WO2011078671A1

WO2011078671A1 - Process for isolating a dechlorophylllized rubisco preparation from a plant material

Info

Publication number: WO2011078671A1
Application number: PCT/NL2010/050881
Authority: WO
Inventors: Fred Van De Velde; Aart Cornelis Alting; Laurice Pouvreau
Original assignee: Nizo Food Research B.V.
Priority date: 2009-12-22
Filing date: 2010-12-22
Publication date: 2011-06-30

Abstract

The invention relates a process for the isolation of RuBisCO (ribulose-1,5-bisphosphate carboxylase oxygenase) from a plant material said process comprising: a.lysing the plant material to release RuBisCO and chlorophyll from the plant cells; b.separating the lysed plant material into a liquid juice and a high solids slurry or a high solids cake, said liquid juice containing RuBisCO as well as chlorophyll; c.contacting the liquid juice with activated carbon to adsorb chlorophyll onto the activated carbon; d.separating the dechlorophyllized liquid juice from the chlorophyll-loaded activated carbon; and e.further processing the dechlorophyllized liquid juice to produce the dechlorophyllized RuBisCO preparation. This present process yields a protein preparation that is essentially free of chlorophyll and in which the bulk of the RuBisCO is undenatured. The invention further provides a particulate dechlorophyllized RuBisCO preparation that is obtained by the aforementioned process, said preparation having a weight averaged diameter of 10-1000 µm; containing at least 30 wt.% of undenatured RuBisCO and containing less than 0.1% of chlorophyll by weight of RuBisCO.

Description

PROCESS FOR ISOLATING A DECHLOROPHYLLIZED RUBISCO PREPARATION FROM A PLANT MATERIAL

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the isolation of RuBisCO (ribulose-1,5- bisphosphate carboxylase oxygenase) from a plant material. More particularly, the present invention relates to such an isolation process that yields a substantially dechlorophyllized RuBisCO preparation.

The invention also provides a particulate dechlorohyllized RuBisCO preparation that can be produced by the aforementioned process. Also provided is a method of preparing a product selected from a foodstuff, a beverage, a pharmaceutical product, a nutrional supplement and animal feed, said method comprising incorporating the aforementioned particulate RuBisCO.

BACKGROUND OF THE INVENTION

RuBisCO is an enzyme that plays a key role in the Calvin cycle that takes place in chloroplasts of photosynthetic organisms. RuBisCO catalyzes the first major step of carbon fixation, the process by which the atoms of atmospheric carbon dioxide are made available to organisms in the form of energy-rich molecules such as sucrose. RuBisCO catalyzes either the carboxylation or the oxygenation of ribulose-1,5- bisphosphate (also known as RuBP) with carbon dioxide or oxygen. RuBisCO is very important in terms of biological impact because it catalyzes the most commonly-used chemical reaction by which inorganic carbon enters the biosphere. RuBisCO is also the most abundant protein in leaves, and it may be the most abundant protein on earth.

Up to 50% of the total protein in green leaves consists of RuBisCO. In C3 plants RuBisCO molecules are found densely packed within chloroplast stroma at concentrations up to 300 mg/mL. The oligomeric protein (MW 550,000) is composed of eight large and eight small subunits which combine to form a compact, nearly spherical molecule.

RuBisCO is easily purified from leaf extracts in the laboratory but, as yet, there is no simple, economical method for isolating large (kilogram) quantities of the plant protein. The protein has potential as an ingredient in e.g. food and beverages. The use of RuBisCo as a food protein has been investigated by Barbeau and Kinsella (Food Review International (1988) vol. 4, no. 1 , 93-127). These authors showed that the essential amino acid composition of RuBisCO is similar to that of other food protein and that it is compatible to the levels recommended by the FAO/WHO for human consumption:

Highly purified RuBisCO is a tasteless, odorless, white powder with a nutritive value reported to be equal to or superior to that of other food proteins. RuBisCO also possesses some desirable functional properties which might enable food processors to successfully incorporate the protein into a number of different food systems. However, more research is needed before RuBisCO becomes an acceptable substitute for other animal or plant proteins.

In the patent literatures processes designed for large scale isolation of RuBisCO from plant parts, notably plant leaves, have been described.

US 3,959,246 describes a process for isolating RuBisCO, which comprises:

• pressing green leafy vegetable material to separate the juice therefrom,

• heating the juice to a temperature of 55-70 °C in a period of 0.6 seconds or less, holding the juice at said temperature for a period of 0-40 seconds, and rapidly cooling it to a temperature of 40-45 °C, whereby to form an agglomerate containing chloroplastic proteins, chlorophyll, carotenoids, and lipids while retaining cytoplasmic proteins in solution in the juice,

• separating said agglomerate from the residual juice,

• cooling the said residual juice to a temperature of 0-5 °C and acidifying it to a pH of 3.5 to 5.5, whereby to precipitate the protein therein,

• separating the precipitated protein from the residual juice, and

• neutralizing the separated protein to a pH of 7.

US 4,006,078 describes process for isolating RuBisCO, which comprises:

• providing a juice containing soluble proteins from green leafy vegetable material, which juice has been treated to remove chloroplastic proteins, chlorophyll, carotenes, and lipids,

• concentrating said juice by applying it under pressure against a membrane which will retain desired proteins having a molecular weight greater than about 20,000- 100,000 but will allow substances of lesser molecular weight to pass therethrough,

• purifying said concentrate by applying it under pressure against a membrane which will retain desired proteins having a molecular weight greater than about 20,000- 100,000 but will allow substances of lesser molecular weight to pass therethrough, the volume of said concentrate being maintained constant by addition of water thereto,

• acidifying said purified concentrate, at ambient temperature, to a pH of 3.5 to 5.5, whereby to precipitate the desired proteins therefrom, and

• separating the precipitated protein from the residual juice.

In the aforementioned process a fraction containing chloroplastic proteins, chlorophyll, carotenoids, and lipids is selectively removed by adjusting the pH of the press juice to a level of 6-7 and then subjecting it to a special heat treatment. Thus, the juice is brought to a temperature of 55-70 °C by application of very rapid heating, i.e., so that the said temperature is reached in 0.6 sec. or less. When the juice has been brought to said temperature, it is held thereat for 0 to 40 seconds and is then rapidly cooled to 40-45 °C. By such treatment there is formed an agglomerate of the chloroplastic proteins, chlorophyll, carotenoids, and lipids which is removed by centrifugation. US 4,268,632 describes a process for obtaining RuBisCO from plant material comprising the leaves of green plants comprising the steps:

• converting the leaves to a pulp comprising a mixture of a solid portion and a liquid portion, said liquid portion containing dissolved RuBisCO;

· heating the liquid portion to a temperature below that at which protein in the liquid portion denatures for a time sufficient to induce the RuBisCO to crystallize when said liquid portion is cooled; and

• cooling said liquid portion to a temperature at which said RuBisCO crystallizes.

It is observed in US 4,268,632 that the heat treatment also causes partial coagulation of the pigmented material which facilitates its removal during further processing e.g. by moderate centrifugal force. It is further said that it is preferred to use a temperature of at least about 48 °C as below that temperature the green pigmented materials do not coagulate sufficiently to permit their easy removal. Best results are said to be obtained by heating the liquid portion to 50 ± 1 °C for 15-20 minutes.

US 4,334,024 describes a method for preparing crystalline RuBisCO from plant material comprising the steps of:

• grinding a sample of said plant material with a suitable buffer solution;

• filtering said solution;

• adding to said solution, while stirring, 5-15% (w/v) of polyethylene glycol having a molecular weight in the range from 5000 to 7000;

• discarding the precipitates formed;

• storing said solution for about 1-8 hours;

• collecting and washing the crystals resulting from said storage; and

• lyophilizing said crystals.

US 4,588,691 describes a method for preparing ribulose 1, 5-bisphosphate carboxylase (RuBisCO) from plant material, said method comprising:

a. comminuting the plant material in an aqueous solution to form a suspension;

b. fractionating the suspension to release the RuBisCO from the comminuted plant material into the suspension;

c. adding a sufficient amount of polyethylene glycol to the suspension so that crystals of RuBisCO are selectively formed;

d. separating the crystals from the suspension, leaving a supernatant; e. adjusting the pH of the supernatant to above 10 to cause precipitation of

phosphorylated sugars;

f. separating the phosphorylated sugars from the supernatant; and

g. recycling the supernatant from step (f) to provide at least a portion of the

polyethylene glycol required in step (c) and repeating step (d) as often as is required to isolate substantially all of the RuBisCO crystals from the supernatant.

Also the scientific literature describes methods for separating protein fractions from plant material. Lamsal et al, Transaction of the ASAE 46(3): 715-720 (2003) describe a process for isolating protein fractions from alfalfa juice, said method comprising maceration, pressing in a screw press, heating to 55°C for 5 minutes under stirring to produce chlorophyll containing aggregates, followed by centrifugation and separation of a soluble juice fraction. This article also describes an alternative method in which, instead of heat treatment, pH reduction by fermentation is used to coagulate green proteins.

Yang et al. J. Agric. Food Chem. (52) 2223-2225, (2004) describe a process for isolating protein from spinach leaves comprising:

• homogenization of fresh spinach leaves with an equal amount of aqueous 0.003N NaOH at 5 °C;

• filtration of the homogenate (pH 11) through two pieces of gauze;

· centrifugation at 13,500 g for 50 min. at 5 °C;

• filtering of the supernatant through filter paper;

• adjustment of pH to 4.5 to precipitate protein;

• successive washing of the precipitate with acetone, ethanol and diethyl ether on a glass filter until the filtrate is colorless.

The above referenced methods for isolating RuBisCO from plant material suffer from a number of drawbacks. First of all some of these methods yield RuBisCO preparations that have a greenish color due to the presence of appreciable levels of chlorophyll. RuBisCO preparations that such a greenish color are generally unsuited for application in foods and beverages.

Other drawbacks of the prior art methods reside in the fact that they rely on precipitation of RuBisCO using organic solvent (polyethylene glycol) and/or acidification. A disadvantage of precipitation is that the precipitated protein usually needs to redissolved for further processing and that it may cause denaturation/aggregation of the protein, thereby lowering its solubility.

SUMMARY OF THE INVENTION

The inventors have designed a simple and robust process for isolating RuBisCO from a plant material that yields a protein preparation that is essentially free of chlorophyll. The process according to the present invention uses as starting material a plant material comprising intact plant cells that hold chlorophyll-containing

chlorop lasts and comprises the steps of:

a. lysing the plant material to release RuBisCO and chlorophyll from the plant cells; b. separating the lysed plant material into a liquid juice and a high solids slurry or a high solids cake, said liquid juice containing RuBisCO as well as chlorophyll;

c. contacting the liquid juice with activated carbon to adsorb chlorophyll onto the activated carbon;

d. separating the dechlorophyllized liquid juice from the chlorophyll- loaded activated carbon; and

e. further processing the dechlorophyllized liquid juice to produce the

dechlorophyllized RuBisCO preparation.

The inventors have unexpectedly discovered that activated carbon very effectively adsorbs the chlorophyll that is contained in the aforementioned liquid juice. Furthermore, the chlorophyll- loaded activated carbon can easily be removed from the liquid juice by means of simple solid- liquid separation techniques, such as decanting. Thus, the present process can easily be scaled up to an economically feasible production process that yields a dechorophyllized RuBisCO preparation that, due to the virtual absence of chlorophyll, can suitable be used as a nutritional or functional protein ingredient in foods, beverages etc.

The present invention also provides a particulate dechlorophyllized RuBisCO preparation that is obtained by the above described process, said preparation having a weight averaged diameter of 10-1000 μιη; containing at least 30 wt.% of undenatured RuBisCO and containing less than 0.1 % of chlorophyll by weight of RuBisCO. This particulate protein preparation can advantageously be applied in foodstuffs, beverages, pharmaceutical products, nutrional supplements and animal feed.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, one aspect of the invention relates to a process for isolating a dechlorophyllized RuBisCO preparation from a plant material, said plant material comprising intact plant cells that hold chlorophyll- containing chloroplasts, wherein the process comprises the steps of:

a. lysing the plant material to release RuBisCO and chlorophyll from the plant cells; b. separating the lysed plant material into a liquid juice and a high solids slurry or a high solids cake, said liquid juice containing RuBisCO as well as chlorophyll; c. contacting the liquid juice with activated carbon to adsorb chlorophyll onto the activated carbon;

e. further processing the dechlorophyllized liquid juice to produce the

dechlorophyllized RuBisCO preparation.

The term "plant" as used herein refers to an organism belonging to the kingdom

Plantae. Examples of plants include trees, herbs, bushes, grasses, vines, ferns, mosses, and green algae.

The term "lysis" as used herein refers the to breaking of the cellular membrane of plant cells, causing the cell contents to spill out.

The term "activated carbon" as used herein is a form of carbon that has an extensively developed internal pore structure. Due to its high degree of microporosity, just one gram of activated carbon typically has a surface area in excess of 250 m², as determined by nitrogen gas adsorption.

Examples of plant materials that can be used as starting material in the present process are leaves, stems, pods, hulls, green algae cells or parts thereof. Preferably, the plant material employed in the present process is derived from leaves and/or stems.

Even more preferably, the plant material is derived from leaves. Typically, at least 40 wt.%, more preferably at least 60 wt.% and most preferably at least 80 wt.% of the dry matter contained in the plant material is derived from plant leaves.

The chlorophyll- containing plant material that is employed as starting material in the present process is suitably derived from a green plant or green algae. Most preferably the plant material is derived from green plants, especially leave bearing green plants. Examples of suitable leave bearing green plants include spinach, alfalfa, beet, tobacco, corn, carrot, pulses.

The present process produces optimum results if the plant material employed as starting material contains high levels of RuBisCo. Preferably, the plant material contains at least 8% RuBisCO by weight of dry matter. More preferably, the plant materials contains at least 10%, even more preferably at least 15% and most preferably at least 20% RuBisCO by weight of dry matter.

The present process offers the advantage that it can suitably be used to produce an essentially color-free protein preparation even when the starting material is a plant material containing high levels of chlorophyll. The benefits of the present process are especially appreciated in case the chlorophyll containing plant material contains at least 5% of chlorophyll by weight of RuBisCO. More preferably, the chlorophyll containing plant material contains at least 10%, most preferably at least 15 wt.% of chlorophyll by weight of RuBisCO. Typically, the chlorophyll content of the plant material does not exceed 50% by weight of RuBisCO.

Lysis of the plant material may suitably be achieved by subjecting it to mechanical, chemical and/or enzymatic lysis. Preferably, the plant material is subjected to mechanical lysis. Examples of mechanical (or physical) lysis include: mechanical agitation, pressure, sonication, nitrogen bomb or nitrogen burst lysis method and ultrasound with small probes. Most preferably, in the present process lysis of plant material is achieved by means of maceration.

Separation of the lysed plant material into a liquid juice and a high solids slurry or a high solids cake may be achieved by any solid-liquid separation techniques known in the art. Examples of such separation techniques include sieving, filtration, centrifugation and decanting. Most preferably, the separation into a low solids liquid juice and a high solids slurry/cake is achieved using sieving, most preferably by using pressure sieving. Prior to the latter separation step, water may be added to the lysed plant material in order to facilitate separation. According to a preferred embodiment, an aqueous buffer is added to the lysed plant material in order to adjust the pH to a level of 6.0- 10.0, more preferably of 6.5-9.0 and most preferably of 6.8-8.5.

Advantageously, the present process includes a step in which proteinaceous components other than RuBisCO are removed from the liquid juice. To this end, the liquid juice is advantageously heated to a temperature of 50-80 °C for at least 1 minute, preferably for 1-100 minutes to coagulate proteinaceous material and the heat coagulated proteinaceous material is removed from the liquid juice prior to the further processing in step e.

The heat treatment of the liquid juice is advantageously conducted under mild temperature conditions. Thus, according to a preferred embodiment, the liquid juice is heated to a temperature of not more than 70 °C, more preferably of not more than 68 °C. The duration of this heat treatment preferably lies in the range of 1.5-60 minutes, most preferably of 2-30 minutes.

The pH of the liquid juice when subjected to this heat treatment preferably exceeds 6.0, more preferably it exceeds 6.5. Even more preferably the pH of the liquid juice is in the range of 6.6-9.0 when it is heated to coagulate proteinaceous material. Most preferably, the pH of the liquid juice is in the range of 6.8-8.5 when it is subjected to the heat treatment.

The removal of proteinaceous material by heat coagulation advantageously occurs prior to the contacting of the liquid juice with the activated carbon in step c. Even more preferably, also the heat coagulated proteinaceous material is removed from the heated liquid juice prior to step c.

The coagulated proteinaceous material may suitably be removed by solid-liquid separation techniques known in the art. Examples of suitably techniques for removing the coagulated material include filtration, sieving, centrifugation and decanting.

Preferably, the removal of the coagulated material from the liquid juice comprises filtration and/or centrifugation. Most preferably, the removal of the coagulated proteinaceous material is achieved by means of microfiltration and/or ultrafiltration.

In the present process the activated carbon is typically added to the liquid juice in a concentration of 0.01-15 wt.%. More preferably, the activated carbon is added to the liquid juice in a concentration of 0.1-10 wt.%, most preferably of 0.5-5 wt.%. Expressed differently, the activated carbon is preferably added to the liquid in a concentration of 5-1500%, more preferably 10-1000% and most preferably 50-500 % by weight of RuBisCO.

The activated carbon was found to be especially effective in removing chlorophyll from the liquid juice if the activated carbon has a surface area in excess of 250 m²/g. Even more preferably, said surface area exceeds 500 m²/g, most preferably it exceeds 750 m²/g. Typically, the surface area of the activated carbon does not exceed 2000 m²/g. The surface area of the activated carbon is suitably determined by means of nitrogen gas adsorption.

The activated carbon is suitably employed in the present process in particulate form. Typically, the activated carbon has a weight averaged diameter of 1-1000 μιη, more preferably of 1-250 μιη and most preferably of 2-150 μιη. Advantageously, at least 80 wt.%, more preferably at least 90 wt.% of the particles contained in the particulate activated carbon has a particle size within the range of 0.5-250 μιη, especially within the range of 1 -200μιη.

The activated carbon employed in the present isolation process typically has an Iodine Number of 400-1400 mg/g. More preferably, the Iodine Number of the activated carbon lies within the range of 600-1300 mg/g, most preferably within the range of 800-1200 mg/g. The Iodine number is commonly used to characterize activated carbon performance. It is a measure of the micropore content of the activated carbon (0 to 20 A) by adsorption of iodine from solution. The Iodine number is defined as the milligrams of iodine adsorbed by one gram of carbon when the iodine concentration in the residual filtrate is 0.02 normal. Basically, iodine number is a measure of the iodine adsorbed in the pores and, as such, is an indication of the pore volume available in the activated carbon of interest.

Molasses Number is a measure of the mesopore content of the activated carbon (greater than 20 A) and is measured by adsorption of molasses from solution. The (European) Molasses Number of the activated carbon used in the present process typically lies in the range of 100-550. More preferably, the Molasses Number of the activated carbon lies within the range of 150-450, most preferably the Molasses Number is within the range of 200-350.

Some activated carbons have a mesopore (20 A to 50 A) structure which adsorbs medium size molecules, such as the dye methylene blue. Methylene Blue Adsorption is reported in g/lOOg. The activated carbon used in the isolation process of the present invention typically has a Methylene Blue Adsorption in the range of 10-30 g/lOOg. More preferably, the activated carbon has a Methylene Blue Adsorption of 12- 28 g/lOOg, most preferably of 14-24 g/100 g.

The liquid juice may suitably be contacted with the activated carbon by adding a particulate activated carbon to the liquid juice or by percolating the liquid juice over a bed of activated carbon. Preferably, the liquid juice is contacted with the activated carbon by adding a particulate activated carbon to the liquid juice.

In case a particulate activated carbon is used, fast adsorption of the chlorophyll onto the activated carbon can be achieved by keeping the carbon particles suspended in the liquid juice. This may be achieved, for instance, by stirring, recirculation, shaking or tumbling,

The chlorophyll-loaded activated carbon can be separated from the

dechlorophyllized liquid juice using any so lid- liquid separation technique known in the art. Examples of suitably techniques for removing the chlorophyll- loaded activated carbon include filtration, sieving, centrifugation and decanting. Preferably, the chlorophyll- loaded activated carbon is removed from the liquid juice using one or more separation techniques selected from filtration, centrifugation and decanting. According to a particularly preferred embodiment, the separation of the carbon is achieved by using centrifugation and/or decanting followed by filtration of the supernatant to remove any carbon fines, said filtration advantageously comprising micro filtration and/or ultrafiltration.

The further processing of the dechlorophyllized liquid juice advantageously comprises removal of water and/or addition of additives in order to stabilize the product against e.g. microbial spoilage. Those additives can be selected from preservatives or components lowering the water activity, for example, salts or sugars. According to a preferred embodiment, the further processing comprises water removal. Water removal may be achieved by different means known in the art, including evaporation, ultrafiltration, reverse osmosis and combinations thereof. Typically, the water content of the dechlorophyllized juice is reduced to less than 60 wt.%, more preferably to less than 50 wt.% and most preferably to less than 40 wt.%

According to a particularly preferred embodiment of the invention further processing of the dechlorophyllized liquid juice comprises the drying of said juice, e.g. after first having reduced the water content of the juice by means of evaporation and/or ultrafiltration.

The dechlorophyllized liquid juice can suitably be dried by means of, for example, spray drying, freeze drying or drum drying. Most preferably, the

dechlorophyllized liquid juice is dried by means of spray dyring.

Advantageously, the present process comprises the drying of the

dechlorophyllized liquid juice to produce a dechlorophyllized RuBisCO preparation having a water content of less than 15 wt.%, more preferably of less than 10 wt.% and most preferably of less than 6 wt.%.

The present process is very efficient in that it enables the isolation of RuBisCO in high yields whilst at the same time effectively removing all chlorophyll contained in the starting material. Accordingly, in the present process the weight ratio of chlorophyll to RuBisCO in the dechlorophyllized RuBisCO preparation typically is at least 100 times lower, more preferably 250 times lower and most prefereably 400 times lower than the same weight ratio in the plant material from which said RuBisCO preparation is isolated.

The chlorophyll content of the RuBisCO preparation obtained by the present process is very low. Typically, the weight ratio of chlorophyll to RuBisCO in the dechlorophyllized RuBisCO preparation is less than 1 : 1000. More preferably, the latter weight ratio is less than 1 : 1500, even more preferably less than 1 :2000 and most preferably less than 1 :2200.

The process according to the present invention enables the preparation of protein preparations containing appreciable levels of RuBisCO. Typically, the dechlorophyllized RuBisCO preparation produced by the present process contains at least 40% of RuBisCO by weight of dry matter. Even more preferably, said protein preparation contains at least 60% of RuBisCO by weight of dry matter. Most preferably, the RuBisCO content is 70% by weight of dry matter.

The protein preparation produced by the present method may include other components besides RuBisCO. Typically, the preparation contains up to 40%> by weight of dry matter of protein other than RuBisCO.

As mentioned herein before, the present process enables the isolation of RuBisCO in high yield. Typically, the yield of the present process in terms of RuBisCO exceeds 20%. More preferably, said yield exceeds 40%, even more preferably it exceeds 50%> and most preferably it exceeds 60%>.

In an advantageous embodiment of the present process the dechlorophyllized liquid juice is dried and optionally agglomerated to produce the dechlorophyllized RuBisCO preparation in the form of a powder having a weight averaged diameter of 10-1000 μιη, more preferably of 20-600 μιη and most preferably of 30-400 μιη.

Advantageously more than 80 wt.% of said powder has a diameter within the range of 10-1000 μιη, more preferably of 15-800 μιη and most preferably of 20-600 μιη.

The present process can be operated in a continuous or batch- wise fashion. Irrespective of whether the process is operated as continuous or batch process, it can be operated on a large scale. In case the process is operated as a batch process, the amount of plant material employed in a single batch typically exceeds 50 kg. Even more preferably one batch of plant material exceeds 200 kg, most preferably it exceeds 1000 kg.

If operated in a continuous fashion, the throughput of plant material in te present process typically exceeds 50 kg/hr. More preferably, said throughput exceeds 200 kg/hr or even 500 kg/hr. Most preferable said throughput exceeds 2000 kg/hr.

An important advantage of the present invention resides in the fact that it can be operated using very mild temperature conditions, thereby avoiding substantial denaturation of the RuBisCO. Advantageously, the present process can be operated under such mild conditions that it yields a dechlorophyllized RuBisCO preparation wherein at least 30 wt.%, preferably at least 60 wt.% and even more preferably at least 90 wt.% of the RuBisCO contained therein is undenatured. Typically, the temperature of the liquid juice remains below 80 °C, preferably below 70 °C and more preferably below 68 °C throughout process steps a. to d.

The present process, unlike some RuBisCO isolation processes of the prior art, preferably does not employ acid precipitation of RuBisCO. Thus, since the iso-electric point of RuBisCO lies at pH 4.1-4.8, it is preferred that the pH of the liquid juice is kept at pH 5.0 or more during processing steps a. to d. Even more preferably said pH is maintained above pH 5.5, most preferably above pH 6.0.

Another advantage of the process of the present invention lies in the fact that it can be operated without making use of any organic solvents. Thus, in accordance with a particularly preferred embodiment the present process does not employ extraction or precipitation with an organic solvent. Even more preferably, the present process does not employ an organic solvent at all.

A further aspect of the present invention relates to a particulate

dechlorophyllized RuBisCO preparation that is obtained by a process as described herein before, said preparation having a weight averaged diameter of 10-1000 μιη; containing at least 30 wt.% of undenatured RuBisCO and containing less than 0.1% of chlorophyll by weight of RuBisCO.

A fundamental difference between the present process and the RuBisCO isolation processes described in the prior art resides in the fact that the present process utilizes activated carbon. The activated carbon in addition to removing chlorophyll also removes substantially all carotenes. Hence, unlike the RuBisCO preparations that have been described in the prior art, the particulate RuBisCO preparation according to the present invention contains less than 1% of carotenes by weight of RuBisCO. More preferably, the particulate RuBisCO preparation contains less than 0.8%, even more preferably less than 0.5% and most preferably less than 0.1% carotenes by weight of RuBisCO. The presence of appreciable levels of carotenes in protein preparations is undesirable as it affects the color of the preparation and because it can adversely affect the flavor of these preparations as carotenes are easily oxidized.

The term carotene is used for several related substances having the formula C₄oH_x, which are synthesized by plants but cannot be made by animals. Carotene is an orange photosynthetic pigment important for photosynthesis. Chemically, carotene is a terpene, synthesized biochemically from eight isoprene units. It comes in two primary forms designated by characters from the Greek alphabet: alpha-carotene (a-carotene) and beta-carotene (β-carotene). Gamma, delta, epsilon, and zeta (γ, δ, ε, and ζ-carotene) also exist.

The particulate dechlorophyllized RuBisCO preparation is suitably produced by e.g. spray drying, freeze drying or drum drying. These drying techniques yield a largely amorphous powder. Consequently, it is preferred that at least 50 wt.%>, more preferably at least 80 wt.% and most preferably at least 90 wt.% of the RuBisCO contained in the present RuBisCO preparation is in an amorphous state.

Yet another aspect of the present invention relates to a method of preparing a product selected from a foodstuff, a beverage, a pharmaceutical product, a nutritional supplement and animal feed, said method comprising incorporating 0.01-50%), preferably 0.1-20% by weight of the product of a particulate RuBisCO preparation as defined herein before. The RuBisCO preparation can advantageously be applied as a foaming agent in aerated products. Likewise, the preparation can suitably be used as gelling agent in products that contain a gelled aqueous phase.

The invention is further illustrated by means of the following non-limiting examples.

EXAMPLES

Comparative Example I

Spinach leaf proteins were extracted using the method described by Yang et al. J.

Agric. Food Chem. (52) 2223-2225, (2004), with some modifications.

Fresh spinach leaves were homogenized with an equal amount of aqueous 0.003N sodium hydroxide at 5°C in a Waring blender. The homogenate obtained was adjusted to pH 11.0 with 1M NaOH and filtered through cheese cloth. The sieved homogenate was then centrifuged at 13,000 g for 30 min at 5°C.

The supernatant so obtained was adjusted with 5% acetic acid to a pH of ~ 4.5 to precipitate the protein. The precipitate was separated by centrifugation at 13000 g for 30 min at 5°C and washed successively with ethanol by mixing (breaking the aggregates created with the centrifugation) and centrifuging at 8000g for 10 min at 5°C. The washing step was repeated until the green color of the pellet had been removed completely. Finally, the pellet was dispersed at a concentration of 2% (w/v) in 0.05M Tris buffer, pH 9.5, containing 0.1 M NaCl and 0.5 mM EDTA and stirred overnight at 5°C.

Subsequently, the solution was centrifuged 15 min at 5°C at 5,000 rpm and the supernatant was freeze-dried. It was found that the juice from the first pressing and the supernatant from the first centrifugation contain appreciable levels of RuBisCO subunits. As expected, the supernatant obtained from the second centrifugation had a reduced protein content due to the preceding acid precipitation. However, the results showed that only about 50 % of the protein had precipitated during acid precipitation. Comparative Example II

Spinach leaf proteins were extracted using the method described by Lamsal et al, Transaction of the ASAE 46(3): 715-720 (2003).

Fresh spinach leaves were macerated in a Waring Blender adding water as necessary to immobilize the leaves to increase the rupture of cells until getting a semi-homogenate. The homogenate was sieved through cheese cloth and the juice was collected. Two alternatives pretreatment procedures were tested:

a) acidification and heat treatment (experiment IIA)

b) heat treatment (experiment IIB).

In experiment IIA, prior to the heat treatment, pH of the juice was adjusted to 4.5 with acetic acid 5%. In experiment IIB, prior to the heat treatment pH was adjusted to 7.0 using 1M NaOH solution. In both experiments the heating step comprised heating of the juice in a water bath at 60°C for 5 min (the temperature of the juice was measured with a sonde to make sure the juice was at 60°C and the bath was previously heated at 69°C to ensure a higher heat transfer at the beginning). Following heat treatment, the suspensions were centrifuged at 13,000 g for 20 min at 21°C. Next, the supernatant was freeze dried.

The freeze dried samples so obtained had a clearly perceptible yellow/green color.

Comparative Example III

Spinach leaf proteins were extracted using the method described by Wildman and Kwanyuen (US 4,268,632) for tobacco leafs. Spinach leafs (1kg) were added to 200 mL cold water and macerated in a food processor (KENWOOD multi pro) at maximum speed. The macerated leafs were transferred to a beaker glass and 5 mL 2- mecraptoethanol was added. The mixture was further homogenized using an

UltraTurrax for 2 min. The homogenate was divided in portion of 400 mL and incubated in a water bath of 50°C during 15 minutes. The heated homogenate was filtered through two layers of cheese cloth. The filtrate was collected within 2 hours after the heating step and subsequently centrifuged at 4500xg for 30 minutes.

The supernatant was stored overnight at 6°C and subsequently for one night in melting ice to induce crystallization of RuBisCO. The thus obtained liquid was filtered through filter paper and some green solid material was obtained. The solid material had an intense green color, indicating that it contains considerably more than 0.1% of chlorophyll by weight of RuBisCO.

Example 1

Fresh spinach (2kg) was washed and was macerated in a Waring blender at a ratio 2: 1 (w/w) with 0.1M Tris buffer, pH 8.0, containing 0.1M NaCl, 0.5 mM EDTA and 2% Na₂S₂0₅ (sodium metabisulfite). The homogenate was sieved through cheese cloth prior to heating for 5 min. at 60°C. Following the heat treatment, the suspension was centrifuged (20 min. at 14,000g and at 5°C). The supernatant was dialyzed overnight using a dialysis tube with a lOkDa cut-off, to remove the sodium metabisulfate.

Subsequently, activated carbon was added in the amount of 19 g/L. The specification of the activated carbon is presented in Table 1 :

Table 1

Following addition of the activated carbon, the suspension was stirred for at least 5 minutes. Subsequently, the supernatant was removed by centrifugation (20 min. at 14,000g and at 5°C). The supernatant so obtained was subjected to two filtration steps. First the supernatant was passed over a 0.45 μηι filter and secondly over a 0.2 μηι filter. The filtrate so obtained was freeze-dried. The freeze-dried material was white, odourless and fully soluble.

Example 2

The amount of soluble protein contained in the freeze dried protein preparations of Examples I, II and 1 was determined. The results are depicted in Table 2.

The protein content was determined using the BCA assay (Interchim) and using bovine serum albumin as a standard protein. The presence of chlorophyll and pigments influences the results of this assay. Thus, the protein concentrations mentioned in Table 2 for the protein preparations of Examples IIA and IIB are higher than the actual soluble protein concentrations of these preparations.

Table 2

These results clearly demonstrate that the isolation process according to the present invention produces a protein preparation in which virtually all protein is water soluble. Comparative example I (Yang et al. J. Agric. Food Chem. (52) 2223-2225, (2004)) failed to produce a particulate dechlorophyllized RuBisCO preparation containing at least 30 wt.% of undenatured RuBisCO. The chlorophyll content of the protein preparation obtained by the process described in Example 1 was approximately 0.04% by weight of RuBisCO. Example 3

Fresh spinach (50 kg) was washed and stored at 4°C one day before the protein isolation experiment described below was conducted. After weighing, spinach was introduced in a Stephan cutter in a ratio 2: 1 (w:v) with 0.1M Tris buffer pH 8.0, containing 0.1M NaCl, 0.5 mM EDTA and 2% Na₂S₂0₅ (sodium metabisulfite).

Maceration was achieved in 5 minutes by operating the Stephan Cutter at full speed. The homogenate obtained from the Stephan Cutter was filtered through cheese cloth (employing the cheese cloth used in Example I, II and 1) using a cheese press. .

Next, the filtrate was subjected to a heat treatment using a water bath and a churn that was provided with a stirrer to enhance heat transfer. The off-set temperature of the water bath was 69°C to ensure high heat transfer at the beginning. The filtrate was kept at 60 °C for 5 minutes and then cooled down to 10 °C. The removal of sodium metabisulfite was performed by ultrafiltration with a lOkDa membrane.

The coagulated protein that had formed during the heat treatment was removed from the heated suspension by passing the suspension over a microfilter having a pore size of 0.2 μιη.

Next, activated carbon was added to the filtrate in an amount of 19 m/L. Following addition of the activated carbon, the suspension was stirred for 5 minutes.

Subsequently, stirring was discontinued and the suspension was left standing for 10 minutes, before the supernatant was removed by decanting.

The supernatant so obtained was subjected to two filtration steps. First, the supernatant was passed over a microfilter having a pore size of 0.2 μιη. Finally, to concentrate the solution, an ultrafiltration setup with a 10 kDa membrane was used and the ultrafiltrate was was freeze dried.

The obtained freeze-dried material was white and odourless. Example 4

Fresh carrot leafs (2kg) were washed and were macerated in a Waring blender at a ratio 2: 1 (w/w) with 0.1M Tris buffer, pH 8.0, containing 0.1M NaCl, 0.5 mM EDTA and 2% Na₂S₂0₅ (sodium metabisulfite). The homogenate was sieved through cheese cloth prior to heating for 5 min. at 60°C.

Following the heat treatment, the suspension was centrifuged (20 min. at 14,000g and at 5°C). The supernatant was dialyzed overnight using a dialysis tube with a lOkDa cut- off, to remove the sodium metabisulfate.

Subsequently, the activated carbon described in Example 1 was added in an amount of 19 g/L. Following addition of the activated carbon, the suspension was stirred for at least 5 minutes. Subsequently, the supernatant was removed by centrifugation (20 min. at 14,000g and at 5°C).

The supernatant so obtained was subjected to two filtration steps. First the supernatant was passed over a 0.45 μιη filter and secondly over a 0.2 μιη filter. The filtrate so obtained was freeze-dried. The freeze-dried material was white, odourless and fully soluble. The amount of soluble protein by weight of freeze-dried material was 82.6% and the amount of soluble protein by weight of carrots leafs starting material was 8.5%.

Example 5

Fresh sugar beet leafs (2kg) were washed and were macerated in a Waring blender at a ratio 2: 1 (w/w) with 0.1M Tris buffer, pH 8.0, containing 0.1M NaCl, 0.5 mM EDTA and 2%> Na₂S₂0₅ (sodium metabisulfite). The homogenate was sieved through cheese cloth prior to heating for 5 min. at 60°C. Following the heat treatment, the suspension was centrifuged (20 min. at 14,000g and at 5°C). The supernatant was dialyzed overnight using a dialysis tube with a lOkDa cutoff, to remove the sodium metabisulfate. Subsequently, the activated carbon described in Example 1 was added in an amount of 19 g/L. Following addition of the activated carbon, the suspension was stirred for at least 5 minutes. Subsequently, the supernatant was removed by centrifugation (20 min. at 14,000g and at 5°C).

The supernatant so obtained was subjected to two filtration steps. First the supernatant was passed over a 0.45 μιη filter and secondly over a 0.2 μιη filter. The filtrate so obtained was freeze-dried. The freeze-dried material was white, odourless and fully soluble. The freeze-dried material contained at least 30 wt.% of undenatured RuBisCO.

Example 6

The freeze-dried, white and odourless RuBisCO material obtained in Example 3 was dissolved at 0.5wt.% concentration in a 0.01 M sodium phosphate buffer at pH 7.0 (buffer A) and in the same buffer containing 0.2 M NaCl (buffer B). Both solutions were stirred for 1 h at 40°C. pH of the solutions was adjusted to pH 4.5 and pH 7.0 using either IM NaOH or IM HCl. The samples were left to settle one hour at room temperature and subsequently centrifuged for 15min at 13,000 rpm. The protein content of the supernatant so obtained was determined using the BCA assay as described in Example 2. The results are depicted in Table 3. Thus at pH 7.0 the solubility of the RuBisCO material obtained in Example 3 is 100%, whereas at pH 4.5 the solubility is below 20%> by weight of protein. Thus the RuBisCO preparation at pH 4.5 can be regarded as denatured material.

Table 3

Solubility (%> by weight of protein)

Solution pH 4.5 pH 7.0

Buffer A 9 100

Buffer B 14 100 Example 7

The freeze-dried, white and odourless RuBisCO material obtained in Example 3 was dissolved at a concentration of 2wt.% (100 mL). Whey Protein Isolate (WPI, BiPRO, Davisco International Inc., La Sueur, MN, USA) and Soy Protein Isolate (SPI, Profam 891 from ADM, Koog aan de Zaan, the Netherlands) were dissolved at a concentration of 2wt.% (100 mL). All the solutions were stirred overnight at 4°C. Afterwards, the pH was adjusted to pH 4.5 or 7.0 using 6M HC1 or 6M NaOH, respectively. Duplicates were performed in the case of WPI and SPI.

Solutions were whipped for 70 s with an Aerolatte mixer connected to a tunable power supply which allows controlling the speed of stirring (settings: intensity of 1.65A and voltage of 10V). The whipped solution was immediately transferred to a 250mL graduated cylinder. Foam volume, coalescence and drainage were visually followed for a period of 2 hours. The results are depicted in Table 4.

Table 4

The undenatured RuBisCO material obtained in Example 3 showed surprisingly high foam volumes and stability compared to denatured RuBisCO (results at pH 4.5), WPI and SPI.

Example 8

The freeze-dried, white and odourless RuBisCO material obtained in Example 3 and the WPI and SPI preparation as described in Example 7 were dissolved at different concentrations, (ranging form 2 to 12.5 wt.%). The pH of the solutions was adjusted to pH 7.0 using 6M HC1 or 6M NaOH. Heat gelation was induced by heating the samples (1.5 mL protein solution in a 2.0 mL Eppendorf tube) at 90°C for 10 minutes and left at room temperature for 30 minutes before measurement took place.

Gel strength measurements were performed on a TA2 Texture Analyzer (Stable Micro Systems Ltd., Godalming, UK) according to the following procedure:

• Measure force in compression with a penetrometer probe with a diameter of 3 mm;

• Test speed 0.3 mm/s;

• Firmness is taken as the maximum of force curve;

• 5 replicates done for each sample;

• The minimum gelation concentration is defined as the minimum concentration at which the gel showed a firmness of above 1 g.

The results so obtained are depicted in Table 5. Table 5

The undenatured RuBisCO material obtained in Example 3 showed surprisingly low minimum gel concentration compared to WPI and SPI.

Claims

1. A process for isolating a dechlorophyllized RuBisCO (ribulose- 1 ,5-bisphosphate carboxylase oxygenase) preparation from a plant material, said plant material comprising intact plant cells that hold chlorophyll- containing chloroplasts, wherein the process comprises the steps of:

d. separating the dechlorophyllized liquid juice from the chlorophyll- loaded

activated carbon; and

e. further processing the dechlorophyllized liquid juice to produce the

dechlorophyllized RuBisCO preparation.

2. Process according to claim 1, wherein the liquid juice is heated to a temperature of 50-80 °C for at least 1 minute to coagulate proteinaceous material and wherein the heat coagulated proteinaceous material is removed from the liquid juice prior to the drying step e.

3. Process according to claim 2, wherein the liquid juice has a pH of at least 6.0 when it is heated to coagulate proteinaceous material.

4. Process according to claim 2 or 3, wherein the liquid juice is heated to coagulate proteinaceous material prior to step c.

5. Process according to claim 4, wherein the heat coagulated proteinaceous material is removed from the heated liquid juice prior to step c.

6. Process according to any one of the preceding claims, wherein the activated carbon is added to the liquid juice in a concentration of 0.01-15 wt.%.

7. Process according to claim 6, wherein the chlorophyll- loaded activated carbon is separated from the dechlorophyllized liquid juice by centrifugation and/or decanting followed by filtration.

8. Process according to any one of the preceding claims, wherein at least 30 wt.%, preferably at least 60 wt.%, even more preferably at least 90 wt.% of the RuBisCO in the dechlorophyllized RuBisCO preparation is undenatured.

9. Process according to any one of the preceding claims, wherein the weight ratio of chlorophyll to RuBisCO in the dechlorophyllized RuBisCO preparation is at least 250 times lower than the same weight ratio in the plant material from which said RuBisCO preparation is isolated.

10. Process according to any one of the preceding claims, wherein the weight ratio of chlorophyll to RuBisCO in the dechlorophyllized RuBisCO preparation is less than 1 : 1000.

11. Process according to any one of the preceding claims, wherein the

dechlorophyllized RuBisCO preparation contains at least 60% of RuBisCO by weight of dry matter.

12. Process according to any one of the preceding claims, wherein the further

processing of the dechlorophyllized liquid juice comprises drying the

dechlorophyllized liquid juice.

13. A particulate dechlorophyllized RuBisCO preparation that is obtained by a process according to claim 1, said preparation having a weight averaged diameter of 10- 1000 μιη; containing at least 30 wt.% of undenatured RuBisCO and containing less than 0.1% of chlorophyll by weight of RuBisCO.

14. Particulate RuBisCO preparation according to claim 13, said preparation being characterized in that it contains less than 1% carotenes by weight of RuBisCO.

15. A method of preparing a product selected from a foodstuff, a beverage, a pharmaceutical product, a nutrional supplement and animal feed, said method comprising incorporating 0.01-50% by weight of the product of a particulate RuBisCO preparation according to claim 13 or 14.