ITMI971754A1 - PROCEDURE FOR ISOLATING ANTIFREEZE PROTEINS FROM CONFECTIONERY PRODUCTS AND FROZEN CONFECTIONERY PRODUCT CONTAINING PROTEIN FOR - Google Patents

Description

DESCRIPTION

The invention relates to a process for the preparation of a food product containing AFP and food products containing AFP. Anti-freezing peptides (AFPs) have been suggested to improve the freezing tolerance of food products.

Anti-freezing proteins have been described in the literature, see for example Marilyn Griffith and K. Vanya Ewart in Biotechnology Advances, Vol 13, No 3, pp 375-402, 1995. Anti-freezing properties generally possess one or more of the following properties: thermal hysteresis , inhibition of ice recrystallization, 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 analyze the presence of AFPs. Thermal hysteresis results from lowering the apparent freezing temperature of a solution that contains active AFP by thermal hysteresis without affecting the melting temperature. The identification of AFP sources by thermal hysteresis analysis is extensively described in the literature, see for example John G Duman in Cryobiology 30, 322-328 (1993).

Inhibition of ice recrystallization is another property of AFPs. This activity is also referred to as suppression of ice crystal growth. This property can be examined by comparing at some point in time the ice crystal sizes of the crystals in the presence of AFP and in the absence of AFP. The application of this procedure in the analysis 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 originates from the selective binding of AFPs to some faces of the ice crystal thus limiting the growth of the crystal in some directions. The presence of ice crystals with a hexagonal bipyramid shape is therefore considered indicative of the presence of AFP. This procedure is described for example for analyzing the AFP activity of extracellular winter rye in WO 92/22581 (University of Waterloo).

A fourth property of AFPs is their ability to inhibit the activity of ice nucleation substances. This interaction between an AFP and an ice nucleator can result in, for example, an increased thermal hysteresis. This property is verified for example in WO 96/40973 (University of Notre Dame du Lac).

AFPs have been suggested to improve the freezing tolerance of products. Many applications have been suggested in this context.

For example, AFPs have been suggested to improve the cryopreservation of biological materials (WO 91/12718, Agouron Pharmaceuticals, WO 91/10361, The Reagents of the University of California). Furthermore, AFPs have been suggested to prevent loss from liposomes, for example in cosmetic or pharmaceutical substances (see WO 96/20695). A further possible application is to increase the tolerance to freezing of plants including within them (or by producing within them transgenetically) an AFP (see J. Celi. Biochem. Suppl. Vol. 14e, 1990, pag 303 XP002030248, Lee et al., abstract R228). Furthermore, the use of fish AFP in food products has been suggested, for example for frozen yogurt or ice cream (US 5,620,732 in the name of Pillsbury and WO 96/11586, HSC Research and development limited partnership).

However, the use of AFPs has not so far been applied on a commercial scale. The applicants believe that one of the reasons for the lack of commercial implementation is that although many AFPs have been described, in practice implementation in actual commercial products encounters serious problems.

Applicants have found that one of the key reasons for these problems is that among the large number of AFPs that have been described in the literature only a limited group of AFPs can be suitably applied for each application; Applicants have also found that this choice of suitable AFP depends on the desired application and / or on the product attributes to be obtained.

WO 90/13571 describes anti-freezing peptides produced chemically or by recombinant DNA techniques. AFPs can suitably be used in food products such as ice cream. Example 3B shows modified ice crystal forms if a sherbet blend is frozen in a film in combination with 0.01 wt% AFP.

WO 92/22581 discloses AFPs derived from plants. This paper further describes a procedure for extracting a polypeptide composition from the extracellular spaces of plants by infiltrating the leaves with an extraction medium without breaking the plant cells.

WO 94/03617 describes the production of AFP from yeast and their possible use in ice cream. WO 96/11586 discloses AFPs derived from fish produced by microbes.

The present invention is aimed at providing solutions to the problems indicated above. In particular, the invention is aimed at providing frozen food products with a relatively soft although fragile consistency, said consistency being preserved even after prolonged storage at low temperatures.

It has surprisingly been found that AFPs can be conveniently incorporated into frozen food products to result in the desired product properties as long as the product and processing conditions are varied so that the shape of the ice crystals meets the specific requirements. .

Consequently, in a first aspect, the invention relates to a process for the production of a frozen food product comprising AFP, in which the conditions are chosen in such a way that the ice crystals in the product have an aspect ratio comprised between 1, 1 and 1.9.

If food is frozen, ice crystals form throughout the product. If AFPs are included in food products to be frozen, this can lead to a change in the recrystallization properties of the ice. Ice crystal aggregation of AFP-containing products can cause product brittleness.

Many consumers are in favor of relatively soft and fragile food products or their ingredients such as ice cream or sorbet. For example a crunchy sherbet or crunchy popsicle can be used as an attractive ingredient in frozen confectionery products, although relatively hard ice cream is enjoyed by a large group of consumers.

Surprisingly we found AFPs offer the opportunity to formulate frozen food products that are relatively soft and brittle on the one hand and retain improved ice recrystallization properties on the other. Applicants have surprisingly found that this advantageous combination of properties can be achieved if the product contains AFP and has a form ratio of the ice crystals in the product between 1.1 and 1.9.

The aspect ratio of ice crystals is defined as the ratio of the length to the width of the ice crystals. A shape ratio between 1.1 and 1.9 corresponds to rounded ice crystals, which are not elongated in shape. The crystal aspect ratio can be established by any suitable process. A preferred method is illustrated in the examples. Preferably the ratio is between 1.2 and 1.8, most preferably between 1.3 and 1.7.

Preferably, the frozen product of the invention is brittle. Preferably the minimum layer thickness at which the fracture behavior can be observed is less than 10 mm, more preferably 1-5 mm. Fracture behavior can be measured by either preparing layers of varying thickness by determining at what minimum thickness fracture behavior occurs or it can be calculated from Young's modulus as described in the examples.

During the formulation and subsequent freezing of food products, various parameters can influence the aspect ratio of the ice crystals to be formed. Examples of factors affecting the aspect ratio are listed below. Applicants believe that it is within the skill of one skilled in the art to choose those conditions for which the ice crystal aspect ratio is within the desired range.

One factor affecting the aspect ratio of ice crystals is the freezing rate of the product. In general terms, increasing the freezing rate decreases the aspect ratio of the ice crystals. In this context, the freezing temperature can influence the freezing rate and thus the aspect ratio of the ice crystals. In this process, freezing processes are sometimes preferred which include a hardening step for example at a temperature below -30 ° F. The storage temperature and storage time can also affect the aspect ratio, whereby higher storage temperatures and / or longer storage times tend to favor the formation of high aspect ratios.

Another factor affecting the aspect ratio of ice crystals is the mobility of the product during freezing. For example, if a liquid sorbet or ice cream mix is to be frozen, coescent freezing leads to a rather high aspect ratio for ice crystals, while stirring leads to a lower aspect ratio. Blending with high shear leads to even lower aspect ratios.

Another factor affecting the aspect ratio of ice crystals is the presence and quantity of the ingredients. For example, the presence of ingredients that tend to form a lattice structure in the product (e.g. gums or fats) can lead to a lower aspect ratio of products without these ingredients. Also other ingredients can lead to lower aspect ratios, such as high solids levels, for example high sugar levels can lead to low aspect ratios.

Finally, the nature and quantity of the AFPs present can lead to variations in the aspect ratios. Some AFPs seem to favor the formation of low aspect ratios while other AFPs seem to induce higher aspect ratios. Suitable text for choosing these AFPs is described in the examples. The variation in the amount of AFP can also lead to a variation in the aspect ratios.

In accordance with a second embodiment, the invention relates to a process for the production of a frozen food product comprising AFP, in which the formulation, freezing and storage conditions are selected in such a way that the ice crystals in the product have an aspect ratio between 1.1 and 1.9.

The process of the invention can be applied to any frozen food product that contains AFP. Examples of suitable products are sauces, ready meals, etc. Preferred food items are frozen confectionery products such as ice cream and sorbet.

The Applicants have found that the AFPs to be used in the process according to the invention can derive from a plurality of sources such as for example plants, fish, insects and microorganisms. Both naturally occurring species and species obtained by genetic modification can be used. For example, microorganisms or plants can be genetically modified to express AFP and the AFPs can then be used according to the present invention.

Genetic manipulation techniques can be used to produce AFP as follows. An appropriate host cell or organism is transformed by a "gene builder" that contains the desired polypeptide. The nucleotide sequence encoding the polypeptide can be inserted into a suitable expression vector which encodes the elements necessary for transcription and translation in such a way that they will be expressed under the appropriate conditions (e.g. in the correct orientation and in the correct reading pattern. and with the desired and appropriate expression sequences). The processes required to construct these expression vectors are well known to those skilled in the art.

A plurality of expression systems can be used to express the coding sequence of the thermostable polypeptides. 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.

A wide variety of plants and plant cell systems can be transformed with the nucleic acid constructs of the desired polypeptides. Preferred embodiments include but not limited to corn, tomato, tobacco, carrots, strawberries, rapeseed and sugar beet.

For the purpose of the invention the preferred AFPs are derived from fish. Particularly preferred is the use of proteins deriving from plants (ie proteins obtained directly from plants or these proteins produced transgenetically by other organisms), derived in particular from winter rye or perennial herbs.

For some natural sources, AFPs may consist of a mixture of two or more different AFPs.

Preferably those AFPs are chosen which have significant ice recrystallization inhibition properties. A suitable test for determining the recrystallization properties is indicated in the examples. Preferably, the AFPs according to the invention provide dimensions of the ice particles after recrystallization - preferably measured according to the examples - less than 20 µm, the range of 5-15 µm more preferred. It is believed that the small size of the ice crystals in combination with the specific aspect ratio is particularly advantageous for obtaining the desirable structural characteristics.

A very advantageous embodiment of the invention relates to product formulations which are selected in such a way that coescent frozen conditions can be used in the preparation of the product, while still obtaining the aspect ratio as described above.

Examples of such frozen food products are: frozen dessert mixes such as ice cream mixes and sorbet mixes which are intended to be stored at room or refrigerator temperatures. Suitable product forms are for example: a powder mixture which is packaged for example in a bag or envelopes. Said mixture being able to form the basis of the frozen food product, for example after the addition of water and optionally other ingredients and - optionally - aeration.

Another example of a suitable blend could be a liquid blend (optionally aerated) which can be frozen if necessary after adding additional components and optional further aeration.

The clear advantage of the blends mentioned above is that the presence of the AFP ingredients allows the blends to be frozen under coescent conditions such as in a shop or home freezer.

In an extremely convenient way, these mixtures are packaged in closed containers (e.g. cartons, bags, boxes, plastic containers, etc.). For single portions the package size will generally range from 10 to 1000 g. For multiple portions pack sizes of up to 500 kg may be appropriate. In general the format of the packaged will be between 10 g and 5000 g.

As indicated above, the preferred products in which AFPs are used are frozen confectionery products such as ice cream or sorbet. Preferably the AFP level is comprised between 0.0001 and 0.5% by weight with respect to the final product. If using dry or concentrated mixes, the concentration may be higher to ensure that the level of the final frozen product falls within the ranges indicated above.

Surprisingly it has been found that combinations of the invention can contain very low amounts of AFP while still having good quality.

Until now, it was generally believed that rather high levels of AFP were necessary to achieve a reasonable improvement in the recrystallization properties. One reason for this is that it was commonly believed that AFPs act on significant portions of the surface of ice crystals and therefore must be present at quite high levels, such as 0.01% or more to achieve a reasonable effect.

Surprisingly it has also been found that for frozen products improved recrystallization properties and higher temperature tolerance can already be achieved if low AFP levels are used.

It has surprisingly been found that AFP levels can be as low as 0.1-50 ppm and yet still provide adequate recrystallization properties and adequate temperature tolerance in frozen confectionery products. Although Applicants do not wish to be limited in any way by a theory, the reason may be that the interaction between the frozen cake solids and the AFPs provides an excellent mechanism for inhibiting crystal growth. In a highly convenient way, the AFP level is between 1 and 40 ppm, in particular 2-10 ppm is preferred.

For the purposes of the invention, the term frozen confectionery products includes frozen desserts containing milk such as ice cream, frozen yogurt, sorbet, ice milk and frozen creams, popsicles, slushes and frozen fruit purees. For some applications, the use of AFP in frozen fermented food products is less preferred

Preferably, the level of solids in the frozen dessert (for example sugar, fat, flavoring, etc.) is greater than 30% by weight, more preferably 40-70% by weight.

In a particularly preferred embodiment of the invention, hard and crunchy frozen dessert formulations are used to create a texture contrast in the ice cream. Preferably, such ice creams contain as discrete elements in their structure the composition containing AFP according to the invention. For example, a relatively hard ice cream core can be coated with a thin layer of the composition of the invention to provide a relatively soft but brittle outer layer surrounding the ice cream core. Another embodiment could be the incorporation of the formulation of the invention as inclusions in ice cream. A third embodiment is to alternate the layers of ice cream with the formulation of the invention to create thin soft but fragile layers alternating with layers of ice cream. Example Z

The isolation of AFPs from winter signal.

The winter rye (Halo variety) was cut in January (the average temperature in that month was 3.5 ° C which ensured acclimatization to the appropriate cold of the plants). The fabric was quickly transported to the laboratory for further handling and thoroughly washed with water to remove dirt.

400 g of small pieces were homogenized at room temperature in a Waring mixer with 800 g of water until the leaf tissue was completely destroyed. The AFP-rich juice was collected by filtering through four layers of muslin.

The AFP-rich juice was then subjected to temperature treatment by boiling the juice for 10 minutes. This resulted in the precipitation of the protein while the AFP to be used according to the invention remained in solution. The supernatant was separated from the precipitate by centrifugation at 15,000 g for 20 minutes or further filtering through muslin.

AFPs can be isolated from the supernatant by lyophilization.

Example ZI

Grass AFP isolation.

Mixed grass fabric (Poa Trivialis, Lolium Perenne, Holcus Lanatus, Bromus Sterilis), was cut in January (average temperature in this month 3.5 "C which ensured acclimatization to the appropriate cold of the plants). grass was quickly transported to the laboratory for further handling and was thoroughly washed with water to remove dirt.

500 g of grass pieces were placed in a 650 Watt microwave oven and heated at full power for 5 minutes, whereby the temperature was raised to 85-100 ° C. The bits of grass were then cooled to room temperature.

After the heating phase, the AFP-rich juice was separated from the small pieces by filtering. The mass was stirred continuously for 5 minutes in the presence of an equal volume of water and then squeezed through three layers of muslin.

The supernatant can be lyophilized to isolate AFP.

Example III

A premix to prepare ice cream was made by mixing:

Ingredient% by weight

Skimmed milk powder 11.39

Sucrose 3.14

Maltodextrin (MD40) 4.00

Corn syrup 63DE 20.71

Butter oil 9.00

Monoglyceride (palmitate) 0.45

Vanillin 0.01

Carob seed gum 0.07

Guar gum 0.05

Carrageenan 0.02

Microcrystalline cellulose 0.24

Gelatin 0.14

AFP (of Example I *) 0.01 or nothing (control) Water the rest

* Note: AFP is added as a concentrated solution; the percentage yes

refers to the amount of AFP.

This mixture can be conveniently stored at temperature

environment for example in a plastic container.

The mixture can be whipped with a conventional household whisk

up to about 100% overrun, followed by quiescent freezing in a

domestic freezer.

After two months of storage, the composition according to the invention

tion had a much better consistency than the sample of

check.

Example IV

A liquid premix for the preparation of ice cream was prepared by mixing

Ingredient% by weight

Skimmed milk powder 10.00

Sucrose 13.00

Maltodextrin (MD40) 4.00

Carob seed gum 0.14

Butter oil 6.00

Monoglyceride (palmitate) 0.30

Vanillin 0.01

AFP (of example II *) 0.01 or nothing (control)

Water the rest

'Note: AFP is added as a concentrated solution; the percentage refers to the amount of AFP.

Example V

Ice creams were prepared by freezing and aerating until an overrun

70% the formulation according to Example IV:

Samples of both products were placed at equilibrium a

-18 ° C in a Prolan environmental cabinet for approximately 12 hours.

Microscope slides were prepared by smearing a thin layer

of ice cream from the center of the thin glass slides.

Each slide was transferred to a microscope stage with ¬

checked in temperature (at -18 ° C) where images of cri¬ were collected

ice stalls (about 400 individual ice crystals) which are ¬

you are sent through a camera to a storage system and to

image analysis.

The stored images of the ice crystals were highlighted

hand-drawn tracing the perimeter which then highlights the entire crì¬

stall. The images of the highlighted crystals were then measured using image analysis software which counts the number of pixels required to complete the longest straight line (length), shortest straight line (width), aspect ratio (length / length).

The average aspect ratio for the crystals was checked.

For the control sample the aspect ratio was 1.45.

For the sample containing AFP the aspect ratio was 1.7.

Example VI

The fragility of the ice cream of Example 2 was determined by calculations on the fracture behavior of the ice cream. Young's modulus was measured using a 3-point bend test.

Young's modulus was measured by preparing strips of ice cream, balancing them for 18 hours in a freezer cabinet and transferring them to a temperature cabinet. The strips were placed on a 3-point bending jig as described in the Handbook of Plastics Test Methods (2nd Edition), ed. R.P. Brown, George Godwin Ltd, 1981. The analysis of the samples was carried out immediately at a strain rate of 50 mm / min. From the force-deformation curve, the inclination or the initial angular coefficient was measured and used to calculate Young's modulus according to the following equation:

- where L = extension of the beam (110 mm), B = width of the sample, w = height of the sample. Usually eight samples were analyzed to give an average value of Young's modulus.

Using the calculations described by Williams 6 Cawood in Polymer Testing 915-26 (1990) the fracture hardness can be calculated.

The results were as follows: For the control sample a thickness of 966 meters was calculated as necessary to obtain a brittle layer. For the sample containing AFP, brittleness (fracture behavior) was already found at a thickness of 3 ml. This clearly shows the better brittleness of the products of the invention. Products with AFP were relatively tough.

Example VII

This example describes a methodology for selecting those AFPs which favor the formation of ice crystal forms as preferred in the invention.

The growth of ice crystals under normal circumstances occurs along the a-axis of the crystal. If AFPs are present, growth changes. This selective influence of crystal shape can be explained by the fact that AFPs tend to bind to some parts of the ice crystals and in doing so inhibit the growth of the ice crystal in some directions. The bond can occur, for example, in correspondence with the planes of the prism (perpendicular to axis a) or in correspondence with pyramidal planes (which protrude from these planes).

Applicants have found that AFPs which favor the formation of shape ratios according to the present invention can be found by selecting for example those AFPs which tend to bond in the plane of the prism. The methodology for selecting these specific binding AFPs can be any suitable methodology. A suitable test employs the so-called single ice crystal hemisphere growth experiment which is based on the technique described in Knight C.A., C.C. Cheng and A.L. DeVries, Biophys. J. 59 (1991) 409-418, Adsorption of a- helical antifreeze peptide s on speci fic ice crystal surface planes.

A well insulated 5 liter plastic beaker was filled with deionized water and placed in a temperature controlled cabinet at -1 ° C. It was then left to freeze slowly at the top. After two days a single ice crystal approximately 4 cm thick covered the beaker. The crystallographic orientation of this crystal was determined using single crystal X-ray diffraction procedures. Ice cubes approximately 2 cm in size were cut from the large single crystal such that one surface was parallel to the prism plane and another parallel to the basal plane. Thus oriented single ice crystals were produced.

An apparatus consisting of a cold brass finger (diameter of approximately 1 cm) was used on which an oriented seed crystal was frozen. The seed was first quarried in such a way that the seed crystal would fit around it. Coolant was then circulated through the finger and the seed froze quickly to it.

The finger with the seed crystal was then immersed in an isolated 100 ml beaker containing a solution of the material being investigated. The initial solution temperature was room temperature (about 18 ° C), and the only cooling was provided by the cold finger. Initially the seed crystal partially melted but then grew into a single hemispherical crystal. After several hours (6-8) a hemisphere with a diameter of 5-7 cm had formed.

The experiment was carried out with various AFP solutions. The AFP solutions used had an AFP concentration of 10-3 mg / ml.

The hemisphere was then removed from the cold finger and taken to a temperature-controlled cabinet at -15 ° C. The surface was scratched and was left in the locker at least overnight (16 hours or more). Air was circulated through the cabinet by an integral fan. During this time there was evaporation of the surface layers of the ice. The surface of the hemisiera of ice then came to have a smooth surface like a mirror. However, for a hemisphere containing A5P, coarse patches are observed on the surface. These correspond to the patches where AFP had bonded on the surface of the hemisphere. Large AFP molecules prevent the ice molecules from evaporating and therefore a coarse intertwining of AFP molecules accumulates on the surface at the surfaces where the preferential bond to ice occurs. Since the orientation of the hemisphere is known, and the angular distance between such coarse patches and the basal and prism directions can be measured by an optical goniometer, the nature of the bond plane can be easily determined.

This test can be used to choose those AFPs that tend to bind to the pyramid planes. For example, AFPs from Example 1 and II tend to tie into pyramid planes. Furthermore, a large number of other plant extracts tend to give rise to pyramidal bonds.

It is within the skill of the skilled person to use the above analysis to determine these AFPs which tend to favor the formation of high ice crystal aspect ratios. To check whether they are suitable in frozen products of the invention, the actual product can be made and the aspect ratio of the crystals in the product can be determined.

Example VIII

Test to determine the size of ice crystals after recrystallization.

A sample of solution containing AFP in water has been adjusted with a sucrose level of 30% by weight (if the starting level of the sample is greater than 30% this can be done by dilution, if the starting level is lower it is added sucrose up to the 30% level).

In general, the test can be applied to any suitable composition including AFP and water. In general, the level of AFP in such a test composition is not particularly critical and can be for example between 0.0001 and 0.5% by weight, more preferably between 0.0005 and 0.1% by weight. weight, highly preferred is between 0.001 and 0.05% by weight, for example 0.01% by weight.

A 3 pL drop of the sample was placed on a 22 mm coverslip. A 16mm diameter coverslip was then placed on top and a 200g weight placed on the specimen to ensure uniform slide thickness. The edges of the coverslip were sealed with clear nail polish.

The slide was placed on a Linkham THM 600 temperature controlled microscope stage. The stage was cooled rapidly (50 ° C per minute) down to -40 ° C to produce a large population of small crystals. The stage temperature was then rapidly increased (50 ° C per minute) down to -6 ° C and held at that temperature.

The ice phase was observed at -6 ° C using a Leica Aristoplan microscope. Polarized light conditions in conjunction with a lambda plate were used to increase the contrast of the ice crystals. The state of the ice phase (size of the ice crystals) was recorded by photomicrography at 35 mm at T = 0 and T = 1 hour. An average particle size (visual determination, numerical average) of less than 20 µm, more preferred between 5 and 15 µm indicates the preferred AFPs to be used in products according to the invention.

Claims (9)

CLAIMS 1. A process for producing a frozen food product comprising AFP, in which conditions are chosen such that the ice crystals in the product have an aspect ratio of 1.1 to 1.9. Process according to claim 1, wherein the conditions for influencing the aspect ratio are selected from the group of: freezing rate, mobility of the product during freezing, storage temperature and storage time, formulation of the product and nature and quantity of AFF and their combinations. Process according to claim 1, wherein the frozen food product is a frozen confectionery product. 4. Frozen confectionery product comprising from 0.0001 to 0.5% by weight of AFP, said product having an ice crystal shape ratio of between 1.1 and 1.9. 5. Frozen confectionery product according to claim 4, wherein the AFP preferably binds to the pyramidal planes of the ice crystals. 6. Frozen confectionery product with a texture contrast, said product comprising discrete elements of a confectionery product according to claim 4. 7. Frozen confectionery product according to claim 6, comprising thin layers of ice cream alternating with thin layers of sherbet or popsicle, wherein the layers of sorbet or popsicle comprise 0.0001 to 0.5% by weight of AFP and have a shape ratio of the ice crystals between 1.9 and 3.0 8. Ice cream mix suitable for use in the preparation of a frozen confectionery product according to claim 4. The ice cream mix according to claim 8, wherein the preparation involves aeration and quiescent freezing.