Background to the invention WO 90/13571 proposes the introduction antifreeze protein (AFP) into plant foodstuffs, either into the entire plant, conferring some degree of general resistance to damage from subfreezing climatic conditions, or into a plant part such as the fruit or vegetable portion to minimise damage specifically to those plant organs upon freezing. This document is completely silent on the means by which the AFP is introduced into plant material.

Griffiths et al. , (Biotech Advances, Vol. 13, No. 3, pp375-402,1995) relates to AFPs in frozen foods. It discloses that for foods, frozen for preservation, AFPs may inhibit re-crystallisation during freezing, storage, transport and thawing, thus preserving food texture by reducing cellular damage and also minimising the loss of nutrients by reducing drip. In so doing the objective is to reduce cellular damage within the material to be frozen.

Cutler et al. , (J. Plant Physiol. Vol. 135 pp 351-354 (1989) ) as<BR> referred to in Griffths et al. , relates to an antifreeze protein isolated from a Winter Flounder and improvement of the cold hardiness of plant tissues. This art discloses a method of vacuum infiltrating a lmg/ml solution of this AFP into leaf samples as a means of depressing the spontaneous freezing temperature. It further describes that cultured cells of bromegrass on exposure to the AFP demonstrated a reduction in the amount of freezable water at any given temperature. Finally this paper discloses a decreased rate of ice crystal formation in the presence of AFP.

JP 11 313605A discloses a process for preparing vegetables and fruit comprising soaking the vegetables and fruit in an antifreezing agent containing aqueous solution after heating for a fixed time, followed by freezing.

W099/06565 discloses plant antifreeze proteins, peptides and polypeptides that bind to ice crystals and inhibit their growth, inhibit ice recrystallation and protect liposome cell membranes and proteins, cells and organisms at low temperatures.

The objective technical problem solved by the present invention relates to improving the texture of frozen vegetables when prepared for consumption. More particularly the problem is to improve the mechanical strength of vegetable material which has been subjected to frozen storage.

Summary of the invention The solution to the identified problem has been found to reside in the disruption of the cell membrane by a blanching step in a process which subsequently infiltrates vegetable material with an AFP before freezing.

It is a first object of the invention to provide a process for preparing vegetables wherein said process comprises the steps of: (i) blanching a vegetable or part thereof; (ii) infiltrating an AFP solution; (iii) freezing; Surprisingly it has been found that a textural improvement in vegetables when prepared for consumption is not reliant on minimising cellular damage as taught by the prior art.

Detailed description Explanation of figures: Figure 1: illustrates mechanical behaviour of raw and blanched carrot cylinders vacuum infiltrated with fish AFP type III, subsequently quiescent frozen, and then thawed.

Figure 2: illustrates a comparison of mechanical behaviour between blanched carrot tissue vacuum infiltrated with fish AFP III and blanched carrot tissue vacuum infiltrated with water, quiescent frozen, and then thawed.

Figure 3: light micrograph of blanched carrot tissue vacuum infiltrated with fish AFP III quiescent frozen, and then thawed.

Figure 4: light micrograph of blanched carrot tissue vacuum infiltrated with water quiescent frozen, and then thawed.

Figure 5: light micrograph of immunocytochemistry studies showing blanched unfrozen carrot tissue vacuum infiltrated with fish AFP III.

Figure 6: light micrograph of immunocytochemistry studies showing blanched carrot vacuum infiltrated with fish AFP III then quiescent frozen.

Figure 7: light micrograph of immunocytochemistry studies showing blanched carrot vacuum infiltrated with ultra-pure water then quiescent frozen.

The expression blanching is used to indicate exposure of vegetable material to either boiling water or a steam bath. The purpose of this exposure is to rupture the cell membranes within the

vegetable material to facilitate the movement of AFP in the subsequent infiltration step. Figure 1 demonstrates the unexpected advantage in mechanical properties gained when the vegetable material is blanched prior to infiltration of AFP.

Typically blanching will take place at a temperature of about 100°C for a period of between 1 and 20 minutes depending on the size of the piece of vegetable being blanched. Preferably blanching will be undertaken for 2 to 10 minutes at this temperature.

Infiltration with the AFP may rely on passive diffusion of the protein throughout the vegetable material by simply soaking the vegetable material in a solution containing AFP. More efficiently however infiltration can be actively driven. Preferably infiltration is vacuum assisted, therefore in a preferred embodiment the invention comprises a process for treating vegetables as described above wherein said infiltration is vacuum assisted.

Any commercially available heat stable or non-heat stable AFP may be used in a process according to the invention because the infiltration occurs after blanching and so AFP activity will not be destroyed prior to freezing. Preferably AFP is selected from the group comprising Lolium derived AFP (described in W09937782), Fish AFP type I and III (W09639878) and carrot AFP (W09822591).

Most preferably the AFP selected is fish AFP type III.

Typically the concentration of AFP solution used in the infiltration will range from 0.01 to lOmg/ml. Preferably 0.1 to l. Omg/ml, most preferably about 0.5 mg/ml.

Immunocytochemistry and light microscopy show that where AFPs are located in a carrot sample the tissue remains intact, however in the absence of AFPs, severe radial cavitation between cells occurs. This radial cavitation is believed to give rise to poor

mechanical properties when the vegetable material is prepared for consumption.

Vegetable material which has been blanched and infiltrated with an appropriate AFP may be frozen by any suitable commercially available means. Typically this will involve the use of a quiescent freezer at a temperature of-18°C for a period of between 12 and 24 hours depending on the size of the material to be frozen.

Suitable vegetables may be selected from the Pisum family such as peas, the family of Brassicae, such as green cabbage, Brussel sprouts, cauliflower, the family of Phaseolus such as barlotti beans, green beans, kidney beans, the family of Spinacea such as spinach, the family of Solanaceae such as potato and tomato, the family of Daucus, such as carrots, family of Capsicum such as green and red pepper. Preferably vegetables of the Solanaceae and Daucus families are selected for use in the process of the present invention and most preferably carrot and potato tubers which have been found to demonstrate particularly improved properties.

Example 1 Carrots were heated for 2 minutes in boiling water and then infiltrated under vacuum with either 0. 5mg/ml fish AFP III solution or ultra-pure water, for 3 hours. They were quiescently frozen for 24 hours at-18°C and then thawed. The carrot tissue infiltrated with fish AFP III solution had a significantly greater stiffness and mechanical strength than the tissue infiltrated with ultra-pure water (figure 2).

Example 2 Light micrographs of blanched carrot tissue vacuum infiltrated with either fish AFP III or water, then quiescent frozen and thawed.

Cylinders were subjected to a standard histological preparation method comprising of several stages, to prepare the potato samples for analysis by light microscopy. Firstly, they were placed in a fixative (90% v/v of 70% ethanol, 5% v/v formaldehyde solution, 5% v/v glacial acetic acid) for 10 days. Next, the tissue was dehydrated using a vacuum infiltration processor (VIP), by subjecting them to a series of increasing alcohol steps (to remove the water), followed by a stage which ensures the alcohol is removed (involving toluene), which allowed the samples to be impregnated into wax.

The samples were embedded into moulds and then cut into 5ym sections, before being incubated at 56°C to dry overnight. The sections were rehydrated through a series of decreasing alcohol steps (to remove the wax) and then stained with 0. 1% Toluidene Blue. The sections were air dried and mounted in a resinous mountant to enhance the resolution and to help preserve the section. (Figures 3 & 4) Example 3 Light micrographs were prepared of immunocytochemistry studies identifying the location of fish AFP III in blanched carrot tissue.

Pre-treat slides with poly-l-lysine (100: 1 in 900ml H20). Add a drop of 2% milk protein in PBS to each section on the multiwell slide. Remove after 10 minutes and repeat blocking. Add a drop of five-fold dilution of the appropriate monclonal antibody culture supernatant in the MP/PBS mixture. Incubate for at least 1 hour at room temperature. Wash six times with MP/PBS. Add a drop of 100 fold dilution of anti-rat IgG linked to FITC in the MP/PBS mixture. Incubate for at least 1 hour in the dark. Wash with six times with PBS and mount in citifluor (glycerol/PBS solution). Put

on coverslip and secure with tippex if required. View using following filters: LEICA fluorescence filter cube"13" excitation filter = 450-490 nm dichromatic mirror = RKP 510 nm suppression filter = LP 515 nm All samples stained with an AFP III antibody at 1/20 dilution, visualised with anti-mouse fitc (1/100).

Results Figure 5-Blanched carrot vacuum infiltrated with fish AFP III (unfrozen). Vascular tissue shows yellow autofluorescence, with positive lime green staining of cell walls and intercellular spaces.

Positive staining seen throughout cell walls on the periphery and in the intercellular spaces in the central pith area.

Figure 6-Blanched carrot vacuum infiltrated with fish AFP III then quienscent frozen. Staining was more abundant around the vascular tissue.

Figure 7-Blanched carrot vacuum infiltrated with ultra-pure water then quiescent frozen. No positive staining seen throughout the tissue.