PHYTOCHEMICALS FROM MACROALGAE

TECHNICAL FIELD

[0001] The present invention relates to a method for the extraction of a phytochemical, e.g., a peptide or protein, from a macroalgae; and to a device for carrying out said method.

BACKGROUND ART

[0002] Global population growth combined with the increase in quality of life in the era of changing climate will increase the demand for food, chemicals and fuels in general, and for plant proteins in particular. Considering the positive impact of plant proteins consumption on sustainability and reduction of land, water, fertilizers and energy consumption, there is a consciously growing interest in exploring different plant sources for direct proteins use in the diet, either directly as entire plant or combined in the processed food products (Tuso et ah, 2013). In the last five decades microalgae biomass gained a lot of interest as a feedstock for proteins production. More recently food proteins production is considered as a valuable co-product with biofuels in the algal biorefineries. The production of microalgae biomass, however, is still cost prohibitive and further advances in the cultivation and harvesting technologies are required. Macroalgae, large multicellular organisms, have been mostly overlooked as a feedstock for protein production for many years. However, many of the marine red and green macroalgae species have shown significantly higher content of proteins in comparison to the terrestrial plant proteins sources such as soy, nuts, and cereals (Fleurence, 2004; Harnedy and FitzGerald, 2011). In addition to their high yields and nutritional properties, marine algae derived proteins and peptides have shown additional value because of their nutraceutical, pharmaceutical and cosmeceutical properties such as antioxidant, antihypertensive, immune-modulatory, anticoagulant and hepeto-protective substances (Fleurence, 2004; Harnedy and FitzGerald, 2011).

[0003] The value of the macroalgae as a protein source depends on the extracted protein yields and the ability to preserve its functional properties. The complex, viscous and often charged macroalgae cell wall and extracellular matrix make the extraction process challenging (Joubert and Fleurence, 2007). Osmotic shock, mechanical grinding, high shear force, ultrasonic treatment, acid and alkaline pretreatment and polysaccharidase aided digestion, and their combination have been used to increase the extraction yields (Barbarino and Lourenco, 2005; Wong and Chikeung Cheung, 2001; Rouxel et ah, 2001; Galland-Irmouli et ah, 2000; Fleurence et ah, 1995; Harnedy and FitzGerald, 2013). Although the mentioned methods were shown to increase the extraction yields, they involve either thermal or chemical procedures that could affect the functionality of the extracted proteins and peptides.

[0004] Pulsed electric field (PEF) is an emerging, non-thermal food processing technology already used for energy-efficient extraction of proteins from microalgae. Although the exact mechanism of biological tissue permeabilisation by PEF is not fully understood, PEF technology is currently used in multiple applications in medicine and biotechnology (Kotnik et ah, 2015; Rubinsky, 2007; Yarmush et ah, 2014). The current theory suggests that the membrane permeabilisation is achieved through the formation of aqueous pores on the cell membrane, a phenomenon known as electroporation (Weaver and Chizmadzhev, 1996).

SUMMARY OF INVENTION

[0005] It has now been found, in accordance with the present invention, that phytochemicals such as proteins may be efficiently extracted from macroalgae, while preserving their chemical structure and functional properties, by a non-thermal PEF treatment, carried out under pressure higher than ambient pressure, which causes to cell membrane rupture due to irreversible electroporation. Such a process can be carried out continuously, e.g., in a fluidized bed chamber, which further allows for direct separation of the extracted product, cutting the costs of downstream purification.

[0006] In one aspect, the present invention thus relates to a method for the extraction of a phytochemical from a macroalgae, said method comprising applying pulsed electric field (PEF)- or continuous electric field (CEF)-treatment, preferably non-thermal PEF or CEF treatment, to said macroalgae in a solvent, under pressure higher than the ambient pressure, thereby extracting said phytochemical from said macroalgae cells into said solvent.

[0007] In another aspect, the present invention relates to a macroalgae phytochemical, e.g., a peptide, protein, sugar, or small molecule, obtained by the method defined above.

[0008] In yet another aspect, the present invention provides a device for the extraction of a phytochemical from a macroalgae, said device comprising: (i) a pulsed electric field (PEF)- or constant electric field (CEF)-treatment chamber comprising at least one inlet and at least one outlet; (ii) a first electrode and a second electrode positioned for creating an electric field in said treatment chamber; (iii) optionally a pressure generating mean for generating pressure within said treatment chamber; (iv) optionally, an agitator and/or air bubbling unit for agitating and mixing the content of said treatment chamber; and (v) optionally, a filtering unit located at the outlet, wherein upon introducing said macroalgae in a solvent into said treatment chamber, and applying PEF- or CEF treatment to said solvent under pressure higher than the ambient pressure, said phytochemical is extracted from said macroalgae into said solvent.

[0009] In still another aspect, the present invention provides a system for the extraction and separation of a phytochemical from a macroalgae, said system comprising: (i) a device as defined above, for the extraction of said phytochemical from said macroalgae into said solvent; and (ii) a separation unit comprising at least one inlet and at least one outlet, for separating said phytochemical from said solvent, wherein upon extracting said phytochemical from said macroalgae into said solvent, said solvent is optionally filtered for removal of macroalgae debris; and said phytochemical is then separated from said solvent in said separation unit.

BRIEF DESCRIPTION OF DRAWINGS

[0010] Fig. 1 shows shape and magnitude of the single electric pulse delivered for macroalgae biomass electroporation. The shape for actually delivered voltage and measured current is shown for the first and last pulse in the series of 75 pulses applied on the biomass.

[0011] Fig. 2 shows PEF and sample resistance behavior during macroalgae biomass treatment. Critical process parameters such as electric field (E), current (A) and biomass resistance (R) were monitored during each pulse. The experiment was done in triplicate. Error bars show + SEM.

[0012] Fig. 3 shows proteins extraction from Ulva. SDS gel electrophoreses (right) and total protein quantification with Bradford (left) process parameters and total yields for 24 kV, 75 pulses applied. Error bars show + SEM. Insert in the top row shows the total water soluble solids extracted from Ulva with PEF.

[0013] Figs. 4A-4C schematically illustrate certain configurations of a device for the extraction of a phytochemical from a macroalgae, as disclosed herein. [0014] Fig. 5 shows a laboratory device for the extraction of a phytochemical from a macroalgae, as disclosed herein, based on the configuration illustrated in Fig. 4C, wherein the PEF/CEF treatment chamber is a centrifuge, and the outlets through which the solvent containing the extracted phytochemical continuously exits the chamber are positioned at the top of the chamber, thereby allowing the solvent containing the macroalgae to first undergo a PEF/CEF treatment under pressure higher than the ambient pressure, and only then move upward and exit the chamber.

[0015] Figs. 6A-6B schematically illustrate certain configurations of a system for the extraction and separation of a phytochemical from a macroalgae, as disclosed herein.

DETAILED DESCRIPTION

[0016] In one aspect, the present invention relates to a method, i.e., process, for the extraction of at least one phytochemical from a macroalgae, said method comprising applying PEF or CEF treatment, preferably non-thermal PEF or CEF treatment, to said macroalgae in a solvent, under pressure higher than the ambient pressure, thereby extracting said at least one phytochemical from said macroalgae cells into said solvent.

[0017] The term "macroalgae" as used herein refer to a macrophyte, i.e., a macroscopic, multicellular (as opposed to microalgae) aquatic plant that grows in or near water and is emergent, submergent, or floating. Generally, macroalgae are divided to three groups known as the green algae, red algae, and brown algae, each one being further divided to families, genus, and species. The macroalgae treated according to the method of the invention may be in the natural form, i.e., a wet macroalgae, or as a dried or semidried material obtained after dehydrating said macroalgae, e.g., in an oven, and optionally cutting and/or grinding.

[0018] A list of macroalgae that may be treated according to the method of the present invention can be found, e.g., in AlgaeBase (a database of information on algae that includes terrestrial, marine and freshwater organisms; htt ://www . al gaebase.org/) . In particular embodiments, the macroalgae being treated according to the method of the invention is a green macroalgae, e.g., Ulva lactuca, which belongs to the genus Ulva, also known by the name sea lettuce. In other particular embodiments, the macroalgae being treated according to the method of the invention is a red macroalgae, e.g., of the genus Porphyra, Kapaphycaus, Gelidium, Gracilaria, or Chondrus; or a brown algae, e.g., of the genus Hizikia or Laminaria (e.g., Laminaria japonica). [0019] The term "pulsed electric field (PEF) treatment" refers to the application of a pulsed electric field characterized by specific voltage, electric field strength, pulse duration, number of pulses, and pulses frequency.

[0020] The term "continuous electric field (CEF) treatment" refers to the application of a single long pulse, e.g., a direct current (DC).

[0021] In certain embodiments, the PEF or CEF treatment applied according to the method of the present invention is homogeneously distributed in said solvent.

[0022] In certain embodiments, the PEF or CEF treatment applied according to the method of the present invention electroporates the cells of the treated macroalgae thereby causing extraction of said phytochemical from said macroalgae cells into the solvent. More particularly, the PEF or CEF treatment applied causes both opening of the membrane of said macroalgae cells and configuration changes in the extracellular matrix of said macroalgae, thereby enabling said extraction of said phytochemical from said macroalgae cells into said solvent.

[0023] In certain embodiments, the method of the present invention comprises applying CEF treatment to said macroalgae in said solvent, under pressure as defined above. In particular such embodiments, said method comprises applying DC previously known as galvanic current, i.e., a unidirectional flow of electric charge. The CEF treatment applied may be characterized by at least one of: (i) electric field strength of from about 10 to about 500 Volt, e.g., from about 10 to about 20 Volt, from about 20 to about 30 Volt, from about 30 to about 40 Volt, about 40 to about 50 Volt, from about 50 to about 60 Volt, from about 60 to about 70 Volt, from about 70 to about 80 Volt, from about 80 to about 90 Volt, from about 90 to about 100 Volt, from about 100 to about 200 Volt, from about 30 to about 300 Volt, from about 300 to about 400 Volt, or from about 400 to about 500 Volt; and pulse duration of from about 1 sec to about 10 min, e.g., from about 1 sec to about 10 sec, from about 10 sec to about 20 sec, from about 20 sec to about 30 sec, from about 30 sec to about 40 sec, from about 40 sec to about 50 sec, from about 50 sec to about 1 min, from about 1 min to about 2 min, from about 2 min to about 3 min, from about 3 min to about 4 min, from about 4 min to about 5 min, from about 5 min to about 6 min, from about 6 min to about 7 min, from about 7 min to about 8 min, from about 8 min to about 9 min, or from about 9 min to about 10 min.

[0024] In certain embodiments, the method of the present invention comprises applying PEF treatment to said macroalgae in said solvent, under pressure as defined above. In particular such embodiments, the PEF treatment applied is characterized by at least one of: (i) pulse number of from 1 to about 10,000, e.g., from 1 to about 500, from 500 to about 1000, from about 1000 to about 2000, from about 2000 to about 3000, from about 2000 to about 4000, from about 4000 to about 5000, from about 5000 to about 6000, from about 6000 to about 7000, from about 7000 to about 8000, from about 8000 to about 9000, or from about 9000 to about 10000; (ii) pulse duration of from about 50 ns to about 10 ms, e.g., from about 50 ns to about 500 ns, from about 500 ns to about 1 ms, from about 1 ms to about 2 ms, from about 2 ms to about 3 ms, from about 3 ms to about 4 ms, from about 4 ms to about 5 ms, from about 5 ms to about 6 ms, from about 6 ms to about 7 ms, from about 7 ms to about 8 ms, from about 8 ms to about 9 ms, or from about 9 ms to about 10 ms; (iii) electric field strength of about 0.1 to about 100 kV/cm, e.g., about 0.1 to about 0.5 kV/cm, about 0.5 to about 1 kV/cm, about 1 to about 5 kV/cm, about 5 to about 10 kV/cm, about 10 to about 20 kV/cm, about 20 to about 30 kV/cm, about 30 to about 40 kV/cm, about 40 to about 50 kV/cm, about 50 to about 60 kV/cm, about 60 to about 70 kV/cm, about 70 to about 80 kV/cm, about 80 to about 90 kV/cm, or about 90 to about 100 kV/cm; and (iv) pulse frequency of from 0.1 to about 10000 Hz, e.g., from 0.1 to about 10 Hz, from 10 to about 100 Hz, from 100 to about 500 Hz, from 500 to about 1000 Hz, from 1000 to about 2000 Hz, from 2000 to about 3000 Hz, from 3000 to about 4000 Hz, from 4000 to about 5000 Hz, from 5000 to about 6000 Hz, from 6000 to about 7000 Hz, from 7000 to about 8000 Hz, from 8000 to about 9000 Hz, or from 9000 to about 10000 Hz.

[0025] As would be clear to any person skilled in the art, the particular characteristics (properties) of the PEF treatment applied, i.e., the combination of particular pulse number, pulse duration, electric field strength and pulse frequency selected, may affect the efficiency of the process, e.g., the electroporation efficiency, and moreover, the temperature developed in the solvent exposed to said PEF treatment. The particular characteristics of the PEF treatment applied should thus be selected such that the extraction process, in terms of yield, would be as efficient as possible, and the solvent's temperature developed during and as a result of said PEF treatment would not be detrimental to said phytochemical. In particular embodiments, the properties of the PEF treatment applied are selected such that the process carried out is considered "non-thermal process", i.e., the solvent's temperature developed during said PEF treatment does not exceed 90°C, e.g., in the range of 25°C-90°C, 25°C-70°C, 25°C-60°C, 25°C-50°C, or 25°C-40°C. Such a nonthermal process might be of high importance in cases said phytochemical is a compound sensitive to relatively high temperatures such as a peptide or protein, or a combination thereof.

[0026] According to the present invention, the PEF or CEF treatment is applied under a pressure that is higher than the ambient pressure, i.e., simultaneously with said pressure. The term "ambient pressure" refers to the pressure of the medium surrounding the treatment zone, i.e., the zone (e.g., vessel or reactor) wherein said PEF or CEF treatment is carried out, and in certain cases actually denotes the standard atmospheric pressure (also referred to as the standard atmosphere, atm) that is 1013.25 millibars or 760 mmHg. A pressure higher than the ambient pressure may thus be any pressure that is higher than the pressure which comes into contact with the treatment zone, e.g., any pressure within the range of about 1 atm to 10 atm or more. According to the invention, pressure can be applied using any technique known in the art, e.g., a mechanical press or centrifugation power. In certain embodiments, the pressure applied during said PEF or CEF treatment is constant during the whole of said treatment. In other embodiments, the pressure applied during said PEF or CEF treatment varies, and may either increase or decrease during said PEF or CEF treatment.

[0027] The solvent used according to the method of the present invention may be any suitable solvent, i.e., either an inorganic solvent such as fresh water, tap water, saline, or salt water (seawater); or an organic solvent such as, without limiting, acetic acid, acetone, acetonitrile, methanol, ethanol, propanol, isopropanol butanol, i-butyl alcohol, hexane, cyclohexane, chlorobenzene, chloroform, 1,2-dichloroethane, ethylene glycol, diethylene glycol, diethyl ether, ethyl acetate, methylene chloride, benzene, or a combination thereof. The solvent in which said macroalgae is submerged and treated may affect the efficiency of the treatment applied to said macroalgae, e.g., the extraction efficiency of said phytochemical from said macroalgae. In particular embodiments, said solvent is selected according to the phytochemical to be extracted. For example, for extraction of soluble peptides or proteins, water can be used as the solvent; and for extraction of phenols, a solvent such as ethanol can be used.

[0028] The method of the present invention may be used so as to extract any particular phytochemical or combination of phytochemicals from the macroalgae treated. Particular such phytochemicals include, e.g., peptides and proteins (including proteins that are naturally occurring in macroalgae as well as recombinant proteins), sugars, small molecules, and any combinations thereof. Phytochemicals occurring in macroalgae may be used, e.g., in the food industry, in the cosmetic industry, or for pharmaceutical purposes.

[0029] In certain embodiments such as those exemplified herein, the phytochemical extracted from said macroalgae is a peptide, protein, e.g., an active site-containing protein (i.e., a non- structural protein including, without being limited to, an antibody, protein antigen, enzyme, protein substrate or inhibitor, receptor, and lectin), or a combination thereof. In particular such embodiments, the treatment applied according to the method of the invention is non-thermal, i.e., the temperature of the solvent developed during said, PEF or CEF treatment is in the range of about 25°C to about 90°C, preferably about 25°C to about 50°C or 40°C, such that said treatment does not affect the functional properties of the product(s) extracted, i.e., said peptide, protein or protein combination does not denature, and its functional properties are preserved.

[0030] In certain embodiments, the method of the present invention further comprises the step of filtering said solvent, after the PEF or CEF treatment, so as to remove macroalgae debris from said solvent. Filtration can be carried out by any technique known in the art, as long as it does not harm the phytochemical being dissolved in said solvent.

[0031] In certain embodiments, the present invention relates to a method as defined in any one of the embodiments above, for the extraction of a peptide or protein, e.g., an active site-containing protein, from a macroalgae, the method comprising applying non-thermal PEF treatment to said macroalgae in a solvent, under pressure higher than the ambient pressure, thereby extracting said peptide or protein from said macroalgae cells into said solvent. In certain particular such embodiments, the non-thermal PEF treatment applied is characterized by at least one of: (i) pulse number of from 1 to about 10,000; (ii) pulse duration of from about 50 ns to about 10 ms; (iii) electric field strength of from about 0.1 to about 100 kV/cm; and (iv) pulse frequency of from 0.1 to about 10000 Hz. In other particular such embodiments, the protein extracted by the method of the invention is an active site-containing protein, and the PEF treatment applied does not affect the functional properties of said active site-containing protein. In further particular such embodiments, the solvent in which said macroalgae is treated is fresh water, tap water, saline, or salt water (seawater).

[0032] In certain embodiments, the method of the present invention, according to any one of the embodiments defined above, further comprises a step of separating said phytochemical from said solvent. Such a separation may be carried out utilizing any procedure or technique known in the art. For example, separation of said phytochemical may be carried out by column chromatography/separation, i.e., passing the solvent containing said phytochemical, after said PEF or CEF treatment, through a separation column comprising binding means having affinity to said phytochemical, to thereby bind said phytochemical; optionally washing said column to remove unspecific bound molecules; and releasing said phytochemical from said binding means.

[0033] Alternatively, the separation may be carried out by introducing binding means having affinity to said phytochemical into said solvent either prior to or after said PEF or CEF treatment, to thereby bind said phytochemical upon extraction from said macroalgae cells; isolating said binding means after said PEF or CEF treatment; and optionally releasing said phytochemical therefrom. In certain embodiments, the binding means referred to herein have functional groups capable of reversibly interacting with functional groups of said phytochemical, thus trapping said phytochemical. In other embodiments, separation of said phytochemical is carried out by introducing binding means having affinity to said phytochemical into said solvent, and said binding means are in the form of beads. In particular embodiments, (i) said phytochemical is a peptide or protein, e.g., an active site-containing protein, and said binding means comprise antibodies to said peptide or protein; (ii) said phytochemical is an enzyme and said binding means comprise a substrate or inhibitor of said enzyme; (iii) said phytochemical is a small molecule and said binding means comprise ionic functional groups capable of interacting with ions of opposite charge of said phytochemical; or (iv) said phytochemical is a sugar and said binding means comprise a lectin.

[0034] The method of the present invention may be carried out within any suitable chamber, vessel or reactor. In certain embodiments, the method of the invention comprises the steps of: (i) introducing said macroalgae submerged in said solvent into a PEF- or CEF- treatment chamber; (ii) subjecting said macroalgae to a PEF- or CEF-treatment under pressure higher than the ambient pressure, thereby electroporating the cells of said macroalgae and consequently extracting said phytochemical from said macroalgae cells into said solvent; and (iii) optionally filtering said solvent to remove macroalgae debris therefrom.

[0035] In one particular such embodiment, said PEF- or CEF-treatment chamber is a fluidized bed chamber. Such a method may further comprise introducing binding means, e.g., in the form of beads, having affinity to said phytochemical, into said solvent either prior to or after step (ii), to thereby bind said phytochemical upon extraction from said macroalgae cells; isolating said binding means from said solvent; and optionally releasing said phytochemical therefrom. Alternatively, such a method may further comprise passing said solvent, after said PEF- or CEF-treatment, through a separation column comprising binding means having affinity to said phytochemical, to thereby bind said phytochemical; optionally washing said column to remove unspecific bound molecules; and releasing said phytochemical from said binding means.

[0036] In another particular such embodiment, said PEF- or CEF-treatment chamber is a centrifugal chamber. In such a method, the pressure inside the treatment chamber that is required so as to extract said phytochemical from said macroalgae into said solvent, i.e., a pressure higher than the ambient pressure, in fact results from the centrifugal force created by the rotation of said centrifugal chamber during said PEF- or CEF-treatment, and the solvent (more specifically part of the solvent) containing the extracted phytochemical is continuously separated during the process as a result of said centrifugal force.

[0037] In another aspect, the present invention relates to a macroalgae phytochemical obtained by the method of the present invention as defined in any one of the embodiments above. The macroalgae phytochemical of the invention may be a peptide, protein, sugar, small molecule, or a combination thereof. In certain embodiments, said phytochemical is a peptide or protein, e.g., an active site-containing protein such as an antibody, protein antigen, enzyme, protein substrate or inhibitor, receptor, and lectin. Considering the characteristics of the process by which the phytochemical, e.g., peptide or protein, is obtained, said phytochemical is expected to be in its natural form, i.e., in the form it exists within the macroalgae cells.

[0038] In yet another aspect, the present invention provides a device for the extraction of a phytochemical from a macroalgae, said device comprising: (i) a pulsed electric field (PEF)- or constant electric field (CEF)-treatment chamber comprising at least one inlet and at least one outlet; (ii) a first electrode and a second electrode positioned for creating an electric field in said treatment chamber; (iii) optionally a pressure generating mean for generating pressure within said treatment chamber; (iv) optionally, an agitator and/or air bubbling unit for agitating and mixing the content of said treatment chamber; and (v) optionally, a filtering unit located at the outlet, wherein upon introducing said macroalgae in a solvent into said treatment chamber, and applying PEF- or CEF-treatment to said solvent under pressure higher than the ambient pressure, said phytochemical is extracted from said macroalgae into said solvent.

[0039] In certain embodiment, the device of the present invention comprises a CEF treatment chamber, and CEF treatment, e.g., constant DC, is applied under pressure higher than the ambient pressure. In other embodiment, the device of the invention comprises a PEF treatment chamber, and PEF treatment, more particularly non-thermal PEF treatment, is applied under pressure higher than the ambient pressure.

[0040] The device of the present invention comprises a first and second electrodes positioned for creating an electric field in said PEF- or CEF-treatment chamber. In particular embodiments, these electrodes are designed to provide different electric field at different locations of the electrodes, so as to save energy during said PEF- or CEF- treatment. In certain embodiments, both of said electrodes are positioned inside said treatment chamber. In other embodiments, one of said electrodes is positioned inside said treatment chamber, and the other one is positioned outside said treatment chamber. In further embodiments, both of said electrodes are positioned outside said treatment chamber. In yet another embodiment, said first and second electrodes are each constructed of a plurality of electrodes positioned such as to create an optimal electric field within said treatment chamber.

[0041] The device of the present invention as defined in any one of the embodiments above may further comprise a thermostat for measuring the temperature of said solvent during said PEF- or CEF-treatment; and a temperature controlling unit associated with said thermostat and capable of either (a) interrupting with said PEF- or CEF-treatment, and/or modulating one or more characteristics of said PEF- or CEF-treatment such as pulse number, pulse duration, electric field strength, pulse frequency, or any combination thereof; or (b) controlling the operation of external cooling means aimed at cooling said treatment chamber.

[0042] Such a temperature controlling unit might be of particular importance in cases PEF treatment, more particularly non-thermal PEF treatment, is applied, and is aimed at controlling the temperature developed during said PEF treatment from exceeding a particular threshold that might be detrimental to the product (phytochemical) extracted, i.e., keeping the solvent temperature in the range of 25°C-90°C, 25°C-70°C, 25°C-60°C, 25°C-50°C, or 25°C-40°C. In cases wherein the solvent temperature constantly measured by the thermostat reaches a particular level, said temperature controlling unit may either interrupt with said PEF- or CEF-treatment, i.e., stop said PEF- or CEF-treatment for a particular time period, until the solvent temperature cools down; or modulate, i.e., alter, one or more of said PEF treatment, e.g., decrease pulse delivery frequency, so as to avoid further increase in the solvent temperature. Alternatively, such a temperature controlling unit may control the operation of external cooling means, which have no connection with the CEF- or PER-treatment and are aimed at cooling the treatment chamber thus reducing the temperature inside the treatment chamber.

[0043] In certain embodiments, the device of the present invention in any one of the embodiments defined above comprises said treatment chamber; said electrodes; said pressure generating mean; optionally said agitator and/or air bubbling unit; and optionally said filtering unit. In one particular such embodiment, the treatment chamber of said device is a fluidized bed chamber.

[0044] In other embodiments, the device of the present invention in any one of the embodiments defined above comprises said treatment chamber; said electrodes; and optionally said filtering unit. In one particular such embodiment, the treatment chamber of said device is a centrifugal chamber, wherein said pressure higher than the ambient pressure results from the centrifugal force created by the rotation of said centrifugal chamber during said PEF- or CEF-treatment, and said solvent containing the extracted phytochemical is continuously separated as a result of said centrifugal force.

[0045] Figs. 4A-4C schematically illustrate certain configurations of a device for the extraction of a phytochemical from a macroalgae, as disclosed herein. Fig. 4A depicts such a device comprising a PEF/CEF treatment chamber having an inlet located at its upper part, for inserting the solvent in which said macroalgae is submerged; an outlet located at its bottom, from which said solvent containing the extracted phytochemical exits upon completion of the PEF/CEF treatment; two electrodes located inside, and on opposite sides of, the chamber, for creating/generating the electric field needed for said PEF/CEF treatment; a mechanical pressure generating mean, e.g., a piston, for generating a pressure higher than the ambient pressure within/inside said chamber during said PEF/CEF treatment; and an agitator for stirring said solvent during said PEF/CEF treatment. Fig. 4B depicts another device comprising a PEF/CEF treatment chamber in which each opposing electrode is, in fact, an electrode array consisting of a plurality of electrodes each capable of generating the same or different electrical current. Such an electrode configuration enables creating different electric fields at different areas of the treatment chamber, which might be beneficial in certain cases, e.g., to reduce the total power consumed by the device. The treatment chamber shown in Fig. 4B lacks an agitator; however, comprises an air inlet through which air is poured into the chamber so as to stir the solvent during the PEF/CEF treatment. Fig. 4C depicts a further device comprising a PEF/CEF treatment chamber that is, in fact, a centrifuge (centrifugal chamber), wherein the PEF/CEF treatment is carried out under pressure higher than the ambient pressure resulting from the centrifugal force created by the rotation of said centrifuge during the PEF/CEF treatment, and solvent containing the extracted phytochemical is continuously separated as a result of said centrifugal force and exits the centrifuge via outlets. The electrode configuration illustrated in the device shown in Fig. 4C comprises a first moving (top-down) electrode, and a second stationary electrode located inside the treatment chamber.

[0046] Fig. 5 shows a laboratory device as disclosed herein, based on the configuration illustrated in Fig. 4C, wherein the PEF/CEF treatment chamber is a centrifuge. As shown, the outlets through which the solvent containing the extracted phytochemical continuously exits the treatment chamber are positioned at the top of the chamber, thereby allowing the solvent containing the macroalgae to first undergo a PEF/CEF treatment under pressure higher than the ambient pressure, and only then move upward and exit the chamber.

[0047] In still another aspect, the present invention provides a system for the extraction and separation of a phytochemical from a macroalgae, said system comprising: (i) a device as defined in any one of the embodiments above, for the extraction of said phytochemical from said macroalgae into said solvent; and (ii) a separation unit comprising at least one inlet and at least one outlet, for separating said phytochemical from said solvent, wherein upon extracting said phytochemical from said macroalgae into said solvent, said solvent is optionally filtered for removal of macroalgae debris; and said phytochemical is then separated from said solvent in said separation unit.

[0048] In certain embodiments, the treatment chamber and the separation unit constituting the system of the present invention are fluidly connected. In particular such embodiments, said treatment chamber and separation unit are fluidly connected via a oneway valve system.

[0049] In certain embodiments, the separation unit of said system comprises binding means having affinity to the phytochemical of interest, for reversibly binding said phytochemical. Such binding means may have functional groups capable of reversibly interacting with functional groups of said phytochemical. Such a separation unit may be, e.g., a chromatography, i.e., separation, column, into which the solvent containing inter alia said phytochemical, obtained from the extraction process carried out in the treatment chamber, is poured. While passing through the column, the phytochemical of interest is adsorbed to the stationary phase, and after washing the stationary phase, said phytochemical is eluted using an appropriate eluent. Alternatively, said binding means having affinity to the phytochemical of interest are in the form of beads. Such beads may be added to the solvent obtained from the extraction process before, during or after introducing into the separation unit, and then treated outside the separation unit so as to release the bound phytochemical; or may be adhered to the wall of said separation unit.

[0050] In certain embodiments, the treatment chamber and the separation unit of the system of the present invention constitute the same chamber.

[0051] In certain embodiments, the separation unit of said system is a centrifuge. The option for separating the phytochemical of interest using a centrifuge might be relevant, e.g., in cases wherein the molecular weight of said phytochemical is substantially different, e.g., substantially higher, than the molecular weights of the other components extracted from said macroalgae cells into the solvent during the CEF- or PEF-treatment.

[0052] In certain embodiments, the separation unit of said system is a filtration system, such as a tangential flow system where the solution obtained from the extraction process flows through a feed channel and along (tangent to) the surface of the membrane as well as through the membrane, as described in detail in, e.g.,

[0053] Figs. 6A-6B schematically illustrate certain configurations of a system for the extraction and separation of a phytochemical from a macroalgae, as disclosed herein, comprising a device for the extraction of a phytochemical from a macroalgae, as defined in any one of the embodiments above including those schematically depicted in Figs. 4A-4C, and a separation unit for separating the extracted phytochemical from said solvent. Fig. 6A depicts such a system, wherein the separation unit is a chamber comprising binding means having affinity to said phytochemical, more specifically wherein said binding means are in the form of beads. According to the present invention, such beads may be introduced into the separation chamber prior to the separation step (as shown in the figure), either before or after introducing the solvent containing the extracted phytochemical; or may be adhered to the wall of the separation chamber (not shown). Fig. 6B depicts a different configuration, wherein the separation unit is a chromatography- or separation-column. The various technologies that might be utilized for separating the extracted phytochemical of interest from the solvent, depending on the particular separation unit used, are well-known to any person skilled in the art.

[0054] Unless otherwise indicated, all numbers expressing, e.g., PEF characteristics such as pulse number, pulse duration, electric field strength, and pulse frequency, pressure applied during the PEF/CEF treatment, or temperature developed in the solvent subjected to the PEF/CEF treatment during said treatment, used in the specification are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification are approximations that may vary by up to plus or minus 10% depending upon the desired properties sought to be obtained by the present invention.

[0055] The invention will now be illustrated by the following non-limiting Examples.

EXAMPLES

Experimental

[0056] Biomass material. Ulva biomass was obtained from Alga Plus Company, Portugal (cultivated in the certified integrated aqyaculture facility). Macroalgae were then stored for 2 days in a 400 L aquarium with a salt concentration of 3.5%.

[0057] PEF treatment. The fresh biomass was centrifuged at about 840 rpm for 3 runs of 1 min each to remove the external water, so that <5 g of water have been removed during the third run. 140 g of Ulva biomass in a 2 L becher was weighted with scale of type KERN 440-49N. This amount of macroalgae was then poured and pushed into the PEF treatment chamber with a volume of 232 cm ³ for the application of a homogeneously distributed pulsed electric field. Water was added to the macroalgae to fill the chamber completely. The chamber was closed to begin the PEF treatment. After the PEF treatment was applied, the macroalgae were collected and returned to the becher. The electroporated macroalgae were weighted again. The treatment parameters were:

average field strength (average was taken between all pulses at all repeats, 225 measurements) 2.964+0.007 kV cm ^-1 , and pulse duration 5.70+0.30 μ8, delivered at 0.5 Hz. These parameters were chosen based on the previous studies with PEF dehydration of various types of biomass with this system (Sack et al., 2009; Sack et al., 2010a; Sack et al., 2010b; Sack et al., 2008). Temperature was measured with a digital thermometer (TFA Type 30.1018). Current and voltage across the electrodes of the treatment chamber during each pulse were measured with a current probe (PEARSON 110 A) and a voltage divider (HILO-Test HVT 240 RCR), both connected to an oscilloscope (Tektronix TDS 640A). The impedance of the treated sample was derived from the current and voltage measurements.

[0058] Energy consumption. The total energy consumed for the PEF treatment was calculated based on the energy stored in the pulse capacitor with the following Eq. 1 :

E _t = 0.5 * C * 10 ^~9 * (V * 10 ³ ) ² * N Eq.l where Et (J) is the total energy consumed for the treatment of one treatment chamber, C is the discharging capacitor capacitance (nF), V (kV) is the applied voltage and N is the total number of pulses. Additional losses of the capacitor charger have not been considered.

[0059] The energy consumed with PEF for protein extraction was than calculated with Eq. 2:

E _P = ¾ /(C _p * m _P£F ) Eq.2

where Ep (kWh kg ^"1 ) is the PEF energy required to extract 1 g of protein, Et (kWh kg ^"1 ) is the total energy consumed to treat the PEF chamber, m (kg) is the raw mass of treated macroalgae, C _p (kg mL ^"1 ) is the concentration of the proteins in the extracted juice, and mPEF (mL) is the volume of the extracted juice.

[0060] Mechanical juice extraction. The electroporated algae were placed in a cloth material that was folded so that the algae could not escape during pressing. The algae wrapped up in the fabric were placed in the mechanical press (HAPA type SPM 2.5S). A force of 45 daN cm ^"2 was applied for a determined time of 5 min using the automatic mode of the press that keeps the pressure applied to the piston constant. Extracted juice from pressing was collected in a 2 L becher and weighted at the end of the pressing. The pressed material was taken out of the press, weighted, reorganized and put back into the press for a second pressing step. The extracted juice and the pressed algae were again weighted.

[0061] Crude protein quantification . Bovine serum albumin (BSA) in DDW calibration curve was done in following concentrations: 1 mg/mL, 500 μg/mL, 250 μg/mL, 200 μg/mL, 150 μg/mL, 100 μg/mL, 50 μg/mL, 25 μg/mL and 0 μg/mL. Every 10 μί BSA concentration mixed with 115 μΐ _^ Bradford buffer. Extracted juice was filtered with 0.2 μιη filter, 10 μΐ _^ of samples mixed with 115 μΐ _^ Bradford buffer. The BSA concentrations and the extracted juice samples with Bradford buffer measured in optical density (OD) 450 nm and 590 nm. The numbers of OD 450 parts 590, were the basis for linear calibration curve. OD detection was done with an EL808, BioTek spectrophotometer (Winooski, VT, USA).

[0062] Gel electrophoresis . Extracted juice from pressing with/without PEF treatment was filtered with a 0.2 μιη filter. Protein precipitation was made: 1 volume of trichloroacetic acid (TCA) 100% (w/v) added to 4 volumes extracted juice samples. Then moved to 1.5 mL tubes. The samples incubated in 10 min at 4°C. The tube spin in microcentrifuge at 14K rpm, 5 min. Supernatant was removed, protein left in the pellet intact. Pellet was washed with 200 μΐ cold acetone. Spin in microfuge at 14K rpm, 5 min. We made total of 2 acetone washes. Pellet was dried by placing tube in 95°C heat block for 5- 10 min. SDS-PAGE, 4X sample buffer (with βΜΕ) and sample boiled for 10 min in 95°C heat block. The samples were loaded onto polyacrylamide Gel. Samples run on SDS gel 12% agarose 200V, 30 min.

Extracted proteins identification quantification with LS-MS/MS.

[0063] Proteolysis . 200 μΐ _^ of the samples was brought to 8M Urea. The protein in 8M Urea was reduced with 2.8 mM DTT (60°C for 30 min), modified with 8.8 mM iodoacetamide in 100 mM ammonium bicarbonate (in the dark, room temperature for 30 min) and digested in 2M Urea, 25 mM ammonium bicabonate with modified trypsin (Promega) at a 1 :50 enzyme-to-substrate ratio, overnight at 37°C. One microgram from each sample was injected into a LC-MS/MS device.

[0064] Mass spectrometry analysis. The tryptic peptides were desalted using C 18 tips (Homemade stage tips) dried and re-suspended in 0.1% formic acid. The peptides were resolved by reverse-phase chromatography on 0.075x180-mm fused silica capillaries (J&W) packed with Reprosil reversed phase material (Dr Maisch GmbH, Germany). The peptides were eluted with linear 60 min gradient of 5-28% 15 min gradient of 28-95% and 15 min at 95% acetonitrile with 0.1% formic acid in water at flow rates of 0.15 μΐ/min. Mass spectrometry was performed by a Q Exactive plus mass spectrometer (Thermo) in a positive mode using repetitively full MS scan followed by collision induces dissociation (HCD) of the 10 most dominant ions selected from the first MS scan.

[0065] Computational analysis. The mass spectrometry data was analyzed using either the MaxQuant software 1.5.1.2 (Mathias Mann's group) or Peaks 7 software (Bioinformatic Solutions). The analyses were done vs. the green algea section of the NCBI-nr database with 1% false discovery rate (FDR). The data was quantified by label free analysis using the same software. Intensity parameter mean: Summed up extracted Ion Current (XIC) of all isotopic clusters associated with the identified AA sequence. For specific protein quantification, the intensity measured for each protein was normalized to the total intensity of all proteins from the same sample.

[0066] Statistical analysis. Statistical analysis was performed with Excel (ver. 13, Microsoft, WA) Data analysis package. All experiments and controls were done in triplicates unless stayed differently. Standard error of the mean (SEM) is shown in error bars. One side Student's t-test was performed for compare the total protein extraction yield to controls. MS statistical analysis for proteins identification was done as described in Computational analysis. The criterion for inclusion was that the same protein was identified in at least two repeats from three.

Study 1. Extraction of proteins extraction from macroalgae Viva with PEF Process of proteins extraction from macroalgae Ulva with PEF

[0067] First we analyzed the shape of the individually delivered electric pulse. Because of the cell membrane electroporation, the resistance of the treated macroalgae biomass reduces. Therefore we expected mild changes in the shape and pick values of each individual pulse. Fig. 1 shows the shape of delivered voltage and current at the first (Ul, II) and last (U_last, I_last) pulse in the delivered series of 75 pulses. The pulse source has been designed such, that a series of pulses of equal energy are applied. A pulse circuit based on a capacitor discharge has been employed. For a series of pulses the charging voltage of the capacitor has been kept constant. Pulses with an aperiodically damped shape are applied to the load. The peak current of the pulse is influenced by the resistance of the electrode system inside the treatment chamber and the stray inductance of the pulse circuit. The resistance of the treatment chamber decreases with the number of applied pulses, as discussed later. As a consequence, the pulse shape changes with the decreasing resistance, and the voltage across the electrode system decreases slightly with increasing number of pulses. PEF parameters and changes in the macroalgae biomass during extraction

[0068] Next, we analyzed the changes in the peak electric field and current during the whole treatment (Fig. 2). We observed the decrease of the actual delivered electric field strength per pulse and increase of the current per pulse. These changes are expected because of treated media conductivity increase (Fig. 2 shows the decrease in the resistance), which follows cell membrane electroporation and release of intracellular cell content. In the application of 75 pulses on the biomass, the actual peak electric field decreased from 3.215+0.033 kV cm ^"1 at the beginning of the treatment (first five pulses) to 2.864+0.040 kV cm ^"1 at the end of the treatment (last five pulses in the series). The current increased from 713.6+16.8 A at the beginning of the pulse series (first five pulses) to 1173.86+30.8 A. These changes in the actual electric field and current are explained by the 46% drop of the sample resistance during the application of pulsed electric fields. Interesting, we observed that the major decrease in the resistance (35%) and increase in the current from 713.6+16.8 A to 1024+26.7 A took place during the first twenty pulses. This is probably the number of pulse required to electroporation the majority of cells in the treated Ulva thalli. Previously, we have observed similar pattern of rapid resistance decrease and current increase in the skin tissue, where electric fields also electroporated cells inside the complex extracellular matrix (Golberg et al., 2013) and also in sugar beet tissue (Bluhm and Sack, 2008).

Extracted proteins yield

[0069] With the treatment parameters used in this study the average electric field strength applied on the macroalgae thalli was 2.964+0.007 kV cm ^"1 , the pulse duration was 5.70+0.30 μ8. The total dry matter consisted 5.52+0.20% of the total extracted juice weight (Fig. 3). PEF increased the total protein extraction yields (p <10 ^"6 ). The total protein concentration in the extracted juice was 59.13+3.82 μg μΐ ¹ , which is in contrast to 23.80+1.33μg μΐ ¹ observed in the control samples (Fig. 3). Importantly, the developed process has almost no thermal effects on the produced proteins as the maximum observed temperature was 35.50+2.02°C.

PEF process energy consumption

[0070] An important parameter in the production of proteins for food and feed applications is the energy consumptions. Non-thermal pulsed electric field has been shown previously to reduce the total energy consumption of the sugar extraction by 30-50% at the industrial scale (Bluhm and Sack, 2008; Sack et al, 2010a; Sack et al, 2010b). Here we analyzed the energetic consumption of the pulsed electric field process for water soluble solids, including proteins, extraction from macroalgae Ulva. The specific energy consumed relative to raw mass of macroalgae was 8.56+0.01 Wh kg ^"1 (30.81+0.03 kJ kg ^"1 ) and the specific energy relative to the extracted proteins was 251+3 kWh kg ^"1 .

PEF extracted proteins identification

[0071] Previous works has shown the use of PEF to extract proteins from microalgae (Goettel et al., 2013; Parniakov et al., 2015), yeast (Ganeva and Galutzov, 1999; Ganeva et al, 2003), bacteria (Haberl Meglic et al, 2015) and plants (Bluhm and Sack, 2008; Doevenspeck, 1961; Sack and Bluhm, 2008; Vorobiev and Lebovka, 2010; Zagorulko, 1958). However, to the best of our knowledge these were reported as crude protein extraction. In this work we identified and quantified specific proteins extracted from Ulva genus with PEF using LC/MS/MS (Table 1 shows all identified proteins in at least one sample). Proteins that have been uniquely identified in the PEF treated samples extracts appear in Table 2 (showing proteins that have been identified in at least two repeats from three). Proteins detected only in samples from Ulva biomass treated only with water, appear in Table 3 (showing proteins that have been identified in both of the two repeats). These proteins were not observed in the PEF treated samples and probably have been damaged by electric fields. Table 4 lists the proteins that have been detected in both PEF treated and non-treated samples. Interesting, we observed that besides Plastocyanin precursor, PEF reduced the quantities of proteins extracted from Ulva biomass with the tap water (based on intensity parameter). Our previous work with DNA showed that specific PEF parameters lead to DNA nicking (Golberg and Rubinsky, 2010). Additional studies showed that specific PEF parameters could lead to either activation or inactivation of enzymes, depending on enzyme type (Ohshima et al., 2007). However, most of the studies until today showed that PEF increases the extraction yields. Indeed, in our study PEF increased about 3 times the extracted proteins yields if measured in a bulk. However, the detailed identification and quantification of individual proteins, reported here for the first time, reveals a more complex scenario. Some proteins are extracted with PEF (Table 2 and Table 4), but some, which can be extracted with the tap water are partially (Table 4) or completely (Table 3) degraded by the treatment. To the best of our knowledge this is a first report that identified proteins extracted from biomass with PEF; previous studies used bulk proteins characterization (Coustets et ah , 2015; Ganeva et ah , 2003). These finding are new and important, as they open a possibility to optimize PEF parameters for the extraction of specific proteins from macroalgae and other biomass. An important future application could be the inactivation of the ingenious proteases during protein extraction processes. Additional future studies will address the effect of PEF extraction on the functional properties of the extracted proteins. PEF provides a potentially unique nonthermal, chemicals-free proteins extraction method that could preserve the functional properties of the proteins, important for food and pharmaceutical applications.

Conclusions

[0072] Macroalgae are promising, but challenging sustainable feedstock for biorefineries. Complete zero waste conversion of macroalgae into food, chemicals and fuels will reduce the burden of the agriculture from arable land. The present study provides a new technology for the extraction of green macroalgae Ulva proteins with electroporation by PEF, which is an emerging, energy efficient technology for biomass processing. The present study shows that PEF increases by about three times the total protein extraction, and is selective, as it increases the extraction yields of some specific proteins but damages others. This study demonstrates the scalable, energy efficient technology for extraction essential for food supply chemicals-proteins, from macroalgae.

Table 1. Proteins identified in at least one sample (+/-PEF treated samples/controls)

acetabulum Table 2. Proteins detected only after PEF treatment

Table 3. Proteins detected with water without PEF treatment Table 4. Proteins detected in samples with PEF and without PEF treatment

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