined for splash inhibition have approximately the same shear viscosity, density, surface tension drop diameter and impact velocity. The solutions differ in extensional viscosity. At high extensional rates the extensional viscosity of the millet solution decreases compared to PEO solutions.

55 Solution drops are impacted on aluminum surfaces roughened with varying grades of sandpaper (500 grit to 40 grit). The initial drop onto the surface does not splash; splashing is observed when a drop hits a thin film of liquid, e.g., when the second drop impacts the wet surface. The impact of a drop is captured by a progressive scan video camera at 50 frames/sec.

The 50%> aqueous glycerol solution and the 8,000 Mwt. PEO (in 35%o aqueous glycerol) solution splashed on all aluminum surfaces tested at both 20° and 25°C. In contrast, solutions containing higher molecular weight PEO (300,000 - 1,000,000 Mwt) do not splash on the tested surfaces at either 20° or 25°C. The millet solution (0.15% millet plant cell gum in 35%> aqueous glycerol) does not exhibit splashing at 20°C , but does exhibit splashing at 25 °C . The millet plant cell gum solution tested does exhibit splash inhibition and can be employed in compositions to provide inhibition of fine mists and satellite droplets.

Those of ordinary skill in the art will appreciate that materials, media components, purification methods, techniques and procedures other than those specifically exemplified herein can be employed in the practice of this invention without departing from its spirit and scope which is defined by the appended claims.

56 Table 1A: Exemplary Plant Sources for Cultured Plant Cell Gums

CLASS: MAGNOLIOPSIDA ORDER 7: RANUNCULALES

(DICOTS)

Family: Ranunculaceae

SUBCLASS 1: MAGNOLIDIAE Ranuncula bulbs

ORDER 1: MAGNOLIALES Family: Lardizabilaceae

Akebia quinata, climber

Family: Annonaceae

Annona muricata (custard apple) Family: Berberidaceae

Berberis spp., shrub

Family: Magnoliaceae Mahonia spp., huckleberry

Magnolia grandiflora

ORDER 8: PAPAVERALES

ORDER 2: LAURALES

Family: Papaveraceae

Family: Lauraceae (Cassythaceae) Papaver somniferum, poppy

Persea americana (avocado) Cassytha melantha, Aust native Family: Fumariaceae Cassytha pubescens, Aust native Fumaria spp., weed herb Cassytha glabella, Aust native

SUBCLASS 2: HAMAMELIDAE

ORDER 3: PIPERALES ORDER 2: HAMAMELIDALES

Family: Piperaceae

Pepperomia obtusifolia Family: Hamamelidaceae

Liquidambar

ORDER 4: ARISTOLOCHIALES Hamamelis spp., witch hazel

Family: Aristolochiaceae Family: Plananaceae

Tropical climbers Planatus spp., plane tree

ORDER S: ILLICIALES ORDER 6: URTICALES

Family: Schisandraceae Family: Cannabaceae

Schisandra spp., climbers Cannabis sativa, hemp Kadsura spp.

Family: Urticaceae

ORDER 6: NYMPHAEALES Boehmeria nivea, ramie

Family: Nymphaeceae

Nymphaea spp., water lilies Brasenia schrebe , water plant

Family: Nelumbonaceae

Nelumbo spp., lotus

57 Table 1A (continued) Family: Chenopodiaceae

Spinacia oleracea, spinach

ORDER 8: JUGLANDALES Family: Basellaceae

Basella alba, San Choy

Family: Juglandaceae Juglans spp., walnut Family: Cactaceae Carya spp., pecan Echinocactus grusonii Neoporteria cormasensis

ORDER 9: MYRICALES Opuntia dillenii, prickly pear

Family: Myricaceae Family: Amaranthacea

Comtonia peregrina, ornamental Amaranthus spp.

ORDER 10: FAGALES Family: Caryophyllaceae

Dianthus caryophyllus

Family: Betulaceae

Beta vulgaris, sugar beet ORDER 2: POLYGONALES Beta vulgaris, beetroot Betula verrucosa, birch Family: Polygonaceae

Polygonum spp., weed

Family: Nothofagaceae Persicaria spp., weed

Nothofagus moorei, Aust. beech

ORDER 3: PLUMBAGINALES

Family: Fagaceae

Fagus sylvatica, Europ. beech Family: Plumbaginaceae

Plumbago spp., shrub

ORDER 11: CASUARINALES Limonium spp., cut flower

Family: Casuarinaceae SUBCLASS 4: DELLENIIDAE

Casuarina spp.

ORDER 1: DILLENIALES

SUBCLASS 3: CARYOPHYLLIDAE

Family: Paeoniaceae ORDER 1: CARYOPHYLLALES Paeony spp., cut flower

Family: Aizoaceae ORDER 2: THEALES

Mesembryanthemum chilense(oύgmaλ) 4 presumed (ID undertaken) varieties Family: Theaceae Aptenia cordii Camellia japonica Carpobrotus acinaciformis Carpobrotus edulis Family: Actinidiaceae Delosperma lehmanni Actinidia chinensis, kiwi fruit Hereroa dyeri Rushia rubricaulis Table 1A (continued) Cucumis sativus, cucumber Crystal Apple Cucumis sativus, cucumber Burpless

Family: Clusiaceae Cucurbita pepa, zucchini

Hypericum perforatum, St. John's Wort Cucurbita maxima, butternut pumpkin Hypercium androsaemum, tutsan Citrullus lanatus, watermelon

ORDER 3: MAL VALES Family: Violaceae

Viiola odorata, violet

Family: Tiliaceae Corchorus spp., jute Family: Begoniaceae Tilia spp., ornamental Begonia spp., begonia

Family: Malvaceae ORDER 7: SALICALES

Gossypium arboreum, cotton Gossypium hirsutum, cotton Family: Salicaceae Hibiscus cannabinus, mesta Populus tremuloides, aspen Hibiscus sabdariffa, rozella Hibiscus esculentus, okra ORDER 8: CAPPARALES Sida rhambifolia, Paddys' lucern Alathaea spp., marshmallow, bioemuls Family: Brassiaceae Abelmoschus glutinotextilis Brassica hirta, yellow mustard Brassica oleracea, cabbage

Family: Sterculiaceae Brassica sinapsis, white mustard

Sterculia urens, Karaya gum Sinapsis alba, mustard Arabidopsis thaliana

ORDER 4: LECYTHIDALES

Family: Mocingaceae

Family: Barringtoniaceae Moringa petrygosperma, horseradish tree

Barringtonia spp., Aust. native

ORDER 9: BATALES

Family: Lecythidaceae

Berthollethia spp., Brasil nut Family: Gyrostemonaceae

Gyrostemon spp., Aust. natives

ORDER 5: NEPENTHALES

ORDER 10: ERICALES

Family: Sarraceniaceae

Sarracenia spp., Pitcher plant Family: Ericaceae

Heath

Family: Droseraceae Rhododendron

Drosers spp., native carnivorous Vaccinium myrtillus, bilberry

ORDER 6: VIOLALES Family: Epacridaceae

Epacris spp., Vic native

Family: Passifloraceae Leucopogon spp., Vic native

Passiflora edulis, passion fruit

Family: Cucurbitaceae

Sechium edule, choko

59 Table 1A (continued) Family: Hydrangeaceae

Hydranges spp.

ORDER 12: EBANALES Family: Grossulariaceae

Ribes uva crispa, gooseberries

Family: Ebenaceae Ribes rubrum, currants

Diospyros virginiana, persimmon Ribes nigrum, blackcurrants

Family: Sapotaceae ORDER 2: FABALES OR

Palaquim spp. guttapercha LEGUMINOSAE Payena spp., guttapercha Chrysophyllum spp., chewing gum Family: Fabaceae Manilkara spp., chewing gum Anthyllis vulneraria, kidney vetch

Astragalus cicer

ORDER 13: PRIMULALES Astragalus glycyphyllos

Astragalus gummifera, tragacanth

Family: Primulaceae Astragalus nuttallianus

Cyclamen euroeum Astragalus sinicus Primula Astragalus tenellus

Centrosoma plumari

SUBCLASS 5: ROSIDAE Certonia siliqua, carob

Cercidium torreyanum, palo verde

ORDER 1: ROSALES Crotalaria incana

Crotalaria intermedia

Family: Rosaceae Crotalaria juncea

Malus pumila, apple Crotalaria lanceolata

Malus domesticus cv Braeburn Crotalaria medicaginea

Rosa glauca Crotalaria mucronato

Prunus avium, sweet cherry Crotalaria retusa

Prunus insitia, damson Crotalaria spectabilis

Prunus domestica, egg plum Crotalaria striata

Prunus cerasus, cherry Cyamopsis tetragonoloba, gar

Prunus virginiana, cherry Delonix regia

Prunus persica, peach Demanthus pulchellum

Prunus amygdalus, almond Desmodium pulchellum

Prunus armenica, apricot Genista raetam

Pyrus communis Genista scoparia

Fragaria ananassa, strawberry Glycine max, soy bean

Vaccinium macrocarpon, cranberry Gymnocladus dioica, Kentucky coffee

Indigofera hirsutum, Indian legume

Family: Cunoniaceae (Baueraceae) Leucaena glauca, Koa hoale

Bauera spp., Aust native Lotus corniculatus

Lotus peduculatus

Family: Pittosporaceae Lotus scoparius

Pittosporum undulatum Lupinus albus Billardiera scandens, Aust. native Sollya heterophylla

60 Table 1A (continued) Family: Caesalpiniaceae

Caesalpinia cacalaco

Family: Fabaceae (cont'd.) Caesalpinia pulcherima

Lupinus angustifolius C. spinosa, Tara

Lupinus luteus Gleditsia amorhoides

Medicago hispida G. Ferox

Medicago Lupulina G. triacantos

Medicago orbicularis Cassia emarginata

Medicago radiata C. fistula

Medicago sativa, alfalfa C. absus

Melilotus albus C. leptocarpa

Melilotus indica C. marylandica

Mimosa scabrella C. nodosa

Mucuna imbricata C. occidentalis

Parkinsonia aculeata C. tora

Phaseolus aureus, mung bean

Phaseolus atropurpureus, Sirato Family: Mimosaceae

Phaseolus vulgaris Mimosa scabrella

Sesbania grandiflora Acacia dealbata

Sesbania speciosa Acacia mearnsii

Sesbania bispinosa Acacia nilotica

Sophora japonica Acacia pycnantha

Stylosanthes humilis, Townsville lucerne Acacia Senegal

Tamarindus indica, tamarind Acacia saligna

Tetragonolobus purpurens Albizia lophantha

Trifolium alexandrinum Albizia julibrissin

Trifolium dubium

Trifolium hirtum ORDER 3: PROTEALES

Trifolium incarnatum

Trifolium repens, white clover Family: Proteacea

Trifolium resupinatum Banksia integ ifolia, gooey fruits

Trifolium pratense, red clover Grevillea, gooey fruits

Trigonella caerulea Hakea spp., gooey fruits

Trigonella cretica Dryandra spp., gooey fruits

Trigonella calliceras Macadamia spp., gooey fruits

Trigonella corniculata Persoonia spp., gooey fruits

Trigonella foenum-graecum, fenugreek

Trigonella hamosa ORDER 5: HALORAGALES

Trigonella monspeliaca

Trigonella polycerata Family: Haloragaceae

Vicia faba Myriophyllum spp., aquarium plant

Vicia sativa

Prosopis velutina, mesquite

Alysicarpus vaginalis

Virgilia oroboides, trees exuding gum

Virgilia divaricata

61 Table 1A (continued) Family: Loranthaceae

Mistletoe amyema ORDER 6: MYRTALES

Family: Viscaceae

Family: Onagraceae Mistletoe dendrophora

Fuchsia spp., fuchsia Korthalsella spp.

Oenothera biennis, evening primrose

ORDER 11: CELASTRALES

Family: Myrtaceae

Eucalyptus camaldulensis Family: Aquafoliaceae Eucalyptus gunni Ilex aquifolium, holly Leptospermum spp. Thriptomene spp. Family: Stackhousiaceae Eugenia spp., Lillipilli Stackhousia monogyna, Aust native Callistemon spp. Kunzea spp. ORDER 12: EUPHORBIALES Mellaleuca spp.

Family: Buxaceae

Family: Punicaceae Buxus spp., European box

Punica granatum, pomegranite

Family: Euphorbiaceae

Family: Combretaceae Euphorbia spp., native weed

Combretum collinum Combretum hartmanniana ORDER 13: RHAMNALES Combretum leonense Anogeissus latifolia, gum ghatti Family: Rhamnaceae Anogeissus leicocargus Rhamnus spp., black juicy fruit

ORDER 7: RHIZOPHORALES Family: Vitaceae

Vitis vinifera, grapes

Family: Rhizphoraceae

Rhizophora spp., mangrove ORDER 14: LINALES

ORDER 8: CORNALES Faimly: Linaceae

Linum usitatissimum, flax

Family: Cornaceae

Cornus spp., dogwood ORDER 15: POLYGALALES Davidia involucrata

Family: Polygalaceae

Family: Garryaceae Polygala spp.

Garrya elliptica

Family: Tremandaceae

ORDER 9: SANTALES Tetratheca spp., Aust. shrub

Family: Santalaceae

Exocarpus spp., Aust native cherry

62 Table 1A (continued) SUBCLASS 6: ASTERIDAE ORDER 1: GENTIANALES

ORDER 16: SAPINDALES

Family: Asclepiadaceae

Family: Aceraceae Araujia hortum

Acer pseudoplatinus Acer saccharum Family: Apocynaceae

Vinca major, blue periwinkle

Family: Meliaceae

Khaya senegalensis, Khaya gum ORDER 2: SOLANALES

Family: Anacardiaceae Family: Solanaceae

Schinus molle, peppercorn tree Nicotiana alata Mangifera spp., mango Nicotiana sylvestris Anacardium spp., cashews Nicotiana tabacum Pistachio spp., pistachio Lycopersicon esculentum, tomato Solanum tuber osum, potato

ORDER 17: GERANIALES

Family: Convolvulaceae

Family: Tropaeolaceae Convolvulus tricolor

Tropaeolum majus, nasturtium Ipomoea muricata

Ipomoea batatas, sweet potato

Family: Balsaminaceae

Impatiens balsamina ORDER 3: LAMIALES

Family: Oxalidaceae Family: Boraginaceae

Oxalis spp., weed Myosotis spp., Forget-me-not Echium vulgare, Paterson's curse

Family: Geraniaceae Borago offinalis, borage

Geranium spp. Symphytum officinale, comfrey Pelargonium spp.

Family: Lamiaceae

ORDER 18: APIALES Mesona procumbens, Hsian tsao Prosanthera spp., Aust native

Family: Apiaceae Westringia spp., Aust native

Carum carvi, carroway Lavandula spp., lavender Daucus carota, carrot Mentha spp., mint

ORDER 5: PLANTAGINALES

Family: Plantaginaceae

Plantago indica Plantago afra, psyllium

63 Table 1A (continued) ORDER 11: ASTERALES

Family: Asteraceae

ORDER 6: SCROPHULARIALES Lactuca sativa, lettuce Helianthes annus sunflower

Family: Buddlejaceae Taraxacum officinale, dandelion

Buddleja spp., butterfly bush Cichorium intybus, chicory Cynara scolymus, Jerusalem artichoke

Family: Acanthaceae Silybum marianum, silymarin

Acanthus spp. Echinacea augustofolia

Family: Myoporaceae Other dicot species of uncertain

Myoporum spp., Aust native classification

Simmondsia chinensis, jojoba

Family: Scrophulariaceae Moraceae

Antirrhinum spp., snapdragons Artocatpus heterophyllus, jackfruit Digitalis spp., foxgloves Nyctaginaceae

Pisonia spp., para para

ORDER 7: CAMPANULALES

CLASS: LILIOPSIDA (MONOCOTS)

Family: Goodeniaceae

Goodenia spp., Aust native SUBCLASS 1: ALISMATIDAE Dampiera spp., Aust native ORDER 3: MAJADALES

Family: Campanulaceae

Wahlenbergia spp., bluebells Family: Zosteraceae

Phyllospadix spp., sea grass

ORDER 8: RUBIALES Zostera marina Zostera annuus

Family: Rubiaceae

Coffea arabica, coffee SUBCLASS 2: ARECIDAE Coprosma spp., Aust native Gardenia spp. ORDER 1: ARECALES

ORDER 9: DIPSACALES Family: Arecaceae (Palmae)

Arenga saccharifera

Family: Caprifoliaceae Borassus flabellifer

Lonicera spp., honeysuckle Cocos nucifera, coconut Viburnum opulus Phytelephas macrocarpa, Ivory nut Abelia spp. Elaeis guineensis, oil palm Hyphaene thebaica, doum palm Phoenix dactyliflora, date palm

64 Table 1A (continued) Edymiom nutans, nursery bulb ORDER 4: ARALES Lilium longiflorum

Scilla nonscripta, nursery bulb

Family: Araceae Aloe vera

Monstera deliciosa, monstera Homesia miniata, cape tulip Amorphophallus konjac

Family: Iridaceae

SUBCLASS 3: COMMELINIDAE Iris ochroleuca, iris Iris sibirica

ORDER 1: COMMELINALES Watsonia pyrimidata Gladiolus spp.

Family: Commelinaceae

Tradescantia albiflora, wandering jew Family: Agaveaceae

Agave sapp., succulent

SUBCLASS 4: ZINGERBERIDAE Agave sisalana, sisal Cordyline indivisa ORDER 1: BROMELIACAE Sansevierra trifasciata Polyanthes tuberosa, tuberose

Family: Bromeliaceae

Ananas comosus, pineapple Family: Haemodoraceae

Anigozanthus spp., kangaroo paw

SUBCLASS 3: COMMELINIDAE

CLASS: GYMNOSPERMS ORDER S: CYPERALES ORDER: CONIFERALES

Family: Poaceae (Graminaceae)

Avena sativa, oats Family: Pinaceae

Hordeum vulgare, barley Abies balsamea, Balsam fir

Lolium multiflorum, rye grass Picea abies, spruce

L. perenne, rye grass Picea sitchenis, Sikta spruce

L. temulentum, rye gass Pinus resinosa, red pine

Oryza sariva, rice Pinus sylvestris, pine

Panicum miliaceum, millet Larix occidentalis, larch

Saccharum officinarum, sugar cane Pseudotsuga menziesii

Triticum aestivum, wheat

Zea mays, corn Family: Cupressaceae

Phleum pratense, Timothy grass Cupressus spp.

Secale cereale, rye

Family: Podocarpaceae

SUBCLASS 5: LILIDAE Podocarpus elatus, plum pine

ORDER 1: LILIALES ORDER: GINKOGOALES

Ginkgo biloba

Family: Liliaceae

Allium cepa, onion Species of uncertain classification Allium sativum, garlic Yew Asparagus officinalis, asparagus Inula helenium, elecampane Tulipa gesneriana, tulip Pouteria cainito, abiu

65 Table IB: Preferred Plant Cells for Cultured Plant Cell Gum Production

Families Lycopersicon Carpobrotus dimidiatus

Actinidiaceae Malus Carpobrotus disparilis

Agavaceae Medicago Carpobrotus dulis

Aizoaceae Mesem bryan themum Carpobrotus edulis

Asteraceae Mimosa Carpobrotus fourcadei

Boraginaceae Neoporteria Carpobrotus glaucescens

Brassicaceae Nicotiana Carpobrotus laevigatus

Caesalpiniaceae Oryza Carpobrotus mellei

Convolvulaceae Panicum Carpobrotus modestus

Cucurbitaceae Passiflora Carpobrotus muirii

Fabaceae Pelargonium Carpobrotus pageae

Geraniaceae Phelum Carpobrotus pillansii

Malvaceae Polyanthes Carpobrotus pulleinii

Mimosaceae Primula Carpobrotus quadrifidus

Myrtaceae Pyrus Carpobrotus rossii

Passifloraceae Rosa Carpobrotus rubrocinctus

Palmae Sida Carpobrotus sauerae

Poaceae Solanum Carpobrotus sublatus

Primulaceae Symphytum Carpobrotus vanzijlae

Rosaceae Taraxacum Carpobrotus virescens

Solanaceae Trifolium Carpobrotus spp.

Tropaeolaceae Trigonella Cichorium intybus

Triticum Cocos nucifera

Genera Tropaeolum Cocos nucifera

Acacia Vaccinium (makapuno mutant)

Actinidia Zea Cucumis sativus

Aptenia Cucurbita maxima

Arabidopsis Species Cynara scolymus

Avena Acacia dealbata Echinacea augustafolia

Cactaceae Acacia Senegal Echinocactus grusonii

Caesalpinia Actinidia chinensis (kiwi Eucalyptus camaldulensis

Carpobrutus fruit) Glycine max

Cichorium Apentia cordifolia Hibiscus esculentus

Cocos Aptenia cordii Hordeum vulgare

Cucumis Arabidopsis thaliana Ipomoea batatas (sweet

Cucurbita Avena sativa potato)

Cynara Caesalpinia pulcherima Lactuca sativa

Echinacea Carpobrotus acinaciformis Lycopersicon esculentum

Echinocactus Carpobrotus aequilaterus Malus domesticus

Eucalyptus Carpobrotus chilense Medicago sativa

Glycine (also Mesembryanthemum Mesembryanthemum

Hibiscus chilense) aitonis

Hordeum Carpobrotus concavus M. alatum

Ipomoea Carpobrotus deliciosus M. albatum

Lactuca M. alborroseum

66 Table IB (Continued)

M. annum M. subtereticaule

M. barklyi M. subtruncatum

M. breve Mesembryanthemum spp.

M. chrysum Mimosa scabrella

M. clandestinum Neoporteria cormasensis

M. cordifolium Nicotiana alata

M. criniflorum Oryza sativa

M. cryocalyx Panicum milliaceum

M. cryptanthum Primula spp.

M. crystallinum Passiflora edulis

M. dejagerae Pelargonium spp.

M. edulis (Carpobrotus Phelum pratense edulis) Polyanthes tuberosa

M. excavatum Primula spp.

M. forsskalei Hochst Pyrus communis

M. galipinii Rosa glauca

M. guerichinanum Sida rhambifolia

M. hypertrophicum Solanum tuberosum

M. inachabense Symphytum officinale

M. inornatum Taraxacum officinale

M. intransparens Trifolium pratense

M. karrooense Trifolium repens

M. latisepalum Trigonella foenum-

M. liebendalense graecum (fenugreek)

M. lincarifolium Triticum aestivum

M. longipapillosum Tropaeolum majus

M. louiseae Vaccinium macrocarpon

M. macrophyllum Zea mays

M. macrostigma

M. nellsonlae

M. nodiflorum

M. pachypus

M. parvipapillatum

M. paucandrum

M. paulum

M. pellitum

M. perlatum

M. purpureo-roseum

M. quinangulatum

M. rhodanthum

M. rubroroseum

M. sedentiflorum

M. setosum

M. squamulosum

M. stenandrum

M. subrigidum

67 Table 2

Droplet size and turbidity measurement

Cone Droplet Turbidity

(& w/v) size (μm) ABS 500 nm Abs-Avr

Gum arabic

0 A Very large 0.049, 0.023 B Very large 0.013, 0.003 0.022

0.2 A Very large 0.006, 0.009 B 10-20 0.058, 0.022 0.023

0.5 A 10-20 0.042, 0.040 B 4-20 0.034, 0.037 0.038

1 A 6-20 0.145, 0.130 B 3-12 0.125, 0.1 12 0.128

5 A 1-8 0.429, 0.366 B 1-6 0.273, 0.277 0.336

10 A 1-10 0.482, 0.508 B 1-6 0.380, 0.384 0.438

20 A 0.5-3 0.505, 0.522 B 1-4 0.404, 0.427 0.464

68 Table 2 (continued)

Pear

0 A Very large 0.049, 0.023

B Very large 0.013, 0.003 0.022

0.2 A 1-20 0.150, 0.136 B 2-20 0.115, 0.110 0.127

0.5 A 1-8 0.187, 0.181 B 2-20 0.173, 0.194 0.183

A 1-8 0.240, 0.223 B 1-6 0.275, 0.270 0.252

A 1-2 0.577, 0.592 some larger

0.653

B 1-3 0.776, 0.670 some larger

A and B are duplicates.

69 Table 3

Shelf life stability

Cone Emulsion Description

(% w/v)

Before After Centrifugation

Gum arabic

0 Oil layer Oil layer Water layer Water layer

0.2 Oil film Oil layer Cream layer Water layer Water layer

0.5 Oil film Oil layer Cream layer Water layer Water layer

1 Cream layer Oil layer Water layer Cream layer Water layer

5 Cream layer Cream layer Water layer Water layer

10 Cream layer Cream layer Water layer Water layer

20 Cream layer Cream layer Water layer Water layer

70 Table 3 (continued)

Cone Emulsion Description

(% w/v)

Before After Centrifugation

Pear

Oil layer Oil layer Water layer Water layer

0.2 Oil film Oil film Cream layer Cream layer Water layer Water layer

0.5 Oil film Oil film Cream layer Cream layer Water layer Water layer

Cream layer Cream layer Water layer Water layer

Cream Cream

Descriptions taken from duplicate emulsions.

71 Table 4: Composition and Selected Properties of Plant Cell Gums RELATIVE PROPORTIONS (%

Carvophyllidae Dilleniidae

Aizoacea Actinidiaceae Malvaceae Cucurbitaceae

COMPONENT

Mesembryanthemum Actinidia deliciosa Hibiscus Sida Cucumis sativus o esculentus rhombifolia

"Pigface" Kiwi fruit Okra Paddy's lucern Cucumber

Protein & Ash² 26 41 30 52 33 s Xyloglucan 27 21 18 14 2

Galactoglucomannan 6

Glucomannan . . 3 3

3.6-Arabinogalactan (Type II) 18 21 17 23 15 ro

CΛ Heteroxvlan tr 2 1 2 5

4-Galactan (Type I) 1 . .

H Arabinan 4 1 1 2 31

Glucuronomannan . . . 4

Rhamnogalacturonan/ 19 (55) 8 36 (32) 3 3 Galacturonan

O Root-slime-like material _ _ _ _ _

Emulsification . 1.5 nt 56 10.6 13 Droϋlet Size 1% Gum⁴

Gelling 1% Gum⁵ NO NO NO NO NO

Viscosity (Centipoise) ⁶:

1/S 1 1 nt nt nt nt o

100/S 7 1.9 2.5 1.7 1.6 H

C vθ 00

TABLE 4: Continued

RELATIVE PROPORTIONS (%)'

Rosidae V© ~O

Roseace Mimosaceae Fabaceae (Λ_^ -4 > VO

Rosa Pyrus Malus Acacia Acacia Trigonella Medicago Trifolium Trifolium Trifolium Glycine VO glauca communis domesticus Senegal Senegal foenum- sativa repens pratense pratense max

COMPONENT eraceum

Rose Brown pear Apple (MS 5) (MS 7) Fenugreek Alfalfa White Red Red Clover Soy Pauls Clover Clover Scarlet

Protein + ash² 86 28 49 60 41 58 56 48 76

Xyloglucan 3 17 17 36 52 9 7 4 26 5 5

Galactoglucomannan .. tr

Glucomannan 1 . . . . 3 .

3,6-Arabinogalactan 5 30 26 37 31 20 27 23 1 1 16 24 (Type II)

Heteroxylan tr 2 2 3 4 5 2 2 1 1 1

4-Galactan tr - - - - - - - - - - fType I)

Arabinan tr 7 . . . 4 5 3 2 7 1

Glucuronomannan . 4 . . _ . . . . 1 1

Rhamnogalacturonan/ 3 6 1 25 2 tr 9 (10) 5 (10) - 6 (7) tr Galacturonan

Root-slime-like material _ _ . _ _ _ _ _ _ _ _

Emulsification⁴ 52 27 9 nt nt 14 16 16 44.1 21.2 35 Droplet size (\ % gum)

Gelling at 1 % gum⁵ NO NO nt nt nt NO NO NO NO NO NO n

Viscosity (Centipoise)⁶: 1/S nt 129 nt nt nt nt nt nt nt nt nt 100/S 4.2 19 nt nt nt 2.1 1.3 1.6 1.7 1.7 1.9 c VO 00

o o

TABLE 4: Continued

RELATIVE PROPORTIONS (%Ϋ

Asteriade

Solanaceae Asteracea VO VO

COMPONENT 5i

Solanum Lycopersicon Nicotiana alata Nicotiana Cichorium Letuca sativa ~o tuberosum esculentum plumbaginiintybus Nt

VO folia VO

Potato Tomato Tobacco Ornamental Chicory Lettuce Tobacco

Protein & Ash² 49 38 55 10 52 70

Xyloglucan 15 9 8 32 5 4

CΛ

Galactoglucomannan 12 10 7 13

H Glucomannan . . . . 3 2

3 ,6- Arabinogalactan 14 16 13 10 20 6 (Type II)

4* Hctcroxylan 3 I 2 1 1 1

CΛ

4-Galactan - - - - 1 - (Type I)

M

H Arabinan 2 1 2 . 3 1

1 Glucuronomannan . . 1 22 (40)

Rhamnogalacturonan/ 3 22 ( 1 1 ) 7 13 (5) 14 (57) 13 w Galacturonan

ON Root-slime-like material _ _ _ _ _ _

Emulsification⁴ Droplet size ( 1 % gum) 35 32 56 15 13.6 7.4

Gelline at 1 % gum⁵ NO NO NO NO NO NO

Viscosity (Centipoise)⁶: TJ Rate l s-1 9 8.7 nt 140 nt nt O

Rate l OOs-l 2.3 4.4 nt 55 1.7 1.9 H

G

VO 00 oo

TABLE 4: Continued

RELATIVE PROPORATIONS (%)'

Commelinidae (Monocot) Lilidae (Monocot)

Poaceae Agavaceae ϋi

COMPONENT

Phleum Panicum Panicum Phalaris Oryza Hordeum Zea Polianthus Polianthus pratense millaceum millaceum aauaticus sativa vulsare Mavs tuberosa tuberosa

Timothy Millet Millet, Phalaris. Rice , Barley Maize (petal) (leaf) grass (MS3V (MS 9)⁷ (MS 6)⁷ Paddv Pelde⁷ Schooner

Protein & Ash² 12 39 35 58 70 66 34 54 51

Xyloglucan 1 1 4 5 2 3 1 3 3

Galactoglucomannan . .

Glucomannan . 6 3 2 2 1 1 2 15

3,6-Arabinogalactan 3 - - - - 4 7 6 20 (Type II)

Heteroxvlan 1 1 7 14 9 4 16 44 2 6

4-Galactan 1 7 12 tr tr tr 3 1 - (Tvpe I)

Arabinan . 3 6 1 2 5 . 1 2

Glucuronomannan . . . . . 25

Rhamnogalacturonan/ - - - - - tr - 2 1 Galacturonan

Root-slime-like material 53 34 25 21 14 _ » _

Emulsification: Droplet size (1% gum) 20 29 17 35 6.9 38.6 9.3 30 49

Gelling at 1% gum⁵ YES NO NO NO NO NO NO NO NO

-d

Viscosity (Centipoise):⁶ O

1/S 4500 1800 1800 10.4 nt nt nt 6.7 nt H

100/S 150 30 25 6.7 1.8 1.5 2 1.9 2.1

C

© o

00

Table 4: FOOTNOTES

Calculated as the sum of mol% of individual monosaccharide residues; tr means trace amounts; the percentage of components listed may add up to only 3 i approximately 100% because the assumptions regarding structure may be inaccurate, such that the presence of minor functional groups such as methyl or acetyl ^ groups may not have been taken into account. Jg

IΛ

Protein and ash values in bold are assumed from total sugar determinations by colorimetric analysis. jj

Rhamnogalacturonan and galacturonan cannot be separated by these analyses; the degree of methyl esterification in parentheses. ^*°

⁴Droplet size measured in μm; nt means not tested. ⁵The presence (YES) or absence (NO) of gelling is determined by visual observation; nt means not tested

Viscosity measured as a function of shear rate in reciprocal seconds; nt means not tested 'Relative proportion of polysaccharide deduced by comparison of linkage analysis from fractionated timothy grass biopolymer

n c vO go

© o

00

Table 5:

Species Explant Medium¹ No. subNo subAge of culture cultures cultures at harvest (callus) (suspension) (davs)

Dicots

Caryophylhdae Aizoaceae

Mesembryanthemum leaf MS2 25 6 13

Dillenπdae Actimdiaccae

Actinidia dehciosa fruit with seed MS9 10+ 4 21

Malvaceae Hibiscus esulentus seedling stem MS9 7 4 13 Sida rhomifoha seedling stem MS9 20 4 14

Rosidae Rosaceae

Pvrus communis³ fruit pear BAL 24 7 1 1

Fabaceae Medicago sativa seed MSI + NOA 20+ 3 6 Trifolium repens seed MS9 10+ 3 14 Trifolium pratense seed MS6 0 3 1 1

Asteπdae Solanaceae

Solanum tuberosum growing points MS6 18 5 18

Lycopersicon fruit esculentum tomato BAL 20+ 2 12

Asteraceae Cichorium intvbus DSM-Gmbh⁴ LS 1 1 + 7 1 1

Monocots

Commelinidae Poaceae

Phleum pratense seed MS + 2,4-D⁵ 0 3 14

Panicum mihaceum seed MS3/MS9 2 4 14

Lihdae Agavaceae

\Polianthes tuberosa leaf MS17 10 3 ..

'MSI = MS powder with 0 5 mg/L 2,4-D (2,4-dιchlorophenoxyacetιc acid), MS2 = MS powder with 2 0 mg/L 2,4-D, MS3 = MS powder with 4 0 mg/L 2,4-D, MS6 = MS powder with 4 0 mg/L 2,4-D and 1 075 mg/L mixed cytokinins, MS9 = MS powder with 4 0 mg/L 2,4-D and 2 150 mg/L mixed cytokinins,

MS 17 = MS powder with 2 0 mg/L NAA (1-naphthaleneacetιc acid) and 2 0 mg/L BAP (6-benzylamιnopuπne), LS = LS powder with 0 2 mg/L 2,4-D and 0 04 mg/1 NAA, see text Example 14, tomato BAL, see text Example 14, NOA = napthoxyacetic acid used at final concentration 0 5 mg/L, Concentration of 2,4-D m Phleum pratense culture is 2 0 mg/L

² "+" means more, 1 e , 10+ means more than 10

³ Brown pear Commercial source of plant cell culture, Deutsche Sammlung von Mikroorgamsmen und Zelkulturen

⁵ Millet has been grown on both MS3 and MS9 with substantially the same results The specific results shown were obtained on MS9

77 Table 6: Polysacchaπde Production in Selected Suspension Cell Cultures

CELL LINE EXPLANT MEDIUM¹ POLYSACCHARIDE² grams/litre

DICOTS

Mesembryanthemum sp leaves MS2 3.1 "vivace

Kiwi Fruit fruit MS I 0.06 Actinidia dehciosa svn chinesis

Okra seedling stem MS9 3.25

Hibiscus esculentts

Paddy's lucern seedling stem MS9 0.5 Sida rhambifoha

Pear fruit Pear BAL 3-4

Pvrus communis

Alfalfa seed MS+NOA 1.25 Medicaso sativa

White clover seeds MS9 0 85 Trifolium repens

Red clover seeds MS6 1 9 Trifolium pratense

Potato - red growing points MS5 and MS6 1 0 Solanum tuberosum

Tomato fruit Tomato BAL 1.75

LvcoDβrsicon esculentum

Nicotiana exisiting CSV 3.5-4.5 Nicotiana culture plumbasinofoha

Chicory DSM-Gmbh⁵ LS 1.6 Cichorium intvbus

Monocots

Timothy Grass seed MS + 2,4-D 6.0 Phleum pratense

Millet seeds MS9

Panicum mihaceum

Tuberose leaf MS17 2.7 Polvanthes tuberosa

Tuberose petal TB 1 1.9

Polvanthes tuberosa

¹ See footnote 1 Table 5 for media descriptions;

CSV see text Example l.B; TB1 see text Example 14.

² The polysacchaπde concentration listed is the maximum concentration obtained in growth cycle measurements;

Polysacchaπde is typically harvested on the culture day on which this maximum occurs; Tuberose petal gum was harvested on day 23.

³ Brown pear

⁴ The Nicotiana plumbaginifolia culture was obtained from Paul Ebert of the School of Botany of the University of Melbourne (February 1989).

Commercial source; Deutsche Sammlung von Mikroorgamsmem und Zelkulturen.

78

Claims

CLAIMS:

1. An isolated plant cell gum produced in suspension cell culture of a plant of the family Aizoaceae.

2. An isolated plant cell gum according to claim 1 wherein the plant is of the genus Mesembryanthemum.

3. An isolated plant cell gum according to claim 1 wherein the plant is of the genus Aptenia or Carpobrotus.

4. An isolated plant cell gum according to claim 2 wherein the plant is Mesembryanthemum spp. or Carpobrotus spp..

5. An isolated plant cell gum according to claim 2 wherein the plant is Mesembryanthemum crystallinum, Mesembryanthemum nodiflorum, Mesembryanthemum cordiflorum, Mesembryanthemum criniiflorum, Mesembryanthemum aitonis,

Mesembryanthemum forsskalei Hochst, Mesembryanthemum barklyi, or Carpobrotus chilense.

6. An isolated plant cell gum according to any of claims 1-5 wherein the gum comprises xyloglucan.

7. An isolated plant cell gum according to any of claims 1-5 wherein the gum comprises rhamnogalacturonan.

8. An isolated plant cell gum according to any of claims 1-5 wherein the gum comprises AGP.

9. An isolated plant cell gum according to any of claims 1-5 wherein the gum comprises xyloglucan and rhamnogalacturonan.

79

10. An isolated plant cell gum according to claim 9 wherein the gum further comprises AGP.

11. A process for manufacturing an industrial, pharmaceutical, veterinary or cosmetic product which process comprises a step of including a viscosifying agent, thickening agent, gelling agent , emulsifying agent, suspending agent, encapsulating agent, flocculating agent, film-forming agent, sizing agent, adhesive agent, binding or coating agent, lubricating agent, water retention agent or coagulation agent or any combination of said agents in the manufactured product wherein a cultured plant cell gum of a plant of the family Aizoaceae is employed as the agent.

12. The process of claim 11 wherein the cultured plant cell gum is that of a plant of the genus Mesembryanthemum.

13. The process according to claim 11 wherein the cultured plant cell gum is that of a plant of the genera Aptenia or Carpobrotus.

14. The process according to claim 11 wherein the cultured plant cell gum is that of Mesembryanthemum spp. or Carpobrotus spp.

15. The process according to claim 11 wherein the cultured plant cell gum is that of the plant Mesembryanthemum crystallinum, Mesembryanthemum nodiflorum, Mesembryanthemum cordiflorum, Mesembryanthemum criniiflorum, Mesembryanthemum aitonis, Mesembryanthemum forsskalei Hochst, Mesembryanthemum barklyi, or Carpobrotus chilense.

16. A process according to any of claims 11-15 wherein the process comprises including an emulsifying agent in the manufactured product and the cultured plant cell gum is employed as the emulsifying agent.

17. A process according to claim 16 wherein the manufactured product is an emulsion of agricultural chemicals.

80

18. A process according to claim 16 wherein the manufactured product is an agricultural pesticide.

19. A process according to claim 18 wherein the cultured plant cell gum is employed to form or stabilize a cloud emulsion.

20. The process of any of claims 11-15 selected from the group consisting of manufacturing processes for sheet paper, an adhesive, an oil and gas well fluid, an ink formulation, a lithography solution, a textile, a textile dyestuff, paint, ceramic glaze, ceramics, cleaning detergent, a pet food, a slow release veterinary composition, a cosmetic lotion and a cosmetic cream.

21. A manufactured product which comprises a cultured plant cell gum.

22. The product of claim 21 which is an ink, a textile, paint, ceramics, an explosive, a cleaning agent or detergent, a cosmetic cream, a shampoo, conditioner or cream rinse, an encapsulated oil, an adhesive, sheet paper, a controlled-release veterinary product, or a canned pet food .

23. A process for making a food product which comprises the step of adding a cultured plant cell gum of a plant of the family Aizoaceae to a food product.

24. A process for making a food product which comprises a step of adding a viscosifying agent, thickening agent, gelling agent, emulsifying agent, an emulsion stabilizing agent, suspending agent, encapsulating agent, enrobing agent, binding or coating agent, or a texture modifier or any combination of thereof to a food product wherein the agent comprises a cultured plant cell gum of a plant of the family Aizoaceae.

25. A process according to either claim 23 or 24 wherein the cultured plant cell gum is that of a plant of the genus Mesembryanthemum.

26. A food product according to claim 23 or 24 wherein the plant cell gum is that of a plant of the genera Aptenia or Carpobrotus.

27. A process according to either of claim 23 or 24 wherein the cultured plant cell gum is that of Mesembryanthemum spp. or Carpobrotus spp.

28. A process according to either of claims 23 or 24 wherein the cultured plant cell gum is that of Mesembryanthemum crystallinum, Mesembryanthemum nodiflorum, Mesembryanthemum cordiflorum, Mesembryanthemum criniiflorum, Mesembryanthemum aitonis, Mesembryanthemum forsskalei Hochst, Mesembryanthemum barklyi or Carpobrotus chilense.

29. A process according to any of claims 23-28 wherein an emulsifying agent is added to the food product.

30. The process according to claim 29 wherein the food product is a soft drink.

31. A food product that comprises a viscosifying agent, thickening agent, gelling agent, emulsifying agent, an emulsion stabilizing agent, suspending agent, encapsulating agent, enrobing agent, binding or coating agent, or a texture modifier or any combination of thereof wherein the agent comprises a cultured plant cell gum of the family Aizoaceae.

32. A food product according to claim 31 wherein the plant cell gum is that of a plant of the genus Mesembryanthemum.

33. A food product according to claim 31 wherein the plant cell gum is that of a plant of the genera Aptenia or Carpobrotus.

34. A food product according to claim 31 wherein the plant cell gum is that of Mesembryanthemum spp. or Carpobrotus chilense.

82

35. A food product according to claim 31 wherein the plant cell gum is that of Mesembryanthemum crystallinum, Mesembryanthemum nodiflorum, Mesembryanthemum cordiflorum, Mesembryanthemum criniiflorum, Mesembryanthemum aitonis, Mesembryanthemum forsskalei Hochst, Mesembryanthemum barklyi or Carpobrotus chilense.

36. A food product according to any of claims 31-35 comprising a plant cell gum that is an emulsifying agent or an emuslion stabilizing agent.

37. A food product according to any of claims 31-36 wherein the plant cell gum is used to form or stabilize a cloud emulsion.

38. A food product according to any of claims 31-36 which is a soft drink.

39. A food product according to any of claim 31-38 which comprises an encapsulated oil powder.

40. A food product according to any of claims 31-36 which is a dietary fiber supplement.

41. In an industrial, food, pharmaceutical or cosmetic manufacturing process in which a plant exudate or plant extract gum is employed as a thickening, emulsifying, suspending, waterproofing, gelling, protective colloid, stabilizing or coating agent, the improvement wherein said plant exudate or extract gum is replaced with a cultured plant cell gum of a plant of the family Aizoaceae.

42. The process of claim 41 wherein the cultured plant cell gum is that of a plant of the genus Mesembryanthemum.

43. The process of claim 41 in which gum arabic is used as a viscosifying, emulsifying or gelling agent, the improvement wherein a cultured plant cell gum of a Aizoaceae plant is substituted for gum arabic.

83

44. A process for manufacturing a food, industrial, veterinary, pharmaceutical or cosmetic product which process comprises a step of including a viscosifying agent, thickening agent, gelling agent , emulsifying agent, suspending agent, encapsulating agent, flocculating agent, film-forming agent, sizing agent, adhesive agent, binding or coating agent, lubricating agent, water retention agent or coagulation agent or any combination of said agents in the manufactured product wherein a cultured plant cell gum of a plant of the families: Actinidaceae, Agavaceae, Aizoaceae, Asteraceae, Cucurbitaceae, Fabaceae, Malvaceae, Mimosaceae, Poaceae, Rosaceae, and Solanaceae.

45. A process as in claim 44 wherein the cultured plant cell gum is of a plant of the genera: Acacia, Actinidia, Chichorium, Cucumis, Glycine, Hibiscus, Hordeum, Letuca,

Lycopersicon, Malus, Medicago, Mesembryanthemum, Carpobrotus, Oryza, Panicum, Phalaris, Polianthus, Sida, Solanum, Trifolium, Trigonella, and Zea.

46. A process as in claim 44 wherein the cultured plant cell gum is that of a plant of the species: Acacia Senegal, Actinidia deliciosa, Chichorium intybus, Cucumis sativus, Glycine max, Malus domesticus, Medicago sativa, Mesembryanthemum spp., Oryza sativa,

Panicum miliaceun, Hibiscus esculentus, Hordeum vulgare, Letuca sativa, Lycopersicon esculentum, Malus domesticus, Phalaris aquaticus, Polianthus tuberosa, Sida rhombifolia, Solanum, Trifolium repens, Trifolium pratense, Trigonella foenum-graceum., and Zea mays.

47. A process according to any of claims 44-46 wherein the process comprises including an emulsifying agent in the manufactured product and the cultured plant cell gum is employed as the emulsifying agent.

48. A process according to any of claims 44-46 wherein the process comprises including a gelling agent in the manufactured product and the cultured plant cell gum is employed as the gelling agent.

49. A process according to any of claims 44-46 wherein the process comprises including a viscosifying agent in the manufactured product and the cultured plant cell gum is employed as the viscosifier.

84

50. A process according to any of claims 44-46 wherein the process comprises including an encapsulating agent in the r.ianufactured product and the cultured plant cell gum is employed as the encapsulating agent.

51. A process according to claim 50 wherein the product is an encapsulated lemon oil for use in detergents and cleaning agents.

52. A process according to any of claims 44-46 wherein the product is an agricultural spray.

53. A process according to any of claims 47-52 wherein the plant cell gum is a gum of a plant of the family Poaceae.

54. A process according to any of claims 47-52 wherein the plant cell gum is a gum of a plant of the genus Panicum.

55 . A process acording to any of claims 44-50 wherein the product is a food product.

56. A process according to any of claims 44-50 wherein the product is a dietary fiber supplement.

57 . A process according to any of claims 44-46 wherein the product is a edible capsule particularly an oil-containing capsule and wherein the plant cell gum is employed to form the capsule.

85 AMENDED CLAIMS

[received by the International Bureau on 25 August 1999 (25.08.99); original claim 21 amended; remaining claims unchanged (lpage)]

21. (Amended) A manufactured product which comprises a cultured plant cell gum of a plant of the family Aizoaceae.

AMENDED SHEET (ARTICLE 19) 86