JPH08252403A - Separating method of sialyllactose - Google Patents
Separating method of sialyllactoseInfo
- Publication number
- JPH08252403A JPH08252403A JP7061361A JP6136195A JPH08252403A JP H08252403 A JPH08252403 A JP H08252403A JP 7061361 A JP7061361 A JP 7061361A JP 6136195 A JP6136195 A JP 6136195A JP H08252403 A JPH08252403 A JP H08252403A
- Authority
- JP
- Japan
- Prior art keywords
- 4glc
- saα2
- smb
- sialyllactose
- 6galβ1
- 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.)
- Granted
Links
- OIZGSVFYNBZVIK-FHHHURIISA-N 3'-sialyllactose Chemical compound O1[C@@H]([C@H](O)[C@H](O)CO)[C@H](NC(=O)C)[C@@H](O)C[C@@]1(C(O)=O)O[C@@H]1[C@@H](O)[C@H](O[C@H]([C@H](O)CO)[C@H](O)[C@@H](O)C=O)O[C@H](CO)[C@@H]1O OIZGSVFYNBZVIK-FHHHURIISA-N 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 17
- 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 claims abstract description 16
- 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 claims abstract description 16
- 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 claims abstract description 10
- 238000013375 chromatographic separation Methods 0.000 claims abstract description 10
- 239000008101 lactose Substances 0.000 claims abstract description 10
- 239000002994 raw material Substances 0.000 claims abstract 2
- 239000003480 eluent Substances 0.000 claims description 10
- 239000003957 anion exchange resin Substances 0.000 claims description 9
- 150000003839 salts Chemical class 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 239000000243 solution Substances 0.000 description 11
- 239000000284 extract Substances 0.000 description 10
- 235000013336 milk Nutrition 0.000 description 10
- 239000008267 milk Substances 0.000 description 10
- 210000004080 milk Anatomy 0.000 description 10
- 239000000843 powder Substances 0.000 description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 239000008351 acetate buffer Substances 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 4
- 239000011347 resin Substances 0.000 description 4
- 229920005989 resin Polymers 0.000 description 4
- 239000013078 crystal Substances 0.000 description 3
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 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
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- 208000007882 Gastritis Diseases 0.000 description 1
- 241000590002 Helicobacter pylori Species 0.000 description 1
- 241000712431 Influenza A virus Species 0.000 description 1
- 206010058780 Meningitis neonatal Diseases 0.000 description 1
- 208000006816 Neonatal Sepsis Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 229920001429 chelating resin Polymers 0.000 description 1
- 210000003022 colostrum Anatomy 0.000 description 1
- 235000021277 colostrum Nutrition 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 235000020247 cow milk Nutrition 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 229940097043 glucuronic acid Drugs 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000006651 lactation Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 125000000962 organic group Chemical group 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000001294 propane Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、シアリルラクトース中
に混在しているシアル酸（ＳＡ）と乳糖（Ｇａｌβ１→
４Ｇｌｃ）とがα２→３結合したＳＡα２→３Ｇａｌβ
１→４Ｇｌｃ及びシアル酸（ＳＡ）と乳糖（Ｇａｌβ１
→４Ｇｌｃ）とがα２→６結合したＳＡα２→６Ｇａｌ
β１→４Ｇｌｃを結合様式の違いにより分離、回収する
方法に関する。TECHNICAL FIELD The present invention relates to sialic acid (SA) and lactose (Galβ1 →
4Glc) and α2 → 3 linked SAα2 → 3Galβ
1 → 4 Glc and sialic acid (SA) and lactose (Galβ1
→ 4Glc) and α2 → 6 linked SAα2 → 6Gal
The present invention relates to a method of separating and recovering β1 → 4Glc depending on the difference in binding mode.
【０００２】[0002]
【従来の技術】シアリルラクトースは、乳中に多く含ま
れていることが知られており、感染防御活性などの生理
活性を有していることが知られている。近年、このシア
リルラクトースについては、結合様式の違いによりその
生理的効果が異なることが解明されつつある。例えば、
シアル酸（ＳＡ）と乳糖（Ｇａｌβ１→４Ｇｌｃ）とが
α２→３結合したＳＡα２→３Ｇａｌβ１→４Ｇｌｃに
ついては、胃炎の原因菌といわれているカンピロバクタ
ー・ピロリ(Campylobacter pylori) の付着を阻害する
こと[Infect Immun., vol.56, pp.2896-2906, 1988] や
新生児の脳膜炎や敗血症の原因菌であるＳ型大腸菌の付
着を阻害すること[Acta Pediatr, vol.82,pp.6-11, 199
3] が報告されている。また、シアル酸（ＳＡ）と乳糖
（Ｇａｌβ１→４Ｇｌｃ）とがα２→６結合したＳＡα
２→６Ｇａｌβ１→４Ｇｌｃについては、Ａ型インフル
エンザ・ウイルスのレセプターであることが報告されて
いる[Nature, vol.333, pp.426-431, 1988] 。2. Description of the Related Art Sialyl lactose is known to be contained in milk in a large amount and is known to have physiological activities such as an infection protective activity. In recent years, it is becoming clear that sialyllactose has different physiological effects depending on the binding mode. For example,
Regarding SAα2 → 3Galβ1 → 4Glc in which sialic acid (SA) and lactose (Galβ1 → 4Glc) are α2 → 3 linked, inhibition of adhesion of Campylobacter pylori , which is said to be a causative bacterium of gastritis [Infect Immun., Vol.56, pp.2896-2906, 1988] or inhibiting the attachment of S-type Escherichia coli which is the causative bacterium of neonatal meningitis and sepsis [Acta Pediatr, vol.82, pp.6-11, 199]
3] has been reported. In addition, SAα in which sialic acid (SA) and lactose (Galβ1 → 4Glc) are α2 → 6 linked
2 → 6Galβ1 → 4Glc has been reported to be a receptor for influenza A virus [Nature, vol.333, pp.426-431, 1988].
【０００３】このシアリルラクトースについては、通
常、牛乳などの乳から分離、回収されることが多い。し
かし、このシアリルラクトース中には、結合様式の異な
るＳＡα２→３Ｇａｌβ１→４ＧｌｃとＳＡα２→６Ｇ
ａｌβ１→４Ｇｌｃとが混在しており、また、このＳＡ
α２→３Ｇａｌβ１→４ＧｌｃとＳＡα２→６Ｇａｌβ
１→４Ｇｌｃとの乳中での存在比率は生物種や泌乳期に
より大きく異なることが知られている。例えば、ヒト初
乳中に含まれるＳＡα２→３Ｇａｌβ１→４ＧｌｃとＳ
Ａα２→６Ｇａｌβ１→４Ｇｌｃとの存在比は約５：１
であるが、牛常乳中に含まれるＳＡα２→３Ｇａｌβ１
→４ＧｌｃとＳＡα２→６Ｇａｌβ１→４Ｇｌｃとの存
在比は約１：６である。The sialyllactose is usually separated and recovered from milk such as milk. However, in this sialyllactose, SAα2 → 3Galβ1 → 4Glc and SAα2 → 6G having different binding modes
alβ1 → 4Glc is mixed, and this SA
α2 → 3Galβ1 → 4Glc and SAα2 → 6Galβ
It is known that the abundance ratio of 1 → 4Glc in milk varies greatly depending on the species and the lactation period. For example, SAα2 → 3Galβ1 → 4Glc and S contained in human colostrum
The abundance ratio of Aα2 → 6Galβ1 → 4Glc is about 5: 1.
However, SAα2 → 3Galβ1 contained in normal cow milk
The abundance ratio of → 4Glc and SAα2 → 6Galβ1 → 4Glc is about 1: 6.
【０００４】本発明者らは、先に擬似移動床式クロマト
分離装置（ＳＭＢ）を用いて牛乳などの乳からシアリル
ラクトースを工業的規模で分離、回収する方法について
提案している [特願平5-252462号] 。The present inventors have previously proposed a method for separating and recovering sialyllactose from milk such as milk on an industrial scale by using a simulated moving bed chromatographic separator (SMB) [Japanese Patent Application No. 5-252462].
【０００５】牛乳などの乳から分離、回収したシアリル
ラクトースについて、さらに、その結合様式の違いによ
り分離する方法としては、樹脂にシアリルラクトースを
吸着させた後、樹脂を水洗し、塩濃度勾配溶出法により
分離する方法[Biochem.Biophys Acta, vol.130, pp.1-1
1, 1966]やシリカゲル樹脂を用い有機溶媒で分離する方
法などが知られているが、これらの方法は操作が非常に
煩雑であり、しかも、工業的規模で連続的に大量処理す
るのに適した方法とはいえない。したがって、工業的規
模で大量かつ簡便にシアリルラクトースを結合様式の違
いにより分離する方法が求められていた。The sialyllactose separated and recovered from milk such as milk can be further separated by the difference in the binding mode. After adsorbing sialyllactose on the resin, the resin is washed with water and then a salt concentration gradient elution method is used. Separation by [Biochem.Biophys Acta, vol.130, pp.1-1
1, 1966] and methods using silica gel resin for separation with an organic solvent are known, but these methods are extremely complicated to operate and are suitable for continuous large-scale processing on an industrial scale. It cannot be said that it was the method. Therefore, there has been a demand for a method for separating sialyllactose on a large scale and conveniently on an industrial scale according to the difference in the binding mode.
【０００６】[0006]
【発明が解決しようとする課題】本発明者らは、シアリ
ルラクトースを結合様式の違いにより分離する方法につ
いて、鋭意研究を進めたところ、擬似移動床式クロマト
分離装置（ＳＭＢ）を用いて処理することにより、従来
なし得なかった工業的規模で大量かつ簡便にシアリルラ
クトースを結合様式の違いにより分離することが出来る
ことを見出し、本発明を完成するに至った。したがっ
て、本発明は、擬似移動床式クロマト分離装置（ＳＭ
Ｂ）を用いてシアリルラクトースを結合様式の違いによ
り分離する方法を提供することを課題とする。DISCLOSURE OF THE INVENTION The inventors of the present invention have made intensive studies on a method for separating sialyllactose by the difference in the binding mode. As a result, it is processed using a simulated moving bed chromatographic separation device (SMB). As a result, they have found that sialyllactose can be easily separated on a large scale and on a commercial scale, which has never been possible before, by the difference in the binding mode, and have completed the present invention. Therefore, the present invention provides a simulated moving bed chromatographic separation device (SM
It is an object of the present invention to provide a method for separating sialyllactose by using B) according to the difference in the binding mode.
【０００７】[0007]
【課題を解決するための手段】本発明者らは、先に擬似
移動床式クロマト分離装置（ＳＭＢ）を用い酸性オリゴ
糖であるグルクロン酸含有オリゴ糖を連続的に分離する
方法について提案した[特開平6-253879号公報] 。ま
た、擬似移動床式クロマト分離装置（ＳＭＢ）を用いシ
アリルラクトースを連続的に分離する方法についても提
案している [特願平5-252462号] 。そして、これらの擬
似移動床式クロマト分離装置（ＳＭＢ）を用い糖質を連
続的に分離する方法を研究することにより蓄積した知見
を基に、シアリルラクトースを結合様式の違いにより分
離する方法について鋭意研究を重ねたところ、擬似移動
床式クロマト分離装置（ＳＭＢ）を用い、アニオン交換
樹脂を充填剤として処理条件を最適化することにより、
ＳＡα２→３Ｇａｌβ１→４ＧｌｃとＳＡα２→６Ｇａ
ｌβ１→４Ｇｌｃとを連続的に分離、回収することがで
きるということを見出した。[Means for Solving the Problems] The present inventors have previously proposed a method for continuously separating oligosaccharides containing glucuronic acid, which is an acidic oligosaccharide, using a simulated moving bed chromatographic separation device (SMB) [ JP-A-6-253879]. Also, a method for continuously separating sialyllactose using a simulated moving bed chromatographic separation device (SMB) is proposed [Japanese Patent Application No. 5-252462]. Based on the knowledge accumulated by studying the method for continuously separating sugars using these simulated moving bed chromatographic separation devices (SMB), the method for separating sialyllactose by the difference in the binding mode is earnestly studied. After repeated research, by using a simulated moving bed chromatographic separation device (SMB) and using anion exchange resin as a packing material to optimize the processing conditions,
SAα2 → 3Galβ1 → 4Glc and SAα2 → 6Ga
It has been found that 1β1 → 4Glc can be continuously separated and recovered.
【０００８】本発明では、シアリルラクトースを結合様
式の違いにより分離する際に擬似移動床式クロマト分離
装置（ＳＭＢ）を用いる。そして、ＳＭＢの充填剤とし
てアニオン交換樹脂を用いる。アニオン交換樹脂につい
ては、特に限定はないが、強塩基性アニオン交換樹脂Ｉ
型であり、好ましくは、架橋度２〜８％で対イオンが酢
酸型のものを用いると良い。なお、この強塩基性アニオ
ン交換樹脂Ｉ型の樹脂としては、アンバーライト XT-50
21（オルガノ社製）、Dowex 1x4 （ダウ・ケミカル社
製）、ダイヤイオン SAIIA (三菱化学社製）などが市販
されている。In the present invention, a simulated moving bed chromatographic separation device (SMB) is used when separating sialyllactose by the difference in the binding mode. Then, an anion exchange resin is used as a filler for SMB. The anion exchange resin is not particularly limited, but a strongly basic anion exchange resin I
It is preferable to use a type having a crosslinking degree of 2 to 8% and a counter ion of acetic acid type. As the strongly basic anion exchange resin type I resin, Amberlite XT-50
21 (manufactured by Organo), Dowex 1x4 (manufactured by Dow Chemical Co., Ltd.), Diaion SAIIA (manufactured by Mitsubishi Chemical Co., Ltd.) and the like are commercially available.
【０００９】ＳＭＢの溶離液については、pHが１〜10の
範囲のものであれば特に限定はないが、好ましくは、pH
が４〜５の酢酸緩衝液を用いると良い。なお、酢酸緩衝
液としては、酢酸ナトリウム緩衝液や酢酸アンモニウム
緩衝液などを例示できる。また、塩濃度が0.01〜1.5Mの
範囲のものであれば特に限定はないが、好ましくは、塩
濃度が0.05〜0.1Mのものを用いると良い。The eluent of SMB is not particularly limited as long as the pH is in the range of 1 to 10, but the pH is preferably pH.
It is advisable to use an acetic acid buffer solution of 4-5. Examples of the acetate buffer include sodium acetate buffer and ammonium acetate buffer. There is no particular limitation as long as the salt concentration is in the range of 0.01 to 1.5M, but it is preferable to use the salt concentration of 0.05 to 0.1M.
【００１０】擬似移動床式クロマト分離装置（ＳＭＢ）
を用い、このような条件で処理することで、工業的規模
で大量かつ簡便にシアリルラクトースを結合様式の違い
により分離することができる。Simulated Moving Bed Chromatographic Separator (SMB)
By using the above and treating under such conditions, sialyllactose can be easily separated on a large scale on an industrial scale according to the difference in the binding mode.
【００１１】以下に実施例を示し、本発明を具体的に説
明する。なお、シアリルラクトースについては、薄層ク
ロマトグラフィー（Art13927、メルク社製）により、ｎ
−プロパノール：アンモニア：水＝120:2.5:57.5の展開
液で展開し、レゾルシノールで発色させてクロマトスキ
ャナー（CS-930、島津製作所製）で定量した。The present invention will be described in detail below with reference to examples. Regarding sialyllactose, n was determined by thin layer chromatography (Art13927, manufactured by Merck).
-Propane: Ammonia: Water = 120: 2.5: 57.5 was developed with a developing solution, color was developed with resorcinol, and quantified with a chromatoscanner (CS-930, Shimadzu).
【００１２】[0012]
【参考例１】限外濾過装置で脱脂乳から蛋白質を除去
し、シーディングにより生成した乳糖結晶を除去した乳
糖結晶母液をエバポレーターで固形率30％となるまで濃
縮したものをＳＭＢ処理液とした。また、脱イオン水を
ＳＭＢ溶離液とした。なお、ＳＭＢは、カラム直径25mm
長さ460mm の８塔型で、各々、カチオン交換樹脂 UBK51
0L（三菱化学社製）の対イオンをＮａ型として充填した
ものを用いた。ＳＭＢの運転条件は、ＳＭＢ処理液供給
量3.4ml/分、ＳＭＢ溶離液供給量5.8ml/分、エキストラ
クト抜き出し量5.0ml/分、ラフィネート抜き出し量4.2m
l/分、カラム温度10℃、ステップ時間7.40分とした。[Reference Example 1] A protein was removed from skim milk by an ultrafiltration device, and a lactose crystal mother liquor from which lactose crystals produced by seeding were removed was concentrated with an evaporator to a solid content of 30%, which was used as an SMB treatment liquid. . Deionized water was used as the SMB eluent. SMB has a column diameter of 25 mm
Eight towers with a length of 460 mm, each with cation exchange resin UBK51
The one filled with 0 L (manufactured by Mitsubishi Chemical Co., Ltd.) of counter ion as Na type was used. The operating conditions of SMB are as follows: SMB processing solution supply rate 3.4 ml / min, SMB eluent supply rate 5.8 ml / min, extract withdrawal rate 5.0 ml / min, raffinate withdrawal rate 4.2 m
l / min, column temperature 10 ° C., step time 7.40 min.
【００１３】以上のＳＭＢ処理条件で乳糖結晶母液濃縮
液46.5リットルを処理したところ、ラフィネートにシア
リルラクトースを含む画分69.0リットルを得た。次に、
このラフィネートをロータリーエバポレーターで固形率
30％となるまで濃縮した後、再びＳＭＢ処理液とし、同
様の条件で処理した。このようにして得られたシアリル
ラクトース画分を濃縮し、凍結乾燥して純度99％のシア
リルラクトース900gを得た。When 46.5 liters of the lactose crystal mother liquor concentrate was treated under the above SMB treatment conditions, 69.0 liters of a fraction containing sialyllactose in raffinate was obtained. next,
Solid content of this raffinate on a rotary evaporator
After concentrating to 30%, it was again used as an SMB treatment liquid and treated under the same conditions. The sialyllactose fraction thus obtained was concentrated and freeze-dried to obtain 900 g of 99% pure sialyllactose.
【００１４】[0014]
【実施例１】参考例１のようにして得られた牛乳由来の
シアリルラクトース中に混在するＳＡα２→３Ｇａｌβ
１→４ＧｌｃとＳＡα２→６Ｇａｌβ１→４Ｇｌｃとの
分離を試みた。なお、ＳＡα２→３Ｇａｌβ１→４Ｇｌ
ｃとＳＡα２→６Ｇａｌβ１→４Ｇｌｃとの存在比は1
3：２であった。Example 1 SAα2 → 3Galβ mixed in sialyllactose derived from milk obtained as in Reference Example 1
An attempt was made to separate 1 → 4Glc and SAα2 → 6Galβ1 → 4Glc. SAα2 → 3Galβ1 → 4Gl
c and SAα2 → 6Galβ1 → 4Glc have an abundance ratio of 1
It was 3: 2.
【００１５】牛乳由来のシアリルラクトースを含む溶液
を 0.07M酢酸緩衝液(pH 4.5)で固形率が10％となるよう
希釈し、ＳＭＢ処理液とした。また、 0.07M酢酸緩衝液
(pH4.5)をＳＭＢ溶離液とした。なお、ＳＭＢは、カラ
ム直径30mm長さ500mm の８塔型で、各々、アニオン交換
樹脂 Dowex 1x4（ダウ・ケミカル社製）を充填したもの
を用いた。ＳＭＢの運転条件は、ＳＭＢ処理液供給量
1.64ml/分、ＳＭＢ溶離液供給量 3.99ml/分、エキスト
ラクト抜き出し量 2.82ml/分、ラフィネート抜き出し量
2.82ml/分、カラム温度15℃、ステップ時間159.56分と
した。A solution containing milk-derived sialyllactose was diluted with a 0.07M acetate buffer (pH 4.5) to a solid content of 10% to obtain an SMB-treated solution. Also, 0.07M acetate buffer
(pH 4.5) was used as the SMB eluent. The SMB was an 8-tower type column having a column diameter of 30 mm and a length of 500 mm, each filled with an anion exchange resin Dowex 1x4 (manufactured by Dow Chemical Co.). SMB operating conditions are SMB processing liquid supply
1.64 ml / min, SMB eluent supply 3.99 ml / min, extract withdrawal 2.82 ml / min, raffinate withdrawal
The column temperature was 2.82 ml / min, the column temperature was 15 ° C., and the step time was 159.56 min.
【００１６】以上のＳＭＢ処理条件でシアリルラクトー
ス含有溶液 7.1リットルを処理したところ、ラフィネー
トにＳＡα２→６Ｇａｌβ１→４Ｇｌｃを含む画分12.2
リットル及びエキストラクトにＳＡα２→３Ｇａｌβ１
→４ＧｌｃとＳＡα２→６Ｇａｌβ１→４Ｇｌｃとを含
む画分12.2リットルをそれぞれ得た。次に、このラフィ
ネート及びエキストラクトそれぞれをロータリーエバポ
レーター（柴田科学機械工業社製）で３リットルとなる
まで濃縮した後、電気透析装置（TS-2型、トクヤマ社
製）で脱塩した。そして、凍結乾燥し、純度99％のＳＡ
α２→３Ｇａｌβ１→４Ｇｌｃ粉末246g及びＳＡα２→
３Ｇａｌβ１→４ＧｌｃとＳＡα２→６Ｇａｌβ１→４
Ｇｌｃとの存在比が３：７の白色粉末320gを得た。When 7.1 liters of the sialyllactose-containing solution was treated under the above SMB treatment conditions, the fraction 12.2 containing SAα2 → 6Galβ1 → 4Glc in the raffinate was treated.
SAα2 → 3Galβ1 in liters and extracts
12.2 liters of fractions containing → 4Glc and SAα2 → 6Galβ1 → 4Glc were obtained. Next, each of the raffinate and the extract was concentrated with a rotary evaporator (Shibata Kagaku Kikai Kogyo Co., Ltd.) to 3 liters, and then desalted with an electrodialyzer (TS-2 type, Tokuyama Corp.). And then freeze-dried and 99% pure SA
α2 → 3Galβ1 → 4Glc powder 246g and SAα2 →
3Galβ1 → 4Glc and SAα2 → 6Galβ1 → 4
320 g of white powder having an abundance ratio with Glc of 3: 7 was obtained.
【００１７】[0017]
【実施例２】実施例１のようにして得られたＳＡα２→
３Ｇａｌβ１→４ＧｌｃとＳＡα２→６Ｇａｌβ１→４
Ｇｌｃとの存在比が３：７の白色粉末を0.5M酢酸ナトリ
ウムで固形率が５％となるよう溶解し、ＳＭＢ処理液と
した。また、0.5M酢酸ナトリウム溶液をＳＭＢ溶離液と
した。なお、ＳＭＢは、カラム直径30mm長さ500mm の８
塔型で、各々、アニオン交換樹脂 XT5021(三菱化学社
製）を充填したものを用いた。ＳＭＢの運転条件は、Ｓ
ＭＢ処理液供給量 0.72ml/分、ＳＭＢ溶離液供給量 3.0
7ml/分、エキストラクト抜き出し量 1.90ml/分、ラフィ
ネート抜き出し量1.90ml/分、カラム温度15℃、ステッ
プ時間 26.04分とした。Example 2 SAα2 obtained as in Example 1 →
3Galβ1 → 4Glc and SAα2 → 6Galβ1 → 4
A white powder having an abundance ratio with Glc of 3: 7 was dissolved with 0.5 M sodium acetate to a solid content of 5% to obtain an SMB-treated solution. A 0.5M sodium acetate solution was used as an SMB eluent. The SMB has a column diameter of 30 mm and a length of 500 mm.
The tower type was filled with anion exchange resin XT5021 (manufactured by Mitsubishi Chemical Corporation). The operating conditions for SMB are S
MB processing solution supply rate 0.72 ml / min, SMB eluent supply rate 3.0
7 ml / min, extract withdrawal amount 1.90 ml / min, raffinate withdrawal amount 1.90 ml / min, column temperature 15 ° C., step time 26.04 min.
【００１８】以上のＳＭＢ処理条件でＳＡα２→３Ｇａ
ｌβ１→４ＧｌｃとＳＡα２→６Ｇａｌβ１→４Ｇｌｃ
とを３：７の割合で含有する溶液 4.0リットルを処理し
たところ、ラフィネートにＳＡα２→６Ｇａｌβ１→４
Ｇｌｃを含む画分 10.56リットル及びエキストラクトに
ＳＡα２→３Ｇａｌβ１→４Ｇｌｃを含む画分 10.56リ
ットルをそれぞれ得た。次に、このラフィネート及びエ
キストラクトそれぞれをロータリーエバポレーターで３
リットルとなるまで濃縮した後、電気透析装置で脱塩し
た。そして、凍結乾燥し、純度99％のＳＡα２→３Ｇａ
ｌβ１→４Ｇｌｃ粉末7.8g及びＳＡα２→３Ｇａｌβ１
→４ＧｌｃとＳＡα２→６Ｇａｌβ１→４Ｇｌｃとの存
在比が１：４の白色粉末 55gを得た。Under the above SMB processing conditions, SAα2 → 3Ga
lβ1 → 4Glc and SAα2 → 6Galβ1 → 4Glc
When 4.0 liters of a solution containing and in a ratio of 3: 7 were treated, SAα2 → 6Galβ1 → 4 was added to the raffinate.
10.56 liters of a fraction containing Glc and 10.56 liters of a fraction containing SAα2 → 3Galβ1 → 4Glc in the extract were obtained. Then, each of the raffinate and the extract was mixed with a rotary evaporator.
After concentrating to 1 liter, it was desalted with an electrodialyzer. And freeze-dried, 99% purity SAα2 → 3Ga
1β1 → 4Glc powder 7.8g and SAα2 → 3Galβ1
55 g of white powder having an abundance ratio of → 4Glc and SAα2 → 6Galβ1 → 4Glc of 1: 4 was obtained.
【００１９】[0019]
【実施例３】実施例２のようにして得られたＳＡα２→
３Ｇａｌβ１→４ＧｌｃとＳＡα２→６Ｇａｌβ１→４
Ｇｌｃとの存在比が１：４の白色粉末を 0.07M酢酸緩衝
液(pH 4.5)で固形率が10％となるよう溶解し、ＳＭＢ処
理液とした。また、 0.07M酢酸緩衝液(pH 4.5)をＳＭＢ
溶離液とした。なお、ＳＭＢは、カラム直径30mm長さ50
0mm の８塔型で、各々、アニオン交換樹脂 Dowex 1x4
（ダウ・ケミカル社製）を充填したものを用いた。ＳＭ
Ｂの運転条件は、ＳＭＢ処理液供給量 1.64ml/分、ＳＭ
Ｂ溶離液供給量 3.99ml/分、エキストラクト抜き出し量
2.82ml/分、ラフィネート抜き出し量 2.82ml/分、カラ
ム温度15℃、ステップ時間159.56分とした。Example 3 SAα2 obtained as in Example 2 →
3Galβ1 → 4Glc and SAα2 → 6Galβ1 → 4
A white powder having an abundance ratio with Glc of 1: 4 was dissolved in 0.07M acetate buffer (pH 4.5) so that the solid content was 10%, to obtain an SMB-treated solution. Also, add 0.07M acetate buffer (pH 4.5) to SMB
The eluent was used. The SMB has a column diameter of 30 mm and a length of 50.
0mm 8 tower type, each anion exchange resin Dowex 1x4
(Made by Dow Chemical Co.) was used. SM
The operating conditions for B are: SMB processing liquid supply rate of 1.64 ml / min, SM
B eluent supply rate 3.99 ml / min, extract withdrawal rate
2.82 ml / min, raffinate withdrawal amount was 2.82 ml / min, column temperature was 15 ° C., and step time was 159.56 min.
【００２０】以上のＳＭＢ処理条件でＳＡα２→３Ｇａ
ｌβ１→４ＧｌｃとＳＡα２→６Ｇａｌβ１→４Ｇｌｃ
とを１：４の割合で含有する溶液 3.0リットルを処理し
たところ、ラフィネートにＳＡα２→６Ｇａｌβ１→４
Ｇｌｃを含む画分5.15リットル及びエキストラクトにＳ
Ａα２→３Ｇａｌβ１→４Ｇｌｃを含む画分5.15リット
ルをそれぞれ得た。次に、このラフィネート及びエキス
トラクトそれぞれをロータリーエバポレーターで２リッ
トルとなるまで濃縮した後、電気透析装置で脱塩した。
そして、凍結乾燥し、純度99％のＳＡα２→６Ｇａｌβ
１→４Ｇｌｃ粉末 22g及びＳＡα２→３Ｇａｌβ１→４
ＧｌｃとＳＡα２→６Ｇａｌβ１→４Ｇｌｃとの存在比
が１：２の白色粉末 25gを得た。Under the above SMB processing conditions, SAα2 → 3Ga
lβ1 → 4Glc and SAα2 → 6Galβ1 → 4Glc
When 3.0 liters of a solution containing and in a ratio of 1: 4 was treated, SAα2 → 6Galβ1 → 4 was added to the raffinate.
5.15 liters of the fraction containing Glc and S in the extract
5.15 liters of fractions containing Aα2 → 3Galβ1 → 4Glc were respectively obtained. Next, each of the raffinate and the extract was concentrated with a rotary evaporator to 2 liters, and then desalted with an electrodialyzer.
And freeze-dried, 99% pure SAα2 → 6Galβ
22g of 1 → 4Glc powder and SAα2 → 3Galβ1 → 4
As a result, 25 g of white powder was obtained in which the abundance ratio of Glc and SAα2 → 6Galβ1 → 4Glc was 1: 2.
【００２１】[0021]
【発明の効果】本発明の方法によると、シアリルラクト
ース中に混在するＳＡα２→３Ｇａｌβ１→４Ｇｌｃと
ＳＡα２→６Ｇａｌβ１→４Ｇｌｃとを工業的規模で大
量かつ簡便に分離することができる。したがって、高純
度のＳＡα２→３Ｇａｌβ１→４Ｇｌｃ及びＳＡα２→
６Ｇａｌβ１→４Ｇｌｃを大量かつ安価に提供できるの
で、これらの素材を飲食品、医薬品、化粧品、飼料など
に利用することが容易となる。According to the method of the present invention, SAα2 → 3Galβ1 → 4Glc and SAα2 → 6Galβ1 → 4Glc mixed in sialyllactose can be easily separated on a large scale on an industrial scale. Therefore, high-purity SAα2 → 3Galβ1 → 4Glc and SAα2 →
Since 6Galβ1 → 4Glc can be provided in a large amount and at low cost, it becomes easy to use these materials for food and drink, pharmaceuticals, cosmetics, feed and the like.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 守田 稔 埼玉県川越市仙波町３丁目２−５ ＹＳハ イツ第二202 (72)発明者 石川 秀敏 北海道札幌市手稲区前田３条３丁目８−３ ─────────────────────────────────────────────────── --- Continuation of the front page (72) Inventor Minoru Morita 3-5, Senba-cho, Kawagoe-shi, Saitama 2-5 YS Heights 202 (72) Hidetoshi Ishikawa 3-chome, Maeda 3-chome, Teine-ku, Sapporo, Hokkaido Three
Claims (2)
含む原料溶液を擬似移動床式クロマト分離装置に供給し
て、シアル酸（ＳＡ）と乳糖（Ｇａｌβ１→４Ｇｌｃ）
とがα２→３結合したＳＡα２→３Ｇａｌβ１→４Ｇｌ
ｃ及び／又はシアル酸（ＳＡ）と乳糖（Ｇａｌβ１→４
Ｇｌｃ）とがα２→６結合したＳＡα２→６Ｇａｌβ１
→４Ｇｌｃを分離することを特徴とするシアリルラクト
ースの分離方法。1. A raw material solution containing sialyllactose having different binding modes is supplied to a simulated moving bed chromatographic separation device to supply sialic acid (SA) and lactose (Galβ1 → 4Glc).
SA α2 → 3Galβ1 → 4Gl in which and α2 → 3 are linked
c and / or sialic acid (SA) and lactose (Galβ1 → 4
Glc) α2 → 6 linked to SAα2 → 6Galβ1
→ A method for separating sialyllactose, which comprises separating 4Glc.
としてアニオン交換樹脂を用い、溶離液として塩濃度が
0.01〜1.5MでpHが１〜10の溶液を用いる請求項１記載の
分離方法。2. An anion exchange resin is used as a separating agent of a simulated moving bed chromatographic separation device, and a salt concentration is used as an eluent.
The separation method according to claim 1, wherein a solution having a pH of 0.01 to 1.5 M and a pH of 1 to 10 is used.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP06136195A JP4082746B2 (en) | 1995-03-20 | 1995-03-20 | Separation method of sialyl lactose |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP06136195A JP4082746B2 (en) | 1995-03-20 | 1995-03-20 | Separation method of sialyl lactose |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH08252403A true JPH08252403A (en) | 1996-10-01 |
| JP4082746B2 JP4082746B2 (en) | 2008-04-30 |
Family
ID=13168957
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP06136195A Expired - Fee Related JP4082746B2 (en) | 1995-03-20 | 1995-03-20 | Separation method of sialyl lactose |
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| Country | Link |
|---|---|
| JP (1) | JP4082746B2 (en) |
Cited By (6)
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|---|---|---|---|---|
| JP2006117597A (en) * | 2004-10-22 | 2006-05-11 | Snow Brand Milk Prod Co Ltd | Agent for suppressing infection/proliferation of human immunodeficiency virus |
| WO2011040360A1 (en) | 2009-09-29 | 2011-04-07 | 雪印乳業株式会社 | Method for separating sialyllactose material |
| WO2011040361A1 (en) | 2009-09-29 | 2011-04-07 | 雪印乳業株式会社 | Method for separating sialyllactose material |
| JP2012505928A (en) * | 2008-10-20 | 2012-03-08 | ベネバイオシス カンパニー リミテッド | Composition for prevention or treatment of eye diseases |
| WO2017086443A1 (en) | 2015-11-18 | 2017-05-26 | 協和発酵バイオ株式会社 | 6'-sialyl lactose sodium salt crystal and method for manufacturing same |
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1995
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006117597A (en) * | 2004-10-22 | 2006-05-11 | Snow Brand Milk Prod Co Ltd | Agent for suppressing infection/proliferation of human immunodeficiency virus |
| JP2012505928A (en) * | 2008-10-20 | 2012-03-08 | ベネバイオシス カンパニー リミテッド | Composition for prevention or treatment of eye diseases |
| WO2011040360A1 (en) | 2009-09-29 | 2011-04-07 | 雪印乳業株式会社 | Method for separating sialyllactose material |
| WO2011040361A1 (en) | 2009-09-29 | 2011-04-07 | 雪印乳業株式会社 | Method for separating sialyllactose material |
| WO2017086443A1 (en) | 2015-11-18 | 2017-05-26 | 協和発酵バイオ株式会社 | 6'-sialyl lactose sodium salt crystal and method for manufacturing same |
| JPWO2017086443A1 (en) * | 2015-11-18 | 2018-08-30 | 協和発酵バイオ株式会社 | Crystal of 6'-sialyllactose sodium salt and method for producing the same |
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| WO2017195743A1 (en) | 2016-05-09 | 2017-11-16 | 協和発酵バイオ株式会社 | Crystal of 3'-sialyllactose sodium salt n-hydrate, and method for producing same |
| KR20190005162A (en) | 2016-05-09 | 2019-01-15 | 교와 핫꼬 바이오 가부시키가이샤 | Crystals of 3'-sialyllactose sodium salt · n-hydrate and method for producing the same |
| US11008355B2 (en) | 2016-05-09 | 2021-05-18 | Kyowa Hakko Bio Co., Ltd. | Crystal of 3'-sialyllactose sodium salt n-hydrate, and process for producing same |
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|---|---|
| JP4082746B2 (en) | 2008-04-30 |
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