CN1284085A - 冷冻食品 - Google Patents
冷冻食品 Download PDFInfo
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- CN1284085A CN1284085A CN98813206.0A CN98813206A CN1284085A CN 1284085 A CN1284085 A CN 1284085A CN 98813206 A CN98813206 A CN 98813206A CN 1284085 A CN1284085 A CN 1284085A
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- afp
- antifreeze protein
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- 108010053481 Antifreeze Proteins Proteins 0.000 claims abstract description 53
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 claims abstract description 4
- 235000009508 confectionery Nutrition 0.000 claims description 10
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 8
- 235000013305 food Nutrition 0.000 claims description 8
- 230000004048 modification Effects 0.000 claims description 5
- 238000012986 modification Methods 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 230000013595 glycosylation Effects 0.000 claims description 2
- 238000006206 glycosylation reaction Methods 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 6
- 102000004169 proteins and genes Human genes 0.000 abstract description 6
- 108010029485 Protein Isoforms Proteins 0.000 abstract 1
- 102000001708 Protein Isoforms Human genes 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 22
- 239000000523 sample Substances 0.000 description 13
- 150000001875 compounds Chemical class 0.000 description 12
- 238000007710 freezing Methods 0.000 description 11
- 230000008014 freezing Effects 0.000 description 11
- 238000012360 testing method Methods 0.000 description 11
- 241000196324 Embryophyta Species 0.000 description 10
- 230000000694 effects Effects 0.000 description 10
- 239000000203 mixture Substances 0.000 description 10
- 239000013078 crystal Substances 0.000 description 9
- 235000015243 ice cream Nutrition 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 9
- 238000001953 recrystallisation Methods 0.000 description 9
- 108090000765 processed proteins & peptides Proteins 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 229930006000 Sucrose Natural products 0.000 description 7
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 7
- 239000005720 sucrose Substances 0.000 description 7
- 239000012505 Superdex™ Substances 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 229920001184 polypeptide Polymers 0.000 description 6
- 102000004196 processed proteins & peptides Human genes 0.000 description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 230000037039 plant physiology Effects 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 239000006059 cover glass Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 230000008034 disappearance Effects 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 239000004570 mortar (masonry) Substances 0.000 description 4
- 238000009928 pasteurization Methods 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 235000020357 syrup Nutrition 0.000 description 4
- 239000006188 syrup Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000012614 Q-Sepharose Substances 0.000 description 3
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000013016 damping Methods 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 235000013399 edible fruits Nutrition 0.000 description 3
- 238000012239 gene modification Methods 0.000 description 3
- 230000005017 genetic modification Effects 0.000 description 3
- 235000013617 genetically modified food Nutrition 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000012723 sample buffer Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 229910052709 silver Inorganic materials 0.000 description 3
- 239000004332 silver Substances 0.000 description 3
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 2
- 241000219194 Arabidopsis Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 235000009337 Spinacia oleracea Nutrition 0.000 description 2
- 244000300264 Spinacia oleracea Species 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 241000003868 Umbilicaria antarctica Species 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 235000009392 Vitis Nutrition 0.000 description 2
- 241000219095 Vitis Species 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 235000011089 carbon dioxide Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011067 equilibration Methods 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 238000013207 serial dilution Methods 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 239000004160 Ammonium persulphate Substances 0.000 description 1
- 208000002109 Argyria Diseases 0.000 description 1
- 240000002791 Brassica napus Species 0.000 description 1
- 235000011293 Brassica napus Nutrition 0.000 description 1
- 235000006008 Brassica napus var napus Nutrition 0.000 description 1
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 1
- 241001674939 Caulanthus Species 0.000 description 1
- 241000020428 Colea Species 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 101150015836 ENO1 gene Proteins 0.000 description 1
- 208000001034 Frostbite Diseases 0.000 description 1
- 101710138460 Leaf protein Proteins 0.000 description 1
- 241001424413 Lucia Species 0.000 description 1
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 1
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 1
- 235000019395 ammonium persulphate Nutrition 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 238000012824 chemical production Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 235000011950 custard Nutrition 0.000 description 1
- 238000002242 deionisation method Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- DBSMLQTUDJVICQ-CJODITQLSA-N onametostat Chemical compound NC1=C2C=CN([C@@H]3C[C@H](CCC4=CC=C5C=C(Br)C(N)=NC5=C4)[C@@H](O)[C@H]3O)C2=NC=N1 DBSMLQTUDJVICQ-CJODITQLSA-N 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000003507 refrigerant Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 229960004418 trolamine Drugs 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 239000011800 void material Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 235000013618 yogurt Nutrition 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G9/00—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor
- A23G9/32—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds
- A23G9/38—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds containing peptides or proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3526—Organic compounds containing nitrogen
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/41—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from lichens
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Inorganic Chemistry (AREA)
- Nutrition Science (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Freezing, Cooling And Drying Of Foods (AREA)
- Confectionery (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Sorption Type Refrigeration Machines (AREA)
- Materials Applied To Surfaces To Minimize Adherence Of Mist Or Water (AREA)
- Compositions Of Macromolecular Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
可以得自地衣属的抗冻蛋白以及该蛋白的修饰型和同种型,所述蛋白的表观分子量为20—28kDa,其N末端氨基酸序列显示与A-P-A-V-V-M-G-D-A-E-S-F-G-A-I-A-H-G-G-L至少80%重叠。
Description
发明技术领域
本发明涉及抗冻蛋白(AFP)和含AFP的冷冻食品。
发明背景
已经提出抗冻蛋白(AFP)用于改善食料的耐冻性。
对于本发明的目的,术语AFP的含义是本领域众所周知的,即这些蛋白显示抑制冰晶生长的活性。参见例如US5,118,792。
WO90/13571公开了化学生产或用重组DNA技术生产的抗冻肽。所述AFP适用于食品。
WO92/22581公开了可用于控制冰淇淋中的冰晶形状的得自植物的AFP。该文献也描述了通过用提取介质浸润叶子而不需要破裂所述植物从植物细胞外间隙提取多肽组合物的方法。
WO94/03617公开了从酵母生产AFP及其在冰淇淋方面的可能应用。WO96/11586描述了微生物生产的鱼AFP。
数个文献也讲述了分离和/或应用植物蛋白用于冷冻保护。冷冻保护蛋白的功能是保护植物膜抵抗霜害。然而,这些蛋白不具有重结晶抑制特性,因此不包含在所述术语AFP中。
Hincha在《植物生理学杂志》(Journal of Plant Physiology),1992,140,236-240中描述了从甘蓝分离冷冻保护蛋白。Volger在Biochimicaet Biophysica Acta,412(1975),335-349中描述了从菠菜分离冷冻保护的叶蛋白。Boothe在《植物生理学》(Plant Physiol)(1995),108:759-803中描述了从欧洲油菜(Brassica napus)分离蛋白。此外,认为这些蛋白是冷冻保护蛋白而不是AFP。Neven在《植物分子生物学》(Plant Molecular Biology)21:291-305,1993中描述了菠菜冷冻保护蛋白的DNA特征鉴定。Salzman在Am.J.Eno1.Vitic.,第44卷,第4期,1993中的ASEV/东部地区的第18届年会的摘要和综述中描述了在葡萄属(Vitis)的芽中存在煮沸稳定的多肽。尽管所述蛋白类似于鱼抗冻肽,但是它们是冷冻保护蛋白而不是AFP。Lin在《生物化学生物物理研究通讯》(Biochemical and Biophysical ResearchCommunication),第183卷,第3期,1992,第1103-1108和Lin在《植物生理学》(Plant Physiology)(1992),99:519-525中描述了得自拟南芥属(Arabidopsis Hakaira)的15kDa冷冻保护多肽。Houde在《植物杂志》(The Plant Journal)(1995)8(4),583-593中讲述了得自小麦的冷冻保护蛋白。然而,直到现在仍然未将AFP应用到市售食品。其原因之一是成本高和获得AFP的方法复杂。另一个原因是至今已经提出用于冷冻食品的AFP不能掺入标准配方混合料中,因为它们往往在加工过程中尤其是在巴氏消毒法步骤中变得不稳定。认为这种不稳定是由所述AFP变性引起的;这是在肽和蛋白质普遍观察到的众所周知的效应。
在我们非预公开的专利申请:WO98/4148中,已经描述了特别好的AFP可以从天然来源例如地衣属(Lichen)分离。
申请人现在已经能确定来自地衣属特别有效的AFP的部分氨基酸序列。
因此本发明涉及可以得自地衣属的AFP,所述AFP的表观分子量约为24kDa,而N末端的氨基酸序列为:A-P-A-W-M-D-A-E-S-F-G-A-I-A-H-G-G-L
具有与上述序列高度相似的序列的蛋白也包括在我们的发明范围内。对于本发明的目的,其氨基酸序列与上述序列至少80%重叠的所有RI活性蛋白也包括在本发明范围内。更优选重叠至少90%最优选重叠高于95%,例如与上述序列相同或仅有一个或二个氨基酸不同的氨基酸序列。
对于本发明的目的,两种(部分)氨基酸序列的重叠程度可以如下计算:
(a)对所述两种氨基酸序列进行序列对比并对相同并以相同顺序出现的氨基酸数(X)进行计数
(b)氨基酸的每个变化、缺失或添加变化记为1点,而总变化、缺失和添加记为(Y)。
(c)现在可以按X*100%/(X+Y)计算重叠度。
例如,N末端的(部分)氨基酸序列:A-P-A-V-V-M-G-D-A-E-S-F-G-A-T-A-H-G-G-L可以如下与对照进行序列对比:A-P-A-V-V-M-G-D-A-E-S-F-G-A-I-A-H-G-G-LA-P-A-W -M- D-A-E-S-F-G-A-I-A-H-G-G-L这产生相同位次相同氨基酸的总数17个。变化数为1个(在第四位W变成V);添加数为2(第五位的V,第七位的G),无缺失。因此总的变化、添加和缺失为3。这导致重叠度为17*100%/(17+3)=85%。
具有N末端(部分)氨基酸序列:A-P-A-V-V-M-G-D-A-E-S-F-G-A-I-A-H-G-G-L的蛋白也因此包括在本发明中。
修饰型的上述蛋白也包括在本发明范围内,因此所述修饰实际上不影响冰重结晶抑制特性,例如其糖基化型。
对于本发明目的,术语约24kDa的分子量是指用标准参比标记在SDS-PAGE上测定的20-28kDa范围内的任何分子量,更优选该分子量为22-26kDa。
本发明的优势AFP可以得自地衣属,尤其是Umbilicaria antarctica物种。
尽管抗冻蛋白开始得自于地衣属,但是用其它方法(例如用基因修饰技术)生产的抗冻蛋白也包括在本发明范围内,因此例如对微生物或植物进行基因修饰以产生上述蛋白。这些蛋白也包括在所述术语“可得自地衣属”。
能够编码上述AFP的核酸序列也包括在本发明范围内。
含能够编码本发明AFP的核酸序列的载体也包括在本发明范围内。
根据以上信息,还可能的是基因修饰其它天然来源,使得它们产生上文鉴定的有益的AFP。
申请人也已经发现,以上序列的AFP改善了冰重结晶抑制特性。测定冰重结晶抑制特性的合适的试验描述于实施例,包括快速冷冻至至少40℃(例如-80℃),然后于-6℃保藏一小时。按照本发明的AFP最好在冰重结晶抑制测定(如实施例中所述)后提供的冰颗粒大小为15μm或15μm以下,更优选5-15μm。
本发明的AFP可以方便地用于食品,优选用于冷冻食品或计划冷冻的食品。在例如冷冻前通过巴氏消毒法或灭菌加热的制品中特别优选使用AFP。在冷冻糖食制品中尤其优选使用。
这类食品的实例为:诸如冰淇淋混合料的冷冻糖食混合料和冷冻前预定进行巴氏消毒的糖水冰糕混合料。这类混合料通常贮藏于室温。合适的制品形式例如:包装于例如袋或小袋中的粉型混合料。所述混合料在例如加入水和任选的其它成分以及可任选充气后能够形成所述冷冻食品的基础。
合适混合料的另一实例可以是:必要时在加入其它组分和任选的其它充气后可以冷冻的液体混合料(任选充气)。
上述混合料的明显优点是,所述AFP成分的存在使得所述混合料可以在静止的条件下冷冻,例如在商店或家用冰箱中冷冻而不会形成不可接受的冰晶形状,因而具有不同于通常通过静止冷冻获得的制品的结构。
非常方便的是,这些混合料可以包装于密封容器中(例如纸盒、袋、盒、塑料容器等)。对于单个等份,所述包装大小一般为10-1000g。对于多个等份,包装大小高至500kg可能是合适的。所述所装大小一般为10-5000g。
如上所述,其中使用所述AFP的优选制品是冷冻糖食,例如冰淇淋或糖水冰糕。优选AFP水平为成品重量的0.00001-0.5%。假如使用干混合料或浓缩物,所述浓度可以较高以保证最终冷冻制品中的水平在上述范围内。
对于本发明目的,术语冷冻糖食包括含乳冷冻糖食,例如冰淇淋、冷冻酸牛奶、冷冻果子露、清凉果子饮料、牛奶冻和冷冻蛋奶甜羹、糖水冰糕、granita和冷冻果糊。对于某些应用,不大推荐在发酵食品中使用。
在所述冷冻糖食品中的固体(例如糖、脂肪、食用香料等)水平最好高于4%(重量),例如高于30%(重量),更优选40-70%(重量)。
按照本发明的冷冻糖食可以用适合生产冷冻糖食的任何方法生产。然而,特别优选的是,在巴氏消毒前和所述冷冻过程开始前,充分混合所有所述配方成分。所述冷冻过程最好可以包括硬化步骤,例如至-30华氏度或更低。
实施例Ⅰ
可如下检测所述AFP的冰重结晶抑制特性:
将含AFP的制品的样品调节至蔗糖水平为30%(重量)(如果所述样品的初始水平高于30%,则通过稀释进行调节,如果所述初始水平较低,则加入蔗糖至30%水平)。
将一滴3μl的所述样品置于22mm盖玻片上。然后在上面放置一个16mm直径盖玻片,在所述样品上放置200g重量以保证均一的玻片厚度。盖玻片的边缘用透明指甲油密封。
将所述玻片放置在Linkham THM 600温控显微镜载物台上。将所述载物台快速降温(每分钟50℃)至-40℃以产生大量小晶体。然后将所述载物台温度快速升高(每分钟50℃)至-6℃并保持于该温度。
于-6℃用Leica Aristoplan显微镜观察到所述冰相。偏振光和λ片用来增强所述冰晶的对比度。用35mm显微镜照相术于T=0和T=1小时记录所述冰相的状态(冰晶的大小)。通过沿所述晶体的周边画线测定冰晶大小(长度)。把一批冰淇淋的每个单独的冰晶的最大长度输入详细记录表,在此表中对所述数据组进行分析以得到平均值和标准差。
测试冰重结晶抑制特性的另一方法如下:
用改进的“splat分析”(Knight等,1988)检测抗冻活性。将在30%(w/w)蔗糖中的待研究的溶液2.5μl转移到洁净、适当标记的、16mm圆形盖玻片上。第二张盖玻片放置于所述溶液滴上,并在指与母指间将所述夹层结构挤压在一起。将所述夹层结构放入在-80℃的干冰盒中保持的己烷浴中。当所有夹层结构制备完备后,用在干冰中预冷的镊子将夹层结构从-80℃己烷浴转移到保持在-6℃的含己烷的观察室内。在转移到-6℃后,可以看到夹层结构从透明变为不透明外观。用摄像机记录图像并用20x目镜取入图像分析系统(LUCIA,Nikon)。在时间=0时记录每个splat的图像,并在30-60分钟后再次记录。比较两个分析中的冰晶大小。如果与t=0时的大小相比,30-60分钟时的大小相似或仅中度增大(即是说增大不足20%,更优选增大不足10%,最优选增大不足5%),这表明冰晶重结晶抑制特性良好。
一般来说,这些试验可用于任何含AFP和水的合适组合物。在这种试验组合物中的AFP水平一般不是非常重要,而可以是例如0.0001-0.5%(重量),更优选0.0005-0.1%(重量),最优选0.001-0.05%(重量),例如0.01%(重量)。
任何含AFP和水的合适组合物均可以用来进行所述试验。然而,获得纯化型的AFP一般是不必要的。对于实际应用,通常制备天然物质的液体提取物或提取汁液,其中可以测试该提取物或汁液,这就应该足够了。
实施例Ⅱ
将在1996年春季从南极洲收集并贮藏于-20℃的9.5g Umbilicariaantarctica在研钵和研杵中的液氮中均化成细粉。将该粉转移到室温下的新研钵和研杵中。加入10ml含10mM EDTA的0.2M Tris HCl后,所述粉再在所述研钵和研杵中研磨,所述匀浆通过二层细绵布过滤。将所述残留物再次置于研钵和研杵中,加入另外的10ml缓冲液,再研磨所述残留物。该物质如上过滤并将滤液与第一次均化步骤的滤液合并。所述滤液于30,000g离心15分钟,收集所述上清液并等份冷冻。
将0.15g NH4SO4溶解于1ml上清液中,该溶液于4℃温育30分钟。于30,000g离心10分钟后,将0.3g NH4SO4溶解于该步骤的上清液中,并将该溶液于4℃温育30分钟。将该溶液于30,000g离心10分钟,并弃去上清液。将沉淀再悬浮于0.2ml水中,连续稀释该溶液,在30%(w/w)蔗糖的水溶液中制备原始提取物用于半定量的splat分析。在所述原始提取物至高于200倍的稀释液以及所述沉淀物至800倍的稀释液中可以检测到(用上述方法)splat活性,表明在所述NH4SO4沉淀物中已经收获到存在于原始提取物中的总splat活性的二分之一以上。
于所述再悬浮的沉淀物中加入200μl 0.1M TrisHCl pH7.5,该溶液于10kDa截留microcon(Amicon)中浓缩至150μl。用SMART层析系统(Pharmacia)以100μl/分钟的流速,将100μl该溶液上样于在50mM Tris HCl pH 7.5预平衡的Q-Sepharose柱,以100μl收集流分。在50mM TrisHCl pH 7.5中洗出800μl后,将1.5ml内的0-0.5MNaCl梯度上该柱,于280nm监测所述洗出液。在30%(w/w)蔗糖中稀释50倍后,按实施例Ⅰ中所述测试流分的splat活性。发现活性与在约0.1M NaCl洗脱、主要收集于流分14中的OD280峰有关。
采用SMART层析系统(Pharmacia),将40μl流分14以流速40μl/分钟上样于在50mM TrisHCl pH 7.5中预平衡的Superdex 75凝胶渗透柱。于OD280和OD215监测洗出液,在上样后从0.6ml开始以80μl收集流分,在1.1-1.6ml之间以50μl收集流分,在1.6-3ml之间以100μl收集流分。将1μl的每一流分在30%(w/w)蔗糖中稀释25倍,并分析splat活性。发现活性与用流分9和10中1.2ml保留物洗脱的OD280和OD215峰有关。通过测定标准蛋白分子量标记(Sigma)的保留体积(Ve)和经蓝葡聚糖上柱测定空隙容积(Vo)为0.91ml,从而校准所述Superdex柱。绘制log10Mr对Ve/Vo的标准曲线,测定与所述地衣splat活性有关的OD280峰的表观分子量为30kDa。
合并从Superdex柱洗脱的32μl流分9和10,并在10kDa截留microcom(Amicon)中浓缩至10μl,将3.5μl 4×SDS-PAGE样品缓冲液加至从Superdex柱洗脱的10μl流分9和10中以及从Q-sepharose柱洗脱的流分12-16中。在于95℃加热5分钟并于10,000g离心3分钟后,将每种样品10μl加样至4%积层凝胶孔中,经12%0.75mm厚的SDS-PAGE微型凝胶(Biorad)电泳分离多肽。电泳后凝胶染色并在考马斯亮蓝中固定,在甲醇∶醋酸∶水(1∶4∶5)w/w中脱色。这显示从Superdex柱洗脱的浓缩合并的流分9和10中的多肽的表观分子量为24kDa。当所述凝胶用Biorad银染色试剂盒按照厂商的说明进行银染时,在从Q-sepharose柱洗脱流分14中和从Superdex柱洗脱的流分9和10中可检测到相同表观分子量的多肽。
在用基本上与上述相同的方法进一步纯化蛋白后,从24kDa多肽获得以下N末端氨基酸序列:A-P-A-V-V-M-G-D-A-E-S-F-G-A-I-A-H-G-G-L。
实施例Ⅲ
如上所述,对按照实施例Ⅱ的粗地衣滤液进行硫酸铵沉淀并再悬浮于0.2M Tris/HCl pH 7.5中,然后按1/10稀释入以下缓冲液之一:0.2M柠檬酸钠pH3.0,0.2M乙酸钠pH4.0,0.2M哌嗪pH5.0,0.2MbisTris pH6.0,0.2M三乙醇胺pH7.0,0.2M Tris pH8.0,0.2M CHESpH9.0,0.2M CAPS pH10.0。然后,将这些样品在适当缓冲液中以1/2连续稀释,如实施例Ⅰ中所述,在按照所述第二个试验进行splat分析前将该稀释液与60%蔗糖以1∶1混合。在pH10和pH6.0之间直至1/320稀释度可以清楚地检测到重结晶抑制活性。在pH3.0-5.0之间,直至1/80稀释度可以清楚地检测到活性,表明尽管所述蛋白在低pH保留部分活性,但是其活性在pH为5.0或5.0以下时下降到原来的1/4。
实施例Ⅳ
按照实施例Ⅱ的纯化地衣抗冻蛋白通过双向电泳分离。含以下组分的凝胶在小玻璃试管(Biorad)中聚合,所述组分为:9.2M尿素、4%丙烯酰胺(2.66ml30%丙烯酰胺0.8%双丙烯酰胺)、2%去离子TritonX100,1%4-7 Bio-lyte两性物(Biorad)、1%3.5-10 Bio-Lyte两性物(Biorad)、0.1%TEMED、0.01%过硫酸铵。所述试管用蒸馏水冲洗并插入能够容纳它们的微型凝胶系统中,上室充以20mM NaOH而下室充以10mM H3PO4。纯化的地衣样品以1∶1与第一向样品缓冲液(9.2M尿素、2.0% Triton X-100,5%β-巯基乙醇、1%4-7 Bio-lyte两性物、0.25%3-10 Bio-Lyte两性物)混合,在上样于一个试管凝胶前温至37℃。对于第二个凝胶圆条,以双向标记蛋白(Biorad)上样,而对于第三个圆条凝胶,用双向标记蛋白和地衣样品的混合物上样。在500V下电泳10分钟以及在750V下电泳4小时后从所述试管挤出所述圆条,上样至3个分开的1mm厚的12%SDS-PAGE微型凝胶(Biorad),覆盖上SDS-PAGE样品缓冲液。电泳后所述凝胶用Biorad试剂盒按照生产商的说明进行银染。该分离在地衣样品的凝胶上显示3个斑点,所有三个斑点的表观分子量约24kDa,而PI低于4.5。
采用与所述双向分离的第一向凝胶组分相同、只是用Biolyte 3-5两性物取代Biolyte 4-7两性物组成的平板凝胶、纯化地衣抗冻蛋白的单向等电聚焦,在银染色后显示一条等电点低于3.6的带。
Claims (9)
1.可以得自地衣属的抗冻蛋白以及该蛋白的修饰型和同种型,所述蛋白的表观分子量为20-28kDa,其N末端氨基酸序列显示与:A-P-A-W-M-D-A-E-S-F-G-A-I-A-H-G-G-L至少80%重叠。
2.具有以下N末端氨基酸序列的权利要求1的抗冻蛋白以及该蛋白的修饰型和同种型,所述氨基酸序列为:A-P-A-V-V-M-G-D-A-E-S-F-G-A-I-A-H-G-G-L。
3.权利要求1或2的抗冻蛋白,其分子量为22-26kDa。
4.权利要求1或2的抗冻蛋白,显示与权利要求1或2的部分序列至少90%重叠。
5.权利要求1或2的抗冻蛋白,显示与权利要求1或2的部分序列100%重叠。
6.权利要求1的抗冻蛋白,其中所述修饰包括糖基化作用。
7.编码一项或多项前述权利要求的抗冻蛋白的核酸序列。
8.包含按照权利要求1或2的抗冻蛋白的食品。
9.为冷冻糖食的按照权利要求8的食品。
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WO1994014472A1 (en) * | 1992-12-22 | 1994-07-07 | The Regents Of The University Of California | Method for inhibition of cell adhesion to receptors containing selectins |
US5676985A (en) * | 1994-10-12 | 1997-10-14 | Hsc Research And Development Limited Partnership | Antifreeze polypeptide-expressing microorganisms useful in fermentation and freezing of foods |
US5620732A (en) * | 1995-06-07 | 1997-04-15 | The Pillsbury Company | Method of making ice cream |
SK9099A3 (en) * | 1996-07-26 | 1999-06-11 | Unilever Nv | Frozen confectionery products |
DK0918863T3 (da) * | 1996-07-26 | 2005-04-04 | Unilever Nv | Frossent levnedsmiddelprodukt indeholdende varmestabilt antifryseprotein |
-
1998
- 1998-01-22 GB GBGB9801420.2A patent/GB9801420D0/en not_active Ceased
- 1998-12-23 ID IDW20001396A patent/ID25509A/id unknown
- 1998-12-23 IL IL13619798A patent/IL136197A0/xx unknown
- 1998-12-23 US US09/582,296 patent/US6774210B1/en not_active Expired - Fee Related
- 1998-12-23 BR BR9814760-9A patent/BR9814760A/pt not_active Application Discontinuation
- 1998-12-23 SK SK1093-2000A patent/SK10932000A3/sk unknown
- 1998-12-23 ES ES98966922T patent/ES2262257T3/es not_active Expired - Lifetime
- 1998-12-23 AU AU26148/99A patent/AU753334B2/en not_active Ceased
- 1998-12-23 CA CA002318869A patent/CA2318869A1/en not_active Abandoned
- 1998-12-23 PL PL98342568A patent/PL342568A1/xx not_active Application Discontinuation
- 1998-12-23 EP EP98966922A patent/EP1049713B1/en not_active Expired - Lifetime
- 1998-12-23 TR TR2000/02105T patent/TR200002105T2/xx unknown
- 1998-12-23 HU HU0100410A patent/HUP0100410A3/hu unknown
- 1998-12-23 JP JP2000528594A patent/JP2002508303A/ja not_active Withdrawn
- 1998-12-23 DE DE69834136T patent/DE69834136T2/de not_active Expired - Lifetime
- 1998-12-23 AT AT98966922T patent/ATE322503T1/de not_active IP Right Cessation
- 1998-12-23 CN CN98813206.0A patent/CN1284085A/zh active Pending
- 1998-12-23 WO PCT/EP1998/008554 patent/WO1999037673A2/en active IP Right Grant
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN100383251C (zh) * | 2002-12-20 | 2008-04-23 | 荷兰联合利华有限公司 | 抗冻蛋白的制备 |
CN101695346B (zh) * | 2009-09-25 | 2011-10-26 | 徐州工程学院 | 一种冷冻食品基料-地衣培养液的制备与应用方法 |
Also Published As
Publication number | Publication date |
---|---|
BR9814760A (pt) | 2000-10-17 |
WO1999037673A3 (en) | 1999-09-16 |
CA2318869A1 (en) | 1999-07-29 |
EP1049713B1 (en) | 2006-04-05 |
US6774210B1 (en) | 2004-08-10 |
ATE322503T1 (de) | 2006-04-15 |
AU2614899A (en) | 1999-08-09 |
DE69834136T2 (de) | 2006-11-09 |
EP1049713A2 (en) | 2000-11-08 |
HUP0100410A2 (hu) | 2001-06-28 |
PL342568A1 (en) | 2001-06-18 |
HUP0100410A3 (en) | 2003-04-28 |
JP2002508303A (ja) | 2002-03-19 |
ES2262257T3 (es) | 2006-11-16 |
AU753334B2 (en) | 2002-10-17 |
TR200002105T2 (tr) | 2000-12-21 |
DE69834136D1 (de) | 2006-05-18 |
SK10932000A3 (sk) | 2001-01-18 |
ID25509A (id) | 2000-10-05 |
GB9801420D0 (en) | 1998-03-18 |
WO1999037673A2 (en) | 1999-07-29 |
IL136197A0 (en) | 2001-05-20 |
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