JP2001516599A - Sequential separation of whey proteins and formulations thereof - Google Patents

Abstract

  (57) [Summary] Disclosed are methods for sequentially separating whey proteins using radial flow chromatography. In the separation method, different buffer systems adjusted to appropriate pH and ionic strength are used. The method separates at least five proteins from whey. In addition, infant nutritional and other food formulations are disclosed that include various proteins in various proportions isolated from whey.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the separation of whey proteins, in particular the sequential separation of whey proteins using chromatography, and to food-related formulations and pharmaceutical preparations using the separated whey proteins.

BACKGROUND OF THE INVENTION The dry content of milk is about 12.5%, of which 3.4% is total protein, 3.5% is fat, 4.7% is lactose and 0.9% is ash Is well known. The protein component mainly consists of casein and whey protein. Other minor components include non-proteinaceous nitrogen compounds, protease peptones, and other minor enzymatic proteins. In the cheese industry, milk protein is separated into casein and whey protein by two main precipitation techniques: rennet precipitation and acid precipitation. In rennet precipitation, rennin is added to warm milk (30-35 ° C). Casein precipitates and whey protein remains in solution. This type of whey is called sweet whey. Acid precipitation is performed at an isoelectric point of milk 4.7 by using a suitable acid. The whey obtained from the acid precipitation is called acid whey. The choice of method depends on the intended cheese product.

[0003] Whey, a by-product of the cheese industry, has a high nutritional value because its composition contains many useful proteins. However, until recently most of the commercially produced whey has been discarded, causing serious environmental pollution problems.
With the emergence of more stringent environmental controls and regulations and the availability of newer technologies such as ultrafiltration, membrane separation such as reverse osmosis, whey proteins and other products formed therefrom, It is becoming increasingly important in meeting the needs of the pharmaceutical, nutritional and food industries. Research efforts with varying degrees of success in the field of isolating individual proteins from whey and formulations formed from whey have become widespread in the dairy and related industries.

The following patents illustrate various approaches in the prior art for isolating individual proteins and other components from whey and food and pharmaceutical products derived from whey.

[0005] US Patent No. 5,077,067, issued December 31, 1991 to Philippe A. Thibault, discloses a method for selectively and quantitatively removing lactoglobulin from whey protein.

[0006] US Pat. No. 5,055,558, issued Oct. 8, 1991 to Emilia Chiancone and Maurizio Gattoni, describes a method for the selective extraction of β-lactoglobulin from whey or milk by subunit exchange chromatography. Has been described.

[0007] US Patent No. 4,791,193, issued December 13, 1988 to Shigeo Okonogi et al., Relates to a method for preparing pure lactoferrin from whey or skim milk.

[0008] US Patent No. 4,668,771, issued May 26, 1987 to Hiroshi Kawakami et al.
Methods for isolating and purifying bovine lactoferrin are provided.

US Pat. No. 4,997,914, issued to Hiroshi Kawakami et al. On Mar. 5, 1991, describes a method for separating and purifying lactoferrin by adsorption chromatography.

[0010] US Pat. No. 4,820,348, issued Apr. 11, 1989 to Matti Harju, relates to a method for separating lactose from milk by chromatography.

US Pat. No. 4,446,164, issued to Roy A. Brog on May 1, 1984, discloses a sweet whey base and soluble protein, edible vegetable oil, skim milk solids, sugar or sugar incorporated therein. It relates to a milky composition formed from additives such as synthetic sweeteners.

US Pat. No. 5,085,881, issued to Hans G. Moeller on Feb. 4, 1992, discloses that a food ingredient or fraction for use as a food or pharmaceutical supplement is separated from dry milk or dairy products. A method is described.

US Pat. No. 5,09, issued Mar. 3, 1992 to Horst Behr and Friedrich Manz
No. 3,143, which mimics milk, relates to a nutritional composition rich in energy and calcium but poor in albumin and phosphorus.

US Pat. No. 4,202,909, issued May 13, 1980 to Harold T. Pederson, Jr., describes a process for treating whey to produce pure lactose and salt products.

US Patent No. 5,008,376, issued April 16, 1991 to Robin C. Bottomley,
A method for producing a whey fraction containing a high concentration of α-lactalbumin by an ultrafiltration technique is disclosed.

US Pat. No. 3,969,337, issued to Karl Lauer et al. On Jul. 13, 1976, discloses a method for fractionating whey by chromatography.

As indicated in the aforementioned patents and other references, various experimental and commercial processes are available for separating, removing, enriching and / or purifying selected whey proteins, Since these prior art methods degrade or discard all but one protein selected from whey, other useful proteins contained in whey are wasted. None of these prior art methods can separate various proteins from whey in one step. Therefore, it is desirable to provide a method for continuously and sequentially separating various proteins from whey in a one or two step separation process.

Therefore, an object of the present invention is to provide a separation technique capable of completely and sequentially separating whey protein in one or two steps.

Another object of the present invention is to provide a radial flow chromatograph.
y) to provide a continuous and sequential separation of whey proteins suitable for experimental as well as commercial use by using the technique;

Yet another object is to provide a variety of buffers that are mild enough to be used for sequential separation of whey proteins without denaturation.

Yet another object is to provide a separation technique applicable to food and pharmaceutical applications of whey protein.

It is another object of the present invention to provide food formulations and pharmaceutical formulations containing different isolated whey proteins in different proportions.

[0023] Further objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will be obvious to those skilled in the art from the following examples, or It can be learned by implementation. The objects and advantages of the invention may be realized and obtained by means of the instruments and combinations particularly pointed out in the appended claims.

SUMMARY OF THE INVENTION In order to accomplish the above and other objects, and in accordance with the objects and principles of the invention set forth herein, the present invention provides a method which is essentially at least 5 watts away from whey.
Methods for sequentially separating different species of proteins and methods for mixing these separated whey proteins into pharmaceutical formulations and food formulations are provided. The method of the present invention is a continuous and sequential separation of whey protein by chromatography, wherein the protein in liquid whey is adsorbed to a suitable separation medium packed in a chromatographic column, and a suitable pH is adjusted using a buffer. And ionic strength of IgG, β-
Sequentially eluting fractions of Lg, α-La, BSA and lactoferrin,
Regarding the separation. Although both axial and radial flow chromatography can be utilized, horizontal flow columns are particularly suitable for the method of the present invention. Whey proteins separated by the method of the present invention include β-lactoglobulin (β-Lg), α-lactalbumin (α-La), bovine serum albumin (BSA), immunoglobulin (Ig-G) and lactoferrin ( L-Fe). These separated proteins are mixed in various proportions in various formulations of the invention for food or pharmaceutical use.

DETAILED DESCRIPTION OF THE INVENTION According to the method of the present invention, sterilized sweet whey, sterilized acid whey, unsterilized acid whey, or reverse osmosis (RO) or obtained as a by-product of cheese production Ultrafiltration
A sample of starting material selected from whey protein concentrate prepared from sterilized or unsterilized whey by known techniques such as (UF),
Add to a chromatographic column, preferably a horizontal flow chromatographic column, packed with an acidic or basic cation or anion exchange resin material such as S or Q. The whey, concentrated whey or whey protein concentrate may be used to separate colloidal and suspended particles such as pre-separation steps, such as demineralization by electrodialysis or ion exchange, clarification to remove casein microparticles, and / or fat residues. It may be subjected to microfiltration or the like.

The various whey components were then eluted and separated according to the protocol described in the examples below.

EXAMPLE 1 Sequential Separation of Sweet Whey Protein Commercially available whey (a by-product from the production of mozzarella cheese) is first clarified to remove casein particulates, centrifuged to remove residual milk fat, and at 162 ° F. About 18
Pasteurized for 2 seconds and cooled to 40 ° F through an HTST plate heat exchanger. The pH of 1000 ml of this defatted commercial sweet whey (pH 6.4 and total solids 6.2%) is adjusted with acetic acid.
Adjusted to 3.8 at 40 ° F. The composition of this whey product used in this example is shown in Table I.

[0028]

[Table 1]

The whey was then filled with a strong S cation exchange resin and charged with 0.05M acetate buffer (pH 3
Passed through a 250 ml radial flow chromatography column equilibrated in .8). All whey proteins bound to the resin matrix, and the effluent containing non-protein components such as lactose, minerals, lactic acid, and non-proteinaceous nitrogenous components passed through the column. The protein-bound resin was then washed with 0.05M acetate buffer (pH 3.8) and returned to the set UV baseline. The bound proteins were then sequentially eluted according to the following protocol:

[0030] Immunoglobulin (IgG) and β-lactoglobulin (β-Lg) were subsequently eluted with a buffer (pH 4.0) containing 0.1 M sodium acetate and 0.5 M sodium chloride. The column was then reconditioned and 0.05M sodium acetate buffer (p
H4.0) and the conductivity was returned to baseline. The α-lactalbumin (α-La) fraction was eluted with a buffer (pH 5.0) containing 0.1 M sodium acetate and 0.1 M sodium chloride. The column was reconditioned with a buffer (pH 5.0) containing 0.05 M sodium acetate to return the conductivity to the default baseline. Next, bovine serum albumin (BSA) was eluted with a 0.05 M phosphate buffer (pH 7.0). Then, lactoferrin (LF) was added to pH 7.5 at pH 7.5.
Elution was carried out with a buffer containing 05M sodium phosphate and 0.5M sodium chloride.

[0031] The column is regenerated by washing the column with a solution containing 0.2 M sodium hydroxide and 1 M sodium chloride, then sterilizing the column by washing with a 20% ethanol (EtOH) solution and using an acetate buffer (pH 3.8). And reused. A flowchart showing the elution protocol is shown in Table II below.

[0032]

[Table 2]

[0033] Each fraction of the eluted protein is collected for the "peak" of the elution, and subjected to further separation, concentration and other processing protocols. UV absorbance at 280 nm The order of elution along with individual protein peaks is shown in FIG. The identity of the protein in each peak was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as known in the art. The protein recovery monitored at various stages of the elution scheme by the bio-rad assay and the gel scanning assay are shown in Tables III and IV.

[0034]

[Table 3]

[0035]

[Table 4]

Example 2 Another Protocol for Eluting Whey Protein A 20 liter RFC column was packed with macro-prep 50S resin. The column was then conditioned, equilibrated, loaded, eluted and reconditioned exactly as in Example 1 above. However, the flow rate and volume of whey and the flow rate and volume of buffer were changed. The protein elution peak was monitored at 280 nm using a UV spectrophotometer. A graphical trace of the eluted protein showing the relative concentration is shown in FIG. The eluted proteins and individual purity (%) are shown in Tables V and VI.

[0037]

[Table 5]

[0038]

[Table 6]

Example 3 Preparation of an anion exchange resin column A 250 ml RFC column was packed with a strongly basic anion exchange resin (macro-prep50Q),
Conditioned with MNaOH and 1M NaCl at a flow rate of 100 ml / min for 10 minutes. The column was then equilibrated with 0.01 M sodium phosphate (pH 6.90) at a flow rate of 100 ml / min for 10 minutes. The column was then used to separate immunoglobulins (IgG) from β-lactoglobulin. Immunoglobulin (IgG) and β-lactoglobulin were eluted as overlapping peaks in Examples 1 and 2 above. This mixture is used in the food formulation (d
ietary formulation) or used to further separate the two protein components.

Example 4 Separation of immunoglobulin (IgG) from β-lactoglobulin Eluate indicated by peak 2 (recovered from the fractionated material obtained by the method described in Example 1, IgG and β-Lg PH 4.0) was passed through a 10,000 molecular weight cut-off UF membrane to concentrate the protein, reduce the salt concentration of the buffer, and reduce the ionic strength of the solution. The protein was further concentrated to five times the initial elution concentration, and the salt concentration of the buffer was diafiltered with distilled water to reduce it to approximately one-fourth the elution concentration. The pH of the diafiltration and concentrated protein solution was adjusted to 6.9 with a 2.0 M NaOH solution. Two liters of this protein solution (pH 6.9) were loaded at a flow rate of 100 ml / min onto a preconditioned RFC column as described in Example 3. The column was treated with a 0.01 M sodium phosphate buffer (pH 6.9).
) To stabilize the UV baseline. The IgG that did not bind to the resin passed through the column by washing, and was collected for further processing. Next, the adsorbed β-Lg was eluted and recovered from the column with 0.05 M sodium citrate buffer (pH 3.0). The column was washed with distilled water, and the remaining protein was stripped with 0.2 M NaOH and 1 M NaCl solutions, then with 20% EtOH and re-equilibrated with sodium phosphate buffer (pH 6.9) (preparation for next cycle) ).

Example 5 Separation and isolation of BSA Eluate indicated by peak 4 (recovered from the fractionated substance obtained by the method described in Example 1 and containing BSA and protease peptone; pH 7.0 ) Was concentrated and diafiltered as described in Example 4 and the pH was further adjusted with acetic acid.
Adjusted to 5.5. A 250 ml RFC column prepared as described in Example 4 was washed with distilled water at a flow rate of 100 ml / min. Two liters of the protein solution was loaded onto the column as described above. The column was again flushed with distilled water at a flow rate of 100 ml / min to elute unadsorbed protease peptone and stabilize the UV baseline. The eluate containing the protease peptone is collected and used further. Then, the adsorbed BSA was
Elution was carried out with a sodium phosphate buffer (pH 7.0) containing sodium chloride. FIG. 3 shows the elution pattern and the position of peak 4.

Example 6 Elution and Separation of β-Lactoglobulin from Liquid Whey A 250 ml radial flow chromatography column packed with a strongly basic anion exchange resin (macro-prep 50Q) was washed and regenerated according to the manufacturer's instructions. . Then the column is adjusted to 0.05
Equilibrated with M sodium phosphate (tribasic; pH 7.5) at a flow rate of 100 ml / min for 10 minutes. 7
There was no significant effect on the elution pattern in the pH range from 0.0 to 8.0. The pH of 2 liters of clarified pasteurized defatted sweet whey (cooled to 40 ° F) obtained during the production of mozzarella cheese is adjusted to 8.0 with 5M sodium hydroxide and circulated at 75 miles / min through a pre-prepared column. Equilibrated. Flow rates in the range of 50-100 ml / min can be used. Next, 1 to 3 liters of the whey sample to be analyzed is loaded onto the column, and the column is loaded with a loading buffer (0.05 M sodium phosphate, pH 7.5).
). Under the conditions used, all whey proteins except for β-lactoglobulin are positively charged. The negatively charged β-lactoglobulin is retained by binding to the anion exchange resin. Unbound protein [α-lactalbumin
(α-La), immunoglobulin (IgG), bovine serum albumin (BSA) and lactoferrin (L-Fe)] passed through the column, collected and stored at 40 ° F.
Further processing is performed.

The adsorbed β-lactoglobulin was then treated with 0.05M sodium phosphate and 0.5M
The column was eluted with a buffer containing sodium chloride (pH 7.5). This eluate containing β-lactoglobulin can be further processed to prepare a self-stable product in a manner similar to Example 8 below.

The column was washed with 1 M sodium chloride at a flow rate of 125 ml / min at approximately 4 times the volume of the column (2 liters), stripped with 1 M sodium hydroxide at the same flow rate, and 100 ml with 1 M sodium chloride. 5 times the volume of the column (2 and 1/2 liter
), Sterilize with 200 ppm sodium hypochlorite at 100 ml / min at approximately 4 volumes (2 liters) of the column, then equilibrate with the loading buffer (for the next cycle). Preparation).

Example 7 Elution and Sequential Separation of Four Proteins from the Non-β-Lactoglobulin Fraction of Liquid Whey Obtained in Example 6 for the purpose of partially concentrating proteins and reducing soluble salts The pass-through fraction (containing 0.55% protein) was passed through a 10,000 molecular weight cut-off helical ultrafiltration membrane and concentrated to 35% of its original volume to remove permeate. With this pretreatment technique, as outlined in Example 1, IgG, α-La, BSA and LSA
-Optimum absorption and subsequent desorption of the Fe protein fraction is facilitated. The pH of the flow-through fraction thus prepared was adjusted to 3.8 with acetic acid, and 1500 ml of this sample was filled with strong S cation exchange resin and pre-equilibrated with 0.05 M sodium acetate buffer (pH 3.8) to 250 ml. Loaded on an RFC column. Next, the washing step, the sequential elution step, and the regeneration step outlined in Example 1 were performed. The eluted protein fractions are individually separated into appropriate molecular weight cutoff membranes (50000 MW cutoff membrane for IgG, L-Fe and BSA, α
The protein was concentrated to remove residual salts by passing through a 10,000 MW cut-off membrane for -La. Further processing was then performed as outlined in Example 8 to obtain the final product.

Example 8 Flow chart showing the preparation of the final product

The whey protein fraction or the separated and purified protein and the non-protein eluate can be added to food and pharmaceutical formulations in appropriate proportions. Such formulations and preparations include, but are not limited to, infant formulas, fat substitutes, foaming agents, egg white substitutes, animal feed substitutes, and the like.

Example 9 Infant Formula In an infant formula constructed in accordance with the present invention, the casein and whey fractions of milk are modified and added to human milk to a much higher degree than prior art compositions and commercial products. A similar composition was obtained. The infant formula of the present invention contains whey proteins at levels comparable to human milk. The infant formula of the present invention was obtained by producing a whey protein component mixture containing the type and proportion of whey protein in human milk.

Commercially available infant formulas are often composed of whole cow milk, which is available in large quantities. Other additives or adjuvants can be included. These formulations are manufactured in powdered, concentrated, or ready-to-feed form. The majority consists of skim milk solids, vegetable oils and lactose, corn syrup solids and carbohydrate sweeteners such as sucrose. These formulations can also be fortified by vitamin C, vitamin D, iron and fluorine. Table VII shows the typical composition of a few representative commercial infant formulas compared to one of the representative formulations of the present invention. The levels of vitamins, minerals and other fortifiers in the formulations of the present invention are adjusted to mimic human milk and meet the nutritional requirements of infants.

[0050]

[Table 7]

However, due to the great difference in protein composition between milk and human milk, some infants show various intolerances to milk and food formulations composed of milk. A comparison of the composition of cow's milk and human milk is shown in Table VIII, and a comparison of their protein contents is shown in Table IX.

[0052]

[Table 8]

[0053]

[Table 9]

Table X shows the proportions of various whey proteins in cow's milk and human milk.

[0055]

[Table 10]

As is evident from the above table, milk contains 3.3% protein, whereas human milk is only 1%. Casein is the main protein component of milk,
On the other hand, human milk contains a high ratio of whey protein to casein (about 2: 1) in human milk. The concentration of β-lactoglobulin in cow's milk is the highest among whey proteins, while it is trace in human milk.
Similarly, lactoferrin is ten times higher in human milk than in milk. The immunoglobulin concentration and serum albumin concentration are about 1.1 in human milk than in milk.
5 times higher.

In the infant formula of the present invention, lactose and fat levels are adjusted to mimic human milk. Milk fat is replaced with vegetable fat. The ratio of casein to whey is also low, similar to human milk. If desired, other additives and supplements such as vitamins, taurine, and minerals may be included. The total solute loading is reduced to levels found in human milk.

To this end, the whey protein fraction obtained from fractionation and elution according to the method of the invention was first mixed in the proportions shown in Table XI below.

[0059]

[Table 11]

The whey protein mixture was then added to a human milk-like composition having the composition shown in Table XII.

[0061]

[Table 12]

The liquid mixture prepared according to the above composition had a total solids content of about 2% (of which salts from the lysis buffer accounted for 90% and total protein content accounted for about 10%), and 98% Contains water. The liquid mixture is then concentrated through a helical ultrafiltration membrane with a molecular weight of 10,000 cut-off to a total protein content of 5-15% and then 0%.
The residual salt was removed by diafiltration with 1.5 to 1.0 times distilled water. The formulation can be further concentrated by methods commonly used to treat chemically susceptible proteins, such as ultrafiltration, reverse osmosis, lyophilization, freeze concentration, spray drying, or any combination thereof. Further, the formulations of the present invention may be fortified by suitable additives and enhancers. Such additives and enhancers include, but are not limited to, skim milk solids, vegetable solids, carbohydrate sweeteners, minerals and vitamins. The solids composition of a representative formulation of the invention is shown in Table XIII.

[0063]

[Table 13]

Example 10 Formulation as a fat substitute β-lactoglobulin shows high water binding properties, α-lactalbumin increases viscosity and absorbs fats and oils in high content. Because of these properties, combining these proteins would be suitable for use as a fat substitute. This product, when added to food in appropriate proportions, improves the quality and properties of the food and can be used as a fat substitute.

The β-lactoglobulin elutes together with the IgG as fraction 2 of the method of the invention,
α-lactalbumin elutes in fraction 3. IgG is separated from β-lactoglobulin as described in Example 7 and mixed with α-lactalbumin fraction at 60% and 40%. The mixture of the two proteins (β-La + α-La) was passed through a 10,000 cut-off ultrafiltration membrane with a molecular weight of 10,000 and a pressure difference of 10 psi at 40 ° F. to remove the permeate consisting of water and soluble salts to a total solids concentration of 40. Remove until ~ 50%. Then, the residual salt is removed by diafiltration with 0.5 times distilled water. The concentrated and purified mixture thus obtained may be frozen, lyophilized, refrigerated or dehydrated for further use. If desired, other additives such as flavor enhancers, vitamins and sweeteners may be added to these formulations.

Thus, it has been shown that the present invention provides a method for continuous and sequential separation of whey protein as well as a blend of eluted fractions that can be used as food additives or substitutes.

The foregoing description of the preferred embodiment of the invention has been presented for purposes of illustration and description, and for a better understanding of the invention. It is not intended to limit the invention to just the precise one disclosed; and obviously, many modifications and variations are possible in light of the above teaching. Having selected certain embodiments to illustrate the principles of the invention and its practical application, those skilled in the art will recognize that, in various embodiments, those skilled in the art will appreciate that various modifications may be made to suit the particular use intended. The invention has been described in some detail to enable its use. The invention is to be defined by the claims.

[Brief description of the drawings]

FIG. 1 is a graph showing elution patterns of various proteins according to the present invention.

FIG. 2 is a graph showing the elution characteristics of separated proteins versus time.

FIG. 3 is a diagram showing an elution pattern and the position of peak 4.

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Miranda, Cyrinas United States 95125 California, San Jose, Jonathan 2097 F term (reference) 4B001 AC05 BC99 EC99 4B018 MD71 MF01 4H045 AA20 AA30 CA43 DA70 DA75 EA01 GA23

Claims (36)

[Claims] 1. A method for the continuous and sequential separation of whey proteins by chromatography, wherein liquid whey is adsorbed to a separation medium packed in a chromatographic column, and immunoglobulin, β-lactoglobin, α-lactalbumin, A method comprising sequentially eluting bovine serum albumin and a fraction of lactoferrin. 2. The liquid whey is sterilized sweet whey, sterilized acid whey,
Selected from the group consisting of unsterilized acid whey, and whey protein concentrate,
The method of claim 1. 3. The method according to claim 1, wherein the separation medium is a cationic resin. 4. Collecting fractions of sequentially eluted immunoglobulin, β-lactoglobin, α-lactalbumin, bovine serum albumin, and lactoferrin,
The method according to claim 1, wherein the concentration is performed by ultrafiltration. 5. The method according to claim 4, wherein the concentrated immunoglobulin, β-lactoglobin, α-lactalbumin, bovine serum albumin, and lactoferrin fractions are further purified by diafiltration. 6. A method for the sequential separation of whey proteins, comprising: a) packing a chromatographic column with a cation exchange resin to prepare a packed chromatographic column; b) equilibrating the packed chromatographic column; c) Providing a whey sample, d) under conditions where whey protein is adsorbed to said packed chromatographic column,
Passing the whey sample through the packed chromatographic column; e) collecting the flow-through fraction from the packed chromatographic column, wherein the flow-through fraction contains lactose, mineral, lactic acid, and non-nitrogenous components; f) Sequentially eluting immunoglobulin and β-lactoglobin from the packed chromatographic column; g) eluting α-lactalbumin from the packed chromatographic column; h) reconditioning the packed chromatographic column; Eluting bovine serum albumin from the chromatographic column, and j) eluting lactoferrin from the packed chromatographic column. 7. The method according to claim 7, wherein the whey is sterilized sweet whey, sterilized acid whey,
Selected from the group consisting of unsterilized acid whey, and whey protein concentrate,
The method of claim 6. 8. The method according to claim 6, wherein said chromatographic column is a radial flow column.
The described method. 9. A method for separating β-lactoglobulin from whey protein, the method comprising: a) packing a chromatographic column with an anion exchange resin to prepare a first packed chromatographic column; b) preparing the first packed chromatographic column; C) preparing a whey sample; and d) loading whey sample on said first packed chromatographic column under conditions in which β-lactoglobulin is adsorbed on said first packed chromatographic column. E) collecting the flow-through fraction from said first packed chromatographic column, wherein said flow-through fraction comprises α-lactalbumin, immunoglobulin, bovine serum albumin and lactoferrin suitable for further processing; And f) removing the adsorbed β-lactoglobulin from the first packed chromatographic column. Obtain the eluate eluted with § chromatography, a method comprising the steps. 10. The first packed chromatographic column is a radial flow column.
The method according to claim 9. 11. The method further comprises: g) packing a second chromatographic column with a cation exchange resin to prepare a second packed chromatographic column; h) equilibrating said second packed chromatographic column; Passing the through fraction through an ultrafiltration membrane with a 10,000 molecular weight cutoff to obtain an ultrafiltrate; j) immunoglobulin, α-lactalbumin, bovine serum albumin and lactoferrin in the second packed chromatographic column Passing said ultrafiltrate through said second packed chromatographic column under conditions that adsorb to said second column; k) eluting immunoglobulin from said second packed chromatographic column; 1) said second packed chromatographic column. Eluting α-lactalbumin from the graph column; m) reconditioning said second packed chromatographic column; Elute bovine serum albumin from the packed chromatography column
And o) eluting lactoferrin from the second packed chromatographic column. 12. The method of claim 12, wherein the second packed chromatographic column is a radial flow column.
The method of claim 11. 13. The composition of claim 12, wherein the through fraction comprises an infant formula.
The described method. 14. The method of claim 13, wherein said infant formula comprises at least 25% lactoferrin and less than 0.5% β-lactoglobulin. 15. The infant formula comprises about 43.5% α-lactalbumin, about 3%.
14. The method of claim 13, comprising 1.6% lactoferrin, about 15.4% immunoglobulin, and about 9.5% bovine serum albumin. 16. The method of claim 14, wherein said infant formula further comprises casein hydrolysate, fat, skim milk solids, carbohydrates, minerals, and vitamins. 17. The method of claim 13, wherein said pass through fraction and said eluate are combined to make a fat substitute. 18. The method of claim 18, wherein the fat substitute comprises about 60% β-lactoglobulin and 40% α.
18. The method of claim 17, comprising lactalbumin. 19. A method for sequential separation of whey proteins, comprising: a) packing a cation exchange resin in a chromatographic column to prepare a packed chromatographic column; b) equilibrating the packed chromatographic column with a buffer; c) preparing a whey sample containing whey protein comprising lactoferrin, immunoglobulin, β-lactoglobin, α-lactalbumin, and bovine serum albumin; d) at least a portion of the whey protein is loaded onto the packed chromatographic column. Passing the whey sample through the packed chromatographic column under conditions of adsorption; e) washing the packed chromatographic column with a buffer; f) sequentially removing immunoglobulin and β-lactoglobin from the packed chromatographic column with a buffer. E) g) Reconditioning the chromatographic column; h) eluting α-lactalbumin with buffer from said packed chromatographic column; i) reconditioning said packed chromatographic column with buffer; j) bovine bovine from said packed chromatographic column with buffer. Elute serum albumin and k) elute lactoferrin with buffer from the packed chromatographic column to obtain a lactoferrin-containing eluate. 20. The method of claim 19, wherein said whey is selected from the group consisting of sterilized sweet whey, sterilized acid whey, unsterilized acid whey, and whey protein concentrate. 21. The method of claim 19, wherein said chromatographic column is a radial flow column. 22. A method for separating β-lactoglobulin from whey protein, comprising: a) packing a radial flow chromatographic column with an anion exchange resin to prepare a first packed chromatographic column; Equilibrating the packed chromatographic column with buffer, c) preparing a whey sample containing whey protein comprising lactoferrin, immunoglobulin, β-lactoglobin, α-lactalbumin, and bovine serum albumin; d) β Passing a whey sample through said first packed chromatographic column under conditions in which lactoglobulin is adsorbed on said first packed chromatographic column and permeate flows out of said first packed chromatographic column; e. ) Collecting the permeate from the first packed chromatographic column,
Said permeate comprises α-lactalbumin, immunoglobulin, bovine serum albumin and lactoferrin suitable for further processing; f) eluted adsorbed β-lactoglobulin from said first packed chromatographic column with a buffer. G) packing a second chromatographic column with a cation exchange resin to prepare a second packed chromatographic column; h) equilibrating said second packed chromatographic column; The permeate collected from the packed chromatographic column is passed through an ultrafiltration membrane to obtain an ultrafiltrate, k) immunoglobulin, α-lactalbumin, bovine serum albumin and lactoferrin are added to the second packed chromatograph. Passing the ultrafiltrate through the second packed chromatographic column under conditions for adsorption to the column; Eluting the immunoglobulin from the second packed chromatographic column with a buffer; m) reconditioning the second packed chromatographic column with a buffer; n) alpha-lacking the buffer from the second packed chromatographic column with a buffer. Eluting albumin; o) reconditioning said second packed chromatographic column with buffer; p) eluting bovine serum albumin with buffer from said second packed chromatographic column; and q) said second packing. Eluting lactoferrin with a buffer from the chromatographic column. 23. The method of claim 22, wherein the permeate is combined with the eluate obtained in step f) to form a fat substitute. 24. The method of claim 23, wherein said fat substitute comprises about 60% of said eluate and 40% of said permeate. 25. The method of claim 22, further comprising combining the α-lactalbumin, immunoglobulin, and bovine serum albumin together to form an infant formula. 26. The method of claim 25, wherein the infant formula further comprises at least 25% α-lactoferrin and less than 0.5% β-lactoglobulin. 27. The method of claim 25, wherein said infant formula further comprises casein hydrolyzate, fat, skim milk solids, carbohydrates, minerals, and vitamins. 28. The infant formula further comprising about 43.5% of α-lactalbumin eluted from the second packed chromatographic column in step n), and about 31.6% of lactoferrin eluted in step q). About 15.4% of immunoglobulin eluted in j)
26. The method of claim 25, comprising about 9.5% of bovine serum albumin eluted in step p). 29. An infant formula comprising at least 25% lactoferrin and less than 0.5% β-lactoglobulin. 30. An infant formula comprising α-lactalbumin, lactoferrin, immunoglobulin, and bovine serum albumin. 31. The infant formula of claim 30, wherein said lactoferrin comprises at least 25%. 32. The composition of claim 3, wherein said formulation further comprises additives and fortifiers.
0. The formulation for infants according to 0. 33. The additive and fortifier are selected from the group consisting of skim milk solids, vegetable solids, carbohydrate sweeteners, minerals, and vitamins.
2. The infant formulation according to 2. 34. An infant formula comprising about 43.5% α-lactalbumin, about 31.6% lactoferrin, about 15.4% immunoglobulin, and about 9.5% bovine serum albumin. 35. A fat substitute comprising about 60% β-lactoglobulin and 40% α-lactalbumin. 36. The fat substitute of claim 35, further comprising an additive and a flavor enhancer.