JPH0795920B2 - Artificial milk for childcare - Google Patents

Description

The present invention relates to an epidermal growth factor (Epidermal Growth Factor) that promotes the growth of normal cells such as human epithelial cells derived from animal milk.
r, hereinafter abbreviated as EGF).

(Prior Art) It is said that infants, especially newborns immediately after birth, are more likely to be infected. The reason for this is considered to be that the immune response mechanism against various antigens has not been completed and that gastrointestinal epithelial cells are immature.

Infectious factors such as lysozyme and lactoferrin are present in breast milk, and it is said that these have an anti-infection effect on immature newborn infants having an infection-preventing ability.
On the other hand, in recent years, it has been attempted to add lysozyme or lactoferrin to infant milk powder for the purpose of imparting an infection protective function of breast milk.

However, it is said that even artificially fed infants fed with infant formula containing lysozyme or lactoferrin are still more susceptible to infections than breast-fed infants.

Human EGF is a polypeptide that is abundantly contained in human milk in the human body, and its physiological effect has been attracting attention in recent years.

It is known that human EGF suppresses gastric acid secretion in the intestinal tract, promotes growth of intestinal epithelial cells, and further promotes secretion of digestive enzymes such as lactose. From these facts, EGF is considered to be a physiologically active substance that functions to improve the growth and digestive ability of the intestine of the newborn.

In addition, the concentration of EGF in breast milk is as high as about 200 ng / ml in colostrum, but gradually decreases thereafter, and changes to about 5 ng / ml in mature milk around 4 weeks. Therefore, especially in the early stages of lactation of newborns,
It is believed to suggest that EGF is important.

In addition to human EGF described above, EGF is also found in mouse saliva.
Is known to exist. Furthermore, in recent years, the presence of EGF-like substances in milk has been suggested, but for this EGF-like substance in milk, the presence or absence of the ability to proliferate human epithelial cells has not been confirmed, and whether or not it is EGF. It was unknown.

On the other hand, recently, baby milk powder containing human EGF has been proposed (JP-A-62-228225), but EGF added to the baby milk powder disclosed in the patent is purified from human urine, or It is obtained by recombinant DNA technology, chemical synthesis method, and cell culture method, and has problems in securing quantitative quantity and application to foods when used as a raw material for artificial milk for child-rearing.

By the way, in the infant milk powder currently on the market, according to the study of the present inventors, the presence of EGF is confirmed only in a very small amount, and the amount thereof is about 1/200 of the colostrum value of colostrum of breast milk. is there.

(Problems to be Solved by the Invention) The present invention has been made in view of the above problems, and it was confirmed that EGF in animal milk has the ability to proliferate human epithelial cells, and a baby formula To enable the use of.

That is, the present invention, by blending EGF derived from animal milk into the artificial milk for child-rearing, to help the growth of the intestine of the artificial feeding infant, with it, anti-allergic, infant formula artificial milk with anti-infective ability added. The purpose is to provide.

(Means for Solving Problems) A feature of the present invention is to add EGF derived from animal milk to artificial milk for feeding.

The infant formula of the present invention is roughly classified into infant formula and infant formula, and as infant formula, infant formula, premature infant formula, follow-up formula, medical special formula and others. Liquid infant formulas that include milk powders used for artificial infant feeding include liquid infant formulas that have been prepared for the purpose of artificial infant feeding.

EGF used in the present invention, animal milk is used as a starting material, but milk, goat milk, buffalo milk are preferable from the viewpoint of quantitative assurance, EGF
The content of each is about 3 ng / ml for milk and about 3 ng / ml for goat milk.
About 5 ng / ml in ml and buffalo milk.

EGF is prepared from these animal milks as follows.

Whey is separated from raw or reconstituted milk, goat milk and buffalo milk. As this separation method, for example, lactic acid bacteria may be allowed to act on milk and then rennet may be reacted to separate whey, but other means can also be used. This whey is passed through a membrane having a molecular weight cut off of 8,000 to 10,000 to obtain a permeate. Then, the permeate is fractionated to a molecular weight.
Further dialysis with a permeable membrane of 500 to 5,000 to obtain a dialysis membrane solution.
This internal solution is a concentrated solution containing about 110 to 900 ng / ml of EGF, and this concentrated solution containing EGF can be used as it is for powdered milk for infants or liquid artificial milk for infants. Exchange column chromatography, or by attaching one or a combination of two fractionation means such as affinity chromatography to increase the EGF-containing concentration,
It is also possible to obtain more pure EGF.

Furthermore, EGF can also be obtained by extracting raw milk or whey with an organic solvent such as ethanol and subjecting this extract to membrane fractionation.

The method of blending the EGF obtained as described above is, in the case of liquid artificial milk for childcare, a solution prepared by sterilizing the raw material, by adding and mixing the sterilized EGF-containing concentrated solution, and cooling according to a conventional method. To do. In the case of milk powder for childcare, the prepared solution obtained by homogenizing the raw materials and sterilizing it, and adding and mixing the sterilized EGF-containing concentrate, and concentrating and drying according to a conventional method may be milk powder, but containing EGF. The sterilized concentrated liquid may be pulverized using a means such as freeze-drying, and then mixed with powdered baby milk.

The amount of EGF to be added to the artificial milk for child-rearing according to the present invention is, in order to impart an effect equivalent to that of breast milk, at the time of preparing a solid content of 13 to 16% by weight, 1.8 to 310 μg with respect to powdered milk for child-rearing. / 100
g, preferably 3 to 300 μg / 100 g, and 3 to 400 ng / ml, preferably 5 to 380 ng / ml, for the artificial milk for feeding.

(Effect of the invention) According to the present invention, it is confirmed that EGF contained in animal milk promotes the growth of normal human cells, and by incorporating this EGF into the artificial infant milk, the intestinal tract of artificial feeding infants. To obtain EGF-containing dairy artificial milk derived from animal milk, which can support the growth of dairy cattle, enhance functions such as anti-allergy, anti-infectious disease and improvement of digestive ability, and have no safety problems. You can

(Examples) Hereinafter, the present invention will be specifically described with reference to Examples.

Example 1 Preparation of EGF Concentrated Powder Lactic acid bacteria were added to 100 l of milk (reduced milk may be used), and the mixture was allowed to act at 37 ° C. for 1 hour, and when the pH reached 6.5 to 6.0, rennet was added at 3 / 100,000 w / w. The mixture was added at a rate of 30 to 35 ° C. and reacted for 30 to 60 minutes. Generated whey at 10,000G 15
After centrifugation for minutes, the supernatant was passed through a membrane having a molecular weight cut off of 10,000 to obtain a permeate. This permeate was concentrated to 1/10 and ethanol was added and mixed.

After further centrifuging this at 3,000 G for 15 minutes, the upper layer liquid was removed and ethanol was distilled off. The residue was dissolved in 0.04 M sodium acetate buffer, pH 3.9, 10 l and equilibrated with the same CM-52 cellulose column (diameter 10 x height 100 cm,
Whatman). After thoroughly washing the column with the same buffer, add 20 l of 1M sodium acetate buffer, pH 3.9.
The EGF-rich fraction was eluted and the eluate was concentrated to 1. This concentrated solution was placed in a dialysis tube having a dialysis cutoff molecular weight of 1,000, dialyzed against pure water for 18 hours, and the dialysis membrane solution was freeze-dried to obtain 3.0 g of bovine EGF concentrated powder.

Furthermore, EGF-concentrated powders derived from goat milk and buffalo milk were obtained by the same method.

Measurement of EGF content: EGF content was obtained for the bovine EGF concentrated powder obtained by the above method, breast milk (colostrum and mature milk after 4 weeks), raw cow milk, goat milk, buffalo milk and commercially available milk powder for parenting. In order to compare the amounts, 1 ml of the solution of each sample was centrifuged at 20,000 G for 30 minutes, and the intermediate layer was measured by a radioreceptor assay according to a conventional method.

The results are shown in Table 1. The solid percentage of bovine EGF concentrated powder is
A sample was prepared by dissolving in pure water so that the concentration was 0.27%.
The commercially available powdered milk for child-rearing was dissolved in pure water so that the solid content was 13%.

Example 2 Test of Growth Promoting Effect on Normal Human Cells Human lung-derived WI-38 cells were cultured in a conferent state in Dulbecco's modified Eagle's medium containing 2% calf serum. To these cells, the solutions of the samples shown in Table 2 were respectively
2.5 μl of each was added, and thymidine labeled with ³ H radioisotope was further added. The cells were cultured at 37 ° C. for 48 hours in the presence of carbon dioxide, the culture medium was removed, and the radioactivity incorporated into the cells was measured by a liquid scintillation counter. For the growth promoting effect, the amount of radioactive thymidine incorporated into cells was used as an index for DNA synthesis.

The results are shown in Table 2.

The control in the table shows the radioactivity when 2.5 μl of only the commercially available Dulbecco's modified Eagle's culture solution was added.

Goat and water buffalo EGF concentrate was prepared by a method similar to the bovine EGF concentrate shown in Example 1. Also,
As the sample in which the commercially available infant milk powder and the bovine EGF concentrate were mixed, those in which 292 μg / 100 g and 3.7 μg / 100 g of bovine EGF were mixed were used.

As can be seen from Table 2, the bovine EGF-containing infant milk powder of the present invention, which is a commercially available infant milk powder blended with bovine EGF, is confirmed to have the same level of cell proliferation ability as that of breast milk (colostrum and adult milk).

Example 3 Preparation of infant formula containing bovine EGF Whey protein concentrate (15.9 kg), lactose (45.9 kg), vitamins and minerals (1 kg) were dissolved in water (650 kg), mixed with desalted skim milk (257 kg), and fresh cream (7 kg) and vegetable oil. 23.9 kg were mixed and homogenized. The obtained solution was sterilized and homogenized by a conventional method. On the other hand, bovine EGF concentrated powder was prepared by the method shown in Example 1, and 0.47 kg of this bovine EGF concentrated powder was added to 20 kg of sterilized water.
After solubilization, the bacteria were sterilized through a sterilization filter, added to the above solution, mixed and spray-dried to obtain 100 kg of infant formula. The infant milk powder contained 22.3 μg / 100 g of bovine EGF.

Example 4 Preparation of Liquid Artificial Milk for Breeding Containing Bovine EGF 15.9 kg of whey protein concentrate, 45.9 kg of lactose, 1 kg of vitamins and minerals were dissolved in 352 kg of water, then mixed with 257 kg of desalted skim milk, and further 7 kg of fresh cream. , 23.9 kg of vegetable oil were mixed and homogenized, and then sterilized according to a conventional method.

On the other hand, as for bovine EGF, 0.47 kg of concentrated powder was dissolved in sterilized water in the same manner as in Example 1, sterilized through a sterilization filter, and added to and mixed with the above sterilized solution to obtain 750 kg of liquid artificial milk for childcare. This liquid artificial milk contained 32.8 ng / ml bovine EGF.

Claims (5)

[Claims] 1. An artificial milk for child-rearing, which comprises an epidermal growth factor derived from animal milk. 2. The artificial milk for childcare according to claim 1, wherein the animal milk is cow milk, goat milk or buffalo milk. 3. The artificial milk for childcare according to claim 1 or 2, wherein the artificial milk for childcare is powdered milk for childcare or liquid artificial milk for childcare. 4. The artificial milk for childcare according to claim 3, which is obtained by incorporating epidermal growth factor into the infant formula at a ratio of 1.8 to 310 μg / 100 g. 5. The artificial milk for childcare according to claim 3, which is obtained by adding epidermal growth factor to the liquid artificial milk for childcare at a rate of 3 to 400 ng / ml.