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WO2002028194A1 - Procede de recuperation de proteines a partir de matieres premieres contenant des proteines de lactoserum - Google Patents

Procede de recuperation de proteines a partir de matieres premieres contenant des proteines de lactoserum Download PDF

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Publication number
WO2002028194A1
WO2002028194A1 PCT/NZ2001/000216 NZ0100216W WO0228194A1 WO 2002028194 A1 WO2002028194 A1 WO 2002028194A1 NZ 0100216 W NZ0100216 W NZ 0100216W WO 0228194 A1 WO0228194 A1 WO 0228194A1
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WO
WIPO (PCT)
Prior art keywords
cmp
wpi
anion exchanger
lactoglobulin
feedstock
Prior art date
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Ceased
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PCT/NZ2001/000216
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English (en)
Inventor
John Stephen Ayers
David Francis Elgar
Kay Patricia Palmano
Mark Pritchard
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Massey University
New Zealand Dairy Board
Original Assignee
Massey University
New Zealand Dairy Board
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Publication date
Priority claimed from NZ51224301A external-priority patent/NZ512243A/xx
Application filed by Massey University, New Zealand Dairy Board filed Critical Massey University
Priority to AU2002211115A priority Critical patent/AU2002211115A1/en
Publication of WO2002028194A1 publication Critical patent/WO2002028194A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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 OR TREATMENT 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
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to processes for recovering acidic peptide fractions, such as CMP (caseino-macropeptide), and ⁇ -lactoglobulin from whey protein containing feedstocks using anion exchangers.
  • CMP caseino-macropeptide
  • anion exchangers for recovering acidic peptide fractions, such as CMP (caseino-macropeptide), and ⁇ -lactoglobulin from whey protein containing feedstocks using anion exchangers.
  • Cation and anion exchange processes have been used to make various purified protein or peptide products from milk raw materials such as skim milk and whey since the late 1970's.
  • Whey protein isolate is produced as a result of whey proteins being adsorbed from the milk raw material by the ion exchanger and then being recovered from the ion exchanger by desorption (elution) after first washing away the treated milk raw material.
  • the latter still contains the lactose, minerals and fat content of the original milk raw material, so the WPI is typically about 90-94% protein.
  • Microfiltration(MF) is also used to produce WPI.
  • the MF membrane retains the fat content of the raw material but passes the whey protein, lactose and minerals.
  • Ultrafiltration(UF) is then used to separate the lactose and minerals from the whey protein.
  • WPI produced by MF and UF is thus high in protein content and, like ion exchange WPI, very low in fat.
  • cation exchangers With cation exchangers, a pH ⁇ 5 is usually used to recover the maximum amount of protein as WPI (cation WPI). With anion exchangers a pH > 5, usually > 6, is used to produce the WPI (anion WPI).
  • CMP caseinomacropeptide, also known as GMP (glycomacropeptide) and CDP (casein derived peptide)
  • GMP glycosylcholine
  • CDP casein derived peptide
  • CMP is rich in sialic acid and has a number of potential therapeutic uses, as well as having functional properties which make it very useful as an ingredient in food compositions.
  • a process to remove CMP from whey to produce a heat and acid stable WPI also produces an isolated CMP fraction.
  • CMP CMP
  • sialic acid rich components are present in human milk at a level of about 3-5 times higher than in bovine milk and are considered to act as an infection protection factor for infants. They are also important for the development of the brain. Therefore it would be useful to be able to produce sialic acid rich food products on an industrial scale.
  • Tanimoto et at in Bioscience, Biotechnology and Biochemistry, 56, 140-141, 1992 describes a process in which a crude CMP powder was first prepared and purified by anion-exchange chromatography on Q-Sepharose. A solution of the crude CMP was loaded onto the Q-Sepharose column at pH 7.5 to adsorb the CMP and then eluted with a linear gradient of sodium chloride. The fractions containing CMP were combined to give a purified CMP with 7.6% sialic acid. Such a process is possible only with CMP that has already been separated from whey proteins and would not be suitable for large scale commercial use.
  • the process described in GB 2188526 involves contacting whey protein containing material with an anion exchanger at a pH of 4 to 6, more particularly at a pH of 4.8 to 5.0, and then eluting the bound proteinaceous material, which, in the case of sweet wheys, is mainly CMP.
  • the process described in GB 2251858 differs from the process described in GB 2188526 in that the anion exchanger is contacted with the milk raw material at a pH of 4 or below instead of a pH of 4 to 6. In the applicants' experience this produces a CMP sub-fraction which is highly glycosylated, particularly acidic and still negatively charged at a pH of 4 or below.
  • WO 98/ 14071 also describes a method for purifying CMP, which involves contacting a CMP containing feedstock with an anion exchanger under conditions which adsorb the CMP, el ting the CMP, removing the impurities therefrom in one or more of a number of alternative ways, and recovering the purified CMP.
  • the examples disclose that when an adsorption pH of 4.9 was used, a high yield of CMP was obtained and that it had a sialic acid content of 5.4% after further purification. A lower pH of 4.2 resulted in a decreased yield of CMP (45%) and increased sialic acid content (12.7%) after further purification.
  • the total CMP was adsorbed initially but then eluted in two stages. Eluate 1 at pH 3.2 gave a CMP sub-fraction with only 2.6% sialic acid, while eluate 2 using salt gave a CMP product with 17.4% sialic acid after further purification.
  • WO 95/ 19714 indicates that it is necessary to first remove CMP, if present as in sweet wheys, for the efficient processing of the whey by anion exchangers. It is suggested that it be removed by preliminary treatment of the whey at pH 5 by anion exchanger as first described in GB 2188526 cited above. However, the processing referred to in WO 95/ 19714 involved the separation of ⁇ -lactoglobulin from ⁇ -lactalbumin and it was this separation which is rendered more efficient by the prior removal of CMP. This had been previously demonstrated in J. Dairy Research 52,167-181, 1985.
  • US 5434250 describes a process for preparing an ⁇ -lactalbumin enriched
  • the present invention provides a process for producing a CMP isolate and an acid and heat stable ⁇ -lactoglobulin enriched WPI from a feedstock containing whey proteins including CMP and ⁇ -lactoglobulin, said process comprising the steps:
  • the feedstock is selected from the group consisting of sweet whey, UF retentate derived sweet whey, or reconstituted WPC (whey protein concentrate) derived from sweet whey.
  • WPC whey protein concentrate
  • feedstocks may optionally be modified so as to reduce their ionic strength by, for example, demineralisation or dilution with water.
  • feedstocks will also contain ⁇ -lactalbumin and BSA and other whey proteins as would be appreciated by a skilled person.
  • step (a) the feedstock is contacted with the anion exchanger at a pH of 5.5-8.5 and more preferably at a pH of about 6-7.5.
  • step (c) the eluate is contacted with the second anion exchanger at a pH of about 4-5.5 and more preferably at a pH of about 4.5- 5.
  • the whey proteins are eluted from the anion exchanger(s) using the following elution conditions:
  • a salt concentration of 0-100 mM may be used and the pH lowered to 1.5-3 by the addition of acid.
  • the anion exchanger may be washed with 20-100 mM acid containing 0-100 mM salt.
  • a 500 mM salt solution may be used at any pH.
  • the whey protein containing feedstock may be contacted with a first anion exchanger at a pH of about 4.5-5 to adsorb acidic peptides and the flow-through contacted with a second anion exchanger at a pH of about 6-7.5 to adsorb both ⁇ -lactalbumin and ⁇ -lactoglobulin.
  • the ⁇ -lactalbumin and ⁇ -lactoglobulin enriched WPI which is acid and heat stable, is then eluted from the second anion exchanger, whilst the acidic peptides (including CMP) may be eluted from the first anion exchanger under conditions set out above.
  • CMP may be eluted at pH 2.5-4 from the first anion exchanger which elutes aglyco-CMP, and this eluate combined with the WPI eluate of the second anion exchanger to form a ⁇ - lactoglobulin enriched WPI containing aglyco-CMP.
  • the second ion exchanger may be replaced with a cation exchanger wherein the flow-through from the first anion exchanger is contacted with said cation exchanger of a pH of 3-4.5 to adsorb both of ⁇ -lactalbumin and ⁇ -lactoglobulin.
  • a ⁇ -lactalbumin and ⁇ - lactoglobulin enriched WPI, which is acid and heat stable, is then eluted from the cation exchanger.
  • the aglyco-CMP eluted at pH 2.5-4 from the anion exchanger is combined with the WPI eluate of the cation exchanger to form an ⁇ -lactoglobulin enriched WPI containing aglyco-CMP which is acid and heat stable.
  • the present invention provides a process for producing a CMP isolate and an acid and heat stable ⁇ -lactoglobulin enriched WPI from a feedstock containing whey proteins including CMP and ⁇ -lactoglobulin, said process comprising the steps:
  • step (b) contacting the MF permeate of step (a) with an anion exchanger under conditions whereby CMP is selectively adsorbed;
  • the feedstock is selected from the group consisting of sweet whey,
  • UF retentate derived sweet whey or reconstituted WPC (whey protein concentrate) derived from sweet whey.
  • WPC whey protein concentrate
  • the MF permeate is contacted with the anion exchanger at a pH of about 4-5.5. More preferably at about pH 4.5-5.
  • the CMP is eluted at step (d) from the anion exchanger.
  • this may conveniently be achieved by using a combination of low pH and low salt concentration, e.g. pH 2 and 50 mM NaCl or a higher salt concentration under neutral conditions, e.g. pH ⁇ 6 with 500 mM NaCl, or points in between.
  • the aforementioned process may be reversed, i.e., the whey protein containing feedstock may be first contacted with an anion exchanger at a pH of about 4-5.5, preferably about 4.5-5 to adsorb acidic peptides and the flow-through processed by microfiltration (MF) to produce a ⁇ - lactoglobulin enriched WPI which is acid and heat stable.
  • the acidic peptides (including CMP) may be eluted from the anion exchanger under the conditions set out above.
  • the present invention provides a process of recovering CMP and ⁇ -lactoglobulin from a feedstock containing whey proteins including CMP and ⁇ -lactoglobulin, the process comprising the following steps:
  • step (c) eluting CMP, or the fraction of the CMP not eluted in step (b), from the anion exchanger.
  • the feedstock is selected from the group consisting of sweet whey, UF retentate derived from sweet whey, or reconstituted WPC (whey protein concentrate) derived from sweet whey.
  • These feedstocks may optionally be modified so as to reduce their ionic strength by, for example, demineralisation or dilution with water.
  • the feedstock is contacted with the anion exchanger at a pH of from about 5 to about 8.5. More preferably at a pH of about 6-7.5.
  • step (a) the adsorption is carried out under conditions so as to achieve a loading of protein on the anion exchanger of at least 50%, more preferably at least 75%, of the protein adsorption capacity of the anion exchanger.
  • step (b) comprises eluting ⁇ -lactoglobulin and substantially no CMP.
  • the ⁇ -lactoglobulin is eluted under conditions of pH about 4 to about 5, and a salt concentration of from 0 to about 100 mM, to produce a first, ⁇ -lactoglobulin-containing, eluate.
  • step (b) comprises eluting ⁇ -lactoglobulin and a fraction of the CMP from the anion exchanger.
  • the ⁇ - lactoglobulin and fraction of CMP are eluted under conditions of pH of less than 4, more preferably at a pH of from about 2.5 to less than 4, and a salt concentration of from 0 to about 50 mM.
  • step (b) will elute from the exchanger a fraction of less glycosylated or non-glycosylated CMP (aglyco-CMP) together with the ⁇ -lactoglobulin, to produce a first eluate containing ⁇ -lactoglobulin and a fraction of the CMP; and step (c) will produce a CMP-containing eluate enriched in more highly glycosylated CMP (glyco-CMP) rich in sialic acid.
  • aglyco-CMP less glycosylated or non-glycosylated CMP
  • the present invention provides a process for producing a sialic acid rich glyco-CMP isolate and a heat and acid stable ⁇ - lactoglobulin enriched WPI containing aglyco-CMP from a feedstock containing whey protein including ⁇ -lactoglobulin and CMP, the process comprising the steps:
  • step (a) eluting the sialic acid rich glyco-CMP of step (a) from the ion exchanger.
  • the feedstock is selected from the group consisting of sweet whey, UF retentate derived sweet whey, or reconstituted WPC (whey protein concentrate) derived from sweet whey.
  • These feedstocks may optionally be modified so as to reduce their ionic strength by, for example, demineralisation or dilution with water.
  • the whey protein containing feedstock is contacted with an anion exchanger at pH 2.5-4.5, preferably pH 3-4, to selectively adsorb the sialic acid rich glyco-CMP.
  • the flow-through may be then treated by known processes (anion exchange or MF) to produce a WPI.
  • This ⁇ -lactoglobulin enriched WPI will be acid and heat stable and will also contain the non-sialic acid bearing CMP, i.e. will comprise a ⁇ -lactoglobulin enriched WPI containing aglyco-CMP.
  • the process of this embodiment may be carried out in reverse order, i.e. wherein a WPI produced by MF or by anion exchange treatment of a whey protein containing solution including ⁇ -lactoglobulin and CMP, produced by known processes as would be appreciated by a person of skill in the art, is used as the feedstock.
  • the WPI feedstock is contacted with the anion exchanger at pH
  • Glyco-CMP may be eluted from the anion exchanger as described above.
  • the present invention provides a process for producing a sialic acid rich glyco-CMP isolate and an acid and heat stable ⁇ - lactoglobulin enriched WPI containing aglyco-CMP from a whey protein containing feedstock including CMP and ⁇ -lactoglobulin comprising the steps: (a) contacting an anion exchanger with an excess of feedstock under conditions whereby sialic acid rich glyco-CMP is selectively adsorbed;
  • step (c) eluting the sialic acid rich glyco-CMP of step (a) from the anion exchanger.
  • the feedstock is selected from the group consisting of sweet whey,
  • UF retentate derived sweet whey or reconstituted WPC (whey protein concentrate) derived from sweet whey.
  • WPC whey protein concentrate
  • the anion exchanger of step (a) is overloaded with respect to CMP so that non-sialic acid bearing aglyco-CMP which may initially bind to the exchanger is displaced by the more acidic sialic acid-rich glyco-CMP.
  • the whey protein containing feedstock is contacted with the anion exchanger of step (a) at a pH of about 4-6, preferably about 4.2-5.5.
  • the breakthrough solution at step (b) may be further processed by anion exchange or MF to produce a WPI by known processes known to a person of skill in the art.
  • a WPI produced by anion exchange or MF may be overloaded with respect to CMP onto an anion exchanger (at pH 4-6, preferably pH 4.2-5.5), whereby sialic acid rich glyco-CMP is selectively adsorbed onto the anion exchanger and ⁇ -lactoglobulin containing non sialic acid bearing aglyco-CMP is collected in the flow-through and further processed to produce a ⁇ -lactoglobulin enriched WPI.
  • the ⁇ -lactoglobulin enriched WPI so produced will be acid and heat stable and will further comprise non sialic acid bearing aglyco-CMP.
  • sialic acid rich glyco-CMP eluted from the first anion exchanger in either the fourth or fifth embodiment may be combined with the flow-through from the second anion exchanger in each case to make an ⁇ -lactalbumin/ sialic acid enriched product.
  • sialic acid rich glyco-CMP eluted from the second anion exchanger may be combined with the flow-through from the first anion exchanger used to make the WPI to produce an ⁇ -lactalbumin/ sialic acid enriched product.
  • the step of eluting CMP from the anion exchanger preferably takes place under conditions of pH lower than those used to elute ⁇ -lactoglobulin, and/or using an eluent with a higher salt concentration than that used to elute ⁇ -lactoglobulin.
  • the eluates containing ⁇ -lactoglobulin and CMP are recovered separately, to produce a ⁇ -lactoglobulin-enriched WPI; a ⁇ -lactoglobulin enriched WPI containing aglyco-CMP; a CMP isolate (comprising aglyco-CMP and glyco-CMP); and a glyco-CMP isolate, respectively by known methods such as ultrafiltration and spray drying.
  • the ⁇ -lactoglobulin enriched WPI's can be neutralized prior to ultrafiltration and spray drying or optionally, they can be ultrafiltered and spray dried without neutralization at a pH in the region of 3.0-3.5 to make a WPI that is ready to use in an acid beverage.
  • the de-proteinised feedstock (flow-through) from an ion exchange step in which both ⁇ -lactoglobulin and CMP are adsorbed may optionally be further processed by ultrafiltration, anion exchange or cation exchange procedures known in the art to recover additional products.
  • step (b) eluting the sialic acid rich glyco-CMP of step (a) from the anion exchanger.
  • the feedstock is overloaded onto the anion exchanger at step (a) at a pH of 4-6, preferably at a pH of 4.2-5.5.
  • the sialic acid rich glyco-CMP bound to the anion exchanger is eluted from the anion exchanger as discussed above. Particularly, in a batch process this may conveniently be achieved by using a combination of low pH and low salt concentration, e.g. pH 2 and 50 mM NaCl or a higher salt concentration under neutral conditions, e.g. pH ⁇ 6 with 500 mM NaCl, or points in between.
  • low pH and low salt concentration e.g. pH 2 and 50 mM NaCl or a higher salt concentration under neutral conditions, e.g. pH ⁇ 6 with 500 mM NaCl, or points in between.
  • the present invention provides a process of producing acid/heat stable WPI from any whey protein containing feedstock comprising the steps of
  • step (d) further processing the solution of step (c) to produce a WPI.
  • the present invention further contemplates a process for producing a ⁇ - lactoglobulin enriched WPI from acid whey.
  • Acid wheys do not contain CMP but they do contain acidic peptides and minor proteins which are highly phosphorylated. Acid wheys may therefore be similarly processed as herein described to produce ⁇ -lactoglobulin enriched WPI and a further isolate enriched in phosphopeptides/ proteins.
  • the more highly phosphorylated peptides bind preferentially to anion exchangers, and at lower pH levels like the glyco-CMP does since both the phosphate and sialylate groups do not lose their negative charge until pH ⁇ 3.
  • the phosphopeptides/proteins are useful as calcium carriers for food ingredients.
  • the anion exchanger(s) used in the processes of the present invention diethylaminoethyl (DEAE) and quarternary amino (QA) exchangers.
  • anion exchangers are of the industrially useful cellulose type comprising a water-insoluble, hydrophilic, water swellable, hydroxy(C 2 ⁇ C ) alkylated and cross-linked regenerated cellulose derivatised with quaternary amino (QA) groups, preferably in granular or beaded form.
  • industrially useful cellulose type comprising a water-insoluble, hydrophilic, water swellable, hydroxy(C 2 ⁇ C ) alkylated and cross-linked regenerated cellulose derivatised with quaternary amino (QA) groups, preferably in granular or beaded form.
  • the level of substitution of the QA groups on the anion exchanger(s) is 1.4 milliequivalents per dry gram of anion exchanger (meq/g) or greater.
  • the level of substitution of QA groups is from about 1.4 to about 2.5 meq/g, more preferably from about 1.5 to about 2.5 meq/g, and most preferably from about 1.7 meq/g to about 2.5 meq/g.
  • the cellulose is hydroxypropylated cross-linked regenerated cellulose.
  • the preferred anion exchanger is QMA SpherosilTM or like ion exchanger.
  • the present invention produces a ⁇ -lactoglobulin enriched WPI product which is heat and acid stable (WPI*) .
  • such a product may further comprise the aglyco fraction of CMP, and thereby comprise a ⁇ -lactoglobulin enriched WPI containing aglyco-CMP (WPI**).
  • the present invention provides a ⁇ -lactoglobulin enriched WPI (WPI*) obtained by or obtainable by a process as defined above.
  • the present invention provides a ⁇ -lactoglobulin enriched
  • WPI containing aglyco-CMP obtained by or obtainable by a process as defined above.
  • WPI** aglyco-CMP obtained by or obtainable by a process as defined above.
  • the present invention provides a CMP isolate obtained or obtainable by a process as defined above.
  • the present invention provides a glyco-CMP isolate obtained or obtainable by a process as defined above.
  • the present invention provides foodstuffs comprising the ⁇ -lactoglobulin enriched WPI (WPI* or WPI**) of the invention, including acid beverages.
  • the present invention provides foodstuffs comprising the CMP or glyco-CMP isolates of the invention, including infant formulas and food formulations for patients suffering from phenylketonuria.
  • FIGS 1 to 7 show schematically the processes of the main embodiments of the present invention.
  • Figure 8 shows HPLC analyses of UF retentate prepared from cheese whey, and of first and second eluates obtained using a process of the invention, showing the individual whey proteins in each sample.
  • FIG 9 shows the acid/heat instability arising when glyco-CMP is added back to acid/heat stable anion WPI (WPI*)
  • Figure 10 shows the displacement of initially adsorbed aglyco-CMP (A and B variants) by glyco-CMP as a column of Macro-PrepTM High Q is overloaded with WPI.
  • the present invention provides new processes useful for recovering separately both ⁇ -lactoglobulin and CMP from a single whey protein-containing feedstock.
  • the applicants have found that up to four products may be made by the processes of the present invention which are enriched in either ⁇ - lactoglobulin, CMP (aglyco-CMP and glyco-CMP or just mainly glyco-CMP) or both from a feedstock containing these proteins /peptides.
  • the products may comprise a ⁇ -lactoglobulin enriched WPI (WPI*); a ⁇ - lactoglobulin enriched WPI containing aglyco-CMP (WPI**); a CMP isolate; and a glyco-CMP isolate.
  • WPI whey protein isolate
  • whey protein isolate is a term used loosely in the industry for whey protein products isolated using ion exchange or microfiltration. It is not well defined but has come to mean a product with ⁇ 1 % fat and greater than about 90% protein. In all cases ⁇ -lactoglobulin is the major protein present, while ⁇ -lactalbumin may vary from very little to an amount giving the same ratio of ⁇ -lactalbumin/ ⁇ -lactoglobulin as found in the original feedstock. Immunoglobulins and CMP may or may not be present depending on whether cation exchange, anion exchange and MF is used.
  • Isolate In cases where ⁇ -lactoglobulin is not the major protein present in the eluate stream from the ion exchanger we have simply referred to the product as an "isolate,” e.g. CMP isolate.
  • ⁇ -lactoglobulin enriched WPI means a WPI with a % ⁇ - lactoglobulin content greater than that found in the whey protein containing feedstock relative to acidic peptides, i.e. peptides with pi ⁇ about 4.5.
  • sweet wheys it is relative to CMP, in particular relative to both glyco- and aglyco-CMP, and is labelled WPI* herein.
  • This WPI* contains ⁇ about 5% total CMP (In the case of acid wheys it is relative to phosphopeptides.).
  • ⁇ -Lactoglobulin enriched WPI containing aglyco-CMP means a WPI with a % ⁇ -lactoglobulin content greater than that found in the whey protein feedstock relative to glyco-CMP but near normal ⁇ -lactoglobulin content relative to aglyco-CMP. In particular, this product contains more than 5% aglyco-CMP and less than 5% Glyco-CMP. It is labelled WPI**.
  • CMP isolate as used herein means an isolate containing CMP as the major protein present. This CMP contains both glyco-CMP and aglyco-CMP in a near normal ratio as found in the whey protein feedstock. It typically has a sialic acid content of 2-5%.
  • Glyco-CMP isolate as used herein means an isolate containing CMP as the major protein present but the CMP in this case has a higher ratio of glyco- CMP to aglyco-CMP than that found in the whey protein feedstock applied to the anion exchanger. It typically has a sialic acid content greater than 5%.
  • the simplest methods of producing a heat/ acid stable WPI by anion exchanger involve removing the acidic peptides and proteins from the process stream either before or after the anion WPI is prepared. To achieve this an additional anion exchange step is used at a pH of about 5. At this pH, the major whey proteins ⁇ -lactoglobulin, ⁇ -lactalbumin, BSA and immunoglobulins are either neutral or positively charged and do not bind to the anion exchanger. However, acidic peptides and some minor whey proteins whose isoelectric point (IEP) is less than bout 4.5, collectively called acidic peptides herein, remain negatively charged and are selectively adsorbed from the process stream.
  • IEP isoelectric point
  • whey is first adjusted to about pH 6-7.5 and passed through a first anion exchanger to adsorb both the ⁇ -lactoglobulin and the acidic peptides.
  • the proteins are then eluted and the eluate contacted with a second anion exchanger at about pH 4.5-5 to remove the acidic peptides.
  • the major whey proteins ⁇ -lactoglobulin, ⁇ -lactalbumin and BSA are then recovered in the flow- through as WPI ( Figure la).
  • WPI labelled as WPI*
  • WPI is also made commercially by microfiltration (MF).
  • MF WPI microfiltration
  • anion WPI is similar to anion WPI in that it contains acidic peptides and is not acid/ heat stable. It can be made stable by passing it through an anion exchanger at about pH 4.5-5 to remove acidic peptides ( Figure 2a). The flow- through containing whey proteins is then further processed to produce acid/heat stable WPI*.
  • the acidic peptides, including CMP may be eluted separately.
  • a further process involves first adsorbing both ⁇ -lactoglobulin and CMP onto an anion exchanger, followed by eluting first the ⁇ -lactoglobulin (optionally together with a fraction of the CMP), and then, by lowering the pH and/ or by increasing the salt concentration, eluting the CMP (or the remaining adsorbed fraction thereof) as shown in Figure 3.
  • This particular process involves the step of contacting the feedstock with an anion exchanger under conditions in which both CMP and ⁇ -lactoglobulin will be adsorbed.
  • the adsorption step is followed by first eluting the ⁇ - lactoglobulin from the exchanger, optionally together with a fraction of the adsorbed CMP, and then eluting the CMP (or the fraction of CMP remaining adsorbed) from the anion exchanger.
  • the feedstock is contacted with the anion exchanger under conditions which will bring about adsorption of CMP and ⁇ -lactoglobulin to the anion exchanger.
  • the feedstock be contacted with the anion exchanger at a pH of about 5 to about 8.5, more preferably at a pH of about 6 to 7.5 ( Figure 3a).
  • ⁇ -lactalbumin in the whey feedstock will be adsorbed and eluted with the ⁇ -lactoglobulin.
  • the exact size of the portion will depend on the ion exchanger and pH used as well as the ratio of feedstock to ion exchanger.
  • the ⁇ -lactalbumin will be spread between the ⁇ - lactoglobulin isolate and the breakthrough stream except in cases where the anion exchanger is substantially under-loaded or over-loaded, in which case it will be present in either the ⁇ -lactoglobulin isolate or breakthrough stream respectively. This is known to those skilled in the art.
  • the step of contacting the feedstock with the anion exchanger to adsorb the CMP and ⁇ -lactoglobulin can be carried out in any convenient manner. Preferred methods are to carry out this step in a stirred bed of anion exchanger or in a column of the anion exchanger. It is generally preferred that the adsorption step be carried out at a temperature of less than about 20°C, more preferably at around 8-15°C, to minimise the growth of mesophilic bacteria. It is also preferred that the contact time of the anion exchanger with the whey protein solution is less than about 2 hours, more preferably less than about 1 hour.
  • the adsorption step is carried out under conditions so as to achieve a loading of protein on the anion exchanger of at least 50%, more preferably at least 75%, of the protein adsorption capacity of the anion exchanger.
  • the ⁇ -lactoglobulin is eluted first.
  • elution of ⁇ - lactoglobulin is carried out under conditions which, while achieving elution of the ⁇ -lactoglobulin (and ⁇ -lactalbumin, if adsorbed together with ⁇ - lactoglobulin), allow all or substantially all of the CMP to remain adsorbed to the anion exchanger.
  • the elution is carried out at a pH of from about 4 to about 5, and at a salt concentration of from 0 to about 100 mM.
  • the elution may conveniently be carried out using sodium chloride, although other salts may also be used. This elution produces a first eluate which contains mainly ⁇ -lactoglobulin.
  • This eluate may then if desired be subjected to further processing including one or more of ultrafiltration, diafiltration, evaporation, freeze-drying and spray drying, to produce ⁇ -lactoglobulin enriched WPI*.
  • the CMP remaining adsorbed to the anion exchanger is eluted. This may conveniently be achieved by using a combination of low pH and low salt concentration, e.g. pH 2 and 50 mM
  • NaCl or a higher salt concentration under neutral conditions, e.g. pH > 6 with 500 mM NaCl, or points in between.
  • the eluted CMP may if desired be subjected to further processing including one or more of ultrafiltration, diafiltration, evaporation, freeze- drying and spray drying, to produce a CMP isolate.
  • the CMP isolate may be further purified to make it suitable as a dietary supplement suitable for persons suffering from phenylketonuria.
  • the process is carried out so as to achieve a fractionation of the CMP, that is, to split the CMP between the first and second eluates.
  • CMP is not a single peptide, but is a complex mixture of macropeptide with varying degrees of glycosylation (covalently attached carbohydrate). About 40% of the macropeptide is not glycosylated at all (referred to as aglycomacropeptide, or aglyco-CMP), while the remainder has carbohydrate groups attached at up to five different sites on the peptide chain (referred to as glycomacropeptide or glyco-CMP). Each of these carbohydrate groups so attached may contain up to two sialic acid units.
  • the adsorption of the CMP and ⁇ - lactoglobulin is carried out in the same manner as described above.
  • the first elution step (b) is carried out under conditions which will also elute a fraction of the CMP. This may conveniently be achieved by carrying out the elution at a pH of less than 4, preferably about pH 2.5 to less than 4, and preferably at a salt concentration of from 0 to 50 mM (Figure 3b). Under these conditions, we have found that a fraction of the CMP.
  • CMP which is mainly aglyco-CMP or less glycosylated CMP
  • WPI** aglyco-CMP
  • the CMP remaining adsorbed to the anion exchanger will mainly be glyco-CMP, which is rich in sialic acid.
  • This fractionation is believed to be achieved by virtue of the negatively charged sialic acid groups associated with the carbohydrate groups, which lower the isoelectric point (IEP) of the glyco-CMP from near pH 4 towards pH 2.
  • IEP isoelectric point
  • a first eluate which contains a desired amount of the CMP from about 20 to about 70% of the CMP
  • a second eluate which contains the balance of the CMP mainly glyco-CMP
  • Sialic acids are contained at higher levels in human milk than bovine milk and are considered to act as an infection protection factor for infants. They are also important for the development of the brain.
  • the first eluate will contain mainly ⁇ -lactoglobulin and some CMP (mainly aglyco-CMP), which may be subjected to further processing if desired, including one or more of ultrafiltration, diafiltration, evaporation, freeze-drying and spray drying, to produce a ⁇ -lactoglobulin enriched WPI containing aglyco-CMP (WPI**).
  • WPI** ⁇ -lactoglobulin enriched WPI containing aglyco-CMP
  • Elution of the fraction of CMP remaining bound to the anion exchanger can conveniently be achieved by using a combination of low pH and low salt concentration, e.g. pH 2 and 50mM NaCl, or a higher salt concentration under neutral conditions, e.g. pH > 6 with 500 mM NaCl, or points in between.
  • low pH and low salt concentration e.g. pH 2 and 50mM NaCl
  • a higher salt concentration under neutral conditions e.g. pH > 6 with 500 mM NaCl, or points in between.
  • the eluted CMP may be further processed as desired, and the resulting product will be an isolate containing mainly glyco-CMP, enriched in sialic acid.
  • Such further processing may include removal of contaminating proteins such as residual ⁇ -lactoglobulin, eg by cation exchange, to further enrich the sialic acid content.
  • the process of the present invention uses a whey protein containing feedstock at pH 2.5-4.5 which is contacted with an anion exchanger to selectively remove the very acidic material from it, e.g. the sialic acid rich glyco-CMP.
  • the glyco-CMP deplete flow-through whey is then contacted with a second exchanger at pH 6-7.5 and a WPI prepared.
  • the flow- through whey stream may be used to prepare MF WPI.
  • This WPI** will again contain the non-sialic acid bearing aglyco-CMP ( Figure
  • the steps of the aforementioned embodiment can be carried in the reverse order, whereby MF WPI, or anion WPI, that is not acid/heat stable is used as a feedstock.
  • the WPI is passed through an anion exchanger at a pH 2.5-4.5 to selectively adsorb the sialic acid rich glyco-CMP.
  • the bulk of the whey protein and non-sialic acid bearing CMP are present in the flow- through. This is recovered as the acid/heat stable ⁇ -lactoglobulin enriched WPI containing aglyco-CMP, WPI**.
  • the sialic acid rich glyco-CMP isolate is eluted from the anion exchanger and recovered ( Figure 4b).
  • a whey protein containing solution is contacted with an anion exchanger at about pH 5 (pH 4-6) to selectively bind the sialic acid rich glyco-CMP by overloading the column such that even if non-sialic acid bearing CMP is bound initially it is eventually displaced by the more tightly binding very acidic peptides e.g. sialic acid rich glyco-CMP (Figure
  • a process to produce a glyco-CMP isolate only is set out in Figure 6, whereby an excess of whey protein containing feedstock is contacted with an anion exchanger at pH 4-6 (preferably pH 4.5-5), whereby sialic-acid rich glyco- CMP is selectively bound to the exchanger, which may then be eluted therefrom.
  • the feedstock used in the processes of the present invention can be any solution containing both CMP and ⁇ -lactoglobulin.
  • CMP is found only in sweet wheys, so suitable feedstocks include sweet whey, UF retentate derived from sweet whey, and reconstituted WPC derived from sweet whey.
  • WPIs, WPI* and WPI** produced by the processes of the present invention are heat stable in the acidic region of pH 3.5 - 4.0. This contrasts with WPI products produced by anion exchange using a conventional, non-selective elution to give a total WPI product comprising ⁇ -lactoglobulin, CMP and other minor components including the very acidic peptides and proteins.
  • Such WPIs suffer the disadvantage of not being heat stable, especially in the acidic region of pH 3.5 - 4.0. This gives solutions of the WPIs a cloudy appearance or forms a sediment which is unacceptable in certain applications such as protein fortification of acidic beverages such as fruit drinks.
  • Anion exchangers suitable for use in the processes of the present invention include diethylaminoethyl (DEAE) and quaternary amino (QA) exchangers.
  • DEAE diethylaminoethyl
  • QA quaternary amino
  • anion exchanger are of the industrially useful cellulosic type comprising a water-insoluble, hydrophilic, water swellable, hydroxy (C 2 -C ) alkylated and cross-linked regenerated cellulose, derivatised with quaternary amino (QA) groups, which is preferably in granular or beaded form.
  • QA quaternary amino
  • the anion exchangers have a substitution level of QA groups of about 1.4 meq/g or higher, more preferably from about 1.5 to about 2.5 meq/g, and most preferably from about 1.7 meq/g to about 2.5 meq/g.
  • QA or "quaternary amino" when used in the context of ion exchangers, means a functional group selected from a group of the formula -ORi-Z, wherein Ri is a lower alkylene group containing 1 to 3 carbon atoms and optionally substituted with a hydroxyl group, and Z is a quaternized amino group of the formula: -N ⁇ Rs ⁇ + OH- or salts thereof, wherein R 2 , R 3 and * are each a lower alkyl group containing 1 to 4 carbon atoms, optionally substituted with a hydroxyl group, or a further group of the formula -R ⁇ -NR2R 3 R + OH _ or salts thereof wherein Ri, R 2 , R 3 and R-t are as defined above.
  • Suitable QA groups are -CH 2 CH2N + R 2 R 3 R4 Cl ⁇ and -OCH 2 CHOHCH 2 N + R 2 R3R4 CF, wherein R 2 , R 3 and R 4 are the same or different and are selected from -CH 3 , -CH 2 CH 3 , -CH2CH2OH, -CH 2 CHOHCH 3 , -CH 2 CH 2 N + R 2 R 3 R 4 Cl ⁇ and -CH 2 CHOHCH 2 N + R 2 R 3 R 4 Cl ⁇ .
  • the QA anion exchangers particularly preferred for use in these processes of the present invention and having a substitution level of 1.4 meq/g or greater may be prepared as disclosed in co-pending application NZ 501644 and NZ 505071.
  • QMA SpherosilTM or like anion exchanger is preferred.
  • QA GibcoCelTM HG2 (1.17 meq/g), an anion exchanger made from granular regenerated cellulose and particularly suited to large scale industrial use, was obtained from Life Technologies Ltd, Auckland, New Zealand. It was suspended in water and then collected on a sintered glass filter where it was washed with 1 M hydrochloric acid, water, 1 M sodium hydroxide and finally de-ionised water. It was then drained of excess water by vacuum filtration. This QA cellulose in its hydroxide form was then further alkylated to raise the density of positively charged QA groups.
  • the QA GibcoCelTM[OH ⁇ ] (450 g) was made up to a thick slurry by the addition of water (310 mL) and 30% (w/v) aqueous sodium hydroxide (50 mL). The mixture was chilled before adding 60 mL of (3-chloro-2- hydroxypropyl)trimethylammonium chloride (60 wt. % solution in water). These ingredients were mixed as a slurry for 17 hours at room temperature followed by 2 hours at 60° C. The QA cellulose product was collected on a filter and washed with water, 1 M hydrochloric acid and water before removing the excess water by vacuum filtration.
  • a small sample of the product was analyzed to determine its substitution levels of QA groups.
  • About 5 g of the moist product was converted to its hydroxide form by washing with 1M sodium hydroxide followed by demineralized water.
  • the sample was then titrated in 1 M sodium chloride with 1.00 M hydrochloric acid to an end-point of pH 4.
  • After titration the sample was collected on a dry tared sintered-glass filter, washed with water and dried overnight at 105° C.
  • the moist QA cellulose (213 g, equivalent to 300 mL settled volume) from Example 1 was placed in an 800 mL reaction vessel fitted with a screen and outlet tap at the bottom, and an overhead stirrer. To this was added 200 g of freshly prepared UF retentate (15.3% protein, 21.5% total solids, pH 6.3) prepared from cheese whey, followed by 400 mL of water. This was stirred for 10 minutes and then the pH was adjusted to 7.0 with 10% sodium hydroxide while stirring for a further 40 minutes. The vessel was drained to the surface of the QA cellulose which was then washed with water (360 mL). The filtrate and washings were combined to give 820 g of flow-through solution.
  • Retentate 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
  • the eluate was concentrated by ultrafiltration, diafiltered and freeze-dried.
  • the resulting WPI powder was found not to be heat stable under acid conditions as described in Example 5.
  • Sialic acid was shown to be present in this WPI powder and amounted to
  • Example 2 This was similar to Example 2 except that the elution was carried out in two stages, a first elution at pH 4 and a second elution with 0.4 M sodium chloride.
  • a second protein fraction was obtained by eluting further protein from the QA cellulose with 0.4 M sodium chloride. Water was added to the vessel to once again bring the total volume up to the 450 mL mark and then 50 mL of 4 M sodium chloride was added to give a final concentration of approximately 0.4 M. This was stirred for 1 hour without any pH adjustment and the vessel was then drained and washed with 0.4 M sodium chloride (360 mL). The protein solution and washings were combined to give a second eluate (547 g). A sample of this CMP containing second eluate was dialysed, freeze-dried and analysed for sialic acid as described in Example 2.
  • Retentate 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
  • Example 3 Samples of the retentate, flow-through, first and second eluates were analysed as in Example 3. The results are shown in Tables 5 and 6. A sample of the second eluate was also dialysed, freeze-dried, analysed for sialic acid content and found to have a sialic acid content of 9% based on powder weight compared with only 4.4% for the second eluate in Example 3.
  • Retentate 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
  • a 3% solution of each of the WPI powders was prepared. Each solution was acidified using 10% phosphoric acid to pH end-points of 4.0, 3.8, 3.6, 3.4 and 3.2. As each end-point was reached a 15 L sample for testing was removed before lowering the pH of the remaining solution to the next point.
  • Table 7 also shows the improved stability of WPIs obtained by anion exchange similar to Example 2 except that the whey solution used was one from which most of the CMP had been removed first (Example 6), or one from which mainly glyco-CMP had been removed (Example 7).
  • compositions of the five WPI products tested are shown in Table 8. Values are given for the glyco and aglyco subfractions of CMP as only a reduction in the former is important to obtain stability as shown by the composition of the WPI from Example 4. (Examples 6d and 10 further confirmed this.)
  • Example 2 A 750 mL sample of this was added to a beaker containing 320 g (450 mL) of moist QA cellulose from Example 1 and stirred for 40 minutes while maintaining the pH at 4.8 with 10% sodium hydroxide. At the end of the mixing period the beaker contents were transferred to the 800 mL reaction vessel (described in Example 2).
  • the second elution of the QA cellulose was carried out in the same manner as for the first except that 40 mL of 4 M sodium chloride was used to make the solution approximately 0.2 M and the pH was lowered to 3.0.
  • the combined filtrate and washings with 0.2 M salt, were neutralized, ultrafiltered and diafiltered.
  • the retentate was analysed by HPLC. The protein was found to be 90% CMP of which 80% was glyco-CMP. A sample was freeze dried for sialic acid analysis.
  • the flow-through retentate from part a) above was adjusted from pH 4.8 to 7.5 by the addition of 10% sodium hydroxide, added to a fresh lot of QA cellulose (320 g, 450 mL) and mixed for 40 minutes while maintaining the pH at 7.5.
  • the anion exchanger was again collected in the 800 mL reaction vessel and washed with water. The filtrate and washings were combined to give 1257 g of whey protein depleted flow-through. Analysis of this solution by HPLC showed that 96% of the ⁇ -lactoglobulin and 61% of the ⁇ -lactalbumin had been adsorbed by the QA cellulose. No attempt was made to separate these two proteins.
  • the WPI retentate from part c), and the glyco-CMP retentate from part b) were recombined in various proportions and diluted with water so as to give 30 mL solutions of 3% total protein in each case, but having a range of 0 to 12% glyco-CMP present in the total protein.
  • the 3% protein solution having 10% of the total protein present as glyco-CMP was made up by combining 6.86 mL of the WPI* retentate (118 mg protein/mL), 6.30 mL of the glyco-CMP retentate
  • glyco-CMP containing fraction which destabilized the protein and not the aglyco-CMP fraction.
  • the 3% protein solution was rendered unstable if it had more than about 4% glyco-CMP but up to about 50% aglyco-CMP could be tolerated without destabilizing the WPI*. Above 50% the instability probably resulted from the small amount (approx. 10%) of glyco-CMP present in the aglyco-CMP fraction.
  • the overall yield of protein in the WPI** was 72% with a further 6% of starting protein in the glyco-CMP fraction.
  • the major losses were ⁇ -lactalbumin and immunoglobulins (7% and 5% of starting protein respectively) in the final flow-through from both anion exchangers ( Figure 7a). Immunoglobulins are not adsorbed by anion exchangers and ⁇ -lactalbumin is known to be difficult.
  • step c) In yet another repeat of steps a, b and c the anion exchanger in step c) was replaced with 600 mL of a cation exchanger, SP GibcoCelTM, and it was contacted with the flow-through retentate from part a) which had been adjusted to pH 3.5 with 10% HC1 ( Figure 7b). The rest of the process was as described in this section e) .
  • the overall yield of protein in the WPI stream increased to 80% as a result of the cation exchanger binding immunoglobulins and more of the ⁇ -lactalbumin.
  • Example 7 a) WPI** by anion exchange from glyco-CMP reduced cheese whey retentate (Figure 5a) This process was the same as example 6 a) except that only half the quantity of QA cellulose was used, i.e. 160 g (225 mL) in order to favour the adsorption of glyco-CMP over aglyco-CMP, and a pH of 4.4 was used instead of 4.8.
  • the treated retentate had a reduced glyco-CMP content and was used to prepare WPI** exactly as described in Example 6 c) for WPI*.
  • the protein composition of the WPI** obtained is shown in Table 8.
  • the improved heat stability of the WPI**, over that from Example 2, is shown in Table 7. The major difference is in the reduction in the amount of glyco-CMP present in the WPI from 8% to about 3% of protein.
  • the protein adsorbed by the QA cellulose in part a) was recovered from it by a single desorption at pH 3.0 in 200 mM NaCl. Water was added to the QA cellulose to give a total volume of 450 mL. Aqueous sodium chloride (22.5 mL of 4 M) was added to make the solution approximately 200 mM and the pH was lowered to 3.0 by the addition of 10% hydrochloric acid. After stirring for 1 hour the QA cellulose was drained and washed with 200 mM sodium chloride. The combined filtrate and washings were neutralized, ultrafiltered and diafiltered. HPLC analysis of the retentate showed that 94% of the protein present was CMP with 64% of this being glyco-CMP. A sample of the retentate was also freeze-dried. Analysis of the powder showed it to have a sialic acid content of 8.7%.
  • the residual protein (32%) in the WPI depleted retentate from part a) was found to still contain 68% of the ⁇ -lactalbumin present in the starting retentate.
  • This ⁇ -lactalbumin made up 40% of the residual protein analysed by HPLC so could be made into a useful WPC.
  • sialic acid rich eluate from b) was combined with this WPI depleted flow- through retentate from a). It was then ultrafiltered to produce a WPC enriched in sialic acid and ⁇ -lactalbumin, as shown in Table 9, both useful ingredients for an infant formula.
  • Glyco-CMP was preferentially adsorbed from cheese whey retentate in much the same manner as for Example 7 a) except that a pH of 5.5 was used.
  • a pH of 5.5 was used.
  • anion exchanger was used to suppress the adsorption of aglyco-CMP as well as ⁇ -lactalbumin and ⁇ -lactoglobulin which would normally be adsorbed at this pH.
  • Cheese whey retentate (2.25 L) was mixed with water (1.6 L) and adjusted to pH 5.5 with 10% HCl. This was then added to 2.25 L (1.6 kg) of QA cellulose (Example 1) and mixed for 40 minutes whilst maintaining the pH at 5.5. The QA cellulose was then drained and washed with water.
  • the treated retentate and wash water were combined, neutralized and further diluted to a protein concentration of 2%.
  • This solution was microfiltered to produce a WPI containing permeate using a Terra- Pak MFS1 system (0.1 micron membrane).
  • a sample of the permeate was then concentrated by ultrafiltration in the laboratory using a Millipore Prep-Scale ultrafilter with 3 kD membrane.
  • the final retentate (WPI** solution) was diluted to give a 3% protein solution which was subjected to the heat stability test as described in Example 5.
  • a sample of the original cheese whey retentate was similarly diluted and processed by microfiltration and ultrafiltration to prepare a standard MF WPI for comparison.
  • the acidic peptides adsorbed by the QA cellulose in part a) were recovered from it as described in Example 7 b).
  • the protein composition of the product is shown in Table 11 and it was found to have a sialic acid content of 5.2%.
  • the moist QA cellulose (213 g, equivalent to 300 mL settled volume) from Example 1 was mixed with 240 g of cheese whey UF retentate and 360 mL of water as described in Example 2 except that the pH was adjusted to 7.4. After mixing for 40 minutes, the retentate was drained and the bed of QA cellulose washed with water. The treated retentate (combined filtrate and washings) was set aside. (Its protein composition is shown in Table 12 and the protein could be recovered as a WPC or further WPI product enriched in ⁇ -lactalbumin by cation or anion exchange treatment as required.)
  • a sample of the solution was dialysed and freeze-dried for analysis.
  • Stage 2 Removal of very acidic peptides from anion WPI
  • a 70 mL fraction of the WPI solution obtained in stage 1 was diluted with an equal quantity of water, to reduce the ionic strength, and the pH lowered to 4.9.
  • This solution with a conductivity of 5.5 mS/cm, was passed through a 7.5 mL column of Macro-PrepTM High Q anion exchanger (Bio-Rad Laboratories) which had been previously washed with 0.5 M NaCl and water.
  • the column was loaded at a flow rate of 2.5 mL/min. At the end of the loading the column was washed with 25 mL of 0.05 M NaCl.
  • the flow- through and washings were collected, neutralized to pH 6.5, dialysed and freeze-dried to recover the WPI.
  • the column was eluted by washing it with 25 mL of 0.15 M NaCl to desorb the acidic peptides, mainly glyco-CMP, which had been removed from the WPI. This eluate fraction was also neutralized to pH 6.5, dialysed and freeze-dried.
  • the anion WPI (Stage 1 product) and the recovered acidic peptides had sialic acid contents of 1.8% and 12.4% respectively.
  • glyco-CMP components made up 74% of the CMP in the very acidic peptides, but only 36% and 17% of the CMP in the initial WPI and flow- through WPI** respectively.
  • stage 2 was carried out at pH 5.3 instead of 4.9 similar results were obtained.
  • Microfiltered whey was prepared by microfiltering rennet whey protein concentrate as described in Example 8.
  • the MF permeate (containing 2.5% protein) was then acidified to pH 5.3 using 10% HCl and passed through a 7.5 mL column of Macro-PrepTM High Q anion exchanger as described in Example 9. Twenty mL fractions were collected and at the finish the column was washed with 0.02 M NaCl. Every alternate fraction was analysed by HPLC to determine the concentration of total CMP, glyco-CMP, and the A and B variants of the aglyco-CMP. The results, shown in Figure 10, clearly show the displacement of the aglyco-CMP variants A and B from the column by the more tightly binding glyco-CMP.
  • the dry weight of the product amounted to about 4% of the starting MF WPI.
  • Samples of the starting WPI, flow-through WPI** and acidic peptides fraction were analysed by HPLC to determine the ratio of individual proteins as described in Example 2. The results are shown in Table 14.
  • the aglyco-CMP components made up 90% of the CMP remaining in the WPI**, whereas the glyco-CMP components made up 76% of the CMP in the eluate.
  • the starting MF WPI and the eluate powder were also analysed for sialic acid as described in Example 2.
  • the sialic acid content of the starting WPI was 0.4% while that of the eluate powder was 10%.
  • Anion WPI was prepared in much the same manner as in Example 9 except that elution of the WPI from the anion exchanger was carried out at pH 2 in 30 mM NaCl, such conditions being useful for the immediate hydrolysis of sialic acid groups from the CMP by heating.
  • Ultrafiltered cheese whey (240 g, 23% total solids) was diluted with 360 g of water and contacted with 214 mL of QA cellulose anion exchanger (152 g) from Example 1.
  • the whey and QA cellulose mixture was adjusted to pH 7.4 with 10% NaOH and stirred for 40 min.
  • the protein-depleted whey was then drained off and the QA cellulose washed to remove residual whey solids.
  • the WPI solution at pH 3.5 from part (a) was passed through a second column (12 mL) of QMA Spherosil (6.2 g) at a flow rate of 1 mL/min. (Analysis of the WPI solution emerging from the column at several points showed that the aglyco-CMP components did not bind to the column at this pH at any stage.)
  • the column was washed with water and the combined flow-through WPI solution and washings neutralized, dialysed and freeze-dried to give 1.52 g of WPI** powder (75% protein yield).
  • the column was washed with 0.5 M NaCl (30 mL) to recover the adsorbed acidic peptides (glyco-CMP).
  • the eluate was neutralized, dialysed and freeze-dried to give 0.13 g of powder (6% of initial protein). Analysis showed this powder to have 8.9% sialic acid.
  • Part a) was repeated and the WPI solution neutralized to pH 6.5, dialysed and freeze-dried for use as a comparative WPI sample.
  • the composition of the WPIs and their heat stabilities are shown in Tables 16 and 17.
  • the decrease in glyco-CMP from 7.6 to 2.4% was the only significant difference resulting in the WPI** being more heat stable.
  • the sialic acid content of the WPI from part a) was 1.2% and dropped to 0.3% after reducing the glyco-CMP content.
  • Freshly prepared rennet whey (0.56% protein) was adjusted to pH 4.9 with 10% HCl and 500 mL passed through a 12 mL column of QMA Spherosil (6.2 g) which had been previously washed with 0.5 M NaCl and water. A flow rate of 0.6 mL/min was used and 25 mL fractions collected. HPLC analysis of 20 DL samples showed that the B and A variants of aglyco-CMP broke through the column after 200 and 250 mL had been loaded respectively and very quickly rose to concentrations greater than that present in the starting whey. Some of the glyco-CMP components had still not broken through even after 500 mL of whey had been loaded. After loading, the column was washed with water. All of the column-passed whey fractions and wash water were combined (520 mL) and pH adjusted to 6.6 with 10% NaOH for further processing to recover WPI (part b, below).
  • the acidic peptides were recovered from the column by passing 50 mL of 0.5 M NaCl through it. This solution was neutralized, dialysed and freeze-dried for further analysis. It was found to have a sialic acid content of 5.6%. Its protein composition is shown in Table 18.
  • glyco-CMP reduced whey from part a) was passed through a 39 mL column of QMA Spherosil (6.2 g) at a flow rate of 3.5 mL/min. The column was washed with 30 mM NaCl. The combined column-passed whey and washings contained only 15% of the protein in the whey used at the start of part a).
  • WPI** was recovered from the column by circulating a further 25 mL of 30 mM NaCl through it whilst maintaining the pH at 2 as described in Example 12 a).
  • the desorbed protein solution was neutralized to pH 6.5 dialysed and freeze-dried for analysis. Its enhanced heat stability is shown in Table 17 and protein composition in Table 18.
  • a sample of ALACENTM 342 (WPC containing 80% protein produced from mineral acid whey) was obtained from the New Zealand Dairy Board. A 10% (w/w) solution (1000 g) was prepared and adjusted to pH 4.8 with 10% HCl. The acidic peptides were then removed from 750 g of this using 450 mL of QA cellulose as described in Example 6 a).
  • the HPLC analysis showed that the protein present in this eluate was composed mainly of PP5 (33%) plus the very hydrophilic peptides with a retention time of 3-7 minutes (as for CMP from sweet whey). About 80% of these hydrophilic peptides present in the starting WPC solution appeared in this eluate.
  • the WPI containing eluate from Example 14 d was neutralized to pH 6 and concentrated by ultrafiltration and diafiltration.
  • a sample of the final retentate was freeze-dried for phosphate analysis.
  • the pH of this was lowered to 5.3 with 10% HCl and then it was passed through a 6.5 mL column of Macro-Prep High Q at 2 mL/min.
  • the flow- through containing WPI* was collected.
  • a sample of it was subjected to the acid/heat stability test of Example 5. The results are shown in Table 19.
  • a further sample of the flow-through was neutralized, freeze-dried and analysed for phosphate content (Table 20).
  • the acidic peptides were desorbed from the column of Macro-Prep High Q by passing 60 ml of 0.5 M NaCl through it.
  • the eluate was neutralized, dialysed, freeze-dried and analysed for phosphate and the result shown in Table 20.
  • the present invention is believed to provide an efficient process of using anion exchange to obtain whey protein containing products from a whey protein-containing feedstocks.
  • WPI's containing mainly ⁇ -lactoglobulin or ⁇ -lactoglobulin and mainly aglyco-CMP which are heat and acid stable are produced.
  • a CMP isolate which may optionally also be enriched in glyco-CMP (and consequently sialic acid) is also provided.

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Abstract

La présente invention concerne des procédés de production d'un isolat protéique de lactosérum enrichi en β-lactoglobuline, d'un isolat protéique de lactosérum enrichi en β-lactoglobuline comportant un aglyco caséinomacropeptide (aglyco-CMP), d'un isolat sous forme de CMP et d'un isolat sous forme de glyco-CMP, à partir de matières premières contenant du lactosérum et par échange anionique.
PCT/NZ2001/000216 2000-10-05 2001-10-05 Procede de recuperation de proteines a partir de matieres premieres contenant des proteines de lactoserum Ceased WO2002028194A1 (fr)

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AU2002211115A AU2002211115A1 (en) 2000-10-05 2001-10-05 Process for recovering proteins from whey protein containing feedstocks

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NZ507356 2000-10-05
NZ50735600 2000-10-05
NZ51224301A NZ512243A (en) 2001-06-08 2001-06-08 Process for recovering proteins from whey protein containing feedstocks
NZ512243 2001-06-08

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003055322A1 (fr) * 2001-12-21 2003-07-10 Wyeth Composition de preparation pour nourrisson comprenant une quantite augmentee d'alpha-lactalbumine
US7651716B2 (en) 2001-12-21 2010-01-26 Wyeth Llc Methods for reducing adverse effects of feeding formula to infants
EP2640483A1 (fr) * 2010-11-15 2013-09-25 Biogen Idec Inc. Enrichissement et concentration d'isoformes de produit choisis par liaison surchargée et chromatographie d'élution
US9055752B2 (en) 2008-11-06 2015-06-16 Intercontinental Great Brands Llc Shelf-stable concentrated dairy liquids and methods of forming thereof
WO2017195221A1 (fr) * 2016-05-11 2017-11-16 Council Of Scientific & Industrial Research Appareil et procédé utilisant l'appareil pour séparer les protéines de petit-lait du petit-lait
EP1811868B1 (fr) 2004-10-13 2018-08-15 MJN U.S. Holdings LLC Preparations et methodes de formulation poour formules enteriques contenant de l acide sialique
WO2019076851A1 (fr) * 2017-10-20 2019-04-25 Groupe Lactalis Concentrat ou isolat de proteines solubles de lait stable au cours des traitements thermiques, et procede d'obtention.
US11490629B2 (en) 2010-09-08 2022-11-08 Koninklijke Douwe Egberts B.V. High solids concentrated dairy liquids
WO2024056840A1 (fr) 2022-09-16 2024-03-21 Univerza V Ljubljani Isolement de l'ostéopontine et du glycomacropeptide à partir du lactosérum
CN117882811A (zh) * 2018-06-27 2024-04-16 阿尔拉食品公司 酸性β-乳球蛋白饮料制品
EP4426128A4 (fr) * 2023-01-13 2025-03-12 Leprino Foods Company Protéines de lait dénaturées et leurs procédés de fabrication

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GB2251858A (en) * 1991-01-21 1992-07-22 Snow Brand Milk Products Co Ltd Production of kappa-casein glycomacropeptides using an anion-exchange resin
WO2001041584A1 (fr) * 1999-12-08 2001-06-14 Massey University Procede de separation de proteines lactoseriques au moyen d'un nouvel echangeur d'anions
WO2001041580A1 (fr) * 1999-12-08 2001-06-14 Fonterra Co-Operative Group Limited Methode permettant d'obtenir un produit appauvri en glyco-macro-peptide a partir de lactoserum

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GB2251858A (en) * 1991-01-21 1992-07-22 Snow Brand Milk Products Co Ltd Production of kappa-casein glycomacropeptides using an anion-exchange resin
WO2001041584A1 (fr) * 1999-12-08 2001-06-14 Massey University Procede de separation de proteines lactoseriques au moyen d'un nouvel echangeur d'anions
WO2001041580A1 (fr) * 1999-12-08 2001-06-14 Fonterra Co-Operative Group Limited Methode permettant d'obtenir un produit appauvri en glyco-macro-peptide a partir de lactoserum

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Title
NAKANO AND OZIMEK: "Purification of glycomacropeptide from non-dialyzable fraction of sweet whey by anion-exchange chromatography", BIOTECHNOLOGY TECHNIQUES, vol. 13, no. 11, 1999, pages 739 - 742 *

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6913778B2 (en) 2001-12-21 2005-07-05 Wyeth Infant formula compositions comprising increased amounts of alpha-lactalbumin
US7651716B2 (en) 2001-12-21 2010-01-26 Wyeth Llc Methods for reducing adverse effects of feeding formula to infants
KR100955408B1 (ko) 2001-12-21 2010-05-06 와이어쓰 엘엘씨 증가된 양의 알파-락트알부민을 포함하는 유아용 조제유
SG169897A1 (en) * 2001-12-21 2011-04-29 Wyeth Corp Infant formula compositions comprising increased amounts of alpha- lactalbumin
WO2003055322A1 (fr) * 2001-12-21 2003-07-10 Wyeth Composition de preparation pour nourrisson comprenant une quantite augmentee d'alpha-lactalbumine
EP1811868B1 (fr) 2004-10-13 2018-08-15 MJN U.S. Holdings LLC Preparations et methodes de formulation poour formules enteriques contenant de l acide sialique
US9055752B2 (en) 2008-11-06 2015-06-16 Intercontinental Great Brands Llc Shelf-stable concentrated dairy liquids and methods of forming thereof
US11490629B2 (en) 2010-09-08 2022-11-08 Koninklijke Douwe Egberts B.V. High solids concentrated dairy liquids
EP2640483A1 (fr) * 2010-11-15 2013-09-25 Biogen Idec Inc. Enrichissement et concentration d'isoformes de produit choisis par liaison surchargée et chromatographie d'élution
US20130331554A1 (en) * 2010-11-15 2013-12-12 Biogen Idec Inc. Enrichment and concentration of select product isoforms by overloaded bind and elute chromatography
US10842165B2 (en) 2016-05-11 2020-11-24 Council Of Scientific & Industrial Research Apparatus and method for separating whey proteins from whey using the same
WO2017195221A1 (fr) * 2016-05-11 2017-11-16 Council Of Scientific & Industrial Research Appareil et procédé utilisant l'appareil pour séparer les protéines de petit-lait du petit-lait
WO2019076851A1 (fr) * 2017-10-20 2019-04-25 Groupe Lactalis Concentrat ou isolat de proteines solubles de lait stable au cours des traitements thermiques, et procede d'obtention.
FR3072542A1 (fr) * 2017-10-20 2019-04-26 Groupe Lactalis Concentrat ou isolat de proteines solubles de lait stable au cours des traitements thermiques, et procede d'obtention.
CN117882811A (zh) * 2018-06-27 2024-04-16 阿尔拉食品公司 酸性β-乳球蛋白饮料制品
WO2024056840A1 (fr) 2022-09-16 2024-03-21 Univerza V Ljubljani Isolement de l'ostéopontine et du glycomacropeptide à partir du lactosérum
EP4426128A4 (fr) * 2023-01-13 2025-03-12 Leprino Foods Company Protéines de lait dénaturées et leurs procédés de fabrication
MA66910A1 (fr) * 2023-01-13 2025-03-28 Leprino Foods Company Protéines de lait dénaturées et leurs procédés de fabrication
US12349701B2 (en) 2023-01-13 2025-07-08 Leprino Foods Company Denatured milk proteins, methods of making, and protein fortified foods
MA66910B1 (fr) * 2023-01-13 2025-09-30 Leprino Foods Company Protéines de lait dénaturées et leurs procédés de fabrication

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