JP7338978B2 - Infant nutrition composition - Google Patents

Description

The present invention relates to infant nutritional compositions.

In recent years, it has been emphasized that various ingredients contained in nutritional compositions for infants should be as close as possible to the ingredients of mother's milk. The Codex Alimentarius Commission (CODEX), which sets international food standards, the European Food Safety Authority (EFSA), and the Ministry of Health, Labor and Welfare, have determined that infant formula contains protein, fat, vitamins, and minerals similar to breast milk. It indicates a certain specification range quantity.

Regarding protein, the protein content of infant formula was previously set higher than that of breast milk, considering that the protein in infant formula is less digestible and absorbable than breast milk (lower utilization efficiency). However, the increased risk of developing lifestyle-related diseases due to excessive protein intake during infancy has become apprehensive, and the current trend is to reduce the amount of protein in infant formula. For example, CODEX specifies a protein content of 1.8 to 3.0 g/100 kcal in infant formula (Non-Patent Document 1), and EFSA specifies a standard range of 1.8 to 2.5 g/100 kcal (Non-Patent Document 2). showing. Infant formula products currently licensed in Japan contain less than 2.4 g/100 kcal.

Breast milk is rich in components that promote healthy growth and growth of infants, and sialic acid has attracted attention as one of the components. Sialic acid is one of the negatively charged 9-carbon sugars and exists in the molecular forms of oligosaccharides, glycoproteins and glycolipids in breast milk. Sialic acid in mother's milk has an effect of preventing infection in infants (Patent Document 1), a neutralizing effect of bacterial toxins (Patent Document 2), a mineral absorption promoting effect (Patent Document 3), and a brain function development effect (Non-Patent Document 3). , and other functions. However, since cow's milk has a low sialic acid content, nutritional compositions for infants made from cow's milk-derived dairy materials have a much lower sialic acid content than breast milk.
Specifically, the amount of sialic acid in breast milk was reported to be 714 mg ± 64 mg/L (mean ± standard error), which is equivalent to 590 mg/100 g solids given the average solids rate of breast milk of 12.1%. (Non-Patent Document 4). Table 1 shows the total sialic acid content per 100 g solids of breast milk calculated from the survey results of general components of breast milk in Japan (Non-Patent Document 5) and sialic acid content (Non-Patent Document 6) by Idota et al. . From these results, although the amount of sialic acid in breast milk decreases with the passage of the lactation period, the amount of sialic acid tends to remain constant at about 260 mg/100 g solid after 120 days of the lactation period. Also for Table 1, the weighted average of total sialic acid concentration from 3 to 482 days of the non-milk period is approximately 330 mg/100 g solids. From the above, in order to make the sialic acid content of the nutritional composition for infants approximate that of breast milk, it is necessary to have a sialic acid content of 260 mg/100 g solid or more, and preferably 330 mg/100 g solid or more, which is the average sialic acid content of Japanese breast milk. is desirable, and more preferably 590 mg/100 g solid or more, which is the average amount of sialic acid in breast milk described in Non-Patent Document 4, is considered desirable.

On the other hand, the amount of sialic acid in general powdered infant formula is 193 mg/100 g solids (Non-Patent Document 7), and the low sialic acid amount in Japanese breast milk, which is 260 mg/100 g solids, is 67 mg. /100g solids is insufficient, and 397mg/100g solids is also insufficient from the average value of 590mg/100g solids of sialic acid in breast milk. Since more than 90% of the sialic acid in cow's milk exists in a protein-bound form, for example, when the amount of protein in infant formula is reduced according to the EFSA standard, the amount of sialic acid is also reduced accordingly. There was a problem that the amount of sialic acid in the infant formula would diverge more and more.

In response to this problem, it has been reported to use sialic acid and sialic acid-containing oligosaccharides prepared using genetically modified microorganisms (Patent Document 4). Regarding the use of sialic acid in nutritional compositions for infants, there is still a sense of reluctance in industrial use from the viewpoint of compliance with the laws and regulations of each country and consumer acceptance of genetic recombination technology itself. is held.

Galacto-N-biose (GNB) is one of oligosaccharides and has recently attracted attention as a prebiotic. Bifidobacterium has an extracellular enzyme that cleaves the core type I O-linked sugar chain GNB of mucin produced from the gastrointestinal epithelial cells, and releases GNB from the mucin (Non-Patent Document 8). The released GNB is taken into the cells by a specific ABC-type transporter, and metabolized by GNB/LNB phosphorylase that phosphorylates GNB in the cells (Non-Patent Document 9), turning GNB into an energy source. It is thought to be used as This metabolic pathway has hardly been found in microorganisms other than Bifidobacteria (Non-Patent Document 10), and it is an oligosaccharide selective to Bifidobacteria. Therefore, GNB is an oligosaccharide that selectively increases bifidobacteria useful for infants.

Although there are no reports on the amount of GNB in general infant formula, GNB is present bound to glycomacropeptide (GMP) in milk. GMP is reported to be 14% of the protein in a typical infant formula (Non-Patent Document 11). GMP also includes GalNAc-OR, Galβ1-3GalNAc-OR, Neu5Acα2-3Galβ1-3GalNAc-OR, Galβ1-3 (NeuAcα2-6) GalNAc-OR, and Neu5Acα2-3Galβ1-3 (Neu5Acα2-6) GalNAc-OR. Five types of sugar chains are bound, and their binding ratios (molar ratios) are reported to be 0.8, 6.3, 18.4, 18.5, and 56%, respectively (Non-Patent Document 12). Furthermore, sialic acid in GMP was reported to be 6%, and based on these reports, the amount of GNB in GMP was estimated to be 5%.
Based on these results, the amount of GNB in common infant formula was considered to be 82 mg/100 g solids. To date, no GNB-fortified infant formula has been marketed. In addition, no report has been made on a production method using genetically modified microorganisms. Furthermore, even if there is a report in the future, regarding the use of GNB prepared using genetic recombination technology in nutritional compositions for infants, the legal compliance status of each country and the genetic recombination technology itself From the standpoint of consumer acceptability, etc., there is still a sense of reluctance in industrial use.

Japanese Patent No. 2514375 Japanese Patent No. 2821770 Japanese Patent No. 3372499 Japanese Patent No. 4318549 Japanese Patent No. 2673828

STANDARD FOR INFANT FORMULA AND FORMULAS FOR SPECIAL MEDICAL PURPOSES INTENDED FOR INFANTS, CODEX STAN 72-1981, Formerly CAC/RS 72-1972. Revision: 2007. COMMISSION DELEGATED REGULATION (EU) 2016/127 of 25 September 2015 Sakai F et al., Journal of Applied Glycoscience, 53(4), 249-254, 2006 Qiao Y et al., The relationship between dietary vitamin A intake and the levels of sialic acid in the breast milk of lactating women. J Nutr Sci Vitaminol (Tokyo). 2013;59(4):347-51. Tadashi Itoda et al.: Journal of the Japanese Society of Pediatric Nutrition and Gastroenterology, 5:145-158, 1991. Tadashi Itoda et al.: Journal of the Japanese Society of Nutrition and Food Science, 47: 357-362, 1994. Infant formula “Sukoyaka M1” (Snow Brand Beanstark Co., Ltd.) can display value Fujita K et al., Identification and molecular cloning of a novel glycoside hydrolase family of core 1 type O-glycan-specific endo-alpha-N-acetylgalactosaminidase from Bifidobacterium longum. J Biol Chem. 2005; 280(45): 37415-22 . Nakajima M et al., Identification of Lacto-N-Biose I Phosphorylase from Vibrio vulnificus CMCP6. Appl Environ Microbiol. 2008; 74(20): 6333-6337. Masafumi Hidaka et al.: Crystal structure of phosphorylase involved in degradation of human milk oligosaccharides by Bifidobacterium, PFNEWS, 27:18-21, 2009. Bruck WM et al., Effects of bovine alpha-lactalbumin and casein glycomacropeptide-enriched infant formula on faecal microbiota in healthy term infants. J Pediatr Gastroenterol Nutr. 2006; 43(5): 673-9. Saito T, & Itoh T et al., Variations and distributions of O-glycosidically linked sugar chains in bovine kappa-casein. J Dairy Sci. 2006; 43(5): 673-9.

In view of the problems described in the background art above, the present invention provides a novel nutritional composition for infants, in which the amount of sialic acid is increased to the level of breast milk while keeping the protein content at less than 2.4 g/100 kcal, which is close to that of breast milk, and its production. The object is to provide a method.

In order to solve the above problems, the present inventors focused on GMP, and subjected it to enzyme treatment and membrane treatment to prepare and blend sialyl glycopeptides with sialic acid content and protein content within specific ranges. The inventors have found that it is possible to prepare a new powdered infant formula with protein and sialic acid levels close to those of mother's milk, and have completed the present invention.
That is, the present invention includes the following configurations.
(1) A nutritional composition for infants, characterized by containing 250 mg/100 g solids or more of sialic acid and less than 2.4 g/100 kcal of protein.
(2) The nutritional composition for infants according to (1), further comprising 90 mg/100 g solids or more of galacto-N-biose.
(3) The nutritional composition for infants according to (1) or (2), wherein the nutritional composition for infants is infant formula.
(4) The nutritional composition for infants according to any one of (1) to (3), which contains a sialylglycopeptide.
(5) Any one of (1) to (4), wherein the sialyl glycopeptide contains 8% by weight or more of sialic acid, 4% by weight or more of galacto-N-biose, and 86% by weight or less of protein. 3. The nutritional composition for infants according to .
(6) The nutritional composition for infants according to any one of (1) to (5), wherein the sialylglycopeptide has a molecular weight of 700 or more and 3,000 or less.
(7) The sialylglycopeptide is a glycopeptide in which a sugar chain is bound to a peptide having a threonine and/or serine residue, wherein the sugar chain is bound to the hydroxyl group of the threonine or serine residue of the peptide, The nutritional composition for infants according to any one of (1) to (6), wherein the chain-constituting sugars consist of sialic acid, N-acetylgalactosamine and galactose.
(8) A method for producing a nutritional composition for infants, comprising adding a sialyl glycopeptide, wherein the sialyl glycopeptide has the following composition.
A glycopeptide in which a sugar chain is bound to a peptide having a threonine and/or serine residue,
The glycopeptide has a molecular weight of 700 or more and 3,000 or less,
the sugar chain binds to the hydroxyl group of the threonine or serine residue of the peptide,
the constituent sugars of the sugar chain are composed of sialic acid, N-acetylgalactosamine and galactose, and the sialic acid is contained in an amount of 8% by weight or more;
Containing 4% by weight or more of the galacto-N-biose,
The protein content of the peptide chain is 86% by weight or less

INDUSTRIAL APPLICABILITY The present invention can provide a novel nutritional composition for infants, in which the amount of protein is less than 2.4 g/100 kcal, which is close to that of mother's milk, while the amount of sialic acid is increased to the level of mother's milk, and a method for producing the same. In addition, it is possible to provide a new nutritional composition for infants in which the amount of GNB is increased as compared with conventional nutritional compositions for infants, and a method for producing the same.

(Nutritional composition for infants)
The nutritional composition for infants of the present invention includes infant formulas and liquid formulas, infant formulas (follow-up milks and growing-up milks), infant formulas for low birth weight infants, allergen-free foods for milk allergy diseases, and lactose. It is a lactose-free food for people with intolerance, etc., and is composed mainly of proteins, fats, carbohydrates, vitamins and minerals. In addition, it may further contain ingredients that can be blended into the nutritional composition for infants.
The nutritional composition for infants of the present invention is characterized by having a sialic acid content of 250 mg/100 g solid or more and a protein content of less than 2.4 g/100 kcal. If the amount of sialic acid is less than 250 mg/100 g solids, it is lower than the low value of sialic acid in Japanese breast milk, so it is poor in nutritional value. It is not appropriate because it is not approved due to concerns about increased risk of developing lifestyle-related diseases due to protein intake.
The amount of sialic acid in the nutritional composition for infants of the present invention is more preferably 330 mg/100 g solids or more, even more preferably 590 mg/100 g solids or more.
Further, the nutritional composition for infants of the present invention desirably contains more GNB than general infant formula powder, preferably 90 mg/100 g solids or more, more preferably 100 mg/100 g solids or more. and more preferably 119 mg/100 g solids or more.
The content of ingredients other than sialic acid and protein is within a certain range of standards similar to breast milk specified by the Codex Alimentarius Commission (CODEX), which establishes international food standards, the European Food Safety Authority (EFSA), or the Ministry of Health, Labor and Welfare. should be adjusted so that

(Method for producing nutritional composition for infants)
The method for producing the nutritional composition for infants of the present invention will be described below by taking powdered milk for infants as an example. First, a composition containing GMP is subjected to enzyme treatment and membrane treatment to prepare sialyl glycopeptides having specific ranges of sialic acid content, GNB content, and protein content. By adding this to general infant formula, it is possible to prepare a new infant formula whose protein content and sialic acid content are close to those of mother's milk.
That is, the protein source, the carbohydrate source, and the sialylglycopeptide obtained as described above were dissolved in water in a tank. The fat sources are then mixed and, after homogenization, concentrated and pulverized by spray drying. Minor ingredients such as vitamins and minerals can be mixed with lactose and then powder-mixed with the spray-dried powder to obtain the infant formula of the present invention.
The enzymatic and membrane treatments of GMP can increase the sialic acid content and GNB content over GMP while keeping the protein content lower than GMP. The balance between the decrease in protein content and the increase in the amount of sialic acid and GNB depends on the enzyme treatment conditions (enzyme type, temperature, time, etc.) and membrane treatment conditions (differences in membrane molecular weight cut off, etc.) to achieve the desired sialyl It can be adjusted appropriately so as to obtain a glycopeptide.
As an example, after treating a composition containing GMP with a protease and/or peptidase, the enzymatic degradation solution is concentrated with an ultrafiltration membrane or a nanofiltration membrane having a molecular weight cut off of 500 to 1000 to reduce the sialic acid content. is 8% or more, the GNB content is 4% or more, and the protein content is 86% or less. 82 mg / 100 g, solid protein: 2.28 g / 100 kcal), the amount of protein is close to breast milk, while infant formula fortified with sialic acid and / or GNB (sialic acid: 250 mg / 100 g solid GNB: 90 mg/100 g solid or more, protein: less than 2.4 g/100 kcal) can be prepared.
In the specification, the term "sialylglycopeptide" refers to a glycopeptide containing sialic acid as a constituent of the sugar chain.

(GMP manufacturing method)
A composition containing GMP can be prepared by the method described in Japanese Patent No. 2673828 or the like. That is, a dairy raw material containing GMP, such as cheese whey, whey protein concentrate, deproteinized cheese whey, etc., is first adjusted to a pH of less than 4, and then a membrane with a molecular weight cutoff of 10,000 to 50,000 is used to limit the pH. A method of obtaining a permeated liquid by external filtration, preferably again adjusting the pH of the permeable membrane to 4 or more, and then desalting and concentrating using a membrane having a molecular weight cutoff of 50,000 or less to adjust GMP. is mentioned. In addition, milk whey was adjusted to pH 6.0 or higher and heat-treated at 40 to 79 ° C., subjected to membrane treatment to remove whey protein, GMP was recovered on the concentrate side, and the resulting concentrate was A method of preparing a composition having a high GMP content can be mentioned by subjecting the pH to 5.5 or lower and then subjecting it to membrane treatment again to collect GMP in the permeate side.
The amount of GMP in the resulting composition can be analyzed, for example, by urea-SDS electrophoresis as shown in Examples of Japanese Patent No. 3,372,499.

(Method for producing sialyl glycopeptide)
A step of treating GMP with an endo-type protease or an exo-type protease, and a treatment step of subjecting the treated product obtained in the above step to ultrafiltration with a molecular weight cutoff of 500 or more and 3,000 or less to obtain the sialyl sugar of the present invention. Peptide compositions can be manufactured.
The type of protease used for glycopeptide production is particularly limited as long as it can hydrolyze peptide bonds to obtain a glycopeptide having a molecular weight and a sugar chain as described above (Sialyl glycopeptide composition). One type or a plurality of endo-type proteases and exo-type proteases can be used. Enzymes that can be used in the production of foods and pharmaceuticals are preferred, such as Actinase E (Kaken Pharma), Actinase AS (Kaken Pharma), Nucleisin (HBI), Orientase AY (HBI), Sumiteam FP (Shinnihon Chemical Industry). , Sumiteam SPP-G (Shin Nihon Kagaku Kogyo), Protease A (Amano Enzyme), Peptidase R (Amano Enzyme), Alcalase (Novozyme), Flavorzyme (Novozyme) and the like can be used alone or in combination.
Although any sialylglycopeptide thus obtained can be used, it is preferable to use one having the following characteristics.
A glycopeptide in which a sugar chain is bound to a peptide having a threonine and/or serine residue,
(1) the glycopeptide has a molecular weight of 700 or more and 3,000 or less;
(2) the sugar chain binds to the hydroxyl group of the threonine or serine residue of the peptide;
(3) Constituent sugars of the sugar chain consist of sialic acid, N-acetylgalactosamine and galactose (4) Contain 8% by weight or more of sialic acid,
(5) contains 4% by weight or more of GNB,
(6) A sialyl glycopeptide having a protein content of 86% by weight or less.

(protein source)
Protein sources include milk protein fractions such as casein, whey protein concentrate (WPC), whey protein isolate (WPI), αs-casein, β-casein, α-lactalbumin, and β-lactoglobulin. , or plant proteins such as soybean protein and wheat protein, peptides obtained by enzymatically decomposing them to various degrees of decomposition, amino acids, and the like can be used.

(fat source)
Fat sources include animal oils such as milk fat, lard, beef tallow, or fish oils including bonito oil, tuna oil, etc., or soybean oil, rapeseed oil, corn oil, coconut oil, palm oil, palm kernel oil, safflower oil. , perilla oil, linseed oil, evening primrose oil, MCT, or cottonseed oil, as well as their fractionated oils, hydrogenated oils, and transesterified oils.

(sugar source)
Sugar sources that can be used include oligosaccharides such as lactose, maltose, glucose, sucrose, dextrin, galactosyl lactose, fructo-oligosaccharides and lactulose, artificial sweeteners, soluble polysaccharides, starch, and the like.

(Vitamin and Mineral Source)
Sources of vitamins and minerals include "Codex Standards for Foods for Special Dietary Uses Including Infant Foods and Related Sanitary Work Regulations, CAC/VOL. 1993), "Handbook of Food Additives Designated in 1993 (Revised 31st Edition) (published by Shokuhin to Kagakusha, 1993)", or "Handbook of Food Additives/Food Ingredients Natural Products (12th Edition)" (Shokudo to Kagakusha, 1992), minerals and vitamins that can be used for infant foods can be used.
Examples of vitamins include vitamins A, B, C, D, E, K, folic acid, pantothenic acid, β-carotene, and nicotinamide. Minerals include calcium, magnesium, and iron. , copper, zinc, iodine, manganese, and selenium.

(Method for measuring sialic acid content)
Dissolve in 50 μL of 50 mM potassium phosphate buffer (pH 5.0) so that the concentration of sialyl glycopeptide is 250 μg/mL and the concentration of sialidase (Cosmo Bio) is 0.2 U/mL. After incubating the solution at 37° C. for 24 hours, it is diluted 2-10 fold with ultrapure water and the concentration of released sialic acid is analyzed by HPLC.
Sialic acid can be measured, for example, using a DIONEX ICS-5000DP system (Thermo Fisher Scientific Co., Ltd.) equipped with a CarboPac PA1 column. An electrochemical detector (pulsed amperometric mode) was used as the detector, and the mobile phase was ultrapure water (A solution), 200 mM sodium hydroxide solution (B solution), and 100 mM sodium hydroxide solution containing 600 mM sodium acetate. (Liquid C) is passed through at a flow rate of 1 mL/min. 45% of B solution and 10% of C solution are passed through for 7 minutes from the start, then B solution is linearly decreased to 37.5% and C solution is linearly increased to 25% by 10 minutes. From 10 minutes to 20 minutes, 37.5% B liquid and 25% C liquid are passed, and from 20.1 minutes to 25 minutes, 45% B liquid and 10% C liquid are passed. The amount of sialic acid in a sample can be calculated from a calibration curve prepared using standards with known concentrations.

(Method for measuring GNB content)
In 50 μL of 50 mM potassium phosphate buffer (pH 5.0), a sialyl glycopeptide concentration of 250 μg/mL, a sialidase (Cosmo Bio) concentration of 0.2 U/mL, and an O-glycanase (Prozyme) concentration of 0.125 U /mL of enzyme and substrate.
After incubating the solution at 37° C. for 24 hours in a thermal cycler (Takara), the reaction was stopped by cooling on ice. After diluting the reaction solution 2 to 10 times with ultrapure water, the concentrations of released sialic acid and GNB are analyzed by HPLC.
GNB measurements can be performed with a DIONEX ICS-5000DP system (Thermo Fisher Scientific, Inc.) equipped with a CarboPac PA1 column. An electrochemical detector (pulsed amperometric mode) was used as the detector, and the mobile phase was ultrapure water (A solution), 200 mM sodium hydroxide solution (B solution), and 100 mM sodium hydroxide solution containing 600 mM sodium acetate. (Liquid C) is passed through at a flow rate of 1 mL/min. After eluting with 50% solution B for 10 minutes from the beginning, the ratio of solution C is linearly increased from 0% to 100% by 25 minutes, and eluted with 100% solution C until 30 minutes. Next, at 30.1 minutes, 50% B solution is added, and then 50% B solution is eluted for 45 minutes. The amount of GNB in the sample is calculated from a calibration curve prepared using standards with known concentrations.

(Method of amino acid analysis)
Arginine, lysine, histidine, phenylalanine, tyrosine, leucine, isoleucine, valine, alanine, glycine, proline, glutamic acid, serine, threonine, and aspartic acid are hydrolyzed with hydrochloric acid, and JLC-500/V fully automated Analyze with a high-speed amino acid analyzer. Cystine and methionine are analyzed with a JLC-500/V fully automatic high-speed amino acid analyzer after hydrolysis with a performic acid solution. Tryptophan is analyzed by HPLC after adding barium hydroxide octahydrate and thiodiethylene glycol to the sample solution, heating, and hydrolyzing with hydrochloric acid.

(Calculation of protein content)
The protein content of GMP and sialyl glycopeptide is defined as the total amino acid content obtained from amino acid analysis.

Examples of the present invention will be described in detail below, but the present invention is not limited to these.

[Example 1] Method for preparing the sialylglycopeptide composition of the present invention 1. Method for producing GMP 1 kg of whey protein concentrate (Sunlacto N-2, manufactured by Taiyo Kagaku) was dissolved in 50 L of water at 50°C and adjusted to pH 3.5 with concentrated hydrochloric acid. This was subjected to ultrafiltration using an ultrafiltration membrane (GR61PP, manufactured by DDS) with a molecular weight cut off of 20,000 at 50°C, a pressure of 0.4 MPa, and an average permeate flow rate of 52.4 L/m ² h. Ta. When the amount of permeated liquid reached 40 L, 40 L of water at 50° C. was added to the concentrate, and ultrafiltration was continuously performed. Continuous operation was carried out as described above, and 160 L of permeated liquid was obtained.
25% caustic soda was added to the obtained permeated liquid to adjust the pH to 7.0, and ultrafiltration was performed again under the same conditions using the same ultrafiltration membrane until the concentrated liquid reached 5 L, followed by desalting and concentration. Subsequently, water at 50° C. was added, and diafiltration was performed with the same ultrafiltration membrane under the same conditions as before while keeping the concentrated liquid volume at 10 L, and desalting was further performed. Stop adding water to the concentrate when the amount of the permeated liquid reaches 80 L by this diafiltration, concentrate by ultrafiltration until the amount of the concentrate reaches 2 L, dry this concentrate, and 54 g of GMP got

2. Method for producing sialyl glycopeptide composition 1 above. 30 kg of 5% GMP solution was prepared using the GMP obtained in . Alcalase (manufactured by Novozyme) and Flavorzyme (manufactured by Novozyme) were added to this and reacted at 50° C. for 4 hours. This was subjected to ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 1,000. Diafiltration was performed by adding water to the concentrate and continuously performing ultrafiltration, and after concentration with water five times, a concentrate fraction was obtained. This concentrate was dried to obtain 300 g of sialylglycopeptide composition A. Furthermore, the product obtained by performing hydroconcentration up to 10 times was designated as sialylglycopeptide composition B (300 g).

3. Analysis of Sialic Acid Amount, GNB Amount, and Amino Acid Composition The sialic acid amount and GNB amount in the prepared GMP and sialyl glycopeptide compositions were measured by the methods described below.
(1) That is, in 50 μL of 50 mM potassium phosphate buffer (pH 5.0), a sialyl glycopeptide concentration of 250 μg / mL, a sialidase (Cosmo Bio) concentration of 0.2 U / mL, O-glycanase (Prozyme) Enzyme and substrate were dissolved to a concentration of 0.125 U/mL. After incubating the solution at 37° C. for 24 hours in a thermal cycler (Takara), the reaction was stopped by cooling on ice. After diluting the reaction solution 2 to 10 times with ultrapure water, the concentrations of released sialic acid and GNB were analyzed by HPLC. A DIONEX ICS-5000DP system (Thermo Fisher Scientific Co., Ltd.) equipped with a CarboPac PA1 column was used. An electrochemical detector (pulsed amperometric mode) was used as the detector, and the mobile phase was ultrapure water (A solution), 200 mM sodium hydroxide solution (B solution), and 100 mM sodium hydroxide solution containing 600 mM sodium acetate. (Liquid C) was passed through at a flow rate of 1 mL/min.
(2) For the measurement of sialic acid, 45% B solution and 10% C solution were passed for 7 minutes from the start, and then the B solution was linearly decreased to 37.5% by 10 minutes, and the C solution was 25%. increased linearly to From 10 minutes to 20 minutes, 37.5% B liquid and 25% C liquid were passed, and from 20.1 minutes to 25 minutes, 45% B liquid and 10% C liquid were passed. The amount of sialic acid in the sample was calculated from a calibration curve prepared using standard products with known concentrations. The amount of sialic acid was calculated as the amount of N-acetylneuraminic acid (Neu5Ac).
(3) Measurement of GNB, after eluting with 50% B solution for 10 minutes from the beginning, linearly increasing the proportion of C solution from 0% to 100% by 25 minutes, and 100% C solution until 30 minutes eluted. Next, at 30.1 minutes, the solution was adjusted to 50% solution B, and then eluted with 50% solution B until 45 minutes later. The amount of GNB in the sample was calculated from a calibration curve prepared using standards with known concentrations.
(4) The amino acid compositions in the prepared GMP and sialyl glycopeptide compositions were analyzed by a general amino acid composition analysis method for food ingredients. The total amount of amino acids was defined as the amount of protein.
(5) Analysis results Sialyl glycopeptide composition A has a sialic acid content of 20% by weight, a GNB content of 11% by weight, and a protein content of 67% by weight. Sialyl glycopeptide composition B has a sialic acid content of 36% by weight. % by weight, the amount of GNB was 19.8% by weight and the amount of protein was 42% by weight (Table 2). The prepared GMP had a sialic acid content of 6.0% by weight, a GNB content of 5.0% by weight, and a protein content of 84% by weight (Table 2).
The sugar chain structure of the glycopeptide contained in the sialylglycopeptide composition was estimated from the MS spectrum and MS/MS spectrum obtained by LC/MS analysis. Many of the detected glycopeptides had the sugar chain structure of Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAc (Glycan types 3, 4, 5, 6, 7) bound with two molecules of sialic acid. Neu5Acα2-3Galβ1-3GalNAc (Glycan type 1) and Galβ1-3(Neu5Acα2-6)GalNAc (Glycan type 2) having one molecule of sialic acid were also detected. Furthermore, N,O-diacetylneuraminic acid (O-Ac-Neu5Ac, Glycan type 4), which is the O-acetyl form of sialic acid, and N,O,O-triacetylneuraminic acid (O-diAc-Neu5Ac, Glycan type 5) was also detected. A glycopeptide (Glycan type 6, 7) in which two sugar chains are bound to one peptide chain was also detected. That is, all the detected glycopeptides are peptides to which a sugar chain containing sialic acid is bound, the sugar chain is bound to the hydroxyl group of the threonine or serine residue of the peptide, and the constituent sugars of the sugar chain are sialic acid, N- It consisted of acetylgalactosamine and galactose. Moreover, the molecular weight of the peptide chain detected by MS/MS spectrum analysis was in the range of 745 to 2,928.

[Example 2] Preparation of infant formula containing sialyl glycopeptide 52.7 kg of whey powder, 239 kg of skim milk, and 340 g of the sialyl glycopeptide composition A prepared in Example 1 were dissolved in 500 kg of water, and 23 kg of vegetable oil was added to the solution. .9 kg was mixed and homogenized. The resulting solution was sterilized, concentrated and dried by a conventional method to obtain 99 kg of powdered milk. 1 kg of powdered milk, vitamins and mineral components were powder-mixed to finally obtain 100 kg of powdered milk for infants. The resulting powdered milk had a sialic acid content of 250 mg/100 g solids, a GNB content of 114 mg/100 g solids or more, and a protein content of 2.29 g/100 kcal.

[Example 3] Preparation of infant formula containing sialyl glycopeptide 52.7 kg of whey powder, 239 kg of skim milk, and 190 g of the sialyl glycopeptide composition B prepared in Example 1 were dissolved in 500 kg of water, and 23 kg of vegetable oil was added to this solution. .9 kg was mixed and homogenized. The resulting solution was sterilized, concentrated and dried by a conventional method to obtain 99 kg of powdered milk. 1 kg of powdered milk, vitamins and mineral components were powder-mixed to finally obtain 100 kg of powdered milk for infants. The resulting milk powder had a sialic acid content of 250 mg/100 g solids, a GNB content of 114 mg/100 g solids or more, and a protein content of 2.27 g/100 kcal.

[Example 4] Preparation of infant formula containing sialyl glycopeptide 52.7 kg of whey powder, 239 kg of skim milk, and 1100 g of the sialyl glycopeptide composition B prepared in Example 1 were dissolved in 500 kg of water, and 23 kg of vegetable oil was added to this solution. .9 kg was mixed and homogenized. The resulting solution was sterilized, concentrated and dried by a conventional method to obtain 99 kg of powdered milk. 1 kg of powdered milk, vitamins and mineral components were mixed to finally obtain 100 kg of powdered milk for infants. The resulting milk powder had a sialic acid content of 590 mg/100 g solids, a GNB content of 300 mg/100 g solids or more, and a protein content of 2.35 g/100 kcal.

[Reference Example 1] Preparation of GMP-containing infant formula 52.7 kg of whey powder, 239 kg of skim milk, and 1,120 g of GMP were dissolved in 500 kg of water, and 23.9 kg of vegetable oil was mixed with this solution and homogenized. The resulting solution was sterilized, concentrated and dried by a conventional method to obtain 99 kg of powdered milk. 1 kg of powdered milk, vitamins and mineral components were powder-mixed to finally obtain 100 kg of powdered milk for infants. The resulting powdered milk had a sialic acid content of 250 mg/100 g solids, a GNB content of 129 mg/100 g solids or more, and a protein content of 2.41 g/100 kcal.

[Reference Example 2] Preparation of GMP-containing infant formula 52.7 kg of whey powder, 239 kg of skim milk, and 6,620 g of GMP were dissolved in 500 kg of water, and 23.9 kg of vegetable oil was mixed with this solution and homogenized. The resulting solution was sterilized, concentrated and dried by a conventional method to obtain 99 kg of powdered milk. 1 kg of powdered milk, vitamins and mineral components were powder-mixed to finally obtain 100 kg of powdered milk for infants. The resulting powdered milk had a sialic acid content of 590 mg/100 g solids, a GNB content of 413 mg/100 g solids or more, and a protein content of 3.35 g/100 kcal.

INDUSTRIAL APPLICABILITY The present invention can provide a novel nutritional composition for infants, in which the amount of protein is less than 2.4 g/100 kcal, which is close to that of mother's milk, while the amount of sialic acid is increased to the level of mother's milk, and a method for producing the same. In addition, it is possible to provide a new nutritional composition for infants in which the amount of GNB is increased as compared with conventional nutritional compositions for infants, and a method for producing the same.

Claims (2)

A nutritional composition for infants containing sialic acid of 250 mg/100 g solids or more, a protein content of less than 2.4 g/100 kcal, and galacto-N-biose of 90 mg/100 g solids or more , wherein the following sialyl The nutritional composition for infants containing a glycopeptide .
sialyl glycopeptide;
A glycopeptide in which a sugar chain is bound to a peptide having a threonine and/or serine residue, wherein the molecular weight of the glycopeptide is 700 or more and 3,000 or less,
the sugar chain binds to the hydroxyl group of the threonine or serine residue of the peptide,
the constituent sugars of the sugar chain consist of sialic acid, N-acetylgalactosamine and galactose,
Containing 8% by weight or more of the sialic acid,
Containing 4% by weight or more of the galacto-N-biose,
The protein content of the peptide chain is 86% by weight or less 2. The infant nutritional composition of Claim 1, wherein the infant nutritional composition is an infant formula.