KR20010030665A - Sequential Separation of Whey Proteins and Formulations Thereof - Google Patents

Abstract

A method for sequential separation of whey protein using radial flow chromatography is disclosed. Different buffer systems adjusted to appropriate pH and ion concentrations are used in the separation method. The method separates at least five different proteins from whey. Baby food and other food formulations are also disclosed in which different varieties of proteins separated from whey are incorporated at different rates.

Description

Sequential Separation of Whey Proteins and Formulations Thereof

The dry content of milk is about 12.5%, 3.4% of which constitutes total protein, 3.5% of fat, 4.7% of lactose and 0.9% of ash. Protein components are mainly composed of casein and whey protein. Other trace components include nonprotein nitrogenous compounds, protease peptones and other trace enzymes.

In the cheese industry, milk proteins are separated into casein and whey proteins mainly by two precipitation methods: rennet precipitation and acid precipitation. In rennet precipitation, lenin is added to warmed milk (30 to 35 ° C). Casein precipitates and only whey protein remains in solution. This type of whey is called sweet whey. Acid precipitation is performed at the isoelectric point of milk (4.7) using the appropriate acid. Whey produced by acid precipitation is called acid whey. The choice of precipitation method depends on what cheese product is desired.

Whey, a byproduct of the cheese industry, is nutritious because it contains many important proteins in its composition. Until recently, however, most of the industrially produced whey was discarded, causing major environmental problems. As environmental controls and regulations become more stringent and more modern technologies are available, such as membrane separations, including ultrafiltration and reverse osmosis, whey proteins and other products made from them meet the needs of the pharmaceutical, dietary and food industries. It is becoming more and more important to make. Research efforts in the area of separating individual proteins from whey and the formulations made therefrom have resulted in many successes in dairy and related industries.

The following patents exemplify several prior efforts to separate individual proteins and other components from whey and food and pharmaceutical products derived therefrom.

US Patent No. 5,077,067 (December 31, 1991, Philippe A. Thibault) discloses a method for the selective and quantitative removal of lactoglobulin from whey protein.

U.S. Patent 5,055,558 (October 8, 1991, Emilia Chiancone and Maurizio Gattoni) describes a method for the selective extraction of β-lactoglobulin from whey or milk by subunit exchange chromatography.

US Pat. No. 4,791,193, issued December 13, 1988 to Shigeo Okonogi et al., Relates to a process for preparing pure lactoferrin from whey or skim milk.

US Patent No. 4,668,771, issued May 26, 1987 to Hiroshi Kawakami et al., Provides a method for the isolation and purification of bovine lactoferrin.

US Pat. No. 4,997,914 (March 5, 1991, Hiroshi Kawakami et al.) Describes a method for separating and purifying lactoferrin by adsorption chromatography.

US Pat. No. 4,820,348 (April 11, 1989, Matti Harju) relates to a chromatographic method for separating lactose from milk.

U.S. Patent 4,446,164 (May 1, 1984, Roy A. Brog) is a milk-like preparation made by adding an additive such as soluble protein, edible vegetable oil, fat-free dry milk solids, sugar or synthetic sweetener to the sweet whey base. It relates to a composition.

US Pat. No. 5,085,881 (February 4, 1992, Hans G. Moeller) describes a method for separating fractions from dry milk or milk products as food ingredients, food supplements, pharmaceutical aids.

U.S. Patent No. 5,093,143, issued March 3, 1992, Horst Behr and Friedrich Manz, relates to a milk mimic nutrient composition that is low in albumin and phosphorus (P) and high in energy and calcium.

US Patent No. 4,202,909 (May 13, 1980, Harold T. Pederson, Jr.) describes a whey treatment method for producing pure lactose and salt products.

U.S. Patent 5,008,376 (April 16, 1991, Robin C. Bottomley) discloses a method for preparing whey fractions with high concentrations of alpha-lactalbumin via ultrafiltration techniques.

U.S. Patent No. 3,969,337 (July 13, 1976, Karl Lauer et al.) Discloses a whey chromatography fractionation technique.

While other experimental and industrial methods may be used to isolate, remove, concentrate, and / or purify a specific whey protein, as demonstrated in the above patents and other documents, these conventional methods may be used for all whey except one protein. Destroying or disposing of the protein results in the loss of other valuable proteins. None of these conventional methods separated several proteins from whey by a one step process. Therefore, there will be a need to provide a method for separating several proteins from whey continuously or sequentially in a one or two stage separation process.

Accordingly, it is an object of the present invention to provide a separation technique that separates whey protein completely sequentially in one or two steps.

Another object of the present invention is to provide a sequential and continuous separation technique of whey protein, suitable for laboratory and industrial use using radial flow chromatography techniques.

Another object is to provide several buffers that are mild enough to be used for sequential separation without denature the whey protein.

Another object is to provide a separation technique that can be applied for food and pharmaceutical use of whey protein.

Another object is to provide a dietary formulation and a pharmaceutical formulation comprising different ratios of whey proteins in different proportions.

Other 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 one skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained through means and combinations particularly pointed out in the appended claims.

<Summary of invention>

In accordance with the objects and principles of the invention described herein, and in order to achieve the above and other objects, the present invention basically separates five or more different proteins sequentially from whey and pharmaceuticals the separated whey protein. Methods of incorporating into formulations and food formulations are provided. The method of the present invention adsorbs proteins in liquid whey on an appropriate separation medium packed in a chromatography column, and sequentially elutes IgG, β-Lg, α-La, BSA and lactoferrin fractions with appropriate buffers of pH and ion concentration. It relates to a method for separating whey protein continuously and sequentially by chromatography. Axial chromatography and radial flow chromatography can be used but horizontal flow columns are particularly suitable for the process of the invention. Whey proteins isolated 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) is included. Various dietary and pharmaceutical formulations of the present invention comprise these isolated proteins in various proportions.

The present invention relates to a method for separating whey protein, in particular a method for sequential separation of whey protein using chromatography and related food and pharmaceutical formulations using isolated whey protein.

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

2 shows the dissolution profile of the separated protein over time.

3 shows the elution pattern and location of peak 4.

According to the method of the present invention, the pasteurized sweet whey, pasteurized acid whey, by-product of cheese production are made from pasteurized or non-cold pasteurized whey through known techniques such as reverse osmosis (RO) or ultrafiltration (UF). A sample of starting material selected from non-sterile acid whey, or whey protein concentrate, obtained by means of a chromatography column filled with an acidic or basic cationic or anionic resin material, such as micro-prep high S or Q, preferably horizontal flow chromatography Load into the chromatography column. Preliminary separation of whey, concentrated whey or whey protein concentrate by electrodialysis or ion exchange, purification to remove casein fines, and / or microfiltration to separate colloidal and suspended particles, including fatty residues Preliminary separation can be achieved through the process.

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

Example 1 Sequential Separation of Sweet Whey Protein

As a byproduct of mozzarella cheese production, commercial whey is first purified to remove casein fines, centrifugation to remove milk fat residue, pasteurized at 162 ° F. (about 72 ° C.) for about 18 seconds, and through a HTST plate heat exchanger. Passed through and cooled to 40 ° F (about 4 ° C). 1000 ml of this skimmed commercial sweet whey (pH 6.4 and 6.2% total solids) were adjusted to pH 3.8 with acetic acid at 40 ° F. (about 4 ° C.). The composition of the whey product used in this experimental example is shown in Table I below.

whey ingredient percentage Protein composition Total solids 6.2 β-lactoglobulin 0.29% Lactose 4.5 α-lactalbumin 0.13% protein 0.8 Serum Casein 0.21% Fat 0.08 Immunoglobulin 0.06% Ash 0.77 Lipoprotein 0.06% Lactic acid 0.05 Bovine Serum Albumin 0.03% Lactoferrin 0.02%

The whey was then passed through a 250 ml radial flow chromatography column prefilled with strong S cation exchange resin and equilibrated with 0.05 M acetate buffer (pH 3.8). All whey proteins bound to the resin matrix and passed through an eluate containing lactose, minerals, lactic acid, as well as nonprotein components and nonprotein nitrogenous components. The protein bound resin was washed with 0.05 M acetate buffer (pH 3.8) to precondition UV baseline. Various bound proteins were then eluted sequentially according to the following protocol.

Immunoglobulins (IgG) and β-lactoglobulin (β-Lg) were eluted sequentially with buffer (pH 4.0) containing 0.1 M sodium acetate and 0.5 M sodium chloride.

The column was then conditioned again and equilibrated with 0.05 M sodium acetate buffer (pH 4.0) to return conductivity to baseline.

α-lactalbumin (α-La) fractions were eluted with pH 5.0 buffer containing 0.1 M sodium acetate and 0.1 M sodium chloride. The column was conditioned again with pH 5.0 buffer containing 0.05 M sodium acetate to return the conductivity back to the initially set baseline. Bovine serum albumin (BSA) was then eluted with 0.05 M phosphate buffer (pH 7.0). Lactoferrin (L-Fe) was then eluted with buffer (pH 7.5) containing 0.05 M sodium phosphate and 0.5 M sodium chloride.

The column was regenerated by washing with a solution containing 0.2 M sodium hydroxide and 1 M sodium chloride and washed with 20% ethanol (EtOH) solution to equilibrate the column with acetate buffer (pH 3.8) for sterilization and reuse.

A flow chart showing the elution protocol is shown in Table II below.

Each fraction of eluted protein was collected as term elution "peak" for further separation, concentration and other processing protocols. The elution sequence with different protein peaks is shown in Figure 1 by UV absorption at 280 nm. Protein identification of each peak was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as is known in the art. Protein recovery is shown in Tables III and IV below by monitoring by Bio-Rad and gel injection assays in several stages of elution schemes.

Summary of Bio-Rad Analysis Data (# 0) Column Load 1.0 L Loaded 4.3 mg / ml Total Protein 4.3 g Loaded (1) column effluent 1.65 L 0.2 mg / ml total protein 0.3 g (7%) (# 2) β-Lg + lgG fraction 2.4 L 1.2 mg / ml total protein 2.9 g (67%) (# 3) α-La fraction 1.25 L 0.5 mg / ml total protein 0.6 g (14%) (# 4) BSA fraction 1.625 L 0.3 mg / ml total protein 0.4 g (9%) (# 5) L-Fe fraction 0.625 L 0.09 mg / ml total protein 0.05 g (1%) (# 6) Wash solution 1 1.6 L 0.03 mg / ml Total protein 0.05 g (1%) (# 7) Wash solution 2 0.4 L 0.09 mg / ml Total protein 0.04 g (1%) Total recovery = 4.3 g = 100% rating

Summary of Gel Injection Data Gel # 1 R2 = 0.98 Column Load 1.0 L Treated Whey 4.5 mg / ml Total Protein 4.5 g Loaded Approximate% Composition 76% β-Lg 17% No protein eluted in α-La effluent 2% BSA 3% IgG 2% Other Gel # 2 R2 = 0.99 Volume = 2.4 L β-Lg and IgG Fraction 1.2 mg / ml Total Protein 2.9 g Recovered (64%) 94% β-Lg 2% IgG Gel # 3 R3 = 0.993 volume = 1.25 L α-La fraction 0.03 mg / ml total protein 0.4 g recovered (9%) 94% α-La Gel # 4 R3 = 0.991 Volume = 1.625 L BSA Fraction 0.625 L L-Fe Fraction 0.08 mg / ml Total Protein in BSA Fraction 0.13 g (3%) 62% BSA Recovery (Min) 0.05 mg / ml in L-Fe Fraction Protein 0.03 g (1%) 45% L-Fe recovery (min)

Example 2 Alternative Protocol for Elution of Whey Protein

A 20 liter RFC column was filled with macro-prep 50 S resin. The column is then conditioned, equilibrated, loaded, eluted and again in exactly the same manner as described in Example 1, except that the flow rate, volume of whey loaded on the column, flow rate and buffer volume are varied. It was conditioned. Protein elution peaks were monitored using a UV spectrophotometer at 280 nm. A graphical record of the eluted protein relative to relative concentration is shown in FIG. 2. Eluted proteins with each ratio of purity are shown in Tables V and VI below.

Protein yield in the eluate fraction (20 L column; flow rate 8 L / min; whey load 80 L) Protein (g / L) Volume (L) Protein yield load (g) % Whey Load 8.8 80 704 - Effluent (P-1) 0.9 97 87 12 β-La + IgG (P-2) 2.8 89 249 35 α-lactalbumin (P-3) 1.1 94 103 15 BSA (P-4) 1.1 102 112 16 Lactoferrin (P-5) 0.9 45 41 6 Rinse solution (P-6) 0.8 22 18 3 Stripping Solution (P-7) 1.2 29 35 5 Protein recovery: 84%; Protein rating: 92%

Protein Purity (gel injection) % β-lactoglobulin 82 Immunoglobulins 11 α-lactalbumin 84 Bovine serum albumin 59 Lactoferrin 52

Example 3 Preparation of Anion Exchange Resin Column

A 250 ml RFC column was charged with strongly basic anion exchange resin (macro-prep 50 Q) and conditioned with 0.2 M NaOH + 1 M NaCl at a flow rate of 100 ml / min for 10 minutes. The column was then equilibrated with 0.01 sodium phosphate (pH 6.90) at a flow rate of 100 ml / min for 10 minutes. This column was then used to separate immunoglobulins (IgG) from β-lactoglobulins eluted as overlapping peaks from Examples 1 and 2 above. This mixture can be mixed into a dietary formulation or used for further separation of the two protein components.

Example 4 Isolation of Immunoglobulins (IgG) from β-lactoglobulin

The eluate, collected from the fractionated material of the method described in Example 1 and represented by peak 2 containing IgG and β-Lg (pH 4.0), was passed through a 10,000 molecular weight filtration UF membrane to concentrate the protein, buffer salt concentration, That is, the ion concentration of the solution was reduced. The protein was further concentrated to 5 times the initial eluted concentration and the buffer salt concentration was diafiltered with distilled water to reduce the eluted concentration to approximately ¼. The diafiltered and concentrated protein solution was adjusted to pH 6.9 with 2.0 M NaOH solution. Two liters of this protein solution (pH 6.9) were loaded at a pre-conditioned RFC column as described in Example 3 at a flow rate of 100 ml / min. The column was washed with 0.01 M sodium phosphate buffer (pH 6.9) to create a UV baseline. IgG that did not bind to the resin was passed through the column with wash and collected for further processing. Adsorbed β-Lg was then eluted from the column with 0.05 M sodium citrate buffer (pH 3.0) and collected. The column was rinsed with distilled water and the residual protein was removed with a 0.2 M NaOH + 1 M NaCl solution followed by 20% EtOH and re-equilibrated again in the preparation for the next cycle with sodium phosphate buffer (pH 6.9).

Example 5 Isolation and Isolation of BSA

The eluate, collected from the material fractionated from the method described in Example 1, represented by peak 4 containing BSA and protease peptone (pH 7.0), was concentrated and diafiltered as described in Example 4, and then the pH with acetic acid. Was adjusted to 5.5. A 250 ml RFC column prepared as described in Example 4 was rinsed with distilled water at a flow rate of 100 ml / min. Two liters of this protein solution was loaded on the column described above. The column was flushed again with distilled water at a flow rate of 100 ml / min to elute nonadsorbed protease peptone and to create a stable UV baseline. Eluates containing protease peptone were collected for further use. The adsorbed BSA was then eluted with sodium phosphate buffer (pH 7.0) containing 0.2 M sodium chloride. 3 shows the elution pattern and position of peak 4.

Example 6 Elution and Separation of β-lactoglobulin from liquid whey

The 250 ml radial flow chromatography column filled with strongly basic anion exchange resin (macro-prep 50 Q) was washed and regenerated according to the manufacturer's instructions. The column was then equilibrated with 0.05 M sodium phosphate (tribasic) (pH 7.5) for 10 minutes at a flow rate of 100 ml / min. The pH range of 7.0 to 8.0 did not significantly affect the elution pattern. 2 liters of clarified, degreasing, pasteurized sweet whey resulting from mozzarella cheese cooling was cooled to 40 ° F. (approx. 4 ° C.), pH adjusted to 8.0 with 5 M sodium hydroxide, pre-prepared at 75 mil / min for equilibrium Through a heated column. Flow rates in the range of 50 to 100 ml / min can be used. One to three liters of whey sample for analysis were then loaded onto the column and the column was eluted with loading buffer (0.05 M sodium phosphate, pH 7.5). Under the conditions used, all whey proteins except β-lactoglobulin were positively charged. The negatively charged β-lactoglobulin was bound and retained by the anion exchange resin. Eluate containing unbound protein [α-lactalbumin (α-La), immunoglobulin (Ig-G), bovine serum albumin (BSA) and lactoferrin (L-Fe)] was collected by passing through a column and further Store at 40 ° F. (about 4 ° C.) for processing.

The adsorbed β-lactoglobulin was then eluted from the column with buffer (pH 7.5) containing 0.05 M sodium phosphate and 0.5 M sodium chloride. The β-lactoglobulin-containing eluate can be further processed in order to further prepare a storage stable product in the same manner as described in Example 8 below.

The column is washed with 1 M sodium chloride at a flow rate of 125 ml / min for about 4 column volumes (2 liters), stripped with 1 M sodium hydroxide at the same flow rate, and 100 ml for about 5 column volumes (2 ½ liters) Regenerated with 1 M sodium chloride at a flow rate per minute and sterilized with 200 ppm sodium hypochlorite at 100 ml / min for about 4 column volumes (2 liters) and then equilibrated with loading buffer in preparation for the next cycle.

Example 7 Elution and Sequential Separation of Four Proteins from Non-β-Lactoglobulin in Liquid Whey

The effluent fraction from Example 6 containing 0.55% protein was passed through a radial ultrafiltration membrane for 10000 molecular weight filtration to 35% of the original volume and removed as permeate for partial protein concentration and reduction of soluble salts. This pre-treatment process facilitates optimal absorption and sequential desorption of Ig-G, α-La, BSA and L-Fe protein fractions as outlined in Example 1. The resulting effluent was adjusted to pH 3.8 with acetic acid and 1500 ml of the sample loaded onto a 250 ml RFC column filled with strong S-cation exchange resin and equilibrated with 0.05 M sodium acetate buffer (pH 3.8). Washing, sequential elution and regeneration steps were performed as outlined in Example 1. The eluted protein fractions are individually passed through appropriate molecular weight filtration (50000 MW filtration) membranes for IgG, L-Fe and BSA and 10000 MW filtration membranes for α-La to concentrate the protein, and the salt residue Removed. It was then further processed to the final product as outlined in Example 8.

<Example 8>

Whey protein fractions, or isolated and purified proteins and non-proteinaceous eluents may be incorporated into dietary and pharmaceutical formulations in appropriate proportions. Such formulations include, but are not limited to, baby food, fatty substitutes, blowing agents, egg white substitutes, animal feed substitutes, and the like.

<Example 9> Baby food

In baby foods constructed in accordance with the present invention, whey fractions of casein and milk were modified to obtain compositions that mimic breast milk much more highly than prior art compositions and commercial products. The baby food of the present invention contained levels of whey protein similar to that of breast milk. This was accomplished by producing a mixture of whey protein components containing the proportion and type of whey protein in breast milk.

Commercially available baby foods are prepared from whole cow's milk because they are mostly sold in large quantities. Other additives or auxiliaries may be included. These formulations are prepared as powders that are concentrated or easy to feed. These mostly consisted of nonfat milk solids, vegetable oils, and carbohydrate sweeteners such as lactose, corn syrup solids and sucrose. In addition, these preparations may be fortified with vitamin C, vitamin D, iron and fluoride. Table VII below shows the typical compositions of some exemplary commercial baby foods compared to one exemplary composition of the present invention. The levels of vitamins, minerals and other nutrients in the formulations of the present invention were adjusted to mimic breast milk and meet the nutritional conditions of infants.

Composition ranges of some commercially available baby food compared to one exemplary formulation of the present invention Nutrients Commercial baby food (Similac, Allmentum, Good Start, etc.) Formulations of the Invention Per 5 oz (~ 12.5% solids) of manufactured food ingredient Per 5 oz (~ 12.5% solids) of manufactured food ingredient protein 2.14-2.75 g NFDM ^* -casein hydrolysate-whey protein composition 1.1-2.50 g Purification, dry or wet mixture of selected proteins Fat 5.1-5.54 g Vegetable oil, coconut oil, soybean oil, palm oil, safflower oil, sunflower oil 4.30-6.48 g Vegetable oil, milk fat carbohydrate 10.2-11.0 g Lactose, sucrose 10.19-10.50 g Lactose water 133-135 g 15-130 g Linoleic acid 850-1600 mg 1200-1300 mg Vitamin A 300 IU 300-350 IU Vitamin D 45-60 IU 50-60 IU Vitamin E 2.0-3.0 IU 2.2-2.7 IU Vitamin c 9 mg 7-9 mg Vitamin K 8-15 mcg 8-10 mcg Vitamin B1 60-100 mcg 25-100 mcg Vitamin B2 90-150 mcg 50-150 mcg Vitamin B6 60-75 mcg 20-60 mcg Vitamin B12 0.22-0.45 mcg 0.10-0.25 mcg Niacin 750-1350 mcg 300-1100 mcg Folic acid 9-15 mcg 7-15 mcg Pantothenic Acid 450-750 mcg 330-450 mcg Biotin 2.2-4.5 mcg 2-4 mcg Choline 8-16 mg 10-16 mg Inositol 4.7-18 mg 4.5-5.5 mg calcium 64-105 mg 47-73 mg sign 36-75 mg 21-56 mg magnesium 6.0-7.5 mg 4.4-6.0 mg iron 0.5-1.8 mg 0.04-1.8 mg zinc 0.75 mg 0.25-0.75 mg manganese 5-30 mcg 5-10 mcg Copper 75-90 mcg 75-90 mcg iodine 8-15 mcg 9-12 mcg salt 24-44 mg 25-40 mg potassium 98-118 mg 75-110 mg chloride 59-80 mg 59-80 mg cholesterol - 18-25 mg * NFDM-fat free dry milk solids

However, due to the large difference in protein composition of milk and breast milk, some infants exhibit different degrees of hypersensitivity to milk and food preparations prepared therefrom. The composition of milk is compared with that of breast milk and is shown in Table VIII below, and the comparison of protein content is shown in Table IX below.

Comparison of cow's milk with human breast milk Per 100 g Cow (Milk) Man (breast milk) Water g 89.99 87.5 Food energy kcal 61 70 Protein (N × 6.38) g 3.29 1.03 Fat g 3.34 4.38 Carbohydrates (total) g 4.66 6.89 Fiber g 0 0 Ash g 0.72 0.2 Mineral Calcium mg 119 32 Iron mg 0.05 0.03 Magnesium mg 13 3 Phosphorus mg 93 14 Potassium mg 152 51 Sodium mg 49 17 Zinc mg 0.38 0.17 vitamin Ascorbic acid mg 0.94 5.00 Thiamine mg 0.038 0.014 Riboflavin mg 0.162 0.036 Niacin mg 0.084 0.177 Pantothenic Acid mg 0.314 0.223 Vitamin B6 mg 0.042 0.011 Folic acid mcg 5 5 Vitamin B12 mcg 0.357 0.045 Vitamin A IU 126 241 Cholesterol mg 14 14

Protein composition g / 100 g (milk and breast milk) protein Cow (Milk) Person (breast milk) Casein (all) 2.6 0.32 β-lactoglobulin 0.32 Can be ignored α-lactalbumin 0.12 0.28 Serum albumin 0.04 0.06 Lysozyme Can be ignored 0.04 Lactoferrin 0.02 0.20 Immunoglobulins 0.07 0.10

Whey proteins in various ratios of milk and breast milk are shown in Table X below.

Whey protein in different proportions in milk and breast milk protein Milk (g / 100 g) %(One) Breast milk % H / C Ratio (2) 1 × 2 Mixture composition α-La 0.12 48 0.28 43.75 2.33 1.10 43.5 L-Fe 0.02 8 0.20 31.25 10 0.80 31.6 lgG 0.07 28 0.10 15.63 1.43 0.39 15.4 BSA 0.04 16 0.06 9.37 1.5 0.24 9.5

As shown in the table above, milk contained 3.3% protein, while breast milk contained only 1%. Casein is the major protein component in milk (about 77% of total protein), while breast milk contains a high ratio (about 2: 1) of whey protein to casein. The β-lactoglobulin concentration in milk was the highest of whey protein, while negligible in breast milk. Similarly, lactoferrin was 10 times higher at the concentration of breast milk than milk. Immunoglobulin and serum albumin concentrations were about 1.5 times higher in breast milk than in milk.

In the baby food of the present invention, lactose and fat levels were adjusted to mimic breast milk. Vegetable fats substituted butter fats. In addition, the ratio of casein to whey was reduced to mimic breast milk. If necessary, other additives and supplements such as vitamins, taurine and minerals may be included. Total solute addition was reduced to the level found in breast milk.

To achieve this goal, the whey protein fractions obtained from fractionation and elution according to the method of the invention were first formulated in the proportions shown in Table XI below.

Dietary Formulation A α-lactalbumin fraction 43.5% Lactoferrin fraction 31.6% Immunoglobulin Fractions 15.4% Bovine Serum Albumin Fraction 9.5%

The whey protein mixture was then incorporated into breast milk-like formulations in the compositions shown in Table XII below.

baby food ingredient Liquid formulation (g / 100 g) Dry formulation base (g / 6 oz liquid) water 87.20 0 β-casein 0.28 0.504 κ-casein 0.04 0.072 Prepared Whey Protein Mixture α-La 0.64 1.152 BSA L-Fe lg-G Lysozyme 0.04 0.072 Lactoperoxidase 750 (activity) 1350 Fat 4.5 8.1 Lactose 7.0 12.6 Ash 0.2 0.36

The liquid mixture prepared according to this composition contained about 2% of total solids (90% of the salt from the elution buffer and about 10% of the total protein), and 98% of water. This liquid mixture was then concentrated through a 10,000 molecular weight filtration spiral ultrafiltration membrane to 5-15% of total protein, followed by diafiltration with distilled water at 0.5-1.0 times to remove remaining salt residues. This formulation may be further concentrated by methods commonly used for the treatment of unstable proteins, such as ultrafiltration, reverse osmosis, lyophilization, freeze concentration, spray drying, or any combination thereof. The formulations of the present invention may be further enhanced with suitable additives and nutrient enhancers. Such additives and fortifying agents include, but are not limited to, nonfat milk solids, vegetable solids, carbohydrate sweeteners, minerals and vitamins. Solid compositions of one exemplary formulation of the invention are shown in Table XIII below.

Solid Compositions of One Exemplary Formulation of the Invention ingredient gm / 16 oz formulation powder protein Casein Hydrolyzate 11.00 α-lactalbumin 9.63 Bovine serum albumin 2.06 Lactoferrin 6.88 Immunoglobulins 3.44 Lysozyme 1.38 Fat Coconut oil 53.56 Sunflower oil 46.08 Corn oil 36.79 Butter fat 14.10 carbohydrate Lactose 236.95 Water content 15.20 Linoleic acid 2.41 vitamin Vitamin A 8288 (IU) Vitamin D 1720 (IU) Vitamin E (Tocopherol) 86 (IU) Vitamin K 0.0003 Vitamin B1 0.0018 Vitamin B2 0.0012 Vitamin B6 0.0004 Vitamin B12 0.0000002 Vitamin C (ascorbic acid) 0.17 Niacin 0.006 Folic acid 0.0001 Pantothenic Acid 0.008 Biotin 0.0001 Choline 0.34 Inositol 0.18 Mineral calcium 1.10 sign 0.48 magnesium 0.10 iron 0.001 zinc 0.006 manganese 0.0003 Copper 0.003 iodine 0.0003 salt 0.59 potassium 1.76 chloride 2.06 cholesterol 0.48

Example 10 Formulation as a Fat Substitute

β-lactoglobulin exhibits high water-binding properties, and α-lactalbumin increases viscosity and also absorbs high amounts of fats and oils. Because of these properties, these protein combinations are used as fat substitutes. Such products, when included in appropriate proportions in some foods, can improve the quality and properties of the product and can be used as a fat substitute.

In the method of the present invention, β-lactoglobulin was eluted with lgG as fraction (2), and α-lactalbumin was eluted as fraction (3). lgG was isolated from the β-lactoglobulin fraction as described in Example 7 and mixed with the α-lactalbumin fraction at 60 and 40% ratio. A mixture of two proteins (β-La + α-La) was passed through a ultrafiltration membrane for filtration of 10,000 molecular weights of 40 ° F (about 4 ° C.) at a partial pressure of 10 psi, and a total solid concentration of 40-50% was obtained. The permeate consisting of water and soluble salts was removed until achieved. The remaining salt residue was then removed by diafiltration with 0.5 times distilled water. The concentrated, purified mixture thus obtained can be frozen, freeze dried, cooled or dehydrated for other uses. Other additives such as flavors, vitamins and sweeteners may be included in these formulations as needed.

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

The foregoing description of the preferred embodiments of the present invention is for the purpose of illustration and description, and for further understanding of the present invention. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and obviously, many modifications and variations are possible in light of the above teaching. In order to explain the principles of the present invention and its industrial uses well and to enable those skilled in the relevant art to utilize the invention in various embodiments, as well as in various modifications as suited to the particular use, specific embodiments have been selected and described in some detail. Described. The invention is defined by the claims appended hereto.

Claims (36)

Whey protein by chromatography, comprising adsorbing liquid whey on a separation medium packed in a chromatography column, and sequentially eluting immunoglobulins, β-lactoglobulins, α-lactalbumin, bovine serum albumin and lactoferrin fractions. Method of continuous sequential separation. The method of claim 1, wherein the liquid whey is selected from the group consisting of pasteurized sweet whey, pasteurized acid whey, unpasteurized acid whey and whey protein concentrate. The method of claim 1, wherein the separation medium is a cationic resin. The method of claim 1, wherein the sequentially eluted immunoglobulin, β-lactoglobulin, α-lactalbumin, bovine serum albumin and lactoferrin fractions are collected and concentrated by ultrafiltration. The method of claim 4, wherein the concentrated immunoglobulin, β-lactoglobulin, α-lactalbumin, bovine serum albumin and lactoferrin fractions are further purified by diafiltration. a) filling the chromatography column with cation exchange resin to obtain a packed chromatography column, b) equilibrate the packed chromatography column, c) feed the whey sample, d) passing the whey sample through the packed chromatography column under conditions that the whey protein is adsorbed to the packed chromatography column, e) collecting an effluent comprising lactose, minerals, lactic acid and non-nitrogenous components from the packed chromatography column, f) immunoglobulin and β-lactoglobulin are eluted sequentially from the packed chromatography column, g) α-lactalbumin is eluted from the packed chromatography column, h) reconditioning the packed chromatography column, i) bovine serum albumin is eluted from the packed chromatography column, j) eluting lactoferrin from the packed chromatography column Sequential separation method of whey protein comprising the step. 7. The method of claim 6, wherein the whey is selected from the group consisting of pasteurized sweet whey, pasteurized acid whey, unpasteurized acid whey and whey protein concentrate. The method of claim 6, wherein the chromatography column is a radial flow column. a) filling the chromatography column with an anion exchange resin to obtain a first packed chromatography column, b) equilibrate the first packed chromatography column, c) feed the whey sample, d) passing the whey sample through the first packed chromatography column under conditions that β-lactoglobulin is adsorbed to the first packed chromatography column, e) collecting an effluent comprising α-lactalbumin, immunoglobulin, bovine serum albumin and lactoferrin suitable for further processing from the first packed chromatography column, f) eluting the adsorbed β-lactoglobulin from the first packed chromatography column with a buffer to obtain an eluate. A method of separating β-lactoglobulin from whey protein comprising the step. The method of claim 9, wherein the first packed chromatography column is a radial flow column. The method of claim 9, g) filling the second chromatography column with a cation exchange resin to provide a second packed chromatography column, h) equilibrating the second packed chromatography column, i) passing the effluent through an ultrafiltration membrane for 10,000 molecular weight filtration to obtain an ultrafiltrate, j) passing the ultrafiltrate through the second packed chromatography column under conditions that immunoglobulin, α-lactalbumin, bovine serum albumin and lactoferrin are adsorbed to the second packed chromatography column, k) eluting said immunoglobulin from said second packed chromatography column, l) eluting the α-lactalbumin from the second packed chromatography column, m) reconditioning the second packed chromatography column, n) eluting the bovine serum albumin from the second packed chromatography column, o) eluting the lactoferrin from the second packed chromatography column The method further comprises a step. The method of claim 11, wherein the second packed chromatography column is a radial flow column. The method of claim 12, wherein the effluent comprises baby food. The method of claim 13, wherein the baby food contains at least 25% lactoferrin and less than 1/2 of the original content of β-lactoglobulin. The method of claim 13, wherein the baby food comprises about 43.5% α-lactalbumin, about 31.6% lactoferrin, about 15.4% immunoglobulin, and about 9.5% bovine serum albumin. 15. The method of claim 14, wherein said baby food further comprises casein hydrolyzate, fat, nonfat milk solids, carbohydrates, minerals and vitamins. The method of claim 13, wherein the effluent is combined with the eluate to produce a fat surrogate. 18. The method of claim 17, wherein said fatty substitute comprises about 60% β-lactoglobulin and 40% α-lactalbumin. a) filling the chromatography column with cation exchange resin to obtain a packed chromatography column, b) equilibrating the packed chromatography column with buffer, c) feeding a whey sample containing whey protein including lactoferrin, immunoglobulin, β-lactoglobulin, α-lactalbumin and bovine serum albumin, d) passing the whey sample through the packed chromatography column under conditions that at least a portion of the whey protein is adsorbed to the packed chromatography column, e) washing the packed chromatography column with buffer, f) sequentially eluting immunoglobulin and β-lactoglobulin from the packed chromatography column into a buffer, g) conditioning the packed chromatography column again, h) α-lactalbumin is eluted from the packed chromatography column with buffer, i) conditioning the packed chromatography column again with buffer, j) bovine serum albumin is eluted from the packed chromatography column with buffer, k) eluting lactoferrin from the packed chromatography column into a buffer to produce an eluate containing lactoferrin. Sequential separation method of whey protein comprising the step. 20. The method of claim 19, wherein said whey is selected from the group consisting of pasteurized sweet whey, pasteurized acid whey, unpasteurized acid whey and whey protein concentrate. The method of claim 19, wherein the chromatography column is a radial flow column. a) filling the radial flow chromatography column with an anion exchange resin to provide a first packed chromatography column, b) equilibrating the first packed chromatography column with buffer, c) feeding a whey sample containing whey protein including lactoferrin, immunoglobulin, β-lactoglobulin, α-lactalbumin and bovine serum albumin, d) passing said whey sample through said first packed chromatography column under conditions that β-lactoglobulin is adsorbed to said first packed chromatography column and a permeate flows through said first packed chromatography column, e) collecting said permeate comprising α-lactalbumin, immunoglobulin, bovine serum albumin and lactoferrin suitable for further processing from said first packed chromatography column, f) eluting the adsorbed β-lactoglobulin from the first packed chromatography column into a buffer to obtain an eluate, g) filling the second chromatography column with a cation exchange resin to obtain a second packed chromatography column, h) equilibrating the second packed chromatography column, i) passing the collected permeate from the first packed chromatography column through an ultrafiltration membrane to obtain an ultrafiltrate, k) passing the ultrafiltrate through the second packed chromatography column under conditions that immunoglobulin, α-lactalbumin, bovine serum albumin and lactoferrin are adsorbed to the second packed chromatography column, l) eluting the immunoglobulin from the second packed chromatography column with a buffer, m) conditioning the second packed chromatography column again with buffer, n) eluting the α-lactalbumin from the second packed chromatography column with a buffer, o) conditioning the second packed chromatography column again with buffer, p) eluting the bovine serum albumin from the second packed chromatography column into a buffer, q) eluting the lactoferrin from the second packed chromatography column into a buffer A method of separating β-lactoglobulin from whey protein comprising the step. The method of claim 22, wherein the permeate is combined with the eluate obtained in step f) to produce a fat surrogate. The method of claim 23, wherein the fat substitute comprises about 60% of the eluate and 40% of permeate. The method of claim 22, further comprising combining the α-lactalbumin, immunoglobulin, and serum albumin of the cow to produce a baby food. The method of claim 25, wherein the baby food further comprises at least 25% of α-lactoferrin and less than 1/2 of the original content of β-lactoglobulin. 27. The method of claim 25, wherein said baby food further comprises casein hydrolyzate, fat, nonfat milk solids, carbohydrates, minerals and vitamins. The elution in 43.5% of the α-lactalbumin eluted in step q), eluting from step 3), eluting from said second packed chromatography column in step n). About 15.4% immunoglobulin and about 9.5% bovine serum albumin eluted in step p). Infant food containing at least 25% lactoferrin and less than half the original content of β-lactoglobulin. Baby food comprising α-lactalbumin, lactoferrin, immunoglobulin and bovine serum albumin. 31. The baby food according to claim 30, wherein said lactoferrin is contained at least 25%. 32. The baby food of claim 30 further comprising an additive and a nutrition enhancer. 33. The baby food according to claim 32, wherein said additives and nutritional supplements are selected from the group consisting of nonfat milk solids, vegetable solids, carbohydrate sweeteners, minerals and vitamins. A baby food comprising about 43.5% α-lactalbumin, about 31.6% lactoferrin, about 15.4% immunoglobulin and about 9.5% bovine serum albumin. A fatty substitute comprising about 60% β-lactoglobulin and about 40% α-lactalbumin. 36. The fatty substitute of claim 35, further comprising an additive and a flavor enhancer.