JP2020124126A - Nutritive composition for baby - Google Patents

Abstract

To provide a new nutritive composition for babies controlling a protein amount to less than 2.4 g/100 kcal close to that of mother's milk and increasing a sialic acid amount up to the mother's milk level, and a method for producing the same.SOLUTION: The nutritive composition for babies includes sialic acid of 250 mg/100 g or more in solid matter and a protein amount of less than 2.4 g/100 kcal; in addition to the foregoing, it further includes galacto-N-biose of 90 mg/100 g or more in solid matter.SELECTED DRAWING: None

Description

The present invention relates to a nutritional composition for infants.

In recent years, it has been emphasized that various components contained in nutritional compositions for infants should be as close as possible to components of breast milk. The Codex Committee (CODEX), which establishes international food standards, the European Food Safety Authority (EFSA), and the Ministry of Health, Labor and Welfare have made the content of proteins, lipids, vitamins and minerals in infant formulas similar to that of human milk. It shows a certain standard range amount.

Regarding protein, it was previously considered that the protein of infant formula was inferior in digestion and absorption (lower utilization efficiency) than that of mother milk, and the amount of protein of infant formula was set higher than that of human milk. However, there is a concern that the risk of developing lifestyle-related diseases due to excessive protein intake during infancy will increase, and the amount of protein in infant formula has now tended to decrease. For example, in CODEX, the amount of protein in infant formula is 1.8 to 3.0 g/100 kcal (Non-patent document 1), and in EFSA, the standard range is 1.8 to 2.5 g/100 kcal (Non-patent document 2). Showing. The amount of infant formula products currently licensed in Japan is less than 2.4 g/100 kcal.

Breast milk contains abundant components that promote healthy growth and growth of infants, and sialic acid has been attracting attention as one of the components. Sialic acid is one of the negatively charged nine carbon sugars, and is present in breast milk in the molecular forms of oligosaccharides, glycoproteins and glycolipids. Sialic acid in breast milk protects infants from infection (Patent Document 1), neutralizes bacterial toxins (Patent Document 2), promotes mineral absorption (Patent Document 3), and develops brain function (Non-Patent Document 3). Has various functionalities such as,. However, since milk has a low amount of sialic acid, the nutritional composition for infants, which uses a milk-derived milk material as a raw material, has a much lower sialic acid content than that of breast milk.
Specifically, the amount of sialic acid in breast milk is reported to be 714 mg±64 mg/L (mean±standard error), which means that the average solid content of breast milk is 590 mg/100 g solid with 12.1%. (Non-Patent Document 4). Table 1 shows the total amount of sialic acid per 100 g of solid of the milk calculated from the results of the investigation of the general components of breast milk in Japan (Non-patent document 5) and the sialic acid content (Non-patent document 6) by Iwata et al. .. From this result, although the amount of sialic acid in breast milk decreases with the progress of the lactation period, the amount of sialic acid after 120 days of the lactation period tends to be constant at about 260 mg/100 g solid. Further, with respect to Table 1, the weighted average value of the total sialic acid concentration during the non-milky period 3 to 482 days is about 330 mg/100 g solid. From the above, in order to make the amount of sialic acid of the infant nutrition composition approximate to that of human milk, 260 mg/100 g solid or more is necessary, and preferably 330 mg/100 g solid or more, which is the average amount of sialic acid in Japanese breast milk. It is considered that the average sialic acid content of the milk described in Non-Patent Document 4 is 590 mg/100 g solid or more, which is desirable.

On the other hand, the amount of sialic acid in general infant formula is 193 mg/100 g solid (Non-Patent Document 7), and 67 mg from 260 mg/100 g solid, which is the low value of the amount of sialic acid in Japanese breast milk described above. /100 g solid is insufficient, and 397 mg/100 g solid is also insufficient from 590 mg/100 g solid, which is the average value of the amount of sialic acid in breast milk. Since 90% or more of sialic acid in milk exists in a form bound to protein, for example, if the amount of protein in infant formula is reduced in accordance with the EFSA standard, the amount of sialic acid will be reduced accordingly, and milk and infants There was a problem that the amount of sialic acid in the modified milk powder for use became more and more different.

In order to solve this problem, it has been reported to use sialic acid or sialic acid-containing oligosaccharide prepared by using a genetically modified microorganism (Patent Document 4), but it was prepared by using a genetic recombination technique. Regarding the use of sialic acid in nutritional compositions for infants, it is still a repulsive factor in industrial use from the viewpoint of compliance with the laws and regulations of each country and the acceptability of consumers for the genetic modification technology itself. Is held.

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

Although there is no report of the amount of GNB in general infant formula, GNB is present in the milk in a state bound to glycomacropeptide (GMP). GMP is reported to be 14% of proteins in general infant formula (Non-patent Document 11). Further, GMP is called 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, but their binding ratios (molar ratios) are reported to be 0.8, 6.3, 18.4, 18.5, and 56%, respectively (Non-Patent Document 12). Further, sialic acid in GMP was reported to be 6%, and the amount of GNB in GMP was estimated to be 5% with reference to these reports.
From these results, it was considered that the amount of GNB in general infant formula was 82 mg/100 g solid. Up to now, infant formulas fortified with GNB have not been put on the market. Further, there is no report on a production method using a genetically modified microorganism. Furthermore, even if there are future reports, regarding the use of GNB prepared using genetic modification technology in nutritional compositions for infants, it is not possible From the viewpoint of consumer acceptance, there is still a sense of evasion 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. Adopted as a worldwide Standard in 1981. Amendment: 1983, 1985, 1987, 2011, 2015 and 2016. 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 Iwada et al.: Journal of Japanese Society of Pediatric Nutrition and Gastroenterology, 5:145-158, 1991. Tadashi Iwada et al.: Journal of Japan Society of Nutrition and Food Science, 47:357-362, 1994. Infant formula "Sukoyaka M1" (Snow Brand Bean Stark 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. Hidaka, H. et al.: Crystal structure of phosphorylase involved in human milk oligosaccharide degradation in Bifidobacterium, PFNEWS, 27:18-21, 2009. Bruck WM et al., Effects of bovine alpha-lactalbumin and casein glycomacropeptide-enriched infant formulae 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 above background art, the present invention provides a new nutritional composition for infants in which the amount of sialic acid is increased to the level of human milk while keeping the amount of protein at less than 2.4 g/100 kcal, which is close to that of human milk, and the production thereof. The challenge is to provide a method.

In order to solve the above problems, the present inventors have focused on GMP, and by subjecting it to enzyme treatment and membrane treatment, a sialylic glycopeptide having a sialic acid content and a protein content in a specific range is prepared and blended. The inventors have found that a new infant formula can be prepared in which the amount of protein and the amount of sialic acid are close to those of breast milk, and have completed the present invention.
That is, the present invention includes the following configurations.
(1) A nutritional composition for infants, wherein sialic acid is 250 mg/100 g solid or more and the amount of protein is less than 2.4 g/100 kcal.
(2) The nutritional composition for infants according to (1), wherein the galacto-N-biose is 90 mg/100 g solid or more.
(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 of (1) to (4), wherein the sialylglycopeptide 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. The nutritional composition for infants as described in 1.
(6) The nutritional composition for infants according to any one of (1) to (5), wherein the sialyl glycopeptide 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, the sugar chain being bound to a hydroxyl group of a threonine or serine residue of the peptide, The nutritional composition for infants according to any one of (1) to (6), wherein the sugar constituting the chain comprises sialic acid, N-acetylgalactosamine and galactose.
(8) A method for producing a nutritional composition for infants, which comprises the step of adding a sialylglycopeptide, wherein the sialylglycopeptide has the following constitution.
A glycopeptide in which a sugar chain is bound to a peptide having a threonine and/or serine residue,
The molecular weight of the glycopeptide is 700 or more and 3,000 or less,
The sugar chain binds to a hydroxyl group of a threonine or serine residue of the peptide,
The sugar constituting the sugar chain consists of sialic acid, N-acetylgalactosamine and galactose, and contains 8% by weight or more of the sialic acid,
Containing 4% by weight or more of the galacto-N-biose,
The protein amount of the peptide chain is 86% by weight or less

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

(Nutritional composition for infants)
The nutritional composition for infants of the present invention includes infant formula and formula liquid milk, infant formula (follow-up milk and glowing-up milk), low birth weight infant formula, milk allergen-relieving food and lactose. Lactose-removing foods for intolerance, etc., which are composed mainly of proteins, fats, sugars, vitamins and minerals. In addition, the composition may further contain components that can be added to the nutritional composition for infants.
The nutritional composition for infants of the present invention is characterized in that sialic acid is 250 mg/100 g solid or more and the amount of protein is less than 2.4 g/100 kcal. When the amount of sialic acid is less than 250 mg/100 g solid, the nutritional value is poor because it is lower than the low value of sialic acid in Japanese breast milk, and when the amount of protein is 2.4 g/100 kcal or more, it is excessive in the infancy. It is not appropriate because approval has not been granted 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 solid or more, and even more preferably 590 mg/100 g solid or more.
Further, the nutritional composition for infants of the present invention preferably further contains GNB in a larger amount than general infant formula powder, preferably 90 mg/100 g solid or more, more preferably 100 mg/100 g solid or more. And even more preferably 119 mg/100 g solid or more.
The content of ingredients other than sialic acid and protein is within a certain standard range similar to that of breast milk as defined by the Codex Commission (CODEX) that establishes international food standards, the European Food Safety Agency (EFSA), or the Ministry of Health, Labor and Welfare. Adjust 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 the infant formula as an example. First, a composition containing GMP is subjected to an enzyme treatment and a membrane treatment to prepare a sialylic glycopeptide having a sialic acid content, a GNB content and a protein content in a specific range. By blending this with a general infant formula, a new infant formula can be prepared in which the amount of protein and the amount of sialic acid are close to those of mother's milk.
That is, a protein source, a carbohydrate source, and the sialyl glycopeptide obtained as described above were dissolved in water in a tank. The fat source is then mixed, homogenized, concentrated and pulverized by spray drying. Minor components such as vitamins and minerals can be mixed with lactose and then powder-mixed with spray-dried powder to obtain the infant formula of the present invention.
By the enzyme treatment and membrane treatment of GMP, the sialic acid content and GNB content can be increased more than GMP while the protein content is lower than GMP. The balance between the decrease in protein content and the increase in sialic acid content and GNB depends on the conditions of enzyme treatment (enzyme type, temperature, time, etc.) and membrane treatment conditions (difference in molecular weight cutoff of membrane, etc.). It can be appropriately adjusted so as to obtain a glycopeptide.
As an example, a composition containing GMP is treated with a protease or/and a peptidase, and then the enzymatic decomposition solution is concentrated with an ultrafiltration membrane or a nanofiltration membrane having a molecular weight cutoff of 500 to 1000 to obtain a sialic acid content. Of 8% or more, GNB content of 4% or more, and protein content of 86% or less are prepared, and this sialylglycopeptide is used for general infant formula (sialic acid: 193 mg/100 g solid, GNB: 82 mg/100 g, solid protein: 2.28 g/100 kcal) to make the amount of protein close to that of breast milk, while enhancing sialic acid and/or GNB for infant formula (sialic acid: 250 mg/100 g solid As described above, GNB: 90 mg/100 g solid or more, protein: 2.4 g/100 kcal or less) can be prepared.
In the specification, the sialyl glycopeptide refers to a glycopeptide containing sialic acid as a sugar chain constituent.

(GMP manufacturing method)
A composition containing GMP can be prepared by the method described in Japanese Patent No. 2673828 and 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 having a molecular weight cutoff of 10,000 to 50,000 is used. A method of adjusting GMP by external filtration treatment to obtain a permeate, and preferably again after adjusting the permeation membrane to pH 4 or higher, followed by desalting and concentration using a membrane having a molecular weight cutoff of 50,000 or less. Is mentioned. In addition, milk whey is adjusted to pH 6.0 or higher and heat-treated at 40 to 79° C. is subjected to a membrane treatment to remove whey protein, GMP is recovered on the side of the concentrated solution, and the obtained concentrated solution is After adjusting the pH to 5.5 or less, a method of preparing a composition having a high GMP content by subjecting it to membrane treatment again to recover GMP on the permeate side can be mentioned.
The amount of GMP in the obtained composition can be analyzed by, for example, a urea-SDS electrophoresis method as shown in Examples of Japanese Patent No. 3372499.

(Method for producing sialyl glycopeptide)
The sialyl sugar of the present invention is obtained by undergoing 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. Peptide compositions can be manufactured.
The type of protease used for glycopeptide production is not particularly limited as long as it can hydrolyze the peptide bond to obtain a glycopeptide having a molecular weight and a sugar chain as described in (Sialyl glycopeptide composition) above. However, one or more endo-type proteases and exo-type proteases can be used. Preferably, an enzyme that can be used for the production of foods and pharmaceuticals is preferable, and actinase E (Kaken Pharma), actinase AS (Kaken Pharma), nuclysin (HBI), orientase AY (HBI), Sumiteam FP (Nippon Chemical) , Sumiteam SPP-G (Nippon Chemical Co., Ltd.), Protease A (Amano Enzyme), Peptidase R (Amano Enzyme), Alcalase (Novozyme), Flavorzyme (Novozyme) and the like can be used alone or in combination.
Any sialyl glycopeptide thus obtained can be used, but one having the following characteristics is preferably used.
A glycopeptide in which a sugar chain is bound to a peptide having a threonine and/or serine residue,
(1) The molecular weight of the glycopeptide is 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) The sugar constituting the sugar chain is composed of sialic acid, N-acetylgalactosamine and galactose. (4) Containing 8% by weight or more of sialic acid,
(5) Containing 4% by weight or more of GNB,
(6) A sialyl glycopeptide having a protein content of 86% by weight or less.

(Protein source)
Examples of 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. Alternatively, plant proteins such as soybean protein and wheat protein, as well as peptides or amino acids obtained by enzymatically degrading these to various degrees of degradation can be used.

(Fat source)
As a fat source, animal fats and oils such as milk fat, lard, beef tallow, or bonito oil, fish oil including tuna oil, etc., or soybean oil, rapeseed oil, corn oil, palm oil, palm oil, palm kernel oil, safflower oil. , Vegetable oils such as perilla oil, linseed oil, evening primrose oil, MCT, or cottonseed oil, and fractionated oils thereof, hydrogenated oils, transesterified oils, and the like can be used.

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

(Vitamin and mineral sources)
As a source of vitamins and minerals, "CODEX standard and special hygiene work rules for special purpose foods including infant foods, CAC/VOL. IX-1st edition and Supplement 1, 2, 3, 4" (issued by the Japan International Dairy Federation, 1993), "1993 Specified Items Food Additives Handbook (Revised 31st Edition) (Published by Food and Science Co., 1993)" or "Notification System Food Additives/Food Material Natural Products Handbook (12th Edition)" Among the minerals and vitamins described in (Food and Science Co., Ltd., 1992), those usable in infant foods can be used.
Examples of vitamins include vitamins A, B, C, D, E, K, folic acid, pantothenic acid, β-carotene, nicotinic acid amide, and the like, and minerals include calcium, magnesium, and iron. , Copper, zinc, iodine, manganese, selenium and the like.

(Method for measuring sialic acid content)
It is dissolved in 50 μL of 50 mM potassium phosphate buffer (pH 5.0) so that the sialylglycopeptide concentration is 250 μg/mL and the sialidase (Cosmo Bio) concentration is 0.2 U/mL. The solution is incubated at 37° C. for 24 hours, diluted 2 to 10 times with ultrapure water, and the concentration of free sialic acid is analyzed by HPLC.
The measurement of sialic acid can be performed, for example, by a DIONEX ICS-5000DP system (Thermo Fisher Scientific Co., Ltd.) equipped with a CarboPac PA1 column. The detector uses an electrochemical detector (pulsed amperometric mode), and the mobile phase is ultrapure water (solution A), 200 mM sodium hydroxide solution (solution B), and 100 mM sodium hydroxide solution containing 600 mM sodium acetate. Three liquids (C liquid) are passed at a flow rate of 1 mL/min. The solution is allowed to flow for 45 minutes from the beginning with the solution B 45% and the solution C 10%, and then the solution B is linearly decreased to 37.5% and the solution C is linearly increased to 25% by 10 minutes. From 10 minutes to 20 minutes, 37.5% of solution B and 25% of solution C are passed, and from 20.1 minutes to 25 minutes, 45% of solution B and 10% of solution C are passed. The amount of sialic acid in a sample can be calculated from a calibration curve prepared using a standard product of known concentration.

(Method of measuring GNB content)
In 50 μL of 50 mM potassium phosphate buffer (pH 5.0), the sialylglycopeptide concentration was 250 μg/mL, the sialidase (Cosmobio) concentration was 0.2 U/mL, and the O-glycanase (Prozyme) concentration was 0.125 U. Dissolve enzyme and substrate to give /mL.
The solution was incubated in a thermal cycler (Takara) at 37° C. for 24 hours, and then the reaction was stopped by cooling with ice. After diluting the reaction solution 2 to 10 times with ultrapure water, the concentrations of free sialic acid and GNB are analyzed by HPLC.
The measurement of GNB can be performed by DIONEX ICS-5000DP system (Thermo Fisher Scientific Co., Ltd.) equipped with the CarboPac PA1 column. The detector uses an electrochemical detector (pulsed amperometric mode), and the mobile phase is ultrapure water (solution A), 200 mM sodium hydroxide solution (solution B), and 100 mM sodium hydroxide solution containing 600 mM sodium acetate. Three liquids (C liquid) are passed at a flow rate of 1 mL/min. After elution with 50% solution B for 10 minutes from the start, the proportion of solution C is linearly increased from 0% to 100% by 25 minutes, and 100% solution C is eluted until 30 minutes. Then, the solution B is made 50% in 30.1 minutes, and then eluated with the solution B 50% until 45 minutes. The amount of GNB in the sample is calculated from a calibration curve prepared using a standard product of known concentration.

(Method of amino acid analysis)
15 kinds of amino acids of arginine, lysine, histidine, phenylalanine, tyrosine, leucine, isoleucine, valine, alanine, glycine, proline, glutamic acid, serine, threonine and aspartic acid are hydrolyzed with hydrochloric acid, and then JLC-500/V fully automatic. Analyze with a high-speed amino acid analyzer. Cystine and methionine are hydrolyzed with a formic acid solution and then analyzed by a JLC-500/V fully automatic high speed amino acid analyzer. For tryptophan, barium hydroxide octahydrate and thiodiethylene glycol are added to a sample solution, heated, hydrolyzed with hydrochloric acid, and analyzed by HPLC.

(Calculation of protein amount)
Regarding the protein amount of GMP and sialylglycopeptide, the protein amount is the numerical value of the total amino acid amount obtained from amino acid analysis.

Hereinafter, examples of the present invention will be described in detail, but the present invention is not limited thereto.

[Example 1] Method for preparing sialylglycopeptide composition of the present invention 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 the pH was adjusted to 3.5 with concentrated hydrochloric acid. This is subjected to ultrafiltration using an ultrafiltration membrane (GR61PP, manufactured by DDS) having 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. It was When the amount of the permeated liquid reached 40 L, 40 L of water at 50° C. was added to the concentrated liquid, and ultrafiltration was continuously performed. Continuous operation was performed as described above to obtain 160 L of permeated liquid.
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 and the same ultrafiltration membrane until the concentrated liquid became 5 L, and desalted and concentrated. Subsequently, water at 50° C. was added, and diafiltration was performed under the same conditions and the same ultrafiltration membrane as before, while maintaining the concentrated liquid amount at 10 L, to further desalt. When the amount of permeated liquid reached 80 L by this diafiltration, stop adding water to the concentrated liquid, concentrated by ultrafiltration until the amount of concentrated liquid reached 2 L, dried this concentrated liquid, and GMP54g Got

2. Method for producing sialylglycopeptide composition 1. 30 kg of 5% GMP solution was prepared using the GMP obtained in (1). To this, Alcalase (manufactured by Novozyme) and Flavorzyme (manufactured by Novozyme) were added and reacted at 50° C. for 4 hours. This was subjected to ultrafiltration using an ultrafiltration membrane having a molecular weight cut off of 1,000. Water was added to the concentrated solution, and ultrafiltration was continuously performed to carry out diafiltration, and after performing 5-fold hydroconcentration, a concentrated solution fraction was obtained. The concentrated solution was dried to obtain 300 g of the sialylglycopeptide composition A. Furthermore, what was obtained by performing hydroconcentration up to 10 times was made into the sialyl glycopeptide composition B (300g).

3. Analysis of Sialic Acid Content, GNB Content, and Amino Acid Composition The sialic acid content and GNB content in the prepared GMP and sialylglycopeptide compositions were measured by the methods described below.
(1) That is, in 50 μL of 50 mM potassium phosphate buffer (pH 5.0), the sialylglycopeptide concentration was 250 μg/mL, the sialidase (Cosmobio) concentration was 0.2 U/mL, and O-glycanase (Prozyme) was added. The enzyme and the substrate were dissolved so that the concentration was 0.125 U/mL. The solution was incubated in a thermal cycler (Takara) at 37° C. for 24 hours, and then the reaction was stopped by cooling with ice. The reaction solution was diluted 2 to 10 times with ultrapure water, and the concentrations of free sialic acid and GNB were analyzed by HPLC. It was performed with a DIONEX ICS-5000DP system (Thermo Fisher Scientific Co., Ltd.) equipped with a CarboPac PA1 column. The detector uses an electrochemical detector (pulsed amperometric mode), and the mobile phase is ultrapure water (solution A), 200 mM sodium hydroxide solution (solution B), and 100 mM sodium hydroxide solution containing 600 mM sodium acetate. Three liquids (C liquid) were passed at a flow rate of 1 mL/min.
(2) For the measurement of sialic acid, the solution B was passed at 45% and the solution C at 10% for 7 minutes from the start, and then the solution B was linearly decreased to 37.5% and the solution C was 25% by 10 minutes. Increased linearly up to. From 10 minutes to 20 minutes, 37.5% of solution B and 25% of solution C were passed, and from 20.1 minutes to 25 minutes, 45% of solution B and 10% of solution C were passed. The amount of sialic acid in the sample was calculated from a calibration curve prepared using a standard product of known concentration. The amount of sialic acid was calculated as the amount of N-acetylneuraminic acid (Neu5Ac).
(3) GNB was measured by elution with 50% B solution for 10 minutes from the start, then linearly increasing the ratio of C solution from 0% to 100% by 25 minutes, and 100% C solution until 30 minutes. It eluted. Next, the solution B was adjusted to 50% in 30.1 minutes, and then the solution B was eluted at 50% until 45 minutes. The amount of GNB in the sample was calculated from a calibration curve prepared using a standard product of known concentration.
(4) The amino acid composition in the prepared GMP and sialylglycopeptide composition was analyzed by the general amino acid composition analysis method for food ingredients. The total amino acid amount was used as the protein amount.
(5) Analysis results The sialylglycopeptide 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. The sialylglycopeptide composition B has a sialic acid content of 36%. % By weight, GNB amount was 19.8% by weight, and protein amount 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).
From the MS spectrum and MS/MS spectrum obtained by LC/MS analysis, the sugar chain structure of the glycopeptide contained in the sialyl glycopeptide composition was estimated. Many of the detected glycopeptides had the sugar chain structure (Glycan type 3, 4, 5, 6, 7) of Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAc with two sialic acid molecules bound, Neu5Acα2-3Galβ1-3GalNAc (Glycan type 1) and Galβ1-3(Neu5Acα2-6)GalNAc (Glycan type 2) having one molecule of sialic acid were also detected. Further, N,O-diacetylneuraminic acid (O-Ac-Neu5Ac, Glycan type 4) which is an O-acetyl form of sialic acid, and N,O,O-triacetylneuraminic acid (O-diAc-Neu5Ac, Glycan type 5) was also detected. In addition, a glycopeptide (Glycan type 6, 7) in which two sugar chains were bound to one peptide chain was also detected. That is, any of the detected glycopeptides is a peptide 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 sugar constituting the sugar chain is sialic acid or N-. It was composed of acetylgalactosamine and galactose. The molecular weight of the peptide chain detected by MS/MS spectrum analysis was in the range of 745 to 2928.

[Example 2] Preparation of infant formula prepared with sialyl glycopeptides Whey powder (52.7 kg), skim milk (239 kg), and sialyl glycopeptide composition A (340 g) prepared in Example 1 were dissolved in water (500 kg). 0.9 kg was mixed and homogenized. The obtained solution was sterilized, concentrated and dried by a conventional method to obtain 99 kg of milk powder. Powdered milk, vitamins and 1 kg of mineral components were mixed by powder to finally obtain 100 kg of infant formula. The amount of sialic acid in the obtained milk powder was 250 mg/100 g solid, the amount of GNB was 114 mg/100 g solid or more, and the amount of protein was 2.29 g/100 kcal.

[Example 3] Preparation of infant formula powder containing sialyl glycopeptide Whey powder (52.7 kg), skim milk (239 kg), and sialyl glycopeptide composition B (190 g) prepared in Example 1 were dissolved in water (500 kg). 0.9 kg was mixed and homogenized. The obtained solution was sterilized, concentrated and dried by a conventional method to obtain 99 kg of milk powder. Powdered milk, vitamins and 1 kg of mineral components were mixed by powder to finally obtain 100 kg of infant formula. The amount of sialic acid in the obtained milk powder was 250 mg/100 g solid, the amount of GNB was 114 mg/100 g solid or more, and the amount of protein was 2.27 g/100 kcal.

[Example 4] Preparation of infant formula containing sialyl glycopeptides Whey powder (52.7 kg), skim milk (239 kg), and the sialyl glycopeptide composition B (1100 g) prepared in Example 1 were dissolved in water (500 kg). 0.9 kg was mixed and homogenized. The obtained solution was sterilized, concentrated and dried by a conventional method to obtain 99 kg of milk powder. Powdered milk, vitamins and 1 kg of mineral components were mixed to finally obtain 100 kg of infant formula. The amount of sialic acid in the obtained milk powder was 590 mg/100 g solid, the amount of GNB was 300 mg/100 g solid or more, and the amount of protein was 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 1120 g of GMP were dissolved in 500 kg of water, and 23.9 kg of vegetable oil was mixed with this solution to homogenize. The obtained solution was sterilized, concentrated and dried by a conventional method to obtain 99 kg of milk powder. Powdered milk, vitamins and 1 kg of mineral components were mixed by powder to finally obtain 100 kg of infant formula. The amount of sialic acid in the obtained milk powder was 250 mg/100 g solid, the amount of GNB was 129 mg/100 g solid or more, and the amount of protein was 2.41 g/100 kcal.

[Reference Example 2] Preparation of infant formula powdered milk containing GMP 52.7 kg of whey powder, 239 kg of skim milk, and 6620 g of GMP were dissolved in 500 kg of water, and 23.9 kg of vegetable oil was mixed with this solution to homogenize. The obtained solution was sterilized, concentrated and dried by a conventional method to obtain 99 kg of milk powder. Powdered milk, vitamins and 1 kg of mineral components were mixed by powder to finally obtain 100 kg of infant formula. The amount of sialic acid in the obtained milk powder was 590 mg/100 g solid, the amount of GNB was 413 mg/100 g solid or more, and the amount of protein was 3.35 g/100 kcal.

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

Claims (8)

A nutritional composition for infants, characterized in that sialic acid is 250 mg/100 g solid or more and the amount of protein is less than 2.4 g/100 kcal. Furthermore, the nutrition composition for infants according to claim 1, wherein the galacto-N-biose is 90 mg/100 g solid or more. The nutritional composition for infants according to claim 1 or 2, wherein the nutritional composition for infants is infant formula. The nutritional composition for infants according to any one of claims 1 to 3, further comprising a sialylglycopeptide. The sialylglycopeptide contains 8% by weight or more of sialic acid, 4% by weight or more of galacto-N-biose, and has a protein amount of 86% by weight or less, for infants according to any one of claims 1 to 4. Nutrition composition. The nutritional composition for infants according to claim 1, wherein the sialylglycopeptide has a molecular weight of 700 or more and 3,000 or less. The sialylglycopeptide is a glycopeptide in which a sugar chain is bound to a peptide having a threonine and/or serine residue, and the sugar chain binds to a hydroxyl group of the threonine or serine residue of the peptide to form the sugar chain. The nutritional composition for infants according to claim 1, wherein the sugar comprises sialic acid, N-acetylgalactosamine and galactose. A method for producing a nutritional composition for infants, which comprises the step of adding a sialylglycopeptide, wherein the sialylglycopeptide has the following constitution.
A glycopeptide in which a sugar chain is bound to a peptide having a threonine and/or serine residue,
The molecular weight of the glycopeptide is 700 or more and 3,000 or less,
The sugar chain binds to a hydroxyl group of a threonine or serine residue of the peptide,
The sugar constituting the sugar chain consists of sialic acid, N-acetylgalactosamine and galactose, and contains 8% by weight or more of the sialic acid,
Containing 4% by weight or more of the galacto-N-biose,
The protein amount of the peptide chain is 86% by weight or less