JP3106256B2 - Method for producing kappa-casein glycomacropeptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for easily producing κ-casein glycomacropeptide having various physiological activities.

[0002]

BACKGROUND OF THE INVENTION κ-Casein glycomacropeptide is
It is conventionally known that sialic acid-binding peptide produced when rennet or pepsin is allowed to act on κ-casein of milk and contained in cheese whey.
Conventionally, as a method for producing κ-casein glycomacropeptide, in a laboratory, after dissolving κ-casein isolated from milk in deionized water, pepsin is allowed to act on the solution.
Trichloroacetic acid is added to precipitate a para-κ-casein fraction, and the resulting supernatant is dialyzed against deionized water, desalted, and then lyophilized to prepare a preparation (Seo et al. Of Food Science "
(Journal of Food Science) 53, 80 (1988)) or dissolving the above κ-casein in deionized water,
After adjusting the pH to 6.7, rennet was allowed to act, and then the pH was further adjusted to 4.6 to precipitate and remove para-κ-casein, and the resulting supernatant was dialyzed and desalted. And a method of preparing by freeze-drying ("Milk Protein" (Milk Protein) pp200, published by Academic Press).

[0003] However, these methods are of a laboratory type and are naturally not suitable for mass production. On the other hand, since the industrial utility of the κ-casein glycomacropeptide has not been known, a method for mass production thereof has not been studied. However, recently, the fact that κ-casein glycomacropeptide has an action to reduce appetite in dogs (Stan et al., “Bullutin of Experimental Biology and Medicine”)
e) 96 , 889 (1983)), indicating that it can be used as a food material for preventing obesity.

[0004] Further, the κ-casein glycomacropeptide has been shown to inhibit the adhesion of Escherichia coli to intestinal cells,
Effect of protecting against influenza virus infection
63-284133) and antiplaque effect (JP-A-63-233911),
It has been found that it has an inhibitory effect on pathogen attachment (Japanese Patent Application No. 2-15285) and a preventive or therapeutic effect on gastric ulcer (Japanese Patent Application No. 2-410695), so that its production on an industrial scale is strongly desired. became.

[0005] A number of methods have been disclosed for industrial separation of kappa-casein glycomacropeptides. These are roughly classified into a method using an ion-exchange resin (Japanese Patent Application Laid-Open No. 2-261343, Japanese Patent Application No. 2-325166, Japanese Patent Application No. 3-19112), a method using ultrafiltration or a reverse osmosis membrane (Japanese Patent Application No. No.58-23400, No.59-27358, JP
63-284199, JP-A-1-68693, JP-A-2-276542,
(Japanese Patent Application No. 2-95686). Among these methods, the method of giving the highest purity κ-casein glycomacropeptide is the method disclosed in JP-A-2-276542,
pH of raw material solution containing κ-casein glycomacropeptide
Is adjusted to less than 4 and the permeate is obtained on an ultrafiltration membrane with a cut-off molecular weight of 10 kDa to 50 kDa, then returned to neutral and concentrated on a membrane with a cut-off molecular weight of 50 kDa or less. The advantage of this method is that high purity can be obtained and the existing whey protein concentrate (W
PC) It is possible to produce κ-casein glycomacropeptide with an ultrafiltration membrane for production, but on the other hand, it has a drawback of low yield. Therefore, these methods have been improved, and the relationship with the ionic strength of the raw material solution has been investigated, thereby completing the present invention.

[0006]

The above-mentioned Japanese Patent Application Laid-Open No. 1-168693 discloses that the pH is adjusted to 4 to 6 before the starting material is subjected to primary ultrafiltration with a 10 kDa to 50 kDa ultrafiltration membrane. Is described, the dissociation of sialic acids is improved, and the membrane permeability is improved. But when it comes to pH,
As described in JP-A-2-276542, the yield is higher when the pH is lower than 4. At a pH of less than 4, it was found that increasing the ionic strength adversely affected the membrane permeability.

The κ-casein glycomacropeptide has a pH
Has the property of performing dependent dissociation-association.
The salt concentration of the solution is highly dependent. That is, the κ-casein glycomacropeptide usually associates at a pH of 4 or more and exists as an oligomer, but partially dissociates at a pH of 4 or less. However, at this time, the electric conductivity of the solution was 4 mS / cm
Above, κ-casein glycomacropeptide has a pH
It does not dissociate even at 4 or less and still exists as an oligomer. Thus, in a solution having an electric conductivity of 4 mS / cm or more, the pH of κ-casein glycomacropeptide was
Dependent dissociation does not occur, and the method described in Japanese Patent Application Laid-Open No. 2-276542 is a great barrier.

In fact, cheese whey, which is one of the main raw materials of κ-casein glycomacropeptide, usually has an electric conductivity of 5 to 6 mS / cm.
-Obtaining casein glycomacropeptide is less than ideal. The present invention is an improvement of the above-mentioned method, which has a kappa-protective effect, an anti-caries effect, a gastric acid secretion controlling effect, etc.
It is an object of the present invention to provide a method for recovering casein glycomacropeptide with high purity and high yield.

[0009]

According to the present invention, a milk material containing κ-casein glycomacropeptide is desalted to obtain a substance having an electric conductivity of 4 mS / cm or less, and the pH is reduced to less than 4. Adjusted, using a membrane having a molecular weight cut-off of 10,000 to 50,000, performing an ultrafiltration treatment to obtain a permeate, and concentrating the permeate using a membrane having a molecular weight cut-off of 50,000 or less, κ-casein. This is a method for producing glycomacropeptide.

[0010] The dairy raw material containing κ-casein glycomacropeptide is not particularly limited. For example, whey protein concentrate (WP) produced by ultrafiltration of cheese whey, rennet casein whey, and cheese whey.
C), whey protein isolate (WPI) produced by bringing cheese whey into contact with an ion exchanger, cheese whey from which whey protein has been precipitated and removed by heating or the like, desalted cheese whey powder, non-desalted cheese whey. Examples thereof include cheese whey powder and lactose mother liquor. When WPC, WPI, and various whey powders are used as raw materials, those reduced with water can be used as starting materials.
It is more preferable to remove fats, insolubles, and the like in the raw material by using a pretreatment such as a cream separator or a clarifier.

[0011] Further, a milk material containing κ-casein glycomacropeptide is brought into contact with a cation exchanger,
The fraction not adsorbed can be used as a raw material. Further, a milk material containing a κ-casein glycomacropeptide having a pH of 4 or less may be brought into contact with an anion exchanger, and a fraction adsorbed on the anion exchanger eluted may be used as the material.

The raw material is desalted and its electric conductivity is 4 mS / cm
In the following steps, any ordinary desalting method may be used, but practically, a desalting method using an ion exchanger, a method using diafiltration, and a method using electrodialysis are exemplified. In the case of desalination using an ion exchanger, the type of ion exchanger used and operating conditions are not particularly limited. When it is necessary to recover the substance adsorbed on the ion exchanger, the κ-casein glycomacropeptide can be effectively recovered using the method disclosed in Japanese Patent Application No. 3-91121. In the case of desalination by diafiltration, reverse osmosis (RO) membrane, ultrafiltration (U
F) Membrane and microfiltration (MF) membrane can be used, and the material, pore size (fraction molecular weight for UF membrane, salt rejection for RO membrane), and operating conditions are not particularly limited.

In the case of preparing κ-casein glycomacropeptide from the thus desalted raw material, in order to obtain particularly high purity, the method described in JP-A-2-276542 is applied. That is, by adjusting the pH of the raw material to less than 4, using a UF membrane having a molecular weight cut-off of 10,000 to 50,000, and performing ultrafiltration, diafiltration, or a combination thereof, the κ-casein glycoprotein is contained in the permeate. This is a method for obtaining a macropeptide. At this time, the electric conductivity of the raw material is 30mS
If it exceeds / cm 2, the yield often decreases. On the other hand, if the electric conductivity of the raw material is 30 mS / cm or less, there is no problem in preparing κ-casein glycomacropeptide. However, considering the subsequent desalination process, the electrical conductivity is 10 mS
/ cm 2 or less is more preferable. Also the molecular weight cut off
A κ-casein glycomacropeptide can be obtained in the permeate using a membrane (UF membrane, MF membrane) larger than 50,000, but in this case, the purity is reduced because other whey proteins are mixed. When the concentrate is used as WPC, the recovery rate may decrease.

In any method, the material of the UF membrane, MF membrane, and RO membrane used in the step of recovering the κ-casein glycomacropeptide in the permeate and concentrating and desalting it is not particularly limited. Materials for these films include:
Examples thereof include polysulfone, polyethersulfone, tetrafluoroethylene, and ceramics. To further increase the yield, a hydrophilic film such as cellulose acetate, nitrocellulose, polyacrylonitrile, or aromatic polyamide, or a charged film can be used. It is preferred to use

Next, in order to concentrate the permeate, see JP-A-2-765.
The method described in No. 42 is used. After returning the pH of the permeate to 4 or more, ultrafiltration, diafiltration, or reverse osmosis filtration is performed using a membrane having a molecular weight cut-off of 50,000 or less. The pH can be adjusted by any alkaline agent, such as sodium hydroxide,
Sodium bicarbonate, aqueous ammonia and the like can be used. Since the κ-casein glycomacropeptide becomes a monomer at a pH of 4 or lower and a monomer (molecular weight of 9,000) at a lower level and a polymer at a higher level (molecular weight of 45,000), the pH is preferably 5 or higher according to the purpose. Any membrane may be used as long as the fractional molecular weight is 50,000 or less, but a membrane exceeding 50,000 will allow the permeation of κ-casein glycomacropeptide. It is most convenient to use a membrane having a molecular weight cut off of about 20,000 used in a general whey protein concentrate manufacturing process.

If the pH of the permeate has not been returned to 4 or higher, the second method is used since κ-casein glycomacropeptide is present as a monomer. That is, molecular weight fraction 1
Ultrafiltration, diafiltration, or reverse osmosis filtration is performed using a membrane of not more than 000, preferably not more than 8,000 to obtain a concentrated solution. In any method, there is no problem in combining the electrodialysis and the desalting step with an ion exchange resin.

[0017] The concentrated solution thus obtained contains essentially κ-
Since only casein glycomacropeptide is contained, it is dried by means such as spray drying or freeze drying.
Incidentally, since the κ-casein glycomacropeptide is stable against heat, it is more desirable to carry out a sterilization step before the drying step. As described above, in the step of concentrating and desalting the κ-casein glycomacropeptide, the time required for the step is obviously shortened as compared with the case of preparing from a non-desalted raw material. Further, it is also possible to apply the above-mentioned known method to the raw material adjusted to have an electric conductivity of 4 mS / cm or less, or a method combining them.

[0018]

According to the present invention, the electric conductivity of the raw material is 4
After adjusting to mS / cm or less, membrane filtration with a constant molecular weight cut-off was performed, so that the recovery of κ-casein glycomacropeptide could be significantly improved, and products produced at lower cost could be marketed. Can be supplied to

[0019]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be specifically described below based on embodiments. Example 1 500 liters of Gouda cheese whey having an electric conductivity of 5.6 mS / cm (Snow Brand Milk Products Co., Ltd.) was placed in a clarifier to remove insolubles, which was heated to 50 ° C. with a plate heater, and the molecular weight cut off was limited to 50,000. Ultrafiltration was carried out using an external filtration membrane (manufactured by DDS, GR51pp) at 50 ° C., a pressure of 0.4 MPa and an average flux of 52.4 l / m ² · h, and concentrated to 100 liters.

Then, while adding deionized water, the filtration was performed until the electric conductivity of the concentrated solution became 1 mS / cm. Hydrochloric acid was added to the concentrated solution to adjust the pH to 2.5 (electrical conductivity: 7.9 mS / cm), followed by ultrafiltration under the above conditions to obtain 80 liters of permeate. This permeate was adjusted to pH 7.0 with 25% caustic soda, concentrated by ultrafiltration as described above, further desalted by diamond filtration, and the concentrate was freeze-dried. 98 g of κ-casein glycomacropeptide powder was obtained. Urea
As a result of analysis by SDS electrophoresis, the purity was 84%.

For comparison, the same Gouda cheese whey 500
When κ-casein glycomacropeptide was prepared from the liter by the above-mentioned method without diamond filtration, the yield was 64 g and the purity was 80%.

Example 2 10 liters of cheddar cheese whey (product of Snow Brand Milk Products) having an electric conductivity of 5.4 mS / cm was passed through a column filled with 100 g of Amberlite (IRA 410) and 100 g of Amberlite (IR 120B). Then, 9.8 liters of desalted whey having an electric conductivity of 0.4 mS / cm was recovered.

This is treated with hydrochloric acid at pH 2.5 (electric conductivity 6.9 mS / cm).
Then, ultrafiltration treatment was carried out in the same manner as in Example 1 using an ultrafiltration membrane having a molecular weight cutoff of 50,000 (manufactured by DDS, GR51pp) to obtain 9 liters of permeate. This was further desalted, concentrated and lyophilized to obtain 2.1 g of κ-casein glycomacropeptide. As a result of analysis by urea-SDS electrophoresis, the purity was 79%.

For comparison, the same cheddar cheese whey 10
When κ-casein glycomacropeptide was prepared from liter without desalting operation, the yield was 1.6 g and the purity was 82
%Met.

Example 3 10 liters of Gouda cheese whey (product of Snow Brand Milk Products) having an electric conductivity of 5.6 mS / cm was applied to an electrodialyzer TS-24 (manufactured by Tokuyama Soda Co., Ltd.) to obtain an electric conductivity of 0.6 mS / cm. The pH was adjusted to 2.0 (electric conductivity: 5.6 mS / cm) with hydrochloric acid. This desalted whey was subjected to ultrafiltration using an ultrafiltration membrane having a molecular weight cutoff of 50,000 (manufactured by DDS, GR51pp) in the same manner as in Example 1 to obtain 9 liters of permeate. The salt was further concentrated to 100 ml with a rotary evaporator, and spray-dried using a Palvis mini spray GA31 (manufactured by Yamato) at an inlet temperature of 150 ° C and an outlet temperature of 85 ° C. κ-casein glycomacropeptide powder 2.
0 g was obtained. As a result of analysis by urea-SDS electrophoresis, the purity was 81%.

For comparison, κ-casein glycomacropeptide was prepared from 10 liters of the same Gouda cheese whey without desalting operation. The yield was 1.7 g and the purity was 80%.

Example 4 50 liters of Camembert cheese whey (manufactured by Snow Brand Milk Products Co., Ltd.) having an electric conductivity of 6.1 mS / cm were heated to 40 ° C. with a plate heater, and an ultrafiltration membrane having a molecular weight cutoff of 50,000 (manufactured by DDS, GR51)
pp), temperature 40 ° C, pressure 0.4MPa, average flux 52.4 l / m
Ultrafiltration was performed for ² h, and the mixture was concentrated to 1 liter.

Thereafter, diafiltration was carried out while adding deionized water until the electric conductivity of the concentrate became 1 mS / cm. Hydrochloric acid was added to the concentrated solution to adjust the pH to 2.5 (electrical conductivity: 5.2 mS / cm), followed by diafiltration while adding deionized water to obtain 8 liters of permeate. The permeate was adjusted to pH 7.0 with 25% caustic soda, concentrated by ultrafiltration as described above, further desalted by diafiltration, and the concentrate was freeze-dried. κ-casein glycomacropeptide powder
49 g were obtained. As a result of analysis by urea-SDS electrophoresis, the purity was 86%.

For comparison, kappa-casein glycomacropeptide was prepared from 50 liters of the same Camembert cheese whey without the first diafiltration by the method described above. The yield was 37 g and the purity was 79%. Met.

──────────────────────────────────────────────────の Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C07K 14 / 47,1 / 34 A23J 1 / 20,3 / 08 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A milk material containing κ-casein glycomacropeptide is desalted, a substance having an electric conductivity of 4 mS / cm or less is adjusted to a pH of less than 4, and a molecular weight cut off of 10,000 to 50,000 is obtained.
A method for producing κ-casein glycomacropeptide, comprising performing an ultrafiltration treatment using the membrane of (1) and concentrating the obtained permeate using a membrane having a molecular weight cut-off of 50,000 or less. 2. The electric conductivity adjusted to pH less than 4
The method for producing a κ-casein glycomacropeptide according to claim 1, which is at most 30 mS / cm.