Frozen food product

Details Frozen food product Frozen food product

2000-12-04

2004-08-10

Tate, Christopher (Department: 1654)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

15 to 23 amino acid residues in defined sequence

C530S300000, C530S327000, C530S328000, C426S565000, C426S139000, C426S104000, C426S101000, C426S100000, C514S001000, C514S002600

Reexamination Certificate

active

06774210

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The invention relates to anti-freeze proteins (AFPs) and frozen food product containing AFPs.
BACKGROUND TO THE INVENTION
Anti-freeze proteins (AFPs) have been suggested for improving the freezing tolerance of foodstuffs.
For the purpose of the invention, the term AFP has the meaning as well-known in the art, namely those proteins which exhibit the activity of inhibit the growth of ice crystals. See for example U.S. Pat. No. 5,118,792.
WO 90/13571 discloses antifreeze peptides produced chemically or by recombinant DNA techniques. The AFPs can suitably be used in food-products.
WO 92/22581 discloses AFPs from plants which can be used for controlling ice crystal shape in ice-cream. This document also describes a process for extracting a polypeptide composition from extracellular spaces of plants by infiltrating leaves with an extraction medium without rupturing the plants.
WO 94/03617 discloses the production of AFPs from yeast and their possible use in ice-cream. WO 96/11586 describes fish AFPs produced by microbes.
Several literature places also mention the isolation and/or use of plant proteins for cryoprotection. Cryoprotective proteins have a function in the protection of plant membranes against frost damage. These proteins, however, do not possess recrystallisation inhibition properties and are, therefore, not embraced within the terms AFPs.
Hincha in Journal of Plant Physiology, 1992, 140, 236-240 describes the isolation of cryoprotective proteins from cabbage. Volger in Biochimica et Biophysica Acta, 412 (1975), 335-349 describes the isolation of cryoprotective leaf proteins from spinach. Boothe in Plant Physiol (1995), 108: 759-803 describes the isolation of proteins from
Brassica napus
. Again, these proteins are believed to be cryoprotective proteins rather than AFPs. Neven in Plant Molecular Biology 21: 291-305, 1993 describes the DNA characterisation of a spinach cryoprotective protein. Salzman in Abstracts and Reviews of the 18th Annual Meeting of the ASEV/Eastern Section in Am. J. Enol. Vitic., Vol. 44, No. 4, 1993 describes the presence of boiling-stable polypeptides in buds of Vitis. Although the proteins are analogous to fish antifreeze peptides, they are cryoprotective proteins and not AFPs. Lin in Biochemical and Biophysical Research Communication, Vol. 183, No. 3, 1992, pages 1103-1108 and in Lin, Plant Physiology (1992) 99, 519-525 describes the 15 kDa cryoprotective polypeptide from Arabidopsis Hakaira. Houde in The Plant Journal (1995) 8(4), 583-593 mentions cryoprotective proteins from wheat.
Up till now, however the use of AFPs has not been applied to commercially available food products. One reason for this are the high costs and complicated process for obtaining AFPs. Another reason is that the AFPs which until now have been suggested for use in frozen food products cannot be incorporated in the standard formulation mix, because they tend to destabilise during processing especially during the pasteurisation step. This destabilisation is believed to be caused by the denaturation of the AFPs; this is a well-known effect commonly observed for peptides and proteins.
In our non pre-published patent application: WO 98/4148 it has been described that particularly good AFPs can be isolated from natural sources such as Lichen.
Applicants have now been able to determine the partial amino acid sequence of a particularly active AFP from Lichen.
Accordingly the invention relates to an AFP which can be derived from Lichen, said AFP having an apparent molecular weight of about 24 kDa and an amino acid sequence from the N-terminus of: A-P-A-W-M-D-A-E-S-F-G-A-I-A-H-G-G-L (SEQ ID NO:1).
Also embraced in the scope of our invention are proteins having a sequence which has a high degree of similarity with the above sequence. For the purpose of the invention all RI active proteins having an amino acid sequence of at least 80% overlap with the above sequence are also embraced in the scope of the invention. More preferred is an overlap of at least 90%, most preferred more than 95%, e.g. those amino acid sequences which differ none or only one or two amino acids with the above sequence.
For the purpose of the invention the degree of overlap of two (partial) amino acid sequences can be calculated as follows:
(a) the two amino acid sequences are aligned and the number of amino acids which are identical and appear in the same order are counted (X)
(b) every change, deletion or addition of an amino acid is counted as 1 point, and the total of changes, deletions and additions is calculated (Y)
(c) the degree of overlap can now been calculated as X*100%/(X+Y).
For example the (partial) amino acid sequence from the N-terminus of: A-P-A-V-V-M-G-D-A-E-S-F-G-A-I-A-H-G-G-L (SEQ ID NO:2), can be aligned with the control as follows:
A-P-A-V-V-M-G-D-A-E-S-F-G-A-I-A-H-G-G-L (SEQ ID NO:2)
A-P-A-W -M- D-A-E-S-F-G-A-I-A-H-G-G-L (SEQ ID NO:1).
This leads to a total number of identical amino acids in the same order of 17. The number of changes is 1 (W into V at the fourth position); the number of additions is 2 (V at fifth position, G at 7th position), while there are no deletions. The total of changes, additions and deletions is therefore 3. This leads to a degree of overlap of 17*100%/(17+3)=85%
The protein having (partial) amino acid sequence from the N-terminus of:
A-P-A-V-V-M-G-D-A-E-S-F-G-A-I-A-H-G-G-L (SEQ ID NO:2) is hence also embraced within the invention.
Also embraced within the scope of the present invention are modified versions of the above described proteins whereby said modification does not materially affect the ice recrystallisation inhibition properties, such as glycosylated versions thereof.
For the purpose of the invention the term about 24 kDa molecular weight means any molecular weight from 20 to 28 kDa as measured on SDS-PAGE using standard reference markers, more preferably the molecular weight is from 22 to 26 kDa.
The advantageous AFP of the present invention can be derived from Lichen especially from the species
Umbilicaria antarctica.
Also embraced within the scope of the present invention are anti-freeze proteins which although originally derived from Lichen are produced by other methods, for example by genetic modification techniques whereby for example microorganisms or plants are genetically modified to produce the above described proteins. These proteins are also embraced within the term “can be derived from Lichen”.
Also embraced within the scope of the present are nucleic acid sequences which are capable to encode the above described AFPs.
Vectors containing a nucleic acid sequence capable of encoding the AFP of the invention are also embraced within the scope of the invention.
Based on the above information it is also possible to genetically modify other natural sources such that they produce the advantageous AFP as identified here-above.
Applicants also have found that AFPs of the above sequence have improved ice-recrystallisation inhibition properties. A suitable test for determining the ice recrystallisation inhibition properties is described in the examples and involves the quick freezing to at least −40° C., for example −80° C. followed by storage for one hour at −60° C. Preferably AFPs in accordance to the invention provide a ice particle size following an ice recrystallisation inhibition assay—as described in the examples—of 15 VM or less, more preferred from 5 to 15 &mgr;m.
The AFP of the invention can conveniently be used in food products, preferably in food products which are frozen or intended to be frozen. Especially preferred is the use of AFPs in products which are heated e.g. by pasteurisation or sterilisation prior to freezing. Especially preferred is the use in frozen confectionery products.
Examples of such food products are: frozen confectionery mixes such as ice-cream mixes and water-ice mixes which are intended to be pasteurised prior to freezing. Such mixes are usually stored at ambient temperature. Suitable product forms are for example: a powder mix which is packe

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