JP2003047402A - Neutral origosaccharide-containing nersery formulated milk - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain nursery formulated milk excellent in infection protective effect by addition of a safe low-molecular substance such as oligosaccharide to milk. SOLUTION: This nursery formulated milk is produced by adding neutral oligosaccharide fractions comprising fucose-containing oligosaccharide, galactosyl lactose, or the like, obtained from animal-originated milk such as breasting cow milk or goat milk, to conventional formulated milk material.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a infant formula containing a neutral oligosaccharide derived from animal milk. More specifically, the present invention relates to a novel modified milk having oligosaccharides obtained from animal milk or an intermediate-treated product thereof and added to a conventional modified milk material to impart an infection protective ability.

[0002]

2. Description of the Related Art Conventionally, cholera bacteria,
Protein-based protective factors such as lactoferrin and lysozyme have been added to protect infants from pathogenic bacteria such as virulent Escherichia coli and Salmonella or toxins thereof.
However, such a protein-based protective substance rarely causes allergies because it is not derived from humans, which leaves a problem. Therefore, there is a demand for a low molecular weight anti-infection substance having no antigenicity, rather than a substance having a large molecular weight such as a protein. As candidates for such substances,
Attempts have been made to add oligosaccharides containing sialic acid (JP-A-59-181497, JP-A-61-12695, JP-A-3-49648). However, sialic acid is an unstable compound, and there is a problem in stability during dissolution storage.

[0003]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a infant formula containing a low-molecular-weight substance free from allergic problems and containing an infection-preventing substance which is stable even for long-term storage. .

[0004]

[Means for Solving the Problems] Human breast milk contains many types of neutral oligosaccharides, mainly oligosaccharides containing fucose, and acidic oligosaccharides, mainly oligosaccharides containing sialic acid. . Of these, oligosaccharides containing sialic acid are known to have the ability to protect against rotavirus, influenza virus, and the like. On the other hand, oligosaccharides containing fucose have been reported to protect against cholera, pathogenic Escherichia coli, Salmonella, etc. (C. Kunz and S. Rud
loff, Acta paediatr. 82, 903-912 (1993), DS New
burg, Current Medicinal Chemistry, 6, 117-127 (199
9)). It is believed that the oligosaccharides in human milk have a protective effect against infection by binding to the receptors of various pathogenic bacteria and preventing the pathogenic bacteria from binding to the living body (EH Beachey, Journal of the Infective Dise
ases, 143, 325-345 (1981)). That is, the more kinds of oligosaccharide, the more kinds of pathogenic bacteria can be handled.

On the other hand, in goat's milk (including colostrum, hereinafter simply referred to as milk), as one component of neutral oligosaccharides,
It has been reported that fucosyl lactose, which has the simplest structure among the oligosaccharides bound with fucose, is contained (T. Urashima, et al, Carbohydrate Research, 262,
173-184 (1994)). It has also been reported that goat milk and bovine colostrum contain various kinds of oligosaccharides contained in human milk, such as galactosyl lactose (T. Saito and T. Urashima, The Chemical Times, 3 -
9 (1998)). The findings to date have not completely revealed the structure of the oligosaccharides contained in bovine colostrum or goat milk, but although it is not as diverse as the oligosaccharides in human milk, it is a considerable variety of oligosaccharides. Is known to exist. In order to exert the protective effect against infection by adding it to the infant formula, it is said that a mixture of various kinds of oligosaccharides obtained from such animal milk is added, rather than addition of one specific kind of oligosaccharide. That makes a lot of sense.

However, cow's normal milk contains almost no neutral oligosaccharides other than lactose, and the infant formula prepared from cow's normal milk also contains neutral oligosaccharides other than lactose. Is currently not included.

The present inventors have focused on neutral oligosaccharides in animal milk (milk, especially colostrum, or goat milk), and have developed a technique for purifying a neutral oligosaccharide mixture from animal milk. did. Furthermore, by adding the mixture of oligosaccharides, infant formula was provided with the protective ability against infection, and the present invention was completed.

That is, the present invention relates to (1) a baby formula containing a neutral oligosaccharide mixture derived from animal milk,
(2) The infant formula as described in (1), wherein the animal milk is goat's milk, (3) The infant formula as described in (1), wherein the animal milk is bovine milk, (4) (1), (2) or (3), wherein the neutral oligosaccharide mixture contains galactosyl lactose. It is a mixture of oligosaccharides (1), (2)
Or (6) the infant formula, (6) the animal milk is colostrum, normal milk, cheese whey or casein whey (1), (2) or (3) the infant formula,
(7) Milk of animals was deproteinized by ultrafiltration membrane, microfiltration treatment, or ion exchange treatment, milk desalted by reverse osmosis membrane treatment or electrodialysis, and lactose was crystallized and centrifuged to remove lactose. Milk (1), (2)
Or (8) the infant formula described in (3), (8) animal whey deproteinized by ultrafiltration membrane, microfiltration treatment, or ion exchange treatment, whey demineralized by reverse osmosis membrane treatment or electrodialysis. The infant formula as described in (1), (2) or (3), which is whey obtained by lactose being crystallized and then centrifuged to remove lactose, (9) Animal milk or its dry powder or an intermediate processed product thereof. Was purified using an activated carbon column, and the resulting neutral oligosaccharide fraction was blended (1),
(2) or (3) infant formula, (10) animal milk or its dried powder or its intermediate-treated product is purified using an anion exchange resin, and the resulting neutral oligosaccharide fraction is obtained. The present invention relates to the infant formula as described in (1), (2) or (3). Hereinafter, the invention will be described in detail.

The infant formula referred to in the present invention includes infant formula (milk formula for infants, formula formula for premature infants, follow-up formula, special formula formula for medical use, etc.) and liquid formula formula for parenting. .

The term colostrum as used in the present invention means that a child is born 2
It refers to milk within a week, but the content of neutral oligosaccharides in colostrum varies somewhat depending on the production area, feed, etc.
Contains more than normal milk. For example, 100 g of dried cow colostrum powder contains about 1-3 g of neutral oligosaccharides. On the other hand, the milk of goats contains about 50-250 mg / L of neutral oligosaccharides. Further, as shown in FIGS. 1 and 3, it can be seen that the neutral sugar fraction obtained from goat milk or bovine colostrum contains many kinds of oligosaccharides. As described above, it is believed that the oligosaccharides in human milk have a protective effect against infection by binding of various kinds of oligosaccharides to receptors of pathogenic bacteria. Therefore, the fact that goat milk or bovine colostrum contains such various kinds of oligosaccharides means that these oligosaccharide mixtures correspond to receptors of many kinds of pathogenic bacteria as well as human milk oligosaccharide mixtures. Therefore, it can be expected to have the same infection defense function.

On the other hand, overseas, formulated milk made from goat milk as a main raw material is also manufactured and sold, and it has been proved that it is safe for infants. Therefore, when taking out the neutral oligosaccharide mixture from goat's milk and adding it to the infant formula, there is no problem in terms of safety. In particular, in the present invention, only neutral oligosaccharides are separated from goat milk and used as a raw material for infant formula, since it does not contain goat-derived proteins that cause allergies. It is clear that it is even safer.

As the raw material for the neutral oligosaccharide, goat milk itself may be used, but if cost is important, cheese or whey produced during casein production may be used. In cheese whey or casein whey, the oligosaccharide fraction is retained as it is, and the neutral oligosaccharide fraction can be obtained by the same method as in the case of producing from milk.

The neutral oligosaccharides are obtained by degreasing and deproteinizing the above-mentioned raw materials as they are or by dissolving them in water or the like and subjecting them to an activated carbon column chromatography, an ultrafiltration method, an ion exchange method or the like. It can be prepared.

When separating neutral oligosaccharides from milk or whey, it is not necessary to separate lactose completely. If the ratio of neutral oligosaccharides and lactose is known, they can be directly used as a "mixture of lactose and neutral oligosaccharides" in the production of infant formula without separating them.

The method for obtaining the neutral oligosaccharide fraction is not limited to the method described above and the examples, and any method of adding the neutral oligosaccharide obtained may be used for the preparation of infants for protection against the present infection. The effects of milk are unchanged. For example, even a neutral oligosaccharide fraction obtained by directly applying milk or whey to an activated carbon column and eluting with ethanol, a neutral oligosaccharide fraction obtained as a pass-through fraction by applying it to an anion exchange resin is used. But it doesn't matter.

The obtained neutral oligosaccharide is appropriately desalted and concentrated by means such as electrodialysis and used as it is, or it is powdered by means such as freeze-drying and spray-drying before use. You can

In the infant formula of the present invention, in the case of the modified milk powder, the neutral oligosaccharide to be added is 1 mg to 100 mg per 100 g of the product.
In the case of liquid modified milk, it is preferable to add 10 g of the neutral oligosaccharide to be added in an amount of 0.1 mg to 1 g per 100 ml of the product.

[0018]

EXAMPLES The present invention will be described below with reference to examples.
The present invention is not limited to this.

Example 1 Separation of Neutral Oligosaccharides from Goat Milk (1) 5 L of goat milk was centrifuged at 15,000 rpm for 60 minutes at 4 ° C. to remove fat. After adding twice the amount of ethanol to the supernatant and allowing it to stand at 4 ° C. overnight, it was centrifuged at 15,000 rpm for 40 minutes to remove proteins. The supernatant was concentrated to dryness, dissolved in 2 L of water, and added to a Q-Sepharose column (Pharmacia, 10 cmf).
x 50 cm). Water was passed through at a flow rate of 40 mL / min to collect 7 L of a flow-through fraction. This flow-through fraction was applied to an activated carbon column (10 cmf x 100 cm). After washing with 20 L of water, lactose was eluted by passing 70 L of 5% ethanol. After that, 30% ethanol was passed through to collect 30 L of oligosaccharide fraction. The oligosaccharide fraction was concentrated and then freeze-dried to obtain 770 mg of neutral oligosaccharide. HPLC of the resulting oligosaccharide mixture
The chart is shown in FIG.

Example 2 Separation of neutral oligosaccharides from whey derived from goat milk (2) 300 g of cheese whey obtained from goat milk was dissolved in 5 L of water and directly applied to an activated carbon column (10 cmf x 100 cm). After washing the activated carbon column with 20 L of water, lactose was eluted by passing 60 L of a 5% ethanol aqueous solution. After confirming that lactose was not produced by the phenol-sulfuric acid method, a 30% aqueous ethanol solution was passed through to collect 1 L fractions.
Fractions developed by the phenol-sulfuric acid method were collected, concentrated and freeze-dried to obtain 720 mg of neutral oligosaccharide. 500 MHz proton N of the obtained oligosaccharide mixture
The MR spectrum is shown in FIG.

Example 3 Separation of Neutral Oligosaccharides from Cow Colostrum Powder 100 g of cow colostrum powder was dissolved in 2 L of water and centrifuged at 10,000 rpm for 5 minutes to remove insoluble matter. The supernatant was applied to an activated carbon column (10 cmf x 120 cm) and washed with 40 L of water. Then, the activated carbon column was washed with a 5% aqueous ethanol solution to elute lactose. From this fraction, 73 g of lactose was obtained. Elution was then performed with a gradient of 5% to 50% ethanol. The eluted fractions of oligosaccharides were collected by coloring each fraction by the phenol-sulfuric acid method. Fractions developed by the phenol-sulfuric acid method were collected and concentrated to obtain 1.84 g of neutral oligosaccharide. The HPLC chart of the obtained oligosaccharide mixture is shown in FIG.

Example 4 A infant formula containing a neutral oligosaccharide derived from goat milk was prepared according to a conventional method by blending the following raw materials. (Basic formulation) Casein 0.5Kg Whey protein concentrate 2.4Kg Lactose 4.3Kg Neutral oligosaccharide derived from animal milk 0.1Kg Minerals and vitamins 0.2Kg Oil / fat raw material 2.5Kg Total 10.0Kg Contains beef colostrum-derived neutral oligosaccharide The infant formula was prepared according to a standard method by blending the following raw materials. (Basic combination) Casein 0.6Kg Whey protein concentrate 2.4Kg Lactose 4.2Kg Neutral oligosaccharide derived from animal milk 0.01Kg Minerals and vitamins 0.2Kg Oil and fat raw material 2.5Kg Total 10.0Kg

[0023]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to easily obtain neutral oligosaccharides from animal milk or an intermediate-treated product thereof and prepare a infant formula having a high infection protective effect.

[Brief description of drawings]

FIG. 1 HPLC chart of neutral oligosaccharide mixture derived from goat milk using Dionex DX500 system. Eluent, 0-
20 minutes: 30 mM NaOH, 20-34 minutes: 30-100 mM NaOH gradient, 34-48 minutes: 100 mM NaOH + 0-30 mM NaOAc gradient, 48-55 minutes: 100 mM NaOH + 30-200 mM NaOAc gradient, 55-60 minutes: 100 mM NaOH + 200 mM NaOAc. Flow rate 1 ml / mi
n. Detection is PAD. Under these conditions, lactose is eluted at approximately 9.5 minutes.

FIG. 2 500 MHz proton NMR spectrum of a mixture of neutral oligosaccharides obtained from goat milk. Measured by Varian,
It was measured with an Inova 500 NMR spetrometer.

FIG. 3: HPLC of neutral oligosaccharides obtained from bovine colostrum
chart. The measurement conditions are the same as in FIG.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Yuichi Yamamoto             1-21-3 Sakaemachi, Higashimurayama, Tokyo Meiji Dairy Co., Ltd.             Shikisha Institute of Nutrition Science F-term (reference) 4B001 AC03 EC05                 4B018 LB10 MD31 ME09

Claims (10)

[Claims] 1. An infant formula containing a neutral oligosaccharide mixture derived from animal milk. 2. The infant formula as claimed in claim 1, wherein the animal milk is goat milk. 3. The infant formula as claimed in claim 1, wherein the animal milk is bovine milk. 4. The infant formula according to claim 1, 2 or 3, wherein the neutral oligosaccharide mixture is an oligosaccharide mixture containing fucose-bound oligosaccharides. 5. The mixture of neutral oligosaccharides as claimed in claim 1, wherein the mixture of oligosaccharides contains galactosyl lactose.
The infant formula described in 3. 6. The infant formula according to claim 1, 2 or 3, wherein the animal milk is colostrum, normal milk, cheese whey or casein whey. 7. Milk obtained by deproteinizing animal milk by an ultrafiltration membrane, microfiltration treatment, or ion exchange treatment,
Milk which has been desalted by reverse osmosis membrane treatment or electrodialysis, and milk which has been lactose crystallized and then centrifuged to remove lactose.
The infant formula described in 1, 2, or 3. 8. An animal whey that has been deproteinized by an ultrafiltration membrane, microfiltration treatment, or ion exchange treatment,
Whey desalted by reverse osmosis membrane treatment or electrodialysis, whey desalted by centrifugation after crystallizing lactose
The infant formula described in 1, 2, or 3. 9. The method according to claim 1, wherein the animal milk or its dry powder or its intermediate-treated product is purified by using an activated carbon column, and the obtained neutral oligosaccharide fraction is blended.
The infant formula described in 3. 10. An animal milk or a dried powder thereof or an intermediate processed product thereof is purified by using an anion exchange resin, and the obtained neutral oligosaccharide fraction is blended,
Or the infant formula described in 3.