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Title:
RECOVERY PROCESS
Document Type and Number:
WIPO Patent Application WO/1999/015023
Kind Code:
A2
Abstract:
The invention provides a process for the separation of a useful substance from a solution. The process includes the steps of: (i) contacting the solution with particles of a composite magnetic resin which comprises magnetic particles embedded in a polymeric matrix which either contains or has attached thereto sites which bind the substance to be recovered; and (ii) separating by magnetic separation the composite magnetic resin particles and bound substance from solution. The process preferably includes the further steps of (iii) dissociating the substance from the composite magnetic resin particles under conditions which substantially maintain the integrity of the substance for further use in the food and/or health industries; and (iv) recovering the dissociated substance. In prefered embodiments, the solution is a natural dairy product or a solution obtained during dairy processing, such as whole milk, skim milk, cream, whey, whey permeate or colostrum.

Inventors:
Bridger, Lynton Alexander (243 Tarata Road RD7 Inglewood, NZ)
Application Number:
PCT/NZ1998/000140
Publication Date:
April 01, 1999
Filing Date:
September 22, 1998
Export Citation:
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Assignee:
KIWI CO-OPERATIVE DAIRIES LIMITED (Whareroa Road Hawera, NZ)
Bridger, Lynton Alexander (243 Tarata Road RD7 Inglewood, NZ)
International Classes:
A23C7/04; A23C9/14; B03C1/01; (IPC1-7): A23C/
Domestic Patent References:
WO1994011103A11994-05-26
Foreign References:
US4144373A1979-03-13
US4554088A1985-11-19
EP0666577A11995-08-09
Attorney, Agent or Firm:
Rutledge, Sue Moira (West-Walker Bennett Mobil on the Park PO Box 17 Lambton Quay Wellington 6001, 344 15, NZ)
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Claims:
WHAT IS CLAIMED IS
1. A process for the separation of a useful substance from a solution containing said substance, which process includes the steps of : (i) contacting the solution with particles of a composite magnetic resin which comprises magnetic particles embedded in a polymeric matrix which either contains or has attached thereto sites which bind the substance to be recovered; and (ii) separating by magnetic separation the composite magnetic resin particles and bound substance from solution.
2. A process as claimed in claim 1, which includes the further steps of : (iii) dissociating the substance from the composite magnetic resin particles under conditions which substantially maintain the integrity of the substance for further use in the food and/or health industries; and (iv) recovering the dissociated substance.
3. A process as claimed in claim 1 or 2 wherein the sites have affinity for and are selective for the substance to be recovered.
4. A process as claimed in any one of claims 1 to 3 wherein the solution is selected from the group consisting of whole milk, skim milk, cream, whey or whey permeate.
5. A process as claimed in any one of claims 1 to 4 wherein the substance to be recovered is a lipid, a fatty acid, a growth factor, an enzyme or other protein, a pigment, a flavouring compound, a vitamin, or a mineral ion.
6. A process as claimed in any one of claims 1 to 5 wherein the substance to be recovered is calcium or magnesium.
7. A process as claimed in any one of claims 1 to 5 wherein the substance to be recovered is riboflavin and the sites selective for riboflavin comprise magnesium silicate.
8. A process as claimed in any one of claims 1 to 3 wherein the solution is a fermentation broth or a mixture of reaction products and the substance to be recovered is a fermentation product or other reaction product.
9. A process as claimed in any one of claims 1 to 8 which further includes the step of regenerating the magnetic resin particles for reuse following dissociation of the recovered substance.
10. A process as claimed in any one of claims 1 to 9 wherein the solution from which the substance has been removed is collected for further processing.
11. A process as claimed in any one of claims 1 to 10 wherein the magnetic particles of the composite magnetic resin comprise a core of a magnetic material surrounded by a mixture of fibrous material and a solid binding agent.
12. A substance recovered by a process as claimed in any one of claims 1 to 11.
13. A composite magnetic resin suitable for use in a process as claimed in any one of claims 1 to 12, said resin comprising magnetic particles embedded in a polymeric matrix which either contains or has attached thereto sites which bind a substance naturally present in whole milk, skim milk, cream, whey, whey permeate or colostrum.
14. A composite magnetic resin as claimed in claim 13 wherein the sites have affinity for and are selective for the substance.
15. A resin as claimed in claim 13 or 14 wherein the sites have affinity for a substance selected from a lipid, a fatty acid, a growth factor, an enzyme or other protein, a pigment, a vitamin, a hormone or a mineral ion.
16. A resin as claimed in any one of claims 13 to 15 wherein the sites have affinity for calcium.
17. A resin as claimed in any one of claims 13 to 16 wherein the sites comprise magnesium silicate and are selective for riboflavin.
18. A resin as claimed in any one of claims 13 to 17 wherein the magnetic particles of the composite magnetic resin comprise a core of a magnetic material surrounded by a mixture of fibrous material and a solid binding agent.
Description:
RECOVERY PROCESS<BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> FIELD OF THE INVENTION This invention relates to a recovery process. More particularly, it relates to a process for recovering minor components from solutions for use in the food and health/pharmaceutical industries. It particularly but not solely relates to a process for recovering minor components from whole milk, skim milk, cream, whey or whey permeate.

BACKGROUND In the dairy industry, there are a number of applications for specific minor components naturally found in mammalian milk and whey solutions. There are also a number of approaches taken to isolate or recover such components. The majority of these involve chromatography, including affinity, ion exchange, size exclusion, hydrophobic interaction and mixed mode chromatographic methods.

Chromatography media used in the dairy industry comprise particles of highly porous material (including cellulose derivatives such as cross linked dextran, or styrene or acrylate polymers). The functional groups are attached generally to glucose units in and around the matrix by stable ether linkages. The affinity of the functional groups to find and bind with the component (s) of interest with such resins is therefore limited by the ability of the influent to be passed around and into the porous spheres. In particular, viscous solutions are difficult to treat, as are solutions in which the minor components it is desired to separate are present in low concentrations and/or bind weakly with the affinity group.

Further, solutions containing suspended solids cannot be treated by passage through beds of porous spheres, the usual method of choice. There are many such solutions of economic interest to the food and pharmaceutical industries and they include unclarified fruit juices, whole fermentation broths, slurries of tea, coffee or hops, and whole blood.

The present invention therefore has as its object the provision of alternative processes for recovering such minor components. The processes of the invention

are believed by the applicants to represent improvements over processes currently employed and as a minimum offer the public a useful choice.

SUMMARY OF THE INVENTION Accordingly, in a first aspect, the invention provides a process for the separation of a useful substance from a solution containing said substance, which process includes the steps of : (i) contacting the solution with particles of a composite magnetic resin which comprises magnetic particles embedded in a polymeric matrix which either contains or has attached thereto sites which bind the substance to be recovered; and (ii) separating by magnetic separation the composite magnetic resin particles and bound substance from the solution.

In a preferred embodiment, the process includes the further steps of : (iii) dissociating the substance from the composite magnetic resin particles under conditions which substantially maintain the integrity of the substance for further use in the food and/or health industries; and (iv) recovering the dissociated substance.

Preferably, the sites which bind the substance to be recovered have affinity for and are selective for the substance.

Conveniently, the solution is whole milk, skim milk, cream, whey, whey permeate or colostrum.

Conveniently, the substance recovered is a mineral ionic species such as calcium or magnesium.

Alternatively, the substance recovered is or includes a protein such as an enzyme. In this embodiment, the conditions of dissociation are selected to ensure that the protein component is not denatured.

In further alternatives, the substance recovered is a vitamin, a flavouring compound, a pigment, a lipid, a fatty acid or a biologically active compound such as a growth factor.

In still further alternatives, the solution may be a fermentation broth or other reaction product mixture and the useful substance recovered is a product of fermentation or other reaction product.

It is preferred that the composite magnetic resin particles be regenerated for reuse following dissociation of the recovered substance.

It is also preferred that the magnetic particles of the composite magnetic resin comprise a core of a magnetic material surrounded by a mixture of a fibrous material and a solid binding agent.

The applicants further contemplate that the solution from which the substance has been removed is collected for further processing. Such further processing may include recovery of a further substance or substances therefrom by a process as defined above but with sites on the composite magnetic resin particles having affinity for and being selective for the further substance (s).

In a further aspect, the invention provides a substance recovered by a process as defined above.

In yet a further aspect, the invention provides a composite magnetic resin suitable for use in a process as defined above, said resin comprising magnetic particles embedded in a polymeric matrix which either contains or has attached thereto sites which bind to a substance naturally present in whole milk, skim milk, cream, whey, whey permeate or colostrum.

It is preferred that the sites have affinity for and are selective for the substance.

Preferably, the sites have affinity for a substance selected from mineral ions (such as calcium), lipids (such as phosphatidylcholine or sphingolipids), fatty acids (such as linoleic acid), enzymes (such as sialyltransferase), other proteins (such as immunoglobulins or lactoferrins), growth factors (such as sialyllactose and IGF), and vitamins (such as riboflavin). In one preferred embodiment, the sites comprise magnesium silicate and are selective for riboflavin.

Preferably, the magnetic particles of the composite magnetic resin comprise a core of a magnetic material surrounded by a mixture of a fibrous material and a solid binding agent.

While the invention is broadly as defined above, it will also be appreciated that it is not limited thereto but that it further includes embodiments of which the following description provides examples.

DESCRIPTION OF THE INVENTION As described above, this invention is broadly directed to a process for the recovery of valuable substances from solutions which contain them. The process has particular application to recovery of substances useful in the food, health and pharmaceutical industries. Solutions from which such useful substances can be recovered include natural dairy products or solutions obtained during dairy processing, including whole milk, skim milk, cream, whey, whey permeate and colostrum. Other solutions to which the process of the present invention is applicable include fruit juices, grape juices, wine and other alcoholic beverages, and extracts of tea, coffee, hops, soy and cocoa beans. The applicants further contemplate that the process may be used to extract useful reaction or fermentation products from reaction mixtures or fermentation broths which contain them in solution. The process may also be used to extract useful substances from hydrolysates, homogenates, blood, blood serum, or urine.

It will be understood that, as used herein, the term"solution"includes mixtures containing suspended solids, but in which the useful substance to be recovered will itself be in solution.

The process of this invention employs a combination of selective ion exchange/affinity binding and magnetic separation. This combination provides particular advantages.

The process of the present invention can be generally characterised as a modification or adaptation of the processes of United States Patent 5,397,476 and EP 0,666,577. The methods of US 5,397,476 and EP 0,666,577 are directed to the removal of pollutant ions from an aqueous solution by a combination of ion exchange and magnetic filtration. The specific focus of those methods is on the removal of pollutants such as heavy metals and radio-nuclides from contaminated solutions to render the solutions safe.

The method of US 5,397,476 employs composite magnetic resin particles generally having a relatively small overall diameter (less than 20 micrometers) to maximise the surface to volume ratio. These particles comprise a composite in which magnetic particles are embedded in a polymeric matrix which either contains, or has attached thereto, sites which are selected for the pollutant ions which are to be removed from the contaminated solution.

The composite magnetic resin particles described in EP 0,666,577 include a core of magnetic particles comprising a magnetic material surrounded by a mixture of a fibrous material and a solid binding agent. In one preferred example of such particles, iron oxide is used as the magnetic material, cellulose fibres as the fibrous material, and agar as the binding agent, preferably cross-linked using a suitable agent such as formaldehyde or epichlorohydrin. The magnetic particles are embedded in a polymeric matrix which contains active sites. The polymeric matrix can be a silicate based polymer or an organic polymer (such as polyacrylamide) or a mixture of polymers.

It will therefore be appreciated that the composite magnetic resin particles disclosed in US 5,397,476 and EP 0,666,577 are similar to those employed in the present process. The essential difference between the composite magnetic resin particles employed by the applicants and those disclosed in US 5,397,476 and EP 0,666,577 is in terms of the affinity or ion selecting component. In the case of US 5,397,476 and EP 0,666,577, this component is selected for pollutant ions, whereas in the present process the selective component has affinity for the valuable substance to be recovered.

It is particularly preferred that the composite magnetic resin particles employed in the present process are similar to those described in EP 0,666,577. These particles have the advantage of having very good durability. In addition, the restrictions on particle size are not so important because the particles have a porous surface dedicated to the affinity binding/ion exchange function.

Those persons skilled in the art will readily appreciate how composite magnetic resin particles suitable for use in the present invention may be prepared, using the preparative methods described in EP 0,666,577. Those persons skilled in the art will also appreciate how polymeric resins can be engineered to contain specific functional groups, which will adsorb selectively a particular substance which it is desired to recover from a solution containing it. It will also be understood how selective adsorbers may be bound to a polymeric matrix. It will further be appreciated that the sites selective for the desired substance will be affinity binding groups where the desired substance is present in the solution in a non-ionic state, and can be either selective ion exchange groups or affinity binding groups where the desired substance is present in the solution in an ionic state.

The following represents a non-exhaustive list of valuable substances species which are present within solutions processed in the dairy industry (such as whole milk, skim milk, cream and whey): Calcium lactate/calcium gluconate Carotene Caseinomorphins linoleic acid and other fatty acids sialyltransferase IGF immunoglobulins lactoferrin lactoperoxidase lactose micellar casein phosphatidylcholine

riboflavin sphingolipids sodium, milk antiinflammatory factor milk oligosaccharides sialyl lactose lactose phosphates creatine.

These components have application in the dairy food and/or health industries.

For example, riboflavin is a vitamin, carotene is a food colouring agent, lactoferrin is a natural antibiotic, lactoperoxidase is a natural food preservative, phosphatidylcholine is a natural emulsifier and sphingolipids are anti- carcinogens.

It will also be appreciated that solutions processed in the dairy industry include other useful substances such as ketones, aldehydes, phenolic compounds, sulphur compounds, fatty acids, alcohols, esters and lactones which may be useful as flavouring compounds.

As outlined above, the process of the invention is not limited to solutions processed in the dairy industry but has application to other solutions containing useful substances. As one example, fruit juices contain anthocyanins which are colouring compounds.

Those persons skilled in the art will also appreciate the various affinity binding/selective ion exchange groups which may be used to recover the useful substances. For example, where it is desired to recover riboflavin from a solution such as whey, the affinity group may conveniently be magnesium silicate.

As another example, to recover nutritionally valuable divalent cations such as Ca2+ or Mg2+, a carboxylic acid ion exchange group is desirable, but for selectivity of Ca2+ or Mg2+ over competing proteins, an iminodiacetic acid (IDA) group is preferable.

Other examples of affinity binding/ion exchange functional groups which may be useful in the processes of the present invention, depending on the substance it is

desired to recover, include the standard carboxylates, sulphonates, diethylaminoethyl and quaternary ammonium ion exchange groups, dye-ligands for recovering immunoglobulins and other proteins, and bound metal ions for metal ion affinity chromatography.

Once the substance to be recovered is selected, and the composite magnetic resin particle is prepared, the process can be performed. The process has two essential steps. The first essential step is that of contacting the solution being processed (usually whole milk or whey) with the composite magnetic resin particles. This can be achieved for example by contacting the particles with a flowing stream of the solution being processed.

The contacting step may be carried out in a reactor operated batchwise or continuously and may take the form of a stirred tank or a fluidised bed or preferably a magnetically stabilised fluidised bed. Agitation of a stirred tank may be by low-shear impeller or by a moving electromagnetic field preferably developed by stationary coils energised by poly-phase electricity. In the particular case of recovery of fermentation product during fermentation, sterilised magnetic resin particles may be injected periodically into a fermenter then captured by electromagnets in the fermenter wall.

During the contacting step, the composite magnetic resin particles mix with the solution and selectively bind the substance to be recovered. Those persons skilled in the art will readily understand how to achieve conditions in which selective adsorption of the desired substance will take place.

For example, where calcium is to be recovered from dairy solutions, the pH of the solution should be less than 7 (preferably less than 6) and the temperature less than 80°C (preferably less than 60°C), to avoid competition with phosphates, hydroxides, sulphates and citrates precipitating. For riboflavin recovery, the pH should preferably be less than 7 to avoid alkaline breakdown of the riboflavin.

Exposure to light should also be avoided.

The second essential step of the process is separation of the magnetic resin particle/bound substance complex from the solution being processed. This is achieved by magnetic separation (such as magnetic filtration) using techniques and equipment which are known in the art. Such known equipment includes

rotating magnetic drum separators, stationary electromagnetic filters with an internal ferritic stainless steel matrix, and electromagnetic filters with continuous discharge achieved by a moving magnetic field.

The immediate result of the process is therefore a depleted solution from which the selected substance has been recovered, and the composite magnetic resin particles carrying the recovered substance.

It will be usual for the magnetic resin particle/recovered substance complex to be dissociated to release the recovered substance. This dissociation will desirably be performed under conditions which are not detrimental to the recovered substance. In particular, where the recovered substance is, or contains, protein (such as a commercially valuable immunoglobulin or enzyme), the dissociation will be effected under conditions which do not denature the protein. Those persons skilled in the art will appreciate how suitable conditions can be achieved.

One particular advantage of the process of the present invention is associated with the fact that the ease by which separation of the magnetic resin from solution can be achieved means that the process need not take place in a chromatography column. The magnetic composite resin particles on which the desired substance is adsorbed can therefore be agitated into a suspension which allows the suspension to be titrated with acid or base en masse to release the bound substance. Unlike in a column, no one portion of the resin is exposed to unduly high or low pH which could destroy or denature the substance. For example, a quaternary ammonium functionalised magnetic composite resin which has been used to adsorb whey proteins can be agitated in water then gradually acidified to facilitate fractional elution of the whey proteins. In a column separation process, salt solutions would generally be used to achieve the required elution. These are expensive on an industrial scale.

Dissociation of the magnetic resin particle/recovered substance complex has two effects. The first, and most obvious effect, is to release the recovered substance.

However, the second effect is to regenerate the particles for reuse. The regenerated particles can then be separated from the recovered substance by a second magnetic filtration step.

It will of course be appreciated that the depleted solution itself can be subjected to further processing. Such processing may involve recovery of a further valuable substance or substances. This can be achieved by essentially repeating the process steps above but using composite magnetic resin particles having an affinity to, and selective for, a further substance.

It will also be appreciated that the process of the present invention may be used to recover more than one substance at the same time, to obtain a mixture of desired substances. In such embodiments of the invention, the composite magnetic resin may contain two or more types of site, namely sites selective for each of the substances it is desired to recover, for example a mixture of riboflavin and calcium may be desired.

Although in the preferred embodiments of the invention described above the composite magnetic resin contains sites having affinity for and selective for one particular substance, the applicants also contemplate that the sites on the composite magnetic resin may bind more than one substance at once. In these embodiments of the invention a desired mixture of substances is bound to the resin. Following binding of the mixture of substances to the resin, the substances can be eluted sequentially from the resin under appropriate conditions, with a magnetic separation step between each elution, in cases where the individual substances are desired. Alternatively, the substances can be eluted simultaneously where what is desired is a mixture of the substances.

For example, where it is desired to recover whey proteins such as lactoferrin and immunoglobulins from a solution containing them, the binding sites on the resin may simply be cation exchange groups which will bind all of the desired proteins under appropriate conditions of pH and ionic strength. Following adsorption of the proteins onto the resin and magnetic separation of the resin from the solution, the desired proteins may be eluted sequentially under appropriate conditions of pH and ionic strength, as is known in the art in relation to conventional (non-magnetic) ion exchange technology. Thus, individual purified protein fractions may be obtained. Alternatively, all of the bound proteins may be eluted simultaneously under appropriate conditions, to obtain a mixture of proteins.

As mentioned above, it is also envisaged that the process of the invention will have particular application in removing useful reaction products from reaction mixtures such as fermentation broths. In particular, fermentation and enzyme reactions are often limited in their yield and productivity by product inhibition. Product inhibition may be palliated by the selective removal of product during reaction, provided the agent of selective removal can be disengaged from the reaction mixture or fermentation broth for regeneration. As one example, the process of the present invention may be used to remove caseinomorphin compounds generated by enzymatic hydrolysis of casein.

The invention will now be described with reference to the following non-limiting examples.

EXAMPLES Example 1: Calcium removal from fresh whey Preparation of resin A magnetic resin was prepared essentially as described in EP 0,666,577 but with active sites (functional groups) of carboxylic acid groups of sufficient density to give a cation exchange capacity of 2.3 meq/g of dry magnetic resin. The resin was washed with 2 bed volumes of 0.1 M NaOH with stirring for 10 minutes. The magnetic resin was held with a strong rare earth magnet (approximately 6 Gauss) while decanting the wash.

This procedure was repeated three times. The resin was further washed with deionised water to neutral (pH 7.0-7.5). The resin was then vacuum filtered to 50- 75% moisture.

Preparation of whey from bovine skimmed milk Bovine skimmed milk (0% fat, 200 ml) was added to a 500 ml beaker fitted with a thermometer probe, pH probe and magnetic follower on a stirrer hot plate. The pH was adjusted to pH 4.6 with 1 M HCl. The coagulum was heated to 40oC and this temperature maintained for 20 minutes with stirring. A metal coffee filter was used to coarsely separate whey from curds. The whey was refiltered with a fine filter (Whatman No 1 filter paper) giving a clear whey solution. This solution (pH 4.9-4.95) was diluted with 200 ml of deionised water and stirred to a uniform mixture.

Extraction of calcium from whey Approximately 400 ml of the diluted whey was added to a 500 ml beaker fitted with a thermometer, pH probe and a paddle stirrer (130-150 RPM). 40 grams of the conditioned resin (50% moisture) was added to the diluted whey and stirred for 15 minutes (pH 6.8-6.9).

Elution of calcium from the magnetic resin The resin was held by a magnet and the treated whey decanted. The resin was washed with deionised water to free non-bound material. The decanted diluted and treated whey was sampled for further analysis. The washed resin was placed in a 500 ml beaker and calcium eluted using two bed volumes of 1 M HN03. The elution was repeated three times. The experiment was repeated using 1 M HC1 for elution. In all cases the eluted resin was washed with three bed volumes of deionised water. This was repeated three times and the wash water added back to the acid eluant.

Calcium determination Method One sample from the 1 M HNO3 eluted/washed resin was reacted with 10 M NaOH to pH 13-14. The precipitated Ca (OH) 2 formed within 15 minutes. This alkali reaction procedure was repeated for the treated whey and raw whey. The

treated whey showed no precipitate, the raw whey slight turbidity and the eluant from the eluted washed beads, heavy precipitate.

Calcium levels in the fresh whey and treated whey from each of the different acid elutions were determined by atomic absorption spectroscopy. The results of four trials using 1 M HNO3 are shown in Table 1 and an abbreviated summary for both acids is given in Table 2.

Table 1: Repeat trials using the same portion of resin (elution with nitric acid) Calcium Concentration (mg/g resin) sal'BiSK ; .......................................................... :........................................................... .......................................................... :.. : :...... :..... 15. 3 2. 2 85. 6 1 15. 3 2. 2 85. 6 2 15.3 1.8 88.2 3 15.3 2.2 85.6 4 15.3 2.2 85.6

Table 2: Trials with elution using two different acids at 1 M concentration Calcium Concentration (mg/g resin) ...,............,.......... : : : : : :....... : : : : :. :..... : : : : : : :'.......................................,.................. : : : : :. : : :. : :. : : : : : :. :.......................... :.........,........................... ........................... .................................................... :........................................................... ............................................................ ...................... .. : :.....;,:::::::::::::::::::::::::::::: ::::::::;::::::.:::::.:::..:::: >:..::::.:::::::.:::: >.:.::...:.::;.....:::::::::::.::.::.: >::,.:...::::............::......:::.:::: >:......::..:::.:::.: >:::::::.::....;:::,:...::.:::;::..:.: >:::.::.:.::..::.:::...:....:::....:...;:.:: : : :.,;.;:: ::: :::::::::::::::.:;::.;: :::::::: :: ::: ::....::::::::...:.::.::.: >....:.::::.::. >:.:::. :: :::. ::::: :::::::::::::. _::::::::::::::.:::.: _::.:::.. ::::::::::.:::: .::: :.: .:- ::::.:::::::::....::::...:::::::...:....:. > :.... : ::...: >...::.:..;;::::::::::::::::::::;::::::::....:::::::::::: ::::::::::::::..:.:::.: ...:.....:::...... >::::::::....,:....::....:...::::..;::..::......:::,.:.:: .: >:. >:::;.:::::...:,:::;.::.:: >::.:::.:.....;.:::.: »:.:.: >::.....:.::.:: »:;::.;:::..:..: : : : : : : : : : : : : :, : :, : ; :. : : ; : : : : : : : : : : : : : : : : : : : : > : : : : :. : < : : ; : : : : : : : : : : :'5c : > : : > : : : : : : : : : : : ; : <.. :... : : :x : : : < : : : : : : : : : : : : : : : : > : :. : », : v : : : : : : :...... :. :. : :. :........................................................... ............ : :.. :.... : :.. : :.......... : :......................... :.. ;.... Nitric (Mean) 15.3 2.1 86.3 Hydrochloric 15.3 1.8 83. S

Magnetic Resin Regeneration The resin was activated twice using 3 bed volumes of 1 M NaOH. The residual NaOH is re-used after re-adjusting the molarity to 1 M. The resin was then washed with deionised water to neutral (pH 7.0-7.5). If the resin is to be used immediately, it was vacuum filtered to 50-75%. The resin could be bottle stored with added preservative (0.8% w/w) for further use at a later time.

Example 2: Calcium recovery from reconstituted whey Preparation of Resin Five different resins were prepared essentially as described but with active sites provided by differing proportions of carboxylic and IDA groups. The resins were washed with 2 bed volumes of 0.1 M NaOH with stirring for 10 minutes then regenerated into the desired form if required. The procedure was repeated three times. The resin was further washed with de-ionised water to neutral then filtered on a Buchner funnel under vacuum. The resins made were: XI carboxylic [2.3 meq/g of 50% moist resin] X1-1 carboxylic [2.7 meq/g] X2 IDA-Na (sodium form] X2 IDA-H [hydrogen form] X3 carboxylic [2.3 meq/g] plus IDA [0.5 meq/g] Preparation of Whey Reconstituted sweet whey 1000 g was prepared by dissolving the sweet whey powder known as Alaway 621 (obtained from New Zealand Milk Products Ltd, Wellington, New Zealand) (50.0 g) in de-ionised water (950 mL). Sodium azide (0.05%) was added as a preservative. The pH was adjusted down to pH 5.92 with 1 M HNO3.

Calcium Removal All five resins (5 g of wet resin each) were contacted with reconstituted sweet whey (50 mL of whey for each) in duplicate. A magnet held to the outside of the

beaker during decanting captured the resins. The resins were washed with deionised water then eluted with two bed volumes of 1M HCI. The eluted resins were then washed with three bed volumes of deionised water, twice, and their washings combined with the acid eluates.

In all cases, calcium recovered from the resin representing greater than 85% of calcium initially present was measured in the combined eluates and washings.

The combined eluates and washings were also analysed for total nitrogen by the Kjeldahl method. Although nitrogen levels were low in all cases they were below the limit of quantification in the case of the two IDA resins.

Example 3: Riboflavin recovery from cheese whey UF permeate Preparation of magnetic core particles Powdered cellulose (100 g) and de-ionised water (100 mL) were manually mixed together into a dough. The dough was transferred to a 1 L covered glass reaction vessel slowly agitated by a large diameter heavy stainless steel agitator and placed in an agitated water bath at room temperature. The vessel had been part filled with deionised water (500 mL) and was further charged with finely powdered Fe304 (100 g) and then adjusted to pH 12 with 50% NaOH. The reactor was heated to 64°C by increasing the water-bath temperature set point. Once the desired temperature had been reached, epichlorohydrin (20 ml) was added and stirring continued for 90 minutes during which the temperature was permitted to drop.

The reaction contents at about 58°C were transferred drop-wise to a 5 L plastic beaker containing 5% nitric acid (4 L) rapidly agitated by an overhead paddle stirrer. Rapid agitation was continued until the majority of particles were between 50 and 100 um diameter. The particles were permitted to settle and the solution, along with fine particles decanted off. The particles were washed five times with 4 L lots of de-ionise water at low agitator speed, settling and decanting after each wash. The magnetic core particles were then blotted on tissue paper and laid out to dry at room temperature.

Preparation of Resin In a 1 L heavy glass vessel were mixed acrylamide (40 g), tetramethyl diamine (0.5 mL), and N, N'methylene-bisacrylamide until dissolved in deionised water (55 mL). Under continued stirring were added 75 g of air-dried magnetic core material prepared as described above and 75 g of magnesium silicate (Synthetic Celite 066 obtained from Celite Corp Lomboc Ca, USA). Care was taken to minimise air introduction during several minutes stirring. When the suspension was homogeneous, 2 mL of 0.5% ammonium persulphate was added to initiate polymerisation and agitation continued until the reaction commenced. Once reaction was well under way, the vessel was transferred to an ice bath.

The block produced was later broken apart, milled. in a coffee mill, and sieved to an average particle size of about 150 im then repeatedly washed in deionised water on a Buchner funnel.

Preparation of Permeate The permeate employed was prepared by collecting the permeate from the ultrafiltration of cheddar cheese whey in a single stage UF plant operated in continuous mode at a volume concentration factor of 4 at 50°C using 4-inch spiral polysulphone membranes of nominal molecular weight cut off 10,000 Daltons. The permeate was collected fresh at pH 5.97 and placed in 500 mL plastic containers and stored in the dark at 4°C.

Riboflavin Removal Permeate samples, (500 mL), were contacted with magnesium silicate composite magnetic particles with gentle agitation by an overhead paddle stirrer for 0,1,5 and 10 minutes in glass 1 L beakers protected from light by aluminium foil. After the appointed time the magnetic beads were drawn to the side of the beaker with a hand-held rare earth permanent magnet and sufficient of the clear solution decanted to approximately fill 250 mL glass measuring cylinders.

From visual inspection, the yellow-green colouration of permeate was markedly reduced even after 1 minute of contact and was completely removed leaving a

water-white solution after 5 minutes contact. This observation was corroborated upon inspection of the measuring cylinders in a darkened room under illumination by a hand-held ultraviolet lamp: the 5 and 10 minute samples showed no fluorescence, the 1 minute sample weak fluorescence and the 0 minute sample strong fluorescence.

Regeneration of Resin and Recovery of Riboflavin The resin that had been contacted for 10 minutes was fully decanted and then washed twice with 200 mL of de-ionised water decanting between washes with the aid of the magnet. The washed resin was divided in two approximately equal portions each placed in a 500 mL glass beaker. One portion was washed with 200 mL of 1 M NaOH then washed with twice 200 mL deionised water. The eluate and washings were combined to give a colourless, cloudy solution.

The second portion of washed resin was washed with 200 mL ethanol then washed twice with 200 mL water and eluted. The combined eluate and washings were yellow and very slightly cloudy, and fluoresced under UV light.

Example 4: Calcium recovery from reconstituted whey pre-treated for riboflavin removal Preparation of Whey Sweet whey (Alaway 621,50.13 g was reconstituted in deionised water (980 mL), preserved with sodium azide (57 mg) and adjusted to pH 6 with 1M HN03. This reconstituted whey was stored in the dark at 4 C.

An aliquot of reconstituted sweet whey (500 mL) was placed in a 1L beaker and agitated with a paddle stirrer at 50-75 RPM. Magnesium silicate composite magnetic particles (50 g) were added and agitation continued for 10 minutes before the solution was decanted with the aid of a magnet giving a clear, colourless solution of pH 6.37.

Extraction of calcium from decolourised whey Three aliquots (150 mL) of each of the reconstituted sweet whey and the magnesium silicate composite magnetic particle-treated reconstituted sweet whey were contacted with 10 g of wet X2-Na resin. Three further portions of resin were contacted with water (150 mL) as blanks. Resin was agitated for 10,15 or 17 minutes, after which the resin was trapped by holding a magnet to the side of the beaker and decanting off the raffinat solution. A sample (15 mL) of raffinat solution was diluted to 50 mL with de-ionised water prior to colorimetric analysis of calcium.

Each resin was washed with de-ionised water, eluted with 1M HNO3 and then washed twice with deionised water. The latter two washings were combined with the eluate and made up to 500 mL with deionised water.

Results: Stan: :. 0. 00 :........................................................... ............................................................ ............................................................ ................. Eluateof untr : :. : :. fiw : : : : :. w : : : : : : n....... 00.......................................................... ............................................................ ............................................................ .......... Standard 0.00 0.025 0.00 Eluate of untreated whey 0.455 0.46 0.46 Raffinate of untreated whey 0.00 0.025 0.00 Eluate of decolourised whey 0. 46 0. 47 0.47 Raffinate of decolounsed whey 0. 00 0. 00 0.025 The pH of the untreated whey after contact with resin X2-Na was 7.81. The pH of the decolourised whey after contact with resin X2-Na was 7.93.

From these results it is clear that pre-extraction of riboflavin by magnesium- silicate-bearing resin had no deleterious effect on calcium extraction.

INDUSTRIAL APPLICATION The above examples demonstrate the ability of the process of the invention to recover selected minor components (calcium or riboflavin) from whey. It is believed that the process will be equally effective in recovering other minor components from whey, from whole milk or cream or other solutions simply through use of different functional groups to bind the selected substances.

The process also offers advantages over conventional chromatographic separation processes as employed in the food industry. For example, because the composite magnetic resin particles can easily be separated from solutions, the process of the present invention permits small sized particles of resin to be used, which could not easily be used in conventional processes. The use of small size particles maximises the surface area to volume ratio and the availability of functional groups for recovery of the desired substance. This is turn allows high influent rates to be used and viscous solutions to be readily treated.

The process of the present invention is particularly suitable for separating substances present in low concentrations in a large volume of solution, especially in cases where it takes a relatively long time for the target substance to bind to the affinity group (ie the binding equilibrium is slow). In conventional, column processes a substantial proportion of the target material will not bind to the column in a first pass of the solution through the column. In the process of the present invention, the composite magnetic resin can be maintained in contact with the solution for a sufficient length of time to enable binding to occur, and then magnetic separation of the resin easily achieved.

A further advantage of the process of the invention is that it enables a fragile or friable substrate to be used in robust fashion, as is often required in food/health /pharmaceutical industry processes. Substrates like silicates, zeolites, acitvated carbons and others are capable of forming strong affinity links but cannot withstand the physical stress of industrial scale applications. They become robust when potted in a polymeric matrix around a magnetic core, in accordance with the present invention. The composite magnetic resins are also robust enough to enable sterilisation with agents such as sodium hydroxide or solvent.

It will be appreciated by those persons skilled in the art that the above description is provided by way of example only and that the invention is not limited thereto.