IE49296B1 - Method of treating liquids containing blood substances - Google Patents

Abstract

1532848 Purifying proteins P G S LINDROOS 19 Nov 1976 [11 Dec 1975] 48376/76 Heading C3H A method for separating iron compounds from the protein in a hemoglobin solution, comprises adding to said solution an organic solvent comprising ethanol in an amount such that the total ethanol content in the solution is at least 40% by volume, adjusting the pH of the liquid to a value lower than 4À5, whereby iron compounds in the solution are agglomerated, and thereafter separating the agglomerated iron compounds from the solution. The solution may be adjusted to a pH value which is less than 4À5 but greater than 2À5. Preferably the pH-value of the solution after agglomeration of the iron compounds is, before or after their separation, raised and wherein any fraction of blood protein precipitated thereby, with iron compounds included therein or adsorbed thereonto, is separated from the mixture. Before or after the agglomeration of iron compounds a minor quantity of broth of blood cells may be added to the solution and any flaky material of fragments of cell membrane formed thereby, together with iron compounds adsorbed thereonto, is then separated from the mixture. The organic solvent preferably includes a minor proportion of glycerin or ethylene glycol or methanol or acetone or ethylacetate or propanol or isopropanol or butanol. The iron compounds may be separated from the solution by centrifuging or a cyclone process or filtering or ultrafiltering or sedimenting or adsorption on to voluminous or solid material, and the protein may be recovered from the solution by precipitation or adsorption on to solid or voluminous material or ultrafiltering or evaporation of the solution or drying or concentration of the solution by means of freezing. The ethanol content in the solution after the agglomeration of iron compounds may be increased before or after their isolation and the fraction of blood protein precipitated thereby, together with any iron compounds included therein or adsorbed thereonto, may then be separated from the mixture. Before addition of ethanol to the solution and lowering of the pH of the solution, the solution is preferably cooled to a temperature of between zero and -20‹ C. and wherein this temperature is maintained during the entire further treatment.

Description

The present invention is concerned with a method of separating the iron compounds from the protein in a liquid that contains blood substances, mainly globin and iron compounds derived from hemoglobin.

The liquid is obtained from slaughtering blood, which is, by means of a simple centrifuge operation, separated into its two blood phases, plasma and broth of red blood cells, the latter one also called waste blood. The waste blood contains about 70 per cent of the total protein 0 content of blood. The main part of this protein is in the form of hemoglobin, which consists of a protein component, globin, and of an iron compound, ferroprotoporphyrin. This iron compound gives the blood its particular taste and colour, red in the liquid form and black in the powder form.

A solution of globin is colourless to slightly yellow, globin in the powder form is white to light yellow.

When ferroprotoporphyrin is cleaved from hemoglobin, the iron is often converted into the ferro form, the chloride of this iron compound is called hemin and characterized by an intensive black colour. - 3 Blood protein, mainly globin, that has been made free from this iron compound and thereby from the taste and colour resulting from same, is used in the food industry because of its nutrient value and, if the functional properties have been retained, as a texturing agent as well.

The iron compound, which is constructed of four pyrrole rings around the iron atom and keeps the iron in a so-called hemo-bound form, is used as an iron enriching agent in the foodstuffs and pharmaceutical industry.

The broth of red blood cells is treated in a way previously known so that the cell membrane oi the red blood cells is exploded and a solution of hemoglobin is obtained. The fragments of cell membrane are possibly separated by centrifuging.

A method is previously known by which, on the laboratory scale, the iron component and the globin are separated chromatographically or by means of extraction, in which connection solvents have been used such as methylethylketone, acetone and dimethylformamide .

The chromatographic method is less suitable on the technical scale, and the extraction by means of solvents is unsuitable in view of the use of the obtained product as fodder or food.

According to the invention, there is provided a method for separating iron compounds from the protein in a hemoglobin solution, comprising adding to said solution an organic solvent comprising ethanol in an amount such that the total ethanol-content in the solution is at least 40 per cent by volume, adjusting the pH of the - 4 litluid to a value lower than 4.5, whereby iron compounds in the solution are agglomerated, thereafter separating the agglomerated iron conpounds from the solution and separating the globin from the solution by raising the pH to a value above 4.5.

Accordingly, the present invention provides a simple and technically and economically practicable method for separating iron compounds from the globin. The globin obtained by this method can be used for fodder and foodstuffs and the iron components can be used for foodstuffs and pharmaceuticals.

According to the method of the invention a hemoglobin solution, prepared in the way described above or in another way, is mixed with ethanol and water and acidified to a pH lower than 4.5. Thus the ferroprotoporphyrin is cleaved from hemoglobin in the form of different iron compounds with a black colour. The chloride of the iron in the ferri form, so-called hemin , probably constitutes the main part of the formed iron compounds, It is also conceivable that polymerization, adsorption on, and reactions with, blood substances, primarily protein, take place.

When the content of iron compounds is sufficiently high and the content of ethanol in the solution is higher than 40 per cent by volume, the iron compounds form agglomerates that can be separated from the bound mixture. The protein, mainly globin, remains in the solution in dissolved form.

When the ion strength is increased, e.g. by addition of salt, an accentuation of the agglomeration of iron compounds is obtained. At the same time, an increased precipitation of protein is obtained.

According to a particularly suitable embodiment of the invention, the liquid is cooled before the addition 49286 - 5 of ethanol to the solution and the lowering of the pH of the solution, to a temperature of between 0 and -20°c, with this temperature being maintained during the entire further treatment. In this way, the functional properties of the protein are retained in addition to its nutrient value.

According to a particular embodiment of the method in accordance with the invention, the pH value of the solution after agglomeration of a major proportion of the iron ccmpounds is, before or after their separation, raised to a value above the pH used to agglomerate said major proportion of the Iran compounds, but having a value less than 4.5, so as to remove substantially all of Said iron conpounds, any fraction of blood protein precipitated thereby, with iron compounds included therein or adsorbed thereinto, is separated from the mixture, Thus a voluminous precipitation of blood substances, mainly protein, and a renewed formation of hemoglobin out of iron compound and globin are obtained. This precipitate adsorbs the finely dispersed ircn compound and can be separated together with this from the solution.

Alternatively, before or after the agglomeration of iron compounds, a minor proportion of broth or red blood cells may be added to the solution and wherein any flaky material of fragments of cell membrane formed thereby, together with iron compounds adsorbed thereonto, is then separated from the mixture.

The ethanol content of the solution, after the agglomeration of iron compounds may be increased before or after their isolation and wherein the fraction of blood protein precipitated thereby, together with any iron compounds included therein or adsorbed thereonto, is then separated from the mixture.

According to another embodiment, the organic solvent may include a minor proportion of methanol or pro4 9 2-9 6 - 6 panol or isopropanol or butanol or ethylene glycol or glycerin or ethyl acetate, or acetone.

With a lower pH, a more complete cleavage of hemoglobin and agglomeration of the iron compounds are obtained, but at the same time, the functional properties of the protein are destroyed and an increased polymerization of the iron compounds is probably produced. With higher pH values, these phenomena are reduced, whereas the precipitation of protein together with iron agglomerations is increased.

The reduction in the pH is achieved, for example, by means of inorganic acids, such as hydrochloric acid and sulphuric acid, or by means of organic acids, such as acetic acid and citric acid.

By maintaining a low temperature in the liquid, preferably lower than -5°C, it is possible to retain the functional properties to a higher extent.

The reaction mixture that is obtained under the conditions indicated in the claims is black to brownish black. When the mixture is centrifuged at 10,OOOxG, an intensive black paste is obtained that contains the major part of the iron compounds in the mixture. The supernatant is light yellow to light brown and contains the major part of the protein in the mixture. The colour of the supernatant is an indication of the extent to which the protein and the iron compound have been separated from each other.

The agglomerates of iron compounds may be separated from the solution by centrifuging or sedimentation or a cyclone process or filtering or ultrafiltering or adsorption onto voluminous or solid material. • s 49296 The protein may be recovered from the solution by precipitation or adsorption onto a voluminous or solid material or evaporation of the solution or drying or concentration of the solution by means of ultrafiltering and freezing.

Depending on the procedure that is selected for the recovery of the protein out of the solution, a protein more or less contaminated with iron compounds is obtained.

The protein in the solution after the separation of the agglomerations of the iron compounds under the conditions indicated in the claims and as illustrated in the examples of processing proved suitable for man/ purposes of foodstuffs as regards the iron content, colour, and taste.

The invention will be illustrated below by means of some exemplifying embodimentsi EXAMPLE 1 Broth of red blood cells obtained from slaughtering blood, with sodium citrate as the anti-coagulating agent, was treated with ethanol and water so that the cell membrane was exploded. The fragments of cell membrane were centrifuged off. The hemoglobin solution obtained in this way contained 15 per cent of dry substance, mainly hemoglobin, 33 per cent by volume of ethanol, and the rest of water, the pH-value being 4.3 and the colour dark red. grams of this hemoglobin solution were cooled down to a temperature of -12°C in a bowl, and, while stirring and cooling, a solution consisting of 0.50 ml of M hydrochloric acid and the 96 per cent by volume ethanol 9 296 - 8 of -12°C was added hereto dropwise. After the addition, the temperature was -12°C and the pH-value 3.1. The content of ethanol in the mixture amounted to 78 per cent by volume.

The pH was measured by diluting samples of the mixture with 3 times the sample volume of water before the measurement.

The mixture, which had a brownish black colour, was centrifuged at 27,OOOxG for 10 minutes in a Sorval centrifuge at -12°C. Hereby a black paste weighing 1.5 g and having a dry-substance content of 2.9 per cent as well as a light brown supernatant were obtained. From the supernatant, a light grey precipitate was precipitated by adding water up to 50 per cent and by adjusting the pH to 7.5. The temperag ture was maintained at -7°C. The precipitate was centrifuged off and 0.95 g of a grey paste with a dry-substance content of 28 per cent were obtained.

After an analysis it was noticed that the grey paste consisted of a protein with an iron content equalling about 5 percent of the iron content in hemoglobin. The black paste evidently contains the major part of the iron compounds derived from hemoglobin. The iron compounds are probably for the major part present in the form of hemin.

EXAMPLE 2.

Grams of a hemoglobin solution (with data as given in Example 1) at a temperature of -7°C were, during stirring and cooling, added dropwise into a bowl containing a solution of 0.45 ml of 1 M hydrochloric acid, 7.5 ml of water, and 10 ml of 96 per cent by volume ethanol at -7°C. After the addition, the temperature was -6°C and the pH value 3.3. The ethanol content in the mixture amounted to 50 per - 9 48296 cent by volume.

The mixture , whose colour was brownish black, was centrifuged at 10,000xG for 10 minutes, whereby a black paste was obtained containing 0.03 g of dry substance, as well as alight brown supernatant.

The supernatant was divided into two equal portions. From one of them, at pH 7.5, a grey precipitate was precipitated, which, after centrifuging and drying, yielded 0,12 g of dry substance.

The other portion was mixed with 5 ml of ethanol and 0.2 ml of 10 per cent sodium chloride in a water solution while stirring and cooling as well as ultrafiltered at -6°C in an Amicon Diaflo (Trade Mark) cell with a membrane XM 300. The filtrate was light yellow, the membrane was coated with a dark film. After dilution with water up to 50 per cent water content and at pH 7.5, from the filtrate was precipitated a white to light grey precipitate that, after centrifuging-off and drying, yielded 0.11 g of dry substance.

EXAMPLE 3 grams of a hemoglobin solution (with data as given in Example 1) at -4°C were, while stirring and cooling, added dropwise into a bowl containing a solution of 0.45 ml of glacial acetic acid, 0.8 ml of water, and 5 ml of 96 per-centby-volume ethanol at -4°C. After the addition, the temperature was -4°C and the pH-value 3.8. The ethanol content in the mixture was 62 per cent by volume.

The brownish black mixture was centrifuged at 27,000xG for 10 minutes, whereupon a black paste with a drysubstance quantity of 0.11 g as well as a light brown supernatant were obtained. 9 296 - 10 From the supernatant, after dilution with water to a water content of 50 per cent and at pH 7.5, a light brown to grey precipitate was precipitated, which, after centrifuging-off and drying, yielded 0.25 g of dry sub5 stance.

EXAMPLE 4 grams of hemoglobin solution (with data as given in Example 1) at -8°C were, while stirring and cooling, added dropwise into a bowl containing a solution of 0.45 ml of 1 M hydrochloric acid, 0.2 ml of 20 per cent sodium chloride in a water solution, and 10 ml of 96 per-cent-byvolume ethanol at -15°C. After the addition, the temperature was -1O°C and the pH-value 3.3, The ethanol content in the solution was 77 per cent by volume.

The mixture, which had a brownish black colour, was centrifuged at 10,000xG for 10 minutes, whereby a black paste containing 0.09 g of dry substance as well as a light yellow to light brown supernatant were obtained. 2o From the supernatant, after dilution with water to a water content of 50 per cent and at pH 7.5, a grey precipitate was precipitated, which, after centrifuging-off and drying, yielded 0.30 g of dry substance.

EXAMPLE 5 6 ml of 96 per-cent-by volume ethanol were added dropwise, while stirring and cooling, into a bowl containing g of a hemoglobin solution (with data as given in Example 1). The temperature was -10°C. Into this hemoglobin suspension were, while stirring and cooling, added dropwise 0.4 ml of 1 M hydrochloric acid in 3 ml of 96 per-cent-by-volume - 11 ethanol. After the addition, the temperature was 10°C and the pH-value 3.4. The ethanol content in the mixture was 77 per cent by volume.

The brownish black mixture was centrifuged at 5 20,000xG for 10 minutes, whereby a black paste containing 0.08 g of dry substance as well as a light brown supernatant were obtained. 0.19 ml of 0.5 M sodium hydroxide in 10 ml of 96 per-cent-by-volume ethanol were added dropwise, while cooling and stirring, to the supernatant. After the addition the temperature was -10°C and the pH-value 4.35. The ethanol content in the mixture was 85 percent by volume.

The precipitate formed, a fraction of blood protein with adsorbed iron compounds, was centrifuged off and yielded a black and red paste with a dry-substance quantity of 0.08 g.

Out of the extremely palely yellow-coloured supernatant, after addition of water to a water-content of 50 per cent and after raising of the pH to 7.5, while cooling, a light grey precipitate was precipitated, which yielded 0.22 g of dry substance, after centrifuging and drying.

EXAMPLE 6. g of a hemoglobin solution (with data as given in Example 1) at a temperature of -8°C were added dropwise, while stirring and cooling, to a bowl containing a solution of 0.45 ml of 1 M hydrochloric acid and 10 ml of 96 percent-by-volume ethanol at a temperature of -8°C. After the addition, the temperature was -8°C and the pH-value 3.3.

The ethanol content in the mixture was 78 per cent by volume. β· 49296 - 12 The brownish black mixture was centrifuged at 10,000xG for 10 minutes, whereby a black paste with a drysubstance quantity of 0.06 g and a light brown supernatant were obtained. 1 ml of a suspension consisting of 0.1 g of broth of red blood cells in equal portions of ethanol and water at a temperature of -8°C and a pH 5.3 was added dropwise, while stirring and cooling, to the supernatant. After the addition, the temperature was -8°C and the ethanol content was 75 per cent by volume.

The mixture was centrifuged at 10,000 x G for 10 minutes, whereby a minor quantity of black paste and a light brown to light yellow supernatant were obtained. In the same way as described in Example 5, from the supernatant a light grey precipitate was precipitated, which yielded 0.25 g of dry substance, after centrifuging and drying.

EXAMPLE 7 g of a hemoglobin solution (with data as given in Example 1) at a temperature of -6°C were added dropwise, while stirring and cooling, into a bowl containing a solution of 0.50 ml of 1 M hydrochloric acid, 8 ml of 96 percent-by volume ethanol, and 2 ml of glycerin at a temperature of -8°C. After the addition the temperature was -8°C and the pH-value 3.1. The total quantity of ethanol and glycerin in the mixture amounted to 82 per cent by volume.

The brownish black mixture was centrifuged at 27,OOOxG for 10 minutes, whereby a black paste with a drysubstance quantity of 0.09 g as well as a light brown to light yellow supernatant were obtained. 49286 In the same way as Example 5, from the supernatant, a light grey precipitate was precipitated, which yielded 0.26 g of dry substance, after centrifuging and drying.

In the same way, experiments were made by adding the same quantity of ethylene glycol, propanol, isopropanol, methanol, acetone, and ethylacetate as that of glycerin.

The result was the same as above.

EXAMPLE 8.

To a solution of 20 ml of 96 per cent by volume ethanol and 1.1 ml of 1 M hydrochloric acid at a temperature of -12°C were added dropwise, while stirring and cooling, 5 g of a hemoglobin solution (with the same data as given in Example 1). After the addition, the pH-value was 2.9 and the temperature -12°C. The ethanol content in the mixture was 80 per cent by volume. The mixture, which is of a brownish black colour, was centrifuged at 27,000xG for 10 minutes, whereby 2.2 g of a black paste with a dry-substance content of 2.7 per cent as well as a light brown supernatant, which later assumed a gel-type consistency, were obtained.

To the supernatent were added dropwise, while stirring and cooling, 70 ml of 96 per-cent-by-volume cold ethanol and then 0.8 ml of 40 per cent ammoniumsulphate solution (water solution) and 8 ml of water. The temperature after the addition was -6°C. The precipitate was centrifuged off at 8,000xG for 10 minutes, whereby 1.7 g of a white paste with a dry-substance content of 36 percent were obtained.

EXAMPLE 9 g of a hemoglobin solution (with data as given in Example 1) at a temperature of +8°C were added dropwise, while stirring, into a bowl containing a solution of 4929 6 - 14 0.80 ml of 1 M hydrochloric acid, 2 ml of water, and 10 ml of 96 per cent by volume ethanol at a temperature of +2°C. After the addition, the temperature was +8°C and the pH-value 2.5. The ethanol content in the mixture amounted to 67 per cent by volume.

The brownish black mixture was centrifuged at 27,000xG for 10 minutes at +8°C, whereby a black paste with 0.06 g of dry substance and a yellow supernatant were obtained.

Prom the supernatant was precipitated in the same way as described in Example 5, a light grey precipitate that yielded 0.27 g of dry substance, after centrifuging and drying.

EXAMPLE 10 3 g of a hemoglobin solution (with data as given in Example 1) at a temperature of -12°C were added dropwise, while stirring and cooling, into a bowl containing a solution of 0.35 ml of 2 M hydrochloric acid and 5 ml of absolute ethanol at a temperature of -12°C. After the addition, the temperature was -12°C and the pH-value 2.9. The ethanol content in the mixture amounted to 71 per cent by volume. The brownish black mixture was centrifuged at 27,OOOxG for 10 minutes at -12°C, whereby a black paste with a dry-substance quantity of 0.07 g as well as a light yellow supernatant were obtained. ml of 96 per cent by volume ethanol at a temperature of -18°C were added dropwise, while stirring and cooling, to the supernatant. After the addition, the temperature was -18°c. The ethanol content in the mixture amoun30 ted to 86 per cent by volume.

After 10 hours at -18°C, the mixture was centri40286 - 15 fuged as described above, whereby a black and red paste was obtained with a dry-substance quantity of 0.08 g.

The extremely vaguely light yellow-coloured supernatant was divided into two equal portions.

From one of the portions, in the same way as in Example 5, a light grey to white precipitate was precipitated, which yielded 0.11 g of dry substance, after centrifuging and drying.

The other portion was ultrafiltered in an Amicon Diaflo cell with a membrane PM 10 at -8°C. The concentrate was mixed with 10 ml of water at 0°C and filtered. The protein solution in this way de-ethanolized was neutralized and dried, and it yielded 0.06 g of dry substance.

Claims (10)

1. CLAIMS:1. A method for separating iron compounds from the protein in a hemoglobin solution , comprising adding to said solution an organic solvent comprising ethanol in an 5 amount such that the total ethanol content in the solution is at least 40 per cent by volume, adjusting the pH of the liquid to a value lower than 4.5, whereby iron conpounds in the solution are agglomerated, thereafter separating the agglomerated iron conpounds from the solution and separating the globin from the 10 solution by raising the pH to a value above 4.5.

2. A method as claimed in Claim 1, wherein the solution is adjusted to a pH value which is less than 4.5 but greater than 2.5.

3. A method as claimed in Claim 1 or Claim 2, wherein the pH-value 15 of the solution after agglomeration of a major proportion of the iron conpounds is, before or after their separation, raised to a value above the pH used to agglomerate said major proportion of the iron conpounds, but having a value less than 4.5, so as to remove substantially all of said iron conpounds, and wherein any fraction of blood protein precipitated 20 thereby, with iron compounds included therein or adsorbed thereonto, is separated from the mixture.

4. A method as claimed in Claim 1 or Claim 2, wherein before or after the agglomeration of iron compounds a minor proportion of broth of blood cells is added to the solution and wherein any flaky material of 25 fragments of cell membrane formed thereby, together with iron conpounds adsorbed thereonto, is then separated from the mixture.

5. A method as claimed in any one of Claims 1 to 4, wherein the organic solvent includes a minor proportion of glycerin or ethylene glycol or methanol or acetone or 30 ethylacetate or propanol or isopropanol or butanol. - 17

6. A method as claimed in any one of Claims 1 to 5, wherein the iron compounds are separated from the solution by centrifuging or a cyclone process or filtering or ultrafiltering or sedimenting or adsorption onto voluminous or solid material.

7. A method as claimed in any of claims 1 to 4, wherein the protein is recovered from the solution by precipitation or adsorption onto solid or voluminous material or ultrafiltering or evaporation of the solution or drying or concentration of the solution by means of freezing.

8. A method as claimed in any one of Claims 1 to 3, wherein the ethanol content in the solution after the agglomeration of iron compounds is increased before or after their isolation and wherein the fraction of blood protein precipitated thereby, together with any iron compounds included therein or adsorbed thereonto, is then separated from the mixture.

9. A method as claimed in any one of Claims 1 to 8, wherein before addition of ethanol to the solution and lowering of the pH of the solution, the solution is cooled to a temperature of between 2ero and -20°C and wherein this temperature is maintained during the entire further treatment.

10. A method as claimed in Claim 1, substantially as herein particularly described in any one of the forgoing examples.