_ <v Europaisches 111111111111111111111111111111 JGV/l Eur°Pean Patent Office <*S Office europeen des brevets (11) EP 0 785 727 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) int. CI.6: A23L 1/03, A23C 9/123, of the grant of the patent: A23C 19/032 12.05.1999 Bulletin 1999/19 (86) International application number: ..... - ...number: 95933279.2 (21) Application PCT/CA95/00568 (22) Date of filing: 11.10.1995 x 7 3 (87)/Q-,X International. 4 4..... publication 4. number: WO 96/11586 (25.04.1996 Gazette 1996/18) (54) PREPARATION OF FROZEN FERMENTED FOODS USING ANTIFREEZE POLYPEPTIDE- EXPRESSING MICROORGANISMS HERSTELLUNG VON GEFRORENEN FERMENTIERTEN NAHRUNGSMITTELN MIT GEFRIERSCHUTZ-POLYPEPTIDE EXPREMIERENDEN MIKROORGANISMEN PREPARATION D' ALIMENTS FERMENTES GLACES EN UTILISANT DES MICRO-ORGANISMES EXPRIMANT UN POLYPEPTIDE ANTIGEL (84) Designated Contracting States: • HEW, Choy, L. AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL Thornhill, Ontario L4J 2A3 (CA) PTSE • JOSHI, Shashikant, B. Toronto, Ontario M5G 1 H4 (CA) (30) Priority: 12.10.1994 US 321991 • WU, Yaling St. John's, Newfoundland A1 A 2V2 (CA) (43) Date of publication of application: 30.07.1997 Bulletin 1997/31 (74) Representative: Thomson, Paul Anthony et al (73) Proprietors: Potts, Kerr & Co. • HSC Research and Development Limited 15, Hamilton Square Partnership Birkenhead Merseyside L41 6BR (GB) Toronto, Ontario M5G 1X8 (CA) • SEABRIGHT CORPORATION LIMITED (56) References cited: St. John's, Newfoundland, A1C 5S7 (CA) EP-A- 0 424 771 EP-A- 0 443 543 WO-A-90/13571 WO-A-94/03617 (72) Inventors: • FLETCHER, Garth, L. • PATENT ABSTRACTS OF JAPAN vol. 16 no. 13 St. John's, Newfoundland A1 A 1T8 (CA) (C-901) & JP,A,03 232896 (SUMITOMO CHEM CO) 16 October 1991, CO r»- Csl 10 CO Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in o a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. Q_ 99(1) European Patent Convention). LU Printed by Xerox (UK) Business Services 2.16.7/3.6 EP 0 785 727 B1 Description FIELD OF THE INVENTION 5 [0001] The present invention relates generally to methods and reagents useful in maintaining the quality of frozen food products during frozen storage, particularly enhanced storage life and the maintenance of consumer accepted quality of dairy products. BACKGROUND OF THE INVENTION 10 [0002] Refrigeration, particularly freezing, is a common and preferred means for storing biological materials. Frozen storage generally arrests or considerably slows the deterioration of the biological product. [0003] Frozen or refrigerated foods are now a mainstay of the human diet in developed nations. Thus extensive research has and is being carried out by food scientists to ensure high quality products for the consumers. This is par- rs ticularly true with regard to frozen vegetables and frozen deserts such as ice cream and yogurt. [0004] Frozen deserts such as ice cream or yogurt are generally eaten in the frozen state. Thus, the texture of the frozen product as well as its flavor is important to consumers. Texture is to a large extent governed by the size of the ice crystals. Producers of these frozen deserts have gone to considerable effort and expense to ensure smooth textured products. However, during frozen storage the ice crystals can grow and thus roughen and spoil this texture. The growth 20 of ice crystals during frozen storage is known as recrystallization. This problem is particularly common when the frozen storage conditions are less than ideal, such as during transportation or storage in modern frost-free home freezers. After a relatively short period of time at above-zero temperatures {i.e., above 0°C), or even at sustained freezing tem- peratures, frozen foods can become less desirable or even unsuitable for human consumption due to the ice recrystal- lization process. 25 [0005] Although manufacturers use a variety of techniques to reduce the damage associated with recrystallization success has been limited and significant problems remain. Thus there is a need for new techniques to reduce or prevent the recrystallization process and improve the characteristics of frozen foods. These techniques and compositions should be inexpensive and completely safe and suitable for human consumption. [0006] It has been clearly demonstrated that antifreeze polypeptides (AFP) can effectively inhibit ice recrystallization 30 at low (ng/ml) concentrations in aqueous solutions and frozen food products (see e.g., Knight et al., 1984, Nature 308:295-296; Knight etal., 1986, Cryobiology, 23:256-262; Knight etal., 1988, Cryobiology, 25:55-60; Warren etal., U.S. Patent No. 5, 1 1 8,792). Warren et al, supra, have suggested adding purified antifreeze polypeptides directly to food products prior to freezing to improve preservation characteristics during frozen storage. [0007] WO-A-901 3571 teaches methods of improving the freeze tolerance of food products by suppressing ice crystal 35 growth or inhibiting ice recrystallization using antifreeze polypeptides in isolated form. Additionally, WO-A-901 3571 dis- closes a microorganism which encodes antifreeze proteins to be expressed into said microorganism for the purposes of maintaining the viability of same on freezing and upon thawing. WO-A-901 3571 does not teach the use of a microor- ganism that is capable of secreting the antifreeze polypeptide into a food product. [0008] At the present time antifreeze proteins are available for commercial use from two sources; the blood serum 40 from a small number of fish species found in cold water, and recombinant DNA techniques such as those described by (but not restricted to) Warren etal., supra. Other sources of antifreeze protein, such as transgenic plants and animals, are currently being explored. Regardless of the source, the antifreeze polypeptides must be isolated from the medium in which they are found or produced, and subject to extensive purification. These purification procedures are expensive to the point where the cost of the antifreeze polypeptide additions could exceed the value of the frozen product. More- 45 over, the purification protocol may introduce contaminants unsafe for consumption. [0009] In the view of the inherent value to producers and consumers of inhibiting ice recrystallization in frozen fer- mented dairy products, and the fact that antifreeze polypeptides are very effective in this regard, it is desirable to develop methods to incorporate the antifreeze polypeptides into the food products in the most efficient and cost effec- tive method possible. so [0010] An ideal method of incorporating antifreeze polypeptides into frozen fermented food products is to have the organism responsible for the fermentation process produce the antifreeze proteins while fermenting the food. A number of antifreeze polypeptides and their genes have been well characterized and sequenced (see, e.g. Anathanarayanan, 1989, Life Chem. Rep. 7:1-32 and Davies etal., 1990, FASEB Journal 4:2460-2467). Several of these genes have been expressed in bacteria, transgenic plants, fish and Drosophila (see, e.g., Fletcher etal., 1988. Can. J. Fish. aquat.Sci 55 45:352- 357; Rancourt et al., 1987, Mol. Cell. Biol. 7:2188-2195; Kenward et al., 1993, Plant Mol.Biol. 23:377-385; Li etal., 1991, Protein Engineering 4:995-1012; and Sonnichsen etal., 1993, Science 259:1154-1157). [001 1 ] In view of the widespread popularity of frozen fermented dairy products such as yogurt it would be desirable to develop methods for producing such products more efficiently at lower cost and with better flavor and texture. The 2 EP 0 785 727 B1 present invention provides methods for achieving this goal. SUMMARY OF THE INVENTION 5 [0012] The present invention provides methods for preparing a frozen fermented food product. This method com- prises the steps of (a) contacting a food product with a microorganism that is capable of secreting a fish antifreeze polypeptide, wherein the microorganism is capable of fermenting the food product to produce the fermented food prod- uct, (b) incubating the food product with the microorganism under conditions in which fermentation takes place so that a fermented food product is produced having the antifreeze polypeptide present in an amount effective at inhibiting 10 recrystallization of the product; and (c) freezing the fermented food product at a temperature below -5°C, so as to pro- duce a frozen fermented food product. [0013] In one embodiment the food product is a dairy product {e.g., milk) which can be fermented to produce yogurt, buttermilk or cheese. [0014] The microorganism of the invention is usually a bacterium {e.g., Lactobacillus bulgaricus; Streptococcus 15 cremoris. Streptococcus lactis; Bifidobacterium bifidum, Bifidobacterium longum) but may also be a fungus such as a yeast {e.g., Saccharomyces fragilis, Saccharomyces cerevisiae, Saccharomyces lactis, and others). According to the invention these microorganisms are genetically engineered so that they are capable of secreting a fish antifreeze polypeptide (or a peptide with substantial sequence similarity to a fish antifreeze polypeptide and with antifreeze prop- erties similar to a fish antifreeze polypeptide). In a preferred embodiment, the antifreeze polypeptide is an animal anti- 20 freeze polypeptide, with a fish antifreeze polypeptide preferred. Most preferred is microorganism capable of expressing an ocean pout type III antifreeze polypeptide (see, e.g.. Hew et al., 1984, J. Comp. Physiol. B155:81-85; Li et al., 1985, J. Biol. Chem. 260:12904-12909). [0015] In a most preferred embodiment the invention comprises incubating milk with bacterial species Lactobacillus balgaricus and Streptococcus lactis that are capable of fermenting milk to produce yogurt and capable of secreting an 25 ocean pout type III antifreeze polypeptide; incubating the bacteria and milk under conditions that produce yogurt; and freezing the yogurt at a temperature below -5°C, so as to produce frozen yogurt.