KR102598193B1 - Manufacturing method of matobionic acid using enzymatic oxidation - Google Patents
Manufacturing method of matobionic acid using enzymatic oxidation Download PDFInfo
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- KR102598193B1 KR102598193B1 KR1020210076795A KR20210076795A KR102598193B1 KR 102598193 B1 KR102598193 B1 KR 102598193B1 KR 1020210076795 A KR1020210076795 A KR 1020210076795A KR 20210076795 A KR20210076795 A KR 20210076795A KR 102598193 B1 KR102598193 B1 KR 102598193B1
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- Prior art keywords
- maltobionic acid
- solution
- exchange resin
- saccharide
- maltose
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- 238000004519 manufacturing process Methods 0.000 title claims abstract description 87
- 238000007254 oxidation reaction Methods 0.000 title claims abstract description 56
- 230000002255 enzymatic effect Effects 0.000 title claims abstract description 24
- 239000002253 acid Substances 0.000 title abstract description 5
- 230000003647 oxidation Effects 0.000 title description 4
- 150000001720 carbohydrates Chemical class 0.000 claims abstract description 156
- JYTUSYBCFIZPBE-UHFFFAOYSA-N Maltobionic acid Natural products OC(=O)C(O)C(O)C(C(O)CO)OC1OC(CO)C(O)C(O)C1O JYTUSYBCFIZPBE-UHFFFAOYSA-N 0.000 claims abstract description 146
- JYTUSYBCFIZPBE-AMTLMPIISA-N lactobionic acid Chemical compound OC(=O)[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O JYTUSYBCFIZPBE-AMTLMPIISA-N 0.000 claims abstract description 146
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims abstract description 94
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims abstract description 94
- 239000007787 solid Substances 0.000 claims abstract description 64
- 238000006243 chemical reaction Methods 0.000 claims abstract description 37
- 239000007795 chemical reaction product Substances 0.000 claims abstract description 26
- 101710128063 Carbohydrate oxidase Proteins 0.000 claims abstract description 18
- 102000016938 Catalase Human genes 0.000 claims abstract description 18
- 108010053835 Catalase Proteins 0.000 claims abstract description 18
- 238000005273 aeration Methods 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 16
- 238000003756 stirring Methods 0.000 claims abstract description 16
- 239000000243 solution Substances 0.000 claims description 152
- 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 description 40
- 239000000047 product Substances 0.000 claims description 26
- 239000003729 cation exchange resin Substances 0.000 claims description 24
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical group [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 22
- 239000003957 anion exchange resin Substances 0.000 claims description 21
- 239000003456 ion exchange resin Substances 0.000 claims description 18
- 229920003303 ion-exchange polymer Polymers 0.000 claims description 18
- 230000002378 acidificating effect Effects 0.000 claims description 16
- 239000011347 resin Substances 0.000 claims description 14
- 229920005989 resin Polymers 0.000 claims description 14
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 12
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 11
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 11
- 239000007921 spray Substances 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 8
- 239000005909 Kieselgur Substances 0.000 claims description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 7
- 238000001914 filtration Methods 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 5
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims 1
- 239000000843 powder Substances 0.000 abstract description 3
- 238000013375 chromatographic separation Methods 0.000 abstract description 2
- 102000004190 Enzymes Human genes 0.000 description 41
- 108090000790 Enzymes Proteins 0.000 description 41
- 229940088598 enzyme Drugs 0.000 description 41
- 235000000346 sugar Nutrition 0.000 description 24
- 102000004316 Oxidoreductases Human genes 0.000 description 15
- 108090000854 Oxidoreductases Proteins 0.000 description 15
- 239000012141 concentrate Substances 0.000 description 15
- 239000012071 phase Substances 0.000 description 15
- 238000002360 preparation method Methods 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 14
- 229910001868 water Inorganic materials 0.000 description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 238000004128 high performance liquid chromatography Methods 0.000 description 11
- 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 10
- 239000008103 glucose Substances 0.000 description 10
- 230000035484 reaction time Effects 0.000 description 10
- 238000001694 spray drying Methods 0.000 description 9
- 150000008163 sugars Chemical class 0.000 description 9
- 238000000746 purification Methods 0.000 description 8
- 230000003078 antioxidant effect Effects 0.000 description 6
- 238000004042 decolorization Methods 0.000 description 6
- 229920001542 oligosaccharide Polymers 0.000 description 5
- 150000002482 oligosaccharides Chemical class 0.000 description 5
- 150000001450 anions Chemical class 0.000 description 4
- 238000002425 crystallisation Methods 0.000 description 4
- 230000008025 crystallization Effects 0.000 description 4
- 238000006116 polymerization reaction Methods 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- PKAUICCNAWQPAU-UHFFFAOYSA-N 2-(4-chloro-2-methylphenoxy)acetic acid;n-methylmethanamine Chemical compound CNC.CC1=CC(Cl)=CC=C1OCC(O)=O PKAUICCNAWQPAU-UHFFFAOYSA-N 0.000 description 3
- 239000004375 Dextrin Substances 0.000 description 3
- 229920001353 Dextrin Polymers 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 235000014633 carbohydrates Nutrition 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 235000019425 dextrin Nutrition 0.000 description 3
- 238000002844 melting Methods 0.000 description 3
- 230000008018 melting Effects 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 2
- AYRXSINWFIIFAE-SCLMCMATSA-N Isomaltose Natural products 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-SCLMCMATSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 208000012839 conversion disease Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- DLRVVLDZNNYCBX-RTPHMHGBSA-N isomaltose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)C(O)O1 DLRVVLDZNNYCBX-RTPHMHGBSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 239000003002 pH adjusting agent Substances 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 238000007711 solidification Methods 0.000 description 2
- 230000008023 solidification Effects 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 238000004148 unit process Methods 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 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 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 239000008406 cosmetic ingredient Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 235000013325 dietary fiber Nutrition 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 239000005417 food ingredient Substances 0.000 description 1
- 239000003365 glass fiber Substances 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- JGJLWPGRMCADHB-UHFFFAOYSA-N hypobromite Inorganic materials Br[O-] JGJLWPGRMCADHB-UHFFFAOYSA-N 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000002075 main ingredient Substances 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- VUZPPFZMUPKLLV-UHFFFAOYSA-N methane;hydrate Chemical compound C.O VUZPPFZMUPKLLV-UHFFFAOYSA-N 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011736 potassium bicarbonate Substances 0.000 description 1
- 235000015497 potassium bicarbonate Nutrition 0.000 description 1
- 229910000028 potassium bicarbonate Inorganic materials 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 235000011181 potassium carbonates Nutrition 0.000 description 1
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 235000017550 sodium carbonate Nutrition 0.000 description 1
- 229910001415 sodium ion Inorganic materials 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N9/0004—Oxidoreductases (1.)
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03004—Glucose oxidase (1.1.3.4)
Abstract
본 발명에 따른 말토비온산 제조방법은 고형분 농도가 15~25 브릭스(Brix)이고 당류 고형분 전체 중량을 기준으로 말토스 함량이 90 중량% 이상인 말토스 함유 당류 용액을 준비하는 단계; 및 상기 말토스 함유 당류 용액에 당류 고형분 중량 대비 카보하이드레이트옥시다제(Carbohydrate oxidase) 4.0~5.0%(v/w) 및 카탈라제(Catalase) 0.1~0.8%(v/w)를 첨가하고 22~35℃의 온도 조건, 6~8의 pH 조건, 0.9~1.2 vvm의 에어레이션(aeration) 속도 조건 및 150~300 rpm의 교반 속도 조건으로 효소 산화반응을 15~30 hr 동안 진행시켜 반응 산물 함유 용액을 수득하는 단계를 포함한다. 본 발명에 따른 제조방법을 사용하는 경우 말토스의 말토비온산으로의 전환율을 99% 이상으로 할 수 있고, 별도의 크로마토그래피 분리 공정을 사용하지 않고도 순도가 95% 이상이고 분말 형태인 고순도 말토비온산을 생산할 수 있다.The method for producing maltobionic acid according to the present invention includes preparing a maltose-containing saccharide solution having a solid concentration of 15 to 25 Brix and a maltose content of 90% by weight or more based on the total weight of saccharide solids; And 4.0-5.0% (v/w) of carbohydrate oxidase and 0.1-0.8% (v/w) of catalase were added to the maltose-containing saccharide solution, based on the weight of saccharide solids, and incubated at 22-35°C. The enzymatic oxidation reaction proceeds for 15 to 30 hr under temperature conditions, pH conditions of 6 to 8, aeration rate conditions of 0.9 to 1.2 vvm, and stirring speed conditions of 150 to 300 rpm to obtain a solution containing the reaction product. Includes steps. When using the production method according to the present invention, the conversion rate of maltose to maltobionic acid can be 99% or more, and high purity maltobi in powder form with a purity of 95% or more can be obtained without using a separate chromatographic separation process. Hot acid can be produced.
Description
본 발명은 말토비온산의 제조방법에 관한 것으로서, 보다 상세하게는 효소적 산화 및 다양한 공정 조건을 통해 말토스 함유 용액으로부터 고순도의 말토비온산을 제조하는 방법에 관한 것이다.The present invention relates to a method for producing maltobionic acid, and more specifically, to a method for producing high purity maltobionic acid from a solution containing maltose through enzymatic oxidation and various process conditions.
당류는 일반적으로 항산화 활성이 낮은 물질이다. Hu 등[Hu et al., In vitro evaluation of the antioxidant activities of carbohydrates, Bioactive Carbohydratesand Dietary Fibre 7 (2016) 19-27]에 따르면 포도당, 과당과 같은 단당 및 올리고당은 약 2,000 ppm의 농도에서도 약 10% 미만의 항산화 활성을 보인다.Sugars are generally substances with low antioxidant activity. According to Hu et al. [Hu et al., In vitro evaluation of the antioxidant activities of carbohydrates, Bioactive Carbohydrates and Dietary Fibre 7 (2016) 19-27], monosaccharides and oligosaccharides such as glucose and fructose are reduced by about 10% even at a concentration of about 2,000 ppm. It shows low antioxidant activity.
당류는 식품 소재나 화장품 소재로 널리 사용되는데, 당류에 높은 항산화 활성이 부여되는 경우 이의 적용 분야는 훨씬 더 다양해질 수 있다. 항산화 활성을 가지는 대표적인 산화 당류로는 글루콘산(Gluconic acid), 말토비온산(Maltobionic acid, MBA)이 있다.Sugars are widely used as food or cosmetic ingredients, and if sugars are given high antioxidant activity, their application areas can become much more diverse. Representative oxidized sugars with antioxidant activity include gluconic acid and maltobionic acid (MBA).
말토비온산은 말토스(맥아당)를 브롬수로 산화시켜 얻는 물질로서 다음과 같은 화학 구조를 갖는다. 미국 공개특허공보 제2016-0081387호에는 전분을 효소 반응을 통해 말토스로 전환하는 단계; 및 말토스를 효소 반응을 통해 말토비온산으로 전환하는 단계를 포함하는 말토비온산 제조방법과 함께 말토비온산이 항산화 효과를 가지는 식품 첨가제로 사용될 수 있다는 점이 개시되어 있다.Maltobionic acid is a substance obtained by oxidizing maltose (maltose) with bromine water and has the following chemical structure. U.S. Patent Publication No. 2016-0081387 includes converting starch into maltose through an enzymatic reaction; It is disclosed that maltobionic acid can be used as a food additive with an antioxidant effect along with a method for producing maltobionic acid, which includes converting maltose into maltobionic acid through an enzymatic reaction.
[말토비온산의 화학 구조][Chemical structure of maltobionic acid]
본 발명은 종래의 기술적 배경하에서 도출된 것으로서, 본 발명의 목적은 말토스 함유 용액으로부터 분말 형태의 고순도 말토비온산을 제조하는 방법을 제공하는데에 있다.The present invention was derived from the conventional technical background, and the purpose of the present invention is to provide a method for producing high-purity maltobionic acid in powder form from a maltose-containing solution.
본 발명의 발명자들은 말토스로부터 고순도의 말토비온산을 상업적 수준으로 제조하기 위해 효소 산화반응 공정, 이온교환수지에 의한 정제공정, 고형화 공정 등과 같이 전체 공정을 구성하는 단위공정들을 조합하고 최종 생산물인 말토비온산의 품질에 영향을 미치는 단위공정들의 다양한 조건들을 탐색한 후 말토스로부터 고순도의 말토비온산을 상업적 수준으로 제조할 수 있는 파라미터들을 확립하였다.In order to produce high-purity maltobionic acid from maltose at a commercial level, the inventors of the present invention combined the unit processes that make up the entire process, such as an enzymatic oxidation reaction process, a purification process using an ion exchange resin, and a solidification process, and produced the final product. After exploring various conditions of unit processes that affect the quality of maltobionic acid, we established parameters that can produce high-purity maltobionic acid from maltose at a commercial level.
상기 목적을 달성하기 위하여, 본 발명의 일 예는 (a) 말토스 함유 당류 용액을 준비하는 단계; 및 (b) 효소 산화반응을 통해 반응 산물 함유 용액을 수득하는 단계를 포함하는 말토비온산 제조방법을 제공한다. 본 발명의 일 예에 따른 말토비온산 제조방법은 바람직하게는 (c) 탈색 및 여과를 통해 말토비온산 함유 당류 용액을 수득하는 단계를 더 포함할 수 있다. 또한, 본 발명의 일 예에 따른 말토비온산 제조방법은 바람직하게는 (d) 이온교환수지 정제를 통해 정제된 말토비온산 함유 당류 용액을 수득하는 단계를 더 포함할 수 있다. 또한, 본 발명의 일 예에 따른 말토비온산 제조방법은 바람직하게는 (e) 분무건조를 통해 분말화된 정제 말토비온산을 수득하는 단계를 더 포함할 수 있다.In order to achieve the above object, an example of the present invention includes the steps of (a) preparing a maltose-containing saccharide solution; and (b) obtaining a solution containing the reaction product through an enzymatic oxidation reaction. The method for producing maltobionic acid according to an example of the present invention may further include the step of (c) obtaining a maltobionic acid-containing saccharide solution through decolorization and filtration. In addition, the method for producing maltobionic acid according to an example of the present invention may further include (d) obtaining a purified maltobionic acid-containing saccharide solution through ion exchange resin purification. In addition, the method for producing maltobionic acid according to an example of the present invention may further include the step of (e) obtaining powdered purified maltobionic acid through spray drying.
이하, 본 발명의 일 예에 따른 말토비온산 제조방법을 각 단계별로 나누어 설명한다.Hereinafter, the method for producing maltobionic acid according to an example of the present invention will be described in each step.
(a) 말토스 함유 당류 용액을 준비하는 단계(a) Preparing a maltose-containing saccharide solution
본 발명의 일 예에 따른 말토비온산 제조방법에서 (a) 단계는 당류 고형분 전체 중량을 기준으로 말토스 함량이 90 중량% 이상인 말토스 함유 당류를 물(water)에 용해하거나 물(water)로 희석하여 고형분 농도를 15~25 브릭스(Brix)인 말토스 함유 당류 용액을 준비하는 것으로 구성된다.In the method for producing maltobionic acid according to an example of the present invention, step (a) is performed by dissolving maltose-containing saccharides having a maltose content of 90% by weight or more based on the total weight of saccharide solids in water or mixing them with water. It consists of preparing a maltose-containing saccharide solution with a solid concentration of 15 to 25 Brix by dilution.
상기 (a) 단계를 통해 준비되는 말토스 함유 당류 용액은 당류 고형분 전체 중량을 기준으로 말토스 90~95 중량% 및 기타 당류 5~10 중량%를 포함할 수 있다. 상기 기타 당류는 포도당(Glucose)이 주성분이고, 포도당(Glucose)을 단위구조로 가지는 중합도 3~12의 올리고당도 소량 포함할 수 있다.The maltose-containing saccharide solution prepared through step (a) may contain 90 to 95% by weight of maltose and 5 to 10% by weight of other sugars based on the total weight of saccharide solids. The main component of the other saccharides is glucose, and may also contain a small amount of oligosaccharides with a degree of polymerization of 3 to 12 that have glucose as a unit structure.
또한, 상기 (a) 단계를 통해 준비되는 말토스 함유 당류 용액은 바람직하게는 당류 고형분 전체 중량을 기준으로 말토스 함량이 95 중량% 이상이다. 예를 들어, 바람직한 일 예에 따른 말토스 함유 당류 용액은 당류 고형분 전체 중량을 기준으로 말토스 95~99 중량%, 이소말토스 0.1~2 중량% 및 기타 당류 0.5~4 중량%를 포함할 수 있다. 상기 기타 당류는 포도당(Glucose)을 단위구조로 가지는 중합도 3~12의 올리고당으로 구성될 수 있다.In addition, the maltose-containing saccharide solution prepared through step (a) preferably has a maltose content of 95% by weight or more based on the total weight of saccharide solids. For example, the maltose-containing saccharide solution according to a preferred example may include 95 to 99% by weight of maltose, 0.1 to 2% by weight of isomaltose, and 0.5 to 4% by weight of other sugars, based on the total weight of saccharide solids. there is. The other saccharides may be composed of oligosaccharides with a degree of polymerization of 3 to 12 that have glucose as a unit structure.
(b) 효소 산화반응을 통해 반응 산물 함유 용액을 수득하는 단계(b) obtaining a solution containing the reaction product through an enzymatic oxidation reaction.
본 발명의 일 예에 따른 말토비온산 제조방법에서 (b) 단계는 말토스 함유 당류 용액에 당류 고형분 중량 대비 카보하이드레이트옥시다제(Carbohydrate oxidase) 4.0~5.0%(v/w) 및 카탈라제(Catalase) 0.1~0.8%(v/w)를 첨가하고 22~37℃의 온도 조건, 6~8의 pH 조건, 0.9~1.5 vvm의 에어레이션(aeration) 속도 조건 및 100~400 rpm의 교반 속도 조건으로 효소 산화반응을 15~30 hr 동안 진행시켜 반응 산물 함유 용액을 수득하는 것으로 구성된다. 상기 (b) 단계에서 카보하이드레이트옥시다제(Carbohydrate oxidase) 첨가량은 효소 산화반응 전환율을 고려할 때 말토스 함유 당류 용액의 당류 고형분 중량 대비 4.1~4.8 중량%인 것이 바람직하고, 4.2~4.5 중량%인 것이 더 바람직하다. 또한, 상기 (b) 단계에서 카탈라제(Catalase)의 첨가량은 효소 산화반응 전환율을 고려할 때 말토스 함유 당류 용액의 당류 고형분 중량 대비 0.15~0.75 중량%인 것이 바람직하고, 0.15~0.5 중량%인 것이 더 바람직하다. 상기 (b) 단계에서 효소 산화반응 온도는 전환율을 고려할 때 24~37℃인 것이 바람직하고 25~35℃인 것이 더 바람직하다. 상기 (b) 단계에서 효소 산화반응 pH는 전환율을 고려할 때 6.5~7.5인 것이 바람직하다. 또한, 효소 산화반응 pH 조건은 약염기성 물질 또는 약염기성 물질 용액에 의해 조절된다. 상기 약염기성 물질의 종류는 효소의 활성을 저하시키는 않는 것이라면 크게 제한되지 않으며, 예를 들어 탄산수소나트륨, 탄산나트륨, 탄산수소칼륨, 탄산칼륨 등이 있고, 이중 탄산수소나트륨인 것이 바람직하다. 상기 (b) 단계에서 효소 산화반응시 에어레이션(aeration) 속도 조건은 전환율을 고려할 때 0.9~1.2 vvm인 것이 바람직하다. 상기 (b) 단계에서 효소 산화반응시 교반 속도 조건은 전환율을 고려할 때 150~350 rpm인 것이 바람직하고, 180~250 rpm인 것이 더 바람직하다. 상기 (b) 단계에서 효소 산화반응 시간은 전환율 및 경제성을 고려할 때 15~28 hr인 것이 바람직하고 18~24 hr인 것이 더 바람직하다. 상기 (b) 단계에서 수득한 반응 산물 함유 용액은 당류 고형분 전체 중량을 기준으로 말토비온산 함량이 90 중량% 이상이고 바람직하게는 95 중량% 이상이다.In the maltobionic acid production method according to an example of the present invention, step (b) is performed by adding 4.0-5.0% (v/w) of carbohydrate oxidase and catalase to the maltose-containing saccharide solution relative to the weight of saccharide solids. Add 0.1 to 0.8% (v/w) and perform enzyme oxidation under temperature conditions of 22 to 37°C, pH conditions of 6 to 8, aeration rate conditions of 0.9 to 1.5 vvm, and stirring speed conditions of 100 to 400 rpm. It consists of allowing the reaction to proceed for 15 to 30 hr to obtain a solution containing the reaction product. In step (b), the amount of carbohydrate oxidase added is preferably 4.1 to 4.8% by weight, and 4.2 to 4.5% by weight, based on the weight of the saccharide solid content of the maltose-containing saccharide solution, considering the enzyme oxidation reaction conversion rate. It is more desirable. In addition, the amount of catalase added in step (b) is preferably 0.15 to 0.75% by weight, and more preferably 0.15 to 0.5% by weight, based on the weight of the saccharide solid content of the maltose-containing saccharide solution, considering the enzyme oxidation reaction conversion rate. desirable. Considering the conversion rate, the enzyme oxidation reaction temperature in step (b) is preferably 24 to 37°C, and more preferably 25 to 35°C. In step (b), the pH of the enzyme oxidation reaction is preferably 6.5 to 7.5 when considering the conversion rate. Additionally, the pH conditions of the enzyme oxidation reaction are controlled by a weak base material or a solution of a weak base material. The type of the weak basic substance is not greatly limited as long as it does not reduce the activity of the enzyme, and examples include sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate, etc., of which sodium bicarbonate is preferable. In step (b), the aeration rate condition during the enzymatic oxidation reaction is preferably 0.9 to 1.2 vvm when considering the conversion rate. Considering the conversion rate, the stirring speed condition during the enzymatic oxidation reaction in step (b) is preferably 150 to 350 rpm, and more preferably 180 to 250 rpm. In step (b), the enzymatic oxidation reaction time is preferably 15 to 28 hr, more preferably 18 to 24 hr, considering conversion rate and economic efficiency. The solution containing the reaction product obtained in step (b) has a maltobionic acid content of 90% by weight or more, preferably 95% by weight or more, based on the total weight of saccharide solids.
(c) 탈색 및 여과를 통해 말토비온산 함유 당류 용액을 수득하는 단계(c) obtaining a saccharide solution containing maltobionic acid through decolorization and filtration.
본 발명의 바람직한 일 예에 따른 말토비온산 제조방법에서 (c) 단계는 반응 산물 함유 용액을 활성탄으로 처리하여 탈색하고 여과하여 말토비온산 함유 당류 용액을 수득하는 것으로 구성될 수 있다. 상기 탈색은 반응 산물 함유 용액에 소정량의 활성탄을 첨가하고 소정의 시간 동안 교반하여 진행될 수 있다. 활성탄의 첨가량은 반응 산물 함유 용액의 고형분 중량 대비 1~6 중량%에서 선택될 수 있다. 상기 여과는 탈색 처리된 반응 산물 함유 용액을 공지의 다양한 여과재에 통과시켜 진행될 수 있다. 상기 여과재는 그 종류가 제한되지 않으며, 예를 들어 여과포, 다공성 금속, 금속 섬유, 유리섬유, 다공성 플라스틱, 다공성 세라믹 등이 있으며, 식품 공업에서 안전하게 사용되는 다공성 규조토 여과재인 것이 바람직하다.In the method for producing maltobionic acid according to a preferred example of the present invention, step (c) may consist of treating the solution containing the reaction product with activated carbon to decolorize and filter it to obtain a saccharide solution containing maltobionic acid. The decolorization can be performed by adding a predetermined amount of activated carbon to the solution containing the reaction product and stirring for a predetermined time. The amount of activated carbon added may be selected from 1 to 6% by weight based on the weight of solids in the solution containing the reaction product. The filtration may be performed by passing the decolorized reaction product-containing solution through various known filter media. The types of filter media are not limited and include, for example, filter cloth, porous metal, metal fiber, glass fiber, porous plastic, porous ceramic, etc., and are preferably porous diatomaceous earth filter media that are safely used in the food industry.
(d) 이온교환수지 정제를 통해 정제된 말토비온산 함유 당류 용액을 수득하는 단계(d) Obtaining a purified maltobionic acid-containing saccharide solution through ion exchange resin purification.
본 발명의 바람직한 일 예에 따른 말토비온산 제조방법에서 (d) 단계는 말토비온산 함유 당류 용액을 강염기성 음이온 교환수지, 강산성 양이온 교환수지 및 혼상 이온교환수지에 순차적으로 통과시켜 정제된 말토비온산 함유 당류 용액을 수득하는 것으로 구성될 수 있다. 상기 혼상 이온교환수지는 강산성 양이온 교환수지 및 강염기성 음이온 교환수지를 혼합한 수지이다. 상기 (d) 단계에서 말토비온산 함유 당류 용액의 통액 속도(space velocity)는 정제된 말토비온산 함유 당류 용액의 pH 또는 전도도를 고려할 때 1~6 ㎖/min인 것이 바람직하고 2~5 ㎖/min인 것이 더 바람직하다. 상기 (d) 단계에서 사용되는 강염기성 음이온 교환수지는 정제용량, 정제된 말토비온산 함유 당류 용액의 pH 또는 전도도를 고려할 때 매크로다공성(Macroporous) 타입인 것이 바람직하다. 또한, 상기 강산성 양이온 교환수지는 정제된 말토비온산 함유 당류 용액의 pH 또는 전도도를 고려할 때 겔(Gel) 타입인 것이 바람직하다. 또한, 상기 (d) 단계에서 강염기성 음이온 교환수지:강산성 양이온 교환수지:혼상 이온교환수지의 부피비는 정제된 말토비온산 함유 당류 용액의 pH 또는 전도도를 고려할 때 1:(0.25~2):(0.25~1)인 것이 바람직하고 1:(0.3~1):(0.3~1)인 것이 더 바람직하다. 또한, 상기 (d) 단계에서 혼상 이온교환수지를 구성하는 강산성 양이온 교환수지:강염기성 음이온 교환수지의 혼합 부피비는 정제된 말토비온산 함유 당류 용액의 pH 또는 전도도를 고려할 때 1:(0.1~0.5)인 것이 바람직하고 1:(0.2~0.3)인 것이 더 바람직하다.In the maltobionic acid production method according to a preferred example of the present invention, step (d) is purified maltobionic acid by sequentially passing the maltobionic acid-containing saccharide solution through a strongly basic anion exchange resin, a strongly acidic cation exchange resin, and a mixed-bed ion exchange resin. It may consist of obtaining a saccharide solution containing acid. The mixed-phase ion exchange resin is a resin that mixes a strongly acidic cation exchange resin and a strongly basic anion exchange resin. In step (d), the space velocity of the maltobionic acid-containing saccharide solution is preferably 1 to 6 ml/min, and 2 to 5 ml/min, considering the pH or conductivity of the purified maltobionic acid-containing saccharide solution. Min is more preferable. The strongly basic anion exchange resin used in step (d) is preferably of the macroporous type, considering the purification capacity and the pH or conductivity of the purified maltobionic acid-containing saccharide solution. In addition, considering the pH or conductivity of the purified maltobionic acid-containing saccharide solution, the strongly acidic cation exchange resin is preferably of a gel type. In addition, in step (d), the volume ratio of strongly basic anion exchange resin:strongly acidic cation exchange resin:mixed phase ion exchange resin is 1:(0.25~2):(, considering the pH or conductivity of the purified maltobionic acid-containing saccharide solution. It is preferably 0.25~1) and more preferably 1:(0.3~1):(0.3~1). In addition, in step (d), the mixing volume ratio of the strongly acidic cation exchange resin:strongly basic anion exchange resin constituting the mixed phase ion exchange resin is 1:(0.1~0.5, considering the pH or conductivity of the purified maltobionic acid-containing saccharide solution. ) is preferable, and 1:(0.2~0.3) is more preferable.
(e) 분무건조를 통해 분말화된 정제 말토비온산을 수득하는 단계(e) Obtaining powdered purified maltobionic acid through spray drying.
본 발명의 바람직한 일 예에 따른 말토비온산 제조방법에서 (e) 단계는 정제된 말토비온산 함유 당류 용액의 고형분 농도를 10~40 브릭스(Brix)로 조절한 후 아토마이저(Atomizer)를 통해 분무건조기 내부 챔버로 주입하여 분말화된 정제 말토비온산을 수득하는 것으로 구성될 수 있다. 상기 (e) 단계에서 정제된 말토비온산 함유 당류 용액의 고형분 농도는 원활한 분말화를 위해 분무건조기에 공급되기 전에 15~35 브릭스(Brix)로 조절되는 것이 바람직하다. 상기 (e) 단계에서 정제된 말토비온산 함유 당류 용액의 주입 속도는 분무건조기의 처리 용량에 따라 다양한 범위에서 선택될 수 있고 예를 들어 500~1,000 ㎖/hour 범위에서 선택될 수 있다. 또한, 상기 (e) 단계에서 정제된 말토비온산 함유 당류 용액을 분무건조기 내부 챔버로 주입하는 통로인 주입구 온도는 원활한 분말화를 위해 135~170℃인 것이 바람직하고, 140~160℃인 것이 더 바람직하다. 또한, 상기 (e) 단계에서 아토마이저(Atomizer) 회전 속도는 원활한 분말화를 위해 600~900 rpm인 것이 바람직하고, 600~800 rpm인 것이 더 바람직하다. 또한, 상기 (e) 단계에서 분무건조기 내부 챔버의 출구 온도는 100~135℃인 것이 바람직하고, 110~130℃인 것이 더 바람직하다.In the maltobionic acid production method according to a preferred example of the present invention, step (e) is performed by adjusting the solids concentration of the purified maltobionic acid-containing saccharide solution to 10 to 40 Brix and then spraying it through an atomizer. It may consist of obtaining powdered and purified maltobionic acid by injecting it into the inner chamber of the dryer. The solid concentration of the maltobionic acid-containing saccharide solution purified in step (e) is preferably adjusted to 15 to 35 Brix before being supplied to the spray dryer for smooth powderization. The injection rate of the maltobionic acid-containing saccharide solution purified in step (e) may be selected from a variety of ranges depending on the processing capacity of the spray dryer, for example, from 500 to 1,000 mL/hour. In addition, the temperature of the inlet, which is the passage through which the maltobionic acid-containing saccharide solution purified in step (e) is injected into the inner chamber of the spray dryer, is preferably 135 to 170 ℃ for smooth powderization, and is more preferably 140 to 160 ℃. desirable. Additionally, in step (e), the atomizer rotation speed is preferably 600 to 900 rpm for smooth powderization, and more preferably 600 to 800 rpm. Additionally, in step (e), the outlet temperature of the inner chamber of the spray dryer is preferably 100 to 135°C, and more preferably 110 to 130°C.
본 발명에 따른 제조방법을 사용하는 경우 말토스의 말토비온산으로의 전환율을 99% 이상으로 할 수 있고, 별도의 크로마토그래피 분리 공정을 사용하지 않고도 순도가 95% 이상이고 분말 형태인 고순도 말토비온산을 생산할 수 있다.When using the production method according to the present invention, the conversion rate of maltose to maltobionic acid can be 99% or more, and high purity maltobi in powder form with a purity of 95% or more can be obtained without using a separate chromatographic separation process. Hot acid can be produced.
이하, 본 발명을 실시예를 통하여 구체적으로 설명한다. 다만, 하기 실시예는 본 발명의 기술적 특징을 명확하게 예시하기 위한 것일 뿐, 본 발명의 보호범위를 한정하는 것은 아니다.Hereinafter, the present invention will be described in detail through examples. However, the following examples are only intended to clearly illustrate the technical features of the present invention and do not limit the scope of protection of the present invention.
1. 말토스 함유 당류 제품을 이용한 말토비온산 제조공정 조건 확립1. Establishment of maltobionic acid manufacturing process conditions using maltose-containing sugar products
제조예 1.Manufacturing Example 1.
말토스 함유 당류 제품(제조사 : 대상주식회사)을 이온교환수로 희석하여 고형분 농도가 각각 20 브릭스(Brix)인 말토스 함유 당류 용액을 제조하였다. 당류 산화효소인 카보하이드레이트옥시다제(Carbohydrate oxidase; 공급자 : Novozymes) 및 카탈라제(Catalase; 공급자 : Novozymes)를 각각 말토스 함유 당류 용액의 당류 고형분 중량 대비 5.2%(v/w) 및 1.3%(v/w)의 양으로 첨가하고 반응 온도 25℃, 에어레이션(aeration) 속도 1 vvm 및 교반 속도 300 rpm의 조건에서 24 hr 동안 효소 산화반응을 진행시켰다. 효소 산화반응이 진행되는 동안 1M 농도의 탄산수소나트륨(NaHCO3) 용액을 이용하여 반응 pH를 약 7로 일정하게 유지시켰다. 효소 산화반응 시간이 완료된 후 반응 산물 용액을 약 80℃로 승온하고 약 2 hr 동안 처리하여 효소를 실활시켰다. 이후, 반응 산물 용액을 실온으로 냉각시킨 후 여과하여 말토비온산 함유 당류 용액을 수득하였다.A maltose-containing saccharide product (manufacturer: Daesang Co., Ltd.) was diluted with ion-exchanged water to prepare a maltose-containing saccharide solution with a solid concentration of 20 Brix. Carbohydrate oxidase (Supplier: Novozymes) and Catalase (Supplier: Novozymes), which are saccharide oxidase enzymes, were added at 5.2% (v/w) and 1.3% (v/ w) and the enzymatic oxidation reaction was carried out for 24 hr under the conditions of a reaction temperature of 25°C, an aeration rate of 1 vvm, and a stirring rate of 300 rpm. While the enzyme oxidation reaction was in progress, the reaction pH was kept constant at about 7 using a 1M sodium bicarbonate (NaHCO 3 ) solution. After the enzyme oxidation reaction time was completed, the reaction product solution was heated to about 80°C and treated for about 2 hr to deactivate the enzyme. Afterwards, the reaction product solution was cooled to room temperature and filtered to obtain a saccharide solution containing maltobionic acid.
제조예 2 내지 제조예 6.Preparation Example 2 to Preparation Example 6.
말토스 함유 당류 용액의 고형분 농도, 효소 산화반응 온도, 에어레이션(aeration) 속도 또는 교반 속도 등의 조건을 다르게 한 점을 제외하고는 제조예 1과 동일한 조건 및 동일한 방법으로 말토비온산 함유 당류 용액을 수득하였다.A saccharide solution containing maltobionic acid was prepared under the same conditions and in the same manner as in Preparation Example 1, except that the conditions such as solid concentration, enzyme oxidation reaction temperature, aeration speed, or stirring speed of the maltose-containing saccharide solution were different. Obtained.
하기 표 1에 제조예 1 내지 제조예 6에서 사용한 제조공정 조건을 정리하였다.Table 1 below summarizes the manufacturing process conditions used in Preparation Examples 1 to 6.
말토스 함유 당류 제품(제조사 : 대상주식회사) 및 제조예 1 내지 제조예 6에서 수득한 말토비온산 함유 당류 용액의 당 성분 및 함량을 HPLC로 분석하였다. HPLC 분석 조건은 다음과 같다.The sugar components and content of the maltose-containing saccharide product (manufacturer: Daesang Co., Ltd.) and the maltobionic acid-containing saccharide solution obtained in Preparation Examples 1 to 6 were analyzed by HPLC. HPLC analysis conditions are as follows.
* 칼럼 및 온도 : Aminex HPX-87C Column, 85℃* Column and temperature: Aminex HPX-87C Column, 85℃
* 이동상 및 유량 : 물(water), 0.6 ㎖/min* Mobile phase and flow rate: water, 0.6 mL/min
* 검출기 : RID* Detector: RID
하기 표 2에 말토스 함유 당류 제품(제조사 : 대상주식회사) 및 제조예 1 내지 제조예 6에서 수득한 말토비온산 함유 당류 용액의 당 성분 및 함량을 정리하였다.Table 2 below summarizes the sugar components and contents of maltose-containing saccharide products (manufacturer: Daesang Co., Ltd.) and maltobionic acid-containing saccharide solutions obtained in Preparation Examples 1 to 6.
* 한계 덱스트린(Limit dextrin) : 전분을 아밀라아제로 분해하였을 때 더 이상 분해되지 않고 남은 덱스트린* Limit dextrin: When starch is decomposed with amylase, the remaining dextrin is not further decomposed.
상기 표 2에서 보이는 바와 같이 말토스 함유 당류 용액을 효소 산화반응에 의해 말토비온산으로 전환하는 경우, 다음과 같은 공정 조건에서 높은 전환율을 보였다. 특히 말토비온산의 전환율은 말토스 함유 당류 용액의 당류 고형분 농도 및 에어레이션 속도에 크게 영향을 받는 것으로 나타났다.As shown in Table 2 above, when a maltose-containing saccharide solution was converted to maltobionic acid by an enzymatic oxidation reaction, a high conversion rate was observed under the following process conditions. In particular, the conversion rate of maltobionic acid was found to be greatly affected by the saccharide solid concentration and aeration rate of the maltose-containing saccharide solution.
* 말토스 함유 당류 용액의 당류 고형분 농도 : 20 브릭스(Brix)* Sugar solid concentration of maltose-containing sugar solution: 20 Brix
* 효소 산화반응 온도 : 25~30℃/경제적 관점에서 25℃* Enzyme oxidation reaction temperature: 25~30℃/25℃ from economical point of view
* 에어레이션(aeration) 속도 : 1 vvm* Aeration speed: 1 vvm
* 교반 속도 : 200 rpm* Stirring speed: 200 rpm
2. 정제 말토스 제품을 이용한 말토비온산 제조공정 조건 확립2. Establishment of maltobionic acid manufacturing process conditions using purified maltose products
제조예 7.Manufacturing example 7.
정제 말토스 제품(제조사 : 대상주식회사)을 이온교환수로 희석하여 고형분 농도가 20 브릭스(Brix)인 정제 말토스 용액을 제조하였다. 당류 산화효소인 카보하이드레이트옥시다제(Carbohydrate oxidase; 공급자 : Novozymes) 및 카탈라제(Catalase; 공급자 : Novozymes)를 각각 정제 말토스 용액의 당류 고형분 중량 대비 5.2%(v/w) 및 1.3%(v/w)의 양으로 첨가하고 반응 온도 25℃, 에어레이션(aeration) 속도 1 vvm 및 교반 속도 200 rpm의 조건에서 24 hr 동안 효소 산화반응을 진행시켰다. 효소 산화반응이 진행되는 동안 1M 농도의 탄산수소나트륨(NaHCO3) 용액을 이용하여 반응 pH를 약 7로 일정하게 유지시켰다. 효소 산화반응 시간이 완료된 후 반응 산물 용액을 약 80℃로 승온하고 약 2 hr 동안 처리하여 효소를 실활시켰다. 이후, 반응 산물 용액을 실온으로 냉각시킨 후 여과하여 말토비온산 함유 당류 용액을 수득하였다.A purified maltose product (manufacturer: Daesang Co., Ltd.) was diluted with ion-exchanged water to prepare a purified maltose solution with a solid concentration of 20 Brix. Carbohydrate oxidase (Supplier: Novozymes) and Catalase (Supplier: Novozymes), which are saccharide oxidase enzymes, were added at 5.2% (v/w) and 1.3% (v/w), respectively, based on the weight of saccharide solids in the purified maltose solution. ) and the enzymatic oxidation reaction was carried out for 24 hr under the conditions of a reaction temperature of 25°C, an aeration rate of 1 vvm, and a stirring rate of 200 rpm. While the enzyme oxidation reaction was in progress, the reaction pH was kept constant at about 7 using a 1M sodium bicarbonate (NaHCO 3 ) solution. After the enzyme oxidation reaction time was completed, the reaction product solution was heated to about 80°C and treated for about 2 hr to deactivate the enzyme. Afterwards, the reaction product solution was cooled to room temperature and filtered to obtain a saccharide solution containing maltobionic acid.
제조예 8.Manufacturing example 8.
정제 말토스 제품(제조사 : 대상주식회사)을 이온교환수로 희석하여 고형분 농도가 20 브릭스(Brix)인 정제 말토스 용액을 제조하였다. 당류 산화효소인 글루코스옥시다제(Glucose oxidase; 공급자 : Novozymes) 및 카탈라제(Catalase; 공급자 : Novozymes)를 각각 정제 말토스 용액의 당류 고형분 중량 대비 5.2%(v/w) 및 1.3%(v/w)의 양으로 첨가하고 반응 온도 25℃, 에어레이션(aeration) 속도 1 vvm 및 교반 속도 200 rpm의 조건에서 24 hr 동안 효소 산화반응을 진행시켰다. 효소 산화반응이 진행되는 동안 1M 농도의 탄산수소나트륨(NaHCO3) 용액을 이용하여 반응 pH를 약 7로 일정하게 유지시켰다. 효소 산화반응 시간이 완료된 후 반응 산물 용액을 약 80℃로 승온하고 약 2 hr 동안 처리하여 효소를 실활시켰다. 이후, 반응 산물 용액을 실온으로 냉각시킨 후 여과하여 말토비온산 함유 당류 용액을 수득하였다.A purified maltose product (manufacturer: Daesang Co., Ltd.) was diluted with ion-exchanged water to prepare a purified maltose solution with a solid concentration of 20 Brix. Glucose oxidase (Supplier: Novozymes) and Catalase (Supplier: Novozymes), which are saccharide oxidase enzymes, are respectively 5.2% (v/w) and 1.3% (v/w) based on the weight of saccharide solids in the purified maltose solution. was added in an amount of , and the enzyme oxidation reaction was carried out for 24 hr under the conditions of a reaction temperature of 25°C, an aeration rate of 1 vvm, and a stirring rate of 200 rpm. While the enzyme oxidation reaction was in progress, the reaction pH was kept constant at about 7 using a 1M sodium bicarbonate (NaHCO 3 ) solution. After the enzyme oxidation reaction time was completed, the reaction product solution was heated to about 80°C and treated for about 2 hr to deactivate the enzyme. Afterwards, the reaction product solution was cooled to room temperature and filtered to obtain a saccharide solution containing maltobionic acid.
제조예 9.Manufacturing example 9.
정제 말토스 제품(제조사 : 대상주식회사)을 이온교환수로 희석하여 고형분 농도가 20 브릭스(Brix)인 정제 말토스 용액을 제조하였다. 당류 산화효소인 카보하이드레이트옥시다제(Carbohydrate oxidase; 공급자 : Novozymes) 및 카탈라제(Catalase; 공급자 : Novozymes)를 각각 정제 말토스 용액의 당류 고형분 중량 대비 5.2%(v/w) 및 1.3%(v/w)의 양으로 첨가하고 반응 온도 25℃, 에어레이션(aeration) 속도 1 vvm 및 교반 속도 200 rpm의 조건에서 24 hr 동안 효소 산화반응을 진행시켰다. 효소 산화반응이 진행되는 동안 1M 농도의 수산화나트륨(NaOH) 용액을 이용하여 반응 pH를 약 7로 일정하게 유지시켰다. 효소 산화반응 시간이 완료된 후 반응 산물 용액을 약 80℃로 승온하고 약 2 hr 동안 처리하여 효소를 실활시켰다. 이후, 반응 산물 용액을 실온으로 냉각시킨 후 여과하여 말토비온산 함유 당류 용액을 수득하였다.A purified maltose product (manufacturer: Daesang Co., Ltd.) was diluted with ion-exchanged water to prepare a purified maltose solution with a solid concentration of 20 Brix. Carbohydrate oxidase (Supplier: Novozymes) and Catalase (Supplier: Novozymes), which are saccharide oxidase enzymes, were added at 5.2% (v/w) and 1.3% (v/w), respectively, based on the weight of saccharide solids in the purified maltose solution. ) and the enzymatic oxidation reaction was carried out for 24 hr under the conditions of a reaction temperature of 25°C, an aeration rate of 1 vvm, and a stirring rate of 200 rpm. While the enzyme oxidation reaction was in progress, the reaction pH was kept constant at about 7 using a 1M sodium hydroxide (NaOH) solution. After the enzyme oxidation reaction time was completed, the reaction product solution was heated to about 80°C and treated for about 2 hr to deactivate the enzyme. Afterwards, the reaction product solution was cooled to room temperature and filtered to obtain a saccharide solution containing maltobionic acid.
정제 말토스 제품(제조사 : 대상주식회사) 및 제조예 7 내지 제조예 9에서 수득한 말토비온산 함유 당류 용액의 당 성분 및 함량을 HPLC로 분석하였다. HPLC 분석 조건은 다음과 같다.The sugar components and content of the refined maltose product (manufacturer: Daesang Co., Ltd.) and the maltobionic acid-containing saccharide solution obtained in Preparation Examples 7 to 9 were analyzed by HPLC. HPLC analysis conditions are as follows.
* 칼럼 및 온도 : Aminex HPX-87C Column, 85℃* Column and temperature: Aminex HPX-87C Column, 85℃
* 이동상 및 유량 : 물(water), 0.6 ㎖/min* Mobile phase and flow rate: water, 0.6 mL/min
* 검출기 : RID* Detector: RID
하기 표 3에 정제 말토스 제품(제조사 : 대상주식회사) 및 제조예 7 내지 제조예 9에서 수득한 말토비온산 함유 당류 용액의 당 성분 및 함량을 정리하였다.Table 3 below summarizes the sugar components and contents of the refined maltose product (manufacturer: Daesang Co., Ltd.) and the maltobionic acid-containing saccharide solution obtained in Preparation Examples 7 to 9.
* 정제 말토스 제품의 기타 당류 : 포도당(Glucose)이 주성분이고, 포도당(Glucose)을 단위구조로 가지는 중합도 3~12의 올리고당도 소량 포함됨* Other sugars in refined maltose products: Glucose is the main ingredient, and a small amount of oligosaccharides with a degree of polymerization of 3 to 12 with glucose as the unit structure are also included.
상기 표 3에서 보이는 바와 같이 당류 산화효소 시스템을 카보하이드레이트옥시다제-카탈라제 조합 대신 글루코스옥시다제-카탈라제 조합으로 변경하는 경우 말토비온산으로의 전환율이 현저하게 감소하였다. 또한, 효소 산화반응 pH 조절제로 약염기인 탄산수소나트륨(NaHCO3) 대신 강염기인 수산화나트륨(NaOH)을 사용하는 경우 효소 활성이 저하되고 말토비온산으로의 전환율이 크게 감소하였다.As shown in Table 3, when the saccharide oxidase system was changed to a glucose oxidase-catalase combination instead of a carbohydrate oxidase-catalase combination, the conversion rate to maltobionic acid was significantly reduced. In addition, when the strong base sodium hydroxide (NaOH) was used instead of the weak base sodium bicarbonate (NaHCO 3 ) as the pH regulator for the enzyme oxidation reaction, the enzyme activity decreased and the conversion rate to maltobionic acid was greatly reduced.
3. 고순도 말토스 제품을 이용한 말토비온산 제조공정 조건 확립3. Establishment of maltobionic acid manufacturing process conditions using high-purity maltose products
(1) 효소 첨가량 조건 확립(1) Establishment of enzyme addition amount conditions
제조예 10.Manufacturing Example 10.
고순도 말토스 제품(제조사 : Junsei Chemical Co.,Ltd.)을 이온교환수로 희석하여 고형분 농도가 20 브릭스(Brix)인 고순도 말토스 용액을 제조하였다. 당류 산화효소인 카보하이드레이트옥시다제(Carbohydrate oxidase; 공급자 : Novozymes) 및 카탈라제(Catalase; 공급자 : Novozymes)를 각각 고순도 말토스 용액의 당류 고형분 중량 대비 2.6%(v/w) 및 0.16%(v/w)의 양으로 첨가하고 반응 온도 25℃, 에어레이션(aeration) 속도 1 vvm 및 교반 속도 200 rpm의 조건에서 24 hr 동안 효소 산화반응을 진행시켰다. 효소 산화반응이 진행되는 동안 1M 농도의 탄산수소나트륨(NaHCO3) 용액을 이용하여 반응 pH를 약 7로 일정하게 유지시켰다. 효소 산화반응 시간이 완료된 후 반응 산물 용액을 약 80℃로 승온하고 약 2 hr 동안 처리하여 효소를 실활시켰다. 이후, 반응 산물 용액을 실온으로 냉각시킨 후 여과하여 말토비온산 함유 당류 용액을 수득하였다.A high-purity maltose product (manufacturer: Junsei Chemical Co., Ltd.) was diluted with ion-exchanged water to prepare a high-purity maltose solution with a solid concentration of 20 Brix. Carbohydrate oxidase (Supplier: Novozymes) and Catalase (Supplier: Novozymes), which are saccharide oxidase enzymes, were used at 2.6% (v/w) and 0.16% (v/w), respectively, based on the weight of saccharide solids in the high-purity maltose solution. ) and the enzymatic oxidation reaction was carried out for 24 hr under the conditions of a reaction temperature of 25°C, an aeration rate of 1 vvm, and a stirring rate of 200 rpm. While the enzyme oxidation reaction was in progress, the reaction pH was kept constant at about 7 using a 1M sodium bicarbonate (NaHCO 3 ) solution. After the enzyme oxidation reaction time was completed, the reaction product solution was heated to about 80°C and treated for about 2 hr to deactivate the enzyme. Afterwards, the reaction product solution was cooled to room temperature and filtered to obtain a saccharide solution containing maltobionic acid.
제조예 11 내지 제조예 21.Production Examples 11 to 21.
당류 산화효소인 카보하이드레이트옥시다제(Carbohydrate oxidase; 공급자 : Novozymes) 및 카탈라제(Catalase; 공급자 : Novozymes)의 첨가량을 다르게 한 점을 제외하고는 제조예 10과 동일한 조건 및 동일한 방법으로 말토비온산 함유 당류 용액을 수득하였다.Maltobionic acid-containing saccharide was prepared under the same conditions and in the same manner as in Preparation Example 10, except that the amounts of carbohydrate oxidase (Supplier: Novozymes) and Catalase (Supplier: Novozymes), which are saccharide oxidase enzymes, were different. A solution was obtained.
하기 표 4에 제조예 10 내지 제조예 21에서 사용한 당류 산화효소 첨가량을 정리하였다.Table 4 below summarizes the amounts of saccharide oxidase added in Preparation Examples 10 to 21.
고순도 말토스 제품(제조사 : Junsei Chemical Co.,Ltd.) 및 제조예 10 내지 제조예 21에서 수득한 말토비온산 함유 당류 용액의 당 성분 및 함량을 HPLC로 분석하였다. HPLC 분석 조건은 다음과 같다.The sugar components and content of the high-purity maltose product (manufacturer: Junsei Chemical Co., Ltd.) and the maltobionic acid-containing saccharide solution obtained in Preparation Examples 10 to 21 were analyzed by HPLC. HPLC analysis conditions are as follows.
* 칼럼 및 온도 : Aminex HPX-87C Column, 85℃* Column and temperature: Aminex HPX-87C Column, 85℃
* 이동상 및 유량 : 물(water), 0.6 ㎖/min* Mobile phase and flow rate: water, 0.6 mL/min
* 검출기 : RID* Detector: RID
하기 표 5에 고순도 말토스 제품(제조사 : Junsei Chemical Co.,Ltd.) 및 제조예 10 내지 제조예 21에서 수득한 말토비온산 함유 당류 용액의 당 성분 및 함량을 정리하였다.Table 5 below summarizes the sugar components and contents of the high-purity maltose product (manufacturer: Junsei Chemical Co., Ltd.) and the maltobionic acid-containing saccharide solution obtained in Preparation Examples 10 to 21.
* 고순도 말토스 제품의 기타 당류 : 포도당(Glucose)을 단위구조로 가지는 중합도 3~12의 올리고당으로 구성됨* Other saccharides in high-purity maltose products: Consists of oligosaccharides with a degree of polymerization of 3 to 12 with glucose as the unit structure.
상기 표 5에서 보이는 바와 같이, 고순도 말토스 용액을 효소 산화반응에 의해 말토비온산으로 전환하는 경우, 카보하이드레이트옥시다제(Carbohydrate oxidase)의 첨가량이 고순도 말토스 용액의 당류 고형분 중량 대비 4.0~5.0%(v/w)이고 카탈라제(Catalase)의 첨가량이 고순도 말토스 용액의 당류 고형분 중량 대비 0.1~0.8%(v/w)일 때 높은 전환율을 보였다.As shown in Table 5 above, when converting a high-purity maltose solution into maltobionic acid through an enzymatic oxidation reaction, the amount of carbohydrate oxidase added is 4.0 to 5.0% based on the weight of saccharide solids in the high-purity maltose solution. (v/w), and a high conversion rate was shown when the amount of catalase added was 0.1 to 0.8% (v/w) based on the weight of saccharide solids in the high-purity maltose solution.
(2) 효소 산화반응 온도 조건 확립(2) Establishment of temperature conditions for enzyme oxidation reaction
제조예 22.Manufacturing Example 22.
고순도 말토스 제품(제조사 : Junsei Chemical Co.,Ltd.)을 이온교환수로 희석하여 고형분 농도가 20 브릭스(Brix)인 고순도 말토스 용액을 제조하였다. 당류 산화효소인 카보하이드레이트옥시다제(Carbohydrate oxidase; 공급자 : Novozymes) 및 카탈라제(Catalase; 공급자 : Novozymes)를 각각 고순도 말토스 용액의 당류 고형분 중량 대비 4.3%(v/w) 및 0.2%(v/w)의 양으로 첨가하고 반응 온도 25℃, 에어레이션(aeration) 속도 1 vvm 및 교반 속도 200 rpm의 조건에서 24 hr 동안 효소 산화반응을 진행시켰다. 효소 산화반응이 진행되는 동안 1M 농도의 탄산수소나트륨(NaHCO3) 용액을 이용하여 반응 pH를 약 7로 일정하게 유지시켰다. 효소 산화반응 시간별로 반응 산물 용액을 샘플링한 후 반응 산물 용액을 약 80℃로 승온하고 약 2 hr 동안 처리하여 효소를 실활시켰다. 이후, 반응 산물 용액을 실온으로 냉각시킨 후 여과하여 말토비온산 함유 당류 용액을 수득하였다. 수득한 말토비온산 함유 당류 용액의 당 성분 및 함량을 HPLC로 분석하였다.A high-purity maltose product (manufacturer: Junsei Chemical Co., Ltd.) was diluted with ion-exchanged water to prepare a high-purity maltose solution with a solid concentration of 20 Brix. Carbohydrate oxidase (Supplier: Novozymes) and Catalase (Supplier: Novozymes), which are saccharide oxidase enzymes, were used at 4.3% (v/w) and 0.2% (v/w), respectively, based on the weight of saccharide solids in the high-purity maltose solution. ) and the enzymatic oxidation reaction was carried out for 24 hr under the conditions of a reaction temperature of 25°C, an aeration rate of 1 vvm, and a stirring rate of 200 rpm. While the enzyme oxidation reaction was in progress, the reaction pH was kept constant at about 7 using a 1M sodium bicarbonate (NaHCO 3 ) solution. After sampling the reaction product solution for each enzyme oxidation reaction time, the temperature of the reaction product solution was raised to about 80°C and treated for about 2 hr to deactivate the enzyme. Afterwards, the reaction product solution was cooled to room temperature and filtered to obtain a saccharide solution containing maltobionic acid. The sugar components and content of the obtained maltobionic acid-containing saccharide solution were analyzed by HPLC.
제조예 23 내지 제조예 27.Production Examples 23 to 27.
당류 산화효소인 카보하이드레이트옥시다제(Carbohydrate oxidase; 공급자 : Novozymes) 및 카탈라제(Catalase; 공급자 : Novozymes)의 첨가량, 효소 산화반응 온도를 다르게 한 점을 제외하고는 제조예 22와 동일한 조건 및 동일한 방법으로 말토비온산 함유 당류 용액을 수득하였다. 수득한 말토비온산 함유 당류 용액의 당 성분 및 함량을 HPLC로 분석하였다.Carbohydrate oxidase (supplier: Novozymes) and catalase (supplier: Novozymes), which are saccharide oxidase enzymes, were added under the same conditions and method as in Preparation Example 22, except that the enzyme oxidation reaction temperature was different. A saccharide solution containing maltobionic acid was obtained. The sugar components and content of the obtained maltobionic acid-containing saccharide solution were analyzed by HPLC.
하기 표 6에 제조예 22 내지 제조예 27에서 사용한 당류 산화효소 첨가량, 효소 산화반응 온도 및 수득한 말토비온산 함유 당류 용액의 당류 고형분 중량 기준으로 말토스(maltose) 함량이 1 중량% 미만이 되었을 때의 효소 산화반응 시간을 정리하였다.Table 6 below shows the addition amount of the saccharide oxidase used in Preparation Examples 22 to 27, the enzyme oxidation reaction temperature, and the maltose content of less than 1% by weight based on the saccharide solid weight of the obtained maltobionic acid-containing saccharide solution. The enzyme oxidation reaction time was summarized.
상기 표 6에서 보이는 바와 같이, 고순도 말토스 용액을 효소 산화반응에 의해 말토비온산으로 전환하는 경우, 카보하이드레이트옥시다제(Carbohydrate oxidase)의 첨가량이 고순도 말토스 용액의 당류 고형분 중량 대비 4.0~4.3%(v/w)이고 카탈라제(Catalase)의 첨가량이 고순도 말토스 용액의 당류 고형분 중량 대비 0.2%(v/w) 수준이고 효소 산화반응 온도 25~35℃일때 전환율이 100% 가까이 되는 효소 산화반응 시간은 15~20 hr 이었다. 반면, 카보하이드레이트옥시다제(Carbohydrate oxidase)의 첨가량이 고순도 말토스 용액의 당류 고형분 중량 대비 3.3~3.8%(v/w)인 경우 다른 조건과 관계없이 전환율이 90%에 미치지 못하였다.As shown in Table 6 above, when converting a high-purity maltose solution into maltobionic acid through an enzymatic oxidation reaction, the amount of carbohydrate oxidase added is 4.0 to 4.3% based on the weight of saccharide solids in the high-purity maltose solution. (v/w), and the amount of catalase added is 0.2% (v/w) compared to the weight of saccharide solids in the high-purity maltose solution, and the enzyme oxidation reaction temperature is 25~35℃, the enzyme oxidation reaction time at which the conversion rate is close to 100%. was 15 to 20 hr. On the other hand, when the amount of carbohydrate oxidase added was 3.3 to 3.8% (v/w) based on the weight of saccharide solids in the high-purity maltose solution, the conversion rate did not reach 90% regardless of other conditions.
(3) 정제 조건 확립(3) Establishment of purification conditions
고순도 말토스 제품(제조사 : Junsei Chemical Co.,Ltd.)을 이온교환수로 희석하여 고형분 농도가 20 브릭스(Brix)인 고순도 말토스 용액을 제조하였다. 당류 산화효소인 카보하이드레이트옥시다제(Carbohydrate oxidase; 공급자 : Novozymes) 및 카탈라제(Catalase; 공급자 : Novozymes)를 각각 고순도 말토스 용액의 당류 고형분 중량 대비 4.3%(v/w) 및 0.2%(v/w)의 양으로 첨가하고 반응 온도 25℃, 에어레이션(aeration) 속도 1 vvm 및 교반 속도 200 rpm의 조건에서 24 hr 동안 효소 산화반응을 진행시켰다. 효소 산화반응이 진행되는 동안 1M 농도의 탄산수소나트륨(NaHCO3) 용액을 이용하여 반응 pH를 약 7로 일정하게 유지시켰다. 이후, 반응 산물 용액을 고형분 중량 대비 4 중량%에 해당하는 양의 활성탄으로 처리하여 탈색하고 0.2 ㎛ 크기의 다공성 규조토로 여과하여 말토비온산 함유 당류 용액을 수득하였다.A high-purity maltose product (manufacturer: Junsei Chemical Co., Ltd.) was diluted with ion-exchanged water to prepare a high-purity maltose solution with a solid concentration of 20 Brix. Carbohydrate oxidase (Supplier: Novozymes) and Catalase (Supplier: Novozymes), which are saccharide oxidase enzymes, were used at 4.3% (v/w) and 0.2% (v/w), respectively, based on the weight of saccharide solids in the high-purity maltose solution. ) and the enzymatic oxidation reaction was carried out for 24 hr under the conditions of a reaction temperature of 25°C, an aeration rate of 1 vvm, and a stirring rate of 200 rpm. While the enzyme oxidation reaction was in progress, the reaction pH was kept constant at about 7 using a 1M sodium bicarbonate (NaHCO 3 ) solution. Thereafter, the reaction product solution was treated with activated carbon in an amount equivalent to 4% by weight based on the solid content to decolorize and filtered through 0.2 ㎛ porous diatomaceous earth to obtain a saccharide solution containing maltobionic acid.
고형분 농도가 20 브릭스(Brix)인 고순도 말토스 용액, 효소 산화반응/탈색/규조토 여과를 거친 말토비온산 함유 당류 용액 및 상업적인 말토비온산 제품(제조사 : Carbosynth)의 고형분 농도가 20 브릭스(Brix)인 용액의 pH, 전도도를 비교하였고 그 결과를 하기 표 7에 정리하였다.High-purity maltose solution with a solids concentration of 20 Brix, a saccharide solution containing maltobionic acid that has undergone enzymatic oxidation/decolorization/diatomaceous earth filtration, and a commercial maltobionic acid product (manufacturer: Carbosynth) with a solids concentration of 20 Brix. The pH and conductivity of the phosphorus solutions were compared, and the results are summarized in Table 7 below.
상기 표 7에서 보이는 바와 같이 고형분 농도가 20 브릭스(Brix)인 고순도 말토스 용액을 효소 산화반응시켜 말토비온산으로 전환하고 탈색 및 규조토 여과를 거쳐 수득한 말토비온산 함유 당류 용액은 상업적인 말토비온산에 비해 상당량의 불순물이 존재하였다.As shown in Table 7, a high-purity maltose solution with a solid content concentration of 20 Brix was converted to maltobionic acid through an enzymatic oxidation reaction, and the maltobionic acid-containing saccharide solution obtained through decolorization and diatomaceous earth filtration was commercially available as maltobionic acid. Compared to that, a significant amount of impurities existed.
수득한 말토비온산 함유 당류 용액을 다양한 조합의 이온교환수지에 통과시켜 정제된 말토비온산 함유 당류 용액을 수득하였고 pH, 전도도 변화 등을 관찰하였다. 사용한 양이온 교환수지 및 음이온 교환수지의 종류 및 정보는 다음과 같다.The obtained maltobionic acid-containing saccharide solution was passed through various combinations of ion exchange resins to obtain a purified maltobionic acid-containing saccharide solution, and changes in pH and conductivity were observed. The types and information on the cation exchange resin and anion exchange resin used are as follows.
* TRILITE SCR-B : 겔(Gel) 타입 강산성 양이온 교환수지* TRILITE SCR-B: Gel type strongly acidic cation exchange resin
* LEWATIT S4268 : 매크로다공성(Macroporous) 타입 강염기성 음이온 교환수지* LEWATIT S4268: Macroporous type strongly basic anion exchange resin
* LEWATIT S6268 : 겔(Gel) 타입 강염기성 음이온 교환수지* LEWATIT S6268: Gel type strongly basic anion exchange resin
말토비온산 함유 당류 용액을 제조하는 과정에서 pH 조절제로 사용한 탄산수소나트륨은 액상에서 나트륨 이온, 이산화탄소 및 물(water)로 존재하기 때문에 말토비온산 함유 당류 용액을을 강산성 양이온 교환수지에만 통과시켜 정제를 실시하였다. 그 결과 정제된 정제된 말토비온산 함유 당류 용액은 상업적인 말토비온산 제품에 비해 pH는 낮고 전도도는 상당히 높았다. 말토비온산 함유 당류 용액을 제조하는 과정에서 pH 조절제로 사용한 탄산수소나트륨 외에도 반응시 생성되는 불순물이 존재할 수 있기 때문에 다양한 조합의 이온교환수지를 사용하여 말토비온산 함유 당류 용액을 정제하였다. 일반적으로 이온교환수지에 의한 정제는 원액을 양이온 교환수지 및 음이온 교환수지에 순차적으로 통과시켜 실시하지만 말토비온산 함유 당류 용액은 양이온 교환수지에 통과시키면 말토비온산이 음이온 형태로 존재할 수 있고, 음이온 형태로 존재하는 말토비온산은 음이온 교환수지에 흡착되어 제거될 여지가 있다. 따라서, 본 발명자는 말토비온산 함유 당류 용액을 음이온 교환수지 및 양이온 교환수지에 순차적으로 통과시켜 정제를 실시하였다. 다만, 양이온 교환수지를 통과한 말토비온산 함유 당류 용액은 다소 낮은 pH 값을 보일 수 있기 때문에 이후 혼상 이온교환수지에 추가적으로 통과시켜 정제하였다. 말토비온산 함유 당류 용액의 통액 속도(Space Volume)은 3 ㎖/min 이었다.Sodium bicarbonate, which was used as a pH adjuster in the process of manufacturing the maltobionic acid-containing saccharide solution, exists as sodium ions, carbon dioxide, and water in the liquid phase, so the maltobionic acid-containing saccharide solution is purified by passing it only through a strongly acidic cation exchange resin. was carried out. As a result, the purified maltobionic acid-containing saccharide solution had lower pH and significantly higher conductivity than commercial maltobionic acid products. In the process of preparing the maltobionic acid-containing saccharide solution, in addition to the sodium bicarbonate used as a pH adjuster, impurities generated during the reaction may exist, so various combinations of ion exchange resins were used to purify the maltobionic acid-containing saccharide solution. In general, purification using an ion exchange resin is performed by sequentially passing the raw solution through a cation exchange resin and an anion exchange resin. However, when a saccharide solution containing maltobionic acid is passed through a cation exchange resin, maltobionic acid may exist in the form of an anion, and the anion may exist in the form of an anion. Maltobionic acid present in the form may be adsorbed on an anion exchange resin and removed. Therefore, the present inventor purified the maltobionic acid-containing saccharide solution by sequentially passing it through an anion exchange resin and a cation exchange resin. However, since the maltobionic acid-containing saccharide solution that passed through the cation exchange resin may have a somewhat low pH value, it was purified by additionally passing through a mixed bed ion exchange resin. The space volume of the maltobionic acid-containing saccharide solution was 3 mL/min.
하기 표 8에 말토비온산 함유 당류 용액을 이온교환수지에 통과시켜 정제한 결과를 정리하였다.Table 8 below summarizes the results of purifying the maltobionic acid-containing saccharide solution by passing it through an ion exchange resin.
* 혼상수지1 : SCR-B 양이온 교환수지 및 S4268 음이온 교환수지를 4:1의 부피비로 혼합하여 제조함* Mixed phase resin 1: Manufactured by mixing SCR-B cation exchange resin and S4268 anion exchange resin at a volume ratio of 4:1.
* 혼상수지2 : SCR-B 양이온 교환수지 및 S6268 음이온 교환수지를 4:1의 부피비로 혼합하여 제조함* Mixed phase resin 2: Manufactured by mixing SCR-B cation exchange resin and S6268 anion exchange resin at a volume ratio of 4:1.
* 정제 용량 : 말토비온산 함유 당류 용액 처리량/이온교환수지 부피* Purification capacity: Maltobionic acid-containing saccharide solution treatment volume/ion exchange resin volume
상기 표 8에서 보이는 바와 같이 정제된 말토비온산 함유 당류 용액의 pH, 전도도 및 정제 용량을 고려할 때 말토비온산 함유 당류 용액을 부피비가 1:1:1인 매크로다공성(Macroporous) 타입 강염기성 음이온 교환수지:겔(Gel) 타입 강산성 양이온 교환수지: 혼상수지(상기 강산성 양이온 교환수지 및 상기 강염기성 음이온 교환수지를 4:1의 부피비로 혼합한 수지)에 순차적으로 통과시켜 정제하는 것이 바람직할 것으로 판단된다.As shown in Table 8, considering the pH, conductivity, and purification capacity of the purified maltobionic acid-containing saccharide solution, the maltobionic acid-containing saccharide solution was used in a macroporous type strongly basic anion exchanger with a volume ratio of 1:1:1. Resin: Gel type strongly acidic cation exchange resin: It is considered desirable to purify by sequentially passing through a mixed phase resin (a resin in which the strongly acidic cation exchange resin and the strongly basic anion exchange resin are mixed at a volume ratio of 4:1). do.
(4) 고형화 조건 확립(4) Establishment of solidification conditions
고순도 말토스 제품(제조사 : Junsei Chemical Co.,Ltd.)을 이온교환수로 희석하여 고형분 농도가 20 브릭스(Brix)인 고순도 말토스 용액을 제조하였다. 당류 산화효소인 카보하이드레이트옥시다제(Carbohydrate oxidase; 공급자 : Novozymes) 및 카탈라제(Catalase; 공급자 : Novozymes)를 각각 고순도 말토스 용액의 당류 고형분 중량 대비 4.3%(v/w) 및 0.2%(v/w)의 양으로 첨가하고 반응 온도 25℃, 에어레이션(aeration) 속도 1 vvm 및 교반 속도 200 rpm의 조건에서 24 hr 동안 효소 산화반응을 진행시켰다. 효소 산화반응이 진행되는 동안 1M 농도의 탄산수소나트륨(NaHCO3) 용액을 이용하여 반응 pH를 약 7로 일정하게 유지시켰다. 이후, 반응 산물 용액을 고형분 중량 대비 4 중량%에 해당하는 양의 활성탄으로 처리하여 탈색하고 0.2 ㎛ 크기의 다공성 규조토로 여과하여 말토비온산 함유 당류 용액을 수득하였다. 이후, 말토비온산 함유 당류 용액을 부피비가 1:1:1인 매크로다공성(Macroporous) 타입 강염기성 음이온 교환수지:겔(Gel) 타입 강산성 양이온 교환수지: 혼상수지(상기 강산성 양이온 교환수지 및 상기 강염기성 음이온 교환수지를 4:1의 부피비로 혼합한 수지)에 순차적으로 통과시켜 정제된 말토비온산 함유 당류 용액을 수득하였다. 정제된 말토비온산 함유 당류 용액은 당류 고형분 전체 중량을 기준으로 말토비온산(Maltobionic acid) 약 96 중량%, 말토스(Maltose) 약 3 중량% 및 당류 부산물 약 1 중량%로 이루어졌다.A high-purity maltose product (manufacturer: Junsei Chemical Co., Ltd.) was diluted with ion-exchanged water to prepare a high-purity maltose solution with a solid concentration of 20 Brix. Carbohydrate oxidase (Supplier: Novozymes) and Catalase (Supplier: Novozymes), which are saccharide oxidase enzymes, were used at 4.3% (v/w) and 0.2% (v/w), respectively, based on the weight of saccharide solids in the high-purity maltose solution. ) and the enzymatic oxidation reaction was carried out for 24 hr under the conditions of a reaction temperature of 25°C, an aeration rate of 1 vvm, and a stirring rate of 200 rpm. While the enzyme oxidation reaction was in progress, the reaction pH was kept constant at about 7 using a 1M sodium bicarbonate (NaHCO 3 ) solution. Thereafter, the reaction product solution was treated with activated carbon in an amount equivalent to 4% by weight based on the solid content to decolorize and filtered through 0.2 ㎛ porous diatomaceous earth to obtain a saccharide solution containing maltobionic acid. Thereafter, the maltobionic acid-containing saccharide solution was mixed with a macroporous type strongly basic anion exchange resin: gel type strongly acidic cation exchange resin: mixed phase resin (the strongly acidic cation exchange resin and the strong acid cation exchange resin) with a volume ratio of 1:1:1. A purified maltobionic acid-containing saccharide solution was obtained by sequentially passing it through a resin mixed with a basic anion exchange resin at a volume ratio of 4:1. The purified maltobionic acid-containing saccharide solution consisted of approximately 96% by weight of maltobionic acid, approximately 3% by weight of maltose, and approximately 1% by weight of saccharide by-products, based on the total weight of saccharide solids.
이후, 정제된 말토비온산 함유 당류 용액의 고형화를 1) 에탄올에 의한 침전, 2) 분무건조(Spray drying) 및 3) 냉각에 의한 결정화와 같이 3가지 방법으로 진행하고 그 결과를 비교하였다.Afterwards, the purified maltobionic acid-containing saccharide solution was solidified using three methods: 1) precipitation by ethanol, 2) spray drying, and 3) crystallization by cooling, and the results were compared.
1) 에탄올에 의한 침전1) Precipitation by ethanol
정제된 말토비온산 함유 당류 용액을 농축하여 고형분 농도가 약 80 브릭스(Brix)인 말토비온산 함유 당류 농축액을 제조하였다. 이후 순도가 약 98%인 에탄올에 말토비온산 함유 당류 농축액을 1:1의 부피비로 첨가하고 진탕 혼합하여 말토비온산의 침전을 유도하고 탈수하였으며 총 10회 이상 반복하였다. 상기 에탄올에 의한 말토비온산의 침전 유도는 에탄올의 사용량이 과다하고 에탄올의 제거도 쉽지 않으므로 경제성이 현저하게 떨어지는 것으로 나타났다.The purified maltobionic acid-containing saccharide solution was concentrated to prepare a maltobionic acid-containing saccharide concentrate with a solid content of about 80 Brix. Afterwards, maltobionic acid-containing saccharide concentrate was added to ethanol with a purity of about 98% at a volume ratio of 1:1 and mixed with shaking to induce precipitation of maltobionic acid and dehydration, which was repeated more than 10 times in total. The induction of precipitation of maltobionic acid by ethanol was found to be significantly less economical because the amount of ethanol used was excessive and ethanol was not easy to remove.
2) 분무건조(Spray drying)2) Spray drying
정제된 말토비온산 함유 당류 용액을 농축하여 고형분 농도가 약 35 브릭스(Brix)인 말토비온산 함유 당류 농축액을 제조하였다. 이후, 분무건조기의 주입구 온도(Inlet temperature)와 아토마이저(Atomizer) 회전 속도를 조절하면서 말토비온산 함유 당류 농축액의 분무건조를 실시하였다.The purified maltobionic acid-containing saccharide solution was concentrated to prepare a maltobionic acid-containing saccharide concentrate with a solid content of about 35 Brix. Afterwards, spray drying of the maltobionic acid-containing saccharide concentrate was performed while controlling the inlet temperature of the spray dryer and the rotation speed of the atomizer.
말토스(Maltose)의 녹는점은 102℃이고 이를 통해 말토비온산의 녹는점을 100℃ 이하로 예상하고, 처음 분무건조 실시 조건을 주입구 온도(Inlet temperature) 125℃, 출구 온도(Outlet temperature) 85~90℃ 및 아토마이저(Atomizer) 회전 속도 500~1000 rpm의 범위로 설정하였다. 그러나, 주입구 온도(Inlet temperature) 125℃의 조건에서 아토마이저(Atomizer)를 통해 분무된 말토비온산 함유 당류 농축액은 분말화가 되지 않고 액상 형태로 분무건조기 내부 챔부 벽에 존재하였다. 이후, 주입구 온도(Inlet temperature)를 말토스의 녹는점보다 훨씬 높은 200℃로 설정하고 출구 온도(Outlet temperature) 145℃ 및 아토마이저(Atomizer) 회전 속도 500 rpm의 조건에서 말토비온산 함유 당류 농축액의 분무건조를 실시하였다. 그 결과 말토비온산 함유 당류 농축액의 분말화가 이루어지지 않았다. 다시, 분무건조 실시 조건을 주입구 온도(Inlet temperature) 150℃, 출구 온도(Outlet temperature) 121℃ 및 아토마이저(Atomizer) 회전 속도 700 rpm 및 말토비온산 함유 당류 농축액 주입속도 700 ㎖/hour으로 설정하였다. 또한, 말토비온산 함유 당류 농축액은 고형분 농도가 15 브릭스(Brix)와 35 브릭스(Brix)인 2가지 종류를 사용하였다. 그 결과 말토비온산 함유 당류 농축액의 분말화가 원활하게 이루어졌다. 최종적으로 말토비온산 함유 당류 농축액의 분무건조 조건을 다음과 같이 결정하였다.The melting point of maltose is 102℃, and from this, the melting point of maltobionic acid is expected to be below 100℃, and the initial spray drying conditions are inlet temperature of 125℃ and outlet temperature of 85℃. It was set at ~90°C and the atomizer rotation speed was in the range of 500~1000 rpm. However, the maltobionic acid-containing saccharide concentrate sprayed through an atomizer at an inlet temperature of 125°C was not powdered but existed in liquid form on the inner chamber wall of the spray dryer. Afterwards, the inlet temperature was set to 200°C, which is much higher than the melting point of maltose, and the saccharide concentrate containing maltobionic acid was purified under the conditions of an outlet temperature of 145°C and an atomizer rotation speed of 500 rpm. Spray drying was performed. As a result, the maltobionic acid-containing saccharide concentrate was not powdered. Again, the spray drying conditions were set to an inlet temperature of 150°C, an outlet temperature of 121°C, an atomizer rotation speed of 700 rpm, and a maltobionic acid-containing saccharide concentrate injection speed of 700 ml/hour. . In addition, two types of maltobionic acid-containing saccharide concentrates with solid concentrations of 15 Brix and 35 Brix were used. As a result, the maltobionic acid-containing saccharide concentrate was smoothly powdered. Finally, the spray drying conditions for the maltobionic acid-containing saccharide concentrate were determined as follows.
* 말토비온산 함유 당류 농축액의 고형분 농도 : 15~35 브릭스(Brix)* Solids concentration of maltobionic acid-containing saccharide concentrate: 15~35 Brix
* 말토비온산 함유 당류 농축액 주입 속도 : 700 ㎖/hour* Maltobionic acid-containing saccharide concentrate injection rate: 700 mL/hour
* 주입구 온도(Inlet temperature) : 150℃* Inlet temperature: 150℃
* 출구 온도(Outlet temperature) : 121℃* Outlet temperature: 121℃
* 아토마이저(Atomizer) 회전 속도 : 700 rpm* Atomizer rotation speed: 700 rpm
3) 냉각에 의한 결정화3) Crystallization by cooling
정제된 말토비온산 함유 당류 용액을 농축하여 고형분 농도가 약 90 브릭스(Brix)인 말토비온산 함유 당류 농축액을 제조하였다. 이후, 말토비온산 함유 당류 농축액에 말토비온산 종정(種晶; seed crystal)을 말토비온산 함유 당류 농축액 중량 대비 20%까지 첨가하고 온도를 90℃에서 15℃까지 서서히 낮추어 결정화를 시도하였다. 그러나, 말토비온산은 흡습력이 강해 상온에서도 녹는 특성이 있고, 이로 인해 높은 농도에서도 냉각에 의한 결정화가 이루어지지 않았다.The purified maltobionic acid-containing saccharide solution was concentrated to prepare a maltobionic acid-containing saccharide concentrate with a solid content of about 90 Brix. Afterwards, maltobionic acid seed crystals were added up to 20% of the weight of the maltobionic acid-containing saccharide concentrate, and crystallization was attempted by gradually lowering the temperature from 90°C to 15°C. However, maltobionic acid has a strong hygroscopic ability and melts even at room temperature, and as a result, crystallization by cooling did not occur even at high concentrations.
이상에서와 같이 본 발명을 실시예를 통해 설명하였지만 본 발명이 반드시 여기에만 한정되는 것은 아니며 본 발명의 범주와 사상을 벗어나지 않는 범위 내에서 다양한 변형실시가 가능함은 물론이다. 따라서, 본 발명의 보호범위는 본 발명에 첨부된 특허청구의 범위에 속하는 모든 실시 형태를 포함하는 것으로 해석되어야 한다.As described above, the present invention has been described through examples, but the present invention is not necessarily limited thereto, and various modifications may be made without departing from the scope and spirit of the present invention. Accordingly, the scope of protection of the present invention should be interpreted to include all embodiments falling within the scope of the patent claims attached to the present invention.
Claims (8)
(b) 상기 말토스 함유 당류 용액에 당류 고형분 중량 대비 카보하이드레이트옥시다제(Carbohydrate oxidase) 4.0~5.0%(v/w) 및 카탈라제(Catalase) 0.1~0.8%(v/w)를 첨가하고 22~37℃의 온도 조건, 6~8의 pH 조건, 0.9~1.5 vvm의 에어레이션(aeration) 속도 조건 및 100~400 rpm의 교반 속도 조건으로 효소 산화반응을 15~30 hr 동안 진행시켜 반응 산물 함유 용액을 수득하는 단계를 포함하는 방법으로서,
상기 pH 조건은 약염기성 물질 또는 약염기성 물질 용액에 의해 조절되는 것을 특징으로 하는 말토비온산 제조방법.
(a) preparing a maltose-containing saccharide solution having a solid concentration of 15 to 25 Brix and a maltose content of 90% by weight or more based on the total weight of saccharide solids; and
(b) Add 4.0-5.0% (v/w) of carbohydrate oxidase and 0.1-0.8% (v/w) of catalase based on the weight of saccharide solids to the maltose-containing saccharide solution and The enzymatic oxidation reaction was carried out for 15 to 30 hr under a temperature condition of 37°C, a pH condition of 6 to 8, an aeration rate of 0.9 to 1.5 vvm, and a stirring speed of 100 to 400 rpm to prepare a solution containing the reaction product. As a method comprising the step of obtaining,
A method for producing maltobionic acid, wherein the pH conditions are controlled by a weakly basic substance or a weakly basic substance solution.
The method for producing maltobionic acid according to claim 1, wherein the weakly basic substance is sodium bicarbonate.
The method for producing maltobionic acid according to claim 1, further comprising the step of (c) treating the solution containing the reaction product with activated carbon to decolorize and filter it to obtain a saccharide solution containing maltobionic acid.
The method for producing maltobionic acid according to claim 3, wherein the filtration is performed using a porous diatomaceous earth filter medium.
상기 혼상 이온교환수지는 강산성 양이온 교환수지 및 강염기성 음이온 교환수지를 혼합한 수지인 것을 특징으로 하는, 말토비온산 제조방법.
The method of claim 3, comprising (d) sequentially passing the maltobionic acid-containing saccharide solution through a strongly basic anion exchange resin, a strongly acidic cation exchange resin, and a mixed-bed ion exchange resin to obtain a purified maltobionic acid-containing saccharide solution. Contains more,
A method for producing maltobionic acid, wherein the mixed-phase ion exchange resin is a resin that is a mixture of a strongly acidic cation exchange resin and a strongly basic anion exchange resin.
The method of claim 5, wherein the strongly basic anion exchange resin is a macroporous type, the strongly acidic cation exchange resin is a gel type, and the strongly basic anion exchange resin: strongly acidic cation exchange resin: mixed bed ion exchange resin. The volume ratio is 1:(0.25~2):(0.25~1), and the mixing volume ratio of the strongly acidic cation exchange resin:strongly basic anion exchange resin constituting the mixed phase ion exchange resin is 1:(0.1~0.5). Method for producing maltobionic acid.
상기 말토비온산 함유 당류 용액을 분무건조기 내부 챔버로 주입하는 통로인 주입구 온도는 135~170℃이고 아토마이저(Atomizer) 회전 속도는 600~900 rpm인 것을 특징으로 하는 말토비온산 제조방법.
The method of claim 5, (e) after adjusting the solid concentration of the purified maltobionic acid-containing saccharide solution to 10 to 40 Brix, it is injected into the inner chamber of the spray dryer through an atomizer and powdered. Further comprising the step of obtaining purified maltobionic acid,
A method for producing maltobionic acid, wherein the temperature of the injection port, which is a passage through which the maltobionic acid-containing saccharide solution is injected into the inner chamber of the spray dryer, is 135 to 170 ° C, and the atomizer rotation speed is 600 to 900 rpm.
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