JPH061800A - Production of kappa-casein glucomacropeptide - Google Patents

Abstract

PURPOSE:To inexpensively obtain a compound useful as a raw material for medicines in an extremely improved recover ratio by desalting a milky raw material substance containing kappa-casein glucomacropeptide, adjusting electrical conductance and pH, ultrafiltering and concentrating the effluent. CONSTITUTION:A milky raw material substance (e.g. Gouda cheese whey) containing kappa-casein glucomacropeptide is subjected to a qualifier to remove insoluble substances, heated to 50 deg.C by a plate heater, desalted with by ultrafiltration with an ultrafilter having 50,000 fractioned molecular weight, concentrated and mixed with deionized water to adjust electrical conductance to <=4mS/cm. Then the resulting material is adjusted to pH<4 with hydrochloric acid, ultrafiltered with a membrane having 10,000-50,000 fractioned molecular weight, the prepared effluent is adjusted to pH7.0 with 25% caustic soda, concentrated with an ultrafilter having <=50,000, further desalted by diafiltration and the concentrate is freeze-dried to inexpensively give kappa-casein glucomacropeptide in high yield.

Description

Detailed Description of the Invention

[0001]

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

[0002]

2. Description of the Related Art κ-Casein glycomacropeptide is
It is a conventionally known 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 the laboratory, after pepsin is allowed to act on a solution of κ-casein isolated from milk in deionized water,
A method in which trichloroacetic acid is added to precipitate a para-κ-casein fraction, and the resulting supernatant is dialyzed against deionized water to desalt and then freeze-dried (see Theo et al. Of Food Science "
(Journal of Food Science) 53, 80 (1988)) or by dissolving the above-mentioned κ-casein in deionized water,
After the pH was adjusted to 6.7, rennet was allowed to act, and then the pH was adjusted to 4.6 to precipitate and remove para-κ-casein, and the resulting supernatant was dialyzed for desalting. The method of preparing by freeze-drying ("Milk Protein" pp200 issued by Academic Press Co.) was used.

However, these methods are not suitable for mass production because they are laboratories. On the other hand, since the industrial applicability of κ-casein glycomacropeptide has not hitherto been known, a method for mass production thereof has not been examined. However, recently, κ-casein glycomacropeptide has a function of reducing appetite in dogs (Stan et al., “Bullutin of Experimental Biologyand Medicin”).
e) 96 , 889 (1983)), it was found that it can be used as a food material for obesity prevention.

Further, thereafter, κ-casein glycomacropeptide was used to prevent the attachment of Escherichia coli to intestinal cells,
Effect of protecting against influenza virus infection
63-284133) and antiplaque effect (JP-A-63-233911),
Since it was found that it has an effect of preventing the adherence of pathogenic bacteria (Japanese Patent Application No. 2-15285) and a preventive or therapeutic effect on gastric ulcer (Japanese Patent Application No. 2-410695), it is strongly desired to produce it on an industrial scale. became.

So far, several methods relating to the industrial separation of κ-casein glycomacropeptide have been disclosed. They are roughly classified into a method using an ion exchange resin (Japanese Patent 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 (special JP-B-58-23400, JP-B-59-27358, JP-A-SHO
63-284199, JP-A 1-168693, JP-A 2-276542,
Japanese Patent Application No. 2-95686). Among these methods, the method of providing 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 to obtain a permeate with an ultrafiltration membrane having a molecular weight cutoff of 10 kDa to 50 kDa, then returned to neutral and concentrated with a membrane having a molecular weight cutoff of 50 kDa or less. The advantage of this method is that a highly pure product is obtained and the existing whey protein concentrate (W
Although it is possible to produce κ-casein glycomacropeptide with an ultrafiltration membrane for production of PC), there is a drawback that the yield is low on the other hand. Therefore, these methods were improved and the relationship with the ionic strength of the raw material solution was investigated to complete the present invention.

[0006]

[Patent Document 1] Japanese Unexamined Patent Publication No. 1-168693 discloses that when the starting material is ultrafiltered with a 10 kDa to 50 kDa ultrafiltration membrane, the pH is adjusted to 4 to 6 before the ionic strength is adjusted. It is described that 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 less than 4. It was found that when the pH is less than 4, conversely, when the ionic strength is increased, the membrane permeability deteriorates.

The κ-casein glycomacropeptide has a pH
It has a property of performing dependent dissociation-association, and its recovery is
The salt concentration of the solution is highly dependent. That is, the κ-casein glycomacropeptide usually associates at pH of 4 or higher and exists as an oligomer, but partially dissociates at pH of 4 or lower. However, at this time, the electric conductivity of the solution was 4 mS / cm.
If the above, the κ-casein glycomacropeptide is
It did not dissociate even at 4 or less, and still existed as an oligomer. Thus, in a solution showing an electrical conductivity of 4 mS / cm or more, the pH of κ-casein glycomacropeptide is
Dependent dissociation does not occur, and the method described in JP-A-2-276542 becomes a large barrier.

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

[0009]

According to the present invention, a dairy raw material containing κ-casein glycomacropeptide is desalted to obtain a substance having an electric conductivity of 4 mS / cm or less, and the pH of the substance is adjusted to less than 4. A κ-casein characterized by being prepared and subjected to ultrafiltration treatment using a membrane having a molecular weight cutoff of 10,000 to 50,000 to obtain a permeate, which is concentrated using a membrane having a molecular weight cutoff of 50,000 or less. It is a method for producing glycomacropeptide.

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 contacting cheese whey with an ion exchanger, cheese whey obtained by precipitating and removing whey protein by a method such as heating, desalted cheese whey powder, non-desalinated Examples 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 the starting raw material.
Further, it is more preferable to remove the fat content, insoluble matter and the like in the raw material by using a pretreatment such as a cream separator and a clarifier.

In addition, a milk material containing κ-casein glycomacropeptide is contacted with a cation exchanger,
The fraction that has not been adsorbed can be used as a raw material. Furthermore, a material obtained by contacting a milk raw material containing a κ-casein glycomacropeptide having a pH of 4 or less with an anion exchanger and eluting a fraction adsorbed on the anion exchanger may be used as a raw material.

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

When a κ-casein glycomacropeptide is prepared from the thus desalted raw material, the method described in JP-A-2-276542 is applied to obtain a particularly high purity. That is, by adjusting the pH of the raw material to less than 4 and using a UF membrane having a molecular weight cut-off of 10,000 to 50,000, ultrafiltration, diafiltration, or a combination thereof, κ-casein glycoside is contained in the permeate. It is a method of obtaining a macropeptide. At this time, the electric conductivity of the raw material is 30 mS
If it exceeds / cm 3, 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 electric conductivity is 10 mS.
It is more preferable that it is not more than / cm. Also the molecular weight cutoff
It is also possible to obtain κ-casein glycomacropeptide in the permeate by using a membrane larger than 50,000 (UF membrane, MF membrane), but in this case, since other whey proteins are mixed, the purity decreases and The recovery rate may decrease when the concentrated liquid is used as WPC.

In any of the methods, the materials for the UF membrane, MF membrane and RO membrane used in the steps of recovering the κ-casein glycomacropeptide in the permeate and concentrating and desalting it are not particularly limited. As the material of these films,
Polysulfone, polyether sulfone, tetrafluoroethylene, ceramics, etc. can be exemplified. To further improve the yield, hydrophilic membranes such as cellulose acetate, nitrocellulose, polyacrylonitrile, aromatic polyamide, or charged membranes can be used. Is preferably used.

Next, in order to concentrate the permeated liquid, JP-A-2-2765
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 with a membrane having a molecular weight cutoff of 50,000 or less. Any alkaline agent can be used to adjust the pH, such as sodium hydroxide,
Sodium bicarbonate, aqueous ammonia, etc. can be used. The pH of the κ-casein glycomacropeptide is a boundary (pH of 9,000) below the pH 4, and a polymer (molecular weight of 45,000) above the pH, so that the pH is preferably 5 or higher depending on the purpose. Any membrane may be used as long as it has a molecular weight cut-off of 50,000 or less, but a membrane having a molecular weight cut off of more than 50,000 allows κ-casein glycomacropeptide to permeate. The simplest method is to use a membrane with a molecular weight cutoff of about 20,000, which is used in the general whey protein concentrate production process.

When the pH of the permeate is not returned to 4 or higher, the second method is used because the κ-casein glycomacropeptide exists as a monomer. Ie molecular weight fraction 1
A concentrated solution is obtained by ultrafiltration, diafiltration, or reverse osmosis filtration with a membrane of 20,000 or less, preferably 8,000 or less. In any method, there is no problem in combining electrodialysis and a desalting step using an ion exchange resin.

The concentrate thus obtained is essentially κ-
Since only casein glycomacropeptide is contained, it is dried by means such as spray drying or freeze drying.
Incidentally, since κ-casein glycomacropeptide is stable against heat, it is more desirable to include 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 where the raw material is not desalted. Further, it is also possible to apply the above-mentioned known methods or a combination thereof to the raw material whose electric conductivity is adjusted to 4 mS / cm or less.

[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 rate of κ-casein glycomacropeptide can be significantly improved, and products produced at lower cost can be marketed. Can be supplied to.

[0019]

EXAMPLES The present invention will be specifically described below based on examples. Example 1 500 liters of Gouda cheese whey (manufactured by Snow Brand Dairy Co., Ltd.) having an electric conductivity of 5.6 mS / cm was put on a clarifier to remove insoluble matters, and the insoluble matter was heated to 50 ° C. with a plate heater to obtain a molecular weight cutoff of 50,000. Using an outer filtration membrane (GR51pp, manufactured by DDS), ultrafiltration was carried out at 50 ° C., pressure 0.4 MPa, average flux 52.4 l / m ² · h, and concentrated to 100 liters.

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 this concentrated liquid to adjust the pH to 2.5 (electrical conductivity 7.9 mS / cm), and subsequently, ultrafiltration treatment was performed under the above-mentioned conditions to obtain 80 liters of permeated liquid. The permeated liquid was adjusted to pH 7.0 with 25% caustic soda, subjected to ultrafiltration by the above-mentioned method to be concentrated, further desalted by diafiltration, and the concentrated liquid 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 liters without diafiltration by the method described above, the yield was 64 g and the purity was 80%.

Example 2 Ten liters of Cheddar cheese whey (manufactured by Snow Brand Milk Products Co., Ltd.) 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), respectively. 9.8 liters of desalted whey having an electric conductivity of 0.4 mS / cm was recovered.

This was added with hydrochloric acid to pH 2.5 (electrical conductivity 6.9 mS / cm)
In the same manner as in Example 1, an ultrafiltration membrane having a molecular weight cut off of 50,000 (GR51pp manufactured by DDS) was used to obtain 9 liters of permeate. Further, this was 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 10 cheddar cheese whey
A κ-casein glycomacropeptide was prepared from a liter without desalting, and the yield was 1.6 g and the purity was 82 g.
%Met.

Example 3 10 liters of Gouda cheese whey (manufactured by Snow Brand Milk Products Co., Ltd.) 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. It was desalted to pH 2.0 (electric conductivity 5.6 mS / cm) with hydrochloric acid. This desalted whey was subjected to an ultrafiltration treatment in the same manner as in Example 1 using an ultrafiltration membrane with a molecular weight cut off of 50,000 (D51, GR51pp) to obtain 9 liters of permeate, which was concentrated and dewatered. The salt was concentrated to 100 ml with a rotary evaporator, and spray-dried using Parbis 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.
I got 0g. As a result of analysis by the urea-SDS electrophoresis method, the purity was 81%.

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

Example 4 50 liters of Camembert cheese whey (manufactured by Snow Brand Dairy Co., Ltd.) having an electric conductivity of 6.1 mS / cm was heated to 40 ° C. with a plate heater, and an ultrafiltration membrane having a molecular weight cut off of 50,000 (DDS, GR51).
pp), temperature 40 ° C, pressure 0.4MPa, average flux 52.4 l / m
Performs ultrafiltration with ² h, concentrated to one liter.

Then, diafiltration was carried out while adding deionized water until the electric conductivity of the concentrated liquid reached 1 mS / cm. Hydrochloric acid was added to this concentrated solution to adjust the pH to 2.5 (electrical conductivity 5.2 mS / cm), and subsequently diafiltration was carried out while adding deionized water to obtain 8 liters of permeate. The permeated liquid was adjusted to pH 7.0 with 25% caustic soda, subjected to ultrafiltration by the above-mentioned method to be concentrated, further desalted by diafiltration, and the concentrated liquid was freeze-dried. κ-Casein glycomacropeptide powder
Obtained 49 g. As a result of analysis by the urea-SDS electrophoresis method, the purity was 86%.

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

Claims (2)

[Claims] 1. A dairy raw material containing κ-casein glycomacropeptide is desalted, and one having an electric conductivity of 4 mS / cm or less is adjusted to a pH of less than 4, and a molecular weight cutoff of 10,000 to 50,000 is obtained.
A method for producing a κ-casein glycomacropeptide, which comprises performing ultrafiltration treatment using the membrane described in 1 above and concentrating the obtained permeate using a membrane having a molecular weight cut off of 50,000 or less. 2. The electrical conductivity of a product adjusted to a pH of less than 4
The method for producing a κ-casein glycomacropeptide according to claim 1, which has a concentration of 30 mS / cm or less.