Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
  • A23C9/00—Milk preparations; Milk powder or milk powder preparations
  • A23C9/14—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment
  • A23C9/146—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment by ion-exchange
  • A23C9/1465—Chromatographic separation of protein or lactose fraction; Adsorption of protein or lactose fraction followed by elution
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
  • A23C11/00—Milk substitutes, e.g. coffee whitener compositions
  • A23C11/02—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins
  • A23C11/04—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing non-milk fats but no non-milk proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
  • A23J1/20—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey
  • A23J1/205—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey from whey, e.g. lactalbumine

Definitions

• the present invention is related to the separation of whey proteins, particularly to the sequential separation of whey proteins using chromatography and to food related and pharmaceutical formulations using separated whey proteins.
• the protein component consists mainly of casein and whey proteins.
• minor components include non-proteinaceous nitrogen compounds, protease peptones, and other minor enzyme proteins.
• milk proteins are separated into caseins and whey proteins, mainly by two types of precipitation techniques - rennet precipitation and acid precipitation.
• rennet precipitation rennin is added to warm milk (30 - 35° C).
• the caseins are precipitated leaving the whey proteins in solution.
• This type of whey is referred to as sweet whey.
• Acid precipitation is carried out at the isoelect c point of milk which is 4.7 by using a suitable acid.
• the whey resulting from acid precipitation is referred to as acid-whey.
• the choice of the method depends on the desired cheese product.
• Whey which is a byproduct of the cheese industry has a high nutritional value because of the many valuable proteins in its composition.
• whey proteins and other products constituted therefrom have become increasingly important in satisfying the needs of the pharmaceutical, dietetic and food industries.
• Research efforts with varying degrees of success in the area of the isolation of individual proteins from whey and formulations constituted therefrom abound in the dairy and related industries.
• Okonogi et al. is directed to a method for the preparation of pure lactoferrin from whey or skim milk.
• U. S. 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.
• U. S. Patent No. 4,997,914 issued March 5, 1991 to Hiroshi Kawakami et al., describes a method for the separation and purification of lactoferrin by adsorption chromatography.
• U. S. Patent No. 4,820,348 issued April 1 1 , 1989 to Matti Harju is directed to a chromatographic method for the separation of lactose from milk.
• U. S. Patent No. 4,446,164 issued May 1 , 1984 to Roy A. Brog relates to milk like compositions constituted from sweet whey base with additives like soluble proteins, edible vegetable oils, non-fat dry milk solids, sugar or synthetic sweeteners included therein.
• U. S. Patent No. 5,008,376 issued April 16, 1991 to Robin C. Bottomley discloses a process for producing a whey fraction with a high concentration of alpha-lactalbumin by ultrafiltration technology.
• U. S. Patent No. 3,969,337 issued July 13, 1976 to Karl Lauer et al. discloses a method for the chromatographic fractionation of whey.
• Another object of the present invention is to provide a separation technique for the sequential and continuous separation of whey proteins which is suitable for laboratory as well as commercial applications using radial flow chromatography technology.
• Yet another object is to provide different buffers which are mild enough to use in sequentially separating whey proteins without denaturing them. Still another object is to provide a separation technique applicable for food and pharmaceutical uses of whey proteins.
• Another object of the invention is to provide dietary and pharmaceutical formulations comprising various separated whey proteins in differing proportions. Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows and in part will become apparent to those 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 attained by means of the instrumentalities and combinations particularly pointed out in the appended claims. Summary Of The Invention
• the present invention basically provides a process for the sequential separation of at least five different proteins from whey and incorporating these separated whey proteins into pharmaceutical and food formulations.
• the process of the invention is directed to the continuous, sequential separation of whey proteins by chromatography, comprising adsorbing the proteins in liquid whey on a suitable separation medium packed in a chromatographic column and sequentially eluting IgG, ⁇ -Lg, ⁇ -La, BSA and lactoferrin fractions with buffers at suitable pH and ionic strength.
• a horizontal flow column is particularly suitable for the process of this invention.
• the whey proteins separated by the process of the invention include ⁇ -lactoglobulin ( ⁇ -Lg), ⁇ -lacta!bumin ( ⁇ -La), bovine serum albumin (BSA), immunoglobulin (Ig-G) and Lactoferrin (L-Fe).
• ⁇ -Lg ⁇ -lactoglobulin
• ⁇ -La ⁇ -lacta!bumin
• BSA bovine serum albumin
• Ig-G immunoglobulin
• Lactoferrin L-Fe
• Figure 1 is a graphic representation of the elution pattern of the various proteins in accordance with this invention.
• Figure 2 presents an elution profile of separated proteins vs. time.
• Figure 3 represents the elution pattern and the location of peak 4.
• RO reverse osmosis
• UF ultrafiltration
• the whey, concentrated whey or whey protein concentrate may be subjected to pre-separation procedures such as de-ashing through electrodialysis or ion exchange, clarification to remove casein fines, and /or microfiltration for separating colloidal and suspended particles including fat residues.
• the whey was then passed through a 250 ml radial flow chromatographic column prepacked with a strong S cation exchange resin and equilibrated with 0.05 M acetate buffer at pH 3.8. All the whey proteins were bound to the resin matrix, and the effluent containing non-protein components including lactose, minerals, lactic acid, and non-protein nitrogenous components is allowed to pass through. The resin with the bound proteins was then washed with 0.05 M acetate buffer at pH 3.8 to a preset UV baseline. The various bound proteins were then sequentially eluted in accordance with the following protocol:
• Immunoglobulin (IgG) and ⁇ -lactoglobulin ( ⁇ -Lg) were eluted in sequential order with a buffer at pH 4.0 containing 0.1 M sodium acetate and
• ⁇ -Lactalbumin ( ⁇ -La) fraction was eluted with a pH 5.0 buffer containing
• lactoferrin (LF) was eluted at pH 7.5 with a buffer containing 0.05 M sodium phosphate and 0.5 M sodium chloride.
• the column was again regenerated by washing it with a solution containing 0.2 M sodium hydroxide and 1 M sodium chloride, followed by a wash with a 20% ethanol (EtOH) solution to sterilize the column and equilibrated with acetate buffer at pH 3.8 for reuse.
• EtOH 20% ethanol
• Whey 1000 ml skimmed sweet whey from mozzareila cheese manufacture - at 0-50°F, pH 6 4) i pH adjusted to 3.8 (with acetic acid) i Loading (on a reconditioned, strong acid cationic exchanger packed in a 250 ml RFC column, @ 100 ml/min) i Row Through ⁇ - - ⁇ - Washing
• a 20 liter RFC column was packed with a macro-prep 50 S resin.
• the column was then conditioned, equilibrated, loaded, eluted and reconditioned in exactly the same manner as described in Example 1 above, except that the flow rates, volume of whey loaded on to the column, flow rates and buffer volumes were varied. Protein elution peaks were monitored at 280 nm using a uv spectrophotometer. A graphical trace of the eluted proteins with their relative concentrations is presented in Figure 2. The proteins eluted with their respective percentages of purity are shown in Tables V and VI.
• Lactoferrin (P-5) 0.9 45 41 6
• Lactoferrin 52 Example 3 - Preparation Of An Anionic Exchange Resin Column
• the eluate represented by peak 2 collected from the fractionated material from the process described in Example 1 , and containing IgG and ⁇ -Lg at pH 4.0, was passed through a 10,000 molecular weight cut-off UF membranes for concentrating the proteins and for reducing the buffer salt concentration and theieby, the ionic strength of the solution
• the proteins were further concentrated to 5x their initial eluted concentrations and buffer salt concentrations were reduced to about one-fourth their eluting concentration by diafiltration with distilled water
• the diafiltered and concentrated protein solution was pH adjusted to 6.9 with a 2 0M solution of NaOH Two liteis of this protein solution at pH 6.9 was loaded on to the pre-conditioned RFC column as described in Example 3, at a flow rate of 100ml/min.
• the eluate represented by peak 4, collected from the fractionated material from the process described in Example 1 , and containing BSA, and protease peptone at pH 7.0 was concentrated and diafiltered as described in Example 4, then pH adjusted to 5.5 with acetic acid.
• 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 the protein solution were loaded onto the column as described earlier. The column was again flushed with distilled water at a flow rate of 100 ml/min to elute the nonadsorbed protease peptone and to establish a stable UV baseline.
• the eluate containing the protease peptone was collected for further use.
• the adsorbed BSA was thereafter eluted with sodium phosphate buffer containing 0.2M sodium chloride at pH 7.0.
• Figure 3 represents the elution pattern and the location of peak 4.
• a 250 ml radial-flow chromatographic column packed with a strong base anionic exchange resin (macro-prep 50 Q) was washed and regenerated according to manufacturer's instructions.
• the column was then equilibrated with 0.05M sodium phosphate (tribasic) at pH 7.5 at a flow rate of 100 ml/min for
• the adsorbed ⁇ -lactoglobulin was then eluted from the column with a pH 7.5 buffer containing 0.05 M sodium phosphate and 0.5 M sodium chloride.
• This eluate containing ⁇ -lactoglobulin may be processed further to prepare a shelf stable product in the same manner as described in Example 8 below.
• the column was washed with 1 M sodium chloride at a flow rate of 125 ml/min for about four column volumes (2 liters), stripped with 1 M sodium hydroxide at the same flow rate, regeneiated with 1 M sodium chloride at a flow rate of 100 ml/min for about five column volumes (2 1/2 liters) sanitized with 200 ppm sodium hypochlorite at 100 ml/mm for about four column volumes (2 liters) and then equilibrated with the loading buffer in preparation for the next cycle.
• Example 6 The flow-through fraction from Example 6, containing 0.55% protein, was passed through a 10000 molecular weight cut-off, spiral ultra-filtration membrane to a 35% of the original volume, removed as a permeate for the purpose of partial protein concentration and also for reduction of soluble salts.
• This pre-treatment procedure facilitates the optimum absorption and sequential desorption of Ig-G, ⁇ -La, BS ⁇ and L-Fe protein fractions as outlined in Example
• Example 1 The prepared flow-through was pH adjusted to 3.8 with acetic acid and 1500 ml sample of it was loaded onto a 250 ml RFC column packed with a strong S- cationic exchange resin and pre-equilibrated with 0.05 M sodium acetate buffer at pH 3.8. Washing, sequential elution and regeneration steps as outlined in Example 1 were followed. The eluted protein fractions were individually passed through the appropriate molecular weight cut-off - 50000 MW cut-off membrane for Ig-G, L-Fe and BSA and 10000 MW cut-off membrane for ⁇ -La - to concentrate proteins and eliminate salt residues. It was then processed further to a finished product as outlined in Example 8.
• whey protein fractions or the separated and purified proteins and the non-proteinaceous eluants may be incorporated into dietary and pharmaceutical formulations in appropriate proportions.
• Such formulations include but are not limited to infant formulas, fat substitutes, foaming agents, egg white substitutes, animal feed substitutes and the like.
• infant formulas constituted in accordance with this invention the casein and whey fractions of cow's milk were modified to achieve a composition simulating human milk to a significantly larger degree than prior art compositions and commercial products.
• the infant formulas of the present invention contain whey proteins at levels similar to those in human milk. This was achieved by producing a whey protein ingredient mix containing the type and ratio of whey proteins of human milk.
• infant formulas are constituted from whole cow's milk, mostly because of its availability on a large scale. Other additives or adjuvants may be included. These formulas are manufactured either in powder, concentrated or ready to feed preparation. They consist, for the most part, of non-fat milk solids, vegetable oils and carbohydrate sweeteners such as lactose, corn syrup solids and sucrose. These formulas may also be fortified with vitamin C, vitamin D, iron and fluoride. Table VII shows the typical compositions of a few exemplary commercial infant formulas in comparison to one exemplary formula of the present invention. Levels of vitamins, minerals and other fortifiers in the formulation of the present invention are adjusted to simulate human milk and to meet nutritional requirements of infants.
• Feed (-12.5 % solid s) Feed (-12.5 % solids)
• Vrt. B2 90 -150 meg 50 -150 meg
• cow's milk contains 3.3% protein while human milk has only 1%.
• Caseins are the major protein components in cow's milk (about 77% of total protein) whereas human milk contains a high ratio of whey proteins to caseins (about 2:1 ).
• ⁇ -Lactoglobulin concentration in cow's milk is the highest of the whey proteins while it is negligible in human milk, similarly, lactoferrin is ten times higher in concentration in human milk than in cow's milk.
• Immunoglobulin and serum albumin concentrations are about 1.5 times higher in human milk than in cow's milk.
• lactose and fat levels are adjusted to simulate human milk.
• Vegetable fat replaces butter fat.
• Casein to whey ratio is also reduced to simulate human milk.
• Other additives and supplements such as vitamins, taurine, and minerals may be included if desired.
• the total solute load is reduced to the level found in human milk.
• whey protein fractions obtained from the fractionation and elution in accordance with the process of this invention were first combined in the ratio shown in Table XI below.
• Lactoperoxidase 750 (activity) 1350
• the liquid mix prepared according to the above composition contained about 2% total solids, (of which salts from the eluting buffers comprise 90% and total proteins comprise about 10%), and 98% water. This liquid mix was then concentrated through a 10,000 molecular weight cut-off, spiral ultrafiltration membrane to 5-15% total proteins, followed by diafiltration with distilled water at 0.5-1. Ox to remove remaining salt residues.
• This formulation may be further concentrated by processes normally utilized in the treatment of labile proteins, such as ultrafiltration, reverse osmosis, freeze drying, freeze concentration, spray drying and the like or any combination thereof.
• the formulations of this invention may be further fortified with suitable additives and fortifiers. Such additives and fortifiers include but are not limited to nonfat milk solids, vegetable solids, carbohydrate sweeteners , minerals and vitamins.
• the solid composition of one exemplary formulation of the present invention is presented in Table XIII.

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• 1997
  • 1997-09-22 EP EP97944384A patent/EP1017286A4/de not_active Withdrawn
  • 1997-09-22 JP JP2000512418A patent/JP2001516599A/ja active Pending
  • 1997-09-22 AU AU45893/97A patent/AU4589397A/en not_active Abandoned
  • 1997-09-22 KR KR1020007003033A patent/KR20010030665A/ko not_active Application Discontinuation
  • 1997-09-22 WO PCT/US1997/016993 patent/WO1999015024A1/en not_active Application Discontinuation

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