JPH03262495A - Production of highly purified sialic acid of liberating type - Google Patents
Production of highly purified sialic acid of liberating typeInfo
- Publication number
- JPH03262495A JPH03262495A JP2059860A JP5986090A JPH03262495A JP H03262495 A JPH03262495 A JP H03262495A JP 2059860 A JP2059860 A JP 2059860A JP 5986090 A JP5986090 A JP 5986090A JP H03262495 A JPH03262495 A JP H03262495A
- Authority
- JP
- Japan
- Prior art keywords
- sialic acid
- sialidase
- milky
- immobilized
- acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 title claims abstract description 45
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 title claims abstract description 44
- 238000004519 manufacturing process Methods 0.000 title claims description 7
- 102000005348 Neuraminidase Human genes 0.000 claims abstract description 24
- 108010006232 Neuraminidase Proteins 0.000 claims abstract description 24
- 239000000126 substance Substances 0.000 claims abstract description 21
- 125000005629 sialic acid group Chemical group 0.000 claims abstract description 11
- 238000000909 electrodialysis Methods 0.000 claims description 6
- 238000011033 desalting Methods 0.000 abstract description 7
- 235000013336 milk Nutrition 0.000 abstract description 6
- 239000008267 milk Substances 0.000 abstract description 6
- 210000004080 milk Anatomy 0.000 abstract description 6
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 4
- 230000007062 hydrolysis Effects 0.000 abstract description 4
- 238000000502 dialysis Methods 0.000 abstract description 3
- 238000000034 method Methods 0.000 description 15
- 239000000843 powder Substances 0.000 description 8
- 239000007858 starting material Substances 0.000 description 8
- 238000000108 ultra-filtration Methods 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 239000005862 Whey Substances 0.000 description 4
- 102000007544 Whey Proteins Human genes 0.000 description 4
- 108010046377 Whey Proteins Proteins 0.000 description 4
- 229920001429 chelating resin Polymers 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 125000000962 organic group Chemical group 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 235000014633 carbohydrates Nutrition 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000003100 immobilizing effect Effects 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 150000007524 organic acids Chemical class 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229930186217 Glycolipid Natural products 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 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 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 235000008452 baby food Nutrition 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 235000013351 cheese Nutrition 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 239000006071 cream Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 235000005985 organic acids Nutrition 0.000 description 2
- 239000012466 permeate Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 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
- 229920001817 Agar Polymers 0.000 description 1
- 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 1
- 231100000699 Bacterial toxin Toxicity 0.000 description 1
- 241000186000 Bifidobacterium Species 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- JOYRKODLDBILNP-UHFFFAOYSA-N Ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 239000003957 anion exchange resin Substances 0.000 description 1
- 239000000688 bacterial toxin Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 235000015155 buttermilk Nutrition 0.000 description 1
- 239000000648 calcium alginate Substances 0.000 description 1
- 235000010410 calcium alginate Nutrition 0.000 description 1
- 229960002681 calcium alginate Drugs 0.000 description 1
- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 235000020247 cow milk Nutrition 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 125000002795 guanidino group Chemical group C(N)(=N)N* 0.000 description 1
- 230000035931 haemagglutination Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 125000002883 imidazolyl group Chemical group 0.000 description 1
- 125000001041 indolyl group Chemical group 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000005373 porous glass Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 125000001174 sulfone group Chemical group 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、結合型シアル酸類を含有する乳質物質を出発
原料とし、固定化シアリダーゼで処理することによって
、高純度の遊離型シアル酸を製造する方法に関する。Detailed Description of the Invention (Industrial Application Field) The present invention is a method for producing highly pure free sialic acid by treating a milky substance containing bound sialic acids as a starting material with immobilized sialidase. Regarding how to.
（従来の技術）
シアル酸はシアリン酸とも称せられ、ノイラ旦ン酸のア
シル誘導体の総称である。(Prior Art) Sialic acid is also called sialic acid, and is a general term for acyl derivatives of neuradanic acid.
シアル酸は、例えばウィルスによる赤血球凝集反応、細
菌毒素の中和、血液蛋白質の血中半減期制御などに関与
することが知られており、生理的に重要な役割を果たし
ている物質である。Sialic acid is known to be involved in, for example, the hemagglutination reaction caused by viruses, the neutralization of bacterial toxins, and the control of the blood half-life of blood proteins, and is a substance that plays a physiologically important role.
また、シアル酸は糖蛋白質や糖脂質のような複合糖質の
非還元末端に結合している酸性糖質であることから、糖
蛋白質や糖脂質の合成原料としても広く利用されるよう
になってきた。In addition, since sialic acid is an acidic carbohydrate that binds to the non-reducing end of complex carbohydrates such as glycoproteins and glycolipids, it has become widely used as a raw material for the synthesis of glycoproteins and glycolipids. It's here.
このシアル酸は、ヒトやウシ等の乳汁中に存在する蛋白
質、脂質及び糖質と結合した結合型シアル酸類として存
在しており、遊離型シアル酸としては殆ど存在しない。This sialic acid exists as bound sialic acids bound to proteins, lipids, and carbohydrates present in the milk of humans, cows, etc., and hardly exists as free sialic acid.
従来、乳質物質を出発原料として用い、遊離型シアル酸
を分離、精製する方法としては、乳質物質に塩酸や硫酸
、或いは有機酸を加えてｐｔ＋を下げ、８０℃前後の温
度で３０〜６０分間程度の加水分解反応を行うことによ
り、遊離型シアル酸を調製する方法が提案されている（
特開昭６４−４０４９１）。Conventionally, the method of separating and purifying free sialic acid using milky substances as a starting material involves adding hydrochloric acid, sulfuric acid, or an organic acid to the milky substance to lower pt+, and then heating at a temperature of around 80°C for 30 to 60 minutes. A method for preparing free sialic acid by carrying out a hydrolysis reaction of a certain degree has been proposed (
Japanese Patent Publication No. 64-40491).
しかし、酸性状態下で長時間反応が行われる為、出発物
質として用いる乳質物質は変性を起こして再利用出来な
くなるという問題がある。However, since the reaction is carried out under acidic conditions for a long time, there is a problem in that the milky substance used as a starting material is denatured and cannot be reused.
一方、ビフィズス菌の増殖促進物質を含み抗原性を減少
させた乳児食の製造方法として、乳、乳製品にシアリダ
ーゼを作用させ、蛋白質に結合しているシアル酸を遊離
、除去する方法が開示されている（特開平２−２０２４
６）。On the other hand, as a method for producing infant food that contains a substance that promotes the growth of bifidobacteria and has reduced antigenicity, a method has been disclosed in which sialidase is applied to milk and dairy products to liberate and remove sialic acid bound to proteins. (Unexamined Japanese Patent Publication No. 2024-2024)
6).
しかし、上記の乳児食の製造方法では、高純度のシアル
酸を効率よく製造する方法については開示しておらず、
分離して除去したシアル酸については全く言及していな
い。However, the above infant food production method does not disclose a method for efficiently producing high-purity sialic acid.
No mention is made of the sialic acid that was separated and removed.
（発明が解決しようとする課題）
本発明は、上述した状況に鑑みなされたものであって、
結合型シアル酸類を含有する乳質物質を出発原料として
用い、固定化シアリダーゼで処理することによって、遊
離型シアル酸を高純度で効率よく、しかも出発原料とし
て用いる乳質物質の再利用を可能とした状態で製造する
ことの出来る方法を提供することを課題とする。(Problem to be solved by the invention) The present invention has been made in view of the above-mentioned situation, and includes:
By using a milk substance containing bound sialic acids as a starting material and treating it with immobilized sialidase, a state in which free sialic acid can be obtained efficiently with high purity, and the milk substance used as a starting material can be reused. The objective is to provide a method that can be manufactured using
（課題を解決するための手段）
本発明は、結合型シアル酸類を含有する乳質物質を固定
化シアリダーゼ（ノイラミニダーゼとも称する）で処理
してシアル酸を遊離させた後、脱塩し、電気透析により
精製処理してシアル酸を得ることを特徴とする。(Means for Solving the Problems) The present invention involves treating milky substances containing bound sialic acids with immobilized sialidase (also referred to as neuraminidase) to liberate sialic acids, and then desalting and electrodialysis. It is characterized by obtaining sialic acid through purification treatment.
本発明において、出発原料として用いることのできる結
合型シアル酸類を含有する乳質物質としては、牛乳、脱
脂乳、バターミルク、ホエーなどを挙げることが出来る
。In the present invention, milky substances containing bound sialic acids that can be used as starting materials include cow's milk, skim milk, buttermilk, whey, and the like.
まず、これらの乳質物質中に結合型シアル酸類として存
在するシアル酸を遊離させる為に、乳質物質を固定化シ
アリダーゼで加水分解する。加水分解に適した温度は２
０〜６０℃、好ましくは４０℃前後である。本発明のよ
うに、酸で加水分解せず、シアリダーゼで加水分解する
ことにより、出発原料の乳質物質を再利用することが可
能となる。また、酵素として固定化シアリダーゼを用い
ることにより、反応液と酵素を速やかに分離できる。First, in order to liberate the sialic acids present in these milky substances as bound sialic acids, the milky substances are hydrolyzed with immobilized sialidase. The temperature suitable for hydrolysis is 2
The temperature is 0 to 60°C, preferably around 40°C. As in the present invention, by hydrolyzing with sialidase instead of with acid, it becomes possible to reuse the milky substance as a starting material. Furthermore, by using immobilized sialidase as the enzyme, the reaction solution and the enzyme can be quickly separated.
ここで用いる固定化シアリダーゼは、通常の酵素固定化
技術を用い、シアリダーゼを固定化したものであればよ
い。The immobilized sialidase used here may be one obtained by immobilizing sialidase using a conventional enzyme immobilization technique.
シアリダーゼを固定化する方法としては、活性炭、セラ
イト、多孔質ガラスピーズ、多孔質セラもンク、ヒドロ
キシアパタイト、キチンなどにシアリダーゼを物理的に
吸着させる方法、イオン交換基を有する水不溶性担体に
シアリダーゼをイオン結合させる方法、シアリダーゼの
アミノ基、スルホン基、カルボキシル基、水酸基、グア
ニジノ基、イミダゾール基、インドール基などの残基に
担体を共有結合させる方法、グルタルアルデヒド、ヘキ
サメチレンジイソシアネートなどを用いてシアリダーゼ
を不溶化する方法、寒天ゲル、に−カラキチン、アルギ
ン酸カルシウム、光硬化性樹脂、ポリアクリルアミドゲ
ル、ウレタンポリマー、セルロースアセテート、ニトロ
セルロースなどにシアリダーゼを包埋する方法などを挙
げることが出来る。Methods for immobilizing sialidase include physically adsorbing sialidase onto activated carbon, celite, porous glass beads, porous seramonk, hydroxyapatite, chitin, etc., and immobilizing sialidase on a water-insoluble carrier having an ion exchange group. A method of ionic bonding, a method of covalently bonding a carrier to a residue such as an amino group, a sulfone group, a carboxyl group, a hydroxyl group, a guanidino group, an imidazole group, or an indole group of sialidase; Examples include a method of insolubilization, a method of embedding sialidase in agar gel, carachitin, calcium alginate, photocurable resin, polyacrylamide gel, urethane polymer, cellulose acetate, nitrocellulose, and the like.
乳質物質と固定化シアリダーゼの接触は、充填槽型カラ
ム、撹拌槽型カラム、回転型カラムなどを用いて行うと
よい。The contact between the milky substance and the immobilized sialidase is preferably carried out using a packed tank type column, a stirred tank type column, a rotating type column, or the like.
加水分解を完了して遊離型シアル酸を含む乳質物質は、
限外濾過装置によって高分子の蛋白質や脂質などと低分
子の遊離型シアル酸や乳糖、無機塩、有機酸などに分画
される。Milky substances that have completed hydrolysis and contain free sialic acid are
An ultrafiltration device separates it into high molecular weight proteins and lipids, and low molecular weight free sialic acid, lactose, inorganic salts, and organic acids.
濃縮液側高分子の蛋白質や脂質などは乳質物質の主成分
であり、しかも限外濾過処理によって脱塩されているの
で、濃縮液のまま、適当な濃度に希釈して、或いは乾燥
粉末化し、食品用素材として再利用することが出来る。Proteins and lipids in the polymers on the concentrate side are the main components of milky substances, and they have been desalted through ultrafiltration, so they can be diluted to an appropriate concentration as a concentrate, or made into a dry powder. It can be reused as a food material.
一方、透過液側低分子の遊離型シアル酸を含む溶液は電
気透析装置によって脱塩、脱酸される。On the other hand, the solution containing low molecular weight free sialic acid on the permeate side is desalted and deoxidized by an electrodialyzer.
即ち、遊離型シアル酸を含む溶液を電気透析装置の脱塩
槽側に供給し、濃縮槽側に脱イオン水を導入して処理を
行うことにより、無機塩及び有機酸が脱塩槽から濃縮槽
側へと移行する。この後、濃縮槽側に脱イオン水を導入
し、更に遊離型シアル酸を含む溶液を電気透析装置によ
って処理することにより、乳糖とも分離することが出来
る。That is, by supplying a solution containing free sialic acid to the desalting tank side of an electrodialysis device and introducing deionized water to the concentrating tank side, inorganic salts and organic acids are concentrated from the desalting tank. Move to the tank side. Thereafter, deionized water is introduced into the concentrating tank, and the solution containing free sialic acid is further treated with an electrodialyzer to separate it from lactose as well.
このようにして電気透析装置によって、処理した遊離型
シアル酸を含む溶液は、シアル酸塩の形で存在する為、
陽イオン交換クロマトグラフィーなどによって脱塩した
後、濃縮、乾燥して遊離型シアル酸粉末とする。Since the solution containing free sialic acid treated by the electrodialysis device in this way exists in the form of sialic acid salt,
After desalting by cation exchange chromatography or the like, it is concentrated and dried to obtain free sialic acid powder.
このようにして得られる遊離型シアル酸の粉末は白色を
呈し、チオバルビッール法による測定で９８〜９９％程
度の純度がある。The free sialic acid powder thus obtained is white in color and has a purity of about 98 to 99% as measured by the thiobarbir method.
（発明の効果）
本発明の方法によれば、高純度の遊離型シアル酸を製造
することができ、また、結合型シアル酸類の加水分解が
温和な条件で行われる為、出発原料として用いる乳質物
質を食品用の素材として、再利用することが出来る。(Effects of the Invention) According to the method of the present invention, highly pure free sialic acid can be produced, and since the hydrolysis of bound sialic acids is carried out under mild conditions, milk quality used as a starting material can be Materials can be reused as food materials.
従って、従来のように遊離型シアル酸を回収した後の乳
質物質の処分について考慮する必要がなくなる。Therefore, there is no need to consider the disposal of the milky substance after recovering the free sialic acid as in the past.
（実施例） 以下、実施例に基づき本発明を具体的に説明する。(Example) Hereinafter, the present invention will be specifically explained based on Examples.
実施例１
シアリダーゼをマクロ多孔性フェノールフォルマリン系
両性イオン交換樹脂（５０〜１００メツシユ）に選択特
異的に吸着させた固定化担体をジャケット付き２００１
カラムに充填した。Example 1 An immobilization carrier in which sialidase was selectively and specifically adsorbed on a macroporous phenol-formalin-based amphoteric ion exchange resin (50 to 100 meshes) was prepared using a jacketed 2001 immobilization carrier.
The column was packed.
チェダーチーズ製造時に生成したホエー４ｔを分離機に
通してクリームを分離した後、固定化担体を充填し、４
０℃に保持された上記のカラムにＳν４で通液してシア
ル酸を遊離させた。この時のｐｉは６．２であった。After passing 4 tons of whey produced during the production of cheddar cheese through a separator to separate the cream, it was filled with an immobilization carrier, and
Sialic acid was liberated by passing liquid through the above-mentioned column maintained at 0° C. at Sv4. The pi at this time was 6.2.
次に、遊離したシアル酸を含む溶液を分画分子量３０．
０００の限外濾過膜（東ソー社製、ＩＦ−３０ＰＳ）を
装着した限外濾過装置（東ソー社製、ＵＰ−１５００Ｍ
Ｅ、１５ｍ”）を用い、運転圧力５ Ｋｇ ｆａｃｔｓ
”、温度６０℃の運転条件で２０倍迄Ｓ縮した。このよ
うに限外濾過膜処理をして得られた遊離型シアル酸を含
む透過液３，８ｔを濃縮機（ヴイーガント社製、蒸発能
力６００１！／ｈ”）で５００ ／迄濃縮した後、電気
透析装置（徳山曹達社製）の脱塩槽側に入れ、電気伝導
度２ａ＋ｓ／ｃ＋＋＋迄脱塩、脱酸してから濃縮槽側の
溶液を廃棄した。次に、濃縮槽側に脱イオン水を入れ、
再度、電気伝導度５０ｐ３／ｃ１）まで透析して、濃縮
槽側に遊離型シアル酸を含む溶液５０１を得た。そして
減圧濃縮によって、５１迄濃縮し、凍結乾燥して約３０
０ｇの白色粉末を得た。Next, a solution containing liberated sialic acid with a molecular weight cutoff of 30.
Ultrafiltration device (manufactured by Tosoh Corporation, UP-1500M) equipped with 000 ultrafiltration membrane (manufactured by Tosoh Corporation, IF-30PS)
E, 15 m”), operating pressure 5 Kg facts
”, S was condensed up to 20 times under operating conditions at a temperature of 60°C. 3.8 tons of the permeate containing free sialic acid obtained by the ultrafiltration membrane treatment was transferred to a concentrator (manufactured by Vigand, evaporator). After concentrating to 500/h with a capacity of 6001!/h, it is placed in the desalination tank side of an electrodialysis machine (manufactured by Tokuyama Soda Co., Ltd.), and desalinated and deoxidized to an electrical conductivity of 2a+s/c+++, and then transferred to the concentration tank side. The solution was discarded. Next, add deionized water to the concentration tank side,
Dialysis was performed again to an electrical conductivity of 50 p3/c1) to obtain a solution 501 containing free sialic acid on the concentration tank side. Then, it was concentrated to 51% by vacuum concentration, and freeze-dried to about 30%
0 g of white powder was obtained.
このようにして得られた遊離型シアル酸粉末の純度は、
チオバルビッール法による測定で約９９％であった。The purity of the free sialic acid powder obtained in this way is
It was about 99% as measured by the thiobarbir method.
一方、限外濾過膜処理して得られた濃縮液を噴霧乾燥し
、粉末化した組成物の成分分析を行ったところ、約８０
％の蛋白質を含んでおり、極めて良好な栄養組成物とな
り得ることが判った。On the other hand, when the concentrated liquid obtained by ultrafiltration membrane treatment was spray-dried and the powdered composition was analyzed, it was found that approximately 80%
% of protein, and it was found that it could be an extremely good nutritional composition.
実施例２
シアリダーゼをカルボキシル基を持つポリスチレン多孔
性樹脂にカルボジイミドカップリング法によって、共有
結合させた固定化担体をジャケット付き１．０００１カ
ラムに充填した。Example 2 An immobilization carrier in which sialidase was covalently bonded to a polystyrene porous resin having a carboxyl group by a carbodiimide coupling method was packed into a jacketed 1.0001 column.
ゴーダチーズ製造時に生成したホエー１０ｔを分離機に
通してクリームを分離した後、固定化担体を充填し、４
０℃に保持された上記のカラムにＳＶ２で通液してシア
ル酸を遊離させた。この時のｐｌ（は５．５であった。After passing 10 tons of whey produced during the production of Gouda cheese through a separator to separate the cream, it was filled with an immobilization carrier, and
The sialic acid was released by passing the solution through the above column maintained at 0° C. at SV2. At this time, pl( was 5.5.
次に、遊離したシアル酸を含む溶液を、アンバーライト
ＩＲ−１２０Ｂ、 Ｈ”型（オルガノ社製）を充填した
容量１．０００１カラム及びアンバーライトｌＲ４００
、ＯＨ−型（オルガノ社製）を充填した容量１．５００
７ｉカラムに、順次ＳＶ５で通液して脱塩を行った。Next, the solution containing the liberated sialic acid was poured into a 1.0001 capacity column filled with Amberlite IR-120B, H” type (manufactured by Organo) and Amberlite 1R400 column.
, capacity 1.500 filled with OH-type (manufactured by Organo)
Desalting was performed by sequentially passing the solution through the 7i column at SV5.
こうして遊離したシアル酸を含む溶液の脱塩を行うと、
シアル酸は陰イオン交換樹脂に吸着されているので、水
酸化ナトリウムによる樹脂の再生の際に溶出させて、遊
離したシアル酸を含むアルカリ再生液１．５ｔを得、１
．５ｔの酸で中和した後、電気透析装置（徳山曹達社製
）の脱塩槽側に入れ、電気伝導度１ ｍｓ／ｃ＋ａ迄脱
塩、脱酸してから濃縮槽側の溶液を廃棄した。次に、濃
縮槽側に脱イオン水を入れ、再度、電気伝導度５０ｐｓ
／ｃｍ迄透析して、濃縮槽側に遊離型シアル酸を含む溶
液１００１を得た。そして減圧濃縮によって１ｏｌ迄濃
縮し、ＨＣＲ−阿２、Ｈ゛型（ダウエックス社製）を充
填した容量５Ｉ！カラムに通液し、凍結乾燥して約５０
０ｇの白色粉末を得た。When the solution containing the sialic acid released in this way is desalted,
Since sialic acid is adsorbed on the anion exchange resin, it is eluted during regeneration of the resin with sodium hydroxide to obtain 1.5 tons of alkaline regeneration solution containing liberated sialic acid.
．． After neutralizing with 5 tons of acid, the solution was placed in the desalting tank side of an electrodialysis machine (manufactured by Tokuyama Soda Co., Ltd.), and the solution was desalted and deoxidized to an electrical conductivity of 1 ms/c+a, and then the solution in the concentrating tank side was discarded. . Next, add deionized water to the concentration tank side and set the electrical conductivity to 50 ps again.
/cm to obtain a solution 1001 containing free sialic acid on the concentration tank side. Then, it was concentrated to 1 liter by vacuum concentration and filled with HCR-A2, H type (manufactured by DOWEX), with a volume of 5 I! The liquid is passed through the column and freeze-dried to approximately 50%
0 g of white powder was obtained.
このようにして得られた遊離型シアル酸粉末の純度は、
エーリッヒ法による測定で約９８．５％であった。The purity of the free sialic acid powder obtained in this way is
It was about 98.5% as measured by Ehrlich method.
なお、遊離したシアル酸を含む溶液を、アンバーライト
ＩＲ−１２０Ｂ、 Ｈ”型（オルガノ社製）を充填した
容量１，０００ Ｎカラム及びアンバーライトＩＲ−４
００、ＯＨ−型（オルガノ社製）を充填した容量１．５
００１カラムに順次ＳＶ５で通液して脱塩を行った、シ
アル酸を含まない溶液を常法によって濃縮、噴霧乾燥し
て、ホエー粉約６００ｋｇを得た。The solution containing the free sialic acid was prepared using a 1,000 N column filled with Amberlite IR-120B, H" type (manufactured by Organo) and Amberlite IR-4.
Capacity 1.5 filled with 00, OH- type (manufactured by Organo)
The sialic acid-free solution, which was desalted by sequentially passing through a 001 column at SV5, was concentrated and spray-dried in a conventional manner to obtain about 600 kg of whey powder.
Claims (1)
アリダーゼ（ノイラミニダーゼとも称する）で処理して
シアル酸を遊離させた後、脱塩し、電気透析により精製
処理してシアル酸を得ることを特徴とする高純度遊離型
シアル酸の製造方法。(1) Milky substances containing bound sialic acids are treated with immobilized sialidase (also referred to as neuraminidase) to release sialic acid, desalted, and purified by electrodialysis to obtain sialic acid. Features: A method for producing high-purity free sialic acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2059860A JPH03262495A (en) | 1990-03-13 | 1990-03-13 | Production of highly purified sialic acid of liberating type |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2059860A JPH03262495A (en) | 1990-03-13 | 1990-03-13 | Production of highly purified sialic acid of liberating type |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH03262495A true JPH03262495A (en) | 1991-11-22 |
Family
ID=13125357
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2059860A Pending JPH03262495A (en) | 1990-03-13 | 1990-03-13 | Production of highly purified sialic acid of liberating type |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH03262495A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2671696A1 (en) * | 1991-01-21 | 1992-07-24 | Snow Brand Milk Products Ltd | Method of manufacture of a composition containing compounds based on sialic acids |
| US5679645A (en) * | 1993-07-23 | 1997-10-21 | Snow Brand Milk Products Co., Ltd. | Sialic acid powder and process for the preparation thereof |
| WO2009000803A1 (en) * | 2007-06-25 | 2008-12-31 | Dsm Ip Assets B.V. | Novel prebiotics |
| WO2013042581A1 (en) * | 2011-09-20 | 2013-03-28 | 国立大学法人和歌山大学 | Process for producing novel sialo-sugar chain |
| JP2014181209A (en) * | 2013-03-19 | 2014-09-29 | Wakayama Univ | Method for producing novel (2→3) linked sialo-sugar chain |
| CN111087432A (en) * | 2020-01-10 | 2020-05-01 | 南京高新工大生物技术研究院有限公司 | Separation and extraction method of N-acetylneuraminic acid |
-
1990
- 1990-03-13 JP JP2059860A patent/JPH03262495A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2671696A1 (en) * | 1991-01-21 | 1992-07-24 | Snow Brand Milk Products Ltd | Method of manufacture of a composition containing compounds based on sialic acids |
| US5679645A (en) * | 1993-07-23 | 1997-10-21 | Snow Brand Milk Products Co., Ltd. | Sialic acid powder and process for the preparation thereof |
| WO2009000803A1 (en) * | 2007-06-25 | 2008-12-31 | Dsm Ip Assets B.V. | Novel prebiotics |
| JP2010531141A (en) * | 2007-06-25 | 2010-09-24 | ディーエスエム アイピー アセッツ ビー．ブイ． | New prebiotics |
| WO2013042581A1 (en) * | 2011-09-20 | 2013-03-28 | 国立大学法人和歌山大学 | Process for producing novel sialo-sugar chain |
| JPWO2013042581A1 (en) * | 2011-09-20 | 2015-07-30 | 国立大学法人 和歌山大学 | Method for producing novel sialo-glycan |
| US9290532B2 (en) | 2011-09-20 | 2016-03-22 | Wakayama University | Process for producing novel sialo-sugar chain |
| EP3009441A1 (en) * | 2011-09-20 | 2016-04-20 | Wakayama University | Process for producing novel sialo-sugar chain |
| US10975346B2 (en) | 2011-09-20 | 2021-04-13 | Wakayama University | Process for producing novel sialo-sugar chain |
| JP2014181209A (en) * | 2013-03-19 | 2014-09-29 | Wakayama Univ | Method for producing novel (2→3) linked sialo-sugar chain |
| CN111087432A (en) * | 2020-01-10 | 2020-05-01 | 南京高新工大生物技术研究院有限公司 | Separation and extraction method of N-acetylneuraminic acid |
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