AU637060B2 - Milk constituents, process for producing them and agents containing them - Google Patents

Description

DPI DATE 23/03/90 APPLN. I D 41944 89 p~T AOJP DATE 26/04I/90 PCT NUMBER PCT/EP89/01021 INTERNATLO E I NTRNAIO....... 4Ltd t flf td~JJ~, tdJLJCd (51) Internationale Patentklassiikatlon 5 (11) Internationale Ver~iffentlichungsnummer: WO 90/02132 C07G 17/00, A23C 9/142, 9/152 Al (43) Internationales A23L 1/30, A61K 35/20, 7/48 Verdffentliungsdatum: 8. Mflrz 1990 (08,03.90) k2l) Internationales Aktenzeichen; PCT/EP89/0 1021 Verilltentllcht Mit intemationalem Recherchenberich.

(22) Internationales Anmeldedatum: 31. August 1989 (31.08.89) Var Ablauf derfllr:4nderungen der Ansprilche zygelassenen Frist. Ver je ntlichung wird wiederholt falls Anderungen eintreffen.

Prioritiitsdaten: P 38 29 552.0 31. August 1988 (3 1.08.88) DE (71X72) Anmelder mid Erflnder: GAURI, Kailash, Kumar [DE/ DE]; Zur Waldburg 13, D-2359 lentf6hrden 676 (74) Anwillte: KINZEBACH-, Werner usw. Postfach 86 06 49, 6 D-8000 Milnchen 86 (DE).

(81) Bestimmungsstaaten: AT (europiiisches Patent), AU, BE (europiiisches Patent), CH (europiiisches Patent), DE (europdisches Patent), FR (europllisches Patent), GB (europltisches Patent), IT (europftisches Patent), JP, LU (eiiropaisches Patent), NL (europliisches Patent), SE (europliisches Patent), US.

(54)Title: MILK CONSTITUENTS, PROCESS FOR PRODUCING THEM AND AGENTS CONTAINING THEM (54) Bezeichnung: MILCHBESTANDTEILE, VERFAHREN ZU IHIRER HERSTELLUNG UND MITTEL, DIE DIESE BE- STANDTEILE ENTHALTEN (57) Abstract Constituents of the n on- protei n- nitrogen fraction of untreated raw milk or fresh milk have a characteristic pharmacological activity.

(57) Zusammenfassung Es werden Bestandteile der Nicht-Protein-Stickstoff-Fraktion von unbehandelter Rohmilch oder Frischmilch beschrieben, Diese Bestandteile haben charakteristische pharmakologische Wirkung._______ tIIv.~ I I haLt 4i0 ~I I tIlifa II II thl,,. II? II, .0

'C)

~.It tIlLrn. I I ~~tV t.L VtA -1- Milk Constituents, Process for their Production and Medicinal Drugs which Contain these Constituents.

The invention concerns constituents of the NPN (non-protein nitrogenfraction of untreated fresh milk, a process for their production, medicinal drugs which contain these constituents and their use in foodstuffs and beverages.

As is well known, the constituents of the milk of mammals are present in the enclosing aqueous phase in coarsely dispersed colloidal or dissolved form. The milk constituents of domestic anima±s have been well investigated and are used for the production of numerous foodstuffs and beverages. The dry matter of cow's milk, as is well known, is on the average about 12.7 Of this about 3.7 are fatty constituents, 3.4 total protein, 4.7 lactose, and about 0.7 ash. The protein component consists mainly of caseins and whey proteins. Beside that, one also speaks of a non-protein -nitrogen (NPN)-fraction, proteo-peptones and minor proteins which are mainly enzymes.

The principal separation step is the separation of the caseins and whey products. This separation is carried out by means of the socalled rennet precipitation, in which rennet enzyme is added to warm milk (30 to 35 0 In this operation so-called caseins are precipitated while the whey proteins remain in solution. The same holds for the so-called acid precipitation of the caseins, which takes place at the iso-electric point (cow's milk pH Caseins are stable to heat while whey proteins are unstable to heat.

Because of legal requirements raw milk is nowadays always subjected to a heating before being processed further, so that all commercial milk qualities and milk products have gone through one of the legally prescribed heating stages (pasteurisation, ultra high heating or sterilisation). It is known that in the milk treatment processes usual today, numerous milk constituents are denatured and altered, especially during heating, rennet and acid precipitation.

Our own investigations of milk and milk products, which have undergone present day prescribed treatments, had shown that completely untreated raw milk contains pharmacologically active components which are found no longer or only partly, in milk which has been subjected to the usual dairy treatments depending on the degree of heat applied.

W086/ 04217 describes protein hydrolysis products which are recovered from whey proteins. For this purpose the whey fraction of untreated raw milk is treated with with proteases and possibly lipases. The mixture of hydrolysates obtained is then separated into individual fractions by means of specific process measures. The fractions obtained in this way have analgetic, antiphlogistic and antimutagenic effects and have glaucoma inhibiting effects.

The invention is based on the task of having to isolate new constituents from milk, especially ones with pharmacological effects.

Surprisingly it has now been found that an active agent can be recovered, especially from the so-called NPN (non-protein-nitrogenfraction) or whey of untreated raw milk, for the treatment of neurodermatitis, allergies, glaucoma and for the stimulation of the immune reaction, as well as a mixture of short chain peptides stimulating cell respiration.

The milk constituents according to the invention have been designated arbitrarily with F and G-a and and product F is characterised by an HPLC-band at RT 4,6; Column: DEA-5PW; Waters Protein Pak; mobile phase: 10 methanol; 0.5 ml/min.; an HPLC-band at RT 26; Column 1-125, Waters Protein Pak, mobile phase KH 2 P0 4 0.05 m; 0.5 ml/min.; HPLC-bands at RT 23.3 (weak); 25.3 (strong); 29.5 (medium); 30.5 (medium); 34.4 (weak); column: 1-125 Waters, Protein Pak, mobile phase: KH 2

PO

4 0.05 m; 0.05 ml/min.; ninhydrin positive reaction; good solubility in chloroform, isopropanol, ethanol and water; and the substance G-a is characterised by HPLC-bands at RT 19.76 (stong); 20.38 (medium); 21.6 (shoulder); 24.3 (medium); column: 1-125, Waters, Protein Pak, mobile phase: KH 2 P04 0.05 m; 0.5 ml/min.; I 4 and the Substance G-b is characterised by: HPLC-bands at RT 21-22 (weak); 23.4 (strong) and 26.5 (strong) conditions as at and the substances G-a and G-b in addition have the following properties: ninhydrine positive reaction (G-a and G-b); good solubility of G-a in 20 to 50 ethanol, especially in 20 to 30 aqueous ethanol; and The milk constituents according to the invention can be obtained by: a) subjecting untreated raw milk or fresh milk to a membrane filtration on a 0.1 to 0.6 U-membrane, b) subjecting the filtrate obtained to a second membrane filtration on a membrane with a separation limit of 6 to 10,000 molecular weight; c) subjecting the filtrate obtained to a third membrane filtration on a membrane with separation limit of 1,000 molecular weight; d) subjecting the filtrate obtained to a reverse osmosis on an open or a closed membrane; e) in case of the use of an open membrane evaporating down the filtrate obtained to about 20 of the volume subjected to reverse osmosis, letting stand at about 2 to and filtering off from the precipitated solids; f) evaporating down the filtrate obtained by about 5 to by volume and adjusting the ethanol content to by volume by means of absolute ethanol and filtering; 1 g) evaporating the filtrate obtained to dryness, extracting the residue with 90 to 95 (vol.) ethanol, filtering and evaporating the filtrate; h) extracting the residue obtained with chloroform, isopropanol or absolute ethanol; i) evaporating the the extract solution to dryness and isolating the residue as product F, and isolating from the residue of the extract solution the product G-a by extraction with 80 ethanol, and the product G-b by extraction with 30 ethanol; j) in case of the use of a closed membrane, subjecting the retained material (instead of the filtrate obtained when using an open membrane) to the processing steps enumerated and obtaining thereby the product F; and recovering from the residue of the extract solution the products G-a and G-b as indicated in step or for obtaining G-a and G-b k) evaporating under vacuum and extracting with 80 (V) ethanol the retained material of the third membrane filtration and/or the retained material IV of the reverseosmosis on an open membrane, or adjusting an ethanol content of 80 by volume by adding absolute ethanol, filtering off insoluble constituents, evaporating to dryness the ethanol solution and thereby obtaining the product G-b as a colourless substance; 1) extracting the components which have remained undissolved after the treatment with ethanol with 25 ethanol, filtering off the undissolved constituents, evaporating the filtrate to dryness in vacuo, and obtaining product G-a in this manner.

The method 'of production according to the invention is explained in detail below. In this reference is made to the diagrams reproduced in figures 1A to 1C which schematically represent the method of production according to the invention.

The following is shown: Figure 1A Figure 1B produc-on oT schematic representaton of the/products F; G-A and Gb, schematic representation of the production of the product F using an ion exchanger, product do schematic representatin of the/product F using lipids; Figure 1C Figure 2 an HLPC of product F (purified by preparative HLPC); column: J-125 Waters, Protein Pak; mobile phase: KH 2

PO

4 0.05 m; 0.5 ml/min; UV-detection 224 nm; extinction: 0-0.2 Figure 3 an HLPC of the product F (purified by preparative HLPC on a C18-reverse phase column of Waters), column: DEAE- Waters, Protein Pak; Mobile phase: 10 methanol; ml/min.; Extinction: 0 to 2, Sample: 0.3 5 ul

I

7 Figure 5 an HLPC of the product G-a, column: J-125 Waters, Protein Pak, mobile phase: KH 2

PO

4 0.05 m, 0.5 ml/min., extinction: 0 to 2, 224 nm, Sample: 2 20 Ul Figure 6 an HLPC of the product G-b, column and mobile phase as with product G-a, extinction: 0 to 0.2, 224 nm, sample: 2 20 Pl Untreated raw milk or fresh milk of a domestic animal which has not been subjected to any of the heat treatments which are usual in dairies nowadays, is preferably used as starting material. Its cream can hati been separated in the usual manner, for example by centrifuging.

However, cream separation can also be carried out together with the subsequently described first membrane filtration.

The said raw milk is subjected to a first membrane filtration on a microporous membrane with a pore size of the order of 0.1 to 0.6 pm, preferably 0.2 pm. In this connection it is of advantage to use an arrangement of membrane filters which makes possible a filtration in tangential flow. A polyvinylidene fluoride membrane has for example proved to be advantageous, for instance a GLVP or HVLP-durapore-membrane of the Millipore company. Further suitable mambranes are described in WO 89/05586 and in the DE-A 39 03 729 of the applicant. The disclosure of these publications is herewith referred to in full.

In this way a clear permeate is obtained (filtrate I) and a viscous retained material I. The filtrate contains all salts, lactose, amino acids, oligopeptides and low molecular polypeptides in genuine, non-denatured form. The retained material contains practically all casein and fatty constituents of the milk. It can, therefore, be u,cd for the production of the usual products on the basis of milk fat and milk casein, whereby it can be of advantage to use this concentrate as starting material. However, cream will frequently be separated prior to the membrane filtration, so that the retained material of the first membrane filtration consists to a great extent of casein.

The dry mass of the filtrate (permeate) is about 6 and the nitrogen content of the dry material about 1.2 Dietetic foodstuffs and food additives can be obtained from it by concentration.

In the framework of the present process the filtrate from the first membrane filtration is subjected to a second nembrane filtration on a membrane with a separation limit of 8 to 10,OC0 molecular weight.

In this, essentially the so-called whey proteins are in the retained material II. The filtrate II, which essentially contains NPN compounds, lactose, short fatty acids and several salts, is passed on to a third membrane filtration on a membrane with a separation limit of 1,000 molecular weight. The retained material III contains the product Its recovery from the retained material is described below.

The filtrate III is passed on to a reverse osmosis. In this either a so-called open or closed membrane can be employed. All usual membranes with corresponding pore sizes are suitable for the filtratrions. As an open membrane an HF-membrane of Millipore t" Mr-3-NF-40, can, for example be used. When using an open membra,the desired product F is mainly in the filtrate.

9 For working up, the filtrate IV is evaporated down to about 15 to of the feed volume to the reverse osmosis, and is then allowed to stand several hours (about 2 to 10 hout preferably 2 to 8 hours) at refrigerator temperature (about 2 to 1000). During this time solids are formed, especially crystallised lactose. Liquid is removed from the solids by suction or filtration. The mother liquor is once more evaporatedpedow to about 5 to 10 by volume and is adjusted with ethanol to an alcohol content of 75 to 80 by volume, especially by volume. This generally requires 8 to 12 times the volume, DoolnO\\k onp nhe especi lly 10 times the volume of absolute ethanol. AotfR Lprecipitate fornnin the coF (2 to 10°C) which is separated by suction or by filtration. The filtrate obtained is evaporated down to dryness and is subsequently extracted with about 8 to 12 times, preferably times the quantity by weight of 90 to 95 of ethanol. The extract obtained is evaporated down to dryness, and the residue is extracted with 5 to 10 times the quantity by weight of ethanol, isopropanol or chloroform. When evaporating the extract to dryness the product F is obtained as a colourless solid or as an oily residue. Three (HPLC) chromatograms of this product, which have been prepared under different conditions, are reproduced in figures 2 and 4.

The product F is ninhydrine-positive, has a good solubility in chloroform, isopropanol, ethanol and water and exhibits the chemical and chromatographic behaviour of short chain peptides.

When using a closed membrane for the reverse osmosis, the product F is recovered from the retained material while the permeate (filtrate) consists practically only of water, several salts and a small quantity of NPN compounds. The said retained material is processed in the same way as descibed above for the filtrate of the reverse osmosis on an open membrane. However, somewhat larger quantities of the organic solvents mentioned above are needed as the retained material contains larger quantites of lactose and salts than the filtrate of the reverse osmosis on an open membrane. By means of the sequence already described the product F is also obtained from the retained material of the reverse osmosis on a closed membrane.

1 11 The product F can be recovered in a simple manner, as represented in figure IB, also using ion exchangers, by i) treating the filtrate obtained in the steps a) and b) according to claim 3 with a cation or anion exchanger and eluting with acid or alkali, ii) evaporating the eluate down to dryness and extracting the residue with absolute ethanol, chloroform or isopropanol, filtering and evaporating the filtrate down to dryness, whereby the product F is obtained in the form of a salt.

Commercial anion and cation exchangers can be employed as ion exchangers, for example Amberlite, Dowex, especially Dowex 50 Wx8 to 100 mesh) or Dowex, type 2x4 (20 to 100 mesh). The quantity of ion exchanger required depends on the peptide and salt concentration in the solution. Usually about 1 kg of ion exchange resin is used for about 20 to 30 g dissolved solids. The treatment with the ion exchanger is carried out in the usual manner, for example by stirring with the ion exchanger or by passing the solution to be treated through an ion exchange column.

The ion exchange resin is then eluted with acid or alkali depending on the form in which it is present. Dilute acid or alkali solutions are used for this purpose, for example 2 to 5 hydrochloric acid or 2 to 5 ammonia.

The eluate is evaporated down to dryness, for example by evaporation, lyophilisation or spray drying. The residue is taken up in absolute ethanol, chloroform or isopropanol and the insoluble constituents are filtered off. The filtrate is evaporated down to dryness. The product F is thereby obtained.

By treating the residue of the extraction with 80 ethanol, filtering and evaporating to dryness a further fraction is obtained. If the residue which is insoluble in 80 ethanol is extracted with 30 to ethanol another additional fraction is obtained.

The product F can also be obtained, as is shown schematically in figure iC, from the filtrates I to III by treating the filtrate in question with lipides and subjecting it to a filtration as described above for the first membrane filtration. The fat containing retained material is evaporated down to dryness in vacuo and is then extracted with 5 to 10 times its quantity by weight of a non-polar solvent, for example, ether or petroleum ether. After removal of the extract solvent the product F is obtained.

0.1 to 10, especially 0.1 to 5 by volume of lipides, based on the volume of the filtrate, are used. Oils of animal or vegetable origin are especially suitable as lipides, preferably milk lipides.

Finally the product F can also recovered from the cream of the fresh or raw milk by pre-treatment with ether or petroleum ether and extraction of the aqueous phase with ethanol. About 100 mg of product F are obtained from one litre of milk.

As already mentioned above the product is recovered from the retained material of the third membrane filtration. The retained material is extracted with 8 to 12 times, preferably about 10 times the volume of 75 to 85 especially 80 ethanol. The ethanolic extract solution is separated by suction or filtration and the residue is rewashed once more with a small amonmt of ethanol of the above concentration. Subsequently it is extracted with about 8 to 12 times, especially 10 times the quantity of of 25 ethanol and also rewashed once more during separation by suction of the extract solution.

The extract solutions obtained are in each case evaporated to dryness in vacuo. In both cases a colourless solid which is here designated as product G is obtained. The two fractions can be combined or used separately. They are distinguished by the fact that the substance G-a, which has been recovered from the 25 ethanol extract, increases the cell respiration to a greater extent than the substance G-b which has been recovered from the 80 ethanol extract. For the fraction from the 25 ethanol extract a strong HPLC band at RT 19/20 is characteristic while bands displaced toward longer retention times are characteristic for the product recovered from the 80 ethanol extract, particularly bands at about RT 23 and 26 (column: 1-125, Waters, Protein pack, mobile phase: KH 2

PO

4 0.05 m; 0.5 ml/min; UV-detection at 224 nm); cf. figures 5 and 6.

The products described can be recovered in the same manner from so-called fresh milk (in the sense given to the term by the Milk Regulation), however, with lower yields.

One can of course also start from whey or from the NPN fraction of a milk which has not been denatured by heat. In this case the 1st and 2nd membrane filtrations are omitted.

Analogous results are obtained with sheep's milk.

The cell respiration increasing effect can be defined with the socalled SCE test (Sister-Chromatide-Exchange-Test); experimental animal: Chinese hamster. A dose of less than 300 mg/kg inhibited 100 the chromatide exchange induced by acetaldehyde. The SCE test is described in Mutation research 56, 169-176 (1977).

The invention also concerns pharmaceutical compositions which conti.ri at least one of the products according to the invention, especially product F, possibly together with a carrier and/or inactive ingredient which is suitable for pharmaceutical purposes.

These compositions can especially find application in the prophylactic and therapeutic treatment of neurodermatitis, allergies, glaucoma and for the stimulation of the immune system. They can, for example,be applied orally, parenterally or locally. Dosage depends in the first place on the specific form of preparation and the purpose of the therapy, respectively the prophylaxis.

With oral application the individual dose given is generally between and 50 mg (for a grown up person with a body weight of about 75 kg) and about 3 to 10 doses per day (24 hours) are given.

In the treatment of neurodermatitis the lower dosage limits can be used, for example at 1.5 3 mg active agent per individual dose.

At this dosage the itch irritation disappears quickly and the skin subsequently becomes normal. In the treatment of rheumatism the daily dose can amount to as much as 500 mg (for a grown up person).

For intravenous application as a rule 0.7 140 mg are given per person (of about 75 kg body weight) per day. As a rule this dose is given only once daily.

In local application as a rule 1 or 2 times the quantity of active agent given above for oral application, is given.

A preparation for oral application can be formulated as solution, for example in water or alcohol, or as tablets, whereby the usual physiologically inert fillers, binders, disintegrants and lubricants can be used for the production of the tablets. Suitable fillers are for example lactose, saccharose, starch or cellulose and their derivatives. Useful binders are for example starch, gelatine, sugar, cellulose ether, polymers, for example polyvinyl pyrrolidone. Starch and starch ethers can be used as disintegrants. Suitable lubricant and form separation agents are for example talcum, stearates or silicones, and highly dispersed silica or talcum can be used as flow regulation agent. The tablets can also be formulated as coated or film covered tablets. The composition can, of course, also be given in a usual soft or hard gelatine capsule.

The usual sterile, isotonic aqueous solutions can be produced for the purpose of injection. The active agent can, however, also be used as lyophilisate being dissolved prior to application in a suitable aqueous dilutant.

A preparation for local application can be available as an aqueous solution, a lotion, a jelly, a solution in oil, a suspension, or as a fatty or emulsion ointment. A preparation in the form of an aqueous solution is, for example, obtained by dissolving the active agents according to the invention in an aqueous buffer solution of pH 4 to 7.5 and, if so desired, by adding a further active agent and/or a polymeric adhesive, for example polyvinyl pyrrolidone, and/or a preservative. The concentration of the active agent is about 1 to 10 by weight.

An oily form of application for local use is, for example, obtained by producing a suspension of the active agents according to the invention in an oil, possibly with the addition of swelling agents such as aluminium stearate, and/or interface active agents (tensides) the HLB value (hydrophilic-lipophilic-balance) of which is below such as fatty acid monoester of polyvalent alcohols, fcr example glycerol monostearate, sorbitol monolaurate, sorbitol monostearate or sorbitol monooleate.

A fatty ointment is, for example, obtained by preparing a suspension of the active agents according to the invention in a fatty base which can be smeared, possibly with the addition of a tenside with an HLB value of below The product G is particularly suitable as an additive for foodstuffs and beverages, especially as addition to diet foods and beverages for allergic patients or as additive for baby foods. With product G, the genuine, heat unstable and non-denatured milk constituents which are necessary for a natural diet, can be returned to milk and milk products which have undergone traditional dairy production processes and therefore heating stages. They are, of course, also of advantage in other foodstuffs such as margarine. They also exhibit an advantageous effet in cosmetics.

(Translators Note: The above paragraph is repeated word for word in the German original.) The following examples explain the invention. Unless indicated otherwise, percentages in the framework of the present invention mean percent by volume.

Eample 1 litres of fresh raw milk (cow's milk) are subjected to a tangential filtration using a polyvinylidene fluoride mebrane with a pore size of 0.2 mun. About 17 L of filtrate I and 3 L of retained material I are obtained. The latter consists essentially of caseins and fat.

(The filtrate (17 dry matter about 6 by weight, and nitrogen content of the dry matter about 1.2 are passed on to a second membrane filtration where a membrane with a separation limit of 6 to 10,000 molecular weight is used. In this operation about to 1 L of retained material I are obtained which contain mainly whey proteins and enzymes. The filtrate II, about 16 L, is passed on to a third membrane filtration where a membrane with a separation limit of 1,000 molecular weight is used. Here also only about co 1 L of retained material III are obtained. From this retained material the product G is recovered in the manner described below.

The filtrate III is passed on to a reverse osmosis. Using an open membrane the filtrate IV is evaporared down to about 3 L and is then allowed to stand in the refrigerator for 4 hours at 6°C. Several products, particularly lactose crystallise out. The solids are filtered off and the filtrate is evaporated down to the extent that an ethanol content of about 80 is adjusted by adding about 10 times the volume of absolute ethanol. The mixture is thoroughly blended, and then the liquid is removed by suction or filtration from the undissolved solids. The filtrate is evaporated down to dryness. The residue obtained in this operation is treated with about 10 times the quantity of chloroform, absolute ethanol or isopropanol. The solution is evaporated down whereby the desired product F is obtained. By means of preparative HPLC (conditions as above and given in figure 3) product F can be further purified. A product is obtained which in a concentration of 1 to 3 mg/kg gives protection against cell damage induced by acetaldehyde. In clinical trials product F proved to be very effective in the treatment of neurodermatitis.

If a closed membrane is used in the reverse osmosis, the desired substance has to be looked for in the retained material, and the retained material has to be processed in the manner described above for the filtrate.

For the recovery of product G (G-a and G-b) about 5 times the quantity of absolute ethanol is added to the retained material III from the third membrane filtration so that a mixture is obtained which is about 80 with respect to ethanol. The whole is stirred thoroughly and is allowed to stand for 5 hours. The undissolved constituents are then filtered off and the filtrate is evaporated down to dryness.

In this manner the product G-b is obtained.

The residue from the extraction is subsequently extractively aftertreated with 25 ethanol (about 10 times the quantity by volume).

The liquid is separated from the undissolved constituents by suction or filtration, the filtrate is evaporated down to dryness and product GCis obtained.

Example 2 100 ml of cream obtained by the centrifugation of raw milk at 3,500 revs./min, 8°C, 20 min. was extracted 3 times with 150 ml ether on each occasion. The aqueous phase remaining after the ether extraction is stirred with 100 ml of ethanol, filtered and the filtrate is evaporated down to dryness in vacuo. The residue is extracted cold with 80 ethanol. After evaporating down in vacuo about 2 g of colourless solids remain from which product F is isolated, by extraction with absolute ethanol, chloroform or isopropanol and evaporating off of the extraction solvent.

Example 3 The filtrate III obtained according to example 1 is allowed to stand for 4 hours in the refrigerator at about 6°C. The precipitate formed is filtered off and the filtrate is evaporated down to dryness. 220 g of the product obtained are dissolved in 200 ml of water and 800 ml of absolute ethanol are added so that the ethanol concentration is 80 The insoluble constituents are filtered off and the filtrate is diluted with the same volume of water. The solution is then passed through an ion exchange column of 1,25 kg Dowex (H-form). The column is at first eluted with 1 L of 5 HC1 and is subsequently washed neutral with water (2.5 The eluate is neutralised and evaporated down to dryness. The residue is taken up in absolute ethanol and is then filtered and evaporated to dryness. 1.4 g of product F (2.8 are thus obtained.

Example4 0.2 by volume of milk lipides are added to 1 L of filtrate III obtained according to example 1. The mixture is subjected to a membrane filtration on a polyvinylidene fluoride membrane with a pore size of 0.2 lpm. The retained material obtained in this manner is extracted with 20 ml of ether, and the ether of the extract is evaporated off.

The residue is extracted with ten times the quantity by weight of ethanol, chloroform or isopropanol. After evaporating off the solvent, product F is obtained.

Claims (2)

1. Milk constituents designated F, G-a and G-b either separately or in combination wherein:- product F is characterised by: an HPLC band at RT 4.6; column: DEAE-5pw, Waters Protein Pak, mobile phase: 10% aqueous methanol; 0.5 ml/min. HPLC bands at RT 26; column 1-125, Waters Protein Pak, mobile phase aqueous KH 2 PO 4 0.05 m; 0.5 ml/min. HPLC bands at RT 23.3 (weak); 25.3 (strong);

29.5 (medium); 30.5 (medium); 34.4 (weak); column 1-125, Waters Protein Pak mobile phase: aqueous KH 2 PO 4 0.05 m; 0.5 ml/min. ninhydrine positive reaction; substantially soluble in chloroform, isopropanol, ethanol and water; and (ii) whereby the substance G-a is characterised by: HPLC bands at RT 19.76 (strong); 20.38 (medium); 21.6 (shoulder); 24.3 (medium); column: 1-125, Waters Protein Pak, mobile phase: aqueous KH 2 PO 4 0.05 m; ml/min. ninhydrine positive reaction; substantially soluble in 20 to 50% aqueous (V/V) ethanol; 25 (iii) and the substance G-b is characterised by: :i HPLC bands at RT 21.22 (weak); 23.4 (strong) and 26.5 (strong) (conditions as with G-a); ninhydrine positive reaction; substantially soluble in 50 to 80% aqueous (V/V) 30 ethanol; 2. Milk constituents F, G-a and G-b as defined in claim 1 wherein the constituents are obtained by the S..:following process:- subjecting untreated raw milk or fresh milk to a 35 membrane filtration on a 0.1 to 0.6 pm-membrane, to 22 obtain filtrate I and retained material I; subjecting the filtrate I to a second membrane filtration on a membrane with a separation limit of 6 to 10,000 molecular weight to obtain filtrate II and retained material II; subjecting the filtrate II to a third membrane filtration on a membrane with a separation limit of 1,000 molecular weight to obtain filtrate III and retained material III; subjecting the filtrate III to a reverse osmosis on an open or closed membrane to obtain filtrate IV and retained material IV with an open membrane and retained material with a closed membrane; in the case where an open membrane is used, evaporating down the filtrate (IV) to about 20% of the volume fed into the reverse osmosis to obtain filtrate R, allowing filtrate R to stand at 2 to 10 0 C for 2 to 8 hours and filtering off from the precipitated solids to obtain filtrate V; evaporating down the filtrate V by 5 to 10% by volume, adjusting to an ethanol content of about 80% by volume with absolute ethanol and filtering to obtain filtrate T; evaporating the filtrate T obtained down to dryness S 25 to obtain residue U, extracting the residue U with 90 to of ethanol, filtering the extract thus obtained to obtain filtrate V and evaporating down the filtrate V to obtain the residue W; extracting the residue W obtained with chloroform, isopropanol or absolute ethanol to obtain an extract solution which after evaporation of the solvent gives constituent F and a residue X; extracting the residue X with 80% V/V aqueous ethanol to give constituent G-a and a residue Y; extracting the residue Y with 30% ethanol to give c constituent G-b; 23 in the case where a closed membrane is used, subjecting the retained material (instead of the filtrate obtained when using an open membrane) to the processing steps given above and obtaining product F in this manner; and recovering the products G-a and G-b as indicated in step or; alternatively for the recovery of G-a and G-b; either evaporating the retained material III from the third membrane filtration and/or the retained material IV from the reverse osmosis on an open membrane in vacuo and extracting with 80% ethanol, or adjusting to an ethanol content of 80% by volume by adding absolute ethanol, filtering off undissolved constituents, evaporating down the ethanol solution to dryness and thus obtaining the product G-b as a colourless substance; extracting the constituents which have remained during the ethanol treatment with 25% ethanol, filtering off the undissolved constituents to obtain filtrate P, evaporating down the filtrate P to dryness in vacuo and obtaining the product G-a in this manner. 3. Process for the production of the milk constituents according to claim 1 and 2, characterised by: subjecting untreated raw milk or fresh milk to a 44: membrane filtration on a 0.1 to 0.6 pm-membrane to obtain filtrate I and retained material I; S 25 subjecting the filtrate I to a second membrane filtration on a membrane with a separation limit of 6 to 10,000 molecular weight, to obtain filtrate II and retained material II; subjecting the filtrate II to a third membrane 30 filtration on a membrane with a separation limit of 1,000 •molecular weight, to obtain filtrate III and retained material III; subjecting the filtrate III to a reverse osmosis on an open or closed membrane to obtain filtrate IV and retained material IV with an open membrane and retained 24 material with a closed membrane; in the case where an open membrane is used, evaporating down the filtrate (IV) to about 20% of the volume fed into the reverse osmosis to obtain filtrate R, allowing filtrate R to stand at 2 to 10 0 C for 2 to 8 hours and filtering off from the precipitated solids to obtain filtrate V; evaporating down the filtrate V by 5 to 10% by volume, adjusting to an ethanol content of about 80% by volume with absolute ethanol and filtering to obtain filtrate T; evaporating the filtrate T obtained down to dryness to obtain residue U, extracting the residue U with 90 to of ethanol, filtering the extract thus obtained to obtain filtrate V and evaporating down the filtrate V to obtain the residue W; extracting the residue W obtained with chloroform, isopropanol or absolute ethanol to obtain an extract solution which after evaporation of the solvent gives constituent F and a residue X; extracting the residue X with 80% V/V aqueous ethanol to give constituent G-a and a residue Y; extracting the residue Y with 30% ethanol to give constituent G-b; in the case where a closed membrane is used, subjecting the retained material (instead of the filtrate obtained when using an open membrane) to the processing steps given above and obtaining product F in this manner; and recovering the products G-a and G-b as indicated in 30 step or; alternatively for the recovery of G-a and G-b; either evaporating the retained material III from e*o. the third membrane filtration and/or the retained material IV from the reverse osmosis on an open membrane in vacuo and extracting with 80% ethanol, or adjusting to an ethanol content of 80% by volume by adding absolute 25 ethanol, filtering off undissolved constituents, evaporating down the ethanol solution to dryness and thus obtaining the product G-b as a colourless substance; extracting the constituents which have remained during the ethanol treatment with 25% ethanol, filtering off the undissolved constituents to obtain filtrate P, evaporating down the filtrate P to dryness in vacuo and obtaining the product G-a in this manner. 4. Process according to claim 3, characterised by the fact that one starts with whey or the NPN fraction of a milk not denatured by heat, or the mother liquor from the lactose production, and starts the process with the second membrane filtration where whey or the mother liquor is used as starting material, and with the third membrane filtration where the NPN fraction is the starting material. Process for the production of the milk constituent F according to claims 1 and 2, characterised by the fact that cream from raw milk or fresh milk is extracted several times with ether or petroleum ether, the residual aqueous phase is stirred with ethanol, filtered, the filtrate being evaporated down, the residue is extracted cold with 80% ethanol, evaporating to dryness in vacuo, and thus obtaining product F. 6. Process for the production of the milk constituent F S 25 according to claims 1 or 2, characterised by the fact that the filtrate I or II obtained according to the steps bcoo. or of claim 3 is treated with a cation or anion :exchanger, and is then eluted with either acid or alkali, (ii) the eluate is evaporated down to dryness and the residue is extracted with absolute ethanol, chloroform or •isopropanol, the whole is then filtered and the filtrate evaporated down to dryness, whereby product F is obtained. boo 0. 7. Process for the production of the milk constituent F according to claims 1 or 2, characterised by the fact that 26 the filtrate I, II or III obtained from steps or of claim 3 is in each case treated with lipides, subjected to a membrane filtration on a 0.1 to 0.6 pm-membrane, the retained material is evaporated to dryness and the residue is extracted with a non-polar solvent, and removing the solvent product F is obtained from the residue by extraction with absolute ethanol, chloroform or isopropanol. 8. Medicinal drugs containing milk constituent F according to one of the claims 1 or 2. 9. Drugs according to claim 8 for the treatment of neurodermatitis, allergies, glaucoma and for immuno-stimulation. Use of a milk constituent F, G-a and G-b according to one of the claims 1 or 2 in infusion solutions. 11. Use of the milk constituent F, G-a and/or G-b in at least a partially purified form according to one of the claims 1 or 3 as additive for foodstuffs and beverages, especially milk products. 12. Use according to claim 11 in diet foods and beverages, especially in diet foode and beverages for allergic patients. 13. Use of a milk constituent F, G-a and G-b according to one of the claims 1 or 2 in cosmetics, especially in 25 skin care products. 14. Any one of the filtrates I, II, III or IV according to claim 2 or 3 when used for the treatment of neurodermatitis, allergies, glaucomas and for immuno-stimulation. 30 15. Milk constituents F, G-a and G-b as hereinbefore described with reference to the accompanying examples. DATED this 1 day of March 1993 KAILASH KUMAR GAURI Patent Attorneys for the 6§TR^j. Applicant: I} 1 F.B. RICE CO.