JP2018165279A - Method for producing sphingoid base-containing extract - Google Patents
Method for producing sphingoid base-containing extract Download PDFInfo
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- JP2018165279A JP2018165279A JP2018140343A JP2018140343A JP2018165279A JP 2018165279 A JP2018165279 A JP 2018165279A JP 2018140343 A JP2018140343 A JP 2018140343A JP 2018140343 A JP2018140343 A JP 2018140343A JP 2018165279 A JP2018165279 A JP 2018165279A
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- Prior art keywords
- ethanol
- sphingoid base
- extraction
- acid
- manufacturing
- Prior art date
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- Granted
Links
- 150000003410 sphingosines Chemical class 0.000 title claims abstract description 108
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 62
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 265
- 238000000605 extraction Methods 0.000 claims abstract description 50
- 239000000243 solution Substances 0.000 claims abstract description 46
- 239000000463 material Substances 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 31
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 28
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims abstract description 27
- 239000005909 Kieselgur Substances 0.000 claims abstract description 27
- 239000003456 ion exchange resin Substances 0.000 claims abstract description 16
- 229920003303 ion-exchange polymer Polymers 0.000 claims abstract description 16
- 239000007864 aqueous solution Substances 0.000 claims abstract description 14
- 239000012530 fluid Substances 0.000 claims abstract description 9
- 238000005903 acid hydrolysis reaction Methods 0.000 claims abstract description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 47
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 34
- 238000006460 hydrolysis reaction Methods 0.000 claims description 26
- 239000006227 byproduct Substances 0.000 claims description 22
- 150000003408 sphingolipids Chemical class 0.000 claims description 18
- 239000001569 carbon dioxide Substances 0.000 claims description 17
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 17
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 14
- -1 ether compound Chemical class 0.000 claims description 14
- 239000004927 clay Substances 0.000 claims description 13
- 230000007062 hydrolysis Effects 0.000 claims description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- 239000003729 cation exchange resin Substances 0.000 claims description 11
- 238000010828 elution Methods 0.000 claims description 11
- 238000001179 sorption measurement Methods 0.000 claims description 11
- 238000004808 supercritical fluid chromatography Methods 0.000 claims description 11
- 239000002253 acid Substances 0.000 claims description 9
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- 238000000194 supercritical-fluid extraction Methods 0.000 claims description 9
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 8
- 150000003839 salts Chemical class 0.000 claims description 7
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 claims description 6
- 241001465754 Metazoa Species 0.000 claims description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 5
- 244000005700 microbiome Species 0.000 claims description 5
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 4
- WWUZIQQURGPMPG-UHFFFAOYSA-N (-)-D-erythro-Sphingosine Natural products CCCCCCCCCCCCCC=CC(O)C(N)CO WWUZIQQURGPMPG-UHFFFAOYSA-N 0.000 claims description 3
- CQKNELOTFUSOTP-HMTIOLNVSA-N 4-hydroxy-8-sphingenine Chemical compound CCCCCCCCC\C=C\CCC[C@@H](O)[C@@H](O)[C@@H](N)CO CQKNELOTFUSOTP-HMTIOLNVSA-N 0.000 claims description 3
- AERBNCYCJBRYDG-UHFFFAOYSA-N D-ribo-phytosphingosine Natural products CCCCCCCCCCCCCCC(O)C(O)C(N)CO AERBNCYCJBRYDG-UHFFFAOYSA-N 0.000 claims description 3
- YRXOQXUDKDCXME-YIVRLKKSSA-N N,N-dimethylsphingosine Chemical compound CCCCCCCCCCCCC\C=C\[C@@H](O)[C@H](CO)N(C)C YRXOQXUDKDCXME-YIVRLKKSSA-N 0.000 claims description 3
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 3
- 125000003277 amino group Chemical group 0.000 claims description 3
- OTKJDMGTUTTYMP-UHFFFAOYSA-N dihydrosphingosine Natural products CCCCCCCCCCCCCCCC(O)C(N)CO OTKJDMGTUTTYMP-UHFFFAOYSA-N 0.000 claims description 3
- 239000001530 fumaric acid Substances 0.000 claims description 3
- 239000004310 lactic acid Substances 0.000 claims description 3
- 235000014655 lactic acid Nutrition 0.000 claims description 3
- 229910017604 nitric acid Inorganic materials 0.000 claims description 3
- AERBNCYCJBRYDG-KSZLIROESA-N phytosphingosine Chemical compound CCCCCCCCCCCCCC[C@@H](O)[C@@H](O)[C@@H](N)CO AERBNCYCJBRYDG-KSZLIROESA-N 0.000 claims description 3
- 229940033329 phytosphingosine Drugs 0.000 claims description 3
- OTKJDMGTUTTYMP-ZWKOTPCHSA-N sphinganine Chemical group CCCCCCCCCCCCCCC[C@@H](O)[C@@H](N)CO OTKJDMGTUTTYMP-ZWKOTPCHSA-N 0.000 claims description 3
- WWUZIQQURGPMPG-KRWOKUGFSA-N sphingosine Chemical compound CCCCCCCCCCCCC\C=C\[C@@H](O)[C@@H](N)CO WWUZIQQURGPMPG-KRWOKUGFSA-N 0.000 claims description 3
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 claims description 3
- QCUHPIBMMDIRKL-VKRJBFOFSA-N (4e,8e)-2-amino-9-methyloctadeca-4,8-diene-1,3-diol Chemical compound CCCCCCCCC\C(C)=C\CC\C=C\C(O)C(N)CO QCUHPIBMMDIRKL-VKRJBFOFSA-N 0.000 claims description 2
- 150000007513 acids Chemical class 0.000 claims description 2
- 239000003792 electrolyte Substances 0.000 claims description 2
- 235000013305 food Nutrition 0.000 abstract description 28
- 230000002378 acidificating effect Effects 0.000 abstract description 12
- 235000013376 functional food Nutrition 0.000 abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 39
- 239000008267 milk Substances 0.000 description 23
- 210000004080 milk Anatomy 0.000 description 23
- 235000013336 milk Nutrition 0.000 description 22
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 150000003904 phospholipids Chemical class 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- 239000012141 concentrate Substances 0.000 description 12
- 239000008346 aqueous phase Substances 0.000 description 10
- 229940106189 ceramide Drugs 0.000 description 10
- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 description 9
- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 description 9
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 8
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 239000003921 oil Substances 0.000 description 7
- 235000019198 oils Nutrition 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 239000003925 fat Substances 0.000 description 6
- 235000019197 fats Nutrition 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 229910052760 oxygen Inorganic materials 0.000 description 6
- 239000001301 oxygen Substances 0.000 description 6
- 238000003815 supercritical carbon dioxide extraction Methods 0.000 description 6
- 235000021323 fish oil Nutrition 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- 235000014121 butter Nutrition 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 150000002632 lipids Chemical class 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 230000001766 physiological effect Effects 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 3
- 238000000909 electrodialysis Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 210000001035 gastrointestinal tract Anatomy 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 238000004809 thin layer chromatography Methods 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 101150012828 UPC2 gene Proteins 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 235000013365 dairy product Nutrition 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 230000006866 deterioration Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 150000002170 ethers Chemical class 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 2
- 239000001307 helium Substances 0.000 description 2
- 229910052734 helium Inorganic materials 0.000 description 2
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 2
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- RTQVJTLVVBJRJG-UHFFFAOYSA-N (2S,3R,4E,8E)-4,8-sphingadienine Natural products CCCCCCCCCC=CCCC=CC(O)C(N)CO RTQVJTLVVBJRJG-UHFFFAOYSA-N 0.000 description 1
- NRGGMCIBEHEAIL-UHFFFAOYSA-N 2-ethylpyridine Chemical compound CCC1=CC=CC=N1 NRGGMCIBEHEAIL-UHFFFAOYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- YDIKCZBMBPOGFT-PWUSVEHZSA-N Malvidin 3-galactoside Chemical compound [Cl-].COC1=C(O)C(OC)=CC(C=2C(=CC=3C(O)=CC(O)=CC=3[O+]=2)O[C@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)=C1 YDIKCZBMBPOGFT-PWUSVEHZSA-N 0.000 description 1
- 102000014171 Milk Proteins Human genes 0.000 description 1
- 108010011756 Milk Proteins Proteins 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- PXUQTDZNOHRWLI-QOPOCTTISA-O Primulin Natural products O(C)c1c(O)c(OC)cc(-c2c(O[C@H]3[C@H](O)[C@@H](O)[C@@H](O)[C@H](CO)O3)cc3c(O)cc(O)cc3[o+]2)c1 PXUQTDZNOHRWLI-QOPOCTTISA-O 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 108010046377 Whey Proteins Proteins 0.000 description 1
- 102000007544 Whey Proteins Human genes 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000012223 aqueous fraction Substances 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 235000015155 buttermilk Nutrition 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 238000005341 cation exchange Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 125000001549 ceramide group Chemical group 0.000 description 1
- 150000001783 ceramides Chemical class 0.000 description 1
- 230000007073 chemical hydrolysis Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002036 chloroform fraction Substances 0.000 description 1
- 238000011208 chromatographic data Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000037319 collagen production Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000000326 densiometry Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 239000002031 ethanolic fraction Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 150000002305 glucosylceramides Chemical class 0.000 description 1
- 150000002313 glycerolipids Chemical class 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 108010071421 milk fat globule Proteins 0.000 description 1
- 235000021239 milk protein Nutrition 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000036284 oxygen consumption Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
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- 238000003860 storage Methods 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
Landscapes
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Extraction Or Liquid Replacement (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
Description
本発明はスフィンゴ脂質含有素材からスフィンゴイド塩基含有抽出物を製造する方法に
関する。より詳しくは、スフィンゴ脂質含有食品素材から、食品素材として許容できる方
法でスフィンゴイド塩基含有抽出物を製造する方法に関する。
The present invention relates to a method for producing a sphingoid base-containing extract from a sphingolipid-containing material. More specifically, the present invention relates to a method for producing a sphingoid base-containing extract from a sphingolipid-containing food material by a method acceptable as a food material.
スフィンゴイド塩基は、スフィンゴ脂質の骨格構造の長鎖塩基である。スフィンゴ脂質
は、動物や植物や微生物に広く存在し、スフィンゴイド塩基を骨格構造とし、それに脂肪
酸が結合したセラミド、さらにセラミドにリン酸や糖が結合したスフィンゴミエリンやグ
ルコシルセラミド等が存在する。
スフィンゴイド塩基は近年様々な食品化学的活性および生理活性を有することが報告さ
れている。
特許文献１には、スフィンゴイド塩基が血糖の組織取り込みを調節するホルモンの分泌
促進作用を有することを記載している。特許文献２には、スフィンゴイド塩基にはコラー
ゲン産生促進作用があることを記載している。
スフィンゴ脂質は経口摂取されると、消化管内で加水分解され、少なくとも一部はスフ
ィンゴイド塩基にまで消化されることが知られている。消化管内で加水分解されて生じた
スフィンゴイド塩基は小腸上皮細胞から体内へ吸収されるものと考えられている。しかし
、スフィンゴ脂質は、グリセロ脂質と比べて消化管内での加水分解を受けにくく、小腸で
の体内への吸収もされにくい。動物細胞に主要なスフィンゴイド塩基と植物細胞に主要な
スフィンゴイド塩基を比較すると、動物細胞に主要なスフィンゴイド塩基の方が小腸上皮
細胞にて吸収されやすいことが知られている。
A sphingoid base is a long-chain base having a skeletal structure of sphingolipid. Sphingolipids are widely present in animals, plants and microorganisms, and include ceramides having a sphingoid base as a skeletal structure and fatty acid bound thereto, and sphingomyelin and glucosylceramide in which phosphoric acid and sugar are bound to ceramide.
Sphingoid bases have recently been reported to have various food chemical and physiological activities.
Patent Document 1 describes that sphingoid bases have a hormone secretion-promoting action that regulates tissue uptake of blood sugar. Patent Document 2 describes that sphingoid bases have a collagen production promoting action.
It is known that when sphingolipids are ingested orally, they are hydrolyzed in the digestive tract and at least partially digested to sphingoid bases. The sphingoid base generated by hydrolysis in the digestive tract is thought to be absorbed into the body from small intestinal epithelial cells. However, sphingolipids are less susceptible to hydrolysis in the gastrointestinal tract than glycerolipids, and are not easily absorbed into the body in the small intestine. Comparing the main sphingoid bases in animal cells and the main sphingoid bases in plant cells, it is known that the main sphingoid bases in animal cells are more easily absorbed by small intestinal epithelial cells.
上記のとおり、スフィンゴイド塩基は、様々な生理活性や食品化学的活性を有すること
が報告されているが、スフィンゴイド塩基の食品への応用はほとんど進んでこなかった。
これまでは、食品素材として使用できるような方法で抽出・精製されたスフィンゴイド
塩基素材がなく、非常に高価な合成品、抽出・精製の過程でメタノールを用いた食品素材
として望ましくない素材（特許文献１、２）、組換え微生物等により製造した素材（特許
文献３）のみであった。メタノールを用いた加水分解によるスフィンゴイド塩基の抽出方
法は、後述する実施例に示すように副生成物の多い方法であることもわかった。
そこで、我々はスフィンゴ脂質含有素材からスフィンゴイド塩基を食品素材として使用
できる方法で安価に簡便に大量かつ副生物の少ない効率的な抽出・精製方法を提供するこ
とを目的とした。
As described above, sphingoid bases have been reported to have various physiological activities and food chemical activities, but application of sphingoid bases to foods has hardly progressed.
Up to now, there has been no sphingoid base material extracted and purified by a method that can be used as a food material, it is a very expensive synthetic product, and an undesired material as a food material using methanol in the process of extraction and purification (patent Documents 1 and 2) and only materials produced by recombinant microorganisms (Patent Document 3). It was also found that the sphingoid base extraction method by hydrolysis using methanol is a method with many by-products as shown in the examples described later.
Therefore, we aimed to provide an efficient extraction / purification method in a simple and inexpensive manner with a large amount and a small amount of by-products using a sphingoid base from a sphingolipid-containing material as a food material.
鋭意検討した結果、酸とエタノール若しくはエタノール水溶液を用いた化学的な加水分
解及び、イオン交換樹脂や超臨界流体抽出や珪藻土や活性白土への吸着等の方法による精
製を組み合わせることにより、食品素材として使用できる方法でスフィンゴイド塩基の抽
出・精製が可能であることを発見し、本発明を完成させた。
すなわち、本発明は、以下の構成を有する。
〔１〕
下記（１）の工程を含む、スフィンゴイド塩基を含む抽出物の製造方法。
（１）スフィンゴ脂質含有素材を０．００１〜１０Ｍの酸性エタノール又は酸性エタノー
ル水溶液に溶解または分散し、３０℃から１００℃で３０分から数日間加熱して酸加水分
解する工程
〔２〕
さらに下記（２）の工程を含む、〔１〕に記載の製造方法。
（２）工程（１）により得られた加水分解物からイオン交換樹脂による抽出、超臨界流体
による抽出、及び珪藻土若しくは活性白土への吸着を利用した抽出からなる群のいずれか
１以上の抽出によりスフィンゴイド塩基を得る工程
〔３〕
工程（１）の酸加水分解で用いる酸は、塩酸、硫酸、酢酸、クエン酸、乳酸、硝酸、フマ
ル酸等の酸から選ばれる少なくとも一種である〔１〕又は〔２〕に記載の製造方法。
〔４〕
工程（２）のイオン交換樹脂は、陽イオン交換樹脂である〔１〕〜〔３〕のいずれかに記
載の製造方法。
〔５〕
陽イオン交換樹脂からの溶出は、塩濃度および／またはｐＨを変化させることにより行う
〔４〕に記載の製造方法。
〔６〕
陽イオン交換樹脂からの溶出は、ＮａＣｌなどの電解質により、イオン強度が0.00001か
ら1までの溶液を用いた溶出液によるものである〔４〕または〔５〕に記載の製造方法。
〔７〕
工程（２）の超臨界流体抽出は、超臨界流体クロマトグラフィーまたは超臨界流体抽出装
置を用いる〔２〕から〔６〕のいずれかに記載の製造方法。
〔８〕
超臨界流体クロマトグラフィーは、超臨界流体として二酸化炭素を用い、エントレーナー
としてエタノールを用いて溶出させる〔７〕に記載の製造方法。
〔９〕
エタノールを用いた溶出が、エタノール濃度を５％から５０％の範囲で変化させる溶出で
ある〔８〕に記載の製造方法。
〔１０〕
エントレーナーを塩基性にして溶出させる〔８〕又は〔９〕に記載の製造方法。
〔１１〕
工程（２）の珪藻土若しくは活性白土への吸着を利用した抽出は、加水分解溶液を珪藻土
若しくは活性白土を含むエタノール水溶液に分散し、不溶物を珪藻土若しくは活性白土に
吸着させることによる抽出である〔２〕又は〔３〕に記載の製造方法。
〔１２〕
エタノール水溶液のエタノール濃度は、６０％以下である〔１１〕に記載の製造方法。
〔１３〕
スフィンゴ脂質含有素材は、動物由来、植物由来又は微生物由来である〔１〕〜〔１２〕
のいずれかに記載の製造方法。
〔１４〕
スフィンゴイド塩基が、ジヒドロスフィンゴシン、スフィンゴシン、N,N−ジメチルスフ
ィンゴシン、フィトスフィンゴシン、４−スフィンゲニン、８−スフィンゲニン、４−ヒ
ドロキシ−８−スフィンゲニン、４，８−スフィンガジエニン、４−ヒドロキシ−８−ス
フィンゲニン、９−メチル−４，８−スフィンガジエニン、４，８，１０−スフィンガト
リエニン、９−メチル−４，８，１０−スフィンガトリエニン、及び、アミノ基が１つ、
ヒドロキシ基が２若しくは３つ結合し、二重結合が一つ以上若しくは存在しない構造を有
するスフィンゴイド塩基、からなる群から選ばれる一種以上である〔１〕〜〔１３〕のい
ずれかに記載の製造方法。
〔１５〕
副生成物としてエタノールとスフィンゴイド塩基のエーテル化合物を生成する〔１〕〜〔
１４〕に記載の製造方法。
〔１６〕
副生成物であるエーテル化合物の総和が、スフィンゴイド塩基の生成量１に対して０．１
以下である〔１〕〜〔１５〕に記載の製造方法。
〔１７〕
〔１〕〜〔１６〕のいずれかに記載の製造方法により得られたスフィンゴイド塩基を含む
抽出物を添加する工程を含む飲食品の製造方法。
〔１８〕
〔１〕〜〔１６〕のいずれかに記載の製造方法により得られたスフィンゴイド塩基を含む
抽出物。
〔１９〕
〔１〕〜〔１６〕のいずれかに記載の製造方法により得られたスフィンゴイド塩基を含む
抽出物を含む飲食品。
As a result of diligent investigation, as a food material by combining chemical hydrolysis with acid and ethanol or ethanol aqueous solution, and purification by ion exchange resin, supercritical fluid extraction, adsorption on diatomaceous earth and activated clay, etc. It was discovered that sphingoid bases can be extracted and purified by a method that can be used, and the present invention has been completed.
That is, the present invention has the following configuration.
[1]
The manufacturing method of the extract containing a sphingoid base including the process of following (1).
(1) A step of dissolving or dispersing a sphingolipid-containing material in 0.001 to 10 M acidic ethanol or an acidic ethanol aqueous solution, and heating it at 30 ° C. to 100 ° C. for 30 minutes to several days for acid hydrolysis [2]
Furthermore, the manufacturing method as described in [1] including the process of following (2).
(2) One or more extractions from the hydrolyzate obtained in step (1) by extraction with an ion exchange resin, extraction with a supercritical fluid, and extraction using adsorption on diatomaceous earth or activated clay. Step of obtaining sphingoid base [3]
The production method according to [1] or [2], wherein the acid used in the acid hydrolysis in step (1) is at least one selected from acids such as hydrochloric acid, sulfuric acid, acetic acid, citric acid, lactic acid, nitric acid, and fumaric acid. .
[4]
The ion exchange resin of a process (2) is a manufacturing method in any one of [1]-[3] which is a cation exchange resin.
[5]
The elution from the cation exchange resin is carried out by changing the salt concentration and / or pH [4].
[6]
The elution from the cation exchange resin is performed by an eluate using a solution having an ionic strength of 0.00001 to 1 with an electrolyte such as NaCl, [4] or [5].
[7]
The supercritical fluid extraction in the step (2) is the production method according to any one of [2] to [6] using supercritical fluid chromatography or a supercritical fluid extraction device.
[8]
Supercritical fluid chromatography uses the carbon dioxide as the supercritical fluid and elutes with ethanol as the entrainer [7].
[9]
The production method according to [8], wherein the elution using ethanol is an elution in which the ethanol concentration is changed in the range of 5% to 50%.
[10]
The production method according to [8] or [9], wherein the entrainer is made basic and eluted.
[11]
The extraction using adsorption on diatomaceous earth or activated clay in step (2) is extraction by dispersing the hydrolysis solution in an ethanol aqueous solution containing diatomaceous earth or activated clay and adsorbing insoluble matter on diatomaceous earth or activated clay. [2] or [3].
[12]
The manufacturing method according to [11], wherein the ethanol concentration of the aqueous ethanol solution is 60% or less.
[13]
The sphingolipid-containing material is derived from animals, plants, or microorganisms [1] to [12]
The manufacturing method in any one of.
[14]
The sphingoid base is dihydrosphingosine, sphingosine, N, N-dimethylsphingosine, phytosphingosine, 4-sphingenin, 8-sphingenin, 4-hydroxy-8-sphingenin, 4,8-sphingadienin, 4-hydroxy-8 -Sphingenine, 9-methyl-4,8-sphingadienine, 4,8,10-sphingatrienine, 9-methyl-4,8,10-sphingatrienine and one amino group,
One or more selected from the group consisting of a sphingoid base having a structure in which two or three hydroxy groups are bonded and one or more double bonds are not present or any one of [1] to [13] Production method.
[15]
[1] to [1] to produce an ether compound of ethanol and sphingoid base as a by-product
14].
[16]
The sum total of ether compounds as by-products is 0.1 for the amount of sphingoid base produced 1.
The production method according to [1] to [15], which is as follows.
[17]
The manufacturing method of food-drinks including the process of adding the extract containing the sphingoid base obtained by the manufacturing method in any one of [1]-[16].
[18]
[1] An extract containing a sphingoid base obtained by the production method according to any one of [16].
[19]
A food or drink comprising an extract containing a sphingoid base obtained by the production method according to any one of [1] to [16].
本発明のスフィンゴイド塩基含有抽出物の製造方法によれば、食品素材として使用可能
な安全かつ簡便な方法によりスフィンゴイド塩基抽出物が得られる。したがって、本方法
により製造された抽出物は食品素材として様々な飲食品、機能性食品に使用できる。
According to the method for producing a sphingoid base-containing extract of the present invention, a sphingoid base extract can be obtained by a safe and simple method that can be used as a food material. Therefore, the extract produced by this method can be used as various foods and functional foods as food materials.
本発明のスフィンゴイド塩基を含む抽出物の製造方法を、以下に詳しく説明する。 The manufacturing method of the extract containing the sphingoid base of this invention is demonstrated in detail below.
本発明のスフィンゴイド塩基を含む抽出物の製造方法に使用する原料は、スフィンゴ脂
質を含む素材であれば特に限定されない。この素材の例としては、牛乳、ウシの脳、卵、
畜肉等の動物由来、小麦、トウモロコシ、豆等の植物由来または酵母等が生産する微生物
由来の素材が挙げられる。特に好ましくは、乳リン脂質濃縮物である。
乳リン脂質濃縮物としては、例えば、乳やホエータンパク質濃縮物（ＷＰＣ）等の乳製
品をエーテルやアセトンで抽出する方法（特開平３−４７１９２号公報）により得られる
乳リン脂質含有画分、バターを加温融解することで得られるバターカードやバターセーラ
ムを含む水性画分、また、バターミルクやバターセーラム中に含まれる乳脂肪球皮膜画分
が挙げられる。そして、これらのリン脂質画分を透析、硫安分画、ゲル濾過、等電点沈殿
、イオン交換クロマトグラフィー、溶媒分画等の手法により精製することにより純度を高
めたリン脂質濃縮物を利用することができる。また、乳リン脂質濃縮物として市販品を利
用することもでき、ミルクセラミドＭＣ−５（雪印メグミルク株式会社製）、ＳＭ−２（
コールマン社製）が挙げられる。
The raw material used for the manufacturing method of the extract containing the sphingoid base of this invention will not be specifically limited if it is a raw material containing a sphingolipid. Examples of this material are milk, bovine brain, eggs,
Examples include materials derived from animals such as livestock meat, plants derived from wheat, corn, beans, or microorganisms produced by yeasts. Particularly preferred is milk phospholipid concentrate.
As the milk phospholipid concentrate, for example, a milk phospholipid-containing fraction obtained by a method of extracting dairy products such as milk and whey protein concentrate (WPC) with ether or acetone (JP-A-3-47192), Examples thereof include an aqueous fraction containing butter curd and butter serum obtained by heating and melting butter, and a milk fat globule membrane fraction contained in butter milk and butter serum. Then, a phospholipid concentrate whose purity is increased by purifying these phospholipid fractions by a method such as dialysis, ammonium sulfate fractionation, gel filtration, isoelectric point precipitation, ion exchange chromatography, solvent fractionation, etc. is used. be able to. Commercially available products can also be used as milk phospholipid concentrate, such as milk ceramide MC-5 (manufactured by Snow Brand Megmilk Co., Ltd.), SM-2 (
Coleman)).
また、本発明で得られるスフィンゴイド塩基は、スフィンゴ塩基構造を有していればよ
く、例えば、ジヒドロスフィンゴシン、スフィンゴシン、N,N−ジメチルスフィンゴシン
、フィトスフィンゴシン、４−スフィンゲニン、８−スフィンゲニン、４−ヒドロキシ−
８−スフィンゲニン、４，８−スフィンガジエニン、９−メチル−４，８−スフィンガジ
エニン、４，８，１０−スフィンガトリエニン、９−メチル−４，８，１０−スフィンガ
トリエニンが挙げられる。その他にも、アミノ基が１つ、ヒドロキシ基が２または３つ結
合し、二重結合が一つ以上もしくは存在しない構造を有するスフィンゴイド塩基でもよい
。すなわち、鎖長、ヒドロキシ基の位置や数、二重結合の位置や数が異なる、様々なスフ
ィンゴイド塩基を得ることができる。
In addition, the sphingoid base obtained in the present invention only has to have a sphingobase structure. For example, dihydrosphingosine, sphingosine, N, N-dimethylsphingosine, phytosphingosine, 4-sphingenin, 8-sphingenin, 4- Hydroxy
8-sphingenine, 4,8-sphingadienine, 9-methyl-4,8-sphingadienin, 4,8,10-sphingatrienin, 9-methyl-4,8,10-sphingatrie Nin is mentioned. In addition, it may be a sphingoid base having a structure in which one amino group, two or three hydroxy groups are bonded, and one or more double bonds are not present. That is, various sphingoid bases having different chain lengths, positions and numbers of hydroxy groups, and positions and numbers of double bonds can be obtained.
本発明のスフィンゴイド塩基を含む抽出物の製造方法は、少なくとも次の（１）の工程
を含み、さらに精製する工程を含む。また、精製する工程は、次の（２）の工程が望まし
い。
（１）スフィンゴ脂質含有素材を０．００１〜１０Ｍの酸性エタノール又は酸性エタノー
ル水溶液に溶解または分散し、３０℃から１００℃で３０分から数日間加熱して酸加水分
解する工程
（２）工程（１）により得られた加水分解溶液からイオン交換樹脂による抽出、超臨界流
体による抽出、及び珪藻土若しくは活性白土への吸着を利用した抽出からなる群のいずれ
か１以上の抽出によりスフィンゴイド塩基を得る工程
The method for producing an extract containing a sphingoid base of the present invention includes at least the following step (1) and further includes a purification step. Moreover, the process of the following (2) is desirable for the process to refine | purify.
(1) A step (2) step (1) wherein a sphingolipid-containing material is dissolved or dispersed in 0.001 to 10 M acidic ethanol or an acidic ethanol aqueous solution and heated at 30 to 100 ° C. for 30 minutes to several days. The step of obtaining a sphingoid base from the hydrolyzed solution obtained by the above method by extraction with an ion exchange resin, extraction with a supercritical fluid, and extraction using adsorption on diatomaceous earth or activated clay.
（１）の「スフィンゴ脂質含有素材を０．００１〜１０Ｍの酸性エタノール又は酸性エ
タノール水溶液に溶解または分散する」方法としては、前記スフィンゴ脂質含有素材から
まずエタノールを用いて、スフィンゴ脂質を含むエタノール可溶性成分を抽出し、次に得
られたエタノール可溶性成分に酸及び必要に応じて水を加えて、酸の最終濃度を０．００
１〜１０Ｍの酸性とする方法がある。他に、あらかじめ０．００１〜１０Ｍの酸性エタノ
ール水溶液を調整し、これにスフィンゴ脂質含有素材を溶解または分散させる方法がある
。
エタノールまたはエタノール水溶液を酸性とする酸としては、例えば塩酸、硫酸、酢酸
、クエン酸、乳酸、硝酸、フマル酸等が挙げられ、これから選ばれる少なくとも一種を用
いることができるが、より好ましくは塩酸である。
酸性エタノール水溶液を用いる場合の水の含有量は、５〜３０％が好ましく、７〜２０
％がより好ましく、１０〜１５％がよりいっそう好ましく、１０％がもっとも好ましい。
次に、このようにして調整した溶解液または分散液を、３０℃から１００℃で３０分から
数日間加熱して加水分解を行う。なお、加水分解後のアルカリ水溶液による中和の有無は
任意である。
As a method of (1) “dissolving or dispersing a sphingolipid-containing material in 0.001 to 10 M acidic ethanol or an acidic ethanol aqueous solution”, ethanol is first used from the sphingolipid-containing material and is soluble in ethanol containing sphingolipid. Extract the ingredients and then add acid and water as needed to the ethanol-soluble ingredients obtained to give a final acid concentration of 0.00.
There is a method of acidifying 1 to 10M. In addition, there is a method in which a 0.001 to 10 M acidic ethanol aqueous solution is prepared in advance, and a sphingolipid-containing material is dissolved or dispersed therein.
Examples of the acid that makes ethanol or ethanol aqueous solution acidic include hydrochloric acid, sulfuric acid, acetic acid, citric acid, lactic acid, nitric acid, fumaric acid, and the like. At least one selected from these can be used, but hydrochloric acid is more preferable. is there.
The content of water in the case of using an acidic ethanol aqueous solution is preferably 5 to 30%, and 7 to 20
% Is more preferred, 10-15% is even more preferred, and 10% is most preferred.
Next, the thus-prepared solution or dispersion is hydrolyzed by heating at 30 to 100 ° C. for 30 minutes to several days. In addition, the presence or absence of neutralization with the aqueous alkali solution after hydrolysis is arbitrary.
（２）の工程は、（１）の工程により得られた加水分解溶液を、イオン交換樹脂による
抽出、超臨界流体による抽出、超臨界流体クロマトグラフィーによる抽出、超臨界二酸化
炭素あるいは液体二酸化炭素による抽出、及び珪藻土若しくは活性白土への吸着を利用し
た抽出からなる群のいずれか１以上の抽出によりスフィンゴイド塩基を分画する工程であ
る。
イオン交換樹脂を用いる場合は、陽イオン交換樹脂が望ましく、溶出液は塩濃度および
／又はｐＨを変化させた溶液を用いる。場合によっては、塩の濃度を段階的に変化させ、
溶出する。溶出液の調製に用いる塩にはＮａやＫやＣａなどを含む塩を用いることができ
る。溶出液のイオン強度は０．００００１から１までの範囲において分画が可能である。
ＮａＣｌを溶出液の塩として用いる場合、スフィンゴイド塩基はＮａＣｌ濃度０．０１ｍ
Ｍから１Ｍまでに溶出される。
また、イオン交換樹脂に対する吸着をよくするために、電気透析を行うことも好ましい
。
In the step (2), the hydrolyzed solution obtained in the step (1) is extracted with an ion exchange resin, with a supercritical fluid, with a supercritical fluid chromatography, with supercritical carbon dioxide or liquid carbon dioxide. It is a step of fractionating sphingoid bases by extraction and extraction of any one or more of the group consisting of extraction using adsorption on diatomaceous earth or activated clay.
In the case of using an ion exchange resin, a cation exchange resin is desirable, and a solution in which the salt concentration and / or pH is changed is used as the eluent. In some cases, the salt concentration is gradually changed,
Elute. As the salt used for preparing the eluate, a salt containing Na, K, Ca or the like can be used. The ionic strength of the eluate can be fractionated in the range of 0.00001 to 1.
When using NaCl as the eluate salt, the sphingoid base has a NaCl concentration of 0.01 m.
Elutes from M to 1M.
Moreover, in order to improve the adsorption | suction with respect to an ion exchange resin, it is also preferable to perform electrodialysis.
超臨界抽出装置を用いる場合は、超臨界抽出装置または超臨界流体クロマトグラフィー
を用いる。超臨界抽出装置または超臨界流体クロマトグラフィーは、超臨界流体として二
酸化炭素を用い、エントレーナーとしてエタノールを用いる。超臨界流体クロマトグラフ
ィーでは、エタノールが５％から５０％の範囲でスフィンゴイド塩基が溶出される。
また、超臨界流体クロマトグラフィーでは、エントレーナーを塩基性にすることで分離
効率を高めることができる。
When a supercritical extraction device is used, a supercritical extraction device or supercritical fluid chromatography is used. The supercritical extraction apparatus or supercritical fluid chromatography uses carbon dioxide as the supercritical fluid and ethanol as the entrainer. In supercritical fluid chromatography, sphingoid bases are eluted with ethanol in the range of 5% to 50%.
In supercritical fluid chromatography, separation efficiency can be increased by making the entrainer basic.
超臨界二酸化炭素抽出装置あるいは液体二酸化炭素抽出装置を用いて抽出を行う場合は
、試料の前処理として、（１）の工程に以下の工程を加える。まず、（１）の工程により
得られた加水分解溶液から減圧濃縮によりエタノールを除去し、アルカリ性水溶液を添加
してｐＨを中性〜塩基性に調整する。次に、これを遠心分離にかけて油層、水層、ゲル状
の中間層の三層に分離し、ろ過により中間層を回収する。このとき、中間層の収率を上げ
るためには、ｐＨを７〜９に調整することが好ましい。回収した中間層は、酸性水溶液を
添加して抽出に適したｐＨに調整し、凍結乾燥を行う。抽出に適したｐＨとは、ｐＨ５〜
９であり、ｐＨ６．６〜８がより好ましく、ｐＨ６．６が特に好ましい。
超臨界二酸化炭素抽出あるいは液体二酸化炭素抽出を行う場合は、エントレーナーとし
てエタノールを使用する。また、超臨界二酸化炭素抽出を行う場合、系内の圧力は７．５
〜３０ＭＰa、特に１５〜２５ＭＰａが好ましく、温度は３２〜６０℃、特に４５〜６０
℃が好ましい。液体二酸化炭素抽出を行う場合、系内の圧力は５〜３０ＭＰａ、特に１５
〜２５ＭＰａが好ましく、温度は１０〜３０℃、特に１５〜２５℃が好ましい。スフィン
ゴイド塩基を高純度に抽出するためには、まず、エタノール比率が０〜５％の移動相で抽
出を行い、その後、エタノール比率を１０〜５０％の範囲で段階的に上げてスフィンゴイ
ド塩基濃縮画分を得る。
When performing extraction using a supercritical carbon dioxide extraction device or a liquid carbon dioxide extraction device, the following steps are added to the step (1) as pretreatment of the sample. First, ethanol is removed from the hydrolyzed solution obtained in the step (1) by concentration under reduced pressure, and an alkaline aqueous solution is added to adjust the pH from neutral to basic. Next, this is centrifuged to separate into three layers, an oil layer, an aqueous layer, and a gel-like intermediate layer, and the intermediate layer is recovered by filtration. At this time, it is preferable to adjust the pH to 7 to 9 in order to increase the yield of the intermediate layer. The recovered intermediate layer is adjusted to a pH suitable for extraction by adding an acidic aqueous solution and lyophilized. PH suitable for extraction is pH 5 to
9, pH 6.6 to 8 is more preferable, and pH 6.6 is particularly preferable.
When supercritical carbon dioxide extraction or liquid carbon dioxide extraction is performed, ethanol is used as an entrainer. When supercritical carbon dioxide extraction is performed, the pressure in the system is 7.5.
-30 MPa, particularly 15-25 MPa, and the temperature is 32-60 ° C, especially 45-60.
° C is preferred. When liquid carbon dioxide extraction is performed, the pressure in the system is 5 to 30 MPa, particularly 15
˜25 MPa is preferable, and the temperature is preferably 10 to 30 ° C., particularly preferably 15 to 25 ° C. In order to extract sphingoid base with high purity, first, extraction is performed with a mobile phase having an ethanol ratio of 0 to 5%, and then the ethanol ratio is gradually increased within a range of 10 to 50% to increase the sphingoid base. A concentrated fraction is obtained.
超臨界流体クロマトグラフィー、超臨界二酸化炭素抽出、液体二酸化炭素抽出を行う場
合、サンプルを圧力容器に仕込む際、吸湿剤、緩衝材として珪藻土や海砂を混合してもよ
い。また、これらの操作により得られた分離された抽出物は、スフィンゴイド塩基を主成
分として含む抽出物であり、エタノールや食塩が混在している場合は、揮発や脱塩操作に
より除くことによって、より純度の高いスフィンゴイド塩基含有抽出物が得られる。
When supercritical fluid chromatography, supercritical carbon dioxide extraction, or liquid carbon dioxide extraction is performed, diatomaceous earth or sea sand may be mixed as a moisture absorbent and a buffer material when the sample is charged into a pressure vessel. In addition, the separated extract obtained by these operations is an extract containing sphingoid base as a main component, and when ethanol and sodium chloride are mixed, by removing by volatilization or desalting operation, A sphingoid base-containing extract with higher purity is obtained.
吸着法を用いた抽出法では、珪藻土や活性白土等の吸着作用のある物質を使用できる。
吸着作用のある物質であれば、珪藻土や活性白土に限らず使用できる。珪藻土を用いる抽
出の場合は、加水分解溶液を珪藻土を含むエタノール水溶液に分散し、不溶物を含む溶解
性の低い物質を珪藻土に吸着させる。珪藻土に不溶物を吸着させることで、スフィンゴイ
ド塩基を含む抽出物と夾雑物が分離できる。この場合のエタノール水溶液のエタノール濃
度は６０％以下の範囲がより好ましい。
スフィンゴイド塩基含有抽出物は、分離や精製度を高度に行った場合には、スフィンゴ
イド塩基を高純度で含むことから、本明細書中では特に断らない限りは、スフィンゴイド
類あるいはスフィンゴイド塩基と同義で用いるものとする。
In the extraction method using the adsorption method, a substance having an adsorption action such as diatomaceous earth or activated clay can be used.
Any substance having an adsorbing action can be used, not limited to diatomaceous earth and activated clay. In the case of extraction using diatomaceous earth, the hydrolysis solution is dispersed in an ethanol aqueous solution containing diatomaceous earth, and a low-solubility substance including insoluble matter is adsorbed on the diatomaceous earth. By adsorbing insoluble matter to diatomaceous earth, the extract containing sphingoid base and impurities can be separated. In this case, the ethanol concentration of the aqueous ethanol solution is more preferably 60% or less.
The sphingoid base-containing extract contains sphingoid bases with high purity when the degree of separation or purification is high, so unless otherwise specified in this specification, sphingoids or sphingoid bases are included. Shall be used interchangeably.
本発明の製造方法は、スフィンゴイド脂質を含有する食品素材から食品として許容でき
るような安全な方法により製造する方法であるから、本製造方法により得られたスフィン
ゴイド塩基含有抽出物は、様々な食品に使用できる。使用する食品に制限はない。例えば
、ヨーグルト、牛乳等の乳製品、ジュース、お茶等の飲料、菓子類、その他にも様々な飲
食品に使用できる。また、抽出物をそのままサプリメントとして、錠剤、カプセル剤、散
剤等に製剤することも可能である。
また、スフィンゴイド塩基が有する食品化学的活性、生理活性の利用を目的として、経
口医薬品、機能性表示食品、特定保健用食品、栄養機能食品、美容用食品として使用する
ことも可能である。
また、本発明の製造方法により得られたスフィンゴイド塩基含有抽出物は、動物用の飼
料や動物用の医薬品としても利用することが可能である。
以下に、実施例および試験例を示し、より詳細に説明するが、本発明はこれらによって
何ら限定されるものではない。
Since the production method of the present invention is a method for producing a sphingoid lipid-containing food material by a safe method that is acceptable as a food, the sphingoid base-containing extract obtained by this production method has various methods. Can be used for food. There are no restrictions on the food used. For example, it can be used for dairy products such as yogurt and milk, beverages such as juice and tea, confectionery, and various other foods and drinks. Moreover, it is also possible to formulate the extract as a supplement as it is in tablets, capsules, powders and the like.
In addition, for the purpose of utilizing food chemical activity and physiological activity possessed by sphingoid bases, it can also be used as oral pharmaceuticals, functional indication foods, foods for specified health use, nutritional functional foods, and beauty foods.
In addition, the sphingoid base-containing extract obtained by the production method of the present invention can be used as animal feed or animal medicine.
Hereinafter, examples and test examples will be shown and described in more detail, but the present invention is not limited thereto.
〔スフィンゴイド塩基含有抽出物の製造／イオン交換樹脂による抽出〕
１．スフィンゴイド塩基含有抽出物の製造方法
乳リン脂質濃縮素材であるミルクセラミドＭＣ−５（雪印メグミルク株式会社製）をエ
タノールに溶解し、エタノール可溶性画分を分取した。エタノール可溶性画分に塩酸を加
えて、０．２Ｍ塩酸／エタノール溶液とした。この塩酸／エタノール溶液を７５℃１２時
間加熱し、加水分解反応を行った。
反応液に水を４０％となるように添加し、水相を分取した。得られた水相を陽イオン交
換樹脂（Ｉｎｅｒｔ Ｓｅｐ ＰＲＳ、ＧＬサイエンス社製）に吸着させ、１００ｍＭの
ＮａＣｌを加えた溶出液にて溶出させた。得られた溶出液を膜処理にて脱塩し、エタノー
ルを揮発させることで、スフィンゴイド塩基抽出物が得られた。得られた抽出物の純度は
８０％であった。
[Production of extract containing sphingoid base / Extraction with ion exchange resin]
1. Production method of sphingoid base-containing extract Milk ceramide MC-5 (manufactured by Snow Brand Megmilk Co., Ltd.), a milk phospholipid concentrate material, was dissolved in ethanol, and an ethanol-soluble fraction was fractionated. Hydrochloric acid was added to the ethanol-soluble fraction to give a 0.2M hydrochloric acid / ethanol solution. This hydrochloric acid / ethanol solution was heated at 75 ° C. for 12 hours to carry out a hydrolysis reaction.
Water was added to the reaction solution so as to be 40%, and the aqueous phase was separated. The obtained aqueous phase was adsorbed on a cation exchange resin (Inert Sep PRS, manufactured by GL Science) and eluted with an eluate added with 100 mM NaCl. The obtained eluate was desalted by membrane treatment and ethanol was volatilized to obtain a sphingoid base extract. The purity of the obtained extract was 80%.
２．スフィンゴイド塩基含有抽出物の塩基組成
得られたスフィンゴイド塩基抽出物について、スフィンゴイド塩基組成を調べた。上記
１．により得られたスフィンゴイド塩基抽出物からクロロホルム／メタノール混合溶液に
て脂質を抽出した。得られたクロロホルム画分を窒素で濃縮後、トリメチルシリル（ＴＭ
Ｓ）化処理をした。ＴＭＳ化処理後の誘導体溶液をＧＣ−ＭＳに導入し、スフィンゴイド
塩基の組成を確認した。
乳リン脂質由来スフィンゴイド塩基のＧＣ−ＭＳクロマトグラムを図１に、各ピークの
同定結果を表１に示す。
2. Base composition of sphingoid base-containing extract The sphingoid base composition of the obtained sphingoid base extract was examined. Above 1. Lipids were extracted from the sphingoid base extract obtained by the above with a chloroform / methanol mixed solution. The resulting chloroform fraction was concentrated with nitrogen, and then trimethylsilyl (TM
S). The derivative solution after the TMS treatment was introduced into GC-MS, and the composition of the sphingoid base was confirmed.
The GC-MS chromatogram of milk phospholipid-derived sphingoid base is shown in FIG. 1, and the identification results of each peak are shown in Table 1.
〔スフィンゴイド塩基含有抽出物の製造／イオン交換樹脂による抽出〕
乳リン脂質濃縮素材であるミルクセラミドＭＣ−５（雪印メグミルク株式会社製）をエ
タノールに溶解し、エタノール可溶性画分を分取した。エタノール可溶性画分に塩酸と水
を加えて、水を１０％の割合で含む０．２Ｍ塩酸／エタノール水溶液とした。この塩酸／
含水エタノール溶液を７５℃２４時間加熱し、加水分解反応を行った。
反応液に水を４０％となるように添加して遠心分離し、油膜を除去することにより水相
を回収した。得られた水相について、実施例１と同様に陽イオン交換と脱塩処理を行い、
エタノールを揮発させることでスフィンゴイド塩基抽出物を得た。得られた抽出物の純度
は９７％であった。
[Production of extract containing sphingoid base / Extraction with ion exchange resin]
Milk ceramide MC-5 (manufactured by Snow Brand Megmilk Co., Ltd.), a milk phospholipid concentrate material, was dissolved in ethanol, and an ethanol-soluble fraction was collected. Hydrochloric acid and water were added to the ethanol-soluble fraction to give a 0.2 M hydrochloric acid / ethanol aqueous solution containing water at a ratio of 10%. This hydrochloric acid /
The hydrous ethanol solution was heated at 75 ° C. for 24 hours to perform a hydrolysis reaction.
Water was added to the reaction solution to 40% and centrifuged, and the oil phase was removed to recover the aqueous phase. The obtained aqueous phase was subjected to cation exchange and desalting as in Example 1,
A sphingoid base extract was obtained by volatilizing ethanol. The purity of the obtained extract was 97%.
〔スフィンゴイド塩基含有抽出物の製造／イオン交換樹脂による抽出〕
乳リン脂質濃縮素材であるミルクセラミドＭＣ−５（雪印メグミルク株式会社製）をエ
タノールに溶解し、エタノール可溶性画分を分取した。エタノール可溶性画分に塩酸を加
えて、０．００５Ｍ塩酸／エタノール溶液とした。この塩酸／エタノール溶液を１００℃
２４時間加熱し、加水分解反応を行った。
反応液に水を４０％となるように添加し、水相を分取した。得られた水相を陽イオン交
換樹脂（Ｉｎｅｒｔ Ｓｅｐ ＰＲＳ、ＧＬサイエンス社製）に吸着させ、１ＭのＮａＣ
ｌを加えた溶出液にて溶出させた。得られた溶出液を膜処理にて脱塩し、エタノールを揮
発させることで、スフィンゴイド塩基抽出物が得られた。得られた抽出物の純度は６０％
であった。
[Production of extract containing sphingoid base / Extraction with ion exchange resin]
Milk ceramide MC-5 (manufactured by Snow Brand Megmilk Co., Ltd.), a milk phospholipid concentrate material, was dissolved in ethanol, and an ethanol-soluble fraction was collected. Hydrochloric acid was added to the ethanol soluble fraction to make a 0.005 M hydrochloric acid / ethanol solution. This hydrochloric acid / ethanol solution
The mixture was heated for 24 hours to conduct a hydrolysis reaction.
Water was added to the reaction solution so as to be 40%, and the aqueous phase was separated. The obtained aqueous phase was adsorbed on a cation exchange resin (Inert Sep PRS, manufactured by GL Sciences), and 1M NaC.
Elution was performed with the eluate to which 1 was added. The obtained eluate was desalted by membrane treatment and ethanol was volatilized to obtain a sphingoid base extract. The purity of the extract obtained is 60%
Met.
〔スフィンゴイド塩基含有抽出物の製造／イオン交換樹脂による抽出〕
乳リン脂質濃縮素材であるミルクセラミドＭＣ−５（雪印メグミルク株式会社製）をエ
タノールに溶解し、エタノール可溶性画分を分取した。エタノール可溶性画分に塩酸を加
えて、１０Ｍ塩酸／エタノール溶液とした。この塩酸／エタノール溶液を３０℃１時間加
熱し、加水分解反応を行った。
反応液に水を４０％となるように添加し、水相を分取した。得られた水相を陽イオン交
換樹脂（Ｉｎｅｒｔ Ｓｅｐ ＰＲＳ、ＧＬサイエンス社製）に吸着させ、０．１ｍＭの
ＮａＣｌを加えた溶出液にて溶出させた。得られた溶出液を膜処理にて脱塩し、エタノー
ルを揮発させることで、スフィンゴイド塩基抽出物が得られた。得られた抽出物の純度は
６０％であった。
[Production of extract containing sphingoid base / Extraction with ion exchange resin]
Milk ceramide MC-5 (manufactured by Snow Brand Megmilk Co., Ltd.), a milk phospholipid concentrate material, was dissolved in ethanol, and an ethanol-soluble fraction was collected. Hydrochloric acid was added to the ethanol-soluble fraction to give a 10M hydrochloric acid / ethanol solution. This hydrochloric acid / ethanol solution was heated at 30 ° C. for 1 hour to carry out a hydrolysis reaction.
Water was added to the reaction solution so as to be 40%, and the aqueous phase was separated. The obtained aqueous phase was adsorbed on a cation exchange resin (Inert Sep PRS, manufactured by GL Science) and eluted with an eluate to which 0.1 mM NaCl was added. The obtained eluate was desalted by membrane treatment and ethanol was volatilized to obtain a sphingoid base extract. The purity of the obtained extract was 60%.
〔スフィンゴイド塩基含有抽出物の製造／イオン交換樹脂による抽出〕
乳リン脂質濃縮素材であるミルクセラミドＭＣ−５（雪印メグミルク株式会社製）をエ
タノールに溶解し、エタノール可溶性画分を分取した。エタノール可溶性画分に塩酸を加
えて、０．２Ｍ塩酸／エタノール溶液とした。この塩酸／エタノール溶液を７０℃１８時
間加熱し、加水分解反応を行った。
反応液に水を４０％となるように添加し、水相を分取した。水相から塩酸を除き、イオ
ン交換樹脂に対する吸着をよくするために、電気透析を実施した。機器は、マイクロアシ
ライザーＳ１（アストム社製）を用いた。電気透析後の反応液を陽イオン交換樹脂（Ｉｎ
ｅｒｔ Ｓｅｐ ＰＲＳ、ＧＬサイエンス社製）に吸着させ、５０ｍＭのＮａＣｌを加え
た溶出液にて溶出させた。得られた溶出液を膜処理にて脱塩し、エタノールを揮発させる
ことで、スフィンゴイド塩基抽出物が得られた。得られた抽出物の純度は８０％であった
。
[Production of extract containing sphingoid base / Extraction with ion exchange resin]
Milk ceramide MC-5 (manufactured by Snow Brand Megmilk Co., Ltd.), a milk phospholipid concentrate material, was dissolved in ethanol, and an ethanol-soluble fraction was collected. Hydrochloric acid was added to the ethanol-soluble fraction to give a 0.2M hydrochloric acid / ethanol solution. This hydrochloric acid / ethanol solution was heated at 70 ° C. for 18 hours to carry out a hydrolysis reaction.
Water was added to the reaction solution so as to be 40%, and the aqueous phase was separated. In order to remove hydrochloric acid from the aqueous phase and improve the adsorption to the ion exchange resin, electrodialysis was performed. The device used was a microacylizer S1 (manufactured by Astom). The reaction solution after electrodialysis is converted into cation exchange resin (In
ert Sep PRS, manufactured by GL Sciences Inc.) and eluted with an eluate containing 50 mM NaCl. The obtained eluate was desalted by membrane treatment and ethanol was volatilized to obtain a sphingoid base extract. The purity of the obtained extract was 80%.
〔スフィンゴイド塩基含有抽出物の製造／超臨界流体クロマトグラフィーによる抽出〕
実施例１と同様にして得られた加水分解反応液を超臨界抽出クロマトグラフィー装置（
ＡＣＱＵＩＴＹ ＵＰＣ２システム、Ｗａｔｅｒｓ社製）に供した。超臨界流体は二酸化
炭素を用い、エントレーナーはエタノールを用いた。カラムはＡＣＱＵＩＴＹ ＵＰＣ２
ＢＥＨ ２−Ｅｔｈｙｌｐｙｒｉｄｉｎｅ １．７μｍ，３．０×１００ｍｍ（Ｗａｔ
ｅｒｓ社製）を用いた。流速は１．２ｍL／分、カラム温度は４０℃、サンプル注入量は
１μLであった。エントレーナーのエタノール濃度を１０％から５０％まで変化させて溶
出した。なお、エントレーナーを塩基性にすることで、さらによく分離できた。得られた
溶出物から二酸化炭素とエタノールを揮発させることで、スフィンゴイド塩基抽出物が得
られた。得られた抽出物の純度は９９％であった。
[Production of extract containing sphingoid base / Extraction by supercritical fluid chromatography]
The hydrolysis reaction solution obtained in the same manner as in Example 1 was subjected to supercritical extraction chromatography (
ACQUITY UPC2 system, manufactured by Waters). Carbon dioxide was used as the supercritical fluid, and ethanol was used as the entrainer. Column is ACQUITY UPC2
BEH 2-Ethylpyridine 1.7 μm, 3.0 × 100 mm (Wat
ers). The flow rate was 1.2 mL / min, the column temperature was 40 ° C., and the sample injection volume was 1 μL. Elution was performed by changing the ethanol concentration of the entrainer from 10% to 50%. In addition, it was able to isolate | separate further better by making an entrainer basic. The sphingoid base extract was obtained by volatilizing carbon dioxide and ethanol from the obtained eluate. The purity of the obtained extract was 99%.
〔スフィンゴイド塩基含有抽出物の製造／超臨界二酸化炭素による抽出〕
実施例１と同様にして得られた加水分解液から減圧濃縮によりエタノールを除去した後
、ＮａＯＨ水溶液を加えてｐＨ ８に調整した。これを遠心分離により三層に分離し、ろ
過によりゲル状の中間層を回収し、凍結乾燥して超臨界二酸化炭素抽出に供した。超臨界
二酸化炭素抽出は、エントレーナーにエタノールを使用し、圧力は２５ ＭＰａ、温度は
６０℃、移動相の流速１０ｍＬ／分で実施した。まず、低極性の夾雑物を除去するために
、エタノール導入率を０〜５％に設定し、抽出を行った。その後、エタノール導入率を１
０〜２０％の範囲で段階的に上げて抽出を行い、得られた溶出物から二酸化炭素とエタノ
ールを揮発させることで、スフィンゴイド塩基抽出物が得られた。スフィンゴイド塩基の
純度は、エタノール１０％の溶出画分が最も高く、８０％であった。
[Production of extract containing sphingoid base / extraction with supercritical carbon dioxide]
After removing ethanol from the hydrolyzed solution obtained in the same manner as in Example 1 by concentration under reduced pressure, an aqueous NaOH solution was added to adjust the pH to 8. This was separated into three layers by centrifugation, the gel-like intermediate layer was recovered by filtration, freeze-dried, and subjected to supercritical carbon dioxide extraction. Supercritical carbon dioxide extraction was carried out using ethanol as an entrainer, a pressure of 25 MPa, a temperature of 60 ° C., and a mobile phase flow rate of 10 mL / min. First, in order to remove low-polar impurities, the ethanol introduction rate was set to 0 to 5%, and extraction was performed. After that, the ethanol introduction rate is 1
Extraction was carried out stepwise in the range of 0 to 20%, and the sphingoid base extract was obtained by volatilizing carbon dioxide and ethanol from the obtained eluate. The purity of sphingoid base was the highest in the elution fraction of ethanol 10%, which was 80%.
〔スフィンゴイド塩基含有抽出物の製造／液体二酸化炭素による抽出〕
実施例１と同様にして得られた加水分解液を減圧濃縮にかけてエタノールを除去した後
、ＮａＯＨ水溶液を加えてｐＨ ８に調整した。これを遠心分離により三層に分離し、ろ
過によりゲル状の中間層を回収した。これにＨＣｌ水溶液を加えてｐＨ ６．６に調整し
、凍結乾燥したものを液体二酸化炭素抽出に供した。液体二酸化炭素抽出は、エントレー
ナーにエタノールを使用し、圧力は２５ ＭＰａ、温度は２５℃、移動相の流速は１０ｍ
Ｌ／分で実施した。まず、低極性の夾雑物を除去するために、エタノール導入率を０〜５
％に設定し、抽出を行った。その後、エタノール導入率を１０〜２０％の範囲で段階的に
上げて抽出を行い、得られた溶出物から二酸化炭素とエタノールを揮発させることで、ス
フィンゴイド塩基抽出物が得られた。スフィンゴイド塩基の純度は、エタノール１５％の
画分が最も高く、７０％であった。
[Production of extract containing sphingoid base / Extraction with liquid carbon dioxide]
The hydrolyzate obtained in the same manner as in Example 1 was concentrated under reduced pressure to remove ethanol, and then an aqueous NaOH solution was added to adjust the pH to 8. This was separated into three layers by centrifugation, and a gel-like intermediate layer was recovered by filtration. An aqueous HCl solution was added thereto to adjust the pH to 6.6, and the lyophilized product was subjected to liquid carbon dioxide extraction. Liquid carbon dioxide extraction uses ethanol as an entrainer, pressure is 25 MPa, temperature is 25 ° C., mobile phase flow rate is 10 m
Performed at L / min. First, in order to remove low-polar impurities, the ethanol introduction rate is set to 0-5.
Extraction was performed with the setting set to%. Thereafter, extraction was carried out stepwise in the range of 10 to 20% in ethanol, and carbon dioxide and ethanol were volatilized from the obtained eluate to obtain a sphingoid base extract. The purity of sphingoid base was the highest in the 15% ethanol fraction, which was 70%.
〔スフィンゴイド塩基含有抽出物の製造／珪藻土による抽出〕
加水分解反応液を、珪藻土を用いた抽出に供した。
実施例１と同様にして得られた加水分解反応液を４０％エタノール水溶液となるように
水で希釈し、これを珪藻土（関東化学製）に分散させて、攪拌して一晩反応させた後、ろ
過により珪藻土とろ液を分離した。その結果、ろ液中にスフィンゴイド塩基が存在した。
ろ液中のエタノールを揮発させることで、スフィンゴイド塩基抽出物が得られた。得られ
た抽出物の純度は４０％であった。
[Production of extract containing sphingoid base / Extraction with diatomaceous earth]
The hydrolysis reaction solution was subjected to extraction using diatomaceous earth.
After the hydrolysis reaction solution obtained in the same manner as in Example 1 was diluted with water so as to be a 40% aqueous ethanol solution, this was dispersed in diatomaceous earth (manufactured by Kanto Chemical Co., Inc.), and stirred and reacted overnight. The diatomaceous earth and the filtrate were separated by filtration. As a result, sphingoid base was present in the filtrate.
A sphingoid base extract was obtained by volatilizing ethanol in the filtrate. The purity of the obtained extract was 40%.
〔スフィンゴイド塩基含有抽出物の製造／珪藻土による抽出〕
加水分解溶液を５％エタノール溶液となるように水で希釈した以外は、実施例７と同様
に珪藻土を用いてスフィンゴイド塩基抽出物を得た。得られた抽出物の純度は３０％であ
った。
[Production of extract containing sphingoid base / Extraction with diatomaceous earth]
A sphingoid base extract was obtained using diatomaceous earth in the same manner as in Example 7 except that the hydrolysis solution was diluted with water so as to be a 5% ethanol solution. The purity of the obtained extract was 30%.
〔スフィンゴイド塩基含有抽出物の製造／珪藻土による抽出〕
加水分解溶液を７０％エタノール溶液となるように水で希釈した以外は、実施例７と同
様に珪藻土を用いてスフィンゴイド塩基抽出物を得た。得られた抽出物の純度は１０％で
あった。
[Production of extract containing sphingoid base / Extraction with diatomaceous earth]
A sphingoid base extract was obtained using diatomaceous earth in the same manner as in Example 7 except that the hydrolysis solution was diluted with water so as to be a 70% ethanol solution. The purity of the obtained extract was 10%.
〔副生成物の検討〕
特許文献２や特許文献３などで知られている従来のスフィンゴ脂質のメタノールを用い
た加水分解では、副生成物が生起することが報告されているため（Chemistry and Physic
s of Lipids 5巻，1970）、対して本願のエタノールを用いた加水分解における副生成物
の生起について検討を行った。
[Examination of by-products]
By-products have been reported to occur in the conventional hydrolysis of sphingolipids known in Patent Document 2 and Patent Document 3 using methanol (Chemistry and Physic
s of Lipids 5 (1970)), and the occurrence of by-products in the hydrolysis using ethanol of the present application was examined.
１．試験方法
１−１．加水分解反応
乳リン脂質濃縮素材であるミルクセラミドＭＣ−５（雪印メグミルク株式会社製）をエ
タノールに溶解し、エタノール可溶性画分を分取した。エタノール可溶性画分に塩酸を加
えて、０．２Ｍ塩酸／エタノール溶液とした。この塩酸／エタノール溶液を７５℃２４時
間加熱し、加水分解反応を行った。また、同量のミルクセラミドＭＣ−５を原料として、
上記の方法のエタノールをメタノールに置換した方法で加水分解反応を行った。
１−２．副生成物の同定
得られた加水分解物からクロロホルム／メタノール混合溶液にて脂質を抽出し、窒素で
濃縮後、薄層クロマトグラフィーにより成分を分離した。薄層クロマトグラフィーの展開
溶媒にはクロロホルム／アセトン／メタノール／酢酸／水＝１０／４／２／２／１を用い
た。展開後のプレートを風乾し、プリムリン試薬を噴霧してUV照射によりスポットを検出
した。スポットをかきとってクロロホルム／メタノール混合溶媒により抽出し、インフュ
ージョン法によりＬＣ−ＭＳ／ＭＳに導入して化合物を同定した。
１−３．副生成物の定量
上記と同様にして、薄層クロマトグラフィーにより成分を分離した。展開後のプレート
を風乾し、硫酸を噴霧して加熱することによりスポットを検出した。副生成物の定量はデ
ンシトメトリー法により行った。
２．試験結果
加水分解反応後の混合物について、スフィンゴイド塩基の生成量１に対して、エーテル
化合物の総和の生成量を表２に示す。本結果によれば、メタノールを用いた加水分解は、
報告のとおり副生成物が０．４も発生したが、これに対して本願のエタノールを用いた加
水分解では０．１にとどまった。一方で、両試験間では、スフィンゴイド塩基の収量には
差がなかった。
定性試験の結果、スフィンゴイド塩基の調製にエタノールを用いた場合の副生成物は図
２に示すエーテル化合物であった。一方、スフィンゴイド塩基の調製にメタノールを用い
た場合の副生成物は図３に示すエーテル化合物であった。なお、スフィンゴイド塩基の調
製にエタノールを用いた場合の副生成物中には図３は検出されなかった。
1. Test method 1-1. Hydrolysis reaction Milk ceramide MC-5 (manufactured by Snow Brand Megmilk Co., Ltd.), which is a milk phospholipid concentrate material, was dissolved in ethanol, and an ethanol-soluble fraction was collected. Hydrochloric acid was added to the ethanol-soluble fraction to give a 0.2M hydrochloric acid / ethanol solution. This hydrochloric acid / ethanol solution was heated at 75 ° C. for 24 hours to carry out a hydrolysis reaction. Also, using the same amount of milk ceramide MC-5 as a raw material,
The hydrolysis reaction was carried out by replacing the ethanol in the above method with methanol.
1-2. Identification of by-products Lipids were extracted from the obtained hydrolyzate with a chloroform / methanol mixed solution, concentrated with nitrogen, and components were separated by thin-layer chromatography. Chloroform / acetone / methanol / acetic acid / water = 10/4/2/2/1 was used as a developing solvent for thin layer chromatography. The plate after development was air-dried, sprayed with a primulin reagent, and spots were detected by UV irradiation. The spot was spotted and extracted with a mixed solvent of chloroform / methanol and introduced into LC-MS / MS by an infusion method to identify the compound.
1-3. Determination of by-products In the same manner as described above, the components were separated by thin layer chromatography. The developed plate was air-dried, and spots were detected by spraying sulfuric acid and heating. The by-product was quantified by densitometry.
2. Test results Table 2 shows the total production amount of ether compounds with respect to the production amount 1 of sphingoid bases for the mixture after the hydrolysis reaction. According to this result, hydrolysis with methanol is
As reported, by-products of 0.4 were generated, but in contrast, hydrolysis using ethanol of the present application remained at 0.1. On the other hand, there was no difference in sphingoid base yield between the two tests.
As a result of the qualitative test, the by-product when ethanol was used for the preparation of the sphingoid base was the ether compound shown in FIG. On the other hand, the by-product when methanol was used for the preparation of the sphingoid base was the ether compound shown in FIG. In addition, FIG. 3 was not detected in the by-product at the time of using ethanol for preparation of a sphingoid base.
〔副生成物の脂質酸化に及ぼす影響評価〕
１．試験方法
実施例１０で確認された副生成物の主成分であるエーテル化合物（図２、又は図３に示
す化合物のいずれかひとつ）１ｍｇを魚油９９ｍｇと混合し供試試料とした。魚油は高度
不飽和脂肪酸であるＤＨＡを１０％含有するものである。
供試試料を分析用バイアル瓶(５ｍＬ)に精秤した後、ブチルセプタムゴムおよびアルミ
シールバイアルで栓をした。５０℃、暗所にてインキュベートした後、一定時間ごとにバ
イアル瓶上部の空気４０μＬを採取して、熱伝導度検出器(ＴＣＤ)を備えたガスクロマト
グラフ装置に注入し、酸素および窒素量を測定した。酸素と窒素のピーク比の変化により
魚油の酸化による酸素消費量を算出した。
保存５０時間後の酸素残存量（％）を表３に示した。
[ＧＣの分析条件]
装置： 島津ＧＣ−１４Ｂ型ガスクロマトグラフ[島津製作所(株)]、インテグレーター：
島津 Ｃ−Ｒ８Ａ 型クロマトデータ処理装置[島津製作所(株)]、電圧機：ＡＭＰ−７Ｂ[
島津製作所(株)] 、検出器：ＴＣＤ、カラム：Ｍｏｌｅｃｕｌａｒ ｓｉｅｖｅｓ−５Ａ(
６０/８０ｍｅｓｈ;３ミリ)、カラム温度：５０℃、注入口温度：１００℃、検出口温度
：１００℃、キャリアガス：ヘリウムガス、ヘリウム圧：５０ｋＰａ
[Evaluation of effects of by-products on lipid oxidation]
1. Test Method 1 mg of an ether compound (any one of the compounds shown in FIG. 2 or FIG. 3), which is the main component of the by-product confirmed in Example 10, was mixed with 99 mg of fish oil to prepare a test sample. Fish oil contains 10% DHA, which is a highly unsaturated fatty acid.
The test sample was precisely weighed into an analytical vial (5 mL), and then capped with a butyl septum rubber and an aluminum sealed vial. After incubating in the dark at 50 ° C, 40 μL of air at the top of the vial is collected at regular intervals and injected into a gas chromatograph equipped with a thermal conductivity detector (TCD) to measure the amount of oxygen and nitrogen did. Oxygen consumption due to oxidation of fish oil was calculated from changes in the peak ratio of oxygen and nitrogen.
Table 3 shows the oxygen remaining amount (%) after 50 hours of storage.
[GC analysis conditions]
Apparatus: Shimadzu GC-14B gas chromatograph [Shimadzu Corporation], integrator:
Shimadzu C-R8A type chromatographic data processor [Shimadzu Corporation], voltage machine: AMP-7B [
Shimadzu Corporation], detector: TCD, column: Molecular sieves-5A (
60/80 mesh; 3 mm), column temperature: 50 ° C., inlet temperature: 100 ° C., detection port temperature: 100 ° C., carrier gas: helium gas, helium pressure: 50 kPa
２．試験結果
表３に示した各試料はいずれも経時的に酸化が進行し、バイアル瓶上部に残存する酸素
量が減少した。
５０時間後の酸素残存量は、副生成物であるエーテル化合物を添加した試料の方が魚油
のみの試料よりも少なかったことから、エーテル化合物には高度不飽和脂肪酸含有油脂で
ある魚油の酸化を促進する作用があることが確認された。なお、エタノール由来のエーテ
ル化合物とメタノール由来のエーテル化合物の酸素残存量は同等であったことから、これ
らの化合物の酸化促進作用はほぼ同等であると考えられた。
スフィンゴイド塩基を食品に配合する際には、油脂が共存する場合が多く、油脂酸化の
進行は異風味等の品質劣化の原因となる。
したがって、本願のエタノールを溶剤としてスフィンゴイド塩基を調製する方法は、従
来法であるメタノールを溶剤として調製する方法と比較して、油脂を酸化させる因子であ
るエーテル化合物の生成が少ないことから、添加する食品の劣化防止に有利である。
2. Test results Each sample shown in Table 3 was oxidized over time, and the amount of oxygen remaining in the upper portion of the vial decreased.
Since the amount of oxygen remaining after 50 hours was less in the sample to which the ether compound as a by-product was added than in the sample containing only fish oil, the ether compound was oxidized with fish oil, which is a highly unsaturated fatty acid-containing fat. It was confirmed that there was an action to promote. In addition, since the oxygen residual amount of the ether compound derived from ethanol and the ether compound derived from methanol was equivalent, it was thought that the oxidation promotion effect | action of these compounds was substantially equivalent.
When a sphingoid base is blended in a food, fats and oils often coexist, and the progress of the oxidation of fats and oils causes quality deterioration such as an unusual flavor.
Therefore, the method of preparing a sphingoid base using ethanol as a solvent of the present application is less likely to produce an ether compound that is a factor that oxidizes fats and oils than the conventional method of preparing methanol as a solvent. This is advantageous for preventing deterioration of food.
本発明の製造方法により得られたスフィンゴイド塩基抽出物は、食品化学的活性、生理
活性を有するので、その効果を得る目的で、飲食品に配合したり、サプリメント等の有効
成分として利用したりすることができる。また、油脂を酸化させる副生成物の含量が少な
いことから、油脂を含む飲食品に配合する添加剤として望ましく利用することができる。
Since the sphingoid base extract obtained by the production method of the present invention has food chemical activity and physiological activity, it can be blended into foods and drinks or used as an active ingredient such as a supplement for the purpose of obtaining its effect. can do. Moreover, since there is little content of the by-product which oxidizes fats and oils, it can utilize desirably as an additive mix | blended with the food-drinks containing fats and oils.
Claims (19)
（１）スフィンゴ脂質含有素材を、酸濃度を０．００１〜１０Ｍとしたエタノール又はエタノール水溶液に溶解または分散し、３０℃〜１００℃で３０分〜数日間加熱して酸加水分解する工程 The manufacturing method of the extract containing a sphingoid base including the process of following (1).
(1) A step of dissolving or dispersing a sphingolipid-containing material in ethanol or an aqueous ethanol solution having an acid concentration of 0.001 to 10 M, and heating it at 30 ° C. to 100 ° C. for 30 minutes to several days for acid hydrolysis.
（２）工程（１）により得られた加水分解物からイオン交換樹脂による抽出、超臨界流体による抽出、及び珪藻土若しくは活性白土への吸着を利用した抽出からなる群のいずれか１以上の抽出によりスフィンゴイド塩基を得る工程 Furthermore, the manufacturing method of Claim 1 including the process of following (2).
(2) One or more extractions from the hydrolyzate obtained in step (1) by extraction with an ion exchange resin, extraction with a supercritical fluid, and extraction using adsorption on diatomaceous earth or activated clay. Step of obtaining sphingoid base
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