JP2001192393A - Endogenous sialidase-resistant lactone derivative- containing composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a nutrition composition or an antiviral agent containing a new lactone derivative of an N-acetylneuraminic acid compound not to be hydrolyzed by sialidase in vivo. SOLUTION: This composition such as powdered milk for infant rearing, diet during the weaning period, a nutrient for intestinal administration, an antiviral agent, etc., is characterized in that the composition contains at least 0.01 wt.% of 3'-N-acetylneuraminyllactose lactone having resistance to hydrolysis with endogenous sialidase or a lactone derivative of N-acetylneuraminic acid compound obtained from casein or casein hydrolyzate as an active ingredient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The invention of this application relates to a composition containing an endogenous sialidase-resistant lactone. More specifically, the invention of this application relates to an endogenous sialidase-resistant lactone-containing composition containing, as an active ingredient, a lactone of an N-acetylneuraminic acid compound having resistance to hydrolysis by endogenous sialidase. .

[0002]

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 (Yamauchi). Kunio et al., "Milk Comprehensive Dictionary", 1st edition, 519
Page, Asakura Shoten, 1992).

Sialic acid is a common name for acyl derivatives of neuraminic acid, meaning, for example, N-acetyl or N-glycolylneuraminic acid and chemicals such as O-acetyl or O-glycolylneuraminic acid. I have.

[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,
Physiological functions are known, such as sialic acid compounds used in foods, pharmaceuticals, etc., as sialic acid-containing milk powder (Japanese Patent Application Laid-Open No. 61-152233). Powdered milk for childcare (JP-A-3-49648), a neutralizing agent for bacterial toxins (JP-A-2805490),
Sialic acid compounds can be used as a substitute for breast milk, functional foods, etc., as in infection-protecting agents (Patent No. 2631470), anti-inflammatory agents (Patent No. 2684179), and powdered milk for infants (Patent No. 2514375) provided with infection-protecting ability. Attempts have been made to use it as a drug material.

However, in any of the above-mentioned prior arts, it is known that the administered sialic acid compound is degraded by not only bacterial and viral sialidases but also animal and human sialidases and loses its physiological functions. (Fumiharu Maruo, Nobuo Tamiya, "Enzyme Handbook", First Edition, p.499, Asakura Shoten, 1986
Year).

[0006] Generally, sialidase is an enzyme that releases α-ketoside-linked sialic acid from a sialic acid compound, and is widely distributed in various tissues such as viruses, bacteria, birds and mammals (Fumiharu Maruo, Nobuo Tamiya). Supervision, Enzyme Handbook, First Edition, p. 499, Asakura Shoten, 1986
Year).

As a method for preventing the decomposition and inactivation of such a physiologically active substance by an enzyme, for example, a method of adding an enzyme inhibitor [Biochemical Pharmacology
(Biochemical Pharmacology), Vol. 36, No. 1035
1039, 1987], or a method of enclosing a physiologically active substance in a liposome having a closed structure or the like (Bioscience and Bioindustry, Vol. 53, No. 130)
~ 133 pages, 1995) are generally known,
In any of the above-mentioned prior arts, a method considering such resistance to sialidase has not been adopted.

The sialic acid compound is not a single substance but a general term for compounds such as glycolipids, oligosaccharides, glycoproteins, etc. in which acetylneuraminic acid or glycolylneuraminic acid is bound in the molecule. Among them, whether or not a specific compound has resistance to sialidase degradation is not described in any of the above-mentioned prior arts.

On the other hand, as a derivative of the sialic acid compound, a compound having a 1-3 lactone structure in the molecule (Japanese Patent Application Laid-Open No.
No. 12695) or a compound having a 1-7 lactone structure (JP-A-63-13913). In addition, the existence of a lactone body of ganglioside having a lactone structure in the molecule is present in the brain [The Journal of Biological Chemistry (The journal
al of Biological Chemistry), Vol. 261, 851
4, 1986], and the presence of 1-4 or 1-2 lactones of N-glycolylneuraminic acid compounds in sheep milk [Biokimica eto biophysica acta (Bio
chimica et Biophysica Acta), Volume 1381, Volume 28
6, p. 1998].

In general, the free carboxyl group of the sialic acid compound does not exist in the lactone form of these sialic acid compounds, and the carboxyl group forms a part of the lactone ring. Is a completely different substance. Naturally, the sialic acid compound is an acidic substance, and is completely different in physicochemical properties from the lactone form of the neutral sialic acid compound.

The lactone form of the sialic acid-binding compound is also different from the conventional sialic acid compound separation method in that electrical properties are different, so that only a very small amount can be separated. , Sialic acid compounds are substances that are distinctly different in nature (chemistry and biology,
37, p. 401, 1999).

It is considered that the lactones of these sialic acid compounds may be resistant to sialidase degradation as described below. However, regarding the 1-3 or 1-7 lactone form of the sialic acid compound, resistance to degradation by sialidase is not known at all. In addition, the sialic acid compound 1-3 or 1-7 lactone is a substance which hardly exists in nature and has a problem that it cannot be administered to humans and animals in large quantities including safety.

On the other hand, ganglioside 1-4 or 1-2
It has been suggested that the lactone forms show resistance to degradation of virus-derived sialidase [glycoconjugate.
Glycoconjugate Journal, Volume 16, Volume 2
P. 23, 1999. Hereinafter, this will be referred to as conventional technology 1]. In addition, 1-4 or 1 of N-glycolylneuraminic acid compound
It has been speculated that the -2 lactone body is also resistant to degradation against bacterial sialidase [Biochimica et Biophysica acta (Biochimica et Bio
physica Acta), volume 1381, page 286, 1998.
Year. Hereinafter, this will be referred to as conventional technology 2]. These prior arts 1
And 2 also have many unclear points regarding the resistance to sialidase as described below, and are currently far from being industrially used in various ways.

[0014]

Even if the lactones of the prior arts 1 and 2 were resistant to viral and bacterial sialidases, they could not be digested and administered to humans and animals. Sialidase present in etc.
That is, it is considered that it is easily decomposed by endogenous sialidase, and the physiological activity as a sialic acid compound is also inactivated. However, at present, these lactones have no known resistance to degradation by human and animal sialidases, as well as no apparent resistance to degradation by viral and bacterial sialidases.

The above-mentioned prior arts 1 and 2 describe the resistance of lactones to sialidase derived from viruses and bacteria, but do not describe the resistance to sialidase which humans and animals originally possess. There are large amounts of endogenous sialidase in various tissues of humans and animals, especially in the digestive tract (tissues and mucous membranes). The endogenous sialidase present in the tissues and gastrointestinal tract of such animals differs depending on the animal species, and also has different characteristics from bacterial and viral sialidases (Metabolism, Vol. 16, p. 37, 197).
9 years).

For example, ganlioside GM2, which is a kind of sialic acid compound, has conventionally been considered to be resistant to sialidase. Animal sialidase degrades this sialic acid compound (metabolism, Vol. 16, p. 37; 1979
Year). Therefore, in the case of sialic acid compounds ingested orally, regarding the stability and effectiveness of the lactone body (meaning protection or prevention of infection), first consider the resistance to sialidase in the digestive tract of humans and animals. And then the bacterial and viral resistance to sialidase must also be considered.

Further, Prior Art 1 describes the resistance to degradation by viral sialidase of only the lactone of ganglioside. However, the lactone form of ganglioside is a substance of natural origin but a substance that is found in trace amounts in the brain such as minke whales (Carbohydrate Research).
307, 281, 1998], and it is neither qualitatively nor quantitatively applicable to foods and pharmaceuticals.

The 1-4 or 1-2 lactone of the N-glycolylneuraminic acid compound of the prior art 2 is a compound present in sheep's milk. No glycolyl neuraminic acid compound is present,
The digestibility and safety when administered are unknown (supervised by Kazutomo Imahori et al., "Biochemical Dictionary", 1st edition, p. 541, Tokyo Kagaku Dojin, 1984).

Further, it has been suggested that the lactone form of N-glycolylneuraminic acid may be used as an antiviral drug and as a powdered milk for childcare to which an infection preventive agent has been added (see Chemistry and Biology, Vol. 37, p. 401, 199
9 years), its safety when administered to humans and certain animals, its resistance to sialidase present in humans and animals is unknown, and foods and lactones of N-glycolylneuraminic acid Application to pharmaceuticals was in an impossible situation.

The inventors of the present application have conducted intensive studies in view of the above-mentioned prior art, and as apparent from the test examples described later, among the lactones of various sialic acid compounds,
It has been found that 1-4 or 1-2 lactones of N-acetylneuraminic acid compounds isolated from milk have remarkable resistance to degradation by endogenous sialidase in animals and humans.

The invention of this application has been made based on the findings as described above, and contains a 1-4 or 1-2 lactone of an N-acetylneuraminic acid compound which is stable in a living body. It is an object to provide a nutritional composition or an antiviral agent.

[0022]

Means for Solving the Problems The invention of this application which solves the above-mentioned problems comprises, first, a lactone form of an N-acetylneuraminic acid compound having resistance to hydrolysis by endogenous sialidase; As at least 0.01
A composition containing an endogenous sialidase-resistant lactone body, characterized in that the composition comprises:
The 1-4 or 1-2 lactone form of the N-acetylneuraminic acid compound is 3'-N-acetylneuraminyl lactose 1-4 lactone, 3'-N-acetylneuraminyl lactose 1-2 lactone, or casein or The above composition, which is a 1-4 or 1-2 lactone form of an N-acetylneuraminic acid compound obtained from a casein hydrolyzate, and thirdly, the composition is a powdered baby milk, a baby food,
The composition is provided as an enteral nutritional or antiviral agent.

[0023]

Embodiments of the present invention will be described below in detail. In the following description, percentages are values by weight unless otherwise specified, except for the adhesion ratio.

First, the 1-4 or 1-2 lactone compound of the N-acetylneuraminic acid compound used in the invention of this application was prepared according to the invention already filed by the applicant (Japanese Patent Application No. 331295). Can be manufactured as follows.

That is, a lactone in a raw material such as milk or whey containing an N-acetylneuraminic acid compound is subjected to ring opening treatment in advance, and the N-acetylneuraminic acid compound in the raw material is separated and lactonized. Contact with an anion exchanger to obtain non-adsorbed fraction. The endogenous sialidase-resistant N-acetylneuraminic acid compound 1-
The 4 or 1-2 lactone form (hereinafter sometimes simply referred to as a lactone form) can be used as it is as a liquid and can be used as a raw material for infant powder, baby food or enteral nutrition. Note that, more specifically, Reference Examples 1 and 2 can be referred to.

As a specific method for incorporating a lactone in a food, an antiviral agent, or the like, the lactone can be mixed in a liquid form or in a powder form. However, since the lactone body may be gradually decomposed in a liquid state, it may be used as it is in the form of powder mixed with food, antiviral agents, etc. on the premise of long-term storage,
Alternatively, it is desirable to use it after dissolving it at the time of use.

When the lactone form is produced as a powder and used as a raw material, the fraction of the lactone form obtained in a liquid form is desalted, rapidly frozen and freeze-dried to improve the storage stability of the lactone form. It can be in an improved powder state.

In the case of producing a powder containing fat in the infant milk powder, baby food or enteral nutrition of the invention of this application, for example, an aqueous phase and an oil phase are separately prepared, and an emulsifier and a stabilizer are prepared. , Homogenize with a homogenizer, concentrate and dry to produce a powdery intermediate product, which contains lactones, if necessary, indigestible sugars, trace metals , Ironing agents and other raw materials can be added and mixed uniformly to produce the final product.

When producing the antiviral agent of the present invention, tablets, pills and capsules are appropriately mixed with pharmaceutically acceptable excipients, extenders, diluents, solubilizing agents and the like. , Suppositories and the like.

[0030] The final product of the present invention, childcare milk,
The content of the lactone of the sialic acid compound in a baby food, enteral nutrition or antiviral agent is at least 0.01%, more preferably 0.1 to 30%. In addition, it is usually desirable to administer a lactone body at a dose of 1 mg / kg / day or more in infant milk, baby food or enteral nutrition. Furthermore, although the antiviral agent of the present invention varies depending on symptoms, age, etc., it is desirable to administer a lactone body at a dose of 10 mg / kg / day or more, as apparent from the test examples described later.

Next, the invention of this application will be described in detail with reference to test examples. Test Example 1 This test was performed to examine the resistance of the lactone compound of the present invention to endogenous sialidase. (1) Preparation of sample 1) Preparation of endogenous sialidase To human large intestine stored at −80 ° C., ice-cold 0.1 mM
PBS buffer (manufactured by Sigma) containing phenylmethylsulfonyl fluoride (manufactured by Roche Diagnostics)
9 volumes were added, homogenized with a Teflon glass homogenizer (manufactured by Tokyo Rika Kikai Co., Ltd.), and 10 g at 1000 × g.
After centrifugation for minutes, the supernatant was separated and diluted with a phosphate buffer to a protein concentration of 3.4 mg / ml to obtain a crudely extracted endogenous sialidase solution. 2) Preparation of each sample 3'-N-acetylneuraminyl lactose (manufactured by Sigma) or 3'-N-acetylneuraminyl lactose lactone prepared by the same method as in Reference Example 1 described below was added at 0.1 mg / ml. At a concentration of 10 mM sodium acetate buffer (pH 4.6).

Each solution was mixed with the endogenous sialidase solution 0.1
and mixed at 37 ° C. to prepare a sample. As a control, 0.1 ml of a sodium acetate buffer was mixed with 0.1 ml of the endogenous sialidase solution, and the mixture was kept in the same manner. (2) Test method The released sialic acid was quantified by the periodate-thiobarbituric acid method (Metabolism, Vol. 16, p. 761, 1979). As described below, this method is a method of generating β-formylpyruvic acid from sialic acid by periodate oxidation and quantifying this by developing color with thiobarbituric acid.

The reagents used in this method are as follows. Periodic acid solution Periodic acid (Wako Pure Chemical Industries, Ltd.) was dissolved in 0.125N sulfuric acid at a final concentration of 25 mM.

Sodium arsenite solution Sodium arsenite (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 0.5N hydrochloric acid at a final concentration of 2%.

Thiobarbituric acid solution 2-thiobarbituric acid (manufactured by Nacalai Tesque, Inc.)
Dissolved in water at a final concentration of 1M and adjusted the pH to 9.0 with sodium hydroxide.

95 ml of acidic butanol n-butanol (manufactured by Nacalai Tesque, Inc.)
5 ml of N hydrochloric acid were mixed.

The endogenous sialidase solution was mixed with each sample and control solution, passed through a cation exchange column AG1X2 (manufactured by Bio-Rad) after 2, 4 and 6 hours to dilute the permeate appropriately. 0.25 ml of periodic acid solution in 5 ml
Was added and kept at 37 ° C. for 30 minutes. Then, add 0.2 ml of sodium arsenite solution to decompose periodic acid,
2 ml of thiobarbituric acid solution was added, capped and kept in a boiling water bath for 7.5 minutes to develop color.

After the color was developed, it was immersed in ice water and cooled, and 5.0 ml of acidic butanol was added and mixed well. The dye was transferred to the butanol layer, and the absorbance at 549 nm was measured. Simultaneously, a sialic acid having a known concentration (manufactured by Sigma) was similarly measured, a standard curve was prepared, and the sialic acid concentration in the sample was quantified.

The amount of sialic acid released from 3'-N-acetylneuraminyl lactose or 3'-N-acetylneuraminyl lactose lactone was determined by the amount of sialic acid obtained by mixing only the control buffer with the crude enzyme. The amount of acid, that is, the background was subtracted from each of them to determine the amount.

The amount of sialic acid released from 3'-N-acetylneuraminyl lactose (A) and 3 '
From the amount of sialic acid released from -N-acetylneuraminyl lactose lactone (B), 3'-N
-The decomposition resistance magnification of acetylneuraminyl lactose lactone was calculated.

Decomposition resistance magnification = A / B (3) Test results The results of this test are as shown in FIG. In the figure, Δ and ○ show the results of 3′-N-acetylneuraminyl lactose and 3′-N-acetylneuraminyl lactose lactone, respectively.

As is evident from FIG. 1, 3'-N-acetylneuraminyl lactose is degraded by sialidase present in the upper cells, whereas 3'-N-acetylneuraminyl lactose lactone is completely degraded. It was found to be not or only slightly degraded and to have strong resistance to digestion by endogenous sialidase.

Further, for example, the degradation resistance magnification after reacting each sample with an endogenous sialidase solution for 4 hours is more than 4, and the lactone form is 4 compared with 3'-N-acetylneuraminyl lactose. It was recognized that the compound exhibited a decomposition resistance that was more than doubled.

From this test result, it was found that the lactone compound of the invention of this application has resistance to endogenous sialidase. The type of endogenous sialidase, the N-acetylneuraminic acid compound and the type of lactone were appropriately changed and tested. In each case, the decomposition resistance magnification of the lactone was 4 or more. It was confirmed that it shows high degradation resistance to human and animal endogenous sialidase. Test Example 2 This test was carried out to examine the effect of the infant formula of the present invention on E. coli adhesion in a test tube. (1) Preparation of sample The infant formula was prepared by adding 3'-N-acetylneuraminyl lactose lactone prepared by the same method as in Reference Example 1 to 0% (control), 0.001%, 0.01%, and 0.1%. Five samples were prepared in the same manner as in Example 1 described below, except that 1% and 1.0% were added. (2) Test method Escherichia coli o11a (ATCC 29552) cultured in a heart infusion medium (manufactured by Nissui Pharmaceutical Co., Ltd.) was used for 10 ⁸ cells.
The cells were suspended in an Eagle medium (manufactured by Gibco BRL) at a ratio of cells / ml. To 0.1 ml of this suspension was added 0.1 ml of a solution prepared by dissolving each of the above-mentioned powdered milk powder samples or control samples containing 3'-N-acetylneuraminyl lactose lactone at a concentration of 13%. 407 strains derived from human small intestine (ATCC CCL-
6) 0.8 ml was added, and each was cultured at a temperature of 30 ° C. for 1 hour.

After the culture, the number of bacteria adhering to the cells of each sample was observed under a microscope, the number of adherent bacteria per 50 cells was counted, and the number of bacteria (C) in the control sample and the number of bacteria in each sample were measured. From (B), the adhesion rate (A) was calculated by the following equation and tested.

A (%) = (B / C) × 100 (3) Test Results The results of this test are as shown in Table 1. As is clear from Table 1, at a concentration of 3% -N-acetylneuraminyl lactose lactone of 0.01% or more, the adhesion ratio was significantly reduced, and at concentrations of 0.1% and 1.0%,
Almost no adhesion was observed, and an effect of preventing E. coli from adhering to cells was observed at a concentration of 0.01% or more.

From this test result, it was found that it is effective to contain at least 0.01% of the lactone compound. In addition, although the test was performed by changing the type of the lactone body, almost the same results were obtained.

[0048]

[Table 1]

Test Example 3 This test was conducted to examine the preventive effect of 3'-N-acetylneuraminyl lactose lactone on bacterial infection in vivo. (1) Preparation of test animals and samples 1) Test animals The animals used in the test are as follows. Weight about 200
25 g g Wistar rats (purchased from Charles River Japan) were randomly divided into 5 groups of 5 rats each, with one group serving as a control group and the remaining 4 groups serving as test groups. 2) Preparation of sample The infant formula was prepared by adding 3'-N-acetylneuraminyl lactose lactone to 0% (control), 0.001%, 0.01%.
Four samples were prepared in the same manner as in Example 1 described below, except that%, 0.1%, and 1.0% were added. (2) Test method Escherichia coli o55 (ATCC12014) at 10 ⁸ cells / ml
To 0.5 ml of the suspension, 9.5 ml of a solution in which each of the samples was dissolved at a concentration of 13% was added, and immediately 2 m / mouse was added.
l was orally administered to rats of each group, and MF solid samples (manufactured by Oriental Yeast Co., Ltd.) and fresh tap water were allowed to freely ingest and bred for 3 days. Thereafter, the onset of diarrhea was observed and tested. (3) Test results The results of this test are as shown in Table 2. As is evident from Table 2, in the group to which the infant milk powder to which 3'-N-acetylneuraminyl lactose lactone was added in an amount of 0.01% or more, the onset of diarrhea was significantly reduced, and the effect of preventing infection was observed. .

From the test results, it was found that administration of the lactone at a rate of at least 1 mg / kg / day is effective in preventing diarrhea caused by Escherichia coli. In addition, although the test was performed by changing the type of the lactone body, almost the same results were obtained.

[0051]

[Table 2]

Test Example 4 This test was conducted to examine the effect of 3'-N-acetylneuraminyl lactose lactone of the present invention in vivo on the prevention of viral infection. (1) Preparation of test animals and samples 1) Test animals As test animals, 27 BALB / C 5-day-old mice (body weight: about 3 to 4 g) were randomly divided into three groups, each consisting of nine mice. 2) Preparation of sample 3'-N-acetylneuraminyl lactose lactone was prepared in the same manner as in Reference Example 1. (2) Test method A solution prepared by dissolving 1 mg or 2 mg of the above 3′-N-acetylneuraminyl lactose lactone in 1 ml of distilled water and 1 human rotavirus (ATCC VP-2104).
^{0 6 FCFFU (Fluorescent Cell Focus Forming} Uni
t) / ml and orally administered to the two groups of the test animals at a rate of 0.01 ml per 1 g of body weight after culturing at 37 ° C. for 30 minutes and bred in the same manner as in Test Example 3. Lactation was carried out from the same kind of mother mice, and a test was conducted by observing the degree of diarrhea symptoms after 3 days. In addition, a mixed solution to which 3′-N-acetylneuraminyl lactose lactone was not added was administered to a control group. (3) Test results The results of this test are as shown in Table 3. As is evident from Table 3, diarrhea was observed in most mice in the control group, whereas diarrhea was significantly suppressed in both test groups to which the lactone body was administered.

From the test results, it was found that the lactone derivative of the present invention was also effective against viruses.
In addition, although the test was performed by changing the type of the lactone body, almost the same results were obtained.

[0054]

[Table 3]

Reference Example 1 15 kg of bovine colostrum was centrifuged at 12,000 g for 20 minutes and defatted to obtain about 10 kg of skim milk. 20 kg of water was added, extracted with a four-fold volume of a 2: 1 mixture of chloroform and ethanol (both manufactured by Wako Pure Chemical Industries, Ltd.), deproteinized, and the obtained aqueous phase was kept at 40 ° C. or lower and rotary-rotated. Approximately 1000 ml (4.0 in terms of solid content) using an evaporator.
%).

The obtained concentrated solution was gradually cooled in a freezer (manufactured by Hitachi, Ltd.) at -10 ° C., frozen, and then left to stand at room temperature to be thawed, thereby opening the lactone form of the N-acetylneuraminic acid compound. Processed. The melted solution was passed through a Wakogel LP-40C18 column (manufactured by Wako Pure Chemical Industries, Ltd.) to obtain a permeate from which the remaining peptide had been removed.

Then, the obtained permeate (3.5% in terms of solid content) was treated at 4 ° C. with an anion exchanger AG1X4 (manufactured by Bio-Rad Co., Ltd., ion type Cl ⁻ , ionic strength 13.5 m).
(S / cm), and the N-acetylneuraminic acid compound adsorbed on the column is eluted with a 0.5 M sodium chloride solution, and the eluate is used with a microacylizer G1 (manufactured by Asahi Kasei Corporation). Then, the mixture was desalted and freeze-dried to obtain about 7.9 g of a powder containing 3'-N-acetylneuraminyl lactose lactone.

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,
The reaction was allowed to take place for a day and lactonized. The obtained solution containing the lactone of the N-acetylneuraminic acid compound is washed with water for 1 hour.
After diluting to 0-fold, the acetic acid was removed with a micro-acylizer G1 (manufactured by Asahi Kasei Kogyo Co., Ltd.), and the concentration was adjusted to 0.1% in terms of solid content. The solution was treated at 4 ° C. with AG1X4 (manufactured by Bio-Rad). Ionic Cl ⁻ , ionic strength 13.5 mS /
cm), a non-adsorbed fraction was separated, and a permeate was collected.

Next, the obtained permeated solution was desalted using a micro-acylizer G1 (manufactured by Asahi Kasei Kogyo Co., Ltd.), and -8 was obtained.
The mixture is rapidly cooled in a 0 ° C. dry ice acetone bath, frozen, freeze-dried, and powdered with about 97% pure 3′-N-acetylneuraminyl lactose lactone, about 5.9.
g was obtained. Reference Example 2 Production Example of Lactone Form of N-Acetylneuraminic Acid Compound Derived from Rennet Whey 200 kg of rennet whey (manufactured by Morinaga Milk Industry Co., Ltd.) was treated with sodium hydroxide (manufactured by Saneigen FFI Co.) to obtain a pH.
The pH was adjusted to 7.0, fine precipitates were removed by a clarifier (APV), concentrated by a concentrator (Asahi Kasei Kogyo), and desalted by an electrodialyzer (Asahi Kasei Kogyo).

The obtained concentrated solution is gradually cooled and frozen by a freezer at -10 ° C. (manufactured by Hitachi, Ltd.), then left to stand at room temperature to be thawed, and the lactone of the N-acetylneuraminic acid compound is opened. Processed.

Next, the melted solution (19% in terms of solid content)
%) At 4 ° C. and a batch method with an anion exchanger DEAE-Sephadex (manufactured by Amersham Pharmacia Biotech Co., Ltd., ion type Cl ⁻ , ionic strength 3 mS / cm), and the anion exchanger gel was washed with water. The adsorbed sialic acid compound was eluted with a 0.8 M sodium chloride solution, and the eluate was desalted with an electrodialyzer (manufactured by Asahi Kasei Corporation).
After freeze-drying, about 900 g of a powder containing the N-acetylneuraminic acid compound was obtained.

The obtained freeze-dried powder was dissolved in glacial acetic acid (manufactured by San-Ei Gen FFI) at a concentration of 2% in terms of solid content, reacted at 37 ° C. for 7 days to form a lactone, and then obtained. The solution containing the lactone form of the sialic acid compound is washed with water for 1 hour.
Dilute to 0-fold, remove acetic acid with an electrodialysis device (manufactured by Asahi Kasei Corporation), adjust to a concentration of 0.1% in terms of solid content,
This solution was treated at 4 ° C. with DEAE-Sephadex (manufactured by Amersham Pharmacia Biotech Co., Ltd .; ionic Cl ⁻ ,
Ionic strength of 3 mS / cm) and a batch method.
The solution of the non-adsorbed fraction was collected.

Then, the non-adsorbed fraction solution obtained was
About 230 g of a freeze-dried powder containing a lactone of an N-acetylneuraminic acid compound derived from rennet whey, which is quenched by freezing in a freezing bath at ℃.
I got

Now, the present invention will be described in further detail with reference to Examples. However, the present invention is by no means restricted to the following Examples.

[0065]

EXAMPLES Example 1 7.5 kg of lactose (manufactured by Mirai, Germany) and 2.5 kg of skim milk powder (manufactured by Morinaga Milk Products) were dissolved in 50 kg of water, and an aqueous phase was prepared in a tank. On the other hand, 1.96 kg of vegetable oil
(Manufactured by Taiyo Yushi Co., Ltd.) and lecithin (manufactured by Ajinomoto Co.) 0.04
kg were melt-mixed to prepare an oil phase. The oil phase was added to the water phase in the tank, mixed by stirring, heated to 70 ° C., homogenized with a homogenizer (manufactured by Sanwa Kikai) at a pressure of 1.47 × 10 ⁷ Pa, and 90 ° C. For 10 minutes, concentrated, and spray-dried to obtain about 10 kg of a powdery intermediate product.

To 7.28 kg of this powdery intermediate product, 0.5 kg of desalted whey (manufactured by Domo, the Netherlands), 0.2 kg of bovine lactoferrin (manufactured by Morinaga Milk Products), and calcium carbonate (manufactured by Wako Pure Chemical Industries) 0.02 kg and 7 g of 3'-N-acetylneuraminyl lactose lactone produced by the same method as in Reference Example 1 were added, and the mixture was mixed uniformly with a mixer to obtain about 8 kg of the final product, powdered milk for childcare. . Example 2 1.5 kg of lactose (manufactured by Mirai, Germany) and 0.5 kg of skim milk powder (manufactured by Morinaga Milk Products) were dissolved in 10 kg of water, and the obtained aqueous phase was stored in a tank. On the other hand, vegetable oil 0.3
92 kg (manufactured by Taiyo Yushi Co., Ltd.) and lecithin (manufactured by Ajinomoto Co.)
8 g was melt-mixed to prepare an oil phase. The oil phase was added to the water phase in the tank, mixed by stirring, heated to 70 ° C., homogenized with a homogenizer (manufactured by Sanwa Kikai) at a pressure of 1.47 × 10 ⁷ Pa, and heated to 90 ° C. For 10 minutes, concentrated, and spray-dried to obtain about 2 kg of a powdery intermediate product.

To 0.5 kg of this powdery intermediate product, 0.034 kg of desalted whey (manufactured by Domo, the Netherlands), 0.013 kg of bovine lactoferrin (manufactured by Morinaga Milk Products), and calcium carbonate (manufactured by Wako Pure Chemical Industries) 1.3 g and 3 g of 3'-N-acetylneuraminyl lactose lactone produced by the same method as in Reference Example 1 were added and uniformly mixed with a mixer to obtain about 0.55 kg of the final product, powdered milk for childcare. . Example 3 1 kg of whey protein enzyme digest (manufactured by Morinaga Milk Industry Co., Ltd.), 3.3 kg of dextrin (manufactured by Showa Sangyo Co., Ltd.), and small amounts of water-soluble vitamins and minerals were dissolved in 18.5 kg of water, and the aqueous phase was placed in a tank. Stored. On the other hand, soybean salad oil (manufactured by Taiyo Yushi Co., Ltd.) 0.27 kg, palm oil (manufactured by Taiyo Yushi Co., Ltd.) 0.78
kg, 0.23 kg of safflower oil (manufactured by Taiyo Yushi Co., Ltd.), 19 g of lecithin (manufactured by Ajinomoto Co.), 19 g of fatty acid monoglyceride (manufactured by Kao Corporation), and a small amount of fat-soluble vitamin were melt-mixed to prepare an oil phase. The oil phase was added to the water phase in the tank, mixed by stirring, heated to 70 ° C., homogenized with a homogenizer (manufactured by Sanwa Kikai) at a pressure of 1.47 × 10 ⁷ Pa, and heated to 90 ° C. For 10 minutes, concentrated, and spray dried to obtain about 5.5 kg of a powdery intermediate product.

To this powdery intermediate product, 0.76 kg of sucrose (manufactured by Hokuren), 5 g of 3'-N-acetylneuraminyl lactose lactone manufactured by the same method as in Reference Example 1, and an amino acid mixed powder (manufactured by Ajinomoto Co.) 0.0185k
g, bovine lactoferrin (manufactured by Morinaga Milk Products) 0.694k
g and ferrous sodium citrate (manufactured by Eisai) 9.
3g are mixed uniformly, about 6.9kg of powdery enteral nutrition
I got Example 4 Each component was uniformly mixed at the following ratio per tablet, granulated by a conventional method, dried, and tableted to obtain a tablet (antiviral agent). In addition, all the raw materials other than 3'-acetylneuraminyl lactose lactone manufactured by the same method as Reference Example 1 used the commercial item.

3′-N-acetylneuraminyl lactose lactone 5 (mg) microcrystalline cellulose 170 corn starch 66 talc 11 magnesium stearate 3 Example 5 Each component was uniformly mixed at the following ratio per tablet and produced by a conventional method. Granulated, dried and tableted to obtain tablets of antiviral agent. In addition, all the raw materials other than 3'-acetylneuraminyl lactose lactone manufactured by the same method as Reference Example 1 used the commercial item.

3′-N-acetylneuraminyl lactose lactone 50 (mg) lactose 170 crystalline cellulose 8.5 carboxymethylcellulose 2 talc 3 Example 6 7.5 kg lactose (manufactured by Mirai, Germany) and skim milk powder (Morinaga Milk Industry) 2.5 kg was dissolved in 50 kg of water, and the aqueous phase was stored in a tank. On the other hand, 1.96 kg of vegetable oil
(Manufactured by Taiyo Yushi Co., Ltd.) and lecithin (manufactured by Ajinomoto Co.) 0.04
kg were melt-mixed to prepare an oil phase. The oil phase was added to the water phase in the tank, mixed by stirring, heated to 70 ° C., homogenized with a homogenizer (manufactured by Sanwa Kikai) at a pressure of 1.47 × 10 ⁷ Pa, and heated to 90 ° C. For 10 minutes, concentrated, and spray-dried to obtain about 10 kg of a powdery intermediate product.

To 7.28 kg of the obtained intermediate product, 0.5 kg of desalted whey (manufactured by Domo of the Netherlands), 0.2 kg of bovine lactoferrin (manufactured by Morinaga Milk Products), 0.02 kg of calcium carbonate, and Reference Example 2 7 g of a lactone of a 3'-N-acetylneuraminic acid compound derived from rennet whey produced by the same method as described above was added and mixed uniformly with a mixer to obtain about 8 kg of the final product, powdered milk for childcare. Example 7 1.5 kg of lactose (manufactured by Mirai, Germany) and 0.5 kg of skim milk powder (manufactured by Morinaga Milk Products) were dissolved in 10 kg of water, and the aqueous phase was stored in a tank. On the other hand, 0.392 kg of vegetable oil
(Taiyo Yushi Co., Ltd.) and 8 g of lecithin (Ajinomoto Co.) were melt-mixed to prepare an oil phase. The oil phase was added to the water phase in the tank, mixed by stirring, heated to 70 ° C., homogenized with a homogenizer (manufactured by Sanwa Kikai) at a pressure of 1.47 × 10 ⁷ Pa, and heated to 90 ° C. For 10 minutes, concentrated, and spray-dried to obtain about 2 kg of a powdery intermediate product.

To 0.5 kg of the obtained intermediate product, 0.034 kg of desalted whey (manufactured by Domo of the Netherlands), 0.013 kg of bovine lactoferrin (manufactured by Morinaga Milk Products), 1.3 g of calcium carbonate, and 5 g of a lactone of a 3'-N-acetylneuraminic acid compound derived from rennet whey produced by the same method was added, and the mixture was mixed uniformly with a mixer.
I got Example 8 1 kg of whey protein enzyme digest (manufactured by Morinaga Milk Industry Co., Ltd.), 3.3 kg of dextrin (manufactured by Showa Sangyo Co., Ltd.), small amounts of water-soluble vitamins and minerals were dissolved in 18.5 kg of water, and the aqueous phase was stored in a tank. did. On the other hand, soybean salad oil (manufactured by Taiyo Yushi Co., Ltd.) 0.27 kg, palm oil (manufactured by Taiyo Yushi Co., Ltd.) 0.78
kg and safflower oil (manufactured by Taiyo Yushi Co., Ltd.) 0.23k
g, 19 g of lecithin (manufactured by Ajinomoto Co.), 19 g of fatty acid monoglyceride (manufactured by Kao Corporation) and a small amount of fat-soluble vitamin were melt-mixed to prepare an oil phase. The oil phase was added to the water phase in the tank, mixed by stirring, heated to 70 ° C., homogenized with a homogenizer (manufactured by Sanwa Kikai) at a pressure of 1.47 × 10 ⁷ Pa, and heated to 90 ° C. For 10 minutes, concentrated, and spray dried to obtain about 5.5 kg of a powdery intermediate product.

Sucrose (manufactured by Hokuren Co., Ltd.) was added to the obtained intermediate product.
0.76 kg, 5 g of a lactone of a 3′-N-acetylneuraminic acid compound derived from rennet whey produced by the same method as in Reference Example 2, 0.0185 kg of mixed amino acid powder (manufactured by Ajinomoto Co.), bovine lactoferrin ( 0.694 kg of Morinaga Milk Industry Co., Ltd.) and 9.3 g of ferrous sodium citrate (Eisai Co., Ltd.) were uniformly mixed to obtain about 6.9 kg of an enteral nutritional supplement. Example 9 Each component was uniformly mixed at the following ratio per tablet, granulated by a conventional method, dried, and tableted to obtain a tablet (antiviral agent). The raw materials other than the lactone of the 3′-N-acetylneuraminic acid compound derived from rennet whey produced by the same method as in Reference Example 2 were all commercially available.

Lactone form of rennet whey-derived 3′-N-acetylneuraminic acid compound 5 (mg) Microcrystalline cellulose 170 Corn starch 66 Talc 11 Magnesium stearate 3 Example 10 Each component is uniformly mixed at the following ratio per tablet. Then, the mixture was granulated by a conventional method, dried, and tableted to obtain a tablet of an antiviral agent. The raw materials other than the lactone of the 3′-N-acetylneuraminic acid compound derived from rennet whey produced by the same method as in Reference Example 2 were all commercially available.

Lactone form of rennet whey-derived 3′-N-acetylneuraminic acid compound 5 (mg) Lactose 170 Crystalline cellulose 8.5 Carboxymethylcellulose 2 Talc 3

[0076]

As described above in detail, the invention of the present application provides a lactone form of an N-acetylneuraminic acid compound which is resistant to hydrolysis by endogenous sialidase in an amount of at least 0.01% by weight as an active ingredient. The present invention relates to a lactone body-containing composition characterized in that the composition is contained in a proportion, and the effects achieved by the invention of this application are as follows. 1) 1-4 or 1- of N-acetylneuraminic acid compound
The 2-lactone is a naturally occurring substance and has higher safety when administered to humans and animals than conventional 1-3 or 1-7 lactones. 2) 1-4 or 1- of N-acetylneuraminic acid compound
The 2-lactone is hardly degraded by endogenous sialidase, and retains some physiological functions of sialic acid. 3) 1-4 or 1- of N-acetylneuraminic acid compound
The composition of the present invention containing a 2-lactone compound can provide a product having a superior physiological function as compared with a conventional nutritional composition containing sialic acid or an antiviral agent.

[Brief description of the drawings]

FIG. 1 shows the relationship between the reaction time and the amount of free sialic acid.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 43/00 111 A61P 43/00 111

Claims (3)

[Claims] 1. An N-acetylneuraminic acid compound having a resistance to hydrolysis by endogenous sialidase of 1-4.
Alternatively, a composition containing an endogenous sialidase-resistant lactone body, comprising at least 0.01% by weight of a 1-2 lactone body as an active ingredient. 2. The N-acetylneuraminic acid compound 1-
The 4 or 1-2 lactone compound is 3'-N-acetylneuraminyl lactose 1-4 lactone, 3'-N-acetylneuraminyl lactose 1-2 lactone, or N-acetyl obtained from casein or casein hydrolyzate. The endogenous sialidase-resistant lactone-containing composition according to claim 1, which is a 1-4 or 1-2 lactone form of a neuraminic acid compound. 3. The lactone body-containing composition according to claim 1, wherein the composition is a powdered baby milk, a baby food, an enteral nutritional agent, or an antiviral agent.