JP2001213894A - Method for producing lactone of sialic acid compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing the lactone of a sialic acid compound, by which the lactone can be produced from the sialic acid component in a high yield and in a high purity. SOLUTION: This method for producing the lactone of the sialic acid compound, characterized by preliminarily applying a ring-opening treatment to the lactone contained in a raw material solution containing the sialic acid compound, separating the sialic acid compound from the raw material solution, converting the separated sialic acid compound into the lactone, allowing the solution to pass through an anion exchanger, and then collecting the non- adsorbed fraction. That the raw material containing the sialic acid compound is milk or a milk processed product, that the milk or the milk processed product is bovine colostrum, bovine skimmed colostrum, whey, KUARUKU acid whey or casein, that the sialic acid compound is sialyl lactose, casein, or a casein hydrolyzate, and that the method for the ring-opening treatment of the lactone is a freezing and thawing method are preferable modes.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a lactone derivative obtained from a sialic acid compound which is mainly obtained from natural raw materials such as milk or milk components in high yield and purity and is useful as a material for foods, feeds, pharmaceuticals or cosmetics. And a method for producing the same.

More specifically, the present invention provides a lactone compound in a raw material solution containing a sialic acid compound in which the sialic acid compound in the raw material solution is separated, lactonized, and passed through an anion exchanger. The present invention relates to a method for producing a lactone compound obtained from a sialic acid compound, comprising liquefying and collecting a non-adsorbed fraction.

[0003]

2. Description of the Related Art Hitherto, it has been known that sialic acid is located at a terminal of a cell surface sugar chain, a glycoprotein sugar chain, or a glycolipid sugar chain and has various physiological functions such as intercellular discrimination.

[0004] Sialic acid is present in milk as a constituent molecule such as oligosaccharides, gangliosides and glycoproteins.
Protective activity against infection and acting as a bifidobacterium (Kunio Yamauchi et al., “Comprehensive Milk Dictionary”, No. 519
P., Asakura Shoten, 1992) and controlling early immunity in infants (dairy science research, Vol. 17, pp. A-55,
1968) is known.

[0005] In addition, breast milk contains a larger amount of sialic acid compounds than cow milk, and attempts have been made to use sialic acid compounds as substitutes for breast milk, functional foods, and pharmaceutical materials (Yamauchi). Kunio et al., "Milk Comprehensive Dictionary", p. 310, Asakura Shoten, 1992).

On the other hand, it has been reported that a lactone obtained from a sialic acid compound having a lactone structure in the molecule exists in the brain and in milk [The Journal of Biological Chemistry (The Journal of Biological Chemistry)]. Biologi
cal Chemistry), vol. 261, p. 8514, 198.
6 years, and Biochimica et Biophysica Acta, Vol. 1381,
286, 1998. ].

A lactone obtained from a sialic acid compound has no free carboxyl group, and the carboxyl group forms a part of a lactone ring, and has a chemical structure completely different from that of a non-lactonized sialic acid compound. is there.

Further, since the lactone obtained from the sialic acid compound has a lactone structure in the molecule, it has a useful property that it is more resistant to degradation of sialic acid by neuraminidase as compared with a non-lactonized sialic acid compound. [Chemical Letters]
s), p. 669, 1997], which is a completely different substance from a non-lactonized sialic acid compound.

Hitherto, as a method for separating a lactone obtained from a sialic acid compound having such useful properties from a natural raw material, a method for separating a sialic acid compound from bovine colostrum, which is a raw material containing the sialic acid compound, has been known. And lactonized,
A method for separating a lactone form obtained from a sialic acid compound by contacting with an anion exchanger to obtain a non-adsorbed fraction [Methods in Enzymolog
y), Volume 242, p. 102, 1994. Hereinafter, this is referred to as Conventional Technique 1. ] Was known. However,
The prior art has the following disadvantages.

[0010]

According to the prior art 1, the lactone form obtained from the sialic acid compound and the non-lactone form (so-called non-lactonized sialic acid compound) have different chemical properties. In the step of separating the sialic acid compound, the lactone form originally present in the raw material cannot be recovered, and the yield of the lactone form obtained from the sialic acid compound that can be finally obtained is poor. Was.

The present inventors have conducted intensive studies in view of the above-mentioned prior art, and as apparent from the test examples described below, the lactone compound in the sialic acid compound-containing raw material is preliminarily subjected to a ring-opening treatment to obtain the raw material. The present inventors have found that the lactone form originally present therein can be easily recovered together with the non-lactone form, and that the lactone form obtained from the sialic acid compound can be produced with high yield and high purity, and completed the present invention. .

An object of the present invention is to provide a method for producing a lactone obtained from a sialic acid compound with high yield and high purity.

[0013]

In order to solve the above-mentioned problems, the present invention provides a lactone compound in a raw material solution containing a sialic acid compound, which is subjected to a ring-opening treatment in advance to separate the sialic acid compound in the raw material solution. A lactone derivative obtained from a sialic acid compound, wherein the sialic acid compound-containing raw material solution is milk or milk. It is a processed product (hereinafter referred to as Aspect 1), and the milk or dairy processed product is bovine colostrum, bovine skim colostrum, whey, quartic acid whey, or casein (hereinafter referred to as Aspect 2). ), The sialic acid compound is sialyl lactose, casein, or a casein hydrolyzate (hereinafter, referred to as embodiment 3), and the ring-opening treatment of the lactone body is a freeze-thaw method (hereinafter, referred to as a thawing method). Aspect 4 Some as a mounting for.) The desirable embodiments. Next, the present invention will be described in detail.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION As a raw material containing a sialic acid compound used in the present invention, any raw material containing a sialic acid compound can be used. Desirably, as shown in the first embodiment of the present invention, milk or a processed milk product is more preferable because it is easy to obtain a large amount of raw materials.

Examples of the milk include cow's milk, human milk, goat's milk, skim milk thereof, and the like. The milk contains a large amount of a lactone of a sialic acid compound and is relatively easily available. Although colostrum is desirable, it is also possible to use bovine normal milk which can be used in large quantities because of the high separation and purification efficiency of the present invention.

Examples of the processed milk include acid casein and rennet casein separated from colostrum or normal milk, whey by-produced during the production of cheese, and the like. For convenience, commercially available casein and whey ( For example, Morinaga Milk Industry Co., Ltd., etc.), quartic acid whey (for example, Mirai Sour Whey Powder, etc.) can be used.

As shown in Embodiment 2 of the present invention, since the milk or the processed milk product is bovine colostrum, bovine skim colostrum, whey, or casein because it contains a relatively large amount of a sialic acid compound. Even more desirably, these can be arbitrarily mixed and used.

Further, as shown in Embodiment 3 of the present invention, the main sialic acid compound of milk or processed milk products is sialyl lactose, casein, or a casein hydrolyzate, so that the sialic acid compound is sialyl lactose, casein, or casein. Desirably, it is a casein degradation product.

The raw material in the form of a solution can be used as a raw material solution as it is, but the raw material in the form of a solid or powder is dispersed and dissolved in water or hot water and used as a raw material solution.
Although there is no particular limitation on the concentration of the dissolving solution, it is usually effective to set the concentration range of about 5 to 15% (weight; hereinafter, the same unless otherwise noted) in terms of solid content, for the efficiency of the subsequent steps. It is desirable in terms of operability and operability.

The raw material solution containing the sialic acid compound is preferably subjected to the following pretreatment in accordance with each raw material solution in terms of efficiency and operability.

The raw material solution containing fat such as bovine colostrum is
It is desirable to perform a degreasing treatment using a centrifuge, a cream separator, or the like.

Raw materials containing proteins such as bovine colostrum, bovine skim colostrum after defatting treatment, or whey excluding rennet whey are removed by ultrafiltration, heating, an organic solvent or a combination thereof. It is desirable to perform protein treatment.
It should be noted that casein, casein hydrolyzate, or rennet whey is preferably present in a state in which the sialic acid compound is also covalently bonded to the protein or peptide, so that the yield is not reduced. .

Examples of the organic solvent used for the protein removal treatment include chloroform, acetone, hexane, ethanol, and mixtures thereof. When the protein removal treatment with an organic solvent is performed, it is necessary to distill the organic solvent from the protein removal treatment solution.

The obtained deproteinized solution is 5% in solid content.
It is desirable to perform concentration treatment by an evaporator or the like to a concentration range of about 15%.

If necessary, an octadecyl group (C1
It is desirable to remove the mixed peptide and the like by hydrophobic column chromatography using a resin or the like having 8) to produce a lactone form obtained from a sialic acid compound with higher purity.

As another treatment of the raw material solution, when salts are contained in the raw material solution, desalting treatment is desirably performed by electrodialysis or the like, and fine precipitates are contained in the raw material solution. In this case, it is desirable to remove these with a clarifier or the like.

The method for ring-opening a lactone in a raw material solution of the present invention may be any method as long as the method allows ring-opening of a lactone. Specifically, a method of raising the pH of a solution with sodium hydroxide, sodium hydrogen carbonate, etc. [Methods in Enzymology, Vol. 242, p. 102, 1994, and tetrahedron. Letter
(Tetrahedron Letter), Volume 35, Page 3321, 1
994. And the like, but as shown in Embodiment 4 of the present invention, the lactone can be opened without adding a substance, and therefore, the freeze-thawing method is preferable.

The freeze-thaw method is carried out, for example, as follows. That is, a raw material solution containing a sialic acid compound is gradually frozen by a freezer or the like at a temperature of -10 ° C or the like over several hours, and then thawed. By this method, the lactone ring of the lactone body existing in the raw material from the beginning can be opened.

The method of the present invention for separating a sialic acid compound from a raw material solution after a ring opening treatment can be used in any method, but a non-lactone form of a sialic acid compound (so-called non-lactonized sialic acid) can be used. An ion exchange method using an anion exchanger that allows efficient separation of the compound) is desirable.

The ion exchange method using an anion exchanger is specifically carried out as follows. That is, the conditions for passing the solution through the ion exchanger are such that the concentration of the raw material solution is in a range where lactose does not precipitate, preferably 0.01 to 30% in terms of solid content,
The temperature is within a range in which lactose does not precipitate, preferably between 4 ° C and 50 ° C.
° C, and ionic strength is 30 mS / cm as electric conductivity.
The following is desirable. Therefore, the concentration, temperature, and ionic strength of the raw material solution after the ring opening treatment are adjusted to the above ranges by adding water, and the solution is passed through an anion exchanger to adsorb the non-lactone of the sialic acid compound and elute. Implemented by separating.

The method of passing the solution through the anion exchanger is not particularly limited, and examples thereof include a column method and a batch method.
Note that a column method in which the solution is passed through an anion exchange resin packed in a column is preferable because the adsorbed fraction and the non-adsorbed fraction can be easily separated.

The anion exchanger used in the step of separating a sialic acid compound of the present invention is not particularly limited.
(Manufactured by Bio-Rad), Macroprep Q Support (manufactured by Bio-Rad), DEAE-cellulose (manufactured by Amersham Pharmacia Biotech), DEAE-Sephadex (manufactured by Amersham Pharmacia Biotech),
Examples thereof include Q-Sepharose (manufactured by Amersham Pharmacia Biotech) and Amberlite IRA400 (manufactured by Organo). The ion type of the ion exchanger is not particularly limited, and examples thereof include a Cl ⁻ type, an OH ⁻ type, a CH ₃ COO ⁻ type and the like obtained by a normal ion exchanger regeneration method.

The non-lactone form of the sialic acid compound adsorbed on the anion exchanger is eluted and separated by washing with water or the like and eluting with a salt solution. Examples of the salt solution include a sodium chloride solution and a sodium acetate solution.

When elution is carried out with a salt solution, the salt concentration is 0.1.
There is no particular limitation as long as the salt concentration is in the range of 01 to 1.5 M. However, when the salt concentration is low, the recovery rate is low, and when the salt concentration is too high, a long time is required for the subsequent desalination treatment. .05
The range of ~ 1.0M is desirable.

The obtained eluate containing the non-lactone of the sialic acid compound is desalted by electrodialysis or the like, and freeze-dried.

The method of lactonizing the non-lactone form of the separated sialic acid compound of the present invention may be any method as long as the sialic acid compound can be lactonized. It is desirable to use lactonization using an acid that is acceptable for foods such as glacial acetic acid, which can be used for foods and the like.

Specifically, a concentrated solution or a lyophilized product containing a non-lactone form of a sialic acid compound is dissolved in glacial acetic acid,
10 hours or more at 25 ° C or more, preferably 7 hours or more at 30 ° C or more
The lactonization is performed by reacting for 2 hours or more. If the concentration of the non-lactone compound of the sialic acid compound is too high, a reaction such as intermolecular dehydration condensation may be caused. Therefore, it is necessary to perform lactonization at a concentration of less than 10% in terms of solid content. .

By repeating the lactonization, the lactone of the sialic acid compound can be obtained with higher purity.

Specifically, the obtained solution containing the lactone form of the sialic acid compound is freeze-dried, the freeze-dried powder is dissolved in glacial acetic acid, and the lactonization is carried out by reacting again as described above.

The intramolecular lactonization of the sialic acid compound can be monitored as appropriate by a nuclear magnetic resonance method or the like as described in the test method described later.

The method of contacting the lactonized sialic acid compound of the present invention with an anion exchanger and collecting a non-adsorbed fraction is carried out in substantially the same manner as the method of separating the sialic acid compound in the raw material solution. The type and ionic type of the anion exchanger that can be used are not limited. Only,
The method is different from the above-described separation method of a sialic acid compound using an anion exchanger in that a non-adsorbed fraction is collected and thus a step of eluting and separating the adsorbed fraction is not required. The anion exchanger used for obtaining the non-adsorbed fraction may be the same as the anion exchanger used in the method for separating the sialic acid compound, and may be a different one. They can be used in combination.

Specifically, a pretreatment of appropriately diluting a solution containing a lactonized sialic acid compound with a neutral aqueous solution (for example, a phosphate buffer or the like), and adjusting the pH to neutral with sodium hydroxide or the like. Pre-adjustment to the vicinity, or pre-treatment to remove acetic acid by electrodialysis, etc., and contact the anion exchanger, the non-adsorbed fraction containing the lactone form obtained from the sialic acid compound with high purity Separate and collect.

The adsorbed fraction of the anion exchanger may contain a small amount of a sialic acid compound in which the lactone has been opened during the operation. Therefore, the adsorption fraction of the anion exchanger is eluted and recovered, desalted, lactonized again, and the lactone obtained from the sialic acid compound is recovered.
Recovery can also be increased.

The non-adsorbed fraction containing the lactone derivative obtained from the sialic acid compound with high purity can be used as it is or after desalting in a solution state. Preferably, it is used in a freezing bath, a dry ice acetone bath, a liquid nitrogen The lactone body can be rapidly frozen and freeze-dried, and stored and used in the form of a solid powder having improved storage stability of the lactone body.

The lactone obtained from the high-purity sialic acid compound produced by the production method of the present invention can be used as a raw material for foods, feeds, pharmaceuticals, cosmetics and the like.

Next, the present invention will be described in detail with reference to test examples. In the present invention, the following test method was adopted.

(1) Method for analyzing intramolecular lactonization of sialic acid compound The method of Nakahara et al. [Tetrahedron Letter
Letter, Vol. 35, p. 3321, 1994], the state of lactonization in the molecule of the sample was analyzed by ¹ H-nuclear magnetic resonance spectroscopy, and the state of formation of the lactone form of the sialic acid compound was monitored. . In order to facilitate understanding of this analysis method, the following representative sialic acid compound, 3
This will be described by way of an example of monitoring the production state of a lactone form of '-sialyllactose.

Incidentally, ¹ H-nuclear magnetic resonance (hereinafter sometimes referred to as NMR) was recorded in heavy water (D ₂ O) at 300 K using a spectrometer INOVA600 (manufactured by Varian Co.), and The shift value (ppm) was determined as a difference from the signal positions of 3- (trimethylsilyl) -1-propane sulfate sodium salt (TPS) and acetone, which are reference substances.

3'-Sialyl lactose (manufactured by Sigma)
Was subjected to lactonization in the presence of glacial acetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) at 25 ° C. for 5 days, and ¹ H-NMR analysis was performed before and after the reaction. The results are shown in FIGS. 1 and 2, respectively. 1 and 2 show NMR spectra.

As a result of the analysis, 3′-sialyl lactose is a kind of 3′-sialyl lactose lactone obtained by lactonization from 3′-sialyl lactose represented by the following formula (Chemical Formula 1), but has a different chemical structure. '-Sialyl lactose 1-
It was confirmed that 2 lactone and 3′-sialyllactose 1-4 lactone represented by the following formula (Formula 3) were produced. Specifically, as is clear from FIGS. 1 and 2, a 2.732 ppm signal derived from equatorial hydrogen (hereinafter, sometimes referred to as H-3eq) located on the 3-position carbon of the sialic acid molecule. But 2.552 each
ppm and 2.581 ppm, which are shifted to the high magnetic field side, lactonization was confirmed, and 3′-sialyl lactose 1-2 lactone and 3′-sialyl lactose 1-4 lactone were generated, respectively. confirmed.

From the above results, when examining the state of formation of the lactone form from the sialic acid compound, it was found that sialic acid H-3e
The intensity of the signal shifted to the high magnetic field derived from q was measured, and it was found that the production of the lactone form could be monitored from the height of the integral curve. In addition, as a result of similarly analyzing other sialic acid compounds, a shift in the signal derived from H-3eq of sialic acid was similarly observed by lactonization.

[0052]

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[0053]

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[0054]

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(2) Method for Quantifying Lactones Obtained from Sialic Acid Compounds According to the thin layer chromatography method (Ryu Shinke, “Science of Carbohydrates”, p. 33, Asakura Shoten, 1996) Were identified and quantified.

In order to facilitate understanding of the present quantification method, a description will be given of an example in which 3′-sialyl lactose, which is a typical sialic acid compound, and 3′-sialyl lactose lactone obtained by lactonizing the same are identified and quantified. I do.

That is, a sample dissolved in distilled water at a concentration of 1% and a known concentration as a standard substance (0.02 per spot)
3'-Sialyl lactose and 3
'-Sialyl lactose lactone was developed using ethanol-n-butanol-pyridine-water-glacial acetic acid (volume ratio 100: 10: 10: 30: 3) as a developing solution. After development is completed, dry and use ethanol as a color former.
Spray sulfuric acid (volume ratio 95: 5) and heat (110 ° C)
And developed the color. Next, the thin-layer chromatogram was captured by a scanner, and the relative concentration and mobility of each spot were determined. As a result of comparison with the standard substance, when the relative mobility of 3′-sialyl lactose was set to 1, the mobility of 3′-sialyl lactose lactone was 1.250, which was easily distinguishable.

The relative concentrations of a standard substance having a known concentration and a spot corresponding to a lactone compound obtained from the sialic acid compound in the sample were compared, and the lactone compound obtained from the sialic acid compound in the sample was quantified.

(3) Method for calculating the purity of the lactone obtained from the sialic acid compound The total amount (A) of the lactone obtained from the sialic acid compound in the sample was measured by the above-mentioned method for quantifying the sialic acid compound. From the value and the value of the total amount (B) of the sample, the purity of the lactone obtained from the sialic acid compound was calculated by the following equation.

Purity (%) = (A / B) × 100

Test Example 1 This test shows that the method for producing a lactone obtained from a sialic acid compound of the present invention is superior in yield compared to the method for producing a lactone obtained from a sialic acid compound of the prior art. Made to show. (1) Preparation of Samples The following two types of samples were each prepared by repeating 5 times. Sample 1: Lactone of sialic acid compound produced by the same method as in Example 1 of the present invention Sample 2: Lactone in the sialic acid compound-containing raw material was subjected to ring opening treatment in advance, as in Prior Art 1. Lactone form of a sialic acid compound produced by the same method as in Example 1 of the present invention except that there is no lactone.

(2) Test Method The yield of the lactone obtained from the sialic acid compound in each sample was measured for each sample by the method for quantifying the lactone obtained from the sialic acid compound, and the average value of five measurements was calculated. did.

(3) Test Results From the results of this test, the rate of increase in the yield (D) of the lactone form of the sialic acid compound of Sample 1 was defined as 100, with the yield (C) of the lactone form of the sialic acid compound of Sample 2 being 100. %) Was calculated by the following equation.

Increase rate (%) = [(D / C) × 100] −100

As a result, the rate of increase by the production method of the present invention was more than 20% as compared with the prior art 1, and it was found that the lactone form could be obtained in high yield. In addition, the purity of the lactone form of the sialic acid compound of Sample 1 and Sample 2 was the same.

The type of raw material, the method of ring-opening lactone, the method of separating sialic acid compound, the method of lactonization,
The test was conducted by appropriately changing the type and ion type of the anion exchanger, and almost the same results were obtained.

Test Example 2 This test was conducted to examine the necessity of anion exchanger treatment after lactonization using the purity of the lactone form obtained from the sialic acid compound as an index. The following three types of samples were prepared by repeating 5 times each. Sample 3: Lactone of sialic acid compound produced by the same method as in Example 1 of the present invention Sample 4: AG2X8 (Bio-Rad), a cation exchanger instead of anion exchanger treatment after lactonization The lactone derivative of the sialic acid compound produced by the same method as in Example 1 of the present invention except that the cation exchanger treatment is carried out using the sample. Sample 5: Do not carry out the anion exchanger treatment after lactonization. Lactone form of a sialic acid compound produced by the same method as in Example 1 of the present invention except for

(2) Test Method The purity of the lactone obtained from the sialic acid compound of each sample was calculated for each sample by the above-described test method, and the average value was calculated five times.

(3) Test Results The results of this test are as shown in Table 1. As is clear from Table 1, in order to produce a lactone derivative obtained from a sialic acid compound having high purity, it was found that treatment with an anion exchanger after lactonization was necessary. In addition, although the kind of raw material, the method of ring-opening treatment of the lactone body, the method of separating the sialic acid compound, the method of lactonization, and the type and ion type of the ion exchanger were appropriately changed, the results were almost the same. Obtained.

[0070]

[Table 1]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0072]

EXAMPLES Example 1 1.5 kg of bovine colostrum (manufactured by Morinaga Milk Industry Co., Ltd.)
The mixture was centrifuged at m / s ² for 20 minutes to defat, thereby obtaining about 1 kg of skim milk. To this, 2 kg of distilled water was added, and the mixture was extracted with a 4-fold volume of chloroform-ethanol (volume ratio 2: 1, all manufactured by Wako Pure Chemical Industries, Ltd.), deproteinized, and obtained water / ethanol phase. Is kept at 40 ° C. or lower and about 100 ml (4.0 in terms of solid content) with a rotary evaporator.
%). The concentrated solution was gradually cooled and frozen in a -10 ° C freezer (manufactured by Hitachi, Ltd.), and then left to stand at room temperature to be thawed, and the lactone was subjected to ring opening treatment in advance. The melted solution was passed through a Wakogel LP-40C18 column (manufactured by Wako Pure Chemical Industries, Ltd.) to remove the remaining peptide.

Next, the obtained eluate (3.5% in terms of solid content) was treated at 4 ° C. with AG1X4, an anion exchanger.
(Manufactured by Bio-Rad, Inc .; ionic Cl ⁻ , ionic strength 1)
3.5 mS / cm. ) Was passed through the column packed with The sialic acid compound adsorbed on the column was eluted with a 0.5 M sodium chloride solution to be separated, and the eluate was desalted using a micro-acylizer G1 (manufactured by Asahi Kasei Corporation), and lyophilized.
About 750 powder containing a non-lactone form of a sialic acid compound
mg was obtained.

This lyophilized powder was dissolved in glacial acetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) at a concentration of 2% in terms of solid content, and reacted at 37 ° C. for 7 days to form a lactone.

The resulting solution containing the lactonized sialic acid compound was diluted 10-fold with distilled water, and then acetic acid was removed using a micro-acylizer G1 (manufactured by Asahi Kasei Corporation). The solution was adjusted to a concentration of 0.1% and the solution was treated at 4 ° C. with an anion exchanger AG1X4 (manufactured by Bio-Rad Co., Ltd., ionic Cl ⁻ , ionic strength 13.5 mS / c).
m. ) Was passed through a column packed with) to collect a permeate, which is a non-adsorbed fraction containing a lactone derivative obtained from a sialic acid compound with high purity.

Next, the permeated liquid was added to the micro-acylizer G
1 (made by Asahi Kasei Corporation), then quenched in a dry ice acetone bath at -80 ° C, frozen, freeze-dried, and freeze-dried containing a lactone obtained from a sialic acid compound in high purity About 450 mg of powder was obtained.

The obtained lyophilized powder was tested by the same test method as described above. As a result, it was found that 3'-sialyl lactose lactone was contained at a purity of 97%.

Example 2 Hydrochloric acid (manufactured by Morinaga Milk Industry Co.) was added to 100 kg of bovine skim colostrum (manufactured by Morinaga Milk Industry Co., Ltd.), and the pH was adjusted to 4.3. The resulting whey was subjected to ultrafiltration. The protein was removed by membrane treatment, and the permeate was adjusted to pH 7.0 with sodium hydroxide (manufactured by San-Ei Gen FFI). The obtained solution was used to remove fine precipitates using a clarifier (produced by API Buoy), concentrated using a concentrator (produced by Asahi Kasei Corporation), and desalted with an electrodialysis device (produced by Asahi Kasei Corporation). The concentrated solution obtained by desalting is gradually cooled and frozen in a freezer (manufactured by Hitachi, Ltd.) at -10 ° C, and then left at room temperature to be thawed.
The lactone was subjected to ring opening treatment in advance.

Next, the melted solution (20 in terms of solid content)
%) At 4 ° C. and the anion exchanger Amberlite I
The solution was passed through a column filled with RA410 (manufactured by Organo Corporation; ion type Cl ⁻ , ionic strength: 1 mS / cm). The sialic acid compound adsorbed on the column is washed with water, separated by elution with a 0.5 M sodium chloride solution, and the eluate is desalted using an electrodialyzer (manufactured by Asahi Kasei Corporation), freeze-dried, and dried. Approximately 130 g of a powder containing the non-lactone compound was obtained.

The lyophilized powder was mixed with glacial acetic acid (San-Ei Gen F.
Dissolved in a concentration of 2% in terms of solid content,
The reaction was carried out at 37 ° C. for 5 days to form a lactone.

The obtained solution containing the lactonized sialic acid compound was diluted 10-fold with distilled water, and then acetic acid was removed using an electrodialyzer (manufactured by Asahi Kasei Corporation). The solution was adjusted to a concentration of 1%, adjusted to pH 6.0 with sodium hydroxide (manufactured by San-Ei Gen FFI), and the solution was heated at 4 ° C at Amberlite I, an anion exchanger.
The solution was passed through a column packed with RA410 (manufactured by Organo; ionic Cl ⁻ , ionic strength: 1 mS / cm), and the permeate, which was a non-adsorbed fraction containing the lactone form obtained from the sialic acid compound with high purity, was passed through the column. Collected.

Next, the permeate was desalted using an electrodialyzer (manufactured by Asahi Kasei Corporation), quenched in a freezing bath at −40 ° C., frozen, freeze-dried, and the lactone obtained from the sialic acid compound. About 50 g of a lyophilized powder containing the body in high purity was obtained.

The obtained lyophilized powder was tested by the same test method as described above, and as a result, it contained 3'-sialyllactose lactone at a purity of 95%.

Example 3 200 kg of rennet whey (manufactured by Morinaga Milk Products Co., Ltd.) was treated with sodium hydroxide (manufactured by San-Ei Gen FFI Co., Ltd.) at pH 7.0.
Adjusted to zero. The obtained solution was used to remove fine precipitates using a clarifier (produced by API Buoy), concentrated using a concentrator (produced by Asahi Kasei Corporation), and desalted with an electrodialysis device (produced by Asahi Kasei Corporation). The concentrated solution obtained by desalting is
The mixture was gradually cooled and frozen in a 10 ° C. freezer (manufactured by Hitachi, Ltd.), then left at room temperature to be thawed, and the lactone was subjected to ring opening treatment in advance.

Next, the melted solution (19% in terms of solid content)
%) At 4 ° C., DEAE-Sephadex (Amersham Pharmacia Biotech, an anion exchanger).
Ionic Cl ⁻ , ionic strength 3 mS / cm. ) In a batch method, the sialic acid compound adsorbed on the anion exchanger gel is washed with water, eluted with 0.8M sodium chloride solution and separated, and the eluate is used with an electrodialyzer (Asahi Kasei Corporation) Then, the resultant was desalted and freeze-dried to obtain about 900 g of a powder containing a non-lactone form of a sialic acid compound.

This lyophilized powder was mixed with glacial acetic acid (San-Ei Gen F.
Dissolved in a concentration of 2% in terms of solid content,
The reaction was carried out at 37 ° C. for 5 days to form a lactone.

The resulting solution containing the lactonized sialic acid compound was diluted 10-fold with distilled water, and then acetic acid was removed using an electrodialyzer (manufactured by Asahi Kasei Corporation). The solution was adjusted to a concentration of 1%, and the solution was passed through a batch method at 4 ° C. through DEAE-Sephadex (manufactured by Amersham Pharmacia Biotech Co., Ltd .; ionic Cl ⁻ , ionic strength 3 mS / cm) as an anion exchanger. Then, a solution of the non-adsorbed fraction containing the lactone derivative obtained from the sialic acid compound with high purity was collected.

Next, the non-adsorbed fraction solution was desalted using an electrodialyzer (manufactured by Asahi Kasei Corporation), quenched in a freezing bath at −40 ° C., frozen, freeze-dried, and recovered from the sialic acid compound. About 230 g of a lyophilized powder containing the obtained lactone compound with high purity was obtained. The obtained lyophilized powder was tested by the same method as the above test method, and as a result, it contained a lactone obtained from a sialic acid compound at a purity of 84%.

Example 4 1.5 kg of bovine colostrum (manufactured by Morinaga Milk Industry Co., Ltd.) was added to 117,600
The mixture was centrifuged at m / s ² for 20 minutes to defat, thereby obtaining about 1 kg of skim milk. To this, 2 kg of distilled water was added, and the mixture was extracted with a 4-fold volume of chloroform-ethanol (volume ratio 2: 1, all manufactured by Wako Pure Chemical Industries, Ltd.), deproteinized, and obtained water / ethanol phase. Is kept at 40 ° C. or lower and about 100 ml (4.0 in terms of solid content) with a rotary evaporator.
%). The concentrated solution was gradually cooled and frozen in a -10 ° C freezer (manufactured by Hitachi, Ltd.), and then left to stand at room temperature to be thawed, and the lactone was subjected to ring opening treatment in advance. The melted solution was passed through a Wakogel LP-40C18 column (manufactured by Wako Pure Chemical Industries, Ltd.) to remove the remaining peptide.

Next, the obtained eluate (3.5% in terms of solid content) was treated at 4 ° C. with an anion exchanger AG1X4
(Manufactured by Bio-Rad, Inc .; ionic Cl ⁻ , ionic strength 1)
3.5 mS / cm. ) Was passed through the column packed with The sialic acid compound adsorbed on the column was eluted with a 0.5 M sodium chloride solution to be separated, and the eluate was desalted using a micro-acylizer G1 (manufactured by Asahi Kasei Corporation), and lyophilized.
About 750 powder containing a non-lactone form of a sialic acid compound
mg was obtained.

This lyophilized powder was dissolved in glacial acetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) at a concentration of 2% in terms of solid content, and reacted at 30 ° C. for 3 days to form a lactone.

The obtained solution containing the lactonized sialic acid compound was diluted 10-fold with distilled water, and then the acetic acid was removed using a micro-acyler G1 (manufactured by Asahi Kasei Corporation), followed by freeze-drying.

This freeze-dried powder was dissolved again in glacial acetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) at a concentration of 2% in terms of solid content,
For 3 days to form a lactone.

The resulting solution containing the lactonized sialic acid compound was diluted 10-fold with distilled water, and then acetic acid was removed using a micro-acylizer G1 (manufactured by Asahi Kasei Corporation). The solution was adjusted to a concentration of 0.1% and the solution was treated at 4 ° C. with an anion exchanger AG1X4 (manufactured by Bio-Rad Co., Ltd., ionic Cl ⁻ , ionic strength 13.5 mS / c).
m. ) Was passed through a column packed with) to collect a permeate, which is a non-adsorbed fraction containing a lactone derivative obtained from a sialic acid compound with high purity.

Next, the permeated liquid was added to the micro-acylizer G
1 (made by Asahi Kasei Corporation), then quenched in a dry ice acetone bath at -80 ° C, frozen, freeze-dried, and freeze-dried containing a lactone obtained from a sialic acid compound in high purity About 510 mg of a powder was obtained.

The obtained lyophilized powder was tested by the same test method as described above. As a result, it was found that 3'-sialyllactose lactone was contained at a purity of 99%.

Example 5 1 kg of sour whey powder (manufactured by Mirai) was added to distilled water 2
The resultant was dissolved in 0 kg, gradually cooled and frozen in a -10 ° C. freezer (manufactured by Hitachi, Ltd.), then left at room temperature to be thawed, and the lactone was subjected to ring opening treatment in advance.

Next, the molten solution (about 5% in terms of solid content)
%) At 4 ° C. and an anion exchanger AG1X4 (manufactured by Bio-Rad Co., Ltd .; ionic Cl ⁻ , ionic strength 13.5 m)
S / cm. ) Was passed through the column packed with The sialic acid compound adsorbed on the column was eluted with a 0.5 M sodium chloride solution and separated.
The salt was desalted using (manufactured by Asahi Kasei Corporation) and freeze-dried to obtain about 11.7 g of a powder containing a non-lactone form of a sialic acid compound.

The freeze-dried powder was dissolved in glacial acetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) at a concentration of 2% in terms of solid content, and reacted at 30 ° C. for 3 days to form a lactone.

The resulting solution containing the lactonized sialic acid compound was diluted 10-fold with distilled water, and then the acetic acid was removed using a micro-acylizer G1 (manufactured by Asahi Kasei Corporation) and freeze-dried.

The freeze-dried powder was dissolved again in glacial acetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) at a concentration of 2% in terms of solid content, and then dissolved at 30 ° C.
For 3 days to form a lactone.

The resulting solution containing the lactonized sialic acid compound was diluted 10-fold with distilled water, and then acetic acid was removed by using a micro-acylizer G1 (manufactured by Asahi Kasei Corporation), and the solid content was reduced to 0. The solution was adjusted to a concentration of 0.1% and the solution was treated at 4 ° C. with an anion exchanger AG1X4 (manufactured by Bio-Rad Co., Ltd., ionic Cl ⁻ , ionic strength 13.5 mS / c).
m. ) Was passed through a column packed with) to collect a permeate, which is a non-adsorbed fraction containing a lactone derivative obtained from a sialic acid compound with high purity.

Next, the permeated liquid was added to the micro-acylizer G
1 (made by Asahi Kasei Corporation), then quenched in a dry ice acetone bath at -80 ° C, frozen, freeze-dried, and freeze-dried containing a lactone obtained from a sialic acid compound in high purity About 5 g of powder was obtained.

The obtained freeze-dried powder was tested by the same method as the above test method, and as a result, it was found to contain 3'-sialyllactose lactone at a purity of 75%.

[0105]

As described in detail above, the present invention provides
The present invention relates to a method for producing a lactone derivative obtained from a sialic acid compound which is useful mainly as a raw material for food, feed, medicine or cosmetics in high yield and high purity from natural raw materials such as milk or milk components, and the present invention. The effects achieved by the above are as follows. 1) According to the method of the present invention, a lactone in a raw material solution containing a sialic acid compound is subjected to ring opening treatment in advance,
The lactone form originally present in the raw material solution can be easily recovered together with the non-lactone form, and the lactone form obtained from the sialic acid compound can be produced in high yield. 2) By the method of the present invention, a lactone compound obtained from a sialic acid compound can be produced with high purity. 3) The lactone form obtained from the sialic acid compound can be produced with high yield and high purity by the method of the present invention, so that the lactone form obtained from the sialic acid compound can be mass-produced, and can be used for foods, feeds, and pharmaceuticals. It can be applied to a wide range of uses as a material for cosmetics. 4) According to the method of the present invention, a lactone obtained from a safe sialic acid compound that is acceptable for food can be produced.

[Brief description of the drawings]

FIG. 1 shows the ¹ H—N of 3′-sialyl lactose
It is an MR spectrum.

FIG. 2 shows the 3′-sialyl lactose lactone.
It is a < ¹ > H-NMR spectrum.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Hiroki Hayazawa 5-1-1, Higashihara, Zama City, Kanagawa Prefecture Inside Morinaga Dairy Co., Ltd. Nutrition Science Research Institute (72) Susumu Teraguchi 5-1-1, Higashihara, Zama City, Kanagawa Prefecture No. 83 Morinaga Dairy Co., Ltd. Nutrition Science Laboratory (72) Inventor Hidefumi Kuwata 5-1-1 Higashihara, Zama City, Kanagawa Prefecture Morinaga Dairy Co., Ltd. Nutrition Science Laboratory F-term (reference) 4C057 AA18 BB02 DD02 KK02 KK25

Claims (5)

[Claims] 1. A lactone compound in a raw material solution containing a sialic acid compound is subjected to ring opening treatment in advance, the sialic acid compound in the raw material solution is separated, lactonized, passed through an anion exchanger, and subjected to non-adsorption. A method for producing a lactone form obtained from a sialic acid compound, comprising collecting a fraction. 2. A raw material solution containing a sialic acid compound,
The method for producing a lactone obtained from the sialic acid compound according to claim 1, which is milk or a processed milk product. 3. The method for producing a lactone obtained from a sialic acid compound according to claim 2, wherein the milk or processed milk product is bovine colostrum, bovine skim colostrum, whey, whey qualic acid, or casein. 4. The method for producing a lactone obtained from a sialic acid compound according to claim 1, wherein the sialic acid compound is sialyl lactose, casein, or a casein hydrolyzate. 5. The method for producing a lactone obtained from a sialic acid compound according to claim 1, wherein the ring opening treatment of the lactone is a freeze-thaw method.