FI60104C - SAETT ATT UR EN BLANDNING ERHAOLLEN VID SPALTNING AV HEMOGLOBINET I DE ROEDA BLODKROPPARNA SEPARERA S K JAERNKONPONENT FRAON PROTEINET - 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

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Ruotsi Sweden (SE) 7513987_3 (71) (72) Paul Göran Sigvard Lindroos, Eriksgatan 37> 20100 Abo 10,

Suomi-Finland (FI) (7 * 0 Oy Kolster Ab. (5U) Ways of separating the hemoglobin in the red blood cells from the protein separates so-called iron component from the protein - Menetelmäk. The present invention relates to a method of separating the iron component from the protein from a mixture containing iron compounds, called the iron component, and blood protein, mainly globin, and the iron component from the protein, whereby the mixture is added to dehydrating agent, suitably an amount such that the total content of dehydrating agent in the liquid is increased to at least 40% by volume, preferably at least 70% by volume, based on the total amount of liquid, and the pH of the mixture is adjusted to a value lower than 4.5, preferably to a value of 2.5 to 4.5, resulting in a precipitation of iron component.

60104

The starting material is obtained from carcass blood which is separated by a simple centrifugation in two liquid phases, plasma and red blood cell porridge, the latter also called waste blood. The waste blood tails about 70% of the blood's total protein content. The majority of this protein is in the form of hemoglobin composed of a protein moiety, globin, and an iron compound, ferroprotoporphyrin. This iron compound gives the blood its special taste and color, red in liquid form and black in powder form.

A solution of globin is colorless to pale yellow in color, globin in powder form is white to light yellow.

Since ferroprotoporphyrin is cleaved by hemoglobin, the iron often transfers into ferric form, the chloride of this iron compound is called hemin and has an intense black color.

Blood protein, mainly globin, which is liberated from this iron compound and thus from the resulting taste and color, finds use in the food industry because of its nutritional value and, if its functional properties are preserved, also as texturing agents.

The iron compound, which is built up of four pyrrole rings around the iron atom and in which the iron is in so-called home-bound form, finds use as an iron enrichment agent in the food and pharmaceutical industry.

Finnish application 75 1643 discloses a method for separating the iron component and the globin. The known process likes to separate the globin from the iron component, which originates from the iron protoporphyrin (hemoglobin). The known separation occurs in two steps. First, a portion of the iron component is filtered off, and then the globin is precipitated so that the remaining iron component is poured into solution in finely divided form. Silunda, the iron component is difficult to recover.

The new process likes to form agglomerates of the iron component of such a nature that in a simple way they can be separated quite quantitatively. According to the new method, adsorption of finely divided particles of the iron component on blood substance, mainly probably solid blood protein, is achieved, whereby easily separable agglomerates arise, whereby the iron component can be recovered fairly quantitatively. It is important from an economic point of view that the yield of the iron component is high.

Characteristic of the new process is that an agglomeration of the precipitate is achieved as follows: the pH of the mixture is increased to a maximum of 4.5, a cell membrane fragment mass obtained in the cleavage of the blood cells is added, or ethanol to a total content of dehydrating agent of no more than 90 % by volume is added and optionally at the same time the temperature is lowered, whereupon the agglomeration is possibly further accentuated by the addition of inorganic or organic salts, such as sodium or potassium salts, e.g. sodium chloride, e.g. in the form of aqueous solutions, and then the agglomerates are separated by the iron component, and the residual globin is stored in a manner known per se.

The iron component agglomerates formed are stable and pourable, and can be centrifuged and filtered on a technical scale, without being broken into small particles that cannot be separated.

Furthermore, since blood protein, which is also referred to as the cell membrane fragments, settles around the iron component, protection against oxidation is obtained (the oxidation leads to an inactivation of the iron component, and is thus undesirable). The iron component, which is believed to consist largely of hemin, is intended to be used as a remedy for iron deficiency, and it is in the manufacturing phase dangerous for oxidation.

To date, there has been no method to separate the iron protoporphyrin from hemoglobin in a technically and economically feasible way, so that hemin and globin are obtained in a quality useful for medicinal and food purposes.

With the new method, the iron component can be separated fairly quantitatively, with up to 99% yield, and there is obtained a super-liquid which is almost colorless and which contains an iron amount lower than 1% of the iron present in the hemoglobin.

Thus, the most important of the process according to the invention is the formation of a stable and easily separable agglomerate: of the precipitate, which is achieved by a) raising the pH of the mixture to a maximum of 4.5, or b) adding the mixture. (c) ethanol is added until the total content of the dehydrating agent settles to a maximum of 90% by volume, possibly possibly lowering the temperature.

The stated increase in the pH of the mixture to a maximum of 4.5 will, of course, occur mainly in cases where the starting mixture pH is low, ie. 2.5-3.5. In case the pH is 4.5 at the outset, agglomeration is achieved by adding cell membrane fragment mass or ethanol.

In the process of the invention, it is convenient to cool the mixture to a temperature of between 0 and -20 ° C before the ethanol addition and the pH reduction. This preserves the functional properties of the protein in addition to its nutritional value.

In the process, smaller portions of the ethanol can be replaced by methanol, propanol, isopropanol, butanol, ethylene glycol, glycerine, ethyl acetate or acetone, giving the same effect.

At lower pH, a more complete cleavage of hemoglobin and agglomeration of iron compounds are phased, but at the same time a destruction of the functional properties of the protein and probably an increased polymerization of the iron compounds. At higher pH, these phenomena decrease while the precipitation of protein along with the iron agglomerates increases.

The reaction mixture's acid phase under the conditions specified in the claims is black to brownish black. When centrifuging the mixture at 10,000 x g phase, an intensely black paste containing the major part of the mixture's iron compounds. The supernatant is light yellow to light brown and tails most of the mixture's protein. The color of the supernatant is an indication of the extent to which protein and iron compound have been separated from each other.

The protein can be recovered from the solution in a conventional manner, such as by precipitation, adsorption on voluminous or solid material, evaporation of the solution, drying, concentration of the solution by ultrafiltration and concentration of the solution by freezing. The well-received protein is suitable for iron content, color and taste for many foods.

5 60104

The invention will be illustrated in the following by means of some embodiments.

Example 1 Red blood cell porridge from offspring blood with Na citrate as anticoagulant was treated with ethanol and water said the cell membranes ruptured. The cell membrane fragments were centrifuged off. The thus obtained hemoglobin solution contained 15% dry substance, mainly hemoglobin, 33% by volume ethanol and the rest water, the pH was 5.3 and the color dark red. 6 ml of 96% v / v ethanol was dropped with stirring and cooling in a beaker with 3g hemoglobin solution,

The temperature was -10 ° C. In this hemoglobin suspension, 0.4 ml of 1 M hydrochloric acid in 3 ml of 96% v / v ethanol was added with stirring and cooling. After the addition, the temperature was -10 ° C and the pH was 3.4. The ethanol content of the mixture was 77% by volume.

The brownish-black mixture was centrifuged at 20,000 x g for 10 min to give a black paste with a dry matter amount of 0.08 g and a light brown supernatant.

0.19 ml of 0.5 M sodium hydroxide in 10 ml of 96% by volume ethanol was dropped with cooling and stirring in the supernatant. After the addition, the temperature was -10 ° C and the pH was 4.35. The ethanol content of the mixture was 85% by volume.

The precipitate formed, a fraction of blood protein with adsorbed iron compounds, was centrifuged to give a black and red paste with a dry matter content of 0.08 g.

From the extremely pale yellow colored supernatant, after water addition to water content 50% and pH increase to 7.5 during cooling, a light green precipitate was precipitated which after 0.centrifugation and drying gave 0.22 g of dry substance.

Example 2 3 g of hemoglobin solution (with data as in Example 1) at a temperature of -8 ° C were dropped with stirring and cooling in a beaker with a solution of 0.45 ml of 1 M hydrochloric acid and 10 ml of 96% v / v ethanol at a temperature of -8 ° C. After the addition, the temperature was -8 ° C and the pH was 3.3. The ethanol content of the mixture was 78% by volume.

60104

The tan mixture was centrifuged at 10,000 x g for 10 min to give a black paste with a dry matter content of 0.06 g and a light brown supernatant.

1 ml of a suspension of 0.1 g red blood cell porridge in equal parts ethanol and the water at a temperature of -8 ° C and pH

5.3 was dropped with stirring and cooling in the supernatant. After the addition, the temperature was -8 ° C and the ethanol content was 75% by volume.

The mixture was centrifuged at 10,000 x g for 10 minutes to give a small amount of black paste and a light brown-to-pale yellow liquid. In the same way, in Example 1, a light green precipitate was precipitated from the supernatant liquid which after 0.centrifugation and drying gave 0.25 g of dry substance.

Example 3 3 g of hemoglobin solution (with data as in Example 1) at a temperature of -12 ° C were dropped with stirring and cooling in a beaker with 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 was 2.9. The ethanol content of the mixture amounted to 71% by volume. The brownish-black mixture was centrifuged at 27,000 x g for 10 min at -12 ° C to give a black paste, with a dry solids content of 0.07 g and a light yellow supernatant.

15 ml of 96% v / v ethanol at a temperature of -18 ° C was dropped into the supernatant with stirring and cooling. After the addition, the temperature was -18 ° C. The ethanol content of the mixture amounted to 86% by volume.

After 10 hours at -18 ° C, the mixture was centrifuged as above to give a black and red paste with a dry solids content of 0.08 g.

The extremely pale light yellow colored supernatant was divided into two equal parts.

From one part, a light gray-white precipitate precipitated in the same way as in Example 1 which after 0.centrifugation and drying gave 0.11 g of dry substance.

The second part was ultrafiltered in an Amicon Diaflo cell with membrane PM 10 at -8 ° C. The conontrate was added with 10 ml of water at 0 ° C and filtered. The thus-ethanolated protein solution was neutralized and dried to give 0.06 g of dry sub-substance.

Claims (3)

8 60104 1. A method of separating the iron component from the protein from a mixture containing iron compounds, called the iron component, and blood protein, mainly globin, and the extracts of the hemoglobin, whereby the mixture is added to dehydration agent preferably ethanol. , in such an amount that the total amount of one of the dehydrating agents in the liquid is added up to at least U volume, preferably at least TO volume, calculated on the total amount of liquid, and the pH of the mixture is adjusted to a value lower than k. , preferably at a value of from 2.5 to U, 5, resulting in a precipitation of iron component, which may be characterized in that an agglomeration of the precipitate is achieved in the following way: the pH of the mixture is raised to a maximum of 5, one at the decomposition. cell membrane fragment mass obtained from the blood cells is added celery ethanol to a total content of dehydrating agents of not more than 90% by volume and optionally at the same time the temperature is lowered, whereupon agg the lomeration may be further accentuated by the addition of inorganic or organic salts, such as sodium or potassium salts, e.g. sodium chloride, e.g. in the form of aqueous solutions, and then the agglomerates are separated by the iron component, and the residual globin is recovered in a manner known per se. 2. A process according to claim 1, characterized in that the iron component is separated from the mixture by centrifugation, cyclone procedure, filtration, ultrafiltration, sedimentation or adsorption on voluminous or solid material. 3. A process according to claims 1 and 2, characterized in that the liquid for the ethanol addition and the pH reduction is cooled to a temperature of between -20 ° C and 0 ° C and that this temperature is maintained throughout the whole operation.