FI59195B - FOERFARANDE FOER SEPARERING AV JAERNPROTOPORFYRIN OCH DESS DERIVAT SAMT AV GLOBIN UR EN VAETSKA INNEHAOLLANDE BLODBESTAONDSDELAR - Google Patents

Description

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νΒ3Γφ lJ 1 V UTLÄGGN I NGSSKRIFT 3 S 1 “0 (^) patent leddelat ^ ^ (51) Kv.lk?/lnt.CI.3 A 23 J 1/06 FINLAND - FINLAND (21) P« t * nulh » k * nni * - PK «ntm6kning 7 5l6U 3 (22) H» k * ml * pllvt —An * ttki »lng * d» g 0 ^ .06.75 (23) Alkuptlvl — Glltlghvtsdag 0U.06.75 (41) Tullut] utklMk * l - Bllvit offantllg 15.12.75

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Patent · och registrstyrelsen 'Ansektn utltgd och utUkrtfun pubhcerad 31 · 03- Si (32) (33) (31) Requested «uoik« ui — Begird priority lU.O6.7U Sweden-Sweden (SE) 7U07882-5 (71) ( 72) Paul Göran Sigvard Lindroos, Eriksgatan 37, Abo, Finland-FI (5U) Oy Kolster Ab (5U) Method for the separation of iron protoporphyrin and its derivatives and globin from a fluid containing blood constituents - Förfarande för separering av järnprotoporfyrt och dess derivat The present invention relates to a process for separating iron protoporphyrin in a liquid and its derivatives or degradation products, i.e. the so-called iron component, from globin from a liquid containing blood constituents, which may contain less than about 33% ethanol.

A method known on a laboratory scale for separating the globin and iron component by chromatography or extraction using organic solvents such as methyl ethyl ketone, acetone and dimethylformamide is known.

However, the chromatographic method is ill-suited to the technical scale and the products obtained by extraction are not suitable for feed or food use.

The method according to the invention is characterized in that the liquid is cooled, suitably to a temperature range of -20 to 0 ° C, the pH value is adjusted to a value below U, 5, 2 59195, suitably to a range of 2.5 · ..U, 5 » an organic solvent having a dehydrating effect, such as ethanol or a mixture of ethanol and glycol, or ethanol and glycerol, is added to the liquid at least 1 + 0, preferably $ 75 of the total volume, maintaining the above temperature, optionally adding a salt such as sodium chloride or sodium citrate, and precipitated the iron component is separated, and finally the globin is precipitated from solution.

The invention thus provides a technically and economically feasible method for isolating an iron component from globin, in which case they are products useful for feed and food use.

According to a preferred embodiment, the organic solvent is added in two portions, first adding a smaller amount and possibly a salt such as sodium chloride or sodium citrate, then separating the formed agglomerate, for example by centrifugation, and then adding the remainder of the organic solvent to precipitate globin, leaving the remaining iron component in solution. and / or in the form of a fine dispersion. The precipitated globin can be separated from the mixture, for example by centrifugation.

The following examples illustrate the invention.

Example 1

To a solution of 36 ml of 9% ethanol, 6 ml of water and 0.55 ml of 1M hydrochloric acid at -12 ° C was added dropwise, with stirring and cooling, 2.5 g of a hemoglobin solution having a dry matter content of 15%. $, temperature -8 ° C and containing about $ 33 ethanol. After the addition, the mixture had a temperature of -12 ° C and a pH of 3.0.

1 + 5 ml of 9l + - $ ethanol was then added and the solution was centrifuged at 8,000 x g for 10 minutes, after which the precipitate obtained weighed less than 0.1 g.

Globin was precipitated by adding 0.1+ ml of 20- $ ammonium sulfate. The pH was adjusted to 3.0 with hydrochloric acid. Centrifuged at 8,000 x g for 10 minutes to give 0.1 + 5 g of a precipitate with a dry matter content of $ 36.

A second aliquot of globin was precipitated from the filtrate by adding 0.1+ ml of 20- $ ammonium sulfate solution to give 0.1+ g of a precipitate with a dry matter content of 1 + 2 $.

The color of the fines was light gray. The filtrate was dark brown and did not form a precipitate upon addition of trichloroacetic acid.

Example 2

To a solution of 20 ml of 9 * + - et ethanol and 1.1 ml of 1-m hydrochloric acid at a temperature of -15 ° C was added dropwise, with stirring and cooling, 5 g of 3,591,95 hemoglobin solution (same as in Example 1). After the addition, the temperature was -15 ° C and the pH was 2.9.

The mixture was centrifuged at 27,000 x g for 20 minutes to give a black precipitate of 0.9 g and a dry matter content of $ 8.3.

To the strong brown filtrate was added, with cooling and stirring, 50 mL of 9% ethanol; the temperature was -15 ° C. Addition of 10 mL of water and 1.2 mL of 20- $ ammonium sulfate solution gave a precipitate which was centrifuged at 8,000 x g for 10 minutes and resuspended in 9k- $ ethanol (~ 15 ° C) and centrifuged as above. 1.6 g of a precipitate with a dry matter content of 3756% were obtained. The precipitate was light gray in color when dry.

No precipitation occurred in the filtrate upon addition of trichloroacetic acid.

Example 3

To a solution of 10 ml of 9 * ethanol and 1.1 ml of 1 M hydrochloric acid at -15 ° C was added dropwise and cooled 5 g of hemoglobin solution (same as in Example 1). After the addition, the temperature was -15 ° C and the pH was 2.9.

To the mixture was added dropwise 5 ml of 20- $ sodium chloride solution, and the mixture was centrifuged at 27,000 x g for 20 minutes to obtain 1.1 g of a black precipitate with a dry matter content of 11%.

To the filtrate were added 12 ml of water and 1 ml of 20- $ ammonium sulfate solution, after which the mixture was centrifuged at 8,000 x g for 10 minutes. 3.8 g of a gray precipitate with a dry matter content of 1 k, $ 6 were obtained.

Example ^ According to this example, extraction of the iron component from the globin precipitate was performed.

To 5 g of a hemoglobin solution (same as in Example 1) at -8 ° C was added dropwise, with stirring and cooling, a solution containing 11 ml of water, U ml of ethanol and 1 ml of 1 M hydrochloric acid at a temperature of -6 ° C. After the addition, the temperature was -U ° C and the pH was 2.9. The pH was adjusted to 3.9 by adding 7 ml of 0.1 M sodium hydroxide solution. Precipitation was performed by adding 0.2 g of ammonium sulfate, followed by centrifugation. The resulting strong brown precipitate was washed several times with a small amount of 9% ethanol solution having a pH of 3 and a temperature of -12 ° C, and finally centrifuged.

This gave 3.05 g of a gray precipitate with a dry matter content of $ 18.8.

Example 5 5 g of a hemoglobin solution (same as in Example 1) was precipitated by adding 10 ml of 9% ethanol with stirring and cooling the solution to -6 ° C.

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The solution was centrifuged at 2,000 x g for 5 minutes to give 6 g of a red precipitate. There was also a small amount of black precipitate at the bottom of the tube.

The red precipitate was added with stirring and cooling to a solution of 70 ml of 9% ethanol and 1.1 ml of 1M hydrochloric acid at -12 ° C. After the addition, the pH was 2.9 and the temperature was -12 ° C. The mixture was centrifuged at 27,000 x g for 10 minutes to give a small amount of black precipitate.

To the centrifuge were added 2.0 ml of 10% ammonium sulfate solution and 10 ml of water. The temperature was -8 ° C and the pH was 3, ¾.

Centrifugation at 8,000 x g for 10 minutes yielded 1.5 g of a gray precipitate with a dry matter content of 39.3% ·

Example 6

To a solution of 20 ml of 9b% ethanol and 1.1 ml of 1M hydrochloric acid at -12 ° C was added dropwise, with stirring and cooling, 5 g of hemoglobin solution (same as in Example 1). After the addition, the pH was 2.9 and the temperature was -12 ° C. The mixture, which was brownish black in color, was centrifuged at 27,000 x g for 10 minutes to give 2.2 g of a black precipitate with a dry matter content of 011 2.7% and a light brown centrifugate which later turned gel-like.

To the centrifuge was added dropwise, with stirring and cooling, 70 ml of 9b% cold ethanol, followed by 0.8 ml of a 10% ammonium sulfate solution and 8 ml of water. The temperature after the addition was -6 ° C.

Centrifuged at 8,000 x g for 10 minutes to give 1.7 g of a white precipitate with a dry matter content of 36%.

Example 7 A solution of 20 ml of 90% v / v methanol and 1 ml of 1M hydrochloric acid was added dropwise to 5 g of a hemoglobin solution (prepared in the same manner as in Example 1) with stirring. After the latter solution was added, the pH of the mixture was 3.0 and the temperature was 5 ° C. The black mixture was centrifuged at 9,000 x g for 10 minutes to give a black precipitate (dry weight 0.1 g) and a light brown solution.

To the resulting solution were added 18 ml of water and 0.8 ml of 10% ammonium sulfate solution to form a light gray precipitate; the precipitate formed was separated by centrifugation and dried; yield 0.5 g of a gray powder.

Example 8 To 5 g of a hemoglobin solution (prepared in the same manner as in Example 1) was added dropwise at -6 ° C with stirring and cooling a solution consisting of 16 ml of 96 ml of ethanol, U ml of glycerol and 1 ml of 1-m hydrochloric acid. After the addition, the pH was 3.2 and the temperature was -6 ° C.

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The resulting black mixture was centrifuged at 12,000 x g for 10 minutes to give a black precipitate. The precipitate was washed with 2 ml of water, and dried; yield 0.15 g (dry weight).

The centrifuged solution was treated with ammonium sulfate solution as in Example 7. A light gray precipitate was obtained which was washed twice with water and then dried; yield 0.1+ g (dry weight).

Example 9 To 5 g of a hemoglobin solution (prepared in the same manner as in Example 1) was added dropwise at -10 ° C and with stirring a solution consisting of 16 ml of 90% by volume of propanol and 1 ml of 1 ml of hydrochloric acid. After the addition, the pH of the solution was 3} 0 and the temperature was -10 ° C. 0.5 ml of 20 ettiin potassium chloride solution was added dropwise to the resulting black mixture. Centrifugation was performed as in Example 1 to give a black precipitate; yield 0.2 g (dry weight). A light gray precipitate was precipitated from the resulting solution; yield 0.1+ g (dry weight).

Example 10

In the same manner as in Example 9, the iron component was separated from globin, but isopropanol was used instead of propanol. The result was exactly the same.

Example 11 A solution of 15 ml of 96% v / v ethanol and 0.7 ml of glacial acetic acid was added dropwise to 5 g of a hemoglobin solution (prepared in the same manner as in Example 1) at -2 ° C with stirring and cooling. After the addition, the pH was 3.6 and the temperature -2 ° C. To the resulting black mixture was added dropwise 0.1+ ml of 10-¾ sodium citrate solution and 20 ml of 50% v / v ethanol (at -2 ° C).

Centrifuged as described in Example 7. The dry weight of the black precipitate obtained was 0.2 g; a light gray precipitate having a dry weight of 0.1+ g was precipitated from the yellow solution phase as described in Example 7.

Claims (2)

6,591 95 1. For the purposes of this Regulation, a mixture of these products with a total content of about 33% ethanol, separated by a specific mixture of protoporphyrinids and derivatives of a specific product, dvs.den sä kallade järnkomponenten, frän globinet the temperature is adjusted to a temperature of -20 ... 0 ° C, the pH is adjusted to a value of less than i +, 5, the temperature is adjusted to 2.5 .-- ^, 5, the temperature is adjusted to an organic solution with a dehydrated solution, is ethanol or a mixture of ethanol and glycol, or ethanol and glycerol, and a mixture of at least 75% of the total volume, under the same temperature at the same temperature, with the addition of sodium chloride to the site, sodium chloride och den utfällda järnkomponenten separeras, och slutligen utfalls globinet ur lösningen. A method of separating iron protoporphyrin and its derivatives or degradation products, i.e. the so-called iron component, from a blood-containing liquid, which may contain less than about 33% ethanol, from globin, characterized in that the liquid is cooled, suitably to a temperature range of - 20 to 0 ° C, the pH is adjusted to less than U, 5, suitably in the range 2.5 --- ^ 5, an organic solvent having a dehydrating effect, such as ethanol or ethanol and glycol, or ethanol is added to the liquid. and a mixture of glycerol, at least UO%, preferably 75% of the total volume, maintaining the above temperature, optionally adding a salt such as sodium chloride or sodium citrate, and separating the precipitated iron component, and finally precipitating the globin from solution. Process according to Claim 1, characterized in that the organic solvent is added in two steps, first adding a smaller amount and optionally a salt such as sodium chloride or sodium citrate, followed by separating the agglomerate formed, for example by centrifugation, and adding more organic solvent in the second step. to separate the globin from the remaining iron component, which then remains in solution. 2. The use of the patented composition 1, which comprises an organic solution of the same type of material, the color of the active ingredient of the present invention, the sodium chloride or the sodium salt being separated from the product. the genomic centrifugation, and the addition of organic lysing agents to the and the separation of the globe from the rest of the other components, the flesh blar kvar i lösningen.