JP3161846B2 - Separation of sialic acid-binding peptides in milk whey - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing improved whey. More specifically, the present invention relates to a method for adsorptive separation of a sialic acid-binding peptide from milk whey using a separating agent. More specifically, the present invention relates to a method for adsorbing a sialic acid-binding peptide in milk whey to an anion exchanger in which a weakly basic exchange group is introduced into a hydrophilic resin matrix, and eluting it with an alkali or a salt.

According to the present invention, sialic acid-binding peptides having various excellent physiological actions can be efficiently separated and collected. Therefore, the present invention provides functional foods, foods for specified health use, health foods, nutrition It plays an important role in the technical field of food and other various food and beverage products, as well as in the technical field of pharmaceuticals and cosmetics.

[0003]

BACKGROUND OF THE INVENTION Milk whey, which is generally produced in cheese making, contains most of the active water-soluble ingredients except fat and casein in milk. Lactose contained in milk whey in large amounts has been easily crystallized from milk whey, separated and used for food and medicine.

[0004] However, milk whey after the separation of lactose is in most cases intact, that is, low-lactose whey, or desalted low-lactose whey obtained by subjecting it to various desalination treatments, or ultrafiltration-treated (UF) whey. It is used only as a food ingredient in the form of protein concentrate. Until now, the use of proteins and peptides in whey by separating them into individual components has been practically used on a commercial scale, except for special cases such as selective separation of lactoferrin from milk. At the present time, there has not been a high degree of effective utilization of the characteristics of various proteins and peptides contained in milk whey.

It is said that milk whey is involved in the metabolism of erythrocytes, the composition of gangliosides that play an important role in the brain, and the reception of certain toxins of pathogenic bacteria (Ogura Haruo, Chemistry and Biology, 29 (4), 248). -251, 1991) sialic acid, most of which is present in a form bound to proteins and peptides. Examples include κ-casein and its rennet degradation products such as glycomacropeptide (GMP) and proteose peptone (PP). Free sialic acid can be easily adsorbed and recovered on a synthetic polymer strong basic anion exchanger used in water treatment and the like (Kouichi Anan et al., Basic Biochemistry Experimental Method, Vol. 5,
p157, Maruzen Co., Ltd., 1976) It has been confirmed that those bound to proteins and peptides are difficult to adsorb.

[0006] Conventionally, many attempts have been made to separate sialic acid-binding peptides from whey, and these known techniques and their problems are listed.

(1) Method using UF 1) Jean-Mari Ustash, published patent publication No. 1983
-23400 Whey is treated with UF having a molecular weight cutoff of 10,000 to 50,000 to separate sialic acid-binding protein on the concentrate side and low molecular weight substances on the permeate side. There is a problem in the purity of sialic acid because proteins containing no fat or sialic acid contain large amounts of lactose and minerals on the permeate side. In order to increase the purity of the sialic acid-binding peptide, a complicated post-process is required.

[0008] 2) Morimasa Tanimoto et al., Published Patent Application No. 2-2
76542 GMP with low molecular weight cut off molecular weight 10,000-50,000
Although it utilizes the property of easily permeating a UF membrane of 0, the GMP purity of the obtained product, that is, the sialic acid content is limited because a part of the whey protein behaves together with GMP.

(2) Method Using Chromatography Examples of the separation of some sialic acid-binding peptides (GMP and PP) by chromatography are described below. These are mainly laboratory-scale experiments and analytical methods,
Industrialization is impossible from the viewpoint of cost and safety. In addition, since various buffers are used, it is not a simple operation.

1) Kuwata et al., Journal of the Japanese Society of Agricultural Chemistry, 43
(3), 183-188, 1969 Weakly basic anion exchanger (DEAE-Cellulos)
e: Whatman) GMP is separated using a column. However, since pyridine is used as a solvent, there is a problem in safety as a food. In addition, it is premised on separation on a laboratory scale, and a gel filtration column or a trichloroacetic acid (TCA) treatment is performed as a pretreatment.

2) C.I. V. Morr et al. Food
Sci. , 53 (1), 80-87, 1988 Gel filtration, weak basic anion exchange (DEAE-Seph
adex: Pharmacia LKB), by combining affinity chromatography,
Although the MP is separated, the operation is complicated. Since a Tris-hydrochloride-imidazole buffer is used during anion exchange, there is a problem in safety as food.

3) Kanno et al., Jpn. J. Zoot
ech. Sci. , 52 (4), 282-296, 19
81 Gel filtration and weak basic anion exchange (DEAE-Cell
ulose DE52: Whatman)
PP is separated. Tris-HCl buffer for gel filtration,
Since a tris-phosphate buffer is used for weakly basic anion exchange, there is a problem in safety as a food. In addition, ammonium sulfate fractionation is performed in the pretreatment, and it takes time to remove it.

4) S. W. Lee et al., Agric. Bi
ol. Chem. , 51 (6), 1535-1540,
1987 Weakly acidic cation exchanger having a carboxymethyl (CM) group introduced therein (CM-Sephadex C-25: Phar
macia LKB) column to separate GMP. Since an imidazole-hydrochloric acid buffer is used as a developing solvent, there is a problem in safety as food.

5) C.I. Olieman et al., Neth. M
ilk Dairy J. , 37, 27-36, 198
3 GMP is separated using gel filtration high performance liquid chromatography. It is intended to detect GMP. As TCA is added to the sample as a pretreatment, there remains a problem in safety as a food.

[0015]

DISCLOSURE OF THE INVENTION The present invention has been made in view of such a state of the art, and has a new system for efficiently and easily separating sialic acid-binding peptides from milk whey. It was made for development.

[0016]

SUMMARY OF THE INVENTION The present invention has been completed as a result of studies from various angles to achieve the above object, and its basic technical idea is to use milk whey of pH 4-6.
After adjusting to a hydrophilic resin matrix, the solution was contacted with an anion exchanger in which a weakly basic exchange group was introduced, and then a solution of an alkali metal salt or an alkaline earth metal salt was passed through the ion exchanger. A method for separating sialic acid-binding peptides in milk whey.

The milk whey of the present invention includes cheese whey, rennet whey, those desalted by electrodialysis or the like, WPC concentrated by UF or the like, and deproteinized whey obtained by heat treatment or the like. And W
PC or one or more selected from those obtained by reducing a dried product thereof with water is used.

In the present invention, the sialic acid-binding peptide is adsorbed on an anion exchanger in which a weakly basic exchange group is introduced into a hydrophilic resin matrix, and the adsorbed sialic acid-binding peptide is an alkali metal salt or an alkali metal salt. The sialic acid-binding peptide in milk whey is separated by elution with a solution of an earth metal salt.

In the present invention, various types of milk whey are used in the pH range.
After adjusting to 4-6, the hydrophilic resin matrix is brought into contact with an anion exchanger in which a weakly basic exchange group is introduced to adsorb only the sialic acid-binding peptide to the anion exchanger. A solution of a metal salt or an alkaline earth metal salt is passed through to separate a sialic acid-binding peptide.

Pre-adjusting milk whey to pH 4-6 in advance is extremely important for the adsorption of sialic acid-binding peptides to an anion exchanger in which a weakly basic exchange group has been introduced into a hydrophilic resin matrix. If the conditions are not satisfied, the adsorption rate of the sialic acid-binding peptide will decrease, and the object of the present invention will not be achieved. The anion exchanger obtained by introducing a weakly basic exchange group into the hydrophilic resin matrix used in the present invention has a number of hydrophilic functional groups such as agarose, dextran, cellulose, natural high molecular polysaccharides such as chitosan, and hydroxyl groups. A carrier whose base is synthetic polyvinyl or the like, into which a weakly basic anion exchange group such as a diethylaminoethyl (DEAE) group or an amino group is introduced, and is generally commercially available, so a commercially available product may be used. . Even a strong basic anion exchanger having a strongly basic exchange group such as a quaternary amine or a quaternary aminoethyl group adsorbs a sialic acid-binding peptide, but the adsorption rate is poor, so the exchange group is weakly basic. Is desirable.

In the present invention, when an anion exchanger in which a weakly basic exchange group is introduced into a hydrophilic resin matrix is used, a column-type or batch-type treatment can be performed in the same manner as in a normal ion-exchange resin. is there. In consideration of work efficiency, continuous operation is preferable to intermittent operation. To increase the adsorption rate, it is preferable to desalt the sample in advance to remove salts that competitively act on the adsorption of the sialic acid-binding peptide to the anion exchanger.

In the case of the batch type, sialic acid-binding peptide is adsorbed only by mixing an anion exchanger having a weakly basic exchange group introduced into a hydrophilic resin matrix and milk whey and adjusting the pH to a predetermined value. Is done. However, whey protein,
For example, when β-lactoglobulin and α-lactalbumin coexist, a part thereof is also adsorbed at the same time, and therefore, a column-type treatment is desirable to increase sialic acid purity.

In the case of the column type, milk whey adjusted to pH 4-6 is passed through a column filled with an anion exchanger in which a weakly basic exchange group has been introduced into a hydrophilic resin matrix, and the sialic acid-binding peptide is simply passed through. Only the adsorbed whey protein is hardly adsorbed. The adsorbed sialic acid-binding peptide is eluted by flowing an alkaline solution such as caustic soda or caustic potassium or a salt solution such as sodium chloride through the column, washing the column, and reusing the milk whey. In particular, when an alkaline solution is used for eluting a sialic acid-binding peptide, the labor for regeneration of the ion exchanger can be omitted.

Next, Test Examples and Examples of the present invention will be described. In addition, the quantification of sialic acid was determined by a colorimetric method using thiobarbituric acid (Kunichi Anan et al., Basic Biochemistry Experimental Method, Vol. 5, p157,
(Maruzen Co., Ltd., 1976).

[0025]

Test Example 1 (Screening of Separating Agent) Separating Agent 10m
EDWP reduced at a concentration of 12% (w / w) to 1
(Gouda cheese whey was desalted by electrodialysis and dried) was mixed in an amount of 50 ml, stirred at a predetermined pH for 20 minutes, and then separated with a filter paper to separate the reaction solution and the separating agent. Was used for quantification of GMP.

The GMP adsorption amounts of various separating agents were compared using (GMP concentration in filtrate / GMP concentration before reaction) as an index, and the results in Tables 1 to 4 were obtained. Although not shown in the table, GMP was scarcely adsorbed to the cation exchanger having a carboxymethyl group or a sulfopropyl group as an exchange group at any pH.

[0027]

[Table 1]

[0028]

[Table 2]

[0029]

[Table 3]

[0030]

[Table 4] Screening of separation agent (4) Note) A test was performed for each separation agent of each manufacturer. Since the lot of EDWP used as the test sample is different, the GMP concentration of the stock solution is different in each experiment. Note) Abbreviation of exchange group DEAE: diethylaminoethyl; QA: quaternary amine; QAE: quaternary aminoethyl; TA: tertiary amine; PA: 1,2 polyamine * 1: 100- (GMP concentration in filtrate / before reaction) GMP concentration in sample) × 100 * 2: Weakly basic anion exchange group * 3: Strongly weak anion exchange group

As is evident from the above results, DE with a DEAE group as an exchange group was used as a separating agent having a large GMP adsorption amount.
AE-Sephadex A25, DEAE-Seph
acel, DEAE-Cellulofine A20
0, same A500, DEAE-Toyopearl 65
0s, Chitopearl E having an amino group as an exchange group
P-03, EP-05 and the like. These are all weakly basic anion exchangers based on hydrophilic natural polysaccharides such as dextran, cellulose and chitosan, and hydrophilic polyvinyl having a hydroxyl group, and quaternary ammonium groups are introduced into the hydrophilic base. It was found that the amount of GMP adsorbed on the whole was larger than that of the strongly basic anion exchanger.

After the column was filled with the above-mentioned separating agent, water and an EDWP reducing solution were passed through the column, and changes in pressure loss and flow rate in the column were examined. DEAE-Chitopearl as a separating agent having excellent operability from the viewpoints of (filterability), an increase in pressure loss in the column due to an increase in the size of the column, and compaction of the separating agent layer.
EP-03, EP-05, DEAE-Sephad
ex A25 was mentioned.

[0033]

Test Example 2 (Effects of PH and Desalting of Samples) The effects of pH and desalting on the amount of GMP adsorbed were examined by the method shown in Test Example 1. The samples were an EDWP reducing solution and a whey powder (WP) reducing solution not subjected to electrodialysis treatment. EDWP,
Table 5 shows the composition of WP and WP. DEAE-Cellu
lofine A200, DEAE-Sephadex
FIG. 1 shows an example of GMP separation using A25 and Chitopearl EP-03. The GMP concentration in the non-adsorbed liquid is large when the pH of the sample is around 4 to 6,
The WP reducing solution was larger than the EDWP reducing solution , indicating that desalting increased the amount of GMP adsorbed.

[0034]

[Table 5]

[0035]

[Test Example 3 (Effect of sample concentration: column treatment)] GMP
The reducing liquids of the two whey materials having different contents are mixed with a separating agent 150
The solution was passed through a column filled with ml, and the treatments were compared. E
Table 6 shows the relative GMP content of DWP and WPC.
G when DEAE-Sephadex A25 is used
Table 7 shows examples of MP separation. By multiplying the sample concentration (A), the processing amount (B), and the relative GMP content (C) in the powder sample before reduction, each G in the case of EDWP and WPC is multiplied.
Comparing the amount of MP adsorption, the amount of GMP adsorption was larger in the WPC reducing solution. This means that if the ash concentration in the sample is low, the GM
This means that the absolute adsorption amount of P does not decrease. From the viewpoint of shortening the column processing time, the sample is more WP than EDWP.
It was confirmed that C was more preferable.

[0036]

[Table 6]

[0037]

[Table 7]

FIGS. 2 and 3 show examples in which GMP was separated from a WPC solution using a column packed with DEAE-Sephadex A25 and Chitopearl EP-05.
Respectively. As a result, it was confirmed that β-lactoglobulin and α-lactalbumin were hardly adsorbed.

[0039]

Test Example 4 (Eluent conditions) A sufficient amount of the WPC solution was passed through a column packed with 50 ml of the separating agent, and when the GMP reached the adsorption saturation, the separating agent was washed with water and then 4 ml of the separating agent was collected. GM by mixing 16 ml of various concentrations of sodium chloride (NaCl) or caustic soda (NaOH) solution.
Batch elution of P was attempted. Table 8 shows the relative GMP elution power under various experimental conditions when the GMP elution power of 0.5 M sodium chloride (pH 6) was 1.00. GM
Preliminary experiments confirmed that P was partially eluted by adjusting the pH to about 3 or 9 with hydrochloric acid or dilute caustic soda, but the elution power was not sufficient. It can be seen from Table 8 that the use of about 0.5 M sodium chloride or about 0.1 N caustic soda significantly increases the GMP elution rate. In order to recover GMP efficiently, a high concentration of caustic soda may be used.
Considering the stability of the solution and the chemical resistance of the separating agent, it is considered that elution should be performed using caustic soda of about 0.1N.

[0040]

[Table 8]

[0041]

EXAMPLE 15 Chitopearl EP-05 was added to an acrylic resin column having an inner diameter of 50 mm and a height of 200 mm.
A multi-stage column filled with 0 ml and stacked in five stages via a wire mesh lamination type filter was produced (total amount of separating agent: 750 ml).
The pH of the WPC 5% (w / w) solution is set to 4.4,
And then centrifuged at 3,000 G for WPC
PH of supernatant, excluding fat and aggregated denatured protein
Was adjusted to 5.5 and the solution was passed through the column (2000 to 2000).
2500 ml / h). After passing 22.5 L of the WPC solution, the column was washed with deionized water, and adsorbed substances were eluted by circulating a total of 4.5 L of 0.1 N caustic soda. Further, the liquid in the column was extruded with deionized water, and the eluate was recovered. After neutralization, the eluate was concentrated 8-fold on a UF membrane having a molecular weight cut off of 10,000, desalted by electrodialysis, and lyophilized. The yield and sialic acid content of the obtained powder were 8.3 and 7.19%, respectively, and most of the recovered sialic acid-binding peptides were GMP and P
P.

[0042]

According to the present invention, for the first time, sialic acid-binding peptides can be industrially separated, and efficient production of sialic acid-binding peptides having excellent physiological functions and sialic acid can be achieved for the first time.

[Brief description of the drawings]

FIG. 1 is a graph illustrating the effect of pH and desalting on the adsorption of GMP.

FIG. 2: G according to DEAE-Sephadex A25
An example of MP separation will be described.

FIG. 3: GM according to Chitopearl EP-05
The example of separation of P is shown.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Tomoko Ueda, Inventor 1-21-3, Sakaemachi, Higashimurayama-shi, Tokyo Meiji Inside Daiichi Dairy Co., Ltd. No. 3 Meiji Dairies Co., Ltd. Central Research Laboratory (72) Inventor Takeshi Matsushita 2-3-2 Marunouchi, Chiyoda-ku, Tokyo Nippon Rensui Co., Ltd. (72) Inventor Takashi Kikuchi 3-2-2 Marunouchi, Chiyoda-ku, Tokyo No. 3 Inside Nippon Rensui Co., Ltd. (72) Inventor Ichiro Kurihara 3-2-3 Marunouchi, Chiyoda-ku, Tokyo Nippon Rensui Co., Ltd. (72) Hitoshi Usami 2-3-2 Marunouchi, Chiyoda-ku, Tokyo Nippon Rensui Co., Ltd. (56) References JP-A-4-243898 (JP, A) JP-A-2-104246 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) ) A23J 1/20 A23L 1/305 C07K 1/16-1/22 A23C 21/00 BIOSIS (DIALOG)

Claims (2)

(57) [Claims] 1. After adjusting the pH of milk whey containing a sialic acid-binding peptide to pH 4 to 6, contacting the milk whey with an anion exchanger having a weakly basic exchange group introduced into a hydrophilic resin matrix. An alkali metal salt on the anion exchanger,
Alternatively, a method for separating a sialic acid-binding peptide in milk whey, comprising passing a solution of an alkaline earth metal salt through the solution. 2. Milk whey is rennet whey, desalinated by electrodialysis or the like, whey protein concentrate (WPC) concentrated by ultrafiltration or the like, or deproteinized protein obtained by heat treatment or the like. The method for separating sialic acid-binding peptides in milk whey according to claim 1, which is at least one selected from whey, WPC, and those obtained by reducing a dried product thereof with water.