JPWO2019009130A1 - ガラクトオリゴ糖の製造方法 - Google Patents
ガラクトオリゴ糖の製造方法 Download PDFInfo
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- JPWO2019009130A1 JPWO2019009130A1 JP2019527643A JP2019527643A JPWO2019009130A1 JP WO2019009130 A1 JPWO2019009130 A1 JP WO2019009130A1 JP 2019527643 A JP2019527643 A JP 2019527643A JP 2019527643 A JP2019527643 A JP 2019527643A JP WO2019009130 A1 JPWO2019009130 A1 JP WO2019009130A1
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- galactosidase
- reaction
- galacto
- sugars
- concentration
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- 235000021255 galacto-oligosaccharides Nutrition 0.000 title claims abstract description 53
- 150000003271 galactooligosaccharides Chemical class 0.000 title claims abstract description 53
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 41
- 108010005774 beta-Galactosidase Proteins 0.000 claims abstract description 61
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 claims abstract description 30
- 229910001425 magnesium ion Inorganic materials 0.000 claims abstract description 30
- 229910001415 sodium ion Inorganic materials 0.000 claims abstract description 26
- 239000000758 substrate Substances 0.000 claims abstract description 20
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 17
- 102000005936 beta-Galactosidase Human genes 0.000 claims abstract 3
- 244000005700 microbiome Species 0.000 claims description 27
- 238000006243 chemical reaction Methods 0.000 abstract description 41
- 102100026189 Beta-galactosidase Human genes 0.000 description 57
- 239000000243 solution Substances 0.000 description 32
- 235000000346 sugar Nutrition 0.000 description 30
- 150000008163 sugars Chemical class 0.000 description 27
- 210000004027 cell Anatomy 0.000 description 24
- 102000004190 Enzymes Human genes 0.000 description 23
- 108090000790 Enzymes Proteins 0.000 description 23
- 229940088598 enzyme Drugs 0.000 description 23
- 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 22
- 239000008101 lactose Substances 0.000 description 22
- 230000000694 effects Effects 0.000 description 20
- 125000002519 galactosyl group Chemical group C1([C@H](O)[C@@H](O)[C@@H](O)[C@H](O1)CO)* 0.000 description 12
- 230000000813 microbial effect Effects 0.000 description 12
- 238000011282 treatment Methods 0.000 description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Inorganic materials [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 230000035484 reaction time Effects 0.000 description 8
- 150000002016 disaccharides Chemical class 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 238000006276 transfer reaction Methods 0.000 description 6
- KUWPCJHYPSUOFW-YBXAARCKSA-N 2-nitrophenyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CC=CC=C1[N+]([O-])=O KUWPCJHYPSUOFW-YBXAARCKSA-N 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 241000235649 Kluyveromyces Species 0.000 description 5
- 239000000370 acceptor Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000010517 secondary reaction Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 241000186000 Bifidobacterium Species 0.000 description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 239000008188 pellet Substances 0.000 description 4
- 150000004043 trisaccharides Chemical class 0.000 description 4
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 3
- 241000193830 Bacillus <bacterium> Species 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241001138401 Kluyveromyces lactis Species 0.000 description 3
- 241000186660 Lactobacillus Species 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 239000012295 chemical reaction liquid Substances 0.000 description 3
- 238000011033 desalting Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 229940039696 lactobacillus Drugs 0.000 description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 description 3
- 241000186012 Bifidobacterium breve Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 235000014663 Kluyveromyces fragilis Nutrition 0.000 description 2
- 241000235650 Kluyveromyces marxianus Species 0.000 description 2
- 235000013960 Lactobacillus bulgaricus Nutrition 0.000 description 2
- 241000186672 Lactobacillus delbrueckii subsp. bulgaricus Species 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 241000194017 Streptococcus Species 0.000 description 2
- 241000194020 Streptococcus thermophilus Species 0.000 description 2
- 150000001242 acetic acid derivatives Chemical class 0.000 description 2
- 238000005377 adsorption chromatography Methods 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical group OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 150000004649 carbonic acid derivatives Chemical class 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 150000001805 chlorine compounds Chemical class 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 238000001641 gel filtration chromatography Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 229940004208 lactobacillus bulgaricus Drugs 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000002934 lysing effect Effects 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000005185 salting out Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- AYRXSINWFIIFAE-ONMPCKGSSA-N (2r,3s,4r,5r)-2,3,4,5-tetrahydroxy-6-[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyhexanal Chemical compound OC[C@H]1O[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)[C@H](O)[C@@H](O)[C@H]1O AYRXSINWFIIFAE-ONMPCKGSSA-N 0.000 description 1
- 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 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 239000005909 Kieselgur Substances 0.000 description 1
- 108010059881 Lactase Proteins 0.000 description 1
- 241001147746 Lactobacillus delbrueckii subsp. lactis Species 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 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
- -1 are produced Chemical class 0.000 description 1
- 125000001488 beta-D-galactosyl group Chemical group C1([C@H](O)[C@@H](O)[C@@H](O)[C@H](O1)CO)* 0.000 description 1
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 235000015140 cultured milk Nutrition 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000013681 dietary sucrose Nutrition 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 235000015094 jam Nutrition 0.000 description 1
- 229940116108 lactase Drugs 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 235000002867 manganese chloride Nutrition 0.000 description 1
- 229940099607 manganese chloride Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- HOVAGTYPODGVJG-UHFFFAOYSA-N methyl beta-galactoside Natural products COC1OC(CO)C(O)C(O)C1O HOVAGTYPODGVJG-UHFFFAOYSA-N 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 235000020138 yakult Nutrition 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
- C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
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- C12P19/12—Disaccharides
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C12P19/00—Preparation of compounds containing saccharide radicals
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- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
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- Polymers & Plastics (AREA)
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- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
Description
[β−ガラクトシダーゼ酵素活性(U)の測定法]
希釈酵素試料0.5mLを試験管に取り、0.1mMとなるように塩化マンガンを加えた100mMのKH2PO4−NaOH緩衝液(pH6.5、以下、「緩衝液」という)0.5mLを加えて混合した後、37℃で3分間保温する。あらかじめ37℃で保温しておいた0.1%のo−ニトロフェニル−β−D−ガラクトピラノシド(以下、「ONPG」という)溶液1.0mLを加えてすばやく混合し、正確に37℃で1分間保温する。0.2Mの炭酸ナトリウム溶液2.0mLを加えてすばやく混合し、反応を停止する(試験系)。別に、希釈酵素試料0.5mLを試験管に取り、緩衝液0.5mLを加えて混合した後、0.2Mの炭酸ナトリウム溶液2.0mLを加え、37℃で3分間保温し、あらかじめ37℃で保温しておいたONPG溶液0.1mLを加えて混合し、正確に37℃で1分間保温する(盲検系)。蒸留水を対照として試験系および盲検系の420nmの吸光度を測定し、次式により酵素活性(U)を算出した。
[数式1]
酵素活性*=(A1−A2)×10×B
A1:試験系の吸光度
A2:盲検系の吸光度
B :希釈倍率
* U/ml
100mL容三角フラスコに局方グレードの乳糖を15g秤量し、脱イオン水で調製した(ナトリウムイオンとマグネシウムイオンが含まれていない)Bis−Tris緩衝液(pH6.8)を85g加えた(乳糖濃度15%)。沸騰水浴中で乳糖を完全に溶解した後に、45℃の恒温水槽中で冷却した。これに2.6Mの塩化ナトリウムをナトリウムイオン濃度が15mMとなるように加え、また0.75Mの塩化マグネシウムをマグネシウムイオン濃度が下記表1に記載された濃度になるように加え、これらにGODO−YNL(クリベロマイセス・ラクチス由来のβ―ガラクトシダーゼ、合同酒精株式会社製)を乳糖1g当たり600U添加して40℃で反応させた。これら反応液を経時的に7時間目までサンプリングし、沸騰水浴中で90℃まで昇温して酵素を失活させた後、残2糖と3糖以上のガラクトオリゴ糖の割合を下記条件に基づくHPLC分析により測定した。各マグネシウムイオン濃度における3糖以上のガラクトオリゴ糖の生成量が最大となったときの測定結果を表1に示す。なお、残2糖には未反応の乳糖及び転移2糖が含まれる。
カラム:Shodex SUGAR KS−802
移動層:精製水
流 速:0.5mL/min
検 出:示差屈折計
2.6Mの塩化ナトリウムをナトリウムイオン濃度が30mMとなるように添加した以外は実施例1と同様にして残2糖と3糖以上のガラクトオリゴ糖の割合を測定した。結果を表2に示す。
反応液中の乳糖濃度を45%とし、2.6Mの塩化ナトリウムをナトリウムイオン濃度が5mMとなるように添加し、GODO−YNLを乳糖1g当たり250U添加して45℃で反応させた以外は実施例1と同様にして残2糖と3糖以上のガラクトオリゴ糖の割合を測定した。結果を表3に示す。
反応液中の乳糖濃度を45%とし、2.6Mの塩化ナトリウムをナトリウムイオン濃度が60mMとなるように添加し、GODO−YNLを乳糖1g当たり250U添加して45℃で反応させた以外は実施例1と同様にして残2糖と3糖以上のガラクトオリゴ糖の割合を測定した。結果を表4に示す。
Claims (3)
- 5〜60mMのナトリウムイオン及び0.5〜8mMのマグネシウムイオンの存在下で、β−ガラクトシダーゼを基質と反応させることを特徴とするガラクトオリゴ糖の製造方法。
- マグネシウムイオンの濃度が1.5〜8mMである請求項1記載の製造方法。
- β−ガラクトシダーゼが、クリベロマイセス属に属する微生物由来のものである請求項1又は2記載の製造方法。
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