This invention relates to a method for retaining the nutritive properties of whey. Specifically, this invention relates to the treatment of whey to enrich the xcex1-lactalbumin fraction.
Whey is the liquid component of milk. During the process of making milk into cheese, whey protein is separated from the curds. Whey is a dilute liquid containing lactose, proteins, salts, and residual fat.
Until recently, a major portion of commercially produced whey was discarded, causing major environmental pollution problems. With the advent of stricter environmental controls and regulations, whey proteins have been reexamined for their utility in the pharmaceutical, dietetic and food industries. Whey proteins have become more heavily incorporated into ice cream, bread, canned soup, infant formulas, and various other food products. Whey is also used as a livestock feed.
Whey proteins possess interesting nutritional, functional, physiological, and pharmaceutical properties. The proteins in whey are divided into two principal groups: 1) the globulin protein fraction containing mainly xcex2-lactoglobulin (xcex2-lg) and immunoglobulins (Ig); and 2) the albumin fraction including xcex1-lactalbumin (xcex1-La) and serum albumin. xcex1-La typically constitutes about 40% by weight of the total proteins in human milk, while cow""s milk contains only about 4-5% xcex1-La by weight of the total proteins.
Whey proteins have been used in infant formula and as a protein source in nutritional mixtures for humans and animals. Since xcex2-lg is not a protein found in human breast-milk, it acts as an allergen to infants. It is therefore desirable for whey proteins in breast-milk substitutes to have either a low concentration of xcex2-lg to reduce the concentration of allergen or a relatively high concentration of xcex1-La to make it more similar to human milk.
Recent methods of preparing xcex1-La enriched fractions from milk or whey have included ultrafiltration, heat precipitation, and ion exchange methods. Ultrafiltration methods use membranes which will allow only molecules up to a given size to pass through into the permeate. U.S. Pat. No. 5,008,376 to Bottomley describes a xcex1-La separation technique using ultrafiltration. Heat precipitation methods involve the application of heat to the whey at a given pH range for a time period sufficient to promote the flocculation of xcex1-La. Such heat precipitation methods are described in U.S. Pat. No. 5,455,331 to Pearce. Ion exchange methods involve contacting the whey with an anion or cation exchanger so as to selectively retain a protein fraction. Such a process is described in U.S. Pat. No. 5,077,067 to Thibault.
The present inventor has now determined that adjusting the pH of the whey to a more acidic level during processing, causes a change in protein conformation and improved retention of the xcex1-La during later processing. This change in comformation and improved retention provides a high concentration of xcex1-La in the protein fraction without the need for heat processing or other expensive separation processes.
Accordingly, it is a primary objective of the present invention to provide a method and means of processing whey to obtain a protein fraction having a high concentration of xcex1-lactalbumin.
It is a further objective of the present invention to provide a method and means of processing whey to obtain a protein fraction having a low concentration of xcex2-lactoglobulin.
It is yet a further objective of the present invention to provide a method and means of processing whey which does not necessitate the application of high temperatures.
It is still a further objective of the present invention to provide a method and means of processing whey to achieve a high concentration of xcex1-La in the protein fraction which is convenient and economical to use.
The method and means of accomplishing each of the above objectives as well as others will become apparent from the detailed description of the invention which follows hereafter.
The invention describes a method of processing whey to obtain a filtrate or precipitate having a high concentration of xcex1-lactalbumin. The method involves the addition of an acid during the processing method to lower the pH of the whey to less than 4.0, with a preferred pH range being from about 3.3-3.8. The pH adjustment results in an enriched xcex1-lactalbumin fraction after further processing. This pH adjustment process may be accommodated to membrane filtration and precipitation methods.
The present invention relates to a method and means for processing whey to obtain a protein fraction having an increased concentration of xcex1-lactalbumin (xcex1-La). The invention consists of the addition of an acid during the whey processing steps to obtain a pH of 4.0 or less.
xcex1-La contains 1 mol of bound calcium per mol of protein. Hiraoka et al. (1980), xcex1-lactalbumin: A calcium metalloprotein. Biochem. Biophys. Res. Comm. 95:1098-1104. This atom of calcium stabilizes the tertiary structure of the protein. It has been found that the binding of calcium to the xcex1-La molecule is pH dependent, especially below pH 5. Braumaud et al. (1995): Thermal Isoelectric Precipitation of xcex1-Lactalbumin from a Whey Protein Concentrate: Influence of Protein-Calcium Complexation. Biotech. and Bioengin. 47:121-130. At pH 1.7, xcex1-La does not bind calcium any longer. Id.
The present inventor has now unexpectedly discovered that when the pH of the xcex1-La molecule is lowered below 4.0, the molecule changes, and behaves as a much larger protein. This phenomenon in turn allows for recovery of an enriched xcex1-La fraction during membrane filtration. This pH lowering also causes the xcex1-La molecule to release calcium. Once this calcium is removed, the xcex1-La can be effectively precipitated to form a xcex1-La concentrate.
As used herein, the term xe2x80x9cwhey protein productxe2x80x9d refers to cheese or acid casein whey, or to a whey protein concentrate obtained from the whey. The whey protein concentrate may also be a whey protein powder. If a whey protein powder is used as starting product, the powder must be brought into solution prior to Applicant""s pH lowering step described below.
Applicant""s process involves the treatment of whey protein concentrate to further concentrate the xcex1-La proteins using either a precipitation or membrane filtration process. The whey protein concentrate starting product may be prepared in any conventional way. For example, the whey protein concentrate may be obtained from skimmed and/or clarified whey. The whey may be concentrated and/or desalted by common means, e.g. by ultrafiltration and/or diafiltration.
Whey is typically obtained during the cheese making or casein process as a result of whey separation and clarification from casein. The whey produced from cheese-making is generally referred to as xe2x80x9csweet wheyxe2x80x9d while whey from cottage cheese sources and acid casein is referred to as xe2x80x9cacid wheyxe2x80x9d. The whey source is preferably bovine, however the milk source may be from any mammal, including goats, sheep, buffalo, water buffalo, yak, rabbit, human, llama, and mouse.
The whey is preferably pasteurized, although it does not have to be pasteurized to be processed in accordance with this invention. Such pasteurization conditions are well known by persons skilled in the art.
In the filtration process, the whey is first concentrated through ultrafiltration using a 10K-100K molecular weight cut-off membrane (MWCO). The portion which passes through the membrane is known as the ultrafiltrate or permeate, while the larger molecules that are retained are known as the retentate. The permeate is processed in accordance with this invention, while the retentate may be dried as whey protein concentrate (WPC). Ultrafiltration of the whey may be performed using any of the ultrafilter housings and membranes known in the art, including hollow fiber or spiral membranes. While temperature is not a critical factor, ultrafiltration is generally conducted at a temperature of between about 5 to 55xc2x0 C., with preferred temperature ranges of 5-10xc2x0 C. or 45-55xc2x0 C. So long as the pH is maintained within its natural range of between about 4.6-6.2, pH is not critical in this step.
Diafiltration is optionally performed following the ultrafiltration process if a high protein WPC is desired. Diafiltration includes the injection of water into the whey stream to remove excess lactose and soluble minerals present into the permeate and further concentrate the proteins. Deionized water is preferred for use in this process. The diafiltration may be performed using known techniques, such as constant volume diafiltration or batch diafiltration.
Applicant""s invention next involves the unique pH-adjustment step which causes the xcex1-La molecule to change its conformation and behave like a much larger molecule. Prior to this step, the pH of the permeate ranges from about 5.5-6.5. This pH is slightly lower if the whey source is acid whey, whereby the whey protein concentrate will have a pH of about 4.6.
In the pH-lowering step, the permeate obtained above is mixed with a sufficient amount and concentration of acid to decrease the pH of the permeate to 4.0 or below. The pH is preferably lowered to a range of between about 3.3-3.8, with about 3.5 being most preferred. The acid is preferably a food grade acid, such as hydrochloric acid, phosphoric acid, citric acid, and sulfuric acid. The preferred acid is hydrochloric acid. Temperature is not critical in this step.
Once the pH of the whey protein concentrate is lowered to 4.0 or below, the present inventor has surprisingly found that, once the pH is lowered, the xcex1-La molecule behaves like a much larger molecule. Specifically, at a pH of 4.0 or below, xcex1-La molecules will be retained on the same ultrafiltration membrane that they originally passed through at the higher pH.
Once the pH of the permeate is lowered to 4.0 or below, the permeate is preferably concentrated through ultrafiltration using a 10K-100K MWCO membrane. The xcex1-La concentrate obtained may then be dried as-is, or neutralized to a pH of 6-7. Acceptable bases for this purpose include NaOH, KOH, and other alkaline bases. Such bases are readily ascertainable by those skilled in the art.
Drying the xcex1-La concentrate allows for easier packaging and the product remains stable for a longer period of time than the raw concentrate in liquid or frozen form. Approximately 25-70% of the protein in the powder is xcex1-La. The xcex1-La concentrate may be included in a variety of foods, supplements, infant formulas, etc. as previously described herein in appropriate concentrations.
In the precipitation technique of this invention, the starting material can be cheese or acid casein whey or the same whey concentrated through ultrafiltration using a 5-50K MWCO membrane to provide a whey protein concentrate having from about 30-80% protein on a solids basis. The pH of the unconcentrated or concentrated whey is then lowered to pH 4.0 or below as already described above. This pH lowering step causes the calcium ions to disassociate from the xcex1-La molecules.
The retentate is then ultrafiltered, and optionally diafiltered with deionized water until the calcium to protein ratio is less than about 0.001, and preferably less than about 0.0004. Removal of the calcium ions in this step allows the xcex1-La molecules to become more concentrated in the precipitation step. Again, while temperature is not critical, the ultrafiltration and diafiltration steps may generally be carried out at a temperature range of between about 5-55xc2x0 C., and preferably at 5-10xc2x0 C. or 45-55xc2x0 C.
Precipitation of xcex1-La and other proteins is induced by diluting the retentate with deionized water. The purpose of dilution is to facilitate precipitate formation and promote subsequent separation. The retentate is diluted until it contains from about 2-12% protein, and preferably from about 4-10% protein. The pH is then adjusted to a range of 4-5, and a preferred range of 4.5-4.7. Many alkali agents, such as NaOH and KOH, can be used for this purpose. The dilution and pH adjustment steps are performed in a temperature range of between about 10-55xc2x0 C., preferably 25-40xc2x0 C. The precipitation is normally complete within 2 hours.
The precipitated proteins (xcex1-La, BSA, Ig) can be separated from the soluble proteins (xcex2-lactoglobulin and casein peptides) by centrifugation or microfiltration. The precipitate stream is xcex1-La-enriched and can be dried or neutralized as described above. The stream containing the soluble proteins is low in fat and can be made into whey protein isolate after further concentration.
The membrane filtration and precipitation processes described offer the advantage of being performed without the necessity for a heat treatment or other expensive processes, while successfully achieving a high concentration of xcex1-La.
The xcex1-La concentrate of this invention may be included in a variety of food products, especially nutritional formulas. Such formulas include infant formulas, adult nutritional formulas, sport formulas, medical formulas, and enteral formulas.
While these food products are conventionally formulated in a liquid form, it is contemplated that they can also be contained in a variety of other oral dosage forms. In general, in addition to the xcex1-La concentrate, the pharmaceutical compositions of this invention may contain suitable excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Oral dosage forms encompass tablets, capsules, powders, suspensions, emulsions, granules, and feed supplements.
The pharmaceutical preparations of the present invention are manufactured in a manner which is itself well known in the art. For example the pharmaceutical preparations may be made by means of conventional mixing, granulating, dissolving, and lyophilizing processes. The processes to be used will depend ultimately on the physical properties of the active ingredient used.
Suitable excipients are, in particular, fillers such as sugars for example, lactose or sucrose mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch, paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired, disintegrating agents may be added, such as the above-mentioned starches as well as carboxymethyl starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are flow-regulating agents and lubricants, for example, such as silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate and/or polyethylene glycol. Dragee cores may be provided with suitable coatings which, if desired, may be resistant to gastric juices.
For this purpose concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate, dyestuffs and pigments may be added to the tablet of dragee coatings, for example, for identification or in order to characterize different combination of compound doses.
Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules can contain the active compounds in the form of granules which may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Possible pharmaceutical preparations which can be used rectally include, for example, suppositories, which consist of a combination of the active compounds with the suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols, or higher alkanols. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the active compounds with a base. Possible base materials include for example liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons. Optionally, the suspension may also contain stabilizers.
The following examples are offered to illustrate but not limit the invention. Thus, they are presented with the understanding that various formulation modifications as well as method of delivery modifications may be made and still be within the spirit of the invention.