EP0586453A1

EP0586453A1 - Stabilisation of plant extracts

Info

Publication number: EP0586453A1
Application number: EP92910632A
Authority: EP
Inventors: Piotr Marek Kalinowski
Original assignee: BTG International Ltd; British Technology Group Ltd
Current assignee: BTG International Ltd
Priority date: 1991-05-28
Filing date: 1992-05-27
Publication date: 1994-03-16
Also published as: GB9211220D0; GB2256600A; AU1765292A; GB9111441D0; WO1992021247A1; JPH06507612A; GB2256600B; CA2110038A1

Abstract

Procédé de production d'un extrait en phase de solvant lipophile stabilisé de tissu végétal cellulaire consistant à ajouter un solvant lipophile non volatil à un jus aqueux obtenu par addition d'un solvant polaire miscible dans l'eau à un tissu végétal cellulaire ou à un jus extrait dudit tissu et à séparer un extrait en phase aqueuse d'un extrait stabilisé en phase de solvant lipophile.Process for the production of an extract in the stabilized lipophilic solvent phase of cellular plant tissue, comprising adding a non-volatile lipophilic solvent to an aqueous juice obtained by adding a polar solvent miscible in water to a cellular plant tissue or to a juice extracted from said tissue and to separate an extract in the aqueous phase from an extract stabilized in the lipophilic solvent phase.

Description

STABILISATION OF PLANT EXTRACTS This invention relates to processes for the production of stabilised extracts from cellular tissues especially plant materials and to the products of such processes.

Considerable research has taken place on methods for the production of a leaf protein concentrate from plants especially those which are too fibrous and/or unpleasant tasting to be suitable for consumption by humans or by onogastric animals. This research centered on green crop fractionation in which a forage crop or vegetable waste was macerated and pressed to produce a partially dewatered fibrous residue potentially useful as a ruminant feedstuff and a proteinaceous juice potentially useful as a foodstuff for human or non-ruminant animals or as a raw material for further processing.

The principle obstacle to the processing of such plant juices is that they are rapidly degraded especially in the presence of oxygen and at ambient temperatures such as those which prevail during the seasons of rapid crop growth. Furthermore water soluble anti-nutrients, e.g. saponins in lucerne (Medicago sativa). oestrogens in clovers (Trifolium spp). alkaloids in potato tops (Solanum tuberosum) oxalates in sugar beet tops (Beta vulαaris) and glucosinolates in brassica vegetables (Brassica spp) are often present and exhibit unacceptable taste properties or toxic properties. For these reasons such juices are of no practical use unless utilised immediately after expression and any antinutrients present rendered harmless and/or tasteless.

There have been a variety of proposals as to how these problems might be overcome and useful stable products isolated. Particular attention has been paid to the production of a proteinaceous material, which is useful as a foodstuff for human or animal use. Commercial processes currently used inject live steam into the plant juice to coagulate a proteinaceous curd which is separated from a supernatant whey, dried and stored under nitrogen. There have been other proposals whereby a proteinaceous product might be separated from a plant juice, e.g. by Huang e± __.. , Canadian Institute of Food Technology, Vol. 4 (3), p85-90 (1970), who added a polar solvent to lucerne plant juice and stored the mixture overnight at 4^βC whereupon a proteinaceous material was precipitated. Huang suggested that the recovery of useful materials from the supernatant liquor would be desirable if only for -economic reasons but gave no indication as to how this might be achieved. In practice, removal of the solvent from such liquors produces a residue which rapidly darkens upon exposure to air and is of no value. This is not unexpected since several of the materials dissolved in the supernatant liquor such as chlorophylls, carotenoids and unsaturated fatty acids are known to be sensitive to heat and atmospheric oxygen.

We have now discovered that the addition of a non-volatile lipophilic solvent to an aqueous liquor obtained b the addition of a water miscible polar solvent to a cellular plant tissue or to a liquor which has been expressed from a cellular plant tissue leads to the separation of an aqueous phase and a lipophilic solvent phase which are relatively stable and which find use either as such or as raw materials from which new and useful products may be obtained. Accordingly from one aspect this invention provides a process for the production of a stabilised extract from a cellular plant tissue which comprises the addition of a non-volatile lipophilic solvent to an aqueous liquor obtained by the addition of a water miscible polar solvent to a cellular plant tissue and separating an aqueous phase extract and a lipophilic solvent phase extract.

The aqueous liquor may be prepared by standard procedures well known in the art. Normally these will involve crushing and/or maceration and/or pressing of plant materials. The methods for obtaining such a liquor, the potentially useful crops from which a liquor could be obtained and the valuable products which could potentially be obtained have been reviewed, e.g. in "Leaf Protein Concentrates" edited by L. Telek and H. D. Graham, 1st Edition, published by the AVI Publishing Company Inc., of Westport, Connecticut, USA (1983) and also in "Proceedings of the 2nd International Conference on Leaf Protein Research" edited by I. Tasaki , Nagoya University, Japan (1986).

In temperate countries potentially useful crops include existing forage crops such as lucerne, clovers and ryegrasses, waste crop materials from vegetable processing plants such as brassicas, under or unutilised waste parts of crops such as sugar beet tops, potato haulm, pea haulm and tomato haulm, and water weeds. Other plants which are not currently grown as food crops may be viable crops when processed according to this invention, examples being Jerusalem Artichoke (Helianthus tuberosus). Fat Hen (Chenopodium album). Cowpea (Vigna spp) and Nettle ⁽Urtica spp). Another plant material which may be useful is tobacco leaf. Lists of plants which may be grown in tropical climates and are potentially useful sources of plant material are presented in the publications which are referred to above, the relevant disclosures of which are hereby incorporated by reference.

An alternative which may be necessary for those tissues from which a stable, aqueous liquor cannot be conveniently expressed comprises the addition of a water miscible polar solvent directly to the plant tissue. The tissue will normally be macerated prior to that addition and may, optionally, be dried. An aqueous liquor may then be separated from the fibrous residue and processed according to this invention.

The process of this invention may also utilise liquors obtained from other sources as a raw material. Examples include plants which are host to heterologous protein material by means of genetic manipulation; plant materials such as garlic cloves, red pepper pods (Capsicum spp). herbs (medicinal or culinary), flower petals and fruit skins; reconstituted plant materials obtained by wetting dried plant material and microscopic plants such as algae, bacteria and yeasts. The term aqueous plant juice is used in this specification to encompass liquors which can be expressed from any such or similar materials either before or after addition of water miscible solvent. The juice may be concentrated prior to the addition of the water miscible solvent. The concentration is preferably carried out whilst maintaining the juice at a temperature below that at which the proteins will coagulate, e.g. by using freeze concentration. The addition of the water miscible polar solvent will normally and also preferably lead to the deposition of a proteinaceous material either as a precipitate in the aqueous liquor or as a deposit within the fibrous residue. Such proteinaceous materials are potentially valuable as foodstuff, or feedstuffs and processes which lead to the production of such materials represent a preferred aspect of the invention. The water miscible solvent is preferably a polar solvent more preferably one having a Hildebrand solubility parameter of from 9.0 to 11.0 or one which leads to the formation of an aqueous liquor having a solubility parameter falling within this range. The solvent will preferably be one which does not form an azeotrope with water, is capable of hydrogen bonding and most preferably is non-toxic to both humans and animals. The solvent is also preferably a relatively volatile solvent since such solvents can be more readily separated from the remainder of the aqueous phase. Examples of preferred solvents include lower alkanones such as acetone, 2-butanone, 2-pentanone and 3-pentanone and the lower alkanols such as ethanol , ethanol , propanol and butanol used either singly or as mixtures.

The quantity of water miscible solvent which is required in order to bring about precipitation of proteinaceous material varies according to the nature of the liquor, the water content of the liquor, the nature of the solvent and the temperature at which the addition is carried out. Typically the ratio of the volume of solvent to the volume of plant juice will be in the range 15:1 to 1:1, preferably within the range 5:1 to 3:1. The quantity of water miscible solvent used should preferably be at least sufficient as will bring a major part or all the lipophilic material into solution. The ability of such polar solvents to extract lipophilic substances from plant material is influenced in part by the water content of the resulting aqueous liquor. Lower water content will generally result in greater extraction. By controlling the level of water content it is possible to leave the non-polar lipophilic substances, such as fatty acids and diglycerides unextracted, if it is considered desirable to do so. Counter current or serial extraction techniques will help minimise the quantity of water miscible solvent required to extract the non-polar lipophilic substances.

The addition of water miscible solvent may take place at any temperature which is above the freezing point of the mixture and preferably below that at which any significant denaturation of the protein will occur, e.g. less than 45°C. Preferably the addition will take place at relatively low temperatures, e.g. from -5^βC to 10°C or from 0°C to 5°C. The liquor is preferably maintained at such a temperature prior to the addition of the solvent in order to minimise degradation of its contents. Conveniently the water miscible solvent may also be pre chilled to a temperature in this range.

The proteinaceous material which is precipitated or deposited may be and preferably is separated from the supernatant liquor at this stage using conventional techniques such as decantation or filtration. This separation may be carried out at any later stage of the process but in general the difficulties in handling a suspension of proteinaceous material in the supernatant liquor mean that this represents a less preferred option. The proteinaceous precipitate may comprise significant proportions, typically up to 50% by weight of the water miscible solvent. The precipitate may be washed with a further quantity of the water miscible solvent and the washings added to the supernatant liquor. The residual content of water miscible solvent may be removed to leave a product which will generally take the form of a grey or brown powder consisting substantially of proteinaceous material or where the liquor was produced from a macerated tissue a fibre rich proteinaceous material which may be useful as a foodstuff or feedstuff for both human or animal use. The water content of the powder may be reduced if it is to be stored for a significant period prior to being utilised.

The supernatant liquor comprises water, water miscible solvent, water soluble salts, water soluble antinutrients, vitamins, carbohydrates lipids and other lipophilic materials. The water miscible solvent may be removed at this stage providing the remaining material is maintained under an inert non-oxidising atmosphere and preferably also at a temperature which is sufficiently low to avoid any substantial degradation of the contents of the liquor until such time as the lipophilic solvent is added. However, in the preferred embodiments the process comprises the addition of a lipophilic solvent to the supernatant iquor.

A wide variety of non-volatile lipophilic solvents may be utilised. The preferred lipophilic solvents are those which we have discovered serve to form a lipophilic solvent phase in which the solute is stabilised against degradation. The most preferred lipophilic solvents are those which are themselves acceptable for use in the food, feed, cosmetic and pharmaceutical industries. Examples of preferred lipophilic solvents are vegetable oils such as corn, sunflower, rapeseed, groundnut, soya, cottonseed, castor, olive, grapeseed, palm and palm kernel oils together with hydrogenated derivatives thereof, animal oils such as fish oils and mineral oils. The most preferred solvents for use in the processes of this invention are the vegetable oils and their hydrogenated derivatives.

A less preferred embodiment comprises the addition of a volatile water immiscible lipophilic solvent such as hexane or pentane to the liquor. Such solvents are effective in extracting the lipophilic material but the solvent phase product is not commercially useful. Such solvent phases may be mixed with a non-volatile lipophilic solvent and the volatile solvent removed from the mixture to produce a non-volatile lipophilic solvent phase. The quantity of lipophilic solvent which is added to the supernatant liquor will be at least that which is required to form a separate phase under the conditions employed. The quantity used may vary through a wide range depending upon the nature of the plant juice and hence the nature of the liquor, the nature of the lipophilic solvent and the nature of, and the proportion of any water miscible solvent which may be present.

The supernatant liquor and the lipophilic solvent are preferably shaken or stirred to ensure thorough mixing. The formation of emulsions is highly undesirable and the mixing is preferably carried out in a manner which minimises the tendency for emulsions to form. The mixing will preferably be carried out at a temperature which minimises the degradation of the contents of the supernatant liquor, e.g. at less than 45^βC and preferably less than 10^βC. The lipophilic solvent may be chilled prior to its addition to the liquor and the solvent selected should be one which is liquid at the temperature at which the mixing is to be effected. The mixture is then allowed to separate into an aqueous solvent phase and a lipophilic solvent phase. These two phases may then be separated. The contents of the supernatant liquor will partition themselves between the aqueous solvent phase and the lipophilic solvent phase and the mixing stage should be carried out under conditions which allow them to do so. The lipophilic content of the liquor will partition itself preferentially in the lipophilic solvent phase according to the partition laws of organic chemistry. The overall proportion of lipophilic material present in the liquor which is extracted into the lipophilic solvent phase may be increased by using multiple step solvent extraction procedures in accordance with known techniques in the art of solvent extraction. These multiple step procedures generally involve using the same overall volume of lipophilic solvent but adding it in a series of increments each of which should be big enough to ensure that a separate lipophilic solvent phase may be formed and separated. In general such multiple step extraction procedures may comprise up to three extraction steps although a greater number may be utilised if the economics of the process justify it. Total separation of lipophilic materials from the aqueous phase cannot be achieved in this embodiment. The aqueous phase will in this embodiment comprise the major portion and usually of all the water miscible solvent. The water miscible solvent is preferably recovered from the aqueous phase and this may be most conveniently achieved by a distillation step. Any remaining lipophilic materials which are heat labile may be degraded by heat distillation as will any heat labile hydrophilic materials. Such degradation is disadvantageous but this disadvantage may be acceptable if it allows the water miscible solvent to be recovered in an economic fashion. An alternative embodiment of the processes of this invention comprises the removal of the water miscible solvent from the mixture of the supernatant liquor and the lipophilic solvent prior to the separation of the aqueous phase and the lipophilic solvent phase. This removal is preferably carried out at low temperatures, e.g. less than 40-45°C, preferably less than 10°C in order to minimise degradation of the contents of the liquor and of the lipophilic solvent phase. It may conveniently be achieved using vacuum distillation or by blowing off the water miscible solvent using an inert gas. The mixture will preferably be agitated during this removal process but again it is preferred to avoid the formation of emulsions wherever possible.

After removal of the water miscible solvent the mixture will separate into two phases. Commonly the relative densities of the phases will be reversed, the lipophilic solvent phase now lying above the aqueous phase.

This embodiment is advantageous insofar as it enables substantially all of the lipophilic material to be removed from the aqueous phase into the lipophilic solvent phase. It enables an aqueous phase which comprises substantially all of the water soluble content of the original plant tissue to be isolated. This material may not have been exposed to elevated temperatures and thus little or no degradation of the heat labile water soluble material need have taken place. Such aqueous phases are believed to be novel and form a further aspect of the invention.

The lipophilic solvent phase isolated from the processes of this invention may exhibit improved properties by virtue of the fact that it comprises little or none of the water soluble contents of the leaf plant concentrate. It may be susceptible to degradation upon exposure to oxygen. We have discovered that a further advantage of the vegetable, animal and mineral oils which are the preferred lipophilic solvents and are mentioned above is that they considerably reduce the susceptibility of the lipophilic solvent phase to such degradation. Where the lipophilic solvent used in the process is not one of these preferred solvents it may be desirable to add a quantity one of these preferred lipophilic solvents. The stability of the lipophilic solvent phase may be increased by the addition of further quantities of the preferred solvents. We have further discovered that the most stable lipophilic solvent phases produced by the processes of this invention are those wherein the oil is a highly saturated oil. These oils have a relatively high melting point and may not be liquid at the temperature at which the process is to be carried out. Accordingly, the degree of stability of the lipophilic solvent phase product of this invention may be limited by the need to employ a relatively unsaturated oil as the lipophilic solvent. In this circumstance, we have discovered that a lipophilic solvent product having improved stability may be produced by adding the lipophilic solvent phase product to a more saturated lipophilic solvent which has been heated to a temperature above its melting point and rapidly cooling the mixture to the desired storage temperature. The product may be a liquid, semi-solid or solid material which is useful in a variety of applications. The lipophilic solvent phase products may comprise a variety of useful materials depending upon the nature of the plant tissue. In particular, those solvent phases which typically may be obtained from green plant tissues may contain unsaturated fatty acids such as linoleic and linolenic acids, fat soluble vitamins such as vitamin A and vitamin E and the provitamin β carotene and fat soluble pigments such as chlorophylls and carotenoids and represent valuable products of this invention. All of the materials mentioned above are of nutritional value and the lipophilic solvent phase may find use directly as a foodstuff or feedstuff or as a component of a foodstuff or feedstuff or as a raw material from which valuable nutrients or other materials may be isolated.

The aqueous phase product may comprise a variety of valuable materials. In particular, certain of the water soluble anti-nutrients such as oestrogens and alkaloids may be useful as raw materials for use in the pharmaceutical industry. The anti-nutrients may also find application either as or in the production of agrochemicals such as pesticides, fungicides or insecticides. The aqueous phase may also be concentrated and the concentrate may find use as a foodstuff or feedstuff. The invention is illustrated by the following Examples: Example 1

Lucerne, which had been chopped in the field by a forage harvester, was introduced into a single worm screw press (Bentall Processor Mark 10), powered by a 15HP variable speed motor. The juice leaving the compression chamber was sieved and cooled immediately to 3°C and stored in a refrigerator. 2 litres of this juice was placed in a glass container, 6 litres of acetone were chilled to a temperature of 3°C and added to the container with stirring. After 20 minutes the mixture was allowed to settle and a brilliant green supernatant liquor was separated from the light green precipitate by decantation. The precipitate was washed with 1 litre of acetone and air dried to remove solvent. The product was a dry light grey powder whose appearance was unchanged after two months storage in a domestic refrigerator.

500mls of the supernatant liquor was shaken with lOmls of sunflower oil at 3°C and left overnight in the dark in an open container at ambient temperature. The acetone evaporated leaving a light brown aqueous phase with no visible green colour lying beneath a brilliant green oil layer when viewed in small quantities in transmitted light. The two layers were separated and the oil phase washed with water in order to remove a small residual quantity of acetone. Example 2

500mls of the supernatant liquor obtained as part of the process of Example 1 were placed in a glass separating flask and 20mls of sunflower oil was added. The mixture was shaken by hand for 20 minutes and allowed to stand for 15 minutes. A brilliant green oil phase lying below the aqueous phase was then separated and removed. A further 20mls of sunflower oil was added to the aqueous phase, which at this stage retained a green colouration and the shaking/settling procedure was repeated. A further oil phase was separated and removed. This had a green colour which was less intense than that produced by the first addition of oil. The extraction was repeated a third time to produce another green coloured oil phase and an aqueous phase which retained a green colouration although this was noticeably less intense than that of the oil layers. Example 3

The procedure of Example 2 was repeated using the following oils; corn oil, sunflower oil, groundnut oil, grapeseed oil and cod liver oil. In each case the products were closely similar in appearance to that obtained in Example 2. A further process utilised an olive oil which at 3°C was in a relatively solid state and found not to function as a lipophilic solvent until the temperature of the mixture was allowed to rise to about 12°C when the oil melted and then gave similar results to the other lipophilic solvents listed above. Example &

The lipophilic solvent phase products made in Example 3 were kept in open containers in the dark in a domestic refrigerator for two months, and observed at weekly intervals. It was found that the cod liver oil sample did not maintain its bright green colour after 3 weeks and became a paler yellowish-green in comparison to the samples based on vegetable oils. Those latter samples essentially retained their original colouration over the two month period, though after this period had elapsed there was some visual reduction in brilliance of green colour when compared with a freshly prepared similar sample.

Example 5

500mls of the supernatant liquor obtained as part of the process of Example 1 were placed in a glass separating flask and

50mls of hexane was added. The mixture was gently shaken by hand for 5 minutes and allowed to stand for 15 minutes. A brilliant green hexane phase, lying above the aqueous phase, was then separated and removed. 10mls of sunflower oil was added to the hexane phase and the resultant mixture was shaken for 1 minute. It was placed in an open container in the dark and at ambient temperature until the hexane evaporated. The oil which remained was a brilliant green colour.

Example 6

Example 5 was repeated but with the hexane and sunflower mixed together beforehand and then added to the supernatant liquor. The hexane/sunflower oil phase, brilliant green in colour, was separated and removed. It was placed in an open container in the dark and at ambient temperature until the hexane evaporated. The oil which remained was a brilliant green colour. Example 7

A sweet red pepper pod (Capsicum annuum) weighing HOg was placed in a blending machine together with 200mls of acetone at ambient temperature. The resulting pulp was pressed through a sieving cloth and the solid material remaining was rewetted with a further 200mls of acetone. The resulting pulp was again pressed through a sieving cloth. The fibrous fraction remaining on the cloth was white in colour with a few red specks. The red liquid products of the two pressings were combined and 5mls of sunflower oil added. The mixture was gently shaken for 2 minutes and allowed to settle. A lower oil phase, bright red in colour, separated from the aqueous phase. It was removed and washed with a small quantity of water to remove any traces of acetone. It tasted strongly of the original sweet red pepper fruit. It was kept for two months in an open container in the dark at ambient temperature and did not visibly change in appearance.

Example 8

Example 7 was repeated using 50g of garlic cloves (Aliiurn sativum) and a total of 200mls of acetone. The resulting oil phase was colourless but tasted and smelled strongly of fresh garlic. The aqueous phase was placed in an open container and the acetone allowed to evaporate at ambient temperature. The resulting liquid tasted and smelled of garlic but much less strongly than the oil product. Example 9 . Example 8 was repeated using a culinary herb Dill (Anethu graveolens). freshly picked. The resulting oil phase was a brilliant green colour with a pronounced dill flavour. The aqueous phase, after evaporation of the acetone, had a very faint dill flavour and was straw-coloured. Example 10

Example 7 was repeated using lOg of Creeping Buttercup flowers (Ranunculus repens) and 40mls acetone. The resulting oil phase was a bright orange-yellow colour. Example 11

Perennial Ryegrass (Lolium perenne) was obtained from a local dairy farm in mid-April. A sample of fresh grass was weighed, oven dried for 2 hrs at 105°C and reweighed. 5Kg of fresh material was chopped into approximately l-2cm lengths and 241 of 99% ethanol added at ambient temperature 11-15^βC. Unless otherwise indicated all subsequent procedures were performed at ambient temperature and undertaken in subdued light conditions. The mixture was left to soak for 12 hours. The grass material was pressed by hand and the resulting bright green liquid filtered. A total of 14.41 of aqueous liquor was recovered. 121 of the liquor were treated in the following manner:

A 21 aliquot was placed in a flask and 25ml of sunflower oil (Flora brand, with added vitamin E) added. The flask was shaken in a standardised manner. The oil phase was allowed to separate for about 5 minutes and was removed and collected. It was termed 1st extraction oil product. Approxi ately 200ml of aqueous liquor was separated and retained for analysis. The remaining 1800ml aqueous phase, termed 1st extraction aqueous liquor, had a further 25ml portion of fresh sunflower oil added to it and the previous process repeated. The resulting oil was termed 2nd extraction oil product and similarly the aqueous phase 2nd extraction aqueous liquor. The process was again repeated using 25ml sunflower oil. The resulting products were termed 3rd extraction oil product and 3rd extraction aqueous liquor. This sequence was repeated a further five times using fresh aqueous liquor. The resultant products of the above procedure were bulked so as to give 1.21 of extraction aqueous liquors and 150ml each of extraction oil products. These samples, together with a 1 litre sample of the original aqueous liquor and refrigerated sample of fresh grass were analysed by an independent analytical consultancy (Aspland and James Ltd.,

Chatteris, Cambs., U. fC). All samples were refrigerated at 3-4°C until analysed within 2 weeks of manufacture.

Table 1 shows the levels of water moisture chlorophyll a, chlorophyll b, 3-carotene, di-hyroxy carotenoids and fatty acids present in the original sample and at each extraction step. Values given are mg of solute per kg of solvent it was analysed in unless otherwise specified.

1 LJL1

Fresh Aqueous 1st Extraction 2nd Extraction 3rd Extraction Bulk Sample Grass Liquor with Aqueous 011 Aqueous Oil Aqueous Oil Aqueous Oil Methanol Liquor Product Liquor Product Liquor Product Liquor Product

10 680 7.6 190 7.1 78 7.6 700 4.7 290 3.1 110

3.3 35 3.4 69

Fresh grass is in wet weight terms

92MAY/ID71

Example 12

A further quantity of the same batch of perennial ryegrass utilised in Example 11 was used. A more rigorous extraction procedure was undertaken on 500g grass in subdued light conditions and at ambient temperature 11-15°C unless otherwise indicated. 125g batches of the grass were liquidised in a blender for 1 minute in the presence of 500mls of 99% acetone. The batch was filtered and pressed in a sieve cloth. The aqueous liquor, now a brilliant green colour, was returned to the blender to treat a second batch, which was similarly liquidised and pressed. The resulting two batches of fibrous solid matter were bulked and the aqueous liquor was placed to one side. This procedure was repeated on the remaining two batches of 125g fresh grass using a further 500mls of fresh 99% acetone. All the previously derived fibrous solid matter was bulked together and treated with a further 500mls batch of fresh 99% acetone. Thereby 500g of fresh grass was treated with 1 ,500mls of acetone.

The resulting fibrous solid matter, light green in colour, was teased apart and allowed to air dry. Once dry it was gently shaken through a sieve with an approximately 1mm mesh to provide a fibre rich fraction retained on the mesh and a fibre poor and consequently protein enriched fraction passing through the mesh. These two fractions were retained for analysis. All the aqueous liquors were bulked together and refrigerated at 3°C for 2 days. 250mls of this liquor was set aside for analysis and 1,250mls used as follows:

250mls batches of the aqueous liquor and 15mls sunflower oil were placed in an amber-tinted flask attached to a Buchi rotary evaporator and a vacuum system. The flask was kept at a constant 20°C in a waterbath. A purge of nitrogen gas was used initially to reduce excessive boiling of the contents of the flask when vacuum was applied. The flask was rotated at the slowest setting available to avoid emulsification of the contents. After approximately 1 hour no further bubbling was observed and the flask was disconnected from the apparatus. A very faint small of acetone was detectable. The oil phase, a brilliant deep green colour, was lying above the aqueous phase, which had a barely visible green colour. The two phases were separated and the process repeated a further five times. The six samples of green oil and of the aqueous liquor were batched. It was noted that some dark green specks about 1mm in diameter or less, were present in the oil but were redispersed by shaking. The oil and aqueous liquor were kept refrigerated at 3-4°C until analysed as in Example 11, with results shown in Table 2. Units are the same as in Table 1.

Fresh grass is In wet weight terms

92MAY/ID71

The lower than expected values for the fatty acids in both this example and the previous one was attributed to insufficient water miscible solvent (acetone or methanol) being added to the leaf protein concentrate. Addition of further solvent should have brought the fatty acids into the aqueous liquor for subsequent extraction into the lipophilic solvent phase.

Claims

CLAIMS 1. A process for the production of a stabilised extract from a cellular plant tissue which is characterised in that it comprises the addition of a non-volatile lipophilic solvent to an aqueous liquor obtained by the addition of a water miscible polar solvent to a cellular plant tissue or to a liquor which has been expressed from a cellular plant tissue and the separation of an aqueous phase extract from a lipophilic solvent phase stabilised extract.

2. A process according to Claim 1 characterised in that the addition of the water miscible solvent to cellular plant tissue results in the production of a fibre rich proteinaceous material and an aqueous liquor.

3. A process according to Claim 1 characterised in that the addition of the water miscible solvent to a liquor which has been expressed from a cellular plant tissue results in the precipitation of a proteinaceous material.

4. A process according to either of Claims 2 or 3 characterised in that the aqueous liquor is separated from the proteinaceous material prior to the addition of the lipophilic solvent.

5. A process according to any of the preceding claims characterised in that the water miscible solvent is removed prior to the addition of the lipophilic solvent and the residue is maintained under an inert atmosphere until such time as the lipophilic solvent is added.

6. A process according to any of Claims 1 to 4 characterised in that at least a part of the water miscible solvent is removed prior to the separation of the lipophilic solvent phase.

7. A process according to Claim 6 characterised in that all the water miscible solvent is removed prior to the separation of the lipophilic solvent phase.

8. A process according to any of Claims 1 to 7 characterised in that the lipophilic solvent is added to the liquor in a series of increments and a series of lipophilic solvent phases are separated.

9. A process according to any of the preceding claims characterised in that a volatile lipophilic solvent is added to the aqueous liquor and the lipophilic solvent phase thus formed is mixed with a non-volatile lipophilic solvent and the volatile solvent is separated from that mixture.

10. A process according to any of the preceding claims characterised in that the water miscible polar solvent has a Hildebrand solubility parameter of from 9.0 to 11.0.

11. A process according to Claim 10 characterised in that the water miscible polar solvent is one which does not form an azeotrope with water.

12. A process according to Claim 11 characterised in that the solvent is non-toxic to humans and animals.

13. A process according to any of Claims 10 to 12 characterised in that the water miscible polar solvent is selected from the group comprising acetone, 2-butanone, 2-pentanone, 3-pentanone, methanol , ethanol , propanol and butanol .

14. A process according to any of Claims 1 and 3 to 13 characterised in that the ratio of the volume of the water miscible solvent to that of the plant juice liquor is in the range of 15:1 to 1:1.

15. A process according to Claim 14 characterised in that the ratio is in the range 5:1 to 3:1.

16. A process according to any of the preceding claims characterised in that the lipophilic solvent is one which is acceptable for use in the food, feed, cosmetic or pharmaceutical industries.

17. A process according to Claim 16 characterised in that the lipophilic solvent is a vegetable oil, a hydrogenated derivative of a vegetable oil, an animal oil or a mineral oil.

18. A process according to Claim 17 characterised in that the lipophilic solvent is a vegetable oil, or a hydrogenated vegetable oil.

19. A process according to Claim 16 characterised in that the oil is a highly saturated oil.

20. A lipophilic solvent phase characterised in that it has been produced by a process according to any of Claims 1 to 19.

21. An aqueous phase extract characterised in that it has been produced by a process according to any of Claims 1 to 19.

22. A proteinaceous material characterised in that it is produced by a process according to any of Claims 1 to 19.

23. A lipophilic solvent phase extract which has been Isolated from a cellular plant tissue or a juice extracted therefrom comprising fatty adds, fat soluble pigments or vitamins which is substantially free from water soluble materials.

24. An extract according to Claim 23 characterised in that the lipophilic solvent is a vegetable oil or hydrogenated vegetable oil .

25. An extract according to either of Claims 23 or 24 characterised in that the extract is solid or semi-solid at ambient temperature.

26. An aqueous extract which has been isolated from a cellular plant tissue or juice thereof which is substantially free of lipophilic substances.

27. An extract according to Claim 26 characterised in that it comprises substantially all the anti-nutrient content of the plant tissue or juice.