EP0595993A1 - Isolation de particules chargees a partir de fluides - Google Patents

Isolation de particules chargees a partir de fluides

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Publication number
EP0595993A1
EP0595993A1 EP92916550A EP92916550A EP0595993A1 EP 0595993 A1 EP0595993 A1 EP 0595993A1 EP 92916550 A EP92916550 A EP 92916550A EP 92916550 A EP92916550 A EP 92916550A EP 0595993 A1 EP0595993 A1 EP 0595993A1
Authority
EP
European Patent Office
Prior art keywords
membrane
process according
ion exchange
lactoferrin
lactoperoxidase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP92916550A
Other languages
German (de)
English (en)
Other versions
EP0595993A4 (en
Inventor
Ian Robert Mitchell
Geoffrey Welsford Smithers
David Alan Dionysius
Paul Anthony Grieve
Geoffrey Owen Regester
Eric Arnold James
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Commonwealth Scientific and Industrial Research Organization CSIRO
Dairy Research and Development Corp
Queensland Department of Primary Industries and Fisheries
Original Assignee
Commonwealth Scientific and Industrial Research Organization CSIRO
Dairy Research and Development Corp
Queensland Department of Primary Industries and Fisheries
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Commonwealth Scientific and Industrial Research Organization CSIRO, Dairy Research and Development Corp, Queensland Department of Primary Industries and Fisheries filed Critical Commonwealth Scientific and Industrial Research Organization CSIRO
Publication of EP0595993A1 publication Critical patent/EP0595993A1/fr
Publication of EP0595993A4 publication Critical patent/EP0595993A4/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J47/00—Ion-exchange processes in general; Apparatus therefor
    • B01J47/014—Ion-exchange processes in general; Apparatus therefor in which the adsorbent properties of the ion-exchanger are involved, e.g. recovery of proteins or other high-molecular compounds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J47/00—Ion-exchange processes in general; Apparatus therefor
    • B01J47/12—Ion-exchange processes in general; Apparatus therefor characterised by the use of ion-exchange material in the form of ribbons, filaments, fibres or sheets, e.g. membranes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • 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

  • This invention relates to a process for the separation of charged molecules from a fluid using ion exchange media.
  • the invention is particularly suitable for the extraction of protein components from biological fluids such as milk and milk products. It will be convenient to hereinafter describe the invention with particular reference to the extraction of protein components from milk and milk products but it is to be understood that the invention is not limited thereto.
  • Milk is a fluid secreted by all species of mammals to supply nutrition, and immune and non-immune protection to the young. Milk consists of water, proteins, fat, carbohydrates, salts, vitamins and a variety of miscellaneous components. Both young and mature humans consume large amounts of bovine milk, and the fluid thus has both nutritional and commercial significance.
  • bovine milk proteins (30 - 35 g/L) consist of caseins (approximately 80%), whey proteins (approximately 20%) and a number of minor protein/enzyme constituents.
  • Whey the yellow-green liquid that separates from the curd during the manufacture of cheese and acid casein, has long been considered a waste by-product in the dairy industry.
  • the protein in whey accounts for about 20% of total milk protein.
  • the primary proteinaceous constituents of whey are ⁇ -lactoglobulin and ⁇ -lactalbumin, two small globular proteins that account for some 70 - 80% of total whey protein.
  • Minor protein components include the glycomacropeptide, serum albumin, lactoferrin, immunoglobulins, phospholipo- proteins, and a number of enzymes (including lactoperoxidase).
  • WPC spray dried whey powder and whey protein concentrate
  • Belgian Patent Specification 901672 describes an alternative ion exchange technique based on a calcium alginate medium in which ion exchange functionality has been obtained by admixture of oxides of zirconium, titanium, silicon (quartz) or aluminium.
  • the milk or whey is mixed with the ion exchange medium in a stirred tank whereby proteins having an isoelectric point above 7.5 are adsorbed to the ion exchange medium.
  • the ion exchange gel is separated mechanically from the milk or whey, washed and diluted with calcium chloride.
  • the Belgian process adopts this unusual methodology because conventional ion exchange columns tend to become quickly fouled using a milk product feedstock. Fat, casein fines, and other particulates, components normally found in pasteurised/separated whey, pose a problem in traditional column based chromatographic procedures as they act as column foulants, both reducing the resin effectiveness and the recovery of the product.
  • a process for the separation of charged molecules from a fluid comprising providing an ion exchange medium disposed on a porous membrane, passing the fluid through the membrane, wherein said charged molecules are preferentially adsorbed on the medium, and eluting the adsorbed molecules from the medium.
  • the above process may be used for the separation of any charged molecules from fluids.
  • molecule when used herein also encompasses aggregates of such molecules.
  • the process is particularly suitable for treatment of biological fluids such as milk or milk products, blood or blood plasma, or other types of fluids such as fermentation fluids, fluids from cell culture, etc.
  • milk or milk product includes milk products such as skim milk, whey, colostrum etc.
  • the process may be used to isolate cationic protein components such as lactoferrin, lactoperoxidase, growth promoting agents and lysozyme from milk or milk products.
  • the process may also be used to isolate other charged molecules such as ⁇ -lactalbumin, glycomacro- peptide, serum albumin, immunoglobulins and enzymes.
  • the process of the present invention may also be used for isolating charged molecules from other biological fluids for instance blood and blood products such as plasma. For example clotting factors, serum albumin and immunoglobulins may be isolated from blood or plasma.
  • the present invention may also be used for processing other fluids such as fermentation fluids or fluids from cell culture wherein pharmaceuticals, vitamins, hormones or other therapeutic proteins may be isolated from the fluid.
  • ion exchange media disposed on membranes with a thickness of the order of microns or millimetres can extract an effective yield of certain protein species from a fluid such as milk.
  • a fluid such as milk.
  • long ion exchange columns and long residence times were required to achieve an effective yield of such species.
  • Suitable pore sizes for the ion exchange membrane range from about 0.1 to about 1.2 microns, preferably about 0.2 to about 0.6 microns with about 0.4 microns being most preferred.
  • the pore size of the membranes used in the process of the present invention may be a little larger than those used in the cross-flow microfiltration process adopted in PCT/SE88/00643 as it is not critical to exclude all particulate matter with the membrane. Larger pores may assist in achieving higher flow-through rates in the present invention.
  • the fluid may be subjected to a microfiltration step prior to being passed through the membrane.
  • the fluid may be passed through the membrane either by means of a dead-end filtration technique or a cross- flow filtration technique.
  • dead-end or static pressure filtration involves forcing a feed material against and through a vertical filter
  • cross-flow filtration involves the passage of a feed material through a narrow gap between two parallel filters, the material passing across the filter surface at a high linear flux.
  • the membranes useable in the present invention can be quantitatively eluted using solutions of successively higher salt concentrations, and/or by successive changes in the pH of an eluting solution to shift the pH of the medium above or below the isoelectric point of the desired charged molecule such as a protein.
  • differences in pi or other binding parameters between the various charged molecules can be exploited to produce relatively pure fractions of each charged molecule as described, for instance in PCT/SE88/00643.
  • Preferential binding of particular proteins may also be exploited to isolate one particular protein from a fluid in preference to other proteins.
  • lactoferrin binds more tightly to the membrane than lactoperoxidase and displaces lactoperoxidase from the membrane. Accordingly the membrane may be saturated with lactoferrin and a relatively pure fraction of lactoferrin may be isolated from a milk product containing both lactoferrin and lactoperoxidase.
  • Elution of the charged molecules can be commenced immediately after passing the fluid through the membrane i.e. before cleaning the upstream surface of the membrane of matter such as fat globules and proteinaceous debris.
  • the membrane can be cleaned of such matter for instance with a wash prior to introducing the eluting medium.
  • the membranes useable in the present invention may avoid problems with swelling or packing of the exchange matrix and maintenance is easier. For example, if an ion exchange membrane runs dry, it does not have to be re-packed. Furthermore sanitisation or sterilisation methods are more diverse and adaptable to existing procedures in the dairy industry such as steaming, dairy detergents or alkaline solutions such as sodium hydroxide.
  • Strong or weak cation or anion exchange medium Either a strong or weak cation or anion exchange medium may be used.
  • strong and weak in the context of ion exchange functional groups refer to the extent of ionisation of the group with pH of the medium. Strong ion exchange functional groups are totally ionised over a wide range of pH values.
  • a strong cation exchange functional group e.g. sulphopropyl
  • the exchange medium may be selected to ensure binding of the desired charged molecules.
  • a strong cation exchange medium should be selected to ensure binding of protein species with high pi values.
  • a suitable strong cation exchange media comprises sulfonic acid functional groups disposed on a symmetrical polya ide support.
  • Such membranes may be prepared in a two step procedure by grafting a polymer onto an inert microporous membrane followed by the introduction of the desired cationic or anionic functional groups. It will thus be apparent that wide variations in pore size and amount of grafted polymer are feasible.
  • the membrane useable in the present invention can be provided in the form of convenient cross-flow cartridges (modules) or dead-end f lters.
  • the effluent which is produced as a by-product of the process of the present invention may also have advantageous properties.
  • extraction of the cationic proteins lactoferrin and lactoperoxidase from cheese whey results in a whey product stream essentially unaltered from that used as the feed material, because these cationic proteins represent less than 3% of the total whey protein.
  • the process also ensures that this whey product stream is free of particulates and very low in microorganisms and fat.
  • this whey product stream may be further processed into a low-fat whey protein concentrate powder with advantageous properties including high solubility.
  • Figure 3 Permeate flux (* ), lactoperoxidase activity (•), and lactoferrin concentration ( ⁇ ) during cross-flow membrane ion exchange filtration of pure proteins in sodium phosphate buffer, pH 6.7. Starting levels in feed material: lactoperoxidase, 5.3 IU/mL; lactoferrin, 100.0 mg/L.
  • Figure 4 HPLC chromatogram of samples from cross- flow membrane ion exchange filtration of pure proteins in sodium phosphate buffer, pH 6.7. a, feed material; b, permeate after processing 107 L; c, 0.2 M NaCl eluate (1:24 dilution).
  • Figure 5 SDS-PAGE electrophoretogram of samples from cross-flow membrane ion exchange filtration of pure proteins in sodium phosphate buffer, pH 6.7.
  • Lane 1 initial feed material; lane 2, retentate during loading; lane 3, permeate during loading; lane 4, 0.2 M NaCl eluate; lane 5, 0.4 M NaCl eluate; lane 6, 1.0 M NaCl eluate; lane 1 , low molecular weight protein markers.
  • Figure 6 Permeate flux ( -), lactoperoxidase activity (•), and lactoferrin concentration ( ⁇ ) during cross-flow membrane ion exchange filtration of microfiltered Cheddar cheese whey, pH 6.0. Starting levels in whey feed material: lactoperoxidase, 7.8 IU/mL; lactoferrin, 81.4 mg/L.
  • Figure 7 HPLC chromatogram of samples from cross- flow membrane ion exchange filtration of microfiltered Cheddar cheese whey, pH 6.0. a, feed material; b, permeate after processing 37 L; c, 1 M NaCl eluate (1:9 dilution).
  • Figure 8 SDS-PAGE electrophoretogram of samples from cross-flow membrane ion exchange filtration of microfiltered Cheddar cheese whey, pH 6.0. Lane 1, initial feed material; lane 2, retentate during loading; lane 3, permeate during loading; lanes 4 and 5, 1 M NaCl eluate; lane 10, low molecular weight protein markers.
  • Figure 9 Permeate flux (A), lactoperoxidase activity (•), and lactoferrin concentration ( ⁇ ) during cross-flow membrane ion exchange filtration of microfiltered Cheddar cheese whey, pH 6.9. Starting levels in feed material: lactoperoxidase, 3.4 IU/mL; lactoferrin, 40.0 mg/L.
  • Figure 10 HPLC chromatogram of samples from cross- flow membrane ion exchange filtration of microfiltered Cheddar cheese whey, pH 6.9. a, feed material; b, permeate after processing 53 L; c, 1 M NaCl eluate (1:12 dilution).
  • Figure 11 SDS-PAGE electrophoretogram of samples from cross-flow membrane ion exchange filtration of microfiltered Cheddar cheese whey, pH 6.9. Lane 6, initial feed material; lane 7, permeate during loading; lane 8, retentate during loading; lane 9, 1.0 M NaCl eluate; lane 10, low molecular weight protein markers.
  • Figure 12 Permeate flux (A ), lactoperoxidase activity (•), and lactoferrin concentration ( ⁇ ) during cross-flow membrane ion exchange filtration of non- microfiltered Cheddar cheese whey, pH 6.2.
  • a cross-flow module with a surface area of 0.6 m 2 was used.
  • Starting levels in feed material lactoperoxidase, 11.0 IU/mL; lactoferrin, 116.0 mg/L.
  • Figure 13 HPLC chromatogram of samples from cross- flow membrane ion exchange filtration of non- microfiltered Cheddar cheese whey, pH 6.2. a, feed material; b, permeate after processing 25 L; c, 1 M NaCl eluate (1:15 dilution).
  • Figure 14 SDS-PAGE electrophoretogram of samples from cross-flow membrane ion exchange filtration of non-microfiltered Cheddar cheese whey, pH 6.2. Lane 1, initial feed material; lane 2, retentate during loading; lane 3, permeate during loading; lanes 4 and 5, 1.0 M NaCl eluate; lane 10, low molecular weight protein markers.
  • Figure 15 Permeate flux (A), lactoperoxidase activity (•), and lactoferrin concentration ( ⁇ ) during cross-flow membrane ion exchange filtration of non- microfiltered Cheddar cheese whey, pH 6.2.
  • a cross-flow module with a surface area of 0.5 m 2 was used.
  • Starting levels in feed material lactoperoxidase, 11.2 IU/mL; lactoferrin, 116.0 mg/L.
  • Figure 16 HPLC chromatogram of samples from cross- flow membrane ion exchange filtration of non-micro- filtered Cheddar cheese whey, pH 6.2. a, feed material; b, permeate after processing 26 L; c, 1 M NaCl eluate (1:5 dilution).
  • Figure 17 SDS-PAGE electrophoretogram of samples from cross-flow membrane ion exchange filtration of non-microfiltered Cheddar cheese whey, pH 6.2. Lane 1, initial feed material; lane 6, retentate during loading; lane 7, permeate during loading; lanes 8 and 9, 1.0 M NaCl eluate; lane 10, low molecular weight protein markers.
  • Figure 18 HPLC chromatogram of samples from dead-end membrane ion exchange filtration of microfiltered Cheddar cheese whey, pH 6.2. a, feed material; b, permeate after filtering 50 mL; c, 1 M NaCl eluate (1:3 dilution).
  • Figure 19 SDS-PAGE electrophoretogram of samples from dead-end membrane ion exchange filtration of microfiltered Cheddar cheese whey, pH 6.2. Lane 1, low molecular weight protein markers; lane 4, initial feed material; lane 5, filtrate (permeate) during loading; lane 6, 1.0 M NaCl eluate.
  • Figure 20 HPLC chromatogram of samples from dead ⁇ end membrane ion exchange filtration of microfiltered Cheddar cheese whey, pH 7.0. a, feed material; b, 1 M NaCl eluate (1:3 dilution).
  • Figure 21 SDS-PAGE electrophoretogram of samples from dead-end membrane ion exchange filtration of microfiltered Cheddar cheese whey, pH 7.0. Lane 1, low molecular weight protein markers; lane 2, filtrate (permeate) during loading; lane 3, 1.0 M NaCl eluate; lane 4, initial feed material.
  • Figure 22 HPLC chromatogram of samples from dead-end membrane ion exchange filtration of dialyzed and microfiltered Cheddar cheese whey, pH 7.0. a, feed material; b, permeate after filtering 30 mL; c, 1 M NaCl eluate (1:3 dilution).
  • Figure 23 SDS-PAGE electrophoretogram of samples from dead-end membrane ion exchange filtration of dialyzed and microfiltered Cheddar cheese whey, pH 7.0. Lane 1, low molecular weight protein markers; lane 2, filtrate (permeate) during loading; lane 3, 1.0 M NaCl eluate; lane 6, initial feed material.
  • Figure 24 Filtrate (permeate) flux during dead-end filtration of microfiltered (A) and non-microfiltered (B) Cheddar cheese whey, pH 6.5 using a membrane ion exchange Sartobind S filter (5.4 cm 2 , 0.45 ⁇ m pore)( ⁇ ) or a conventional Minisart N filter (5.3 cm , 0.2 ⁇ m pore)( ⁇ ). Flux rates were determined at 50 ⁇ C at a constant applied pressure of 50 kPa.
  • Dairy whey was a byproduct of Cheddar cheese production, and was obtained fresh either from commercial cheese manufacturers or prepared "in-house” at the CSIRO Dairy Research Laboratory.
  • the whey was separated (40°C) and pasteurized (72°C, 15 sec) prior to use.
  • Non-fat milk was prepared by separation (35°C - 40°C) and pasteurization (72 C C, 15 sec).
  • cytochrome c equine heart
  • bovine lactoferrin and lactoperoxidase were isolated from cheese whey essentially as described previously (Law, B.A., and Reiter, B. (1977) The isolation and bacteriostatic properties of lactoferrin from bovine milk whey,
  • Milk ultrafiltrate was prepared by collecting the permeate stream during ultrafiltration (18,000 molecular weight cutoff membrane) of non-fat milk at 50°C.
  • the cheese whey and non-fat milk raw materials were pretreated using microfiltration prior to membrane ion exchange.
  • Sartorius Minisart N 5.3 cm 2 , 0.2 ⁇ m pore
  • non-fat milk using Nalgene cellulose acetate (3.8 cm 2 , 0.45 ⁇ m pore) at 20°C.
  • Binding and recovery data were collected at ambient temperature and at 50"C using a controlled flow-rate of 10 mL/min. Flux data were collected at 50 ⁇ C using a constant pressure of 50 kPa. Filters were pre-equilibrated prior to use and washed with 10 mM sodium phosphate, pH 7.0 (10 mL), and bound protein was eluted with 1 M NaCl in 10 mM sodium phosphate, pH 7.0 (10 mL).
  • the areas of peaks, representing proteins of interest were determined by electronic integration using the Delta Junior data analysis software package (Digital Solutions Pty. Ltd., Australia). Standard lactoperoxi- dase and lactoferrin eluted from the column, under the stated conditions, with retention times of 9.7 min and 18.9 min, respectively.
  • Polyacrylami e gel electrophoresis Determination of the identity, purity and molecular size of protein components in the raw materials and in samples following membrane ion exchange processing was carried out by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (denaturing conditions) (SDS- PAGE) using a vertical slab gel apparatus as described previously (Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4, Nature 277, 680-685). Proteins in samples (50 ⁇ L) were separated in a linear 10 - 15% gradient gel, and protein bands were stained with Coomassie Brilliant Blue R.
  • SDS- PAGE sodium dodecyl sulphate
  • Low molecular weight marker proteins were used to calibrate the gel. Markers included: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa) and ⁇ -lactalbumin (14.4 kDa). Under the conditions used, lactoferrin and lactoperoxidase, appear on the gel at an equivalent molecular weight of 80 kDa.
  • Spec tropho tome try Activity of the enzyme lactoperoxidase was determined spectrophotometrically in a continuous assay using the artificial substrate 2,2'- azino-Jbis(3-ethyl-benzthiazoline-6-sulphonic acid)(ABTS), essentially as described previously (Putter, J. , and Becker, R. (1983) Peroxidases. In Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), Vol. 3 (3rd Edition), pp. 286-293, Verlag Chemie, Weinheim). Assay mixtures (2.38 mL) contained 1.67 mM ABTS and 0.18 mM H 2 ⁇ 2 in 100 mM citrate buffer, pH 5.5.
  • the reaction was initiated by the addition of enzyme containing solution (20 ⁇ L), and the rate of change of absorbance at 405 nm was measured on a recording spectrophotometer at 25°C.
  • One unit of enzyme activity catalyzes the oxidation of 1 ⁇ mole of ABTS per min under the stated conditions (IU).
  • Example 1 In a trial to determine the binding capacity of the membrane ion exchanger (cross-flow configuration) for pure proteins, 200 L of 10 mM sodium phosphate (pH 6.7) containing 30 mg/L lactoperoxidase and 100 mg/L lactoferrin was used as feed material. The Sartocon II plant was configured to recycle the retentate stream. Samples of permeate and retentate were collected during loading for later analysis by HPLC, SDS-PAGE and enzyme assay. After loading, the membrane was washed with 10 L of 10 mM sodium phosphate pH 6.7, and elution of the proteins was carried out at 20°C with three 10-L batches of the sodium phosphate buffer containing increasing concentrations of NaCl, viz.
  • Example 2 Cheddar cheese whey at pH 6.0 was microfiltered prior to membrane ion exchange.
  • the microfiltered whey (120 L) was passed over the narrow channel cross-flow module (0.6 m 2 ) using the Sartocon II plant configured to recycle the retentate stream.
  • Samples of permeate and retentate were collected during the trial to enable subsequent determination of the lactoferrin and lactoperoxidase content by HPLC, SDS-PAGE, and enzyme assay.
  • the membrane was washed with 45 L of 10 mM NaCl. Elution was subsequently effected with 20 L of 1 M NaCl at 20°C, with the retentate stream in non-recycle mode.
  • Example 3 In this trial, experimental conditions were as described above for Example 2 with the exception that the whey was adjusted to pH 6.9 with a concentrated solution of NaOH prior to membrane ion exchange. Following pH adjustment, the whey (97 L) was micro- filtered and then subjected to cross-flow membrane ion exchange as described for Example 2 , with the exception that elution was carried out with 10 L of IM NaCl at 20°C. Data describing permeate flux and "breakthrough" of the proteins of interest in the permeate stream during the trial are shown in Figure 9. Results of HPLC and
  • Example 4 Cheddar cheese whey at pH 6.2 was used, as described above for Example 2, with the exception that the whey was not microfiltered prior to membrane ion exchange.
  • the whey (80 L) was passed over the narrow channel cross-flow module (0.6 m 2 ) using the Sartocon II plant configured to recycle the retentate stream.
  • Other processing conditions were as described for Example 2 , with the exception that elution was carried out with 10 L of IM NaCl at 20 ⁇ C.
  • Data describing permeate flux and "breakthrough" of the proteins of interest in the permeate stream during the trial are shown in Figure 12. Results of HPLC and SDS-PAGE analysis of samples from the trial are depicted in Figures 13 and 14, respectively.
  • Example 5 This trial was a repeat of that described for Example 4 , with the exception that the whey (pH 6.2, 76 L, same batch as that used in Example 4 ) was passed over the wide channel cross-flow module (0.5 m 2 ). Other processing conditions were as described for Example 2, with the exception that elution was carried out with 10 L of IM NaCl at 20 ⁇ C. Data describing permeate flux and "breakthrough" of the proteins of interest in the permeate stream during the trial are shown in Figure 15. Results of HPLC and SDS-PAGE analysis of samples from the trial are depicted in Figures 16 and 17, respectively.
  • Example 6 In a trial to determine the binding capacity of the membrane ion exchanger (dead-end configuration, Sartobind S, 5.4 cm 2 ) for pure proteins, 20 mL of 10 mM sodium phosphate (pH 7.0) containing 0.6 mg/mL cytochrome c, or 0.6 mg/mL lactoperoxidase, or 0.6 mg/mL lactoferrin were used as feed materials. The experiment was carried out at 20°C. Samples of the feed, filtrate, wash and eluate were collected for later analysis by HPLC and spectrophotometry.
  • Example 8 In this trial, the binding capacity of the membrane ion exchanger (dead-end configuration, Sartobind S, 5.4 cm 2 ) for lactoferrin and lactoperoxidase dissolved in 10 mM sodium phosphate (pH 7.0), when presented as a solution containing both proteins, was determined at both 20°C and 50"C. The experiment was carried out as described for Example 6 with the exception that the feed material (60 mL) contained 0.03 mg/mL lactoperoxidase and 0.15 mg/mL lactoferrin, concent ⁇ rations that mimic those found in milk and whey.
  • Example 9 microfiltered Cheddar cheese whey at pH 6.2 (150 mL) was presented to the membrane ion exchange filter (Sartobind S, 5.4 cm 2 ) at 20°C. Samples of the feed, filtrate, wash and eluate were collected for later analysis by HPLC, SDS-PAGE and spectrophotometry. Results of HPLC and SDS-PAGE analysis of samples from the trial are shown in Figures 18 and 19, respectively. Data describing binding capacity of the membrane for lactoferrin and lactoperoxidase, and recovery of these proteins following elution, are presented in Table 9.
  • Example 10 This trial was a repeat of that described in Example 9, with the exception that the whey was adjusted to pH 7.0 with 1 M NaOH prior to membrane ion exchange. Results of HPLC and SDS-PAGE analysis of samples from the trial are shown in Figures 20 and 21, respectively. Data describing binding capacity of the membrane for lactoferrin and lactoperoxidase, and recovery of these proteins following elution, are presented in Table 10.
  • Example 11 This trial was a repeat of that described in Example 9 , with the exception that the whey (50 mL) was dialyzed against 10 mM sodium phosphate, pH 7.0 prior to membrane ion exchange. Results of HPLC and SDS-PAGE analysis of samples from the trial are shown in Figures 22 and 23, respectively. Data describing binding capacity of the membrane for lactoferrin and lacto ⁇ peroxidase, and recovery of these proteins following elution, are presented in Table 11.
  • Example 12 filtrate (permeate) flux rates for dead-end membrane ion exchange filtration (Sartobind S, 5.4 cm 2 , 0.45 ⁇ m pore) were compared with flux rates for conventional filtration (Minisart N, 5.3 cm 2 , 0.2 ⁇ m pore) using both microfiltered and non-microfiltered Cheddar cheese whey, pH 6.5.
  • the experiments were conducted at 50°C using a constant applied pressure of 50 kPa. Results are reported in Figure 24.
  • Non-fat milk containing lactoferrin (75.5 mg/L) and lactoperoxidase (9.7 IU/mL), was presented to the Sartobind S dead-end filter (5.4 cm 2 ) at 20°C. b Definition is provided in Figure 2.

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Abstract

Procédé de séparation, à partir de fluides tels que des fluides biologiques, et à l'aide d'un milieu échangeur d'ions placé sur une membrane poreuse, de molécules chargées telles que les protéines. On empêche les globules adipeux et les matières particulaires de traverser la membrane afin d'éviter que le milieu échangeur d'ions ne se bouche.
EP9292916550A 1991-07-25 1992-07-24 Isolation of charged particles from fluids Withdrawn EP0595993A4 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
AUPK743691 1991-07-25
AU7436/91 1991-07-25
PCT/AU1992/000381 WO1993002098A1 (fr) 1991-07-25 1992-07-24 Isolation de particules chargees a partir de fluides

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EP0595993A4 EP0595993A4 (en) 1994-08-17

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EP0744901B1 (fr) * 1994-02-16 2001-12-05 Pharming Intellectual Property BV Isolation de la lactoferrine du lait
JP4263932B2 (ja) * 2003-04-01 2009-05-13 雪印乳業株式会社 ラクトフェリンの製造方法
JP4511847B2 (ja) * 2004-02-16 2010-07-28 積水化学工業株式会社 ヘモグロビンＡ１ｃの測定方法
DE102007012439A1 (de) * 2007-03-15 2008-09-18 Emsland-Stärke GmbH Verfahren zur Gewinnung pflanzlicher Proteine und/oder Peptide, danach hergestellte Proteine und/oder Peptide und Verwendung derselben
US10048262B2 (en) 2012-06-13 2018-08-14 Asahi Kasei Kabushiki Kaisha Method for detecting specific substance in milk
EP3451845B1 (fr) * 2016-05-11 2020-03-11 Council of Scientific & Industrial Research Appareil et procédé utilisant l'appareil pour séparer les protéines de petit-lait du petit-lait
US11109604B2 (en) 2019-05-09 2021-09-07 Memtec LLC Dairy processing systems and methods
GB201906722D0 (en) * 2019-05-13 2019-06-26 Ttp Plc A method of preparing a sample for a diagnostic assay

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BE901672A (fr) * 1985-02-07 1985-08-07 Oleofina Sa Procede de purification de proteines du lait et de ses derives.
FR2584727B1 (fr) * 1985-07-11 1988-06-17 Roussel Uclaf Procede d'extraction de proteines du lait, produits, application du procede, et compositions pharmaceutiques
SE458818B (sv) * 1987-11-27 1989-05-16 Svenska Mejeriernas Riksforeni Foerfarande foer utvinning av rena fraktioner av laktoperoxidas och laktoferrin ur mjoelkserum

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See also references of WO9302098A1 *

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EP0595993A4 (en) 1994-08-17
JPH07502016A (ja) 1995-03-02
NZ243727A (en) 1995-03-28
WO1993002098A1 (fr) 1993-02-04

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