Method for Processing Plant Protein

In a broad sense all green plants having photosynthetic activity are considered suitable for processing according to the invention. The protein content thereof after isolation and, in given cases, after concentration can be utilized on a commercial scale.

The difficulty in recovery of green plant protein is in the separation of fractions having different value from accompanying substances and the adaptation of the valuable fractions for human or animal consumption. There is therefore a need to provide more economical methods on an industrial scale to improve the separation of green plant proteins.

It is known that the pressed juice obtained via pressing of green plants contains protein fractions coagulable under heat treatment and having different values. In the framework of these methods a separation of the chloroplastic and more valuable cytoplasmic protein fractions represents a rather complicated task. An improvement in the separation, however, is of decisive importance because the cytoplasmic protein fraction contains the most valuable protein fractions inherent in the plant material. These are not bound to chloroplastids and contain only low amounts of lipids and mineral accompanying substances. The cytoplasmic protein fraction is therefore suitable for use in human consumption because of its lack of odorous and aromatic substances which provide for a more appealing and therefore edible fraction. A main task in the processing of green plant protein is to increase the ratio of cytoplasm protein fraction to the amount of the protein available.

The chloroplastic protein fraction contains the major part of the minerals present in the accompanying substances, the major part of the lipid content, and practically all of the lipid soluble pigment substances of which chlorophyll, xantophyll and beta-carotene are the most significant. The processing of the latter protein fraction represents an independent technical task in the working up of the green plant protein. On the one hand, the separation of the accompanying substances from the protein which are present in the chloroplastic protein fraction should be solved in order to increase the protein content and to improve the quality. On the other hand, the processing of the lipid-soluble dye substances, the pigments for foddering purposes, should be elaborated. The importance of pigment substances has recently increased in closed livestock farming because the pigments are indispensable components in breeding certain kinds of animals in order to bring about their natural color.

The complicated process of fractionating by heat treatment for the separation of chloroplastic and cytoplasmic protein fractions has been suggested. It is, however, disadvantageous because the two fractions having different value but physically very analogous properties cannot be separated sharply. In the course of the heat treatment a part of the protein precipitate is damaged and the pigments and other accompanying substances in chloroplast fraction which detrimentally influence the properties necessary for human consumption, cannot be removed. Different organic solvents or a combination thereof have been proposed for the extraction of the pigments present in the chloroplastic protein fraction. The hitherto known processes, however, can be realized only on a laboratory scale.

An object of the present invention is to increase the amount of the cytoplasmic protein fraction which can be obtained on processing green plant protein from the pressed juice via pressing; a treatment of the chloroplastic protein fraction to improve the separation of pigment substances, and an improvement in the efficiency of the extraction, an improvement of the properties of the chloroplastic protein fraction which render the fraction more appealable for human consumption--after--fraction.

Summing up an increase in the ratio of the green plant protein inside the total protein mass having a quality identical to the cytoplasmic protein fraction on processing a given plant protein is a main object of the invention. A further important object is the processing of the pigment substances which can be obtained from the chloroplastic fraction via solvent extraction into a pigment concentrate suitable for industrial purposes.

According to the present invention for processing green plant protein, especially for the improvement of separation and purification of obtainable protein substances the following measures should be taken:

(a) the chloroplastic protein fraction is flocculated in the presence of inorganic agents promoting the flocculation at a temperature below 50.degree. C. from the pressed juice which is obtained via pressing of the green plant material and is separated preferably in two steps;

(b) the extent of the heat treatment necessary to coagulate the protein and therewith effect the separation of the chloroplastic and cytoplasmic protein fractions is controlled by measuring the pigments concentration of the juice flowing off the separator with light absorption method;

(c) the separated chloroplastic protein fraction is treated in an aqueous suspension with a protease enzyme, if desired, after a treatment with a polar solvent;

(d) the chloroplastic protein fraction treated with the enzyme is extracted in a wet state with a polar solvent or a mixture of said solvents, the solvent is recirculated after regeneration into the process;

(e) the extracted pigment and lipoid concentrate is utilized after mixing it with an inert carrier or a carrier having a stabilizing effect;

(f) after separation of the chloroplastic protein fraction the remaining pressed juice containing the cytoplasmic protein fraction is treated to coagulate the protein at about 80.degree. C. or the precipitation of the is carried out by treating the juice with a solvent;

(g) the coagulated proteins precipitated are utilized separately or mixed with each other after drying as animal fodder or for human nourishment.

The juice obtained by pressing the plant material is either heated to certain temperatures or treated with inorganic flocculation agents, such as the calcium, aluminum and iron salts or the hydroxides respectively, which are applied based on the green plant juice in an amount not exceeding 70 ppm or preferably based on the dry matter content of the pressed juice in an amount not exceeding 800 ppm to coagulate the chloroplastic protein fraction. The coagulated chloroplastic protein fraction is separated preferably in separators coupled in series. The first stage of the two-step-process is carried out in a decanter type separator, the second step in a self-discharging disc separator. In addition, there is a possibility to backfeed the separated dilute sludge into the first stage thereby ensuring that a sludge discharge should occur only in the first stage. Completing the separation of the chloroplastic protein fraction in two steps the chloroplyll content of the juice flowing off is decreased to a negligible extent by measuring the light absorption value of the juice with a photometer inserted in the take-off pipeline of the second separation stage. Thus the time-period necessary for the heat treatment can be adjusted. The separated chloroplastic protein fraction is treated in an aqueous suspension in an alkaline or acidic medium with a proteolytic enzyme. In case the pigment fraction is isolated the protein precipitate is treated in a mildly alkaline medium with an alkaline protease which is a protease prepared with fermentation from a fungus or a bacterium. Another variation of this method is that the precipitated chloroplastic protein reaction is extracted at once, in still wet state, with polar solvents and after extraction is treated in a mildly acidic medium with pepsine or papaine, or with a fungus bacterial protease functioning in acidic medium. The enzymatic treatment is realized in a suspension having a dry matter content of about 15-25% by weight and is continued until at most 5% by weight of the protein value present is decomposed. The solvent applied in the extraction is methanol, ethanol, propanol, isopropanol, acetone, methyl ethyl ketone or dimethyl sulfoxide. After separation of the lipid soluble pigment fraction with extraction, the solvent is regenerated from the extraction liquid through an atmospheric distillation, as a rule in the presence of an antioxidant, the remaining pigment concentrate is in turn dried, if desired, after mixing with an edible consumable carrier. It is important to start the extraction always with a mixture of solvent and water, the starting ratio of water and solvent being at least 80:20% by volume. This ratio is adjusted by continuously increasing the solvent ratio and decreasing the water content.

After separating the chloroplastic protein fraction the remained pressed juice is treated at about 80.degree. C. to coagulate the remaining proteins. The coagulation can be carried out by treating the mother liquor, with a solvent. The mother liquor, however, is evaporated to 1/3 to 1/4 part of its original volume prior to solvent coagulation.

The extracted or enzymatically treated extracted chloroplastic protein with respect to its edible properties. comes near to the quality of the cytoplasmic protein fraction consequently, if desired, it can be utilized in admixture with a cytoplasmic protein fraction in the practice.

It has been recognized that the coagulation of the chloroplastic protein fraction can be improved and the applied temperature can be lowered by using flocculation agents. This makes possible the separation of the two protein fractions to a sharper extent and the chloroplastic protein fraction coagulated at a lower temperature is more suitable for concentration and for removing the accompanying substances. The normally difficult extraction of pigment substances can be made easier if the precipitated chloroplastic protein fraction is treated enzymatically. The enzymatic treatment modifies the structure of the composite protein and the treated precipitate still in a wet state can be extracted more easily and with a smaller solvent quantity than otherwise. The coagulation of the chloroplastic protein fraction at a lower temperature can be considered surprising because the applied flocculation agents exert their effect selectively and the ratio of the cytoplasmic protein fraction inside the total quantity of the protein mass can be increased thereby. According to the invention a coagulation of the cytoplasmic protein fraction can be hindered at a lower temperature. The favourable effect, however, is due not only to the action of the flocculation agents but to the fact that the separation process can be followed according to the invention by measuring the pigment concentration in a physical way by means of the light extinction of the juice flowing off the separator.

According to our experience on processing the same green plant a substantial shift in favour of one protein fraction can be attained within narrow temperature limits, for example, applying the same coagulation time, e.g. a pressed juice of Spinacea oleacea green plant with a dry matter content amounting to 8% by weight is heat treated at 48.degree. C. and 54.degree. C. for 120 seconds. The relative weight percent of the chloroplastic protein fraction based on the total amount of the protein recovered via coagulation is at 48.degree. C. 75.3% by weight, at 53.degree. C. already 80.0%. by weight, and, naturally, by an extent proportional to these amounts the ratio of the more valuable cytoplasmic protein fraction is decreased. These experimental data show therefore that an increase of the cytoplasmic protein fraction depends on the temperature used on coagulating the chloroplastic protein fraction. At 46.degree. C., the chloroplastic fraction cannot be precipitated to a whole extent therefore the flocculating agents according to the invention are used for promoting the precipitation.

Other experimental data prove that on processing other green plant material, as Spinacea, if the coagulation is carried out at 46.degree. C. then about 80% by weight of the total protein amounted is precipitated in form of the chloroplastic protein fraction. Carrying out, however, the coagulation in the presence of a 0.2% by weight aqueous AlCl.sub.3 and/or calcium hydroxide solution, the amount of the chloroplastic protein fraction is decreased from 80.0% by weight to 74.0 and 64.0% by weight respectively, that is, the amount of the more valuable cytoplasmic protein fraction can be increased at the expense of the chloroplastic fraction by coagulating this fraction at a lower temperature.

Further important result is that on treating the chloroplastic protein fraction enzymatically the dye substances characterstic to this fraction and other components influencing unfavourably the edible properties of the protein can be removed in a more complete manner. Thus the quality of the chloroplastic protein fraction comes near to the favourable properties of the cytoplasmic fraction after the enzymatic treatment and extraction.

The process of the invention is illustrated further on basis of the following Examples:

1000 kg of alfalfa (Medicago sativa) at the beginning of the green bud state having 28% by weight of dry matter and 4.8% by weight protein content was disintegrated. After disintegration the ground product was pressed in a continuous screw press at first under a pressure not exceeding 15 atm. thereafter at 80 atm. As a result of the pressing 800 kg of pressed juice with 1.0 weight % dry matter content was obtained which had a crude protein content based on dry matter amounting to 36%.

600 ppm calcium hydroxide per kg dry matter content of pressed juice was added in form of a saturated aqueous solution to the juice and was heated to 46.degree. C. The heating was effected by injecting steam with 2 atm pressure. The injection of steam was controlled by the temperature of the juice removed from the system. The chloroplastic protein precipitate was separated quantitatively and a fluffy precipitate was formed. The obtained sludge was separated in two steps. In the first step the sludge was conducted in a screw separator of decanter type where the bulk of the coagulated protein fraction was separated. The juice leaving the separator containing 0.5-1.0 volume % dilute sludge was added directly into a self discharging disc separator which is the second apparatus of the separation unit. The second juice is led off the separator under pressure in a way that the light extinction value of the clear juice leaving the separator was measured in 1:1 dilution with a photometer at 600 nm wavelength visible light with the aid of cuvettes of 1 cm size. The discharge time periods of the separator was controlled in a manner that between two discharge periods the sludge was filled not more than 50% of its whole volume with juice. In the pipeline of the cleared juice containing chlorophyll an inserted photometer measures the reduction of the light intensity. In the presence of chlorophyll, therefore, the photocurrent intensifies and at an extinction value higher than 0.05 (at wavelength 600 nm) this photometric regulation decreases the efficiency of the feeding pump of the separator unit and simultaneously a forced discharge takes place in the second separator. Thus the separation of the chloroplastic protein fraction can be controlled accurately.

The dilute sludge obtained periodically in the second separator has an increased mother liquor content therefore it is recirculated into the buffer tank which is placed before the feeding pump of the separator unit. Sludge is removed only from the first decanter separator with a controlled water content. The sludge obtained from the first separator is adjusted to a dry matter content of 30.0 to 35.0 % whereas the dry matter content of the sludge obtained from the disc separator was 10 to 15% by weight.

The chloroplastic protein fraction separated in the above manner amounts to 142 kg with a 30% by weight dry matter content. 45.0 mg of butyl hydroxy anisole is added to each dry matter kg of the chloroplastic protein fraction and after homogenization the obtained product is dried in a flash type drying apparatus, inlet air temperature 250.degree.-280.degree. C. and outlet air temperature 105.degree.-110.degree. C.

The weight of the dried product amounts to 45 kg and had a following composition:

After the separation of the chloroplastic protein fraction the obtained purified juice was heated with direct steam injection (2 atm) to 80.degree. C. and the coagulated cytoplasmic protein fraction is separated from the juice in an apparatus identical with the separator for chloroplastic protein fraction. The amount of the protein precipitate in wet state separated in two steps amounts to 45 kg, had a yellowish white colour and a dry matter content of about 30% by weight. The drying was effected in a flash drier apparatus inlet air temperature 220.degree. C., outlet air temperature 105.degree. C. The formed 14.5 kg of cytoplasmic protein fraction had a following composition:

Alternatively the separation can be carried out in one step in a decanter separator. The yield of the cytoplasmic protein fraction is then decreased.

The utilization of the chloroplastic protein fraction is advantageous e.g. in the foddering of egg-laying poultry where the protein demand is limited and thus a complete substitution of carotenoid pigments is possible. The cytoplasmic protein fraction is suitable without any limitation to substitute optional protein carrier. After refinement, utilization in human nourishment is possible as well.

After separation of the chloroplastic and cytoplasmic protein fractions--the weight of the condensed steam included--690 kg of mother liquor is formed. This was evaporated in a multistage vacuum evaporator until a dry matter content of about 50% by weight of the residue and this concentrate can be mixed in a proper proportion to the fibrous presscake prior to drying thereof.

100 kg of alfalfa was processed according to Example 1. The chloroplastic and cytoplasmic protein fractions were separated.

The green sludge isolated at the first separator of the chloroplastic protein fraction separation is contacted in a stirring apparatus coupled to the decanter with a solvent. A fivefold amount (220 kg) of acetone and 25.0 mg/kg dry matter butyl hydroxy toluene is added to the sludge based on its dry matter content in a ratio proportional to its formation rate. The wet sludge is stirred for 15 minutes with solvent, thereafter in the decanting separator the sludge was isolated. The extraction liquid amounting to 265 kg is conducted to a tank and the residue free from solvent amounting to 100 kg was suspended in 200 liter of water and the pH value thereof was adjusted to 1.8 and heated to 37.degree. C. 1 g of crystalline pepsine was added to the mixture and treated therewith for 3 hours. Thereafter the sludge was separated in a decanter separator and the mother liquor, in a nearly neutralized state was intermixed with dilute juice to be vacuum evaporated. 90 kg of sludge was extracted with 2.times.150 kg of acetone. After the extractions took place the solvent was separated and after regeneration was recirculated into the process. The extraction residue was washed free from the solvent and dried in a flash drier apparatus air inlet temperature 220.degree. C. and air outlet temperature 105.degree. C.

The product weighing 31 kg is pale brownish-yellow with the following composition:

265 kg of extraction liquid is conducted into a distilling apparatus and 2.0 g butyl hydroxy toluene is added based on 10 kg of dry matter content. The solvent was distilled at atmospheric pressure and thus regenerated. The regenerated acetone was recirculated into the extraction step. 25 kg of distillation residue was adjusted with sodium carbonate to 7.5 pH value and was mixed in a homogenizing apparatus with 5 kg of dextrine, 0.1 kg of sodium alginate and 0.9 kg of gelatine containing solution.

The homogenized mixture was dried in a spray drier apparatus furnished with a spray nozzle, air inlet temperature 200.degree. C. and air outlet temperature 105.degree. C. The composition of the obtained product weighing 14 kg was the following:

After separation of the cytoplasmic protein fraction the obtained mother liquor was processed according to Example 1.

The extracted chloroplastic protein fraction is suitable to substitute in animal foddering with an optional protein carrier in any quantity and using in foddering optional animal species.

The cytoplasmic protein fraction can be utilized as well without limitation as a substitute for any protein carrier for human nourishing purposes. The chloroplastic protein concentrate prepared by enzymatic hydrolysis combined with extraction can be dried together with the cytoplasmic protein fraction and the two fractions can be used in combination. The pigment concentrate is suitable for substituting carotenoid pigments in broiler and egg-layer poultry fodder and can be employed independently from the chloroplastic protein fraction in animal breeding.

1000 kg of Amaranthus mantegazzianus having a dry matter content amounting to 18.0% by weight and a crude protein content of 3.9%.

The chloroplastic protein fraction was precipitated according to Example 1. 47 kg of wet sludge was diluted with water to 100 kg total weight. The temperature of the mixture was adjusted to 40.degree. C. and the pH value thereof with sodium hydroxide to 7.8. 32 g of a suspension of an alkaline protease enzyme having an Anson-activity of 4-5 was added to the mixture and mixed for 30 minutes. After the protease treatment the pH value was adjusted with a mineral acid to 4.5 and the mixture was separated. As a result of the separation 42 kg of sludge and 60 kg of mother liquor was obtained, the latter being processed according to Example 1.

The sludge was extracted three times with 100 kg of isopropanol. The extraction liquids obtained in each extraction step were united, the solvent was distilled off and recirculated into the process. The distillation residue was processed according to Example 2, as additive 2 kg of dextrine, 0.1 kg of sodium alginate and 0.5 kg of gelatine were employed dissolved in a solution. The obtained product amounted to 8 kg and contained 1600 mg/kg of beta-carotene and 3500 mg/kg of xantophyll.

The extracted residue was united with the cytoplasmic protein fraction. This fraction was precipitated by means of treating it with a solvent.

The separated juice obtained after removing of the chloroplastic protein fraction was evaporated in a vacuum evaporator until 30% by weight dry matter content and the concentrate thus obtained weighing 100 kg was added together with the extracted chloroplastic protein fraction into 150 kg of isopropanol and stirred for an hour. The mixture was thereafter separated, washed twice with water and the solvent and aqueous solvent were distilled at atmospheric pressure. The regenerated solvent was backfed into the process. The protein precipitate washed with water free from solvent and was dried. The weight of the protein concentrate was 27 kg, with the following composition:

The preparation steps of the pigment concentrate were combined to obtain a product suitable to intermix it with the usual fodders and to obtain a product having satisfactory shelf life. The carriers of the pigment concentrate were selected and the drying of the suspension prepared with these carriers and the pigments was controlled in a manner that the dried endproduct should be free-flowable with a proper size distribution. In the course of the preparation of the concentrate such measures were carried out that the surface of the prepared granules should be covered with a gastight material layer that is a product in a quasi-capsulated state was prepared. This gastight layer prevents the penetration of the oxygen into the body of the product and ensures and increases protection in addition to the use of an antioxidant.

The proper additives satisfying the above requirements are dextrine, gelatine, sodium caseinate and sodium alginate. Using these additives the pigment concentrate can be obtained in a quasi-capsulated form. Another advantage of these additives is that they form without vacuum evaporation a sludge having proper viscosity which can be dried into a product having a preferable size distribution by means of a spray drier furnished with a spray nozzle or by means of a centrifugal spray drier.

The sludge prepared for spray drying is adjusted to a dry matter content amounting to 30-40% by weight. The viscosity of the suspension obtained is about 28.degree.-32.degree. E (Engler Grade) (at 40.degree. C.). This suspension obtained was dried with a spray drier furnished with a single or two nozzles, an inert gas was used for spray drying having a pressure of 2.0-2.5 atm. In case a centrifugal spray drier is used then the inlet air temperature was 210.degree.-230.degree. C. and the outlet temperature 100.degree.-105.degree. C.

The size distribution of the dried product was that it passed in its bulk through a sieve having a 0.2-0.35 aperture size. The pigment concentrate is stable, stored at room temperature far more then a year less than 10% by weight of its original carotenoid pigment content was lost.