JPS6219523A - Extraction of protein from milk, product, use of process andpharmaceutical composition - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The subject matter of the present invention is a method for the extraction of proteins, in particular iron-fixing proteins, from milk, the use of said method in particular for the production of lactotransferrin, the products obtained by said extraction method and the A pharmaceutical composition comprising a product.

Methods for producing proteins, particularly lactotransferrin and/or immunoglobulins, have already been reported. In particular, French Painting Patent No. 2,505,615 and CheyoH
paper (C, R, Aead, Sc, Paris
, t284 (February 14, 1977)].

Such methods cannot produce lactotransferrin of good quality or materials suitable for industrial production with reasonable yields.

The method of the present invention produces milk proteins, particularly lactotransferrin, in a single step of adsorption and elution on an ion exchanger under non-denaturing conditions, particularly mild conditions, and a constant pH, in a short time, and using convenient and industrial materials. make it possible.

The subject of the invention is thus a method for extracting proteins from milk, in particular iron-fixing proteins, from milk which has previously been substantially freed of caseins and fatty substances, by adsorption on an ion exchanger and subsequent elution of the adsorbed proteins. The method is characterized in that the adsorption and elution are carried out at a constant pH.

The ion exchanger used may be an anion exchanger, but
Preferably it is a cation exchanger. To speed up and simplify elution to avoid protein denaturation, ring cationic resins such as CM) lithacrylic (1, BF) or 0M
Preferably, a carboxymethyl resin commercially available under the trade name Sepharose (Pharmacia) is used.

When using cationic resins, the adsorption and elution are at a lower PR than lactotransferrin at the isoelectric point, preferably 5
It is carried out at a pH of ~8.5, more preferably 6.5-8.3 and most preferably 7-8.

The starting material is preferably unpasteurized or raw milk from which the casein and most of the fatty substances have been extracted beforehand, such as mildly acidic whey (lactoserum).
is used. The milk should not have undergone any protein destruction or degradation operations or cream separation under harsh conditions, such as pasteurization at 90° C. for several minutes. This raw milk can, for example, be weakened by precipitation with hydrochloric acid.

Separation of curds and whey is carried out according to techniques commonly used for liquid-solid separation such as decantation, centrifugation or preferably filtration.

In the case of filtration, the membrane chosen should preferably not immobilize proteins, for example by adsorption - cellulosic membranes are used, for example.

In preferred operating conditions, thick whey is used to start with.

Whey can be processed by conventional methods such as evaporation, e.g.
The vine is concentrated at a ratio of 5 times, preferably about 5 times.

This concentrated whey is preferably desalted to reduce its ionic strength.

These concentration and ionic strength conditions are advantageously met in a single step when performing ultrafiltration. The type of ultrafiltration membrane must be selected so as not to excessively adsorb or retain milk proteins. The selectivity limit is, for example, I
QOOO~7Q,000 preferably 25. OOO~5
Q. It will be selected in the 000 range. The ultrafiltration membrane may have a planar or tubular shape, or may have a hollow or spiral fiber shape. As an ultrafiltration membrane that does not immobilize milk proteins, especially lactotransferrin, or only slightly immobilizes it, in particular, polysulfone type membranes commercially available under the trade name Millipor*, such as PS
ED OHV 10 is mentioned.

Thus, salt removal and concentration are carried out by the same means using the same equipment. In this case, the salt removal is preferably carried out by a dialysis pass (ultrapass under slight pressure), e.g.
．． It is carried out with 5-2 volumes of distilled water.

The adsorption of milk proteins and their elution are determined by the selected p)(
This is carried out by using a buffer solution of the same concentration and only changing the ionic strength of the acid solution by adding sodium chloride to maintain the same concentration. The different ionic strengths are selected to first elute the undesired proteins and then only the desired proteins. For example, the elution of iron-fixing proteins, especially lactotransferrin, is advantageously preceded by 1 to 5 low ionic strength elutions, preferably 1 to 3 times. The elution buffer is preferably anionic, such as a phosphate buffer.

It is then advantageous to desalt the relevant eluate in order to exclude small molecules with a molecular weight below 1000. This salt removal is accomplished in conventional manner, such as by transient or gel permeation chromatography (eg G25) on Millipore ultrafiltration membranes.

The process according to the invention carries out a complete production cycle of pure lactotransferrin starting from whey in 9 hours by ultratransfer and separation on a cation exchanger, whereas in the Ch@rom article cited above, Since the time required for the purcess as described is 24 hours, or about three times longer, the nearly pure milk proteins forming the O 2 solution, which is extremely efficient in the process of the present invention, can be advantageously dried, especially by freeze-drying.

Iron-fixing proteins can be advantageously subjected to the action of complexing agents, preferably chelating agents, which have a higher affinity for iron than the protein exhibits, in order to increase their iron-containing capacity. For example, EDTA phosphate buffer is used.

The complexing agent is removed by salt removal according to the techniques previously described.

The present invention also includes as its subject matter the substances obtained by the above method, in particular lactotransferrin and immunoglobulins. The above method is advantageously used for the production of such materials. The invention thus also includes as its subject the application of the above-mentioned methods for producing these substances.

Therapeutic benefits of milk proteins are well known, especially the bacteriostatic activity of transferrin.

For this reason, the present invention also includes as its subject a pharmaceutical composition containing the product obtainable by the above method. In fact, the lactotransferrin obtained by the above method retains its bacteriostatic properties.

As a medicament, the product obtainable by the above method can be incorporated into pharmaceutical compositions intended for the digestive or parenteral route.

The pharmaceutical composition may be, for example, solid or liquid, and in any pharmaceutical form used today for human drugs, such as sugar-coated or plain tablets, capsules, granules, herbal medicines, eye drops, nasal liquid forms. provided by. These are prepared by conventional methods. Thus, the active ingredient or ingredients may be combined with excipients commonly used in these pharmaceutical compositions, such as talc, acacia, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous vehicles, animal or vegetable sources. It can be formulated with fatty substances, halaffin derivatives, glycols, various wetting, dispersing or emulsifying agents and preservatives.

The following examples are illustrative of the invention and are not intended to limit it.

Example 1: Extraction phase of lactotrans7erin Milk 151 with 57-39% hydrochloric acid 15.8-
was added, precipitated at 40°C for 1 hour, and passed through a cellulose membrane. 2,100 ml of the whey thus obtained was filtered through dialysis using the same volume of distilled water to a separation limit of 25,000 ml.
polysulfone type Millia membrane (PSED OHV)
10) F''l outside the upper limit was added and concentrated to the next step 420d.

The pH of the concentrated solution was adjusted to zO with sodium hydroxide and the ionic strength was adjusted to 0.15M with sodium chloride (measured with a conductance meter). The solution 400- was then passed through a column with an inner diameter of 25 mm filled with 100 id of CM Sepharose CL6B (7Armacia) equilibrated with α15M pH 7, 0 m salt buffer at a rate of 5 m/min.
Elution with IM pH 7,0 phosphate buffer at 3 m/m
It was done at a speed of im. The ionic strength of the buffer solution was determined by sodium chloride (Ll 5M Te270114, α25M 145mg).

It was adjusted according to the gradient of 1451 at α30M. Lactotransferrin was eluted with 45Od at α4M. Salt removal was performed on 7 Armacia Gel G25. When the obtained liquid was freeze-dried, 84 ml of lactotransferrin was obtained.

Example 2: Extraction of lactotransferrin The same work as described in Example 1 was carried out starting from milk tSl.

150 d of liquid was passed through the column at a speed of 1 m/min. p of α15M with ionic strength of α25M
After an initial elution with 150 d of H7,0 phosphate buffer, lactotransferrin was eluted with PNZO buffer 8011114 having an ionic strength of 43M.

After salt removal and lyophilization, 40IR9 rough)) Lance 7 Erin was obtained.

Example 3: Lactotrans7erin bbm Whey was prepared and deposited onto the ring cation exchanger as described in Example 1.

Next, the caraft exchanger was washed with 200 d of α1M pI(7,CI phosphate buffer).The ionic strength of the buffer was adjusted to [115M] with sodium chloride.

Then, add lactotransferrin to 100% of the same buffer.
It was eluted at mJ. However, the ionic strength was adjusted to 1M with sodium chloride.

Salt removal was carried out in the same manner as in the previous example (7 Armacia Gel G2
5). After freeze-drying, the working yield obtained is approximately 90 mg for milk&5
lactotransferrin.

Example 4 Whey was prepared as described in the previous example. The pH was adjusted to 7.0 with sodium hydroxide. Next, bruise at a rate of 1 d for every whey! Saturation of J (AZARI) lactotransferrin in ferric ions was allowed to occur for 2 hours at ambient temperature. After this, the solution was centrifuged and
It was then ultrafiltered as described in the previous example.

The material 400111 to be retained on the filter membrane was passed over the ring cation exchanger 100. After washing with 200 g of 5M pH 7.0 phosphate buffer, the saturated lactotransferrin was eluted with 100 g of the same buffer but with the ionic strength adjusted to 1 M with sodium chloride.

The preparation was desalted by filtration on gel (G25);
It was then freeze-dried.

Saturated and salt-free lactotransferrin [105
The solution was dissolved in a pH 4.5 phosphate/citrate buffer of M at a rate of 111 Jg per liter of buffer.

Desaturation was carried out at 4° C. for 12 hours. Next, the desaturated solution of lactotransferrin was filtered through G625 gel to remove salt, and then freeze-dried.

Prepare CLlM sodium citrate solution and α1M sodium bicarbonate solution.

To 100 g of this preparation was added 232 da of ferric chloride hexahydrate.

Example 5 Whey was prepared and concentrated as described in the previous example.

The pH of the concentrated solution was adjusted to zO with sodium hydroxide.

This preparation 107 was prepared beforehand at 0.15M (7) pH 7.0.
0M Sepharose 7 Earth Flow (7 Armacia) 60011 equilibrated with phosphate buffer! The mixture was passed through a column with a diameter of 10 cm that could be packed with 61/br at a rate of 61/br.

When this was done, the column was washed with one portion of 0.15M pH 10 phosphate buffer, followed by one portion of the same buffer with the ionic strength adjusted to 0.3M with sodium chloride.

Lactotransferrin was then eluted with 600 ml of the same buffer, the ionic strength of which was adjusted here to 1M.

Salt removal was performed by filtration on a G25 gel and the preparation was then lyophilized. Approximately 30~30 lactotransferrin/11 base materials were obtained.

Example 6: Pharmaceutical composition

Claims (14)

[Claims] (1) A method for extracting proteins from milk, in which iron-fixing proteins in particular are extracted from milk from which casein and fatty substances have been almost completely removed by adsorption on an ion exchanger and then elution of the adsorbed proteins. , a method characterized in that the adsorption and elution are carried out at a constant pH. (2) The method according to claim 1, wherein the ion exchanger is a cation exchanger. (3) The method according to claim 2, wherein the exchanger is a weak cation resin. (4) The method according to claim 2 or 3, characterized in that adsorption and elution are carried out at a pH of 5 to 8.5. (5) The method according to claim 4, characterized in that adsorption and elution are carried out at a pH of 7 to 8. (6) The milk according to any one of claims 1 to 5, wherein the milk from which casein and fatty substances have been substantially removed is whey that can be concentrated about 5 times. Method. (7) The method according to claim 6, wherein the concentration is carried out by ultrafiltration. (8) Cutoff limit of ultrafiltration membrane is 25,000 to 50,000
8. A method according to claim 7, characterized in that 00. (9) The method according to claim 7 or 8, wherein the ultrafiltration membrane is a polysulfone type. (10) The method according to any one of claims 1 to 9, characterized in that the elution of the iron-fixing protein is preceded by 1 to 5 low ionic strength elutions. (11) The method according to any one of claims 1 to 10, characterized in that iron complexing occurs subsequent to elution of the iron-fixing protein. (12) A method for producing lactotransferrin using the method according to any one of claims 1 to 11. (13) A method for producing immunoglobulin using the method described in any one of claims 1 to 11. (14) A pharmaceutical composition containing at least one product obtainable by the method according to any one of claims 1 to 11.