JP4465164B2 - Method for producing oligosaccharide containing sialic acid - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a production method for easily and efficiently separating and extracting sialic acid-containing oligosaccharides having various physiological activities from milk with high purity.
[0002]
[Prior art]
Sialic acid-containing oligosaccharides are those in which sialic acid is bound to galactose, N-acetylglucosamine, or sialic acid itself, which is a constituent sugar constituting oligosaccharides. Typical examples include sialyl lactose and sialyl lactosamine. It has been known. It is known that these sialic acid-containing oligosaccharides further exist in two types depending on the binding mode of galactose and sialic acid, which are neutral sugar chains serving as the basic skeleton.
In recent years, oligosaccharides to which these sialic acids are bound not only have physiological functions such as cell-to-cell discrimination, but also have anti-infection activity, act as a growth factor for bisids, and have an immune control function for infants. In addition to research that promotes the elucidation of such physiological functions, it can be used in foods and drinks for the purpose of imparting various physiological functions by incorporating oligosaccharides containing sialic acid. It has been attracting attention as a useful food material that can be expected.
[0003]
So far, various methods for producing sialic acid-containing oligosaccharides by chemical synthesis have been reported. However, it is difficult to produce in large quantities with a complicated synthesis method. Since it is used, it is not always a preferable method in consideration of use in foods.
[0004]
On the other hand, in recent years, it has been clarified that sialic acid-containing oligosaccharides that have been reported to exist only in trace amounts in the natural world are relatively contained in milk such as cows, sheep, and goats. As a method for producing an oligosaccharide containing an acid, a separation and extraction method using milk such as milk as a raw material has been studied. For example, (1) after removing protein and lactose from a milk raw material containing a sialic acid-containing oligosaccharide fraction, the solution is passed through a simulated moving bed chromatographic separation apparatus using a cation exchange resin as a separating agent, A method for separating and extracting sugar (Patent Document 1), (2) a method for concentrating a supernatant liquid from which protein has been removed from skim milk powder, and then crystallizing and removing lactose (Patent Document 2), and (3) a sialic acid compound A method in which a lactone body in a raw material solution is previously subjected to ring-opening treatment, a sialic acid compound in the raw material solution is separated, lactonized, passed through an anion exchanger, and a non-adsorbed fraction is collected (Patent Document) 3) and the like.
[0005]
[Patent Document 1]
Japanese Patent No. 3368389 [Patent Document 2]
JP 2001-240599 A [Patent Document 3]
JP-A-2001-213894 gazette
[Problems to be solved by the invention]
However, these conventional methods are not necessarily preferable methods. For example, in the method of Patent Document 1, it is not possible to selectively separate and extract only sialic acid-containing oligosaccharides contained in milk. In the method of Patent Document 2, oligosaccharides to which sialic acid is bound from milk raw materials are not extracted. Although it can be extracted, it is not possible to selectively extract only sialic acid-containing oligosaccharides having a specific structure. The method of Patent Document 3 is very useful as a method for selectively separating and extracting a sialic acid-containing oligosaccharide that forms a lactone body and a sialic acid-containing oligosaccharide that does not form a lactone body. In order to extract the contained oligosaccharide, many complicated steps must be performed, and the working efficiency is poor. Further, among sialic acid-containing oligosaccharides, there is a problem that only those that can form a lactone structure can be separated.
[0007]
Therefore, an object of the present invention is to provide a production method for selectively separating and extracting sialic acid-containing oligosaccharides having various physiological functions selectively, with high purity and high yield, and without complicated steps.
[0008]
[Means for Solving the Problems]
Accordingly, the present inventors have intensively studied a production method for easily separating and extracting sialic acid-containing oligosaccharides from milk with high purity. As a result, the oligosaccharide fraction obtained by removing lipids and proteins from milk is subjected to a specific column chromatography treatment. As a result, it was found that sialic acid-containing oligosaccharides can be selectively extracted, and the present invention was completed.
[0009]
The present invention provides a method for producing a sialic acid-containing oligosaccharide, characterized in that an oligosaccharide fraction obtained by removing lipids and proteins from milk is subjected to activated carbon column chromatography.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
The sialic acid-containing oligosaccharide separated and extracted from the milk of the present invention has N-acetylneuraminic acid or N-glycolylneuraminic acid as sialic acid bonded to the non-reducing end of the sugar constituting the oligosaccharide. It is mainly composed of neuraminyl lactose or N-glycolyl neuraminyl lactose. Specifically, α-2,3-N-acetyl-neuraminyl lactose, α-2,6-N-acetyl-neuraminyl lactose, α-2,3-N-glycolyl-neuraminyl lactose or α-2 , 6-N-glycolyl-neuraminyl lactose.
[0011]
Any milk containing sialic acid-containing oligosaccharides can be used as the milk used in the present invention, but α-2,3-N-acetyl, which is a sialic acid-containing oligosaccharide that is separated and extracted. -Rich in neuraminyl lactose, α-2,6-N-acetyl-neuraminyl lactose or α-2,3-N-glycolyl-neuraminyl lactose, α-2,6-N-glycolyl-neuraminyl lactose Raw milk is preferable, and milk such as cow, sheep, goat and the like is particularly preferable.
[0012]
Α-2,3-N-acetyl-neuraminyl lactose or α-2,6-N-acetyl-neuraminyl lactose in which N-acetylneuraminic acid is bonded as a sialic acid to the non-reducing end of the sugar constituting the oligosaccharide When the sialic acid-containing oligosaccharide is obtained, it is preferable to use cow and goat milk containing a relatively large amount thereof, and N-glycolylneuraminic acid as sialic acid is present at the non-reducing end. When obtaining sialic acid-containing oligosaccharides which are conjugated α-2,3-N-glycolyl-neuraminyl lactose or α-2,6-N-glycolyl-neuraminyl lactose, sheep containing a relatively large amount of them The milk is preferably used as a raw material.
[0013]
Moreover, since sialic acid-containing oligosaccharides contained in milk such as milk, sheep milk or goats decrease as the number of days passes after delivery, in order to obtain sialic acid-containing oligosaccharides in high yield These colostrums are preferably used as raw milk, and in particular, colostrum milked within 24 hours after delivery is preferably used as raw milk.
[0014]
The first step of the method of the present invention is a step of removing lipids and proteins from milk. Lipid removal is performed by separating milk into a cream layer and a skim milk layer using a high-speed centrifuge, a cream separator, or the like, and separating the skim milk layer.
Centrifugation conditions in the case of using a high-speed centrifuge are not particularly limited, but it is preferable to carry out the conditions under which the cream layer and the skim milk layer can be sufficiently separated. For example, the temperature is 0 to 10 ° C., more preferably 2 to 5 ° C., and the rotational speed is 1000 to 100,000 r / min, more preferably 3000 to 10000 r / min, for 5 to 60 minutes, and further for 10 to 40 minutes. preferable. This operation may be repeated as necessary in order to perform sufficient layering. After the layering, the skim milk layer may be separated by a conventional method.
[0015]
The protein is then removed from the skim milk layer. For protein removal, the pH of the separated skim milk layer is adjusted to the vicinity of the isoelectric point (pH 4.6) of casein to precipitate the protein, and then casein is used using a conventional method such as a high-speed centrifuge. The white precipitate is separated and removed.
[0016]
The layer from which protein is removed from the skim milk layer (hereinafter sometimes referred to as whey) is preferably obtained by treating in this process from colostrum such as cattle and sheep, but even if commercially available whey is used. Good. Examples of commercially available whey include acidic whey obtained when a casein is produced by precipitating curd from skim milk with acid, cheese whey discharged when producing cheese, and the like.
[0017]
The whey obtained in this way was adjusted to a pH of around 7 in order to further sufficiently remove protein, and then 1 to 3 times by weight, preferably about 2 times by weight of the lower alcohol, based on the total amount of whey. In addition, a solvent such as acetone, for example, 99% unmodified ethyl alcohol is added and mixed, and the mixture is allowed to stand overnight at a low temperature, preferably 0 to 10 ° C., particularly preferably 3 to 5 ° C., and then a high-speed centrifuge is used. Thus, a white precipitate of protein is separated to obtain a supernatant. The supernatant is concentrated, preferably concentrated to about 1/5 to 1/10, or lyophilized to obtain an oligosaccharide fraction.
[0018]
The reason why whey is adjusted to around pH 7 is to prevent sialic acid from dissociating from sialic acid-containing oligosaccharides under low pH conditions. When pH is adjusted, it is usually used for pH adjustment. Any of these may be used as long as they are acceptable as food, and it is preferable to use acetic acid, an aqueous sodium hydroxide solution, or the like.
In addition, as a solvent for precipitating proteins in skim milk, a solvent such as acetone can be used. However, it can be used for food and activated sugar column chromatography using oligosaccharide prepared from raw milk using ethyl alcohol. In consideration of the process of attaching to the solvent, it is most preferable to use ethyl alcohol.
[0019]
Since the oligosaccharide fraction thus obtained contains sugars and minerals other than sialic acid-containing oligosaccharides, an insoluble precipitate may be produced during the concentration treatment. Therefore, it is preferable to remove these precipitates as much as possible before repeating the above operation and subjecting to activated carbon column chromatography.
[0020]
Next, the oligosaccharide fraction was dissolved in an aqueous solution containing a solvent such as lower alcohol, acetonitrile, etc. and subjected to activated carbon column chromatography. The elution fraction containing the sialic acid-containing oligosaccharide is collected, the sialic acid-containing oligosaccharide elution fraction is detected by the sialic acid detection method described below, and the sialic acid-containing oligosaccharide is obtained by fractionation. .
Here, examples of the solvent to be used include lower alcohols such as ethyl alcohol and isopropyl alcohol, acetonitrile, and the like, but ethyl alcohol is most preferable when producing sialic acid-containing oligosaccharides as food materials for food and the like.
[0021]
The concentration of the elution solvent in the production of the sialic acid-containing oligosaccharide by activated carbon column chromatography is not particularly limited, but it is preferably carried out with a 5-90 wt% concentration gradient. When the solvent is ethyl alcohol, it is preferably carried out by concentration gradient using 1 to 50% by weight ethyl alcohol aqueous solution. More specifically, first, a 3 to 7% by weight ethyl alcohol aqueous solution is passed, and then a 10 to 50% by weight ethyl alcohol aqueous solution is passed stepwise in order from a low concentration. And it is preferable to fractionate the fraction eluted with 15 to 35% by weight ethyl alcohol aqueous solution. Furthermore, in the case of milk and goat milk, 15 to 25% by weight ethyl alcohol aqueous solution and 25% in the case of sheep milk. It is preferable to fractionate an elution fraction of a ~ 35 wt% aqueous ethyl alcohol solution. The liquid passing rate of the solvent is preferably 1 to 20 mL / min, more preferably 3 to 10 mL / min, from the viewpoint of production efficiency of the sialic acid-containing oligosaccharide.
[0022]
The sialic acid-containing oligosaccharide obtained by the production method of the present invention has a sialic acid / hexose content weight ratio of 0.5 to 0.96, more preferably 0.6 to 0.96. This is preferable because the sugar content is low.
[0023]
Examples of the detection and quantification method for sialic acid include the periodate resorcinol hydrochloride method. In this method, a sample is dissolved in distilled water, reacted with periodic acid, then reacted with resorcinol solution, then cooled, added with t-butanol, further mixed, and centrifuged. The supernatant is collected using, and the absorbance at 630 nm is measured with a spectrophotometer. At this time, when the absorption is measured at 630 nm, it indicates that the sample contains sialic acid, and the content is measured from the absorbance by a conventional method.
[0024]
Examples of the hexose detection and quantification method include the phenol sulfate method. In this method, a phenol reagent is added to a sample and stirred, and concentrated sulfuric acid is further added and stirred. The sample is cooled to room temperature, and the absorbance at 490 nm is measured using a spectrophotometer. At this time, when the absorption is measured at 490 nm, it indicates that the sample contains a neutral sugar (hexose), and the content is measured from the absorbance by a conventional method.
In addition, the content of neutral sugars can be calculated by comparing with a calibration curve using standard sugars (eg, glucose, lactose, etc.).
[0025]
In the method for producing a sialic acid-containing oligosaccharide of the present invention, a sialic acid-containing oligosaccharide in which N-acetylneuraminic acid is bound in a 15 to 25 wt% ethyl alcohol flow-through fraction when bovine colostrum is used as a raw material In particular, α-2,3-N-acetyl-neuraminyl lactose can be selectively and easily separated and extracted. In addition, even when sheep colostrum is used as a raw material, sialic acid-containing oligosaccharides containing N-glycolylneuraminic acid as a main component are selectively and easily separated in a 25-35 wt% ethyl alcohol flow-through fraction. Can be extracted. Even when goat's colostrum is used as a raw material, the sialic acid-containing oligosaccharide to which N-acetylneuraminic acid is bonded is selectively obtained by passing 15 to 25 wt% ethyl alcohol through activated carbon column chromatography. It can be easily separated and extracted.
[0026]
The sialic acid-containing oligosaccharide produced by separation and extraction from milk by the method for producing a sialic acid-containing oligosaccharide of the present invention may be used as it is in an ethyl alcohol solution, or may be concentrated and dried according to a conventional method. It may be used after being powdered.
[0027]
A sialic acid-containing oligosaccharide produced by the production method of the present invention, particularly a sialic acid-containing oligosaccharide having a sialic acid / hexose weight ratio of 0.5 to 0.96, has a cell-to-cell discrimination function, an infection-protecting activity, It is used for research materials that promote the elucidation of various physiological functions such as growth factors and immune control functions of infants, foods and drinks that impart physiological functions, and the like. It can also be used as a material for feed, pharmaceuticals, cosmetics, and the like.
[0028]
【Example】
EXAMPLES Next, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these Examples.
[0029]
Example 1
(1) Production of oligosaccharide fraction from milk Approximately 1000 mL of milk collected immediately after parturition from dairy cows (20 Holsteins) was centrifuged at 4 ° C. and 5000 r / min for 15 minutes to remove the lipid fraction. The obtained skim milk is adjusted to pH 4.6 with 0.1 mol / L acetic acid to cause isoelectric precipitation of casein, and then treated with a high-speed centrifuge to separate the white precipitate of casein, and whey. Got. The whey was further adjusted to pH 7 by adding 0.1 mol / L sodium hydroxide, 2 times by weight of 99% by weight unmodified ethyl alcohol was added, and the mixture was allowed to stand at 4 ° C. overnight, followed by centrifugation. The generated insoluble fraction was removed by filtration, and the filtrate was concentrated and freeze-dried to obtain 24.2 g of an oligosaccharide fraction.
[0030]
(2) Production of oligosaccharide containing sialic acid Oligosaccharide fraction of milk obtained in (1) on a glass column (φ5.0 × 23.0 cm, 450 mL) packed with activated carbon (manufactured by Wako Pure Chemicals, for chromatography) A 6 g portion was dissolved in a 5 wt% aqueous ethyl alcohol solution and applied. Next, 5 wt%, 10 wt%, 20 wt%, 30 wt% and 50 wt% of 5 types of ethyl alcohol aqueous solutions were sequentially passed at a flow rate of 5 mL / min, and fractionated by 100 mL, respectively. Hexose and sialic acid were detected by the resorcinol iodate method. The result is shown in FIG.
[0031]
From FIG. 1, it was confirmed that the sialic acid-containing oligosaccharide was selectively eluted in the fraction eluted with 20% by weight aqueous ethyl alcohol solution. The yield of the fraction eluted with this 20% by weight aqueous ethyl alcohol solution was 280 mg.
The content weight ratio of sialic acid / hexose was 0.92.
[0032]
Detection of oligosaccharides containing sialic acid 10 mg of a fraction eluted with a 20% by weight aqueous ethyl alcohol solution was weighed and dissolved in distilled water and subjected to HPLC (high performance liquid chromatography) under the following conditions. Was purified. The result is shown in FIG.
HPLC: TSK Amide 80 column (4.6 mm ID x 25 cm)
Flow rate 1mL / min
Eluate a: acetonitrile / 15 mmol / L potassium phosphate buffer (pH 5.2) = 80: 20
Liquid b: acetonitrile / 15 mmol / L potassium phosphate buffer (pH 5.2) = 50: 50
[0033]
From FIG. 2, it was confirmed that the sialic acid-containing oligosaccharide eluted with a 20 wt% aqueous ethyl alcohol solution was α-2,3-N-acetyl-neuraminyl lactose.
[0034]
Example 2
(1) Manufacture of oligosaccharide fraction from sheep milk 6.8 g of oligosaccharide fraction was obtained in the same manner as in Example 1 (1) using about 440 mL of milk collected immediately after delivery from sheep (4 heads). .
[0035]
(2) Production of sialic acid-containing oligosaccharide 25 mL of the oligosaccharide fraction of sheep milk obtained in (1) was dissolved in a 5 wt% aqueous ethyl alcohol solution in the same manner as in Example 1 (2). Next, 5 wt%, 20 wt%, 30 wt% and 50 wt% of four kinds of ethyl alcohol aqueous solutions were sequentially passed at a flow rate of 5 mL / min, fractionated by 100 mL, and phenol sulfuric acid method and periodate acid resorcinol hydrochloride. Hexose and sialic acid were detected by the method. The result is shown in FIG.
[0036]
From FIG. 3, it was confirmed that the sialic acid-containing oligosaccharide was selectively eluted in the fraction eluted with 30 wt% aqueous ethyl alcohol. The yield of the sialic acid-containing oligosaccharide fraction eluted with this 30% by weight aqueous ethyl alcohol solution was 64.3 mg.
The content weight ratio of sialic acid / hexose was 0.85.
[0037]
【The invention's effect】
The method for producing a sialic acid-containing oligosaccharide of the present invention is simpler and can obtain a sialic acid-containing oligosaccharide with high purity and high yield. In addition, a plurality of compounds that are sialic acid-containing oligosaccharides can be easily separated and extracted, and can be applied to various research materials, foods, etc. as purified products with high purity.
[Brief description of the drawings]
FIG. 1 is a diagram showing elution of sialic acid-containing oligosaccharides in an activated carbon column chromatography method.
FIG. 2 is a diagram showing the results of HPLC measurement of a fraction eluted with a sialic acid-containing oligosaccharide.
FIG. 3 is a diagram showing elution of sialic acid-containing oligosaccharides in an activated carbon column chromatography method.

Claims (3)

The oligosaccharide fractions obtained by removing lipids and proteins from milk and with the activated charcoal column chromatography, and then subjected to elution by the density gradient with 1-50 wt% of ethyl alcohol aqueous solution, 15 to 35 wt% of ethyl alcohol aqueous solution A method for producing a sialic acid-containing oligosaccharide having a sialic acid / hexose weight ratio of 0.5 to 0.96, wherein an elution fraction obtained by the step is collected . The method for producing a sialic acid-containing oligosaccharide according to claim 1, wherein the sialic acid-containing oligosaccharide obtained contains N-acetyl-neuraminyl lactose or N-glycolyl-neuraminyl lactose . The method for producing a sialic acid-containing oligosaccharide according to claim 1 or 2 , wherein the milk is colostrum of cow, sheep or goat.

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