JP2000270858A - Lysozyme-saccharide conjugate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a lysozyme-saccharide conjugate that further enhances the antimicrobial activity that the egg white lysozyme intrinsically has and shows also the antimicrobial activity against the gram-negative bacteria and excellent heat resistance, thus is useful as medicines, cosmetics, food products by isolating saccharides from egg white and purifying them. SOLUTION: The objective lysozyme saccharide conjugate is separated from the egg white liquid, for example, raw egg white that contains one or two or more kinds of disaccharides selected from sucrose, lactose, maltose, trehalose and cellobiose, is sterilized at such a degree that the egg white may not be thermally denatured. This lysozyme-saccharide conjugate forms its structure through the covalent bonds, ionic bonds, coordinate bonds, hydrogen bonds and the like and is separated and purified by the gel filtration, the cationic ion-exchange chromatography, the salting out or the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a lysozyme saccharide complex separated and purified from egg white liquor containing one or more saccharides, which enhances the antibacterial activity inherent in egg white lysozyme and broadens the antibacterial spectrum. A lysozyme saccharide complex with improved heat resistance, which is used as a novel antibacterial agent in the fields of medicine, cosmetics, food, feed and the like.

[0002]

2. Description of the Related Art Lysozyme is also called muramidase or mucopeptide hydrolase and has a molecular weight of about 14,
The isoelectric point is 10.7 at 300 to 14,600, and β-1,4 between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) existing in the peptidoglycan layer of the bacterial cell wall. It is an enzyme protein that hydrolyzes bonds. Lysozyme has an antibacterial activity because it has the action of cutting bacterial cell walls by its enzymatic action and lysing bacteria. Particularly, lysozyme derived from egg white has been extensively studied as egg white lysozyme, and its amino acid sequence has been determined.

Lysozyme is widely distributed in the animal and plant kingdoms. At present, lysozyme derived from mammalian tears, saliva, urine, milk, avian egg white, fish body surface mucus, microorganisms, bacteriophage T4, and the like are particularly known. Lysozyme derived from egg white can be mass-produced and separated by cation chromatography, interfacial transfer electrophoresis, salting out, or the like. Its antibacterial activity is anti-inflammatory, in the fields of medicine, cosmetics, food and feed.
It is used for preservation, keeping freshness, antibacterial, sterilization, etc.
It is known that the inactivation of lysozyme by heat depends on pH and temperature. For example, if lysozyme is dissolved in 5 M phosphate buffer and the pH is 9, no inactivation occurs at 63 ° C. for 10 minutes. However, 6
At 5 ° C., the activity of lysozyme in a pH 9 buffer is 1
About 70% decrease in 0 minutes. Lysozyme is said to be about 50 times more heat sensitive when in egg white than in buffer.

[0004] The antibacterial activity of lysozyme is particularly strong against gram-positive bacteria, and against gram-negative bacteria.
It is known that it has almost no antibacterial activity. Gram-positive bacteria are bacteria that are stained purple by Gram staining.
Cocci (Micrococcus, Staphylococcus, Streptococcus, etc.), Spore-forming bacilli (Bacillus, Clostridium, etc.), Lactic acid bacteria (Lactobacillus, etc.), Coryneform bacteria (Corynebacterium, Nocardia, etc.) , Actinomycetes (such as Streptomyces genus), and yeast are known. Its cell wall is 20
Consists of a ~ 80 nm thick peptidoglycan layer and is highly sensitive to lysozyme. That is, lysozyme easily hydrolyzes the peptidoglycan layer and exhibits the action of lysing gram-positive bacteria.

[0005] On the other hand, Gram-negative bacteria are bacteria that are not stained by Gram staining, such as photosynthetic bacteria (Rhodopseudomonas sp.), Pseudomonas sp., Intestinal bacteria (Escherichia sp., Salmonella sp., Etc.), and chemolithotrophic bacteria (Nitrophy). Bacter spp., Thiobacillus spp.), Methanogenic bacteria, and some cocci (Niceria spp.) Are known. The surface layer of the cell has a thin peptidoglycan layer of about 2 to 3 nm outside the cell membrane (inner membrane), and further has an outer membrane containing lipopolysaccharide outside. Therefore, lysozyme cannot easily cleave the peptidoglycan layer, and does not exhibit lysozyme sensitivity unless the outer membrane lipopolysaccharide is damaged by ethylenediaminetetraacetic acid or the like.

Some attempts have been made to make the antibacterial activity possessed by lysozyme act not only on Gram-positive bacteria but also on Gram-negative bacteria, that is, attempts to expand the antibacterial spectrum of lysozyme. For example, it has been disclosed that lysozyme bound to a polysaccharide such as dextran using the Maillard reaction has an antibacterial activity against Gram-negative bacteria by heat treatment at 50 ° C. (Agric. Bio).
l. Chem. , 54, 3057-3059, 1990.
Year). Palmitic acid (J. Agric. Food)
Chem. , 39, 2077-2082, 1991.
Years), stearic acid, myristic acid (J. Agric.
Food Chem. , 41, 1164-1168, 1
Lysozyme bound to a fatty acid such as lysozyme (993), but these lysozyme derivatives have the disadvantage that the solubility is significantly poor. In addition, lysozyme obtained by introducing a hydrophobic peptide to the C-terminal of lysozyme by a genetic engineering technique (Biosci. Biotech.
Biochem. , 56,1361-1363,199
2 years), it is disclosed that it has a strong antibacterial activity against Gram-negative bacteria, but it requires a complicated operation and has a practically difficult problem.

[0007]

Lysozyme has a strong antibacterial activity, particularly against Gram-positive bacteria, and has little antibacterial activity against Gram-negative bacteria. Therefore, its use is limited to a shelf life improver for foods, a preservative for cosmetics, an anti-inflammatory drug, etc. for the purpose of suppressing the growth of Gram-positive bacteria. However, in these applications, it is desired to suppress the growth of gram-positive bacteria and gram-negative bacteria such as Escherichia coli and Salmonella, which have more adverse effects on the lives and health of humans and animals.

[0008] An object of the present invention is to provide a practical lysozyme having heat resistance and also having an antibacterial activity against Gram-negative bacteria.

[0009]

Means for Solving the Problems The present inventors have conducted intensive studies on egg white lysozyme which also has antibacterial activity against Gram-negative bacteria. As a result, it was found that egg white liquor containing one or more saccharides selected from one or more kinds was used. The isolated and purified lysozyme saccharide complex has increased heat resistance and, by using heat treatment, significantly enhances its antibacterial activity against Gram-positive bacteria.In addition, by using heat treatment together,
The present inventors have found that they exhibit strong antibacterial activity against gram-negative bacteria, and completed the present invention. That is, the present invention relates to a lysozyme sugar conjugate having significantly increased heat resistance and antibacterial activity.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The egg white lysozyme of the present invention is
-Refers to an enzyme protein that hydrolyzes a β-1,4 bond between acetylmuramic acid and N-acetylglucosamine;
The origin is not particularly limited, but lysozyme derived from chicken egg and egg white, which can be easily prepared in large quantities, is preferred. Usually, lysozyme has a reduced enzymatic activity when mixed directly into an aqueous system. Regarding the application of lysozyme, methods of keeping it dry until just before use, mixing it with an aqueous solution at the time of use, and microencapsulation are used, but this is not an essential solution. In addition, when lysozyme separated and purified from egg white liquid is dissolved in an aqueous system, a method of adding a saccharide such as polyhydric alcohol, maltose, or sorbitol has a stabilizing effect on a group of enzymes such as proteolytic enzymes and lipolytic enzymes. However, no satisfactory stabilizing effect has been obtained for lysozyme.

The saccharide of the present invention is not particularly limited, but is preferably selected from the following disaccharides: sucrose, lactose, maltose,
One or more selected from the group consisting of trehalose and cellobiose. Preferred are sucrose, lactose, maltose and trehalose, and more preferred are sucrose, maltose and trehalose.

The lysozyme sugar complex of the present invention is obtained from egg white liquor to which one or more saccharides have been added. That is,
In the prior art, stabilization has been attempted by mixing an egg white lysozyme separated and purified from egg white with an aqueous system of saccharides, but the present invention provides one or more kinds of egg white lysozyme in an egg white liquid before separation and purification. The present invention relates to a lysozyme sugar complex in which egg white lysozyme before separation and purification specifically forms a complex with the sugar by mixing the sugar, and the egg white lysozyme sugar complex is separated and purified from egg white liquid.

[0013] The egg white liquid of the present invention is raw egg white obtained by breaking eggs, furthermore, raw egg white and frozen egg white which have been heat-sterilized to the extent that they do not denature by heat, and are egg white liquid in which egg white powder is rehydrated. There is no limitation on the type of egg white liquid, but it is preferably a liquid at the time of treatment and has little denaturation of egg white protein, that is, an egg white liquid composed of an undenatured protein, a heat sterilized to the extent that it does not heat denature, a liquid such as raw egg white, frozen egg white, etc. Refers to the egg white. The one or more saccharides of the present invention are usually referred to as disaccharides, and sucrose, trehalose, fructose and the like are selected.

Next, the method for producing the lysozyme conjugate of the present invention will be described in detail below. It begins by mixing one or more saccharides with the egg white solution comprising the above-mentioned native protein. It is desirable that the mixing ratio of the saccharide to the egg white liquor is 1/2 to the equivalent amount of the protein in the egg white, and the amount of the protein is about 10% to the egg white liquor. Is about 10 parts, and the amount of saccharide added is not particularly limited, but it is desirable to add 5 to 30 parts, more preferably 5 to 10 parts of saccharide to be added. Addition of a sufficient amount of saccharide adds a saccharide to native lysozyme or other native protein to form a complex, and it is necessary to add a sufficient amount of saccharide to the egg white solution. The formation of the complex is desirably performed at a low temperature in consideration of the growth of microorganisms and the like.
It is prepared in a temperature range of ° C., and the time is not particularly limited as long as the saccharide is completely dissolved.

The lysozyme sugar complex in the present invention is not particularly limited, but a complex formation by a covalent bond, an ionic bond, a coordination bond, a hydrogen bond, or the like is conceivable. A lysozyme saccharide complex is formed by a combination of the type of the functional group of the saccharide and the type of the functional group of the constituent amino acid of lysozyme. To give an example, a complex is formed by an aminocarbonyl reaction generated by the carbonyl group of the disaccharide and the amino group in the constituent amino acids of lysozyme, and the molecular weight of the egg white lysozyme is increased because the disaccharide is bonded. About 14,3
It will be larger than 00 to 14,600.

The lysozyme saccharide complex is formed even in an aqueous system in which disaccharide is added to egg white lysozyme separated and purified from egg white liquor by cation chromatography, interfacial transfer electrophoresis, salting-out method or the like. Unlike the lysozyme sugar complex of the present invention, a disaccharide is added to the egg white solution of the present invention, and a lysozyme sugar complex separated and purified by a cation chromatography method or an interfacial transfer electrophoresis method, a salting-out method, and the like. In comparison, there was a remarkable difference in heat resistance and antibacterial activity. That is, there is a difference in the binding site between the complex obtained by adding disaccharide to lysozyme in the egg white solution before separation and purification, and the complex obtained by adding disaccharide to lysozyme after separation and purification from egg white solution,
It is presumed that this difference affects heat resistance and antibacterial activity.

Next, the lysozyme sugar complex can be separated and purified by a general method for separating and purifying lysozyme, for example, gel filtration, cation exchange chromatography or salting out. Among them, the method using the salting out method is simple. In order to separate the lysozyme saccharide complex as a salted out product, usually, sodium sulfate or ammonium sulfate may be added so as to have a saturation degree of 50% or more.

In gel filtration, the molecular weight of the lysozyme sugar complex is about 14,300 to 14,600 or more,
For example, using Sephadex G15 or G25, a neutral pH value that does not affect the enzyme activity of the lysozyme glycoconjugate is used.
H4-8 can be used. In ion exchange chromatography, the isoelectric point of egg white lysozyme is around 11, and in consideration of this, it is possible to adsorb and separate the lysozyme saccharide complex using a cation exchanger.

In the salting-out method, usually, lysozyme sugar complex can be obtained as a salted-out product by adding sodium sulfate or ammonium sulfate to the solution after the reaction so as to have a saturation degree of 50% or more. It can be purified by separation and drying by a general method such as separation. Of these, the salting-out method is the simplest, and its use is preferred. The lysozyme sugar complex of the present invention can be used in the same manner as ordinary lysozyme.
That is, it is used for food products such as kamaboko, custard cream, pickles, sausage, ham, cooked rice, prepared foods, processed egg products such as powdered eggs and liquid eggs, cosmetics such as rinses and shampoos, mouthwashes, toothpastes, etc. Can be.

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not particularly limited thereto.

[0021]

Example 1 Preparation and Purification of Lysozyme Sugar Complex A lysozyme sugar complex derived from trehalose, a disaccharide, and egg egg white was prepared and purified as follows.

A. Preparation of Lysozyme Sugar Complex The undenatured egg white liquid separated from the egg is mixed into a 1 L egg white liquid sufficiently homogenized by a tabletop homomixer (manufactured by Nippon Tokusyu Kika Kogyo Co., Ltd.). 0.1 kg of trehalose was added and stirred at a temperature of 5 ° C. for 1 hour to prepare a lysozyme saccharide complex.

B. Purification of Lysozyme Sugar Complex Next, the pH was adjusted to 6.5 with 5N-hydrochloric acid while stirring.
A cation exchange resin is added to this solution to adsorb the lysozyme sugar complex. Before adding the cation exchange resin Amberlite IRC-50 (manufactured by Rohm and Hearst) to the egg white solution, use caustic soda or the like. PH of about 6.5 as a cation (cation such as Na, K, NH4, etc. excluding H) substitution type of about 1.2 to 1.4 mg equivalent / ml (resin) Used after adjustment. Amberlite I thus adjusted to pH 6.5
200 ml of RC-50 is added to the above-mentioned egg white solution containing the pH adjusted lysozyme sugar complex, and the lysozyme sugar complex is adsorbed while stirring for 1.5 hours.

If the mixture is allowed to stand after completion of the stirring, the cation exchange resin sediments in 10 to 20 minutes. Discard the supernatant egg white solution, wash the cation exchange resin with 5 L of purified water, and repeat until the washing solution is clear. After sufficiently removing the washing solution, 1000 ml of a 3% saline solution is added, and while stirring at a low speed,
A 1N NaOH solution was added at a rate of about 0.6 ml / min for 10 minutes to elute the lysozyme saccharide complex while maintaining the pH at about 6.5.

Next, the above cation exchange resin-containing solution was filtered through an 80-mesh filter cloth, and the obtained filtrate was added to a 0.5 mesh filter cloth.
The pH was adjusted to 9.5 by adding an NNaOH solution, and sodium chloride was added thereto to adjust the salt concentration to 5%, and the lysozyme sugar complex was recovered by a salting-out method. The recovered lysozyme sugar complex weighed 9.5 g in a cake state. Further, the crystals were dried with a freeze dryer to obtain 3.1 g of crystals of a lysozyme sugar complex.

Comparative Example 1 The following adjustments were made to Example 1 in a system to which trehalose was not added.

A. Preparation of egg white lysozyme 1 L of egg white liquid was prepared by homogenizing the undenatured egg white liquid separated from the broken eggs using a tabletop homomixer (manufactured by Nippon Special Kika Kogyo Co., Ltd.).

B. Purification of egg white lysozyme Next, the pH was adjusted to 6.5 with 5N-hydrochloric acid while stirring.
A cation exchange resin is added to this solution to adsorb egg white lysozyme. Before adding the cation exchange resin Amberlite IRC-50 (manufactured by Rohm and Hearst) to the egg white solution, use caustic soda or other alkali. About 1.2 to 1.4 mg equivalent / ml (resin) after treatment with an agent (cations such as Na, K and NH4 excluding H)
As a substitution type, it was used after adjusting the pH to around 6.5. Amberlite IRC- thus adjusted to pH 6.5
50 ml of the egg white lysozyme is added to the pH-adjusted egg white lysozyme, and the egg white lysozyme is adsorbed while stirring for 1.5 hours.

If the mixture is allowed to stand after completion of the stirring, the cation exchange resin sediments in 10 to 20 minutes. Discard the supernatant egg white solution, wash the cation exchange resin with 5 L of purified water, and repeat until the washing solution is clear. After sufficiently removing the washing solution, 1000 ml of a 3% saline solution is added, and while stirring at a low speed,
A 1N NaOH solution was added at a rate of about 0.6 ml / min for 10 minutes to elute lysozyme egg white while maintaining the pH at about 6.5.

Next, the above cation exchange resin-containing solution was filtered through an 80-mesh filter cloth, and the obtained filtrate was subjected to 0.5 ml of filtration.
The pH was adjusted to 9.5 by the addition of an NNaOH solution, and salt was added thereto to adjust the salt concentration to 5%, and egg white lysozyme was recovered by salting out. The recovered egg white lysozyme weighed 9.2 g in the form of a cake. Furthermore, it dried with the freeze dryer and obtained 3.0 g of egg white lysozyme crystals.

Comparative test

Test Example 1 Enzyme Activity of Example 1 and Comparative Example 1 Lysozyme Sugar Complex Crystal Obtained in Example 1 and Comparative Example 1
Was compared with the egg white lysozyme crystals. The enzyme activity of lysozyme was determined by the activity of Micrococcus rhizodeticus (M.l.
ysodeikticus). The lysozyme sugar complex crystal obtained in Example 1 and the egg white lysozyme crystal of Comparative Example 1 were mixed at a final concentration of 1 μg / ml.
The cell suspension (initial turbidity A600nm = 0.75 to 50 mM) in a 50 mM phosphate buffer (pH 6.2)
0.80) and its turbidity change was measured at 25 ° C. As a result, when the amount of change in turbidity of the egg white lysozyme crystal of Comparative Example 1 was 100%, that of the lysozyme sugar complex crystal was 40%, and Example 1 was very high in comparison with the enzyme activity of egg white lysozyme. It had enzyme activity. Further, the lysozyme sugar complex crystal and the egg white lysozyme crystal of Comparative Example 1 were added to a final concentration of 10 μg / ml.
The mixture was adjusted to 90 ml with 0 mM phosphate buffer (pH 6.2), subjected to a heat treatment at a temperature of 80 ° C. for 20 minutes, and then subjected to a logarithmic growth period of Micrococcus resodictica (M. lys).
odeikticus) at 1 × 10 ³ cells / ml at 10 m
1 and the turbidity change was measured at 25 ° C. As a result, the turbidity change amount of the lysozyme sugar complex crystal of Example 1 was 100
%, That of egg white lysozyme crystals was 250%, and enrichment was observed in egg white lysozyme crystals. As a result, it was found that the activity of the egg white lysozyme crystal of Comparative Example 1 was decreased by heating at 80 ° C., and that the enzyme activity of the lysozyme sugar complex crystal of Example 1 was maintained even by heating at 80 ° C. Was observed.

Test Example 2 Antibacterial activity of lysozyme glycoconjugate against gram-negative bacteria Crystal of lysozyme glycoconjugate obtained in Example 1, Test Example 2
The antibacterial activity of the egg white lysozyme obtained in Example 1 against Gram-negative bacteria was compared. Escherichia coli (Esc
herichia coli K-12 strain). Escherichia coli in the logarithmic growth phase was suspended in 10 mM phosphate buffer (pH 7.0) so as to be 1 × 10 ⁶ cells / ml. The lysozyme sugar complex crystal obtained in Example 1 and the egg white lysozyme crystal of Comparative Example 1 were added to an E. coli suspension at a final concentration of 1
After adding to a concentration of 00 μg / ml and incubating at 37 ° C. for 1 hour, a certain amount of the suspension was inoculated on MacConkey agar medium, and the respective viable cell count after culturing at 35 ° C. for 24 hours was measured. . As a result, assuming that the viable cell count of the egg white lysozyme crystal of Comparative Example 1 was 100%, the respective viable cell counts were compared. As a result, the lysozyme sugar complex crystal was 25%. The antibacterial activity against Escherichia coli of the present invention was found to be higher for lysozyme sugar complex crystals.

Test Example 3 Antibacterial Activity of Lysozyme Glycoconjugate Against Gram-Positive Bacteria Lysozyme Glycoconjugate Crystal Obtained in Example 1, Test Example 2
The antibacterial activities of the egg white lysozyme obtained in the above for Gram-positive bacteria were compared. Gram-positive bacteria include Staphylococcus aureus I
FO14462 strain). 1x bacteria in logarithmic growth phase
The cells were suspended in a 10 mM phosphate buffer (pH 7.0) so as to be 10 ⁶ cells / ml. The lysozyme sugar complex crystal obtained in Example 1 and the egg white lysozyme obtained in Test Example 2 were added to the bacterial suspension to a final concentration of 100 μg / ml, and incubated at 37 ° C. for 1 hour. An aliquot of the suspension was inoculated on MacConkey agar and incubated at 35 ° C for 2 hours.
After culturing for 4 hours, the number of viable cells was measured. As a result, the viable cell count of the egg white lysozyme crystal was set to 100%, and the viable cell count was compared. As a result, that of the egg white lysozyme sugar complex crystal was 5%. The body crystals were found to be high.

The following can be considered as embodiments of the present invention. (1) A lysozyme saccharide complex separated and purified from an egg white solution containing one or more saccharides. (2) The lysozyme saccharide conjugate according to (1), wherein the saccharide is a disaccharide. (3) The lysozyme sugar according to (1) above, wherein the egg white liquid is raw egg white obtained by breaking eggs, and further separated and purified from raw egg white, frozen egg white, and the like, which have been heat-sterilized to the extent that they do not denature. Complex. (4) The lysozyme saccharide complex according to (1), wherein the saccharide is selected from one or more disaccharides such as sucrose, lactose, maltose, trehalose, and cellobiose. (5) The lysozyme saccharide conjugate according to (1), wherein the β-1,4 bond between N-acetylmuramic acid and N-acetylglucosamine is hydrolyzed. (6) The lysozyme saccharide conjugate according to (1), wherein the mixing ratio of the saccharide to the egg white liquor is equal to or more than の of the protein content in the egg white. (7) The lysozyme sugar complex according to (1) above, wherein the lysozyme sugar complex in the egg white solution is separated by gel filtration, cation exchange chromatography, salting-out method or the like. (8) A kamaboko comprising the lysozyme sugar complex obtained in the above (1) to (7). (9) A custard cream containing the lysozyme sugar complex obtained in the above (1) to (7). (10) A pickle comprising the lysozyme sugar complex obtained in (1) to (7). (11) A sausage characterized by incorporating the lysozyme sugar complex obtained in (1) to (7) above. (12) A ham, comprising the lysozyme sugar complex obtained in (1) to (7). (13) Cooked rice characterized by containing the lysozyme sugar complex obtained in the above (1) to (7). (14) A processed egg product comprising the lysozyme sugar complex obtained in the above (1) to (7). (15) A side dish comprising the lysozyme sugar complex obtained in the above (1) to (7). (16) An animal drug comprising the lysozyme sugar complex obtained in (1) to (7). (17) A shampoo characterized by containing the lysozyme sugar complex obtained in (1) to (7). (18) A rinse comprising the lysozyme sugar complex obtained in the above (1) to (7). (19) A mouthwash comprising the lysozyme sugar complex obtained in the above (1) to (7). (20) A dentifrice comprising the lysozyme sugar complex obtained in (1) to (7) above.

[0036]

In the past, the antibacterial activity of lysozyme was limited to that against gram-positive bacteria, and its intended use was limited. The lysozyme sugar complex of the present invention has the effect of enhancing the antibacterial activity of conventional egg white lysozyme against Gram-positive bacteria. In addition, the antibacterial spectrum of conventional egg white lysozyme has been limited to Gram-positive bacteria, but the lysozyme glycoconjugate of the present invention has the effect of expanding its antibacterial spectrum, and also exhibits antibacterial activity against Gram-negative bacteria. . That is, the lysozyme sugar complex of the present invention has an effect of reducing the effective use concentration of the conventional antibacterial compound. The lysozyme sugar complex of the present invention has the above-mentioned effects, and is used as a medicine for anti-inflammatory and antibacterial purposes, as cosmetics such as shampoos and rinses for the purpose of suppressing the causative bacteria of dandruff and caries, and washing. As a quasi-drug such as mouth liquid and toothpaste, as a shelf life improver for foods such as kamaboko, custard cream, pickles, sausage, ham, cooked rice, and chicken egg products for the purpose of preventing rot and sterilizing livestock and marine animals It can be used as a feed additive or animal drug for the purpose of disease prevention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (reference) // A23K 1/165 A61K 37/54 F term (reference) 2B150 AA01 AA07 AB10 DC13 DF11 4B018 LB03 LB05 LB06 LB07 MD29 MD72 MD90 ME13 4B050 CC07 DD11 FF11E KK08 LL01 LL02 LL10 4C083 AD201 AD211 AD212 AD471 AD472 BB48 EE01 FF01 4C084 AA02 AA07 CA41 DC01 NA03 NA05 ZB352

Claims (2)

[Claims] 1. A lysozyme saccharide complex separated and purified from egg white liquor containing one or more saccharides. 2. The lysozyme sugar conjugate according to claim 1, wherein the saccharide is a disaccharide.