JPH07203863A - Production of beta-lactoglobulin, alpha-lactoalbumin and lactoferrin originated from milk whey - Google Patents

Abstract

PURPOSE:To efficiently obtain the subject compounds in high purity substantially without denaturating the compounds by treating milk whey with a hydrophobic chromatography resin, thus largely reducing the prices of the compounds which have been highly expensive food raw materials. CONSTITUTION:Milk whey is adjusted to a pH of 4.4-4.6, a protein concentration of 0.5-10%, and a Nail concentration of 1M. The adjusted milk whey is brought into contact with a hydrophobic chromatography resin and subsequently fractionated with a 0.75 Nacl aqueous solution and with a 40% (V/V) ethanol. Thus, 3-lactoglobulin (beta-Lg) is obtained from the non-adsorption fraction, and alpha- lactoalbumin(alpha-La) and lactoferrin (Lf) are obtained from the ethanol elusion fraction. Concretely, e.g. the non-adsorption fraction of the resin is desalted with a ultrafiltration module having a fractionation mol.wt. of 6K-14K to obtain the highly pure beta-Lg. Further, the ethanol elusion fraction is subjected to the removal of the ethanol, and subsequently treated with an ultrafiltration module having a fractionation mol.wt. of 50K and then with an ultrafiltration module having a fractionation mol.wt. of 60K to obtain the highly pure alpha-La and the crude Lf, respectively.

Description

Detailed Description of the Invention

[0001]

This invention relates to β-derived from milk whey.
Regarding a method for producing lactoglobulin (hereinafter referred to as β-Lg), α-lactalbumin (hereinafter referred to as α-La) and lactoferrin (hereinafter referred to as Lf), more specifically, β-Lg in milk whey, The present invention relates to a method for fractionating α-La and Lf by bringing them into contact with a hydrophobic chromatography resin.

[0002]

BACKGROUND OF THE INVENTION Since whey contains many proteins having high nutritional value and properties, many methods for isolating individual proteins have been reported. They can be roughly classified into a method utilizing the physicochemical properties of each protein and a method utilizing the molecular weight. As a typical method of them, the former method is to add iron chloride to the whey and adjust the pH to 3.0 to obtain β-L.
Method of precipitating and fractionating proteins other than g (Kuwata et al.
Food Sci. , Vol. 50, No. 3,605
~ 609, 1985), a method of specifically isolating β-Lg using immobilized β-Lg by utilizing the unique property that β-Lg easily forms a dimer (Tiancone Emilia). , Gattoni Margio, JP-A-64-63598), and β-Lg using a polymer heavy electrolyte such as CMC.
Coprecipitation (Jie Hidalgo et al., J. Dair
y Sci. , 54.1270, 1970) and the ion-exchange chromatographic method (Paul J. Skudder, Chemist), which utilizes the difference in charge of each protein.
ry and Industry J. , 7, 810,
1983), and the latter includes gel filtration and ultrafiltration (Lee and Merson, J. Food).
Sci. , 41, 402-410) have been proposed.

Although each of the above methods has its advantages, each of them is irreversible with the exception of the protein of interest due to the complexity of the process, energy cost, extremely low yield, use of a large amount of salts and the like. When it is attempted to industrially obtain each protein of whey in a high yield,
It has not been scaled to a viable way.

[0004]

When the final product is to be used in a product of the food industry while having the functional properties of each protein, each protein has few contaminants, that is, its physical properties are as natural. It is essential to obtain a product with a high protein content, in a non-denatured state with functional properties intact. Among the whey proteins, this invention must prevent β-irreversible denaturation due to its properties.
By utilizing Lg, α-La and Lf by utilizing the difference in hydrophobicity of these proteins, and introducing some conventional techniques,
It is intended to efficiently produce each of the above proteins by a series of operations with high purity and without denaturation.

[0005]

According to a first aspect of the present invention, milk whey has a pH of 4.4 to 4.6 and a protein concentration of 0.
Adjusted to 5-10%, NaCl concentration 1.0M, contact with hydrophobic chromatography-resin, 0.75M NaCl
It is characterized by fractionation with 1 and 40% (V / V) ethanol.

According to the second aspect of the invention, milk whey is added to the p
Adjust to H3.0, protein concentration 0.5-10%, NaCl concentration 0.5M, contact with hydrophobic chromatography resin and fractionate with 0.75M NaCl and 40% (V / V) ethanol. It is characterized by

In the invention of claim 3, the milk whey is one or more selected from acid whey, sweet whey, and processed whey such as concentrated whey. Regarding the method.

[0008]

From various milk whey of the present invention, β-Lg, α
-To efficiently fractionate La and Lf, in order to separate β-Lg and α-La having similar molecular weights and isoelectric points, NaCl is added to the starting material, whey, and β-Lg is added. Lg
By adsorbing proteins other than the above to the hydrophobic chromatography resin, β-L occupying about 50% of the whey protein
g and other proteins were first fractionated, then α-La and Lf adsorbed on the resin were eluted and fractionated by the ultrafiltration method, the operating conditions were very gentle and found in the prior art. Such irreversible denaturation of the final product does not occur, and each protein can be efficiently produced by a series of operation steps.

[0009]

EXAMPLES Some examples of the present invention will be listed below. Example 1: Whey (21) obtained from fresh skim milk by a conventional method (adjusting to pH 4.5 and removing casein by centrifugation)
NaCl concentration of 1.0M. 300 ml of a hydrophobic chromatography resin such as butyltoyopar equilibrated with a 1.0 M NaCl solution was mixed with whey (21) having a NaCl concentration of 1.0 M, and the mixture was gently stirred at room temperature for 20 to 30 minutes. This was filtered through a filter paper or a glass wool filter membrane, and 1. until absorption at 280 nm could not be detected.
Wash with 0M NaCl solution (ca. 1.51). Then 1
All material was eluted with 1 40% (V / V) ethanol. Butytoyopar non-adsorbed fraction was fractionated with a molecular weight of 6K to 14
It was desalted using K ultrafiltration module (manufactured by Asahi Kasei Co., Ltd.) to obtain 9.4 g of highly pure β-Lg (FIG. 1). Again 4
The ethanol fraction of the fraction eluted with 0% (V / V) ethanol was removed under reduced pressure, and the mixture was fractionated using an ultrafiltration module (Asahi Kasei Co., Ltd.) having a molecular weight cutoff of 50K, and the filtrate was obtained. And non-filtrate were respectively collected, desalted and concentrated with an ultrafiltration module (made by Asahi Kasei Co., Ltd.) having a molecular weight cut off of 6K, and then 2.5 g of highly pure α-La (Fig. 2) and
1.1 g of crude Lf (Lf content about 30%) (FIG. 3) was obtained.

Example 2: Clarified Godatype Cheesehoe (made by Kyodo Dairy Co.) 11 prepared by centrifugation and NaC
l was added to 1.0 M, and 1.0 M NaCl was added.
Mix with 150 ml of a hydrophobic chromatography resin (butyl toyopar, manufactured by Tosoh Corporation) equilibrated with the solution,
The mixture was gently stirred at room temperature for 20 to 30 minutes. The following operation was performed according to the above-mentioned Example 1, and high-purity β-Lg 4.7 g
(FIG. 1), α-La2.1g (FIG. 2) and coarse Lf (Lf
1.9 g (FIG. 3) was obtained with a content of about 30%.

Example 3: Acid whey protein (manufactured by Kyodo Dairy Co., Ltd.) 11 concentrated to a concentration of about 10 times by the ultrafiltration method was adjusted to pH 6N
The mixture was adjusted to 3.0 with HCl and mixed with 1.51 of a hydrophobic chromatography resin (Butyl Toyopar, Toso Co.) equilibrated with a NaCl solution. The mixture was stirred at room temperature for 20 to 30 minutes, stirred and filtered. 0.75 M adjusted to pH 3.0 with HCl until no absorption at 280 nm can be detected
After washing the resin with NaCl, the protein adsorbed on the resin was eluted with 0.5 M NaCl adjusted to the same pH. Next, all adsorbed substances were eluted with 40% (V / V) ethanol, and each was carried out according to the operation method described in the above Example 1, and highly pure β-Lg 45.2 g, α-La8.
5 g and crude Lf 5.5 g were obtained.

Example 4: pH of the whey (21) obtained in Example 1 above
Was adjusted to pH 3.0 with 1N HCl, and NaCl was added to 0.5M. 150 ml of hydrophobic chromatography resin (Butyl Toyopar, Toso Co.) equilibrated with 0.5 M NaCl whose pH was adjusted to 3.0
In addition, the mixture was gently stirred at room temperature for 20 to 30 minutes.
Subsequent operations are performed according to the method described in Example 3,
High-purity β-Lg 9.5 g, α-La 2.1 g and crude L
f1.1g was obtained.

Example 5 Godatype cheesehoe (manufactured by Kyodo Daigaku Co., Ltd.) 11 concentrated about 10 times by the ultrafiltration method was treated according to the method described in Example 3 above. 38.2 g and 5.5 g of high-purity β-Lg and α-La, respectively, and crude Lf
5.1 g was obtained.

[0014]

Industrial Applicability The method of the present invention comprises whey-β-L.
In order to efficiently produce g, .alpha.-La and Lf through a series of operating steps, the difference in hydrophobicity of each protein is utilized. For example, the above-mentioned Tiancone and Gattoni (Japanese Patent Laid-Open No. 64-63598). Β-L as reported by
It is a well-known fact that β-Lg can be specifically obtained by using the bioaffinity chromatogram method in which g is immobilized. However, such a method requires a high degree of pretreatment of the starting material (such as clarification and desalting), and the immobilization carrier itself is very expensive. In addition, the immobilized β-Lg itself is vulnerable to microbial contamination, lacks acid resistance, base resistance, pressure resistance, etc., and cannot be used for a long period of time. However, the functional group of the hydrophobic chromatography-resin used by the present inventors is a chemical butyl group, a phenyl group, etc., and is excellent in acid resistance, base resistance, pressure resistance, microbial resistance,
Since it can be used even at room temperature, it can be used on an industrial scale, and β has been regarded as a very expensive food material in the past.
It is possible to significantly reduce the prices of -Lg, α-La and Lf.

[Brief description of drawings]

FIG. 1 shows a molecular weight cut-off of 6K for butytoyopar non-adsorbed fraction.
The β-Lg obtained by desalting with an ultrafiltration module of 14K is shown.

[Fig. 2] Ethanol of a fraction eluted with 40% (V / V) ethanol was removed under reduced pressure, and fractionation was performed using an ultrafiltration module having a molecular weight cutoff of 50K, and the fraction was separated from the filtrate. The α-La obtained by desalting and concentrating the filtrate with an ultrafiltration module having a molecular weight cut off of 6K and then drying is shown.

FIG. 3 shows rough Lf obtained by the above method.

[Explanation of symbols]

 1 β-Lg (Example 1) 2 β-Lg (Example 2) 3 α-La (Example 1) 4 α-La (Example 2) 5 Crude Lf (Example 1) 6 Crude Lf (Example 2) M Molecular weight marker Car

Claims (3)

[Claims] 1. Milk whey is adjusted to pH 4.4 to 4.6, protein concentration 0.5 to 10%, NaCl concentration 1.0 M, and brought into contact with a hydrophobic chromatography resin to obtain 0.75.
A method for producing β-lactoglobulin, α-lactalbumin and lactoferrin derived from milk whey, which comprises fractionating with M NaCl and 40% (V / V) ethanol. 2. Milk whey is adjusted to pH 3.0, protein concentration 0.5 to 10%, NaCl concentration 0.5M, and brought into contact with a hydrophobic chromatography resin to obtain 0.75M N
Milk whey-derived β-lactoglobulin, α, characterized by fractionation with aCl and 40% (V / V) ethanol
-A method for producing lactalbumin and lactoferrin. 3. The milk-derived β-lacto according to claim 1 or 2, wherein the milk whey is one or more selected from acid whey, sweet whey and processed whey such as concentrated whey. A method for producing globulin, α-lactalbumin and lactoferrin.