AU2521200A - Frozen food product - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT TITLE OF INVENTION FROZEN FOOD PRODUCT Name and Address of Applicant: UNILEVER PLC of Unilever House, Blackfriars, London EC4P 4BQ, England The following statement is a full description of this invention, including the best method of performing it known to me:- F7389 AU2(C) -1- Frozen Food product Technical Field of the Invention The invention relates to frozen food products containing AFPs.

Background to the Invention 10 Antifreeze proteins have been described in the literature, S. :see for example Marilyn Griffith and K. Vanya Ewart in Biotechnology Advances, Vol 13, No 3, pp 375-402, 1995.

Antifreeze proteins generally possess one or more of the following properties: thermal hysteresis, inhibition of ice 15 recrystallisation, control of ice crystal shape and interaction with ice nucleators.

*Thermal hysteresis is the best known property of AFPs and the property is normally used to test for the presence of 20 AFPs. Thermal hysteresis results from.a lowering of the apparent freezing temperature of a solution containing a thermal hysteresis active AFP without affecting the melting temperature. The identification of sources of AFP by thermal hysteresis tests is widely described in the literature, see for example John G. Duman in Cryobiology 30, 322-328 (1993).

Inhibition of ice recrystallisation is another property of AFPs. This activity is also referred to as ice crystal growth suppression. This property can be tested by comparing at a certain point in time the ice crystal size of crystals in the presence of AFP and in the absence of AFP. The F7389 AU2(C) 2 application of this method in the testing of fish AFPs is described in US patent 5,118,792 (DNA Plant Technology Corporation) A third property of AFPs is their ability to influence the shape of ice crystals. This property stems from the selective binding of AFPs to certain faces of the ice crystal and therewith limiting crystal growth in certain directions. The presence of ice crystals having an hexagonal 10 bipyramid shape is then considered indicative of the S. presence of AFP. This method is for example described for testing the activity of extracellular winter rye AFPs in WO 92/22581 (University of Waterloo).

A fourth property of AFPs is their ability to inhibit the activity of ice nucleating substances. This interaction between an AFP and an ice nucleator may for example result in increased thermal hysteresis. This property is for example tested in WO 96/40973 (University of Notre dame du Lac) AFPs have been suggested for improving the freezing tolerance of products. Many applications have been suggested in this context.

For example AFPs have been suggested for enhancing the cryopreservation of biological materials (WO 91/12718, Agouron Pharmaceuticals, WO 91/10361, The Regents of the University of California). Also AFPs have been suggested to prevent leakage from liposomes e.g. in cosmetic or pharmaceuticals (see WO 96/20695). A further possible F7389 AU2(C) 3 application is to increase the freezing tolerance of plants by including therein (or transgenetically producing therein) an AFP (See J. Cell. Biochem. Suppl. vol. 14e, 1990, page 303 XP002030248, Lee et al, abstract R228). Also fish AFPs have been suggested for use in food products for example in frozen yoghurt or ice cream ('US 5,620,732 Pillsbury and WO 96/11586, HSC Research and development limited partnership).

10 Up till now, however the use of AFPs has not been applied on a commercial scale. Applicants are of the opinion that one of the reasons for the lack of commercial implementation is that although many AFPs have been described, in practice the implementation in actual commercial products encounters serious problems.

Applicants have found that one of the key reasons for these problems is that out of the great number of AFPs that have been described in the literature only a limited set of AFPs can suitably be applied for each application; also applicants have found that this selection of suitable AFPs is dependent on the desired application and/ or product attributes to be achieved.

A particular desirable source of AFPs is from the lichen family. Lichen can fairly easily be obtained in relatively large quantities and relatively simple isolation procedures can be used for obtaining an AFP containing concentrate.

Furthermore the use of AFPs from lichen is believed to be favoured by consumers who tend to prefer natural vegetable sources to e.g. fish AFPs.

F7389 AU2(C) 4 Applicants have now found that if a specific application for the use of lichen AFPs is selected, this creates the need for a specific test to select the AFPs which can advantageously be applied in this application.

The object of the present invention is therefore to provide those lichen AFPs which can advantageously be used in frozen confectionery products.

Accordingly in a first aspect the invention relates to frozen confectionery products comprising one or more AFPs derived from lichen, wherein the AFPs in water have an ice crystal size after quick freezing to -40 °C followed by storage for 1 hour at -6 °C (measured as described below) of less than 15 um.

Background to the invention A number of literature places have suggested that AFPs may potentially be used for favourably influencing the textural properties of frozen confectionery products such as ice cream. However most of these documents do not provide a teaching how these favourable properties can actually be achieved in practice.

A further set of documents describes the use of fish AFPs.

WO 96/11586 teaches the application of fish antifreeze polypeptides in frozen fermented food products. This F7389 AU2(C) 5 document does not teach the use of specific AFPs derived from lichens in these products.

WO 96/39878 describes the application of AFP in ice-cream.

Suitable AFPs for this application may be derived from blood and muscle tissue of antartic fish, artic fish, worms and insects. Again no teaching is provided that lichen AFP can be used.

0* 0 US 5,118,792 describes in example 3B the inhibition of recrystallisation by a purified A-Saf5 fusion protein in a popsicle mixture. Again this document does not teach the use of AFPs derived from lichens.

15 WO 92/22581 describes a plurality of polypeptides derived from the extracellular spaces of winter rye. Several possible applications of these polypeptides are described in general, among these is ice-cream. However no teaching is provided which of the polypeptides should be selected to obtain a good quality ice-cream. This document does not teach the use of AFPs derived from lichens.

Applicants have found that a great number of contain a significant level of AFPs. Applicants .now aim at providing a particular novel selection of lichen sources which surprisingly provide on the one hand good AFP properties and on the other hand are capable of favourably influencing the textural properties of ice cream.

F7389 AU2(C) 6 In particular it has been found that suitable AFPs can be selected by taking a composition comprising AFP in an aqueous composition, quick freezing this composition to or less followed by storage for 1 hour at -6 °C.

Suitable AFPs result in an ice crystal size after storage for 1 hour at these conditions of less than 15 um, more preferably 5-14 mr, most preferred 8 to 12 um. The temperature of quick freezing is advantageously -40 to -100 oC, preferably -80 °C.

1 0 A detailed description of a suitable test to determine this characteristic is given in example I.

Generally the test can be applied to any suitable composition comprising AFP and water. Generally the level of AFP in such a test composition is not very critical and can for example be from 0.0001 to 0.5 wt%, more preferred 0.0005 to 0.1 wt%, most preferred 0.001 to 0.05 wt%, for example 0.01 wt%. The water level in the aqueous composition is 20 advantageously 30 wt% or more, for example 50 wt% to 99.9999 wt% Any suitable composition comprising AFP and water can be used to carry out the test. If desired additives may be present, e.g. sucrose or buffering agents. Generally, however, it will not be necessary to obtain the AFP in purified form. For practical applications normally it would suffice to prepare a liquid extract or juice of lichen material, wherein this extract or juice can then be tested.

A suitable method to prepare suitable liquid compositions is given in example II.

F7389 AU2(C) 7 Normally lichen to be tested for suitable AFPs have been subjected to cold. For example lichen can be tested which grow in cold climates, for example Antarctic lichen.

Alternatively lichen can be harvested during the winter period, preferably December to March, more preferred January to February, most preferred in January (northern hemisphere) or June to September, more preferred July to August, most preferred July (southern hemisphere).

Applicants have subjected a number of lichen to the above described test. The results are given in example III.

Preferred sources of AFPs are derived from Alectoria S 15 nigricans, Caloplaca regalis, Himantormia lugubris, Hypogymnia physodes, Parmeilia subrudecta, Ramalina farinaceae, Stereocaulon glabrum, Umbilicaria antactrica and SUsnea subfloridana.

0o 20 Applicants are of the opinion that based on the guidelines given in the description of the invention the skilled person will be well able to select further AFPs which can suitably be derived from lichen. The use of these AFPs is also embraced within the scope of the-present invention.

These AFPs show particularly good properties in frozen confectionery products.

For some applications those AFPs are selected which maintain their ability to limit ice crystal growth (as evidenced by the above test) even after heat treatment above a F7389 AU2(C) 8 temperature of 600°C, most preferably from 80 to 105 °C for a period of at least 30 seconds, more preferred more than 1 minute, 10 minutes or even more than 1 hour. Suitable lichen sources which are heat stable are for example Ramalina farinaceae and Usnea subfloridana.

Detailed description of the invention 9 Frozen products in accordance to the present invention comprise at least one AFP, which can be derived from lichen sources.

The AFPs can be obtained from the lichen sources by any 15 suitable process, for example the isolation processes as 9 described in the above mentioned documents. Alternatively the AFPs can be extracted from the lichen, for example by preparing an extract from the lichen followed by an optional concentrating step. Examples of suitable methods for 20 obtaining said extracts are given in the examples.

Also micro-organisms or plants may be genetically modified to express AFPs and the AFPs may then be used in accordance to the present invention.

Genetic manipulation techniques may be used to produce AFPs having at least 80%, more preferred more than 95%, most preferred 100% homology to the AFPs directly obtained from lichen sources which naturally contain the AFPs. For the purpose of the invention these AFPs possessing this high level of homology are also embraced within the term "AFP F7389 AU2(C) 9 derived from lichen". For the purpose of the invention the term "AFP derived from lichen" preferably does not include AFPs which naturally occur in non-lichen sources e.g. fish and which by transgenetic routes are produced by lichen.

The genetic manipulation techniques may be used as follows: An appropriate host cell or organism would be transformed by a gene construct that contains the coding region for the desired polypeptide.

The nucleotide sequence coding for the polypeptide can be inserted into a suitable expression vector. Said vector encoding the necessary elements for transcription and translation, and in such a manner that they will be expressed under appropriate conditions in proper orientation and correct reading frame and with appropriate targeting and expression sequences).

The methods required to construct these expression vectors 20 are well known to those skilled in the art.

A number of expression systems may be utilised to express the polypeptide coding sequence. These include, but are not limited to, bacteria, yeast, insect cell systems, plant cell culture systems and plants all transformed with the appropriate expression vectors.

AFPs obtainable from the above mentioned sources can be used in any suitable frozen confectionery product. For the purpose of the invention the term frozen confectionery product includes milk containing frozen confections such as F7389 AU2(C) 10 ice-cream, frozen yoghurt, sherbet, sorbet, ice milk and frozen custard, water-ices, granitas and frozen fruit purees. For some purposes the use in fermented frozen food products is less preferred.

Preferably the level of AFPs in the frozen confectionery product is from 0.0001 to 0.5 wt% based on the final product, more preferred 0.0005 to 0.3 wt%, most preferred 0.001 to 0.2 wt%.

Preferably the level of solids in the frozen confection sugar, fat, flavouring etc) is more than 2 wt%, more preferred from 4 to 000 15 If desired the frozen confectionery products of the invention may be aerated, for example to an overrun of from 50 to 500%.

*see The method of preparing the frozen confectionery product of 00 00 20 the invention can be selected from any suitable method for the preparation. The AFPs can generally be added at various stages of the preparation, for example it can be added in the first pre-mix of ingredients or can later be added during a later stage of the preparation process. For some applications it is sometimes preferred to add the AFPs at a relatively late stage of the production process, for example after (partial) pre-freezing of the product.

The freezing process for frozen confectionery products can be selected from any suitable freezing process and may optionally comprise an aeration step for example to an F7389 AU2(C) 11 overrun of 50 to 300 For some purposes it is advantageous that the freezing process involves a cold hardening step, for example at a temperature of -30 Fahrenheit or lower.

For some applications it may be advantageous to include a mixture of two or more different AFPs into the frozen confectionery product. One reason for this can for example be that the lichen source for the AFPs to be used, contains more than one AFP and it is more convenient to add these, for example because they are both present in the lichen extract to be used. Alternatively it may sometimes be desirable to add more than one AFP from different sources.

The invention will now be illustrated by means of the 15 following examples Example

I

Test for determining ice crystal particle size after quick 20 cooling followed by storage at -6 °C for 1 hour.

The preferred method is as follows: Ia: Anti-freeze activity was measured using a modified "splat assay" (Knight et al, 1988). 2.5 p1 of the solution under investigation in 30% sucrose was transferred onto a clean, appropriately labelled, 16 mm circular coverslip. A second coverslip was placed on top of the drop of solution and the sandwich pressed together between finger and thumb. The sandwich was dropped into a bath of hexane held at -80 0 C in a box of dry ice. When all sandwiches had F7389 AU2(C) 12 been prepared, sandwiches were transferred from the -80 °C hexane bath to the viewing chamber containing hexane held at -6 °C using forceps pre-cooled in the dry ice. Upon transfer to -6 sandwiches could-be seen to change from a transparent to an opaque appearance. Images were recorded by video camera and grabbed into an image analysis system (LUCIA, Nikon) using a 20x objective. Images of each splat were recorded at time 0 and again after 60 minutes.

Alternatively (less preferred) the properties can be measured as follows: Ib: A sample of an AFP containing product containing water is adjusted to a sucrose level of 30 wt% (If the starting 15 level of the sample is more than 30% this was done by dilution, if the starting level was lower sucrose was added to the 30% level).

A 3 pL drop of the sample is placed on a 22 mm coverslip.

A

20 16 mm diameter cover-slip is then placed on top and a 200 g weight is placed on the sample to ensure a uniform slide thickness. The edges of the coverslip are sealed with clear nail varnish.

The slide is placed on a Linkham THM 600 temperature controlled microscope stage. The stage is cooled rapidly °C per minute) to -40 °C to produce a large population of small crystals. The stage temperature is then raised rapidly °C per minute) to -6 °C and held at this temperature.

F7389 AU2(C) 13 The ice-phase is observed at -6 OC using a Leica Aristoplan microscope. Polarised light conditions in conjunction with a lambda plate were used to enhance the contrast of the icecrystals. The state of the ice phase (size of ice-crystals) is recorded by 35 mm photomicrography at T=0 and T=l hour.

Whereby an average particle size (visual determination, number average) of below 15 pm indicates a suitable AFP for use in frozen confectionery products.

10 Example II Methods to obtain compositions containing AFP and water.

A. Fresh tissue of lichen can be ground with a pestle and 15 mortar (cooled to 4 oC) in an equal volume buffer A (for example 10mM EDTA, 20 mM Ascorbic acid, buffered with Tris to pH 7.4) held on ice. The homogenates are filtered through .one or more layers of muslin and kept on ice prior to further use.

This method can generally be applied to most lichen and provide fresh lichen juice containing the AFP.

B. Method to extract AFPs from sources which are capable of withstanding heat such as Ramalina farineae and Usnea subfloridana. This method is exemplified by using mixed grass. However it will be apparent that this method may equally be applied to heat stable lichen.

Mixed grass tissue (Poa Trivialis, Lolium Perenne, Holcus Lanatus, Bromus Sterilis) was cut in January (mean F7389 AU2(C) 14 temperature in that month was 3.5 °C ensuring the appropriate cold acclimatization of the plants). The grass tissue was rapidly transported into the laboratory for further handling and washed thoroughly with water to remove dirt.

OOQOO

o o 5 *o *o 15 500 g of grass clippings was placed in a 650 Watt microwave oven and heated at full power for 5 minutes, whereby the temperature was raised to 85 to 100 The grass clippings were then cooled to ambient temperature.

After the heating step the AFP rich juice was separated from the clippings by filtering. The mass was stirred continuously for 5 minutes in the presence of an equal volume of water and then squeezed through 3 layers of muslin.

F7389 AU2(C) 15 Example III Screening of various lichen. Non Antarctic lichen were harvested in January (mid winter). The Antarctic lichen were harvested mid summer (February-March).

An AFP containing juice was prepared according to the method as decribed in Example IIa.

o• 10 oo r The samples were subjected to the test of Example Ia.

Suitable AFPs for application in frozen confectionery products are indicated with a positive sign LICHEN NAME RI test Alectoria nigricans Caloplaca regalis Himantormia lugubris Hypogymnia physodes Parmelia subrudecta Ramalina farinaceae Stereocaulon glabrum Umbilicaria antartica Usnea subfloridana F7389 AU2(C) 16 Example IV A liquid pre-mix for preparing ice-cream was made by mixing: Ingredient Skimmed milk powder Sucrose Maltodextrine (MD40) Locust bean gum Corn Syrup 63DE Guar Gum Genulacta L100 Butter Avicel RC581 15 Gelatin e Monoglyceride (palmitate) Vanillin AFP (of example IIb) Water by weight 11.390 3.410 4.000 0.072 20.705 0.048 0.020 9.015 0.240 0. 140 0.450 0.010 0.100 or none balance (control) *Note: AFP is added as concentrated AFP solution using some of the added water as a diluent, percentage refers to amount of AFP.

This mix can conveniently be pasteurised at 85 °C for seconds and stored chilled in a can.

The mixes can be used in the preparation of a ice-cream by whipping with a conventional house-hold mixer to an overrun of about 100%, followed by quiescently freezing into a house-hold freezer.

F7389 AU2(C) 17 The composition according to the invention had a markedly better texture than the control sample.

Similar results can be obtained by using the following lichen sources: Ramalina farinaceae and Usnea subfloridana.

*9 9 e 9*

Claims (4)

1. A frozen confectionery product comprising one or more anti-freeze polypeptide derived from lichen, wherein the anti-freeze polypeptide in an aqueous composition have an ice crystal size after quick freezing to -40 °C or less, followed by storage for 1 hour at -6 oC of less than 15 pm. 10

2. Frozen confectionery product according to claim 1, wherein the anti-freeze polypeptide is derived from Alectoria nigricans, Caloplaca regalis, Himtormica,lugubris, Hypogymnia physodes, Parmelia subrudecta, Ramaline farinaceae, Sterocaulon glabrum, 15 Umbilicaria antartica, Usnea subfloridana.

3. Frozen confectionery product according to claim 2 wherein the anti-freeze polypeptide maintain their ability to limit ice crystal growth after heat 20 treatment above a temperature of 60 0 C for a period of at least 30 seconds, more preferred more than 1 minute.

4. Frozen confectionery product according to claim 3, wherein the anti-freeze polypeptide is derived from Ramalina farinaceae, Usnea subfloridana. DATED 9n Signed nd on behalf of UNILEVER PLC by Un ver Australia Limited B. JO pany Secretary.