DE2821392A1 - METHOD OF EXTRACTING PROTEINS FROM MILK - Google Patents

Description

BK. ING. F. WtTKSTHOI-F I) HK ν. 1'K ( ¹ IIMANX DK. ΙΝ <ί. I>. ItKHItKNS DIPI ... IN «. IJ. «OIiTZ PATENT APPLICATION LTE

HOOO MÜrcCirKJV OO KCIIWKICIKHSTIIASSK TKI.KKON (080) 00 20 TKI.EX 5 24 070

TKI.EIIUUIMKI ΡΗΟΤΚΟΤΓΑΤΚΚΤ MÜHOSBX

1A-50 757

Patent application

Applicant: RHÖNE-POULENC INDUSTRIES

22, avenue Montaigne, 75 Paris (8βΊηβ), France

Title: Method of extracting proteins from milk

809848/0799

DR. ING. Τ · \ "WITESTIIO Kl '' IJIi-E. ν. PECH MAN YOU. ING. I). BEHITICNS DIPL. ING. 11. (JOICTZ PATJENTANWÄr / TJB

ROOO MUNICH OO SOHU'HKJ KISTKASSE S τι: ι, εγον (089) 00 20 01 TELHX Π 24 070

ΤΚΙ, ΚΚΗΛΜΜΚ t ΙΜΙΟΤΚΟΤΙ'ΛΤΚΝΤ

1-50 757

Note j Rhone-Poulenc Ind.

description

The invention relates to a new extraction method for Milk proteins.

The treatment of skimmed milk is generally that first the casein by means of acidic or enzymatic Coagulation is separated; The proteins of the milk serum are then thermally coagulated by means of ultrafiltration or ion exchange and finally the lactose which are hydrolyzed is separated or isolated can.

However, this usual treatment has various disadvantages: the separated casein is precipitated and partially degraded and may contain other entrained proteins. The other proteins produced by thermal coagulation or are separated by means of ultrafiltration, lose some of their biological properties because of the duration of the necessary operation generally makes pasteurization of the milk serum necessary. The separation by means of ion exchange again, it is difficult to carry out on an industrial scale because the known ion exchangers

based on cellulose or dextran do not have sufficient mechanical strength.

Ion exchangers for the separation of the proteins of the milk serum, which do not have these disadvantages, have already been used

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in FR-OSes 75 26 530 and 76 22 985 described. But also In these processes, the casein is still precipitated, partially broken down and can carry away (other) proteins contain.

The invention is based on the object with the help of a new method for fractionating or separating the milk to avoid known disadvantages and, on an industrial scale, the production of pure proteins in the native state and with all biological properties to allow.

The object is achieved with the aid of the method according to the invention, in which all proteins of the skimmed milk are extracted or are separated and a solution of mineral salts and lactose remains; the procedure is characterized by that you first separate all other proteins except casein by adding the skimmed milk with at least one after the other an anion exchange resin and then silica or with successively first silica and at least one anion exchange resin brought into contact, the proteins are fixed and then eluted and that one that remains in solution Casein then separates from the mineral salts and lactose.

Besides casein or milk serum proteins, lactalbumins, serum albumin, lactoglobulins and immunoglobulins are used as proteins designated.

The anion exchange resins consist of a carrier: alumina or silica coated with less than 20 mg / m of a film made of a crosslinked polymer containing functional tertiary amino groups or quaternary ammonium salt groups of the general formula -CH ₂ -N-CH ₂ - or -CH ₂ -N ^ (R), X ^ "^

in which R is the same or different and each represents an alkyl or hydroxyalkyl group having 1 to 4 carbon atoms and X is a mineral (inorganic)

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or is an organic anion, for example the chloride, sulfate, nitrate, phosphate or citrate anion. The exchange capacity of these anion exchange resins is below 2 meq / g.

The silica and the carrier of the anion exchange resins have a particle size distribution of 4 / um to 5, a specific surface area in the range from 50 to 150 in / g, a pore volume of 0.4 to 2 ml / g and a pore diameter from 25 to 250 nm (250 to 2500 Å), i.e. smaller than the casein molecules and larger than the molecules of the other proteins.

The crosslinked polymers that form the surface of the carrier materials Coating are products known per se obtained from monomers: for example starting from epoxy compounds with polyamines as Catalysts crosslink, starting from formaldehyde by polycondensation with urea, melamine, or polyamines Cross-linked phenols; starting from vinyl monomers such as vinyl pyridine, styrene and derivatives with polyfunctional Crosslink monomers, for example with mono- or polyalkylene glycol diacrylates or dimethacrylates, divinylbenzene, Vinyltrialkoxysilane, vinyltrihalosilane or bismethylene acrylamide in the presence of an initiator or UV rays.

The mineral support is coated or coated with the crosslinked polymer by the fact that the support material impregnated with a solution of the monomer or monomer mixture and optionally initiator in a solvent then the solvent evaporates and the monomers are crosslinked according to processes known per se. As a solvent all solvents are suitable for the monomers and the initiator whose boiling point is preferably as low as possible, to facilitate subsequent evaporation. These include, for example, methylene chloride, ethyl ether, benzene and acetone and ethyl acetate.

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If the crosslinked polymer on the surface of the support does not contain any functional groups in its chain, it must be modified. This is especially true for crosslinked polymers based on styrene and styrene derivatives and the polymers of formaldehyde and urea, melamine, polyamines or
Phenols.

In the case of polymers based on styrene or phenol-formaldehyde condensation products, this modification consists of
that chloromethyl groups are fixed on the polymer, which are then reacted with a secondary or tertiary amine in any known manner.

To fix the chloromethyl groups on the polymer
In the case of the styrene polymers, it is advantageous to disperse the polymer-coated mineral carrier in warm chloromethyl ether in the presence of a Lewis acid. In the case of a phenol-formaldehyde resin, on the other hand, the polymer-coated mineral carrier is dispersed in epichlorohydrin and then reacted in the heat.

In the case of the polymers of formaldehyde and polyamines, urea or melamine, this modification consists in that
the primary amino groups present in the chain are converted into groups of tertiary amines or quaternary ammonium salts in any known manner, for example by reaction with a sulfate or an alkyl halide.

The skim milk is contacted with the anion exchange resin or the anion exchange resins and silica without modifying the pH of the milk at temperatures of from 0 to 5O ⁰ C, preferably from 0 to 15 C in contact.

5 to 15 g of anion exchange resin and 2 to 7 g of silica are required for each g of the total amount of protein that is to be separated off used.

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The proteins retained by the anion exchange resins can be ß-lactoglobulins, tiL-lactalbumins, serum albumin as well a small part of the immunoglobulins. The silica fixes most of the immunoglobulins. The proteins retained by the ion exchange resin and silica are either eluted with a solution of high ionic strength or, in the case of anion exchange resins, with an acidic one Solution and in the case of silica with a basic solution. The acidic pH solution is a solution of a mineral or organic acid, for example hydrochloric acid, acetic acid, nitric acid, sulfuric acid, lactic acid; the Basic pH solution is a solution of alkali hydroxides such as ammonia, caustic soda or potassium hydroxide.

In order to achieve a selective separation, the milk can be treated successively with several anion exchange resins that are identical or different from one another and that before or after the treatment with the silica. In the event of of two anion exchange resins, the solution obtained when the first resin is eluted is very rich in ß-lactoglobulins, while the solution that is obtained when the second resin is eluted, the c -lactalbumins and serum albumin and in very small amounts ß-lactoglobulins and Contains immunoglobulins.

The extraction of the proteins from the skimmed milk can be discontinuous or semi-continuous with identical results be made in columns or continuously in columns. The continuous working method is particularly suitable good for implementation on an industrial scale, as the resins easily fill the columns, a large throughput and allow easy elution.

The protein solutions obtained only contain traces of lactose and mineral salts. They can be used as such or else the proteins can be known as desired

* cascaded

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Manner, in particular separated or obtained by atomization.

After extraction or separation with the aid of the anion exchange resins and the silicic acid does not contain the remaining solution consisting of casein, lactose and mineral salts more proteins. The casein can with the help of exclusion chromatography (exclusion chromatography) or in particular be separated by means of ultrafiltration, any known working methods being adapted to this particular case will. The extraction or separation can be carried out batchwise or continuously.

In this way a solution of native casein is obtained in water, the concentration of which depends on the extraction process used, and a solution of lactose and Mine rals alζ en

The native, non-coagulated casein solution only contains still traces of lactose and can be used as such or the casein can be separated from the solution, especially by spraying.

According to a variant of the method according to the invention, the lactose and the mineral salts are first extracted from the skimmed milk separated and then the other proteins with the exception of casein, by the milk successively with at least one Anion exchange resin and silica or successively with silica and at least one anion exchange resin in Brought into contact, the proteins are fixed and then eluted. The casein remains in this mode of operation in solution or liquid phase.

The separation of the lactose and the mineral salts from the skimmed Milk is made by extraction using exclusion chromatography or ultrafiltration.

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The extraction of the proteins with the anion exchange resin or the anion exchange resins and the silica are carried out as described above. After the extraction of the other proteins In addition to casein, a solution made from native, non-coagulated casein is obtained.

According to another variant, the method according to the invention applied to milk serum, i.e. skimmed milk from which the casein has already been separated. In this case the milk serum is brought into contact with at least one anion exchange resin and silicic acid in succession, or with successively silica and at least one exchange resin; the proteins are fixed and then eluted.

The separation of the proteins of the milk serum with the help of Anion exchange resins and cation exchange resins have already been described in FR-OS 76 22 985. The usage of silica instead of a cation exchange resin, however, allows much easier elution, so that more concentrated proteins are obtained. As a result, less water needs to be removed when the proteins are dried to become; the treatment is shorter and consequently less detrimental to the proteins.

In addition, the silica is a simpler product than cation exchange resins and is used in the food industry used.

The extraction or separation of the proteins of the milk serum with the anion exchange resin or the anion exchange resins and the silica is carried out as described for milk. However, the contact of milk serum with the resin or the resins and the silica takes place at a pH value above 4, preferably in the range from 5.5 to 7.5, at temperatures from 0 to 50 ^° C. and preferably from 0 to 30 C. After extracting proteins with the anion exchange resin and the silica contains the remaining

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Solution still lactose and mineral salts, but no more proteins.

Regardless of its origin, the lactose in solution can be hydrolyzed chemically or enzymatically, see above that a solution of glucose and lactose is obtained.

The method according to the invention is used in the dairy industry for separating proteins, including casein and is particularly suitable for purposes of the food industry, the production of diet foods, pharmaceuticals Area or veterinary area.

The following examples serve to explain the invention in more detail.

example 1

In a first column with a diameter of 2.5 cm, 20 g of anion exchange resin was placed given, consisting of a silica with a particle size distribution of 100 to 200 μm, specific surface

2
24 m / g, mean pore diameter 140 nm and pore volume 1 ml / g, which was coated with 3.3 mg / m of a copolymer of styrene and vinyltriethoxysilane, the functional groups

CH,

CH _P - N <i> CH, α ^(-) CH,

contained. This exchange resin had the following characteristics:

Carbon content 4.8 %

Chlorine content 2 %

Nitrogen content 0.9 %

Exchanger capacity 0.6 meq / g

In a second column with a diameter of 2.5 cm, 10 g of silica beads with a particle size distribution of

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100 to 200 / µm, with a specific surface of 25 m / g, a pore diameter of 140 nm and a pore volume of 1.1 ml / g.

The two columns were connected in series; then the resin and silica were made by bubbling 500 ml of water through them washed.

Then 250 ml of skimmed milk containing 7 g of casein, Containing 1.6 g of other proteins and 12 g of lactose, successively applied to column 1 and then to column 2 and although at a rate of 100 ml / h. Then resin and silica were in the two columns with 200 ml Water washed.

The proteins fixed in the first column were eluted with a n / 100 hydrochloric acid solution, containing 33 ml of the solution 1.3 g of proteins were obtained. These proteins were the * <lactalbumins, the ß-lactoglobulins, serum albumin and a small part of the immunoglobulins.

The proteins fixed in the second column were eluted with a 1N ammonium carbonate solution; 12 ml of solution were obtained, containing 0.3 g of immunoglobulins.

Both protein solutions contained less than 1 % by weight of fat and lactose o The electrophoretic migration of the proteins was identical to the migration in milk. The proteins were therefore not denatured.

The solutions (milk + washing water) emerging from the second column, which no longer contain any proteins other than casein were ultrafiltered. The retentate contained about 7 g of native casein at a concentration of 20 wt .- #.

The ultrafiltrate contained practically the entire amount of lactose in a concentration of about 42 g / l as well as the mineral salts.

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Example 2

Example 1 was repeated, but the treatment of the skimmed Milk made with anion exchange resin and with silica at 50 C. The results obtained were identical to those in the example 1.

Example 3

Example 1 was repeated in that the first column was preceded by a column A with a diameter of 1 cm, the 3 g of the same Anion exchange resin as in column 1 contained. Column A was eluted with n / 100 hydrochloric acid solution, whereby 15 ml of a solution was received which contained 0.3 g of proteins consisting almost exclusively of ß-lactoglobulins.

Column No. 1 was eluted with / 100 hydrochloric acid solution 33 ml of solution containing 1 g of proteins, which mainly consisted of ot-lactalbumin and serum albumin and to a very small extent from ß-lactoglobulins and immunoglobulins. The elution of column no. 2 gave the same results as in Example 1 «

Example 4

In a column 1 with a diameter of 2.5 cm, 10 g of silica beads were placed with a particle size distribution of 100 to 200 μm, a specific surface area of 25 m / g, a pore diameter of 140 nm and a pore volume 1.1 ml / g given.

15 g of anion exchange resin were placed in a column 2 with a diameter of 2.5 cm given consisting of a silica with a grain size of 100 to 200 / um, specific surface 24 m / g, medium Pore diameter 140 nm and pore volume 1 ml / g, with 6 mg / m of a copolymer of styrene and vinyltriethoxysilane was coated, the following functional groups

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and had the following characteristics:

Carbon content 7.5 %

Nitrogen content 1.5 %

Exchange capacity 1.07 meq / g

The two columns were connected in series, then were Washed silica and exchange resin with 500 ml of water.

Then 250 ml of skimmed milk, which contained 7 g of casein, 1.6 g of other proteins and 12 g of lactose, with a Speed of 100 ml / h first through the column ^ and then through the column 2 percolated. Thereupon were silica and exchange resin in the two columns washed with 200 ml of water.

The proteins fixed in column 1 were then also used a n / 100 ammonia solution eluted. 12 ml of solution were obtained which contained 0.4 g, i.e. the majority of the immunoglobulins.

The proteins fixed in column 2 were eluted with a n / 10 hydrochloric acid solution: 23 ml of solution were obtained, the 1.2 g Proteins, namely et-lactalbumins, ß-lactoglobulins, serum albumin as well as traces of immunoglobulins.

Both protein solutions contained less than 1% by weight lactose. The electrophoretic migration of the proteins was identical to the migration in milk. So the proteins had not been denatured _e

The exiting from the column 2 solution, about 260 ml, containing casein except no further P _r oteine more and was percolated through a column 3 to the casein by means of size exclusion chromatography to separate. This column 3 with a diameter of 3 cm contained 450 g of silica with a grain size of 100 to 200 μm, a specific surface area of 400 m / g, an average pore diameter of 8 nm and a pore volume of 1 ml / g and was eluted with water.

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330 ml of a solution were obtained which contained practically the entire amount of native casein and a solution which contained lactose and which contained mineral salts.

Example 5

In a column 1 with a diameter of 2.5 cm, 15 g of the same anion exchange resin as in the column 2 of the example were placed 4th

In a column 2 with a diameter of 2.5 cm, 10 g of silica beads were placed with grain size 100 to 200 / µm, specific surface

50 m / g, pore diameter 80 nm and pore volume 1.1 ml / g.

Both columns were connected in series and resin and silica washed by passing 500 ml of water therethrough.

300 ml of milk serum of pH 6.5, which contained 1.5 g of protein and 11 g of lactose, were then successively added at room temperature percolated through column 1 and through column 2 at one rate of 400 ml / h. The resin and silica of the two columns were then washed with 200 ml of water.

The proteins bound in column 1 were eluted with a n / 100 hydrochloric acid solution. 33 ml of solution were obtained 1.25 g protein. These proteins are composed of the dl-lactalbumin, ß-lactoglobulins, the serum albumin and a small part of immunoglobulins.

The proteins bound in column 2 were eluted with a n / 100 ammonia solution; 10 ml of solution were obtained, which contained 0.25 g Contained proteins; the proteins consisted almost exclusively of immunoglobulins.

Both protein solutions contained less than 1% by weight of fatty substances and lactose. The electrophoretic migration of the proteins was identical to their migration in the milk serum. the

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Proteins were therefore not denatured. Example 6

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Example 5 was repeated, but carried out the treatment of the milk serum with anion exchange resin and silica at 50 ⁰ C. The results were identical to Example 5.

Example 7

Example 5 was repeated in that column 1 was preceded by a column A with a diameter of 1 cm, the 3 g of the same Anion exchange resin such as column 1 contained. The column A was eluted with n / 100 hydrochloric acid, 15 ml of a solution received, which contained 0.3 g of protein consisting almost exclusively of ß-lactoglobulins.

Elution of column 1 with n / 100 hydrochloric acid gave 33 ml of a solution containing 0.95 g of proteins, the majority of which from o ^ -lactalbumin and serum albumin and at a very low level Part consisted of ß-lactoglobulins and immunoglobulins.

When column 2 was eluted, the same results as in Example 5 were obtained.

Example 8

In a column 1 with a diameter of 2.5 cm, 10 g of silica beads were placed with a grain size of 100 to 200 μm, specific surface area 25 m / g, pore diameter 140 nm and pore volume 1.1 ml / g given. In a column 2 with a diameter of 2.5 cm, 20 g of the same anion exchange resin as in column 1 of the Example 1 given.

Both columns were connected in series; then silicic acid and resins were added by bubbling 500 ml of water through them washed.

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Λ?

Then 300 ml of milk serum pH 6.5 were applied, the Contained 1.5 g protein and 11 g lactose; the milk serum was at room temperature at a rate of 600 ml / h first percolated through column 1 and then through column 2. Subsequently, silica and resin were in the two Columns washed with 200 ml of water.

The proteins fixed in column 1 were eluted with a n / 100 ammonia solution. 12 ml of solution were obtained, which contained 0.4 g, i.e. contained the majority of the immunoglobulins.

The proteins fixed in column 2 were eluted with n / 10 sulfuric acid. 20 ml of solution consisting of 1.1 g of proteins were obtained from aL-lactalbumin, ß-lactoglobulins, serum albumin and traces passed by immunoglobulums.

The two protein solutions contained less than 1% by weight of lactose. The electrophoretic migration of the proteins was identical to the migration in the milk serum. The proteins were thus not been denatured.

Example 9

20 g of the same anion exchange resin as in column 2 of Example 1 were placed in a column 1 with a diameter of 2.5 cm. In a column 2 with a diameter of 2.5 cm, 10 g of silica spheres with a grain size of 100 to 200 μm, specific surface area

ρ /

25 m / g, pore diameter 140 mn and pore volume 1.1 ml / g given.

The two columns were connected in series and the resin and silica were then washed with 500 ml.

Then 500 ml of milk serum, adjusted to pH 7.5 by adding 0.1 η sodium hydroxide solution and filtered to remove the insoluble Separate components, issued. The milk serum

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contained 2.5 g protein and 18 g lactose and was at room temperature percolated successively through column 1 and then through column 2 at a rate of 300 ml / h. The resin and silica of the two columns were then washed with 100 ml of water.

The proteins bound in column 1 were treated with n / 100 hydrochloric acid eluted to give 35 ml of solution containing 2.05 g of proteins. These proteins were theoC-lactalbumins, ß-lacto-

globulins, serum albumin and, to a small extent, immunoglobulins .

The proteins bound in column 2 were washed with a 1M ammonium carbonate solution eluted; 12 ml of solution were obtained containing 0.45 g of proteins, almost exclusively from immunoglobulins passed.

For comparison, this example was repeated, but instead of the 10 g of silica in column 2, 10 g of a cation exchange resin used; this cation exchange resin consisted of a silica with a grain size of 100 to 200 μm,

2 '

specific surface 25 m / g, mean pore diameter 140 mn and pore volume 1.1 ml / g coated or coated with 7.2 mg / m of a copolymer of acrylic acid and diethylene glycol dimethacrylate, the had functional -COOH groups. The resin was characterized by the following features:

Carbon content 10.55 %

Exchange capacity 1.05 meq / g

Eluting the first column gave the same results as in example 5.

When the second column was eluted, 17 ml of solution were obtained, the 0.45 g of proteins consisting almost exclusively of immunoglobulins contained.

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This comparison shows that when using silica, a much simpler material than the cation exchange resin , a more concentrated immunoglobulin solution is obtained, namely:

with the exchange resin 2.65 g / 100 ml, with the silica 3.75 g / 100 ml, corresponding to 41.5 % more P _r oeine.

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Claims (9)

Claims 1. method of extracting proteins from milk, characterized in that one initially the other proteins besides casein are extracted by mixing skimmed milk with at least one anion exchange resin in succession and silica or successively with silica and at least one anion exchange resin in Brings contact, fixes and elutes the proteins and then the casein remaining in the liquid phase separates from the mineral salts and the lactose. 2. The method according to claim 1, characterized in that one uses anion exchange resins which are coated from alumina or silica as a carrier material with less than 20 mg / m of a film of a crosslinked polymer, the functional tertiary amino groups or quaternary ammonium salt groups of the general formula -CH ₂ -N-CH ₂ - or -CH ₂ N (± ^ (RJ ₃ X * "', where R is the same R.
or is different and represents an alkyl group or hydroxyalkyl group with 1 to carbon atoms and X is a mineral or inorganic anion, and which have an exchange capacity below 2 meq / g. 3. The method according to claim 1 or 2, characterized in that one anion exchange resin and silica with a grain size of 4 / µm up to 5 mm, a specific surface area of 5 to 150 m / g, a pore volume of 0.4 to 2 ml / g and a pore diameter of 25 to 250 nm are used. 809848/0799 ORiGiNAL INSPECTED 1A-50 757 4. The method according to any one of claims 1 to 3, characterized in that bringing the skimmed milk with the anion exchange resin and the silica at a temperature of 0 to 50 ⁰ C in contact. 5. The method according to any one of claims 1 to 4, characterized in that per g of protein that is to be extracted, 5 to 15 g of anion exchange resin and 2 to 7 g of silica are used. 6. The method according to any one of claims 1 to 5, characterized characterized in that the proteins retained by the exchange resin and the silica with either a solution of high ionic strength or with a solution of acidic pH in the case of the anion exchange resin or eluted with a solution of basic pH in the case of silica. 7. The method according to any one of claims 1 to 6, characterized in that after the extraction the other proteins, the native casein extracted by means of exclusion chromatography or ultrafiltration. 8. Modification of the method according to one of claims 1 to 7, characterized in that the lactose and the mineral salts are first obtained from the skimmed milk by means of ultrafiltration or exclusion chromatography and then the other proteins besides casein by contacting the milk with at least one after the other an anion exchange resin and silica or successively with silica and at least one anion exchange resin, Fixation of the proteins and elution, whereby the native casein remains in the liquid phase. 9. Modification of the method according to one of claims 1 to 7 for the extraction of the proteins of the milk serum, characterized in that one milk serum 809848/0799 - 3 - 1A-50 757 with at least one anion exchange resin and with silicic acid in succession or with silicic acid and in succession brings at least one anion exchange resin into contact at a pH value above 4 and fixes the proteins and eluted. 8098A8 / 0799