EP0042856A1

EP0042856A1 - A method for precipitating protein from an aqueous protein-containing liquid and apparatus therefore

Info

Publication number: EP0042856A1
Application number: EP19810900222
Authority: EP
Inventors: Inger Lise Christensen
Original assignee: Individual
Current assignee: Individual
Priority date: 1980-01-02
Filing date: 1981-01-02
Publication date: 1982-01-06
Also published as: WO1981001943A1

Abstract

La proteine est precipitee a partir d'un liquide aqueux contenant la proteine en mettant le liquide en contact avec un fluide qui ne peut pas se melanger audit liquide, en maintenant le contact dans des conditions suffisantes pour provoquer la precipitation de la proteine contenue dans le liquide, et en separant la phase aqueuse du fluide. Le fluide est notamment de l'huile comestible ou de la graisse fondue maintenue a une temperature elevee, en particulier 70-85 C, sous forme de couches (4) laminaires, et le liquide aqueux contenant la proteine est constitue particulierement par du jus de plante telle que la luzerne et est mis en contact avec la graisse ou l'huile dans la couche (4) en passant goutte a goutte au travers de la couche. Le procede permet une precipitation selective de la fraction de proteines de chloroplaste (proteine verte) (9) ainsi que son extraction efficace du jus vert, ce qui permet d'obtenir un jus blanc (en 19) dont on peut precipiter une proteine blanche convenant a l'alimentation humaine. Un dispositif permettant la mise en oeuvre du procede comprend des moyens (1, 3) pour mettre le liquide aqueux en contact avec le fluide, des moyens (6) pour separer la phase aqueuse du fluide et des moyens (11) pour separer la proteine precipitee. Les moyens de contact comprennent notamment un recipient (3) contenant une couche stationnaire (4) de fluide et un distributeur de liquide (1) servant a faire passer au travers de la couche des gouttelettes et/ou de minces jets d'un fluide non miscible avec le premier fluide.The protein is precipitated from an aqueous liquid containing the protein by bringing the liquid into contact with a fluid which cannot mix with said liquid, maintaining contact in conditions sufficient to cause precipitation of the protein contained in the liquid, and separating the aqueous phase from the fluid. The fluid is in particular edible oil or melted fat maintained at a high temperature, in particular 70-85 C, in the form of laminar layers (4), and the aqueous liquid containing the protein is constituted in particular by juice of plant such as alfalfa and is brought into contact with the fat or oil in the layer (4) by passing dropwise through the layer. The process allows selective precipitation of the chloroplast protein fraction (green protein) (9) as well as its efficient extraction of green juice, which gives a white juice (in 19) from which a suitable white protein can be precipitated. has human food. A device allowing the implementation of the process comprises means (1, 3) for bringing the aqueous liquid into contact with the fluid, means (6) for separating the aqueous phase from the fluid and means (11) for separating the protein rushed. The contact means include in particular a container (3) containing a stationary layer (4) of fluid and a liquid distributor (1) serving to pass through the layer droplets and / or thin jets of a non-fluid. miscible with the first fluid.

Description

A METHOD FOR PRECIPITATING PROTEIN FROM AN AQUEOUS PROTEIN CONTAINING LIQUID AND APPARATUS THEREFORE

The present invention relates to a method and an apparatus for precipitating" protein from an aqueous protein-containing liquid.

_*

In the present context, the term "precipitation" is intended to designate an operation by which protein which is present in. a liquid in dissolved form or in the form of such small particles that it cannot be separated by any filtration or normal industrial cen- trifugation technique, is brought into a form which can be sepa¬ rated by filtration and normal industrial centrifugation techniques . Hence, in its present utilization, the term "precipitation" covers such phenomena as flocculation , coagulation , conglomeration , etc . , provided that these phenomena or combinations thereof will convert the protein contained in the liquid into a flocculate, coagulate, conglomerate, etc. which may easily be separated from the liquid by filtration or centrifugation . A special phenomenon covered by the term "precipitation" in the present context is the attraction of lipophilic proteins to oil droplets, which droplets with attracted protein may, if desired, be separated by centrifugation .

In the present context, the term "protein" is intended to designate not only proteins according to a strict chemical interpretation of that term, but also proteinaceous substances which, in addition to protein-characterizing moieties , may contain other chemical princi¬ ples . One major utilization of the present invention is in the field of separation of proteins and proteinaceous substances from aqueous liquids of vegetable origin, and one typical type of pro- tein precipitated by the process of the invention is the "chloro¬ plastic fraction" of the vegetable protein , that is , a fraction com¬ prising ruptured and unruptured chlorophyll granules and the single components associated therewith , including proteins , hydro¬ carbons , lipids , chlorophyll, carotinoids , etc . In the following specification and claims , this chloroplastic fraction , which has green colour, is termed "green protein" .

OMPI ,. V, IPO •* The main aspect of the present invention concerns the precipitation of protein by heating.

In the known art, the precipitation of protein from a protein-con - taining liquid by heating has been performed by indirect heating or by steam injection.

Precipitation by indirect heating incurs problems involving coagu¬ lation of the proteins on the heating surfaces with consequent reduced heat transfer and destruction of the protein burnt onto the heating surfaces . Precipitation by steam injection introduces large amounts of condensate which, again, requires large amounts of energy for the subsequent removal thereof, and in steam in¬ jection, it is generally difficult to control the temperature. Espe- cially in connection with the utilization of press juice from green plants as protein source (which is a utilization of increasing im¬ portance, also for the production of proteins for human consump¬ tion, considering that compared to any other agricultural utili¬ zation, the highest protein yields per area are obtained by cul- tivation of green plants, e.g. , lucerne or clover), it has been difficult to obtain an effective separation of the green chloroplast protein fraction . The green chloroplast- containing fraction is a valuable product as animal feed, but is not immediately suitable for human consumption because of its bitter taste. If the green protein is not effectively separated from the juice, the quality and pro¬ perties of that fraction of the proteins contained in the juice which is suitable for human consumption, the so-called "white protein" , will be deteriorated. However, the green protein is especially difficult to handle technically as it shows an excessive tendency, even at very moderate temperature, to clot and deposit as a rubber-like substance on heating surfaces , thus virtually rende¬ ring the further use of the equipment in question impossible. It has been attempted to remove the green proteins from vegetable juices by means of various solvents , but this is undesirable be- cause of the necessity of later removing the solvents from the green protein fraction and from the juice from which the green protein fraction has been separated, and because there is a risk of contamination of both fractions with residues of the solvents . Steam injection is not a useful alternative for this particular pur¬ pose as it does not seem possible to control the temperature con¬ ditions generated by the steam injection in such a manner that the green proteins are selectively precipitated, leaving the - white pro¬ tein unprecipitated.

According to the present invention, an effective precipitation of proteins from an aqueous protein-containing solution is obtained under controllable conditions, avoiding deposit problems and per- mitting desired fractionation of the proteins contained in the pro¬ tein-containing liquid. The method of the invention comprises con¬ tacting the aqueous protein-containing liquid with a fluid which is immiscible with the said aqueous protein-containing liquid, main¬ taining the contact under conditions sufficient to cause precipi- tation of protein from the said protein- containing liquid, separating the aqueous phase from the said fluid, and, if desired, separating the precipitated proteins from the aqueous phase and/or the fluid phase.

The aqueous protein-containing liquid subjected to the method of the invention is typically a green juice from vegetables as dis¬ cussed above, but it is also within the scope of the invention to precipitate protein from other aqueous protein- containing liquids where separation or fractionation of protein or a fraction thereof may present similar problems , including fermentation broths con¬ taining microorganisms (bacteria or fungi) , blood, whey, and waste water from starch production and from other processes processing cereals or vegetables .

Aqueous protein-containing liquids for which the method of the invention is particularly suited are liquids where coagulation of protein occurs very readily even at a relatively low temperature such as , e.g. , 40 C .

In the present context, the term "a fluid which is immiscible with the aqueous protein- containing liquid" (in the following often simply termed "fluid" for brevity) is intended to designate a fluid which, when contacted with the aqueous protein-containing liquid under the conditions prevailing during the contact, will not to any substantial extent become dissolved in the aqueous phase of the protein-containing liquid, but will, in contrast, exist as a separate phase different from the aqueous phase of the protein- containing aqueous liquid. The fluid is typically an edible oil or an edible fat which is fluid under the conditions prevailing during the contact, but also fluids (for example, water-immiscible inert non- toxic ingestible oils or fats such as paraffin oil) which may not normally be considered edible are suitable for the purpose of the present invention, provided that they are not of a character which would incur risk of contamination of the end products with con¬ stituents that are physiologically unacceptable.

In the method of the invention, the fluid is suitably maintained at an elevated temperature which is effective to cause the desired degree of precipitation of proteins from the said protein-containing liquid, but typically, the temperature will not be so high as to result in a product having any "burnt" or "fried" character; on the contrary, the product precipitated by the method of the in- vention is preferably denatured to as low a degree as possible, thus rendering the product available for desired subsequent processing. Hence, the temperature of the fluid will normally be below the boiling point of the aqueous protein -containing liquid, and typical temperatures of the fluid are in the range of 60 - 95 C, most often 70 - 85 C. However, depending upon the ratio between the fluid and the aqueous liquid and the character of the liquids to be processed, fluid temperatures which are above the boiling point of the liquid processed cannot be precluded. Typically, the conditions prevailing during the contact will be conditions which are insufficient to cause any substantial boiling of the aqueous liquid.

The conditions sufficient to cause precipitation of protein from the protein-containing liquid are primarily represented by the combi- nation of the temperature of the fluid, the ratio of the amount of fluid to the amount of aqueous liquid contacted therewith, and the time period during which the contact is maintained. However, also other factors will influence the precipitation, such as the pH of

OMPI the aqueous protein-containing liquid, the degree of agitation during the contact, and the affinity between the fluid and the protein . In most embodiments of the invention, it is desired to maintain only a low degree of agitation during the contact in order to allow formation of as large particles or agglomerates of preci¬ pitated protein as possible. The desirable pH of the aqueous pro¬ tein-containing liquid will depend on the nature of the liquid and may vary within relatively wide limits . When the liquid is green juice from plants such as lucerne or clover, a pH in the neutral range (5 - 8) will often be most suitable .

According to a preferred embodiment, the contact between the fluid and the aqueous liquid is performed by maintaining the fluid as a stationary layer in a reaction vessel and passing the protein- containing liquid through the layer dropwise or as a fine jet. This embodiment is illustrated in the drawing and in the examples and is discussed in greater detail below. The layer will tj ically be the top layer of a 2-ρhase system where the lower layer is constituted by an aqueous phase where the protein-containing liquid with precipitated protein collects . In such case, the aqueous protein- containing liquid is suitably contacted with the fluid by being added dropwise or as a fine jet on top of the fluid phase. When the fluid phase is kept under laminar conditions , that is , with slight or moderate agitation, the droplets of the liquid will pass substantially vertically through the layer, and if the walls of the reaction vessel are vertical at the levels where they contact the fluid layer, any contact between the protein-containing liquid and the walls of the vessel is minimized, thus minimizing deposits on the walls . The depth of the fluid layer is adapted to obtain satis- factory precipitation of the desired fraction during the passage of the droplets of the protein -con taining liquid through the layer. Normally, this depth will be between 10 and 50 cm . It is suitable to distribute the droplets of the aqueous liquid substantially uni¬ formly over the surface of the fluid layer. This may be obtained, e . g. , by means of suitable spraying devices or distributing devices with perforated bottoms .

O PI If the fluid has a density which is higher than that of the aqueous liquid, the fluid is suitably arranged on top of a distributing unit generating droplets of the aqueous liquid. The droplets of the aqueous liquid will then pass upwardly through the fluid layer, and the aqueous phase will collect on top of the fluid layer after having passed the layer.

According to another embodiment of the method of the invention, the aqueous protein-containing liquid constitutes a stationary phase through which the heated fluid is passed. When the fluid has a lower density than the aqueous liquid, it is introduced from the bottom of the aqueous phase.

When the contact has been maintained under conditions sufficient to cause precipitation of protein from the protein- containing liquid, the aqueous phase is separated from the fluid. The separation may be performed in a manner known per se for separating phases which are mutually immiscible, including allowing the phases to separate effectively by gravity and withdrawing one of the phases .

Depending upon the character of the precipitated proteins, they will collect in the aqueous phase or in the fluid phase, or in both. In the above-discussed precipitation of green proteins from vege¬ table juice, the predominant proportion of the green protein will follow the aqueous phase, although a minor proportion thereof will sometimes collect in the fluid phase. The separation of the precipi¬ tated protein from the aqueous phase and/or from the fluid phase is performed by filtration, decantation, centrifugation, etc. , in a manner known per se .

In the method of the invention, a very intimate contact between the fluid serving as heating medium and the aqueous protein-con¬ taining liquid is obtained with a minimum consumption of energy. Because the heating medium is a liquid, it is very easy to control the temperature to a level which is optimal for the protein fraction in question . Furthermore, any problems arising from "burning" of proteins onto heating surfaces are obviated as the heat transfer takes place at the interface between the heating fluid and the

OM aqueous liquid. The heat transfer is easy to control, in particular when the heating fluid is maintained as a stationary layer where the temperature and thickness of the layer can be optimally ad¬ justed. In the operation of the method of the invention it has been experienced that the green proteins tend to have a lipophilic cha¬ racter in the contact with the fluid. Thus , when the method is performed by passing droplets of green juice through the fluid layer, the precipitated green protein collects at the interface be¬ tween the _.aqueous droplets and the heating fluid.

When the method is performed by converting a heated fluid, e.g. a heated fat or oil, into microfine droplets and passing the droplets upwardly through an aqueous protein-containing liquid, a product consisting of oil droplets surrounded by attracted proteins will be obtained. This embodiment of the method of the invention will not necessarily incur coagulation of the proteins . Rather, the desired effect in certain cases is an effect where the lipophilic character of the proteins is utilized to "extract" the proteins from the aqueous phase onto or into the oil droplets . The "cream-like" product ob¬ tained in this manner may be used for human or animal consump¬ tion, depending upon the identity of the oil and the proteins . After separation of this cream phase from the aqueous liquid, the cream phase may be used per se, or may be redispersed in water or other suitable liquid. Depending upon the identity of the oil and the proteins in the aqueous protein -containing liquid, it may or may not be necessary to perform any heating of the oil passed into the aqueous liquid .

During recent years , the utilization of protein from microorga¬ nisms , "single cell protein" , has attracted increasing interest. These microorganisms may, e. g. , utilize nutrients in waste water or waste products . It has been a great problem to separate these microorganisms after completion of their cultivation, the reason for this being that the concentration of the microorganisms is so small and/or the microorganisms themselves are so small that normal filtering equipment cannot be used. Furthermore, the difference between the density of the microorganisms and the density of the fermentation liquid in which they are present is usually so small that it is extremely difficult to obtain effective separation by cen¬ trifugation. Such microorganism-containing fermentation liquid is an interesting aqueous protein-containing liquid subjected to the me¬ thod of the present invention .

When the method of the invention is used for precipitating green protein from a green juice, the filtrate, after removal of the pre¬ cipitated green protein therefrom, is a liquid contain a high pro¬ portion of. protein which can be separated and used for human consumption, the so-called "white protein" , which, when uncon- taminated with green protein, is a substantially tasteless and odourless protein of optimum nutritional composition. This white protein can be separated from the filtrate by acidification, heating, etc. , the separation by acidification, e. g. , to pH 3, presently being preferred because it results in a white protein fraction which is easily soluble. The white protein precipitated in this manner may be separated by centrifugation or filtration. The centrifugate or filtrate, the so-called "brown juice" , is extremely suitable as a substrate for fermentation of microorganisms or as an addition to substrates for the fermentation of microorganisms . The brown juice is also useful as a basis for "liquid supplement" (li¬ quid feed for ruminants) .

The method of the invention is particularly attractive for this fractionated precipitation of protein from juice from green plants .

Firstly, when the fluid is an edible fat or oil, the method does not introduce any undesired substances into the protein fractions , considering that any absorbed fat or oil in fact increases the nu- tritional value of the end products . Secondlj*^*, due to the effective temperature control rendered possible through the method of the invention, the lipophilic green chloroplast fraction may be preci¬ pitated at such low temperatures that only a minimum of the white protein fraction is entrained with the green protein fraction. At the same time, the green protein fraction may be removed so efficiently that the white protein fraction will be immediately useful for human consumption. Thus , it is a particular feature of the method of the present in¬ vention that it permits efficient, but selective removal of green protein from green juices , thus permitting the obtainment of both a high yield of green protein, and a high yield of white protein which is substantially uncontaminated with green protein .

In connection with the treatment of green juice, it has been expe¬ rienced that some green juices may have a character which makes them less suitable for the immediate fractionation by the method of the invention. A green juice which is less suitable for immediate treatment by the method of the invention is typically characterized by a somewhat elevated viscosity, but a general indicative expe¬ riment for assessing the suitability for the green juice for treat¬ ment by the method of the invention is to subject the green juice to high speed centrifugation (centrifugation at about 25.000 rpm. ) .

If it is impossible, in such high speed centrifugation, to obtain a clear supernatant, the green juice is indicated as less suitable for treatment by the method of the invention . Such green juice can be rendered suitable for the treatment by the method of the invention by treatment with a base such sodium hydroxide to pH 8 - 10, e.g. for about half an hour, and subsequent readjustment of the pH, that is , to about 6. After such pH cycling, the green juice will be found to give a clear supernatant in the high speed cen¬ trifuge treatment ans will be suitable for the method of the in- vention .

Reference is made to the drawing which diagram atically illustrates an apparatus of the invention for performing the method of the invention .

From a reservoir (not shown) for protein-containing aqueous liquid (the protein- containing aqueous liquid being used as example in this description of the drawing being green juice, for example from lucerne) , the protein-containing aqueous liquid is passed to a liquid distributor 1, exemplified as distributor vessel having a bottom 2 provided with uniformly spaced perforations . From the distributor, the droplets or thin jets passing through the perfo¬ rations enter into a precipitation vessel 3 having an upper part

OMPΓ defined by a vertically extending cylindrical wall and a bottom part defined by a conical wall. The vessel 3 contains an oil phase 4 consisting of, e . g. , vegetable oil or molten lard, etc. , located in the upper part of the vessel. The oil phase is heated to. a suitable protein precipitation temperature by means of a heating and ther¬ mostating unit 24 immersed in the oil phase, e.g. , a thermostating unit with pumping or stirring means for circulating the oil and ope¬ rating at a rate which will not induce turbulent conditions in the oil phase. The oil phase 4 floats on a water phase 5 which, at the upstart of the apparatus, may be tap water, but which eventually will be constituted by the aqueous phase of the droplets having passed the oil phase. Upon passage through the oil phase, the droplets of the juice will substantially entrain precipitated green proteins which will then fall down through the aqueous phase 5 as the droplets "break" substantially immediately upon contact with the aqueous phase. The aqueous phase with precipitated protein is withdrawn, either continuously or at suitable intervals, through a valve 6 and is passed into a sedimentation vessel 7 in which no stirring is performed. In the sedimentation vessel 7, the aqueous phase divides into an upper, substantially clear liquid 8 and a sediment-containing lower aqueous phase 9. The aqueous phase 9 with sedimented green protein is passed through a pump 10 to a separating unit 11 such as a filter, a centrifuge, or a decanter. The protein separated in the separator 11 is passed to a dehy- drator 12, e .g. drum drier, a spray drier, or a fluid bed drier, and is withdrawn as dehydrated green protein concentrate through a conduit 13. The liquid 8 is passed through a conduit 14 to a filter 15 where any content of precipitated protein is separated from the liquid. From the separator 11, the liquid separated from the green protein is passed through a conduit 17 and is combined with the liquid 8 subjected to the filtration . The protein filtered off from the liquid in the filter 15 is passed through a conduit 18 to the pump 10 and is combined with the main sediment of green protein. The filtrate from the filter 15 is withdrawn through a conduit 19 as a white protein solution which can be further pro¬ cessed in a manner known per se to obtain white, protein and brown juice.

-OMPI Depending upon the character of the protein precipitated by the contact with the oil phase 4, the protein may to a higher or lesser degree be retained in the oil phase. For operation with such types of proteins, the apparatus suitably further comprises . separating means (not shown) , such as filters or centrifuges , for separating precipitated protein from the oil phase.

The invention is further illustrated in the following examples . All percentages stated in the examples are by weight. The crude pro- tein contents referred to in the examples were determined by the

Kjeldahl method using a "Kjel-Foss Automatic" instrument from A/S N. Foss Electric, Hillerød, Denmark.

Example 1.

Green juice from lucerne, prepared by desintegration and subse¬ quent pressing in an industrial leaf protein concentrate-producing plant was frozen immediately after preparation and was thawn prior to this experiment. The juice was sprayed dropwise into a vessel containing an aqueous phase of a depth of about 2_ cm and, on top of the aqueous phase, a phase of molten lard of a depth of 12.5 cm. The lard was heated in a thermostat bath outside the reaction vessel, and from this bath, it was continuously recycled into the reaction vessel, thereby maintaining the depth of the lard phase and controlling its temperature to 70 C . The temperature of the juice added was 5 C. After passage of all the juice through the fat layer, the temperature of the water phase was 38 - 39 C . The water phase with precipitated green protein was sucked off and filtered. The filtrate was heated to 58°C which resulted in the precipitation of a voluminous white precipitate. After removal of this precipitate by filtration, the filtrate was heated to about 95 C, and an additional white protein coagulate was obtained.

The above procedure was repeated, but in this case, the white protein was precipitated by passing the filtrate from the removal of the green protein through the lard layer heated to 85 C .

^* gT3REA O PI Example 2.

Fresh green juice, obtained from lucerne by desintegration in a desintegrator and subsequent pressing in a juice press , was used as starting material after storage overnight. The properties of the juice were as ollows :

Dry matter content: 6.73% by weight

Crude protein content: 2.20% by weight pH: about 6.

In a laboratory reaction vessel, 500 ml of tap water constituting a layer of a depth of 3 cm, was kept at 36°C by means of an exterior water bath and was covered with a 13 cm deep layer of edible vegetable oil. The temperature of the vegetable oil was controlled by continuously pumping the oil through a thermostat immersed in the oil phase and was maintained between 63 and 76 C .

385 ml of the green juice at a temperature of 17 C was added dropwise to the oil phase in the course of about half an hour.

After passage of all of the green juice, the aqueous phase contain¬ ing precipitated protein had a temperature of 40 C . 770 ml of the aqueous phase containing protein sediment was withdrawn and was centrifugated at 2000 rpm . for 10 minutes in a laboratory centri- fuge. The sedimented green protein was separated as a green sludge (78.8 g) by this centrifugation .

The green sludge contained 12.8% of dry matter including 5.75% of crude protein. The dry matter content in the green sludge corre- sponds to 28.6% of the total dry matter in the original green juice, and the crude protein content in the green sludge corresponds to 48% of the total crude protein content in the original green juice.

The supernatant was subjected to acid precipitation bj^τ addition of HCl to pH 3 and centrifugation at 2000 rpm . for 10 minutes in a laboratory centrifuge and was thereafter freeze-dried. The yield of the white protein was 3.83 g having a dry matter content of 89.6%, corresponding to 10.5% of the total dry matter content in the ori- ginal green juice. The content of crude protein in the white pro¬ tein was 47.8%, corresponding to 20% of the crude protein content in the original green juice. In the second centrifugation , a super¬ natant of 710 g of brown juice having a dry matter content of 2.8% was obtained. The dry matter content of the brown juice was 60.9% of the total dry matter content of the original green juice . The brown juice contained 0.4% of crude protein, corresponding to 32% of the total crude protein content in the original green juice .

The above experiment was repeated, but with a lower temperature

(52 - 57 C) of the oil phase. From a starting juice having a dry matter content of 5.2% _.and a crude protein content of 1.94%, the following fractions were obtained:

A green sludge containing 32% of the dry matter present in the original green juice and having a crude protein content of 3.7%, corresponding to 46% of the crude protein content in the original green juice,

A "white protein" fraction (slightly green) having a dry matter content corresponding to 12.8% of the dry matter content of the original green juice and a crude protein content of 7.0%, corres¬ ponding to 20% of the crude protein content of the original green juice,

A brown juice having a dry matter content corresponding to 56% of the dry matter content of the original green juice and a crude protein content of 0.47%, corresponding to 34% of the crude protein contained in the original green juice.

Example 3.

Fresh green juice, obtained from lucerne by mincing in a mincing machine and subsequent pressing in a juice press , was used as starting material. The properties of the juice were as follows : Dry matter content: 8.39% by weight

Crude protein content: 3.71% by weight. pH: about 6

In a laboratory reaction vessel, 500 ml of tap water, constituting a layer of a depth of 3 cm, was kept at 36°C by means of an exterior water bath and was covered with a 13 cm deep layer of freshly molten lard having a temperature at 80 C. The temperature of the lard was maintained at 80 C by continuously pumping the lard through a thermostat immersed in the lard phase.

400 ml of the green juice at a temperature of 21°C was added drop¬ wise to the lard phase in the course of about 25 minutes . As soon as the droplets of the juice had passed through the lard phase and entered into the water phase, the droplets "broke" , and the preci¬ pitated proteins from the droplets fell down through the aqueous phase. After passage of all of the green juice, the aqueous phase had a temperature of 43 C and was allowed to stand for one hour for sedimentation. 704.9 g of aqueous phase containing protein sediment was withdrawn and was centrifugated at 2000 rpm for 10 minutes in a laboratory centrifuge. The sedimented green protein was effectively separated as a green sludge (85,9 g) by this cen¬ trifugation .

The green sludge contained 9.4% of dry matter and 4.9% of crude protein. The dry matter content in the green sludge corresponds to 34% of the total dry matter in the original green juice, and the crude protein content in the green sludge corresponds to 44% of the total crude protein content in the original green juice .

The supernatant was subjected to acid precipitation by addition of HCl to pH 3 and standing for 20 hours at 5 C . A pure white pro¬ tein precipiate was obtained. The white protein was separated by centrifugation at 2000 rpm for 10 minutes in a laboratory centri¬ fuge and Λvas thereafter freeze-dried. The yield of white protein was 3.74 g having a dry matter content of 95%, corresponding to 15% of the total dry matter content in the original green juice. The content of crude protein in the white protein was 49.3%, corres¬ ponding to 20% of the crude protein content in the original green juice. In the second centrifugation , a supernatant of 619 g of brown juice having a dry matter content of 1.97% was obtained. The dr ¹" matter content of the brown juice was 51% of the total dry matter content of the original green juice . The brown juice contained 0.56% of crude protein, corresponding to 36% of the total crude protein content in the original green juice.

Examples 4 - 8.

In the same manner as described in Example 2, a series of further experiments were performed. In all the experiments , the green juice added had room temperature, and after passage of all of the green juice through the oil phase, the aqueous phase had a tempe¬ rature in the range of 40 - 44 C. Examples 4 - 8 were performed with green juice from the same batch and illustrate the influence of the oil phase temperature on the yield of white and green proteins. The results appear from Table I below where all percentages are by weight, and where the term "% tot. orig. " designates percent by weight of the total amount of the substance in question in the original fresh green juice:

Table I

Fresh Green White . Brown green sludge protein juice juice

Example 4.. Lard, 60 C

Amount 435 g 51.3 g 3.34 g* 874 g

Dry matter, % 6.73 13.7 95 2.1

Dry matter, % tot.orig. 24.5 11.1 64.4

Crude protein, % 2.2 6.63 45.7 0.36

Crude protein, % tot.orig. 42 18.9 39

*Dis coloured

Example 5. Lard, 66 >-> C _*

Amount 447g 80.0 g 2.75 g* 869 g

Dry matter, % 6.73 13.8 95 2.5

Dry matter, % tot.orig. 31.4 7.5 62

Crude protein, % 2.2 7.26 44.9 0.35

Crude protein, % tot.orig. 58 12.3 30.4

* Discoloured

Example 6. lard 71.5 C

Amount 453 g 57.4 g 2.13 g 899 g

Dry matter, % 6.73 15.2 95 2.2

Dry matter, % tot.orig. 28.5 6.6 64.9

Crude protein, % 2.2 7.75 45.2 0.33

Crude protein , % tot.orig. 53.1 11.4 35

OMF Table I continued

Fresh Green White- Brown green sludge protein juice juice

Example 7. Lard, 76 C

Amount 422 g 89.2 g 4.13 771 g

Dry matter, % 6.73 10.1 95 2.1

Dry matter, % tot.orig. 30.9 13.4 55.7

Crude protein, % 2.2 4.79 38 0.31

Crude protein, % tot. orig. 51.9 19.3 28.8

Example 8. Lard, 80 C

Amount 427 g 77.7 g 2.36 g 806 g

Dry matter, % 6.73 12.1 95 2.0

Dry matter, % tot.orig. 33.8 8.0 59.3

Crude protein, % 2.2 6.14 42 0.30

Crude protein, % tot.orig. 58.5 12.2 29.3

OMPI IP

Claims

Claims .

1. A method for precipitating protein from an aqueous protein-con¬ taining liquid, comprising contacting the aqueous protein-contain- ing liquid with a fluid which is immiscible with the said aqueous protein-containing liquid, maintaining the contact under conditions sufficient to cause precipitation of protein from the said protein- containing liquid, separating the aqueous phase from the said fluid, and, if desired, separating the precipitated proteins from the aqueous phase and/or the fluid phase.

2. A method according to claim 1 wherein the protein-containing liquid is selected from the group consisting of green juice from plants , fermentation liquids containing microorganisms, blood, whey, and waste water from the processing of cereals or vege¬ tables .

3. A method according to claim 1 or 2 wherein the fluid is an edible oil or molten fat.

4. A method according to any of the preceding claims where the contact is maintained under conditions which are insufficient to cause any substantial boiling of the aqueous liquid.

5. A method according to claim 4 wherein the fluid has a tempe¬ rature below the boiling point of the aqueous liquid.

6. A method according to claim 5 wherein the fluid has a tempe- rature in the range of 60 - 95 C .

7. A method according to claim 6 wherein the fluid has a tempe¬ rature of 70 - 85°C .

8. A method according to any of the preceding claims in which the contact is maintained under conditions which will precipitate a fraction of the protein contained in the liquid, but which will not precipitate all of the protein contained in the liquid .

OT.-I

9. A method according to claim 8 wherein the fluid is green juice from plants , and the contact is maintained under conditions which will precipitate the green protein contained in the juice, but which will not precipitate the white protein contained in the juice.

10. A method according to any of the preceding claims wherein the fluid which is immiscible with the aqueous protein-containing liquid is maintained as a layer in a vessel, and the protein-containing liquid is contacted therewith by being passed dropwise through the said layer.

11. A method according to claim 9 wherein the fluid layer is kept under laminar conditions .

12. A method according to any of the preceding claims wherein the precipitated protein is dehydrated subsequent to the separation.

13. A method according to any of claims 9 - 12 wherein the white protein is precipitated from the juice after the separation of the green protein from the juice.

14. A method as claimed in any of claims 1 - 5 in which the con¬ tact between the aqueous protein-containing liquid and the fluid immiscible therewith is obtained by passing droplets of the fluid through a layer of the aqueous protein-containing liquid.

15. An apparatus for precipitating protein from an aqueous pro¬ tein-containing liquid, comprising means for contacting the aqueous protein-containing liquid with a fluid which is immiscible with the said aqueous protein-containing liquid, means for separating the aqueous phase from the said fluid, and optionally means for sepa¬ rating the precipitated proteins from the aqueous phase and/or the fluid phase.

16. An apparatus according to claim 15 wherein said contacting means comprise a vessel containing a stationary layer of a fluid and a liquid distributor for passing droplets and/or fine jets of a fluid immiscible .with the said first fluid through said layer.

CΪ.-PI