WO2009031160A1 - An improved process for the preparation of natural vanilla extract - Google Patents
An improved process for the preparation of natural vanilla extract Download PDFInfo
- Publication number
- WO2009031160A1 WO2009031160A1 PCT/IN2008/000529 IN2008000529W WO2009031160A1 WO 2009031160 A1 WO2009031160 A1 WO 2009031160A1 IN 2008000529 W IN2008000529 W IN 2008000529W WO 2009031160 A1 WO2009031160 A1 WO 2009031160A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- vanilla
- beans
- extract
- tea
- enzyme
- Prior art date
Links
- 235000009499 Vanilla fragrans Nutrition 0.000 title claims abstract description 131
- 235000012036 Vanilla tahitensis Nutrition 0.000 title claims abstract description 129
- 239000000284 extract Substances 0.000 title claims abstract description 65
- 238000002360 preparation method Methods 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims description 26
- 230000008569 process Effects 0.000 title claims description 23
- 244000263375 Vanilla tahitensis Species 0.000 title description 3
- 244000290333 Vanilla fragrans Species 0.000 claims abstract description 130
- 102000004190 Enzymes Human genes 0.000 claims abstract description 78
- 108090000790 Enzymes Proteins 0.000 claims abstract description 78
- 244000046052 Phaseolus vulgaris Species 0.000 claims abstract description 73
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims abstract description 73
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 58
- 244000269722 Thea sinensis Species 0.000 claims abstract description 55
- MWOOGOJBHIARFG-UHFFFAOYSA-N vanillin Chemical compound COC1=CC(C=O)=CC=C1O MWOOGOJBHIARFG-UHFFFAOYSA-N 0.000 claims abstract description 19
- FGQOOHJZONJGDT-UHFFFAOYSA-N vanillin Natural products COC1=CC(O)=CC(C=O)=C1 FGQOOHJZONJGDT-UHFFFAOYSA-N 0.000 claims abstract description 19
- 235000012141 vanillin Nutrition 0.000 claims abstract description 19
- 239000000796 flavoring agent Substances 0.000 claims abstract description 18
- 235000019634 flavors Nutrition 0.000 claims abstract description 17
- 239000007790 solid phase Substances 0.000 claims abstract description 14
- 239000000470 constituent Substances 0.000 claims abstract description 13
- 239000007791 liquid phase Substances 0.000 claims abstract description 11
- 230000009471 action Effects 0.000 claims abstract description 7
- 238000005352 clarification Methods 0.000 claims abstract description 7
- 238000003306 harvesting Methods 0.000 claims abstract description 6
- 229940088598 enzyme Drugs 0.000 claims description 73
- 230000000694 effects Effects 0.000 claims description 24
- 239000000843 powder Substances 0.000 claims description 24
- 235000019441 ethanol Nutrition 0.000 claims description 20
- 239000002002 slurry Substances 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 230000036961 partial effect Effects 0.000 claims description 10
- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 claims description 8
- 108010059892 Cellulase Proteins 0.000 claims description 7
- 230000003750 conditioning effect Effects 0.000 claims description 7
- 108010031186 Glycoside Hydrolases Proteins 0.000 claims description 5
- 102000005744 Glycoside Hydrolases Human genes 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 244000013123 dwarf bean Species 0.000 claims description 5
- 235000021331 green beans Nutrition 0.000 claims description 5
- 238000000227 grinding Methods 0.000 claims description 5
- 230000002829 reductive effect Effects 0.000 claims description 5
- 108010093031 Galactosidases Proteins 0.000 claims description 4
- 102000002464 Galactosidases Human genes 0.000 claims description 4
- LPHGQDQBBGAPDZ-UHFFFAOYSA-N Isocaffeine Natural products CN1C(=O)N(C)C(=O)C2=C1N(C)C=N2 LPHGQDQBBGAPDZ-UHFFFAOYSA-N 0.000 claims description 4
- 108700023158 Phenylalanine ammonia-lyases Proteins 0.000 claims description 4
- 229960001948 caffeine Drugs 0.000 claims description 4
- VJEONQKOZGKCAK-UHFFFAOYSA-N caffeine Natural products CN1C(=O)N(C)C(=O)C2=C1C=CN2C VJEONQKOZGKCAK-UHFFFAOYSA-N 0.000 claims description 4
- 229930002875 chlorophyll Natural products 0.000 claims description 4
- 235000019804 chlorophyll Nutrition 0.000 claims description 4
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
- 235000013824 polyphenols Nutrition 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 3
- 150000008442 polyphenolic compounds Chemical class 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 230000003203 everyday effect Effects 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000008371 vanilla flavor Substances 0.000 abstract description 14
- 239000002243 precursor Substances 0.000 abstract description 6
- 239000000203 mixture Substances 0.000 abstract description 5
- 238000005549 size reduction Methods 0.000 abstract description 4
- 239000007858 starting material Substances 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 2
- 235000013616 tea Nutrition 0.000 description 43
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 238000000605 extraction Methods 0.000 description 6
- 238000003860 storage Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 230000002255 enzymatic effect Effects 0.000 description 4
- 235000009569 green tea Nutrition 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000003960 organic solvent Substances 0.000 description 4
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
- 235000013599 spices Nutrition 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 238000011282 treatment Methods 0.000 description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- 102000030523 Catechol oxidase Human genes 0.000 description 3
- 108010031396 Catechol oxidase Proteins 0.000 description 3
- 241000233855 Orchidaceae Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000013019 agitation Methods 0.000 description 3
- 238000001952 enzyme assay Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 238000005191 phase separation Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 230000035900 sweating Effects 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 102000006995 beta-Glucosidase Human genes 0.000 description 2
- 108010047754 beta-Glucosidase Proteins 0.000 description 2
- 235000019658 bitter taste Nutrition 0.000 description 2
- 235000012206 bottled water Nutrition 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 229940106157 cellulase Drugs 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 230000002354 daily effect Effects 0.000 description 2
- 239000003651 drinking water Substances 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000011536 extraction buffer Substances 0.000 description 2
- 239000004744 fabric Substances 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
- 239000004570 mortar (masonry) Substances 0.000 description 2
- 229910000160 potassium phosphate Inorganic materials 0.000 description 2
- 235000011009 potassium phosphates Nutrition 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000004576 sand Substances 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- PFTAWBLQPZVEMU-DZGCQCFKSA-N (+)-catechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-DZGCQCFKSA-N 0.000 description 1
- 239000001100 (2S)-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chroman-4-one Substances 0.000 description 1
- WBYWAXJHAXSJNI-VOTSOKGWSA-M .beta-Phenylacrylic acid Natural products [O-]C(=O)\C=C\C1=CC=CC=C1 WBYWAXJHAXSJNI-VOTSOKGWSA-M 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 240000000467 Carum carvi Species 0.000 description 1
- 235000005747 Carum carvi Nutrition 0.000 description 1
- WBYWAXJHAXSJNI-SREVYHEPSA-N Cinnamic acid Chemical compound OC(=O)\C=C/C1=CC=CC=C1 WBYWAXJHAXSJNI-SREVYHEPSA-N 0.000 description 1
- 244000037364 Cinnamomum aromaticum Species 0.000 description 1
- 235000014489 Cinnamomum aromaticum Nutrition 0.000 description 1
- 244000223760 Cinnamomum zeylanicum Species 0.000 description 1
- 240000002943 Elettaria cardamomum Species 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- QUQPHWDTPGMPEX-UHFFFAOYSA-N Hesperidine Natural products C1=C(O)C(OC)=CC=C1C1OC2=CC(OC3C(C(O)C(O)C(COC4C(C(O)C(O)C(C)O4)O)O3)O)=CC(O)=C2C(=O)C1 QUQPHWDTPGMPEX-UHFFFAOYSA-N 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 240000008474 Pimenta dioica Species 0.000 description 1
- 235000006990 Pimenta dioica Nutrition 0.000 description 1
- 108010059820 Polygalacturonase Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 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 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000001447 alkali salts Chemical class 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000019606 astringent taste Nutrition 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- QUQPHWDTPGMPEX-UTWYECKDSA-N aurantiamarin Natural products COc1ccc(cc1O)[C@H]1CC(=O)c2c(O)cc(O[C@@H]3O[C@H](CO[C@@H]4O[C@@H](C)[C@H](O)[C@@H](O)[C@H]4O)[C@@H](O)[C@H](O)[C@H]3O)cc2O1 QUQPHWDTPGMPEX-UTWYECKDSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical group OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 235000005300 cardamomo Nutrition 0.000 description 1
- ADRVNXBAWSRFAJ-UHFFFAOYSA-N catechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3ccc(O)c(O)c3 ADRVNXBAWSRFAJ-UHFFFAOYSA-N 0.000 description 1
- 235000005487 catechin Nutrition 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 229950001002 cianidanol Drugs 0.000 description 1
- 229930016911 cinnamic acid Natural products 0.000 description 1
- 235000013985 cinnamic acid Nutrition 0.000 description 1
- 235000017803 cinnamon Nutrition 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- APSNPMVGBGZYAJ-GLOOOPAXSA-N clematine Natural products COc1cc(ccc1O)[C@@H]2CC(=O)c3c(O)cc(O[C@@H]4O[C@H](CO[C@H]5O[C@@H](C)[C@H](O)[C@@H](O)[C@H]5O)[C@@H](O)[C@H](O)[C@H]4O)cc3O2 APSNPMVGBGZYAJ-GLOOOPAXSA-N 0.000 description 1
- 229940110456 cocoa butter Drugs 0.000 description 1
- 235000019868 cocoa butter Nutrition 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000011143 downstream manufacturing Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 108010093305 exopolygalacturonase Proteins 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 108010002430 hemicellulase Proteins 0.000 description 1
- 229940059442 hemicellulase Drugs 0.000 description 1
- 229940025878 hesperidin Drugs 0.000 description 1
- VUYDGVRIQRPHFX-UHFFFAOYSA-N hesperidin Natural products COc1cc(ccc1O)C2CC(=O)c3c(O)cc(OC4OC(COC5OC(O)C(O)C(O)C5O)C(O)C(O)C4O)cc3O2 VUYDGVRIQRPHFX-UHFFFAOYSA-N 0.000 description 1
- QUQPHWDTPGMPEX-QJBIFVCTSA-N hesperidin Chemical compound C1=C(O)C(OC)=CC=C1[C@H]1OC2=CC(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO[C@H]4[C@@H]([C@H](O)[C@@H](O)[C@H](C)O4)O)O3)O)=CC(O)=C2C(=O)C1 QUQPHWDTPGMPEX-QJBIFVCTSA-N 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- WBYWAXJHAXSJNI-UHFFFAOYSA-N methyl p-hydroxycinnamate Natural products OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- ARGKVCXINMKCAZ-UHFFFAOYSA-N neohesperidine Natural products C1=C(O)C(OC)=CC=C1C1OC2=CC(OC3C(C(O)C(O)C(CO)O3)OC3C(C(O)C(O)C(C)O3)O)=CC(O)=C2C(=O)C1 ARGKVCXINMKCAZ-UHFFFAOYSA-N 0.000 description 1
- 239000002420 orchard Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000003495 polar organic solvent Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000004202 respiratory function Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- -1 sulfhydryl compound Chemical class 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000007669 thermal treatment Methods 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 239000005418 vegetable material Substances 0.000 description 1
- 239000011345 viscous material Substances 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/10—Natural spices, flavouring agents or condiments; Extracts thereof
- A23L27/11—Natural spices, flavouring agents or condiments; Extracts thereof obtained by solvent extraction
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/10—Natural spices, flavouring agents or condiments; Extracts thereof
Definitions
- the present invention relates to an improved process for the preparation of natural vanilla extract from fresh green beans.
- vanilla planifolia Andr. is herbaceous, perennial vine a tropical orchid. This spice crop is grown for its pleasant aromatic essence "Vanillin”. Annual world production is estimated at around 3000-4000 MT. In India vanilla crop is getting established as an important spice crop (Sreekrishna Bhat and Sudharshan 2002). The genus vanilla belongs to the monocotyledonous family Orchidaceae, and perhaps is the only orchid crop, which is of economic importance as a spice, the others being valued as ornamentals. Over 100 species of vanilla have been described in scientific literature, but only three if these are considered important for commerce and cultivation.
- vanilla planifolia is the most valued fruits flavor qualities and is therefore widely cultivated and used (Persleglove 1981).
- Vanilla pompna and Vanilla tahitensis yield vanilla beans of poorer quality. Green vanilla pods are almost odourless and the characteristic flavour of vanilla develops only when the mature beans are subjected to a controlled curing and conditioning process.
- vanilla extract is produced by extraction of cured vanilla pods with aqueous alcohol followed by desolventisation to get a brown/red coloured, viscous material called the vanilla extract /concentrate.
- the extract/concentrate contains the constituents responsible for flavour namely vanillin, and other soluble components such as fats, fiber and waxes.
- Vanillin/ vanilla extract is useful as a flavourant in foods and confectionary items.
- the industry aims at producing the pure natural vanillin/vanilla extract with a high flavor value, but the quality gets restricted due to the natural composition of the vanilla with respect to flavour.
- the first critical phase is vanilla process is known as curing.
- a number of procedures have been evolved for curing of vanilla but they are all characterized by four phases whereby the green mature beans are transferred into a commercially desirable product. These phases are called killing, sweating, drying and conditioning.
- Killing or wilting brings about physiological death of the living tissues by respiratory function of the disrupting the cells.
- the cell-disruption can be carried out by subjecting the fresh beans to any one of the treatments such as hot water scalding, sun and oven wilting, scarification treatment with ethylene gas or freezing. Sweating helps the beans to develop their characteristic colour, aroma and flavouring properties.
- the drying step the moisture is reduced and beneficial chemical changes take place. Due to conditioning stage, the beans are stored in closed boxes and this rests for several months. Various chemical and biochemical reactions take place in this step, producing various volatile aroma constituents enhancing the overall flavour quality of the cured beans (Purseglove 1981 , Randire 1984).
- JP83085278 which relates to the preparation of vanilla extract from cured beans using water and /or a water- soluble organic solvent in the presence of an alkali or alkali salt.
- JP8168355 which recites the preparation of vanilla extract from cured beans employing water and /or aqueous organic solvent using the irradiation of ultrasonic waves.
- JP2002038188 relates to the preparation of vanilla extract from cured vanilla beans employing cocoa butter to enhance the vanilla flavour.
- GR3022203T relates to a process for the production of natural vanilla extract, consisting of processing crushed green vanilla pods by means of an enzymatic system capable of destroying plant cell membrane systems and hydrolysing glycoside precursors of volatile compounds.
- JP2001181671 relates to preparing vanilla extract by treating cured vanilla beans or its extract with at least one enzyme selected from protease, astringency / bitterness splitting enzyme and hesperidin splitting enzyme.
- WO2004091316 relates to a process for the preparation of a vanilla extract, the process consisting of subjecting green vanilla beans to accelerated browning followed by extractive / enzymatic treatment.
- US4956192 relates to the preparation of natural vanilla flavour comprising freezing green vanilla beans at 5 degree and -30 degree, thawing and then extracting the flavour constituents from the beans.
- US5705205 relates to enzymatic treatment of a hydrated slurry of ground dehydrated green vanilla pods, addition of enzyme consisting of 10-1000 units of beta-glucose activity per gram of green vanilla pods followed by incubation and phase separation to obtain vanilla extract.
- vanillin US5279950
- vanillin is produced through biOconversion of vanilla precursor with a tissue culture of undifferentiated callus cells derived from a vanilla plant and / or enzymes obtained therefrom in the presence of a water soluble sulfhydryl compound and an assimilable carbon source.
- Production of novel vanilla extract using Co2 of a supercritical state JP4214799 wherein, preparation of vanilla extract from cured beans initially by supercritical carbon dioxide and subsequently with a polar organic solvent is reported.
- JP58056654 relates to the preparation of vanilla flavour with enhanced fragrance by extracting a mixture of pulverized vanilla beans or dried material with spice vegetable material of allspice, caraway, cardamom, cassia or cinnamon with a water- miscible organic solvent.
- vanilla extract for the preparation of vanilla extract.
- enzymes such as protease, astriningency / bitterness splitting enzyme and hesperdins splitting enzyme and ⁇ -glucosidase
- chemical flavour quality as assayed by gas chromatographic method and also organoliptic quality for sensory acceptance are considered two important quality attributes.
- this aspect does not appear to be adequately addressed in the above patents. It is desirable to produce a vanilla extract similar or better in quality as compared to the commercially manufactured extracts, wherein cured vanilla beans are used as the starting material.
- the present invention involves a novel approach wherein a natural bioactive preparation made from green tea leaves which has enzymatic activity capable of action on phenolic precursors of green vanilla beans and forming characteristic vanilla flavour compounds, is made use of.
- the flavour quality of the extracts so prepared has been assessed chemically and organically as seen to be highly acceptable in tune with to conventional cured vanilla extracts.
- a method is provided for the preparation of the vanilla extract from green vanilla pods by using enzymes obtained from fresh tea leaves (one bud two leaves).
- the main object of the present invention is thus to provide an improved process for the preparation of vanilla extract from green vanilla beans which obviates the drawbacks of the prior art.
- vanilla extract is prepared without using any chemicals / commercial enzymes.
- Yet another object is to enhance the extractability of vanilla constituents with acceptable flavour by a combination of fresh vanilla pulp with an enzyme preparation derived from fresh tea leaves in a crude or purified form.
- Fresh green vanilla beans of a recent harvest, or partially cured vanilla beans or preserved green vanilla beans are used as the starting material.
- a preparation containing natural tea enzyme is made from fresh tea leaves.
- Fresh/cured/preserved vanilla beans, after proper size reduction, are mixed in a suitable proportion with the tea enzyme preparation and incubated to facilitate action of natural enzymes on vanilla flavour precursors present in the beans.
- the fermented vanilla & tea enzyme mix is treated with ethanol to inactivate enzymes and to extract the vanilla flavour constituents into ethanol. This is followed by separation of solid phase from liquid phase and clarification, to obtain vanilla extract having characteristic flavour, appearance and acceptable vanillin content.
- the present invention provides an improved process for the preparation of natural vanilla extract, wherein the process comprises the steps of: a) grinding partially cured fresh vanilla pods/partially cured stored vanilla beans in water [in the ratio of 1 :0.25 to 1 :1 w/v] to obtain a paste; b) simultaneously grinding fresh tea leaves separately with ice-cold ethyl alcohol [in the ration of 1 :2 to 1 : 4 w/v] to obtain a slurry; c) separating solid phase from liquid phase of the slurry as obtained in step (b) followed by repeated washings of the solid phase with ethanol to remove tea constituents such as caffeine, polyphenols, and chlorophyll; d) drying the solid phase as obtained from step (c) at a temperature below
- step (e) mixing the powder obtained in step (d) with the paste as obtained in step (a) in a ratio of 5 : 85 to 20 : 80 w/w to obtain a slurry; f) incubating the slurry obtained in step (e) at a temperature in the range of 40 to 50 degree Celsius for about 2 - 48 h to get maximum yield of vanillin; g) adding absolute alcohol to the slurry obtained in step (f) in a ratio of
- step (g) separating solid phase from liquid phase of the slurry as obtained in step (g) followed by clarification to obtain natural vanilla extract with characteristic aroma , brown color and vanillin content not less than
- vanilla extract as obtained in step (h) to partial desolventization at reduced pressure followed by clarification by filtration to obtain vanilla extract containing 0.2 - 0.4 % w/v vanillin and having desirable characteristic flavor and brown color.
- partially cured vanilla beans are prepared by controlled heat treatment and conditioning of green vanilla beans of a recent harvest which is used for extraction.
- partially cured vanilla beans are prepared by controlled heat treatment and conditioning of stored mature green vanilla beans.
- mature green vanilla beans are preserved by storing them in a cold room for up to 6 months at a temperature of 0- 8 degree Celsius.
- the mature green beans are preserved by storing in a deep freezer for up to 12 months, at a temperature of - 40 to -80 degree Celsius.
- partial curing of fresh or stored green vanilla beans is effected by holding the green beans in a hot air drier at a temperature of 40-70 degree Celsius for 1-3 hrs every day for a period of 5-15 days and holding the warm, vanilla beans in an insulated chamber to preserve the heat, immediately after the hot air exposure and till the next hot air exposure.
- fresh or stored green vanilla beans subjected to partial curing by known art involving components of holding the beans in hot water/steam, repeated exposure to hot sun, and repeated sweating of the warm beans in wooden box is mixed, after size reduction, with the enzyme preparation made from green tea leaves as described under claim 1 , to be followed by incubation and extraction as described.
- natural tea enzymes has an activity of 7000-28,000 units of PPO per gram , 150-300 units of galactosidase activity per gram, 1000-2000 units phenylalanine ammonia lyase activity per gram and 700- 1500 units glycosidase activity per gram when as determined by standard methods of enzyme assay.
- 10 gms of the powder containing the tea enzymes is mixed with an extraction buffer prepared with potassium phosphate 0.05M, pH 6.8; containing 0.35M KcI, 0.25% Triton X-100 and 0.01 mM
- the enzyme containing powdery preparation from tea leaves is treated with commercial cellulase enzyme with an activity of 5000 to 7500 S Units, at a concentration of 0.2 -1.0% by weight of the tea enzyme powder with the moisture level maintained at 40-70% by addition of water, maintaining the pH at 4-5, and incubated at 35-40 degree Celsius for 1 to 3 hours to facilitate release of the tea enzymes for action on vanilla bean
- the enzyme powder was dispersed in water and treated with commercial cellulase by holding at 40-50 degree Celsius for 1-3 hrs to facilitate release of enzymes adhering to the matrix
- the natural enzymes containing preparation from green tea leaves is made by using the known art of employing acetone as solvent but subsequently rinsing with ethanol to remove the adsorbed acetone, followed by gentle drying and grinding to obtain a powdery enzyme containing preparation, which can be used as a source of enzymes.
- the vanilla extract from step 1(i) is subjected to partial desolventization at reduced pressure and clarified by filtration to obtain vanilla extract containing 0.2 - 0.4 % vanillin and having desirable characteristic flavour and brown color.
- Fresh green vanilla pods were collected from the region of Koppa, Chikmagalur District, Karnataka, India and were procured from Mr.Darryl Colaco, Kalsapur Estate, Local producer, before the curing process was begun.
- the vanilla beans were first subjected to a partial curing process.
- Mature green vanilla beans of a recent harvest or green beans stored in a cold room or in a deep freezer can be used for partial curing, wherein beans were subjected to controlled heat treatment and conditioning for up to 15 days.
- the beans were held in a hot air dryer at a temperature of 40-70 degree Celsius for short spells of 1-3 hrs over 3 - 15 days. For rest of the period in between the thermal treatments, the beans were stored in an insulated chamber.
- Freshly harvested tea leaves (one bud two leaves) were subjected to thorough pre-cleaning by visual inspection to sort out and remove any extraneous matter and washing with potable water.
- the cleaned tea leaves were macerated with ice-cold ethanol to obtain fine slurry of the leaves in ethanol.
- the slurry was processed to separate the solid phase from the liquid phase.
- the solid phase was rinsed well with ethanol to remove tea constituents such as caffeine, polyphenols and chlorophyll and dried gently to remove the adsorbed solvent and obtain a free-flowing powder preparation containing enzymes from green tea leaves, especially polyphenol oxidase and ⁇ -primeeraridase. This powder retained 80% of its enzyme activity after 3 months storage in a refrigerator.
- vanilla beans were macerated to a paste or cut into bits and mixed with the tea enzyme preparation made as described above from fresh tea leaves.
- the mixed mass was incubated at 35-50 degree Celsius for a period of 2 - 48 hrs, wherein enzymes helped the development of characteristic vanilla flavour constituents by acting upon the vanilla flavour precursors.
- the fermented mass was treated with ethanol to inactivate the enzymes and extract vanilla flavour.
- the solid phase was separated from the liquid phase to obtain natural vanilla extract.
- vanilla extract can be desolventized partially under reduced pressure and clarified to obtain vanilla extract of characteristic brown colour, aroma and having vanillin strength to meet regulatory requirements.
- the enzyme containing powdery preparation from tea leaves is treated with commercial cellulase enzyme with an activity of 5000 to 7500 S Units, at a concentration of 0.2 -1.0% by weight of the tea enzyme powder with the moisture level maintained at 40-70% by addition of water, maintaining the pH at 4-5, and incubated at 35-40 degree Celsius for 1 to 3 hours to facilitate release of the tea enzymes for action on vanilla bean.
- the natural tea enzymes has an activity of 7000-28,000 units of PPO per gram , 150-300 units of galactosidase activity per gram, 1000-2000 units phenylalanine ammonia lyase activity per gram and 700-1500 units glycosidase activity per gram when as determined by standard methods of enzyme assay.
- the enzyme containing powdery preparation from tea leaves is optionally treated with commercial cellulase enzyme with an activity of 5000 to 7500 S Units, at a concentration of 0.2 -1.0% by weight of the tea enzyme powder with the moisture level maintained at 40-70% by addition of water, maintaining the pH at 4-5, and incubated at 35-40 degree celsius for 1 to 3 hours to facilitate release of the tea enzymes for action on vanilla bean.
- vanilla extract of good flavour quality is obtained avoiding the prolonged traditional curing step.
- Fully mature green vanilla beans (10 kg) were procured from an orchard in a local growing area and 5 kg batch each of the beans were stored in a cold room (0 - 4 degree Celsius) for up to a period of 4 months, and in a deep freezer maintained at -40 degree Celsius for up to a period of 12 months.
- Natural Enzyme powder preparation Freshly harvested tea leaves (two leaves and a bud) processed within 48 hours of the harvest. Tea leaves were thoroughly cleaned with potable water. Tea leaves (50Og) were blended with ice-cold 80% ethanol (2 liters) using a blender to get a homogenous paste. This paste was charged into a Buckner funnel and repeatedly washed with ice-cold ethanol. The creamy whitish powder was dried below 30 degree Celsius and to obtain the tea enzyme powder (100g). The powder contains natural tea enzymes.
- Fresh tea leaves (two leaves and a bud) were collected and kept overnight for freezing. Frozen crispy tea leaves (100gms) were transferred to a blender, and chilled aqueous alcohol (300 ml) was added. This was ground to a pulp and transferred to a buckner funnel. The pulp was washed repeatedly (1.5L of 80% ethanol)) to remove caffeine, chlorophyll and other pigments present in the material. Finally it was washed with 100% ethanol (500 ml) to remove the moisture present in the material. The residue was dried at ambient conditions (27 ⁇ 5°C) to remove all the adsorbed solvent. A creamy white powder containing tea enzymes was obtained.
- Extraction buffer was prepared with potassium phosphate 0.05M, pH 6.8; containing 0.35M KCI, 0.25% Triton X- 100, and 0.01 mM PMSF. 10gms of enzyme powder was weighed and transferred to a mortar. To this 20 g of acid washed sand , 10 g of PVP and 100 ml of buffer were added and ground. Homogenate was filtered through a muslin cloth to get the first extract containing tea enzymes. Volume of first extract was 60 ml. Similarly second and third extract were prepared and the three extracts pooled (230ml), were centrifuged at 10,000 rpm, 20 min at 4°C. Aliquots of the clear enzyme extract were used for activity evaluation ad detailed below.
- Polyphenol oxidase A reaction mixture was prepared with 5OmM phosphate buffer of pH-6.8, which contains 1OmM catechin as substrate. An aliquot of enzyme extract was added and absorbance measured at 42OnM. Change in 1 unit of absorbance is one unit activity of PPO.
- Glycosidase In a total volume of 0.5ml, the reaction mixture contained
- Galactosidase Same assay method as used for glycosidase. Lactose was used as the specific substrate.
- Phenylalanine ammonia lyase Assay of the enzyme was done at 273nm with phenylalanine as substrate. One unit of activity is defined 1mM cinnamic acid formed/hr by 1g enzyme powder.
- vanilla beans in requisite quantity were procured from vanilla gardens. And beans were converted to a paste.
- An aqueous slurry pf the tea enzyme powder was made to which a commercial cellulase enzyme (5000 units/ml/min.) at 1% wt/volume on the basis of the tea enzyme powder was added and incubated at 45 degree Celsius for 2 hours and this was mixed with the vanilla bean paste followed by incubation at 45 degree Celsius with agitation for 48 hours. This was followed by alcohol addition and liquid phase separation to obtain vanilla extract.
- Table.1 The results of examples 2 & 3 are presented in Table.1
- vanilla beans in requisite quantity were withdrawn from cold storage and from frozen storage separately and subjected to partial curing by holding in a hot air drier at 50 degree Celsius for 2 hrs, daily for a period of 3 days.
- the beans were converted to a paste.
- Tea enzyme extract preparation from tea enzyme powder and having an activity of 4300 polyphenol oxidase units /ml/ min was added to the vanilla paste at 10% and 20% by weight of the paste and incubated at 45 degree celsius with agitation for 2 hours followed by alcohol addition and liquid phase separation and clarification to obtain vanilla extract.
- Table.2 Recovery of vanillin under different experimental conditions
- vanilla beans in requisite quantity were withdrawn from cold storage and from frozen storage separately and subjected to partial curing by holding in a hot air drier at 50 degree Celsius for 2 h, daily for a period of 3 days.
- the beans were converted to a paste with added water and mixed with the tea enzyme powder (8% w/w on vanilla paste).
- the mixture was incubated at 45 degree Celsius with agitation for 48 h and then stirred gently (2 h) with equal volume of ethanol.
- the solid phase was separated from liquid phase and clarified to obtain vanilla extract. Advantages of this process are :
- Green vanilla beans stored from 3 months to 1 year can be used for processing into extract as and when needed.
- vanilla extract prepared with out using any chemicals / commercial enzymes.
Landscapes
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Fats And Perfumes (AREA)
- Tea And Coffee (AREA)
Abstract
Fresh green vanilla beans of a recent harvest, or partially cured vanilla beans or preserved green vanilla beans are used as the starting material. A preparation containing natural tea enzyme is made from fresh tea leaves. Fresh/cured/preserved vanilla beans, after proper size reduction, are mixed in a suitable proportion with the tea enzyme preparation and incubated to facilitate action of natural enzymes on vanilla flavour precursors present in the beans. The fermented vanilla & tea enzyme mix is treated with ethanol to inactivate enzymes and to extract the vanilla flavour constituents into ethanol. This is followed by separation of solid phase from liquid phase and clarification, to obtain vanilla extract having characteristic flavour, appearance and acceptable vanillin content.
Description
"AN IMPROVED PROCESS FOR THE PREPARATION OF NATURAL VANILLA EXTRACT"
Field of the Invention The present invention relates to an improved process for the preparation of natural vanilla extract from fresh green beans.
Background of the Invention and Description of prior Art
Vanilla planifolia Andr. is herbaceous, perennial vine a tropical orchid. This spice crop is grown for its pleasant aromatic essence "Vanillin". Annual world production is estimated at around 3000-4000 MT. In India vanilla crop is getting established as an important spice crop (Sreekrishna Bhat and Sudharshan 2002). The genus vanilla belongs to the monocotyledonous family Orchidaceae, and perhaps is the only orchid crop, which is of economic importance as a spice, the others being valued as ornamentals. Over 100 species of vanilla have been described in scientific literature, but only three if these are considered important for commerce and cultivation. There are Vanilla fragrans Salish Ames also known as Vanilla planifolia Andrews b) Vanilla pompna Shiede and c) Vanilla tahitensis J.W Moore, of these, Vanilla planifolia is the most valued fruits flavor qualities and is therefore widely cultivated and used (Persleglove 1981). Vanilla pompna and Vanilla tahitensis yield vanilla beans of poorer quality. Green vanilla pods are almost odourless and the characteristic flavour of vanilla develops only when the mature beans are subjected to a controlled curing and conditioning process.
For Vanilla producing countries, it is mainly an export oriented flavouring material and the international market is very competitive and quality conscious for vanilla beans and vanilla based products. Hence, it will be important for the growers/processors to adopt suitable methods for processing of vanilla. In the vanilla/ flavour industry, vanilla extract is produced by extraction of cured vanilla pods with aqueous alcohol followed by desolventisation to get a brown/red coloured, viscous material called the vanilla extract /concentrate. The extract/concentrate contains the constituents responsible for flavour namely vanillin, and other soluble components such as fats, fiber and waxes. Vanillin/ vanilla extract is useful as a flavourant in foods and confectionary items. The
industry aims at producing the pure natural vanillin/vanilla extract with a high flavor value, but the quality gets restricted due to the natural composition of the vanilla with respect to flavour.
The first critical phase is vanilla process is known as curing. A number of procedures have been evolved for curing of vanilla but they are all characterized by four phases whereby the green mature beans are transferred into a commercially desirable product. These phases are called killing, sweating, drying and conditioning. Killing or wilting brings about physiological death of the living tissues by respiratory function of the disrupting the cells. The cell-disruption can be carried out by subjecting the fresh beans to any one of the treatments such as hot water scalding, sun and oven wilting, scarification treatment with ethylene gas or freezing. Sweating helps the beans to develop their characteristic colour, aroma and flavouring properties. During the drying step the moisture is reduced and beneficial chemical changes take place. Due to conditioning stage, the beans are stored in closed boxes and this rests for several months. Various chemical and biochemical reactions take place in this step, producing various volatile aroma constituents enhancing the overall flavour quality of the cured beans (Purseglove 1981 , Randire 1984).
Reference may be made to a Japanese patent (JP8308528) which relates to the preparation of vanilla extract from cured beans using water and /or a water- soluble organic solvent in the presence of an alkali or alkali salt. Reference may be made to a Japanese patent (JP8168355) which recites the preparation of vanilla extract from cured beans employing water and /or aqueous organic solvent using the irradiation of ultrasonic waves. Another patent JP2002038188 relates to the preparation of vanilla extract from cured vanilla beans employing cocoa butter to enhance the vanilla flavour.
Reference may be made to the patents JP9111285 and KR255037 that relate to preparation of vanilla perfume by treating vanilla pods with specific microorganisms followed by extraction with an organic solvent.
Reference may be made to a series of patents involving the use of enzymes for vanilla extraction. GB812443 relates to green vanilla beans being extracted in substantial absence of oxygen and the extract cured. An enzyme system can be added during or prior to the curing and concentration of the extract that preferably precedes curing; W09325088 deals with bringing vanilla beans in contact with enzymes of the pectinase, cellulase and /or hemicellulase type and making a beta-glucosidase enzyme react and extracting natural vanilla flavour thus obtained.
GR3022203T relates to a process for the production of natural vanilla extract, consisting of processing crushed green vanilla pods by means of an enzymatic system capable of destroying plant cell membrane systems and hydrolysing glycoside precursors of volatile compounds. JP2001181671 relates to preparing vanilla extract by treating cured vanilla beans or its extract with at least one enzyme selected from protease, astringency / bitterness splitting enzyme and hesperidin splitting enzyme. WO2004091316 relates to a process for the preparation of a vanilla extract, the process consisting of subjecting green vanilla beans to accelerated browning followed by extractive / enzymatic treatment.
US4956192 relates to the preparation of natural vanilla flavour comprising freezing green vanilla beans at 5 degree and -30 degree, thawing and then extracting the flavour constituents from the beans. US5705205 relates to enzymatic treatment of a hydrated slurry of ground dehydrated green vanilla pods, addition of enzyme consisting of 10-1000 units of beta-glucose activity per gram of green vanilla pods followed by incubation and phase separation to obtain vanilla extract.
Reference may be made to a bioconversion process for the production of vanillin (US5279950) wherein vanillin is produced through biOconversion of vanilla precursor with a tissue culture of undifferentiated callus cells derived from a vanilla plant and / or enzymes obtained therefrom in the presence of a water soluble sulfhydryl compound and an assimilable carbon source. Production of novel vanilla extract using Co2 of a supercritical state (JP4214799) wherein,
preparation of vanilla extract from cured beans initially by supercritical carbon dioxide and subsequently with a polar organic solvent is reported.
Preparation of enhanced vanilla flavour has also been reported. JP58056654 relates to the preparation of vanilla flavour with enhanced fragrance by extracting a mixture of pulverized vanilla beans or dried material with spice vegetable material of allspice, caraway, cardamom, cassia or cinnamon with a water- miscible organic solvent.
Relevant earlier patents (as indicated above) mention use of enzymes such as protease, astriningency / bitterness splitting enzyme and hesperdins splitting enzyme and β-glucosidase for the preparation of vanilla extract. For any vanilla extract chemical flavour quality as assayed by gas chromatographic method and also organoliptic quality for sensory acceptance are considered two important quality attributes. However, this aspect does not appear to be adequately addressed in the above patents. It is desirable to produce a vanilla extract similar or better in quality as compared to the commercially manufactured extracts, wherein cured vanilla beans are used as the starting material.
However, keeping in view the drawbacks of the reported prior art the present invention involves a novel approach wherein a natural bioactive preparation made from green tea leaves which has enzymatic activity capable of action on phenolic precursors of green vanilla beans and forming characteristic vanilla flavour compounds, is made use of. The flavour quality of the extracts so prepared has been assessed chemically and organically as seen to be highly acceptable in tune with to conventional cured vanilla extracts. In this application a method is provided for the preparation of the vanilla extract from green vanilla pods by using enzymes obtained from fresh tea leaves (one bud two leaves). Objects of the Invention The main object of the present invention is thus to provide an improved process for the preparation of vanilla extract from green vanilla beans which obviates the drawbacks of the prior art.
Another object is to provide a process wherein the vanilla extract is prepared without using any chemicals / commercial enzymes.
Yet another object is to enhance the extractability of vanilla constituents with acceptable flavour by a combination of fresh vanilla pulp with an enzyme preparation derived from fresh tea leaves in a crude or purified form.
Summary of the Invention
Fresh green vanilla beans of a recent harvest, or partially cured vanilla beans or preserved green vanilla beans are used as the starting material. A preparation containing natural tea enzyme is made from fresh tea leaves. Fresh/cured/preserved vanilla beans, after proper size reduction, are mixed in a suitable proportion with the tea enzyme preparation and incubated to facilitate action of natural enzymes on vanilla flavour precursors present in the beans. The fermented vanilla & tea enzyme mix is treated with ethanol to inactivate enzymes and to extract the vanilla flavour constituents into ethanol. This is followed by separation of solid phase from liquid phase and clarification, to obtain vanilla extract having characteristic flavour, appearance and acceptable vanillin content.
Accordingly, the present invention provides an improved process for the preparation of natural vanilla extract, wherein the process comprises the steps of: a) grinding partially cured fresh vanilla pods/partially cured stored vanilla beans in water [in the ratio of 1 :0.25 to 1 :1 w/v] to obtain a paste; b) simultaneously grinding fresh tea leaves separately with ice-cold ethyl alcohol [in the ration of 1 :2 to 1 : 4 w/v] to obtain a slurry; c) separating solid phase from liquid phase of the slurry as obtained in step (b) followed by repeated washings of the solid phase with ethanol to remove tea constituents such as caffeine, polyphenols, and chlorophyll; d) drying the solid phase as obtained from step (c) at a temperature below
30 degree Celsius to obtain a powder containing natural tea enzymes; e) mixing the powder obtained in step (d) with the paste as obtained in step (a) in a ratio of 5 : 85 to 20 : 80 w/w to obtain a slurry; f) incubating the slurry obtained in step (e) at a temperature in the range of 40 to 50 degree Celsius for about 2 - 48 h to get maximum yield of vanillin;
g) adding absolute alcohol to the slurry obtained in step (f) in a ratio of
1:0.5 to 1 :2 w/v and providing a contact time of 1-6 hr to inactivate the enzyme and to extract the constituents; h) separating solid phase from liquid phase of the slurry as obtained in step (g) followed by clarification to obtain natural vanilla extract with characteristic aroma , brown color and vanillin content not less than
0.2% w/v; i) optionally subjecting the vanilla extract as obtained in step (h) to partial desolventization at reduced pressure followed by clarification by filtration to obtain vanilla extract containing 0.2 - 0.4 % w/v vanillin and having desirable characteristic flavor and brown color.
In an embodiment of the invention, partially cured vanilla beans are prepared by controlled heat treatment and conditioning of green vanilla beans of a recent harvest which is used for extraction.
In another embodiment of the invention, partially cured vanilla beans are prepared by controlled heat treatment and conditioning of stored mature green vanilla beans. In still another embodiment of the invention, mature green vanilla beans are preserved by storing them in a cold room for up to 6 months at a temperature of 0- 8 degree Celsius.
In yet another embodiment of the invention, the mature green beans are preserved by storing in a deep freezer for up to 12 months, at a temperature of - 40 to -80 degree Celsius.
In a further embodiment of the invention, partial curing of fresh or stored green vanilla beans is effected by holding the green beans in a hot air drier at a temperature of 40-70 degree Celsius for 1-3 hrs every day for a period of 5-15 days and holding the warm, vanilla beans in an insulated chamber to preserve the heat, immediately after the hot air exposure and till the next hot air exposure.
In another embodiment of the invention, fresh or stored green vanilla beans subjected to partial curing by known art involving components of holding the beans in hot water/steam, repeated exposure to hot sun, and repeated sweating of the warm beans in wooden box, is mixed, after size reduction, with the enzyme preparation made from green tea leaves as described under claim 1 , to be followed by incubation and extraction as described.
In yet another embodiment of the invention, natural tea enzymes has an activity of 7000-28,000 units of PPO per gram , 150-300 units of galactosidase activity per gram, 1000-2000 units phenylalanine ammonia lyase activity per gram and 700- 1500 units glycosidase activity per gram when as determined by standard methods of enzyme assay.
In yet another embodiment of the invention 10 gms of the powder containing the tea enzymes is mixed with an extraction buffer prepared with potassium phosphate 0.05M, pH 6.8; containing 0.35M KcI, 0.25% Triton X-100 and 0.01 mM
PMSF and transferred to a mortar. To this 20 g of acid washed sand, 10 g of PVP and 100 ml of buffer were added and ground. Homogenate was filtered through a muslin cloth to get the first extract containing tea enzymes. Volume of first extract was 60 ml. Similarly second and third extract were prepared and the three extracts pooled (230 ml), were centrifuged at 10, 000 rpm, 20 min at 40C. Aliquots of the clear enzyme extract are used for activity evaluation.
In still another embodiment of the invention, the enzyme containing powdery preparation from tea leaves is treated with commercial cellulase enzyme with an activity of 5000 to 7500 S Units, at a concentration of 0.2 -1.0% by weight of the tea enzyme powder with the moisture level maintained at 40-70% by addition of water, maintaining the pH at 4-5, and incubated at 35-40 degree Celsius for 1 to 3 hours to facilitate release of the tea enzymes for action on vanilla bean
In another embodiment of the invention, the enzyme powder was dispersed in water and treated with commercial cellulase by holding at 40-50 degree Celsius for 1-3 hrs to facilitate release of enzymes adhering to the matrix,
In yet another embodiment of the invention, the natural enzymes containing preparation from green tea leaves is made by using the known art of employing acetone as solvent but subsequently rinsing with ethanol to remove the adsorbed acetone, followed by gentle drying and grinding to obtain a powdery enzyme containing preparation, which can be used as a source of enzymes.
In yet another embodiment of the invention, as an optional step, the vanilla extract from step 1(i) is subjected to partial desolventization at reduced pressure and clarified by filtration to obtain vanilla extract containing 0.2 - 0.4 % vanillin and having desirable characteristic flavour and brown color.
Detailed Description of the Invention
Fresh green vanilla pods were collected from the region of Koppa, Chikmagalur District, Karnataka, India and were procured from Mr.Darryl Colaco, Kalsapur Estate, Local producer, before the curing process was begun. The vanilla beans were first subjected to a partial curing process. Mature green vanilla beans of a recent harvest or green beans stored in a cold room or in a deep freezer can be used for partial curing, wherein beans were subjected to controlled heat treatment and conditioning for up to 15 days. The beans were held in a hot air dryer at a temperature of 40-70 degree Celsius for short spells of 1-3 hrs over 3 - 15 days. For rest of the period in between the thermal treatments, the beans were stored in an insulated chamber. Freshly harvested tea leaves (one bud two leaves) were subjected to thorough pre-cleaning by visual inspection to sort out and remove any extraneous matter and washing with potable water. The cleaned tea leaves were macerated with ice-cold ethanol to obtain fine slurry of the leaves in ethanol. The slurry was processed to separate the solid phase from the liquid phase. The solid phase was rinsed well with ethanol to remove tea constituents such as caffeine, polyphenols and chlorophyll and dried gently to remove the adsorbed solvent and obtain a free-flowing powder preparation containing enzymes from green tea leaves, especially polyphenol oxidase and β-primeeraridase. This powder retained 80% of its enzyme activity after 3 months storage in a refrigerator. Partially cured vanilla beans were macerated to a paste or cut into
bits and mixed with the tea enzyme preparation made as described above from fresh tea leaves. The mixed mass was incubated at 35-50 degree Celsius for a period of 2 - 48 hrs, wherein enzymes helped the development of characteristic vanilla flavour constituents by acting upon the vanilla flavour precursors. The fermented mass was treated with ethanol to inactivate the enzymes and extract vanilla flavour. The solid phase was separated from the liquid phase to obtain natural vanilla extract. In order to enhance the vanillin content, vanilla extract can be desolventized partially under reduced pressure and clarified to obtain vanilla extract of characteristic brown colour, aroma and having vanillin strength to meet regulatory requirements.
The enzyme containing powdery preparation from tea leaves is treated with commercial cellulase enzyme with an activity of 5000 to 7500 S Units, at a concentration of 0.2 -1.0% by weight of the tea enzyme powder with the moisture level maintained at 40-70% by addition of water, maintaining the pH at 4-5, and incubated at 35-40 degree Celsius for 1 to 3 hours to facilitate release of the tea enzymes for action on vanilla bean.
The natural tea enzymes has an activity of 7000-28,000 units of PPO per gram , 150-300 units of galactosidase activity per gram, 1000-2000 units phenylalanine ammonia lyase activity per gram and 700-1500 units glycosidase activity per gram when as determined by standard methods of enzyme assay.
The enzyme containing powdery preparation from tea leaves is optionally treated with commercial cellulase enzyme with an activity of 5000 to 7500 S Units, at a concentration of 0.2 -1.0% by weight of the tea enzyme powder with the moisture level maintained at 40-70% by addition of water, maintaining the pH at 4-5, and incubated at 35-40 degree celsius for 1 to 3 hours to facilitate release of the tea enzymes for action on vanilla bean.
Novelty
The partially cured mature green vanilla beans, after size reduction are treated with an enzyme preparation made from fresh tea leaves which facilitate better formation of the natural vanilla flavour constituents which is a novel inventive step.
Vanilla extract of good flavour quality is obtained avoiding the prolonged traditional curing step.
The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.
Example 1
Fully mature green vanilla beans (10 kg) were procured from an orchard in a local growing area and 5 kg batch each of the beans were stored in a cold room (0 - 4 degree Celsius) for up to a period of 4 months, and in a deep freezer maintained at -40 degree Celsius for up to a period of 12 months.
Natural Enzyme powder preparation: Freshly harvested tea leaves (two leaves and a bud) processed within 48 hours of the harvest. Tea leaves were thoroughly cleaned with potable water. Tea leaves (50Og) were blended with ice-cold 80% ethanol (2 liters) using a blender to get a homogenous paste. This paste was charged into a Buckner funnel and repeatedly washed with ice-cold ethanol. The creamy whitish powder was dried below 30 degree Celsius and to obtain the tea enzyme powder (100g). The powder contains natural tea enzymes.
Example 2
Fresh tea leaves (two leaves and a bud) were collected and kept overnight for freezing. Frozen crispy tea leaves (100gms) were transferred to a blender, and chilled aqueous alcohol (300 ml) was added. This was ground to a pulp and transferred to a buckner funnel. The pulp was washed repeatedly (1.5L of 80% ethanol)) to remove caffeine, chlorophyll and other pigments present in the material. Finally it was washed with 100% ethanol (500 ml) to remove the moisture present in the material. The residue was dried at ambient conditions (27±5°C) to remove all the adsorbed solvent. A creamy white powder containing tea enzymes was obtained. Extraction buffer was prepared with potassium phosphate 0.05M, pH 6.8; containing 0.35M KCI, 0.25% Triton X- 100, and 0.01 mM PMSF. 10gms of enzyme powder was weighed and transferred to a mortar. To this 20 g of acid washed sand , 10 g of PVP and 100 ml of buffer were
added and ground. Homogenate was filtered through a muslin cloth to get the first extract containing tea enzymes. Volume of first extract was 60 ml. Similarly second and third extract were prepared and the three extracts pooled (230ml), were centrifuged at 10,000 rpm, 20 min at 4°C. Aliquots of the clear enzyme extract were used for activity evaluation ad detailed below.
Enzyme assay
Polyphenol oxidase: A reaction mixture was prepared with 5OmM phosphate buffer of pH-6.8, which contains 1OmM catechin as substrate. An aliquot of enzyme extract was added and absorbance measured at 42OnM. Change in 1 unit of absorbance is one unit activity of PPO.
Glycosidase: In a total volume of 0.5ml, the reaction mixture contained
100mMol/l sodium acetate buffer, pH 4, 5 mM of sucrose as substrate, and an aliquot of the enzyme. Incubation was for 15 min at 32 0C. The reaction was stopped by adding 2.0ml of 0.1 mol/L NaOH and the absorbance of released pNP was measured at 410 nm.
Galactosidase: Same assay method as used for glycosidase. Lactose was used as the specific substrate.
Phenylalanine ammonia lyase: Assay of the enzyme was done at 273nm with phenylalanine as substrate. One unit of activity is defined 1mM cinnamic acid formed/hr by 1g enzyme powder.
TableA: Enzyme profile of natural enzyme powder
Vanilla beans in requisite quantity were procured from vanilla gardens. And beans were converted to a paste. An aqueous slurry pf the tea enzyme powder was made to which a commercial cellulase enzyme (5000 units/ml/min.) at 1% wt/volume on the basis of the tea enzyme powder was added and incubated at 45 degree Celsius for 2 hours and this was mixed with the vanilla bean paste followed by incubation at 45 degree Celsius with agitation for 48 hours. This was followed by alcohol addition and liquid phase separation to obtain vanilla extract. The results of examples 2 & 3 are presented in Table.1
Table.1 Effect of added tea enzyme preparation on vanillin recovery in extracts
Example 4
Vanilla beans in requisite quantity were withdrawn from cold storage and from frozen storage separately and subjected to partial curing by holding in a hot air drier at 50 degree Celsius for 2 hrs, daily for a period of 3 days. The beans were converted to a paste. Tea enzyme extract preparation from tea enzyme powder and having an activity of 4300 polyphenol oxidase units /ml/ min was added to the vanilla paste at 10% and 20% by weight of the paste and incubated at 45 degree celsius with agitation for 2 hours followed by alcohol addition and liquid phase separation and clarification to obtain vanilla extract. The results are presented in Table.2.
Table 2 : Recovery of vanillin under different experimental conditions
Example 5
Vanilla beans in requisite quantity were withdrawn from cold storage and from frozen storage separately and subjected to partial curing by holding in a hot air drier at 50 degree Celsius for 2 h, daily for a period of 3 days. The beans were converted to a paste with added water and mixed with the tea enzyme powder (8% w/w on vanilla paste). The mixture was incubated at 45 degree Celsius with agitation for 48 h and then stirred gently (2 h) with equal volume of ethanol. The solid phase was separated from liquid phase and clarified to obtain vanilla extract.
Advantages of this process are :
1. Green vanilla beans stored from 3 months to 1 year can be used for processing into extract as and when needed.
2. Down stream processing time for making the extract starting from partially cured beans is very low (2 to 3 days) compared to traditional extraction methods from cured beans, which takes many weeks.
3. Vanilla extract prepared with out using any chemicals / commercial enzymes.
Claims
1. An improved process for the preparation of natural vanilla extract, wherein the process comprises the steps of: a) grinding partially cured fresh vanilla pods/partially cured stored vanilla beans in water [ in the ratio of 1:0.25 to 1:1 w/v] to obtain a paste; b) simultaneously grinding fresh tea leaves separately with ice-cold ethyl alcohol [in the ration of 1 :2 to 1 : 4 w/v] to obtain a slurry; c) separating solid phase from liquid phase of the slurry as obtained in step (b) followed by repeated washings of the solid phase with ethanol to remove tea constituents such as caffeine, polyphenols, and chlorophyll; d) drying the solid phase as obtained from step (c) at a temperature below
30 degree Celsius to obtain a powder containing natural tea enzymes; e) mixing the powder obtained in step (d) with the paste as obtained in step
(a) in a ratio of 5 : 85 to 20 : 80 w/w to obtain a slurry; f) incubating the slurry obtained in step (e) at a temperature in the range of
40 to 50 degree Celsius for about 12 - 48 h to get maximum yield of vanillin; g) adding absolute alcohol to the slurry obtained in step (f) in a ratio of
1 :0.5 to 1:2 w/v and providing a contact time of 1-6 hr to inactivate the enzyme and to extract the constituents; h) separating solid phase from liquid phase of the slurry as obtained in step
(g) followed by clarification to obtain natural vanilla extract with characteristic aroma , brown color and vanillin content not less than
0.2% w/v; i) optionally subjecting the vanilla extract as obtained in step (h) to partial desolventization at reduced pressure followed by clarification by filtration to obtain vanilla extract containing 0.2 - 0.4 % w/v vanillin and having desirable characteristic flavour and brown color.
2. A process according to claim 1 , wherein partially cured vanilla beans are prepared by controlled heat treatment and conditioning of green vanilla beans of a recent harvest/ stored mature green vanilla beans.
3. A process according to any preceding claims, wherein the mature green vanilla beans are preserved by storing them in a cold room for up to 6 months at a temperature of 0-8 degree Celsius or by storing in a deep freeze for up to 12 months at a temperature of -40 to -80 degree Celsius.
4. A process according to any preceding claims, wherein partial curing of fresh or stored green vanilla beans is effected by holding the green beans in a hot air drier at a temperature of 40-70 degree Celsius for 1-3 hrs every day for a period of 5-15 days and holding the warm, vanilla beans in an insulated chamber to preserve the heat, immediately after the hot air exposure and till the next hot air exposure.
5. A process according to any preceding claims, wherein the natural tea enzymes have an activity of 7000-28,000 units of PPO per gram, 150-300 units of galactosidase activity per gram, 1000-2000 units phenylalanine ammonia lyase activity per gram and 700-1500 units glycosidase activity per gram.
6. A process according to any preceding claims, wherein the enzyme preparation from tea leaves is optionally treated with commercial cellulase enzyme with an activity of 5000 to 7500 S Units, at a concentration of 0.2 -1.0% by weight of the tea enzyme powder with the moisture level maintained at 40-70% by addition of water, maintaining the pH at 4-5, and incubated at 35-40 degree Celsius for 1 to 3 hours to facilitate release of the tea enzymes for action on vanilla bean.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IN1895DE2007 | 2007-09-07 | ||
IN1895/DEL/2007 | 2007-09-07 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2009031160A1 true WO2009031160A1 (en) | 2009-03-12 |
Family
ID=39933922
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/IN2008/000529 WO2009031160A1 (en) | 2007-09-07 | 2008-08-25 | An improved process for the preparation of natural vanilla extract |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2009031160A1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102550768A (en) * | 2010-12-21 | 2012-07-11 | 津田彻 | Spices combination for black tea beverage |
CN103289821A (en) * | 2012-03-05 | 2013-09-11 | 广州市名花香料有限公司 | Preparation method of Vanilla extract |
CN105037378A (en) * | 2015-07-30 | 2015-11-11 | 潍坊友容实业有限公司 | Method for preparing and extracting iron chlorophyll and prepared iron chlorophyll |
CN105037377A (en) * | 2015-07-30 | 2015-11-11 | 潍坊友容实业有限公司 | Extraction preparation method for sodium ferrous chlorophyllin and sodium ferrous chlorophyllin prepared through extraction preparation method |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004091316A1 (en) * | 2003-04-15 | 2004-10-28 | Indena S.P.A. | A process for the enzymatic preparation of vanilla flavor |
US20050074519A1 (en) * | 2003-10-01 | 2005-04-07 | Sensient Flavors Inc. | Method for the production of natural botanical extracts |
-
2008
- 2008-08-25 WO PCT/IN2008/000529 patent/WO2009031160A1/en active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004091316A1 (en) * | 2003-04-15 | 2004-10-28 | Indena S.P.A. | A process for the enzymatic preparation of vanilla flavor |
US20050074519A1 (en) * | 2003-10-01 | 2005-04-07 | Sensient Flavors Inc. | Method for the production of natural botanical extracts |
Non-Patent Citations (1)
Title |
---|
RUIZ-TERAN F ET AL: "Enzymatic extraction and transformation of glucovanillin to vanillin from vanilla green pods", JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, AMERICAN CHEMICAL SOCIETY. WASHINGTON, US, vol. 49, 1 January 2001 (2001-01-01), pages 5207 - 5209, XP002275039, ISSN: 0021-8561 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102550768A (en) * | 2010-12-21 | 2012-07-11 | 津田彻 | Spices combination for black tea beverage |
CN102550768B (en) * | 2010-12-21 | 2014-07-23 | 津田彻 | Spices combination for black tea beverage |
CN103289821A (en) * | 2012-03-05 | 2013-09-11 | 广州市名花香料有限公司 | Preparation method of Vanilla extract |
CN105037378A (en) * | 2015-07-30 | 2015-11-11 | 潍坊友容实业有限公司 | Method for preparing and extracting iron chlorophyll and prepared iron chlorophyll |
CN105037377A (en) * | 2015-07-30 | 2015-11-11 | 潍坊友容实业有限公司 | Extraction preparation method for sodium ferrous chlorophyllin and sodium ferrous chlorophyllin prepared through extraction preparation method |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP5887470B1 (en) | Barley stems and / or leaves from Aso | |
KR20200094392A (en) | Method for producing Kombucha using Camellia sinensis and Petasites japonicus and Kombucha produced by the same method | |
KR101874161B1 (en) | Anti-obesity compositions and methods of manufacturing the same as a main component of raspberry leaf and stem extract | |
WO2009031160A1 (en) | An improved process for the preparation of natural vanilla extract | |
KR100907037B1 (en) | Cultivation method of Ganoderma lucidum or shiitake mushroom mycelium containing green tea ingredients and food prepared using the same | |
CN103981029A (en) | Vanilla legume extract as well as preparation method and application thereof | |
KR20190042433A (en) | A vinegar composition comprising camellia flower extract or the powder of camellia flower extract and a method for preparing the same | |
KR20190060023A (en) | Process for producing beverages containing active ingredients of Omija and Aronia extract and beverages produced therefrom | |
KR101837937B1 (en) | Production method of soy source and soybean paste using extract of Dendropanax morbifera Lev | |
KR102073393B1 (en) | Methdo for preparing soybean sauce and paste using fermented chaga mushroom powder and bamboo salt | |
JPH06502685A (en) | Method for producing natural vanilla flavor by enzymatic treatment of green vanilla pods, and the resulting flavor | |
KR20160099214A (en) | A method for preparing beer containing blueberry | |
KR102443519B1 (en) | Manufacturing method of the fermented tea using melon | |
KR102005795B1 (en) | Method for producng functional tea using Cudrania tricuspidata | |
KR101708052B1 (en) | Freezing concentrates the aroma excellent way sugar peach wine and ice wine production method | |
KR101870419B1 (en) | Method for preparing chungkookchang comprising nipa fruticans wurmb and chungkookchang by the method | |
KR101788801B1 (en) | MANUFACTURING METHOD FOR PEPPER POWDERS USING Rubus coreanus Miquel AND PEPPER POWDERS USING Rubus coreanus Miquel MANUFACTURED BY THE SAME | |
KR20220081786A (en) | Cudrania tricuspidata fruit-fermented vinegar with enhances antiobesity activity and producing method thereof | |
KR102426674B1 (en) | Kadsura coccinea liquid tea and Manufacturing method thereof | |
KR100422064B1 (en) | A manufacturing method of green tea extracts | |
KR102732422B1 (en) | Bean sprout with excellent retention of freshness and high content of saponin ingredient, growing method of the same bean sprout and dish including the same bean sprout | |
KR102596424B1 (en) | Manufacturing method of mandarin dessert | |
KR102734377B1 (en) | Method for producing fermented pear wine comprising pear peel component | |
JP2005278596A (en) | Process for producing processed sweet potato leaves | |
KR102308716B1 (en) | Process for preparing cold noodle with blueberry |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 08808165 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 08808165 Country of ref document: EP Kind code of ref document: A1 |