The present invention relates to an improved method for extracting oil from oil bodies, and to an oil extracted using the method.

Oil bodies are subcellular droplets of oil ( 1 -3 μιη in diameter), covered with an oleosin-protein-rich half unit membrane . The oleosin proteins, in addition to a hydrophobic domain that associates with the entrapped oil, have hydrophilic N- terminal and C-terminal regions. These regions are enriched in basic amino acids that appear to associate with acidic phospholipids in the half unit membrane, thus forming a protective coat over much of the oil body surface. Tocopherol molecules (and other bioactive micronutrients) are also intrinsically associated with oil bodies. It is likely that these molecules are positioned at the interface between the oil body and the cytosol of the oilseed cell.

Oil bodies can be recovered intact from seeds etc by using a simple, wet milling protocol. The physical stability of the natural oil bodies once isolated is controllable to a certain extent. Intact oil bodies can be used directly in applications that require an oil dispersed system, or they can be destabilised/disrupted to yield the bulk oil. This invention is concerned with destabilisation/disruption of the oil bodies and release of the oil contained therein.

As mentioned above oil bodies can be easily recovered using, for example, a wet milling protocol; once recovered the oil bodies form an emulsion, and it is from this emulsion that the oil bodies are disrupted and the oil recovered.

Oil bodies ex-vivo, and in emulsion, are prone to thermodynamic changes with time that can result in the oil slowly leaking out from the system. This process can be accelerated by adding enzymes. However, the use of enzymes can be expensive and it can be difficult, and expensive, to recover the enzymes. Unless recovered the enzymes may be a contaminant in the recovered oil.

Another method to disrupt recovered oil bodies to release the oil involves a solvent extraction step. Hexane is usually the preferred solvent. Hexane based processes for oil recovery have been in commercial use for many years. The solvent extraction method recovers almost all the oils and leaves behind only 0.5% to 0.7% residual oil in the raw material. This is in comparison to mechanical pressing where the residual oil left in the oil cake may be anywhere from 6% to 14%. However, whilst hexane is efficient and effective for the extraction of oil from oil bodies, it is a harsh and environmentally unfriendly chemical. Organic solvents such as hexane contribute to the industrial emissions of volatile organic compounds which can react in the atmosphere with other pollutants to produce ozone and other harmful photochemical oxidants. Other reasons to avoid hexane include reduced safety risk with regard to fire and explosion during the extraction process, and reduced damage to the extracted oil.

The present invention provides an alternative method for the extraction of oil from oil bodies which avoids the need for harsh chemicals such as hexane or expensive enzymes.

According to a first aspect the invention provides a method for releasing oil from oil bodies comprising:

i) providing oil bodies;

ii) treating the oil bodies with one or more of

a) salt;

b) a weak acid and/or an agent to reduce the pH of the oil bodies to between about 3.5 and about 5.5 ; and

c) an organic acid or salt thereof with lipophilic moieties;

iii) releasing the oil from within the oil bodies.

The skilled man will appreciate that oil bodies provided for use in the method of the present invention may be obtained by any suitable method, and such methods are well known in the art. The oil bodies may be provided in an emulsion/milk or in a cream.

In step (ii) two or more of a), b) and c) may be used to treat the oil bodies. Preferably in step (ii) oil bodies are treated with a), b) and c) .

In step (ii) the treatments may be carried out in any order or any combination. Preferably in step (ii) the steps are carried out in the order listed. In step (ii) part c) preferably the organic acid or salt thereof with lipophilic moieties is one or more of sodium benzoate, potassium sorbate and sodium salicylate . In step (ii) part c) sodium benzoate and/or potassium sorbate and/or sodium salicylate may be used alone or in combination of two or more. Preferably the total concentration of organic acid or salt thereof with lipophilic moieties in the aqueous phase of the oil body dispersion is between about 0. 1 M and about 1.2 M.

In step (ii) part a) the salt treatment may be applied by performing a salt wash of the oil bodies, or by adding solid salt to the oil bodies.

In step (ii) the salt may be NaCl, KC1 or other suitable salt, preferably the salt is NaCl. The salt may be used at a concentration of about 2M. In step (ii) part b) the agent to reduce the pH of the oil bodies to between about 4.5 and about 5.0 may be a weak acid.

In step (ii) part b) the pH may be reduced to between 3.5 and 5.0, or between about 4.0 and 5.5, or between about 4.0 and about 5.0, more preferably between 4.5 and 5.0.

A weak acid may include one or more of acetic acid, oxalic acid and lactic acid, in particular one or more of acetic acid and lactic acid. In an embodiment 2% lactic acid (w/w) may be used. The method of the invention may comprise a further step of recovering the released oil. The released oil may be recovered by one or more of centrifugation, decanting or filtration.

The recovered oil from the method of the invention may or may not need to be refined before use.

The recovered oil may be pure enough to use or it may are require further refining before use. Further refining may involve one or more of degumming, bleaching, winterising, de-waxing, deodorising or polish filtering. The method of the invention preferably does not use enzymes in the oil recovery process. The method of the invention preferably does not use enzymes to disrupt the oil bodies and effect the release of the oil contained therein. The method of the invention may cause the oil bodies to burst and release at least about 50%, 60%, 70%, 75%, 80%, 85%, 90% or more of the oil in less than 60 minutes, less than 30 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, or less than 1 minute . Preferably at least about 90% of the oil is released from the oil bodies in 60 minutes or less.

The method of the invention may be performed at a temperature between about 20°C to about 60°C, in a preferred embodiment the method of the invention may be performed at or near, room temperature. The rate of oil release may increase with temperature.

The method of the invention may offer a cheaper and/or greener method for the recovery of oil from oil bodies. The oil bodies for use in the method of the invention may be recovered from a material containing them into a wet preparation by grinding the material in a water based medium in which the pH, viscosity and ionic strength can be controlled, filtering out the larger material, and then centrifuging the filtrate. The oil bodies will float on the surface of the filtrate forming a thick, cream-like pad (crude oil bodies) that can be easily removed.

The removed oil bodies (crude oil bodies - emulsion/milk) may be washed several times by dispersing the oil bodies in a washing medium (in which the pH, viscosity and ionic strength can be controlled, which may or may not contain a chaotropic agent such as salt or urea), and re-centrifuged and recovered, to clean the oil bodies and/or to remove contaminants. Further washing of the oil bodies with water may be necessary to remove any undesirable chaotropic agents such as urea.

The oil bodies may be washed in salt and/or urea prior to use in the method of the invention. The recovered oil body material may then be a concentrated oil-in-water emulsion (thick cream) with a solids content of between about 35% and about 75%, mostly made up of triacylglycerol. This cream may be dispersed to form a more dilute emulsion if required.

The material containing the oil bodies may be selected from one or more of seeds, pollen, flowers, roots and stems of flowering plants, the spores and vegetative organs of non-flowering plants, algae, microalgae, animal cells, fungi and protists such as Euglena. Preferably in this invention the oil bodies are extracted from seeds or algae, more preferably from seeds.

The seeds may be seeds or kernels from one or more of the following plants, sunflower, soybean, oil palm, safflower, almond, macadamia, cotton seed, ground nut, coconut, oil seed rape, echium, borage, linseed/flax/hemp, evening primrose, rice, wheat, oat, maize, barley, olive, peanut, acai palm, Nigella sativa, blackcurrant, carob, amaranth, apricot, apple, Argania spinosa, avocado, A tialea speciosa, Moringa o lei f era, Shorea, cape chestnut, cacao, Xanthium, poppy, cohume palm, coriander, date, Irvingia gabonensis, Camelina sativa, grape, kapok, kanaf, mafura, marula, meadowfoam, mustard, nutmeg, okra, papaya, perilla, persimmon, pequi, pili nut, pomegranete, prune, quinoa, Niger pea, rice, Prinsepia utilis, Sacha inchi, sapote, Jessenia bataua, shea, argula, tea, thistle, Cyperus esculentus, tobacco and tomato .

According to a further aspect the present invention provides oil released by the method of the invention.

According to yet further aspect the present invention provides a food product or food ingredient comprising oil released by the method of the invention. In a further embodiment, the oil bodies in the method of the invention may be replaced with other liquid droplets obtained from, or in, an emulsion and the method may be used to destabilise the droplets allowing the release of the liquid contents. For example, in this embodiment the method of the invention may be used to recover the liquid contents from droplets in an emulsion stabilised with complex lipids and proteins. The droplets in the emulsion may be recovered by any known method, for example by filtration or by centrifugation and the recovery of a cream or pad of droplets. The method of the invention may be used to release the contents of milk fat globules or LDL (low density lipoprotein) particles, or any other liquid droplets recovered from, or in, an emulsion.

The method of the invention may be used to fractionate an emulsion into its constituent parts.

The skilled man will appreciate that the preferred features of the invention discussed with respect to the release of oil from oil bodies may be applied to the method for the release of the contents of any droplet recovered from, or in, an emulsion.

The skilled man will appreciate that the preferred features of any aspect of the invention can be applied to all aspects of the invention.

Embodiments of the invention will now be described, by way of example only, with reference to the following figures.

Figures 1A to 1C - are micrograph images illustrating the effect of a 'lactate/sorbate/benzoate ' mix on 2M NaCl-washed hemp seed oil bodies. Oil body disintegration is observed after - A: 30 seconds; B: 1 Minute; C: 5 minutes after addition of this organic acid cocktail;

Figure 2 - illustrates the release of oil soon after the addition of a 'lactate/sorbate/benzoate ' mix to 2M NaCl-washed hemp seed oil body material;

Figure 3 - is a table illustrating the effect of lactic acid and Microcare SB addition to salt washed hemp seed oil bodies;

Figure 4 - demonstrates the complete and rapid release of oil from urea-washed sunflower seed oil bodies Recovery of Oil Bodies

The biochemistry of oil bodies has been studied since the early 1970's and therefore the methods used to recover them are not new. In principle, the seed containing the oil bodies is ground in water/buffer, and filtered to produce a milk emulsion. The filtrate/milk may then be centrifuged until a pad (or cream) of oil bodies floats to the surface . This crude preparation can be cleaned by resuspension in chaotropic agents, such as salt in this case, followed by further centrifugation. This assists in removing proteins that are not intrinsic to the oil bodies. For this current invention the milk emulsion, the cream or the salt-washed oil bodies may be used.

In one example, oil body (OB) material was extracted from oilseeds (e.g. hemp) by blending (liquidiser) at max speed, 100 g of seed with 500 mL deionised water for 2 min. The solution/milk was filtered under vacuum through cheese cloth. The solid residue was discarded and the filtrate isolated and centrifuged. The OB material was recovered by centrifuging for 20 min at 7500 g, 5 ^° C. The process of centrifugation allows the oil bodies to form a layer like a thick cream on the surface, and the heavier solid residue is pelleted out. The OB material/cream was removed from the surface and placed into a clean bottle; this was classed as the crude oil bodies (COB) .

Rapid disruption of oil bodies

In one embodiment, a specific mixture of acids is added to salt-washed oil body material to elicit a rapid disintegration of oil bodies accompanied by the release of oil, which separates from the aqueous phase .

In a particular example, the following methods steps were followed in order to release the oil contained within the COB material: i) Combine l Og COB with 40ml 2M NaCl and vortex for 3 minutes.

11) Centrifuge mixture for 20 min at 7500 g, 5 ^° C

in) Recover the salt-washed OB material/cream

iv) Add 2% w/w lactic acid to the OB material/cream (equivalent to 0.35 M lactic acid in the aqueous phase) and vortex for 2 minutes or until fully dispersed. v) Add 3 % w/w Microcare SB (combination of sodium benzoate and potassium sorbate) and vortex for 2 minutes or until fully dispersed.

Figure 1 illustrates the effect of a lactate/sorbate/benzoate mix on the integrity of recovered oil bodies. 30 seconds after addition (Figure 1A) discrete oil bodies are clearly visible, after 1 minute the disruption of oil bodes is clear (Figure IB), and after 5 minutes (Figure 1 C) nearly all the oil bodies have been disrupted and have released their oil. Figure 2 illustrates how the release of oil can be clearly seen in a bottle .

Effect of salt washing-and-disruption procedure on oil body integrity

Experiments were carried out to determine the importance of reagent combination effects on the tendency of hemp oil bodies to rapidly disintegrate. The results are shown in Figures 3.

Effect of oil body washing procedure on oil release

Oil bodies recovered from different seeds carry varying amounts of extraneous proteins; these can be removed through a range of washing procedures. Urea-washing tends to remove more protein from, for example, sunflower seed oil bodies, than a salt-washing protocol. The effect of washing rigour on the rate of oil release was therefore tested. The results (below and Figure 4) clearly indicate that the more rigorous the oil body washing procedure, the more rapid the rate of oiling out. The results also suggest that crude oil body preparations that are naturally low in extraneous proteins (e .g . hemp) will tend to be more easily disrupted by the method of the invention.

No washing - just crude oil bodies

This is compared to crude oil bodies

o CRUDE OIL BODIES + SALT (added as a solid to the oil body preparation and mixed to dissolve in the aqueous phase, providing an equivalent of 2 M in the aqueous phase, solid salt was added in order to obtain the same conditions as the salt washed oil bodies, which were already recognised as prone to oiling out in the presence of 'reagent') = no oiling out CRUDE OIL BODIES + ACIDIFYING ACID (4% w/w 80% lactic acid [equates to 0.7 M in continuous aq. phase]) + 'REAGENTS ' (potassium sorbate and/or sodium benzoate and/or sodium salicylate [ 1.2 M total concentration in the aqueous phase]) = no oiling out

CRUDE OIL BODIES + SALT + ACIDIFYING ACID + 'REAGENTS ' (using the conditions described above) = slow oiling out

Salt washing

With respect to salt washing, two different salts were tested, KC1 and NaCl, at concentrations ranging from 0.5M to 3M. The rate of oiling increased with increasing salt concentration up to 2 M, then remained constant. The salt remains in the aqueous phase of the salt-washed oil body preparation, presumably at the same concentration as the washing solution. These are the results from oil bodies washed with 2 M NaCl: o SALT-WASHED OIL BODIES = no oiling out;

o SALT-WASHED OIL BODIES + ACIDIFYING ACID (4% w/w 80% lactic acid [equates to 0.7 M in continuous aq. phase]) + 'REAGENTS' (potassium sorbate and/or sodium benzoate and/or sodium salicylate [ 1.2 M total concentration in the aqueous phase]) = oiling out.

The oiling out of the salt washed oil bodies is quicker and more thorough than that of crude oil bodies.

Urea washing

With respect to urea washing, crude oil bodies were resuspended (washed) in a 9M urea solution. The oil bodies were concentrated through centrifugations then resuspened in water to remove urea; this water-washing step was repeated twice to remove any residual urea.

o UREA WASHED OIL BODIES + SALT (added as a solid to the oil body preparation and mixed to dissolve in the aqueous phase, providing an equivalent of 2 M in the aqueous phase, solid salt was added in order to obtain the same conditions as the salt washed oil bodies, which were already recognised as prone to oiling out in the presence of 'reagent') = no oiling out o UREA WASHED OIL BODIES + ACIDIFYING ACID (4% w/w 80% lactic acid [equates to 0.7 M in continuous aq. phase]) + 'REAGENTS ' (potassium sorbate and/or sodium benzoate and/or sodium salicylate [ 1.2 M total concentration in the aqueous phase]) = no oiling out o UREA WASHED OIL BODIES + SALT + ACIDIFYING ACID + REAGENTS (using the conditions described above) = oiling out rapid and complete.

The urea wash significantly increases the rate of oiling out, even above that of the salt washed material. This is probably due to the low protein content of urea washed oil bodies.

Effect of salt type and acidification acid on oil release

Two types of acid were tested, lactic acid (80% in water) and acetic acid (glacial) in different percentages ranging from 0.5% to 4% w/w.

An ideal concentration for each acid was demonstrated to be a final concentration (in the aqueous phase of the oil body dispersion) of 0.35 M for lactic acid and 0.67 M for acetic acid. Increasing the concentration of acid did not have a significant effect on the oiling out rate.

The minimum effective percentage of each acid is preferably 2% (w/w).

NaCl/lactic acid, KCl/lactic acid and NaCl/acetic acid were demonstrated to be effective salt/acid combinations.

Effect of 'Reagent' on oil release

Microcare SB is a proprietary food grade preservative used in a range of applications. The active agents (sodium benzoate and potassium sorbate) are not a commercial secret. A range of organic acid salts, with lipid soluble moieties, i.e. 'reagents ' were tested for their ability to trigger oil body destabilisation (as demonstrated by the release of oil) in the presence of salt. The reagents tested were:

Potassium Sorbate

Sodium Benzoate

Sodium Salicylate

All were demonstrated to be effective on their own, or in different combinations with each other, at total concentrations (in the aqueous phase) of 0. 1 M. The rate increased in a concentration dependent manner, but no benefits, in terms of rate, was perceived above a concentration of 1.2 M.

Effect of temperature on oil release

Temperature may dramatically affect the oiling out rate. Increasing the temperature may increase the rate . Three temperatures were tested, 20°C, 40°C and 60°C and, as expected, the maximum effect was observed at 60°C.

Conclusion :

Oil bodies will rapidly disintegrate and release their oil cargo after salt-washing (or in the presence of salt having been washed by another method) followed by a treatment with an acidifying agent (e.g. acetic acid) and an organic acid (or its salt) with a lipophilic moiety (e.g. sorbic acid). This phenomenon, a rapid release of oil from oil bodies, does not occur with a crude oil body preparation, nor does it occur with salt- washed oil bodies with the addition of just lactic acid or just an organic acid with an lipophilic moiety. The rate of release may increase with temperature and with increasing concentrations of salt, acidifying agent and 'reagent' (organic acid or salt thereof with lipophilic moiety).