WO1992003468A1 - Method for isolating alpha-lactalbumin from whey - Google Patents

Method for isolating alpha-lactalbumin from whey Download PDF

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Publication number
WO1992003468A1
WO1992003468A1 PCT/US1991/006047 US9106047W WO9203468A1 WO 1992003468 A1 WO1992003468 A1 WO 1992003468A1 US 9106047 W US9106047 W US 9106047W WO 9203468 A1 WO9203468 A1 WO 9203468A1
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whey
lactalbumin
solvent
product
supernatant
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PCT/US1991/006047
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French (fr)
Inventor
Willi E. Heine
Peter D. Klein
Chicko Miyashita
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Baylor College Of Medicine
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/20—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey
    • A23J1/205—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey from whey, e.g. lactalbumine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/04—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from milk
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to a method for separating proteins from their sources. It more particularly relates to a method of isolating ⁇ -lactalbumin and other proteins from whey.
  • ⁇ -Lactalbumin is a component of whey (about 19%) or milk (about 4%). It is an exceptionally valuable protein.
  • the special advantage of ⁇ -lactalbumin is its high concentration of tryptophan (about 6%), cystine (about 5.9%), lysine (about 11.4%) and other essential amino acids. This composition makes it superior to all other food proteins as a dietary constituent. Present prices of lactalbumin at $115/gram, however, have prevented its use in dietetics.
  • ⁇ -lactalbumin examples include low protein diets designed to minimize the excretory function of kidneys in pat' nts with chronic renal failure.
  • ⁇ -Lactalbumin also may st v*e as a protein source for reducing the urea synthesis rate in chronic liver diseases.
  • the amino acid composition of infant formulas can be modified to reproduce the pattern of amino acids in human milk protein. It also can be used as a source of protein in preterm infant formulas. Due to its low molecular weight, ⁇ -lactalbumin also may serve as a protein source for dietary treatment of individual allergies.
  • ⁇ -lactalbumin enrichment within whey protein fractions has been obtained by ultraf ⁇ ltration using small pore filters.
  • Commercial products produced in this way contain up to 21% lactalbumin besides high amounts of ⁇ -lactoglobulin and lesser amounts of serum albumin and immunoglobulins.
  • These ⁇ -lactalbumin enriched whey protein preparations may contain 2.8% tryptophan. Thus, they have a comparatively high biological value approaching 40-50% of the chemical score or purity of ⁇ -lactalbumin.
  • Two detrimental aspects of these ⁇ -lactalbumin enriched whey protein preparations are their relatively high amounts of lactose (milk sugar) and minerals, which narrow their domain of application.
  • the prior art also includes methods for chemical separation of ⁇ -lactalbumin from cow's milk using ammonium sulfate precipitation (Gordon and Ziegler 1953; Aillesburg and Drewy 1957). Alvesburg and Drewy improved their method further by separating ⁇ -lactoglobulin from the whey protein (Alvesburg 1963). The method requires approximately 17 different steps and, therefore, is too complicated for practical use. Also, it uses trichloracetic acid and high amounts of ammonium sulfate or anhydrous sodium sulfate, which may be unsuitable in processing human foods. A modified method Armstrong et al. (1967) has the same shortcomings, i.e., use of ammonium sulfate and trichloroacetic acid. These chemicals must be removed before use in human foods.
  • Bleumink (1966) used an acetone precipitation of milk whey in the preparation of ⁇ -lactalbumin.
  • the production disadvantage of his method is that it involves the vacuum distillation of large volumes of pure acetone added during preparation. There are no substitutes for acetone in the prior art methods. From a human food standpoint, use of acetone also is undesirable.
  • ⁇ -lactalbumin is extracted from other whey fraction by water diluted acidified acetone. Further, ethanol and methanol can be substituted for acetone in human food and non-human food applications, respectively.
  • Still another object of the present invention is a method of separating ⁇ -lactalbumin from the whey fraction of milk that avoids the use of large amounts of chemical substances that are potentially injurious to human health and the environment.
  • a method of isolating ⁇ -lactalbumin from acidified and heat-treated whey isolating ⁇ -lactalbumin from acidified and heat-treated whey.
  • the ⁇ -lactalbumin is extracted from the acidified, heat-treated whey using organic solvents and precipitated by adding a base.
  • the ⁇ -lactalbumin then may be further processed by separating the solvent from the precipitated' ⁇ -lactalbumin and drying the ⁇ -lactalbumin.
  • a feature of this method is the production of ⁇ -lactalbumin that has more than 85% protein purity and is 99% lactose and salt free.
  • the resulting product is soluble and almost tasteless. It has all the advantages of its unique amino acid composition.
  • the ⁇ -lactalbumin obtained from this method and its hydrolysis product are highly suitable for maintaining the nitrogen balance in protein metabolism on a low level of protein-nitrogen intake.
  • This invention provides an improved method of production of ⁇ -lactalbumin that has 85% to 97% protein purity and is 99% salt and lactose free.
  • the method provides for the simple, cost-effective and rapid separation of ⁇ -lactalbumin from the whey in milk.
  • the method involves differential heat denaturization of proteins of acidifying whey. Specifically, the method renders ⁇ -lactoglobulin, serum albumin, and immunoglobulins insoluble while _.naintaining the ⁇ -lactalbumin in a soluble form. All sources of whey can be used but bovine whey is the most practical.
  • the ⁇ -lactalbumin is extracted with organic solvents from acidified (preferably less than pH 4.6, most preferably pH 4.1 to 4.3) and heat-treated whey.
  • Organic solvents such as ketones, esters, and alcohols such as acetone, ethylacetate, ethanol, propanol, and methanol can be used.
  • a volatile, food compatible organic solvent is preferred.
  • the solvent is ethanol.
  • the volume of solvent used will depend on the volume of whey. The lower limit on the amount of solvent is dictated by the ability to move the paste- or pulp-like material formed through the processing. The upper limit on the mount of solvent is determined by solvent recovery and disposal problems.
  • the whey is acidified and heat treated before solvent extraction.
  • any inorganic acid may be used.
  • the acid and a base when later neutralized, the acid and a base form a water soluble salt.
  • hydrochloric acid is used because, when neutralized with sodium hydroxide, it forms the most innocuous product.
  • the amount of acid treating time has no influence on yield and purity of the product. The smallest amount of acid or base necessary to reach the desired pH is the most desirable.
  • the desired result of heat treatment is a maximum differentiation of the proteins through maximum denaturization of the ⁇ -lactoglobulin, serum albumin, and immunoglobulins with minimal loss of the ⁇ -lactalbumin.
  • Heat treatment is preferred at about 50 to lOOoC, most preferably about lOOoC. Ultra high temperatures for very short periods of time could be used. The amount of time for heat treatment, however, will be highly depend on the configuration and volume of the vessel, the volume of whey, and the rate and source of heat transfer.
  • the solvent-insoluble whey components are separated from the solvent extract by centrifugation, filtration or other standard separation methods. For the examples described below, separation was usually done using cheese cloth. Centrifugation forces on the order of 2500 x G are used.
  • bases including sodium hydroxide, potassium hydroxide, calcium hydroxide, and mixtures of bases can be used.
  • the acid and a base preferably form a water soluble salt. Most preferably sodium hydroxide is used.
  • the precipitated ⁇ -lactalbumin then is separated from the organic solvent by centrifugation, filtration, lyophilization or other standard separation techniques. Centrifugation forces on the order of 2500 x G can be used.
  • the ⁇ -lactalbumin is then dried and ground using standard methods in the art.
  • the particle size of the product is not critical to the instant invention. Grinding of the product, however, is recommended in order to increase the digestibility of residual ⁇ -lactoglobulin and serum albumin.
  • the solvent-insoluble whey components include ⁇ -lactoglobulin, serum albumin, and immunoglobulins.
  • the protein components of whey lose their ability to dissolve in acidic, organic solvents because of heat denaturation during heat treatment of the acidified whey.
  • the solvent-insoluble whey components primarily the ⁇ -lactoglobulin and serum albumin, have commercial value. They can be recovered as end by-products from the organic solvent. After the organic solvent is recovered, the solvent-insoluble whey components can be further processed by repeated rinsing with water to produce 99% mineral-free and lactose-free products. They can then be dried and ground using standard methods in the art to increase digestibility.
  • Contamination of the extracted ⁇ -lactalbumin by undenatured ⁇ -lactoglobulin and serum albumin can be minimized by fractionation achieved by differential precipitation of these proteins near their isoelectric points (about 4.1 to 4.3 for ⁇ -lactalbumin, about pH 4.7 to 4.9 for bovine serum albumin and about 5.1 or 5.3 for ⁇ -lactoglobulin, respectively).
  • alkali treatment at about pH 8.0-9.0 (preferably 8.3) and refrigerated storage for at least three weeks, ⁇ -lactoglobulin contamination disappears completely. This is due to an alkali-induced decay of ⁇ -lactoglobulin.
  • the bases and mixtures of bases used during ⁇ -lactalbumin precipitation are used for alkali treatment.
  • the order of the steps in the process can be varied to some extent. As shown in the following examples, the variations include (a) simultaneous acid treatment i d solvent extraction and (b) the addition of a base prior to heat treatment.
  • EXAMPLE 1 Reconstituted whey (about 100 g Alacen * , a whey protein concentrate obtained from the New Zealand Milk Products, Inc., suspended in about 285 ml water to correspond to about a 26% whey solution) was acidified with about 12.5 ml of 10 N HC1. The acidified whey was heated to and maintained at about 100 ⁇ C for not more than about two minutes.
  • the heat-treated, acidified whey was cooled by quenching to between 40oC and ambient (room) temperature and then extracted with 1250 ml ethanol. Cooling, however, is not required for extraction. Cooling does not change the purity of the ⁇ -lactalbumin but minimizes amount of ⁇ -lactalbumin lost through heat denaturing the ⁇ -lactalbumin.
  • the insoluble whey components were removed by centrifugation or filtration. The supernatant was adjusted to about pH 4.5 by adding IN NaOH to precipitate the ⁇ -lactalbumin. The precipitated ⁇ -lactalbumin was separated by centrifugation or filtration.
  • the method yielded 4.0 g ⁇ -lactalbumin.
  • the ethanol-insoluble whey components were rinsed with water to remove mineral and lactose. The insoluble remainder was then filtered, air-dried and ground using standard methods in the art.
  • EXAMPLE 3 100 g of Alacen" was suspended in a mixture of 1250 ml 90% ethanol and 12.5 ml ION HC1. The mixture was stirred more than 10 minutes. The supernatant was separated from the ethanol-insoluble remainder and then adjusted to about pH 4.5 to 5.5, most preferably 5.0, with IN NaOH to precipitate the whey proteins. The whey proteins were resuspended in about 1250 ml water and adjusted to about pH 6.0 by adding IN NaOH. They were then heated to and maintained at about lOOoC for not more ian about two minutes to coagulate ⁇ -lactoglobulin and serum albumin.
  • the supernatant was adjusted to about pH 7.0 to 8.0 with IN NaOH for storage at temperatures between about 4 and 25.0 « C for not more than 3 weeks. Thereafter the supernatant was adjusted to about pH 4.5 to precipitate the ⁇ -lactalbumin. The method yielded 3.1 g ⁇ -lactalbumin.
  • EXAMPLE 4 About 100 g of Alacen" was suspended in about 125 ml 1 N hydrochloric acid, heated to about lOOoC and kept at this temperature for about 2 minutes. The acidified and heat-treated whey was then extracted with about 1250 ml 80% acetone. After removing the insoluble remainder by centrifugation, the supernatant was adjusted to about pH 4 by adding about 10 ml lN-NaOH. T precipitated pure ⁇ -lactalbumin was separated by centrifugation. Yield was about 4 g ⁇ -lactalbumin. A second fraction was obtained at about pH 5 by further addition of 10 ml 1 N NaOH. Yield was about 2 g about ⁇ -lactalbumin. This fraction, however, was contaminated with around 30% ⁇ -lactoglobulin.
  • the ⁇ -lactalbumin has a 85 to 97% protein purity, and is 99% salt and lactose free. This can be compared to the ultrafiltration method which results in a maximum of 50% protein purity and relatively high in lactose and mineral salts. After alkali treatment and refrigeration for at least three week, all ⁇ -lactoglobulin contamination disappears from the ⁇ -lactalbumin produced from the instant methods.
  • the ⁇ -lactalbumin separated by ethanol extraction from acidified and heat-treated whey was analyzed by SDS gel electrophoresis and stained with Comassie blue. Contamination by other whey protein was minimal.
  • the ⁇ -lactalbumin band was located in the region of low molecular proteins corresponding to a molecular weight of 14.7 kiloDaltons. Its electrophoretical mobility was identical with that of a commercial ⁇ -lactalbumin standard ( ⁇ -lactalbumin, Sigma, St. Louis, MO, Product No. L6010). Its absorption curve in 0.05 mm Tris was identical with the standard.
  • the immunological purity of the ⁇ -lactalbumin fraction was checked by an antiserum to bovine whey proteins (Sigma, St. Louis, MO, Product No. W3501) and a bovine serum albumin antiserum (Sigma, St. Louis, MO, Product No. B3515) using the Ouchterlony assay. Both bovine whey protein antiserum and bovine serum albumin antiserum precipitated the ⁇ -lactalbumin standard.
  • the ⁇ -lactalbumin produced by ethanol extraction from acidified whey reacted with the antibody to bovine serum albumin forming a precipitation band identical with that of the ⁇ -lactalbumin standard.
  • the antibody to bovine whey protein exhibited 2 additional precipitation bands that were identical with those displayed by the ⁇ -lactalbumin standard when it had been previously subjected to acidification, ethanol extract, heated to 70°C and precipitated at pH 4.5.

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Abstract

A method for separating α-lactalbumin from the other protein fractions of whey. The method includes extracting α-lactalbumin from acidified, heat-treated whey using an organic solvent. The α-lactalbumin is then precipitated from the solvent using a base. The method can be varied to produce purified β-lactoglobulin and serum albumin.

Description

METHOD FOR ISOLATING ALPHA-LACTALBUMIN FROM WHEY
FIELD OF THE INVENTION The present invention relates to a method for separating proteins from their sources. It more particularly relates to a method of isolating α-lactalbumin and other proteins from whey.
BACKGROUND OF THE INVENTION α-Lactalbumin is a component of whey (about 19%) or milk (about 4%). It is an exceptionally valuable protein. The special advantage of α-lactalbumin is its high concentration of tryptophan (about 6%), cystine (about 5.9%), lysine (about 11.4%) and other essential amino acids. This composition makes it superior to all other food proteins as a dietary constituent. Present prices of lactalbumin at $115/gram, however, have prevented its use in dietetics.
Examples of potential dietary uses of α-lactalbumin are many. They include low protein diets designed to minimize the excretory function of kidneys in pat' nts with chronic renal failure. α-Lactalbumin also may st v*e as a protein source for reducing the urea synthesis rate in chronic liver diseases. Using α-lactalbumin, the amino acid composition of infant formulas can be modified to reproduce the pattern of amino acids in human milk protein. It also can be used as a source of protein in preterm infant formulas. Due to its low molecular weight, α-lactalbumin also may serve as a protein source for dietary treatment of individual allergies. In addition, enzymatic breakdown of α-lactalbumin can be used for the production of amino acid oligopeptide mixtures with high proportions of essential amino acids. α-Lactalbumin enrichment within whey protein fractions has been obtained by ultrafϊltration using small pore filters. Commercial products produced in this way contain up to 21% lactalbumin besides high amounts of β-lactoglobulin and lesser amounts of serum albumin and immunoglobulins. These α-lactalbumin enriched whey protein preparations may contain 2.8% tryptophan. Thus, they have a comparatively high biological value approaching 40-50% of the chemical score or purity of α-lactalbumin. Two detrimental aspects of these α-lactalbumin enriched whey protein preparations are their relatively high amounts of lactose (milk sugar) and minerals, which narrow their domain of application.
Separation of α-lactalbumin from whey protein by preparative chromatography (polyacrylamide gel electrophoresis) h.as been done and yields a highly purified product (Boodbeck, U. et al., 1967; Kronman, M.J. et. al., 1981). This method, however, is very expensive and not feasible for large scale production.
The prior art also includes methods for chemical separation of α-lactalbumin from cow's milk using ammonium sulfate precipitation (Gordon and Ziegler 1953; Aschaffenburg and Drewy 1957). Aschaffenburg and Drewy improved their method further by separating β-lactoglobulin from the whey protein (Aschaffenburg 1963). The method requires approximately 17 different steps and, therefore, is too complicated for practical use. Also, it uses trichloracetic acid and high amounts of ammonium sulfate or anhydrous sodium sulfate, which may be unsuitable in processing human foods. A modified method Armstrong et al. (1967) has the same shortcomings, i.e., use of ammonium sulfate and trichloroacetic acid. These chemicals must be removed before use in human foods.
Bleumink (1966) used an acetone precipitation of milk whey in the preparation of α-lactalbumin. The production disadvantage of his method is that it involves the vacuum distillation of large volumes of pure acetone added during preparation. There are no substitutes for acetone in the prior art methods. From a human food standpoint, use of acetone also is undesirable. In the instant invention, where acetone is used for non-human food uses, α-lactalbumin is extracted from other whey fraction by water diluted acidified acetone. Further, ethanol and methanol can be substituted for acetone in human food and non-human food applications, respectively.
CITED AND OTHER PRIOR ART Gordon, WG, Semmet WF. Isolation of crystalline α-lactalbumin from milk. J. Amer Chem Soc 1953;75:328.
Gordon, WG, Ziegler J. α-Lactalbumin. Biochem Prep 1955;4:16. Larson BL, Rolleri GD. Heat denaturation of the oecific serum proteins in milk. J. Dairy Sci 1955;38:351. Korner A, Debro JR. Solubility of albumin in alcohol after precipitation by trichloracetic acid: A simplified procedure for separation of albumin. Nature, London 1956;4541:1067. Aschaffenburg R, Drewy J. Improved method for the preparation of crystalline B-lactoglobulin and α-lactalbumin from cow's milk. Biochem J 1957;65:273. .Aschaffenburg R, Preparation of α-lactalbumin from cow's or goat's milk: A method improving the yield. J Dairy Sci 1963;51:1295. Larson BL, Hageman EC. Determination of α-lactalbumin in complex systems. J Dairy Sci 1965;46:14. Sen A, Chaudhuri S. Non casein proteins of goat's milk. Nature,
London 1962;195:286. Robbins FM, Rronman, MJ. A simplified method for preparing α-lactalbumin and β-lactoglobulin from cow's milk. Biochim Biophys Acta 1964;82:186.
Groves ML. Preparation of some iron-binding proteins and α-lactalbumin from bovine milk. Biochim Biophys Acta 1965;100:154. Bleumink E. Preparation of α-lactalbumin from milk whey by precipitation with acetone. Netherlands Milk Dairy J
1966;20:13. Fox KK, Holsinger VH, Postai LP, Pallansch MJ. Separation of β-lactoglobulin from other milk serum proteins by trichloracetic acid. J Dairy Sci 1967;50:1363. Armstrong J McD, McKenzie HA, Sawyer WH. On the fractionation of β-lactoglobulin and α-lactalbumin. Biochim Biophys Acta
1967;147:60. Ebner KE, Brodbeck W. Biological role of α-Iactalbumin: A review. J.
Dairy Sci 1968;51:317. Hiraoka V, Sagawa T, Kuwajima K, Sugai S, Murai N. α-Lactalbumin:
A calcium metalloprotein. Biochim Biophys Acta 1980;95:1098. Kronman M J, Sinha SK, Brew K. Characteristics of the binding of Ca! 2+ and other divalent metal ions to bovine α-lactalbumin. J Biol
Chem 1981;256:8582. Renner E. Milk and Dairy Products in Human Nutrition. 1983.
W-GmbH, Volkswirtsch.aftlicher Verlag, Munchen pp. 91-101,
119, and 283-285. (Volkswirtschaftlicher Verlag Munchen,
Verlag TH. Mann, KG. Gelsenkirchen-Buer). Eigel WN, Butler JE, Ernstrom CA, Farrel HM Jr, Harwalkar VR, Jennes R, Whitney R McL. Nomenclature of proteins of cow's milk: Fifth revision. J Dairy Sci 1984;67:1599. Loennerdal B, Glazier C. Calcium binding by a α-lactalbumin in human milk and bovine milk. J Nutr 1985;115:1209.
Berliner LJ, Koga K, Nishikawa H, Scheffler JE. High resolution proton and laser photochemically induced dynamic nuclear polarization NMR studies of cation binding to bovine α-lactalbumin. Biochemistry 1987;26:5769. Forsum E, Hambraeus L. Siddiqi I H. Large scale fractionation of whey protein concentrates. J Dairy Sci 1974; 54:659. Forsum E. Nutritional evaluation of whey protein concentrates and their fractions. J Dairy Sci 1973; 57:665. Sawar G, Botting H G, Peace R W. Amino acid rating method for evaluating protein adequacy of infant formulas. J Assoc Off Anal
Chem 1989; 72:622. Janas L M, Picciano M F, Hatch T F. Indices of protein metabolism in term infants fed either human milk or formulas with reduced protein concentration and various whey/casein ratios. J Pediatr 1987; 110:838.
SUMMARY OF THE INVENTION An object of the present invention is an improved method of isolating and purifying α-lactalbumin from milk or more specifically the whey fraction of milk. Another object of the present invention is developing the inexpensive, rapid method of separating α-lactalbumin from the whey fraction of milk that is feasible for large scale production.
Still another object of the present invention is a method of separating α-lactalbumin from the whey fraction of milk that avoids the use of large amounts of chemical substances that are potentially injurious to human health and the environment.
Thus in accomplishing these objects, there is provided according to one aspect of the present invention a method of isolating α-lactalbumin from acidified and heat-treated whey. The α-lactalbumin is extracted from the acidified, heat-treated whey using organic solvents and precipitated by adding a base. The α-lactalbumin then may be further processed by separating the solvent from the precipitated' α-lactalbumin and drying the α-lactalbumin.
A feature of this method is the production of α-lactalbumin that has more than 85% protein purity and is 99% lactose and salt free. The resulting product is soluble and almost tasteless. It has all the advantages of its unique amino acid composition. The α-lactalbumin obtained from this method and its hydrolysis product are highly suitable for maintaining the nitrogen balance in protein metabolism on a low level of protein-nitrogen intake.
Another feature of this method is that the solvent-insoluble whey components, primarily β-lactoglobulin and serum albumin, can be recovered as end by-products. Alternatively, modifications of the procedure can be used to produce purified β-lactoglobulin. Other and still further objects, features and advantages of the present invention will be apparent from the following description of a presently preferred embodiment of the invention.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENT This invention provides an improved method of production of α-lactalbumin that has 85% to 97% protein purity and is 99% salt and lactose free. The method provides for the simple, cost-effective and rapid separation of α-lactalbumin from the whey in milk. The method involves differential heat denaturization of proteins of acidifying whey. Specifically, the method renders β-lactoglobulin, serum albumin, and immunoglobulins insoluble while _.naintaining the α-lactalbumin in a soluble form. All sources of whey can be used but bovine whey is the most practical.
According to the invention, the α-lactalbumin is extracted with organic solvents from acidified (preferably less than pH 4.6, most preferably pH 4.1 to 4.3) and heat-treated whey. Organic solvents such as ketones, esters, and alcohols such as acetone, ethylacetate, ethanol, propanol, and methanol can be used. A volatile, food compatible organic solvent, however, is preferred. Most preferably, the solvent is ethanol. The volume of solvent used will depend on the volume of whey. The lower limit on the amount of solvent is dictated by the ability to move the paste- or pulp-like material formed through the processing. The upper limit on the mount of solvent is determined by solvent recovery and disposal problems. Preferably the whey is acidified and heat treated before solvent extraction. Any inorganic acid may be used. Preferably, when later neutralized, the acid and a base form a water soluble salt. Most preferably hydrochloric acid is used because, when neutralized with sodium hydroxide, it forms the most innocuous product. The amount of acid treating time has no influence on yield and purity of the product. The smallest amount of acid or base necessary to reach the desired pH is the most desirable.
The desired result of heat treatment is a maximum differentiation of the proteins through maximum denaturization of the β-lactoglobulin, serum albumin, and immunoglobulins with minimal loss of the α-lactalbumin. Heat treatment is preferred at about 50 to lOOoC, most preferably about lOOoC. Ultra high temperatures for very short periods of time could be used. The amount of time for heat treatment, however, will be highly depend on the configuration and volume of the vessel, the volume of whey, and the rate and source of heat transfer.
The solvent-insoluble whey components are separated from the solvent extract by centrifugation, filtration or other standard separation methods. For the examples described below, separation was usually done using cheese cloth. Centrifugation forces on the order of 2500 x G are used.
The α-lactalbumin precipitates immediately from the solvent extract by adding bases and adjusting to the pH to about 3.8 to 5.5, most preferably 4.2 to 4.8. A number of bases, including sodium hydroxide, potassium hydroxide, calcium hydroxide, and mixtures of bases can be used. As discussed above regarding acids for acid treatment, the acid and a base preferably form a water soluble salt. Most preferably sodium hydroxide is used.
The precipitated α-lactalbumin then is separated from the organic solvent by centrifugation, filtration, lyophilization or other standard separation techniques. Centrifugation forces on the order of 2500 x G can be used. The α-lactalbumin is then dried and ground using standard methods in the art. The particle size of the product is not critical to the instant invention. Grinding of the product, however, is recommended in order to increase the digestibility of residual β-lactoglobulin and serum albumin.
The solvent-insoluble whey components include β-lactoglobulin, serum albumin, and immunoglobulins. The protein components of whey lose their ability to dissolve in acidic, organic solvents because of heat denaturation during heat treatment of the acidified whey. The solvent-insoluble whey components, primarily the β-lactoglobulin and serum albumin, have commercial value. They can be recovered as end by-products from the organic solvent. After the organic solvent is recovered, the solvent-insoluble whey components can be further processed by repeated rinsing with water to produce 99% mineral-free and lactose-free products. They can then be dried and ground using standard methods in the art to increase digestibility.
Contamination of the extracted α-lactalbumin by undenatured β-lactoglobulin and serum albumin can be minimized by fractionation achieved by differential precipitation of these proteins near their isoelectric points (about 4.1 to 4.3 for α-lactalbumin, about pH 4.7 to 4.9 for bovine serum albumin and about 5.1 or 5.3 for β-lactoglobulin, respectively). Further, by subjecting the α-lactalbumin to alkali treatment at about pH 8.0-9.0 (preferably 8.3) and refrigerated storage for at least three weeks, β-lactoglobulin contamination disappears completely. This is due to an alkali-induced decay of β-lactoglobulin. The bases and mixtures of bases used during α-lactalbumin precipitation are used for alkali treatment. The order of the steps in the process can be varied to some extent. As shown in the following examples, the variations include (a) simultaneous acid treatment i d solvent extraction and (b) the addition of a base prior to heat treatment. EXAMPLE 1 Reconstituted whey (about 100 g Alacen*, a whey protein concentrate obtained from the New Zealand Milk Products, Inc., suspended in about 285 ml water to correspond to about a 26% whey solution) was acidified with about 12.5 ml of 10 N HC1. The acidified whey was heated to and maintained at about 100©C for not more than about two minutes. The heat-treated, acidified whey was cooled by quenching to between 40oC and ambient (room) temperature and then extracted with 1250 ml ethanol. Cooling, however, is not required for extraction. Cooling does not change the purity of the α-lactalbumin but minimizes amount of α-lactalbumin lost through heat denaturing the α-lactalbumin. The insoluble whey components were removed by centrifugation or filtration. The supernatant was adjusted to about pH 4.5 by adding IN NaOH to precipitate the α-lactalbumin. The precipitated α-lactalbumin was separated by centrifugation or filtration.
The method yielded 4.0 g α-lactalbumin.
The ethanol-insoluble whey components were rinsed with water to remove mineral and lactose. The insoluble remainder was then filtered, air-dried and ground using standard methods in the art.
EXAMPLE 2
100 g of Alacen" was suspended in 112.5 ml 4% hydrochloric acid. The acidified whey concentrate was heated to and maintained at about 100°C for not more than about three minutes. The heat-treated, acidified whey was cooled by quenching to between 40oC and ambient temperature and then extracted with 500 ml ethanol. As described in the previous example, cooling is not required for extraction. The insoluble components of the whey were removed by centrifugation or filtration. The supernatant was adjusted to about pH 5.0 by adding IN NaOH to precipitate the α-lactalbumin. The precipitated α-lactalbumin was separated by centrifugation or filtration. The method yielded 5.7 g α-lactalbumin.
EXAMPLE 3 100 g of Alacen" was suspended in a mixture of 1250 ml 90% ethanol and 12.5 ml ION HC1. The mixture was stirred more than 10 minutes. The supernatant was separated from the ethanol-insoluble remainder and then adjusted to about pH 4.5 to 5.5, most preferably 5.0, with IN NaOH to precipitate the whey proteins. The whey proteins were resuspended in about 1250 ml water and adjusted to about pH 6.0 by adding IN NaOH. They were then heated to and maintained at about lOOoC for not more ian about two minutes to coagulate β-lactoglobulin and serum albumin. After the β-lactoglobulin and serum albumin were separated from the supernatant, the supernatant was adjusted to about pH 7.0 to 8.0 with IN NaOH for storage at temperatures between about 4 and 25.0« C for not more than 3 weeks. Thereafter the supernatant was adjusted to about pH 4.5 to precipitate the α-lactalbumin. The method yielded 3.1 g α-lactalbumin.
EXAMPLE 4 About 100 g of Alacen" was suspended in about 125 ml 1 N hydrochloric acid, heated to about lOOoC and kept at this temperature for about 2 minutes. The acidified and heat-treated whey was then extracted with about 1250 ml 80% acetone. After removing the insoluble remainder by centrifugation, the supernatant was adjusted to about pH 4 by adding about 10 ml lN-NaOH. T precipitated pure α-lactalbumin was separated by centrifugation. Yield was about 4 g α-lactalbumin. A second fraction was obtained at about pH 5 by further addition of 10 ml 1 N NaOH. Yield was about 2 g about α-lactalbumin. This fraction, however, was contaminated with around 30% β-lactoglobulin.
PROOF OF PURITY The α-lactalbumin has a 85 to 97% protein purity, and is 99% salt and lactose free. This can be compared to the ultrafiltration method which results in a maximum of 50% protein purity and relatively high in lactose and mineral salts. After alkali treatment and refrigeration for at least three week, all β-lactoglobulin contamination disappears from the α-lactalbumin produced from the instant methods.
The α-lactalbumin separated by ethanol extraction from acidified and heat-treated whey was analyzed by SDS gel electrophoresis and stained with Comassie blue. Contamination by other whey protein was minimal. The α-lactalbumin band was located in the region of low molecular proteins corresponding to a molecular weight of 14.7 kiloDaltons. Its electrophoretical mobility was identical with that of a commercial α-lactalbumin standard (α-lactalbumin, Sigma, St. Louis, MO, Product No. L6010). Its absorption curve in 0.05 mm Tris was identical with the standard. The immunological purity of the α-lactalbumin fraction was checked by an antiserum to bovine whey proteins (Sigma, St. Louis, MO, Product No. W3501) and a bovine serum albumin antiserum (Sigma, St. Louis, MO, Product No. B3515) using the Ouchterlony assay. Both bovine whey protein antiserum and bovine serum albumin antiserum precipitated the α-lactalbumin standard.
The α-lactalbumin produced by ethanol extraction from acidified whey reacted with the antibody to bovine serum albumin forming a precipitation band identical with that of the α-lactalbumin standard. The antibody to bovine whey protein exhibited 2 additional precipitation bands that were identical with those displayed by the α-lactalbumin standard when it had been previously subjected to acidification, ethanol extract, heated to 70°C and precipitated at pH 4.5. These changes in immunologic reactivity apparently were due to reversible molecular changes in α-lactalbumin associations or partial denaturation.
One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as others inherent therein. While the presently preferred embodiments of the invention
Figure imgf000015_0001
been described for the purpose of disclosure, numerous change in the details of the formulations and their methods of synthesis may be made without departing from the spirit of the present invention and the scope of the appended claims. For example, one skilled in the art will readily appreciate that the concentration of a reagent, e.g., an acid, can influence the speed of a reaction and volumes of reagent required. It should be understood, however, that there is no intention to limit the invention to the specific methods disclosed, but on the contrary, the intention is to cover all modifications, alternative methods and equivalents falling within the spirit of the present invention and the scope of the appended claims.
What is claimed is:

Claims

1. A method of isolating α-lactalbumin from the whey in milk, comprising the steps of: acidifying said whey; heat treating said acidified whey; adding an organic solvent to said heat-treated whey wherein said solvent extracts α-lactalbumin and leaves solvent- insoluble whey components; separating said solvent-insoluble whey components from said organic solvent; and thereafter precipitating said α-lactalbumin by adding a base to said solvent extract.
2. The method of claim 1 further comprising the steps of: separating said α-lactalbumin precipitate from said organic solvents; and thereafter drying said α-lactalbumin precipitate.
3. The method of claim 1 further comprising the steps of: subjecting said α-lactalbumin to alkali treatment; and refrigerating said treated α-lactalbumin for at least three weeks.
4. The method of claim 1 further comprising the step of cooling said heat-treated whey before said separation step.
5. The method of claim 1 wherein said whey is acidified with hydrochloric acid.
6. The method of claim 1 wherein said organic solvent is a volatile, food compatible solvent.
7. The method of claim 6 wherein said organic solvent is ethanol.
8. The method of claim 1 wherein said base is sodium hydroxide.
9. The method of claim 1 wherein said heat treating step comprises heating said whey at about 50 to 100« C for not more than about 3 minutes.
10. The method of claim 9 wherein said heat treating step compr es heating said whey at about 100°C for not more than about 3 mimr 3.
11. The method of claim 1 further comprising the steps of: removing lactose and minerals from said solvent-insoluble whey components with water and; thereafter drying and grinding said solvent-insoluble whey components.
12. The product of claim 1.
13. The product of claim 12, wherein the product has at least a 85% protein purity.
14. The product of claim 12, wherein the product is 99% salt and lactose free.
15. A method of isolating α-lactalbumin from whey, comprising the steps of: acidifying said whey; heat treating said acidified whey; adding an organic solvent to said heat-treated whey wherein said solvent extracts α-lactalbumin and leaves solvent- insoluble whey components; separating said solvent-insoluble whey components from said organic solvent; and thereafter precipitating said α-lactalbumin by adding a base to said solvent extract; separating said α-lactalbumin precipitate from said organic solvent; and thereafter drying said α-lactalbumin precipitate.
16. The method of claim 15 wherein said whey is acidified with hydrochloric acid.
17. The method of claim 15 further comprising the steps of: subjecting said α-lactalbumin to alkali treatment; and refrigerating said treated α-lactalbumin for at least three weeks.
18. The method of claim 15 further comprising the step of cooling said heat-treated whey before said separation step.
19. The method of claim 15 wherein said organic solvent is a volatile, food compatible solvent.
20. The method of claim 19 wherein said organic solvent is ethanol.
21. The method of claim 15 wherein said base is sodium hydroxide.
22. The method of claim 15 wherein said heat treating step comprises heating said whey at about 50 to lOOoC.
23. The method of claim 22 wherein said heat treating step comprises heating said whey at about lOOoC for not more than about 3 minutes.
24. The method of claim 15 further comprising the steps of: removing lactose and minerals from said solvent-insoluble whey components by repeatedly rinsing said solvent-insoluble whey components with water; and thereafter drying and grinding said solvent-insoluble whey components.
25. The product of claim 15.
26. The product of claim 25, wherein the product has at least a 85% protein purity.
27. The product of claim 25, wherein the product is 99% salt and lactose free.
28. A method of isolating α-lactalbumin from whey, comprising the steps of: forming a mixture of said whey, an acid and an organic solvent; stirring said mixture and then allowing said mixture to form a solvent insoluble remainder and a first supernatant; separating said first supernatant from said solvent insoluble remainder; adding base to said first supernatant and forming a whey protein precipitate; separating said whey protein precipitate from said solvent; resuspending said whey protein precipitate in water and adjusting pH to about 6.0 with base; heating said resuspended whey protein precipitate and forming a second supernatant and a coagulate, said coagulate including β-lactoglobulin and serum albumin; thereafter separating said second supernatant from said coagulate; adjusting the pH of said second supernatant to about 4.5 to 5.5 and forming a α-lactalbumin precipitate and a third supernatant; and thereafter separating said α-lactalbumin precipitate from said third supernatant.
29. The method of claim 28 further comprising the steps of: subjecting said α-lactalbumin to alkali treatment; and refrigerating said treated α-lactalbumin for at least three weeks.
30. The method of claim 28 wherein said heat treating step comprises heating said whey protein precipitate to and maintaining said whey protein precipitate at about lOOoC for not more than about 2 minutes.
31. The method of claim 28 wherein said organic solvent is a volatile, food compatible solvent.
32. The method of claim 31 wherein said organic solvent is ethanol.
33. The method of claim 28 wherein said acid is hydrochloric acid.
34. The method of claim 28 wherein said base is sodium hydroxide.
35. The method of claim 28 further comprising the step of: purifying said β-lactoglobulin and said serum albumin.
36. The product of claim 28.
37. The product of claim 36, wherein the product has at least a 85% protein purity.
38. The product of claim 36, wherein the product is 99% salt and lactose free.
39. A method of isolating α-lactalbumin from whey, comprising the steps of: forming a mixture of said whey, an acid and an organic solvent; stirring said mixture no less than about 10 minutes; allowing a solvent insoluble remainder and a first supernatant to form from said mixture; separating said solvent insoluble remainder from said first supernatant of said mixture; thereafter adjusting the pH of said first supernatant to about 4.5 to 5.5 with a base and forming a whey protein precipitate; separating said whey protein precipitate from solvent; resuspending said whey protein precipitate with water and adjusting pH to about 6.0 with base; heating said resuspended whey protein precipitate to and maintaining said whey protein precipitate at about 100°C for not more than about 2 minutes and forming a second supernatant and a coagulate, said coagulate including β-lactoglobulin and serum albumin; thereafter separating said second supernatant from said coagulate; adjusting the pH of said second supernatant to about 4.5 to 5.5 and forming a α-lactalbumin precipitate and a third supernatant; and thereafter separating said α-lactalbumin precipitate from said third supernatant.
40. The method of claim 39 further comprising the steps of: subjecting said α-lactalbumin to alkali treatment; and refrigerating said treated α-lactalbumin for at least three weeks.
41. The method of claim 39 wherein said solvent is an organic, volatile, food compatible solvent.
42. The method of claim 41 wherein said solvent is ethanol.
43. The method of claim 39 wherein said acid is hydrochloric acid.
44. The method of claim 39 wherein said base is sodium hydroxide.
45. The method of claim 39 further comprising the step of purifying said β-lactoglobulin and said serum albumin with differential precipitation at different pH values.
46. The product of claim 39.
47. The product of claim 46, wherein the product has at least a 85% protein purity.
48. The product of claim 46, wherein the product has a 85 to 97% protein purity.
49. The product of claim 46, wherein the product is 99% salt and lactose free.
PCT/US1991/006047 1990-08-24 1991-08-23 Method for isolating alpha-lactalbumin from whey WO1992003468A1 (en)

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