WO2025170901A1 - Methods and systems for extracting photopigments - Google Patents

Abstract

Methods and systems for extracting phycobiliproteins from seaweed, such as phycoerythrin, are generally described. In some embodiments, the phycobiliprotein extracted from seaweed is phycoerythrin, and the methods and systems described by this disclosure may extract phycoerythrin from seaweed while maintaining other compounds within the seaweed, such as bromoform.

Description

METHODS AND SYSTEMS FOR EXTRACTING PHOTOPIGMENTS

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 63/549,964, filed February 5, 2024, entitled “Methods and Systems for Extracting Photopigments,” incorporated herein by reference in its entirety.

TECHNICAL FIELD

Methods and systems for extracting phycobiliproteins, such as phycoerythrin, from seaweed are generally described. In some embodiments, the phycobiliprotein extracted from seaweed comprises phycoerythrin, and the methods and systems described by this disclosure may be used to extract phycoerythrin from seaweed while maintaining other compounds within the seaweed, such as bromoform.

BACKGROUND

Phycoerythrin (PE) extraction conventionally involves breaking down cell walls of red algae or cyanobacteria through methods like mechanical disruption, enzymatic treatment, or solvent extraction. Once released, the pigment is purified, opening avenues for diverse applications. However, these cellular disruption methods, while effective in liberating phycoerythrin, can pose challenges for the utilization of other bioactive compounds within the cells. Said in another way, the harshness of these conventional methods can lead to the degradation or unwanted modification of coexisting compounds within cells containing the phycoerythrin.

In the case of seaweed, such as Asparagopsis taxiformis, Asparagopsis armata, or Halymenia hawaiiana, one bioactive compound of interest is bromoform. This is due to its methanogenic reducing properties. Conventional phycoerythrin extractions techniques greatly affect the final content of bromoform in the harvested biomass, typically being much lower than what is desired. Accordingly, improved extraction methods for seaweed are desired.

SUMMARY

Methods and systems for extracting phycobiliproteins from seaweed, such as phycoerythrin, are generally described. In some embodiments, the phycobiliprotein extracted from seaweed is phycoerythrin, and the methods and systems described by this disclosure may extract phycoerythrin from seaweed while maintaining other compounds within the seaweed, such as bromoform. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.

In one aspect, a method for extracting phycoerythrin from seaweed is described, the method comprising freezing seaweed in a confined volume; thawing the seaweed; and extracting phycoerythrin from the thawed seaweed.

In another aspect, a method for extracting a phycobiliprotein from biomass is described, the method comprising freezing the biomass in a confined volume; thawing the biomass; extracting phycobiliprotein from the thawed biomass; and forming the thawed biomass into a solid mass.

Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the figures:

FIG. 1 is a plot showing the bromoform content of different treatments, where the baseline is the harvested biomass with no further manipulation, the control is the biomass subjected to the freeze-thaw cycle but without preceding with the PE extraction methods, and where PE is the biomass subjected to all the processes mentioned above, according to some embodiments; and

FIG. 2 shows a schematic diagram of one embodiment of a freezing arrangement that slows or retards the rate of freezing, where such an arrangement may provide a simple and economic way in some cases to slow down the freeze rate during that stage of the cycle in the extraction process.

DETAILED DESCRIPTION

The following disclosure describes methods and systems for extracting one or more phycobiliproteins, such as phycoerythrin, from biomass (e.g., seaweed) without damaging, or otherwise degrading, other compounds in the biomass. As mentioned elsewhere herein, conventional methods for extracting phycobiliproteins from biomass may disadvantageously degrade other compounds within the biomass. However, the Inventors have recognized and appreciated that certain phycobiliproteins, such as phycoerythrin, can be extracted from seaweed while preserving desirable compounds within the seaweed, such as bromoform. Advantageously, by extracting phycoerythrin from the seaweed while preserving the bromoform (or other compounds) in the seaweed, the phycoerythrin can be concentrated used for a variety of application, while the bromoform that remains in the seaweed may provide useful properties to the seaweed. For example, without wishing to be bound by any particular theory, it is believed that improved levels of bromoform in the seaweed may beneficially reduce an amount of methane produced that results from consumption of the seaweed, for example, by cattle. This can reduce the amount of methane released into the atmosphere by replacing feedstocks that result in increased concentrations of methane in the atmosphere with seaweed (or other bromoform-containing feedstocks).

The Inventors have appreciated that applying freezing and thawing to the biomass (e.g., seaweed) may enhance the extraction of a phycobiliprotein (e.g., phycoerythrin) while preserving other compounds within the biomass (e.g., bromoform). In some embodiments, freezing and/or thawing is applied to the biomass one or more times and may be combined with other processes, such as centrifugation, filtration, and so forth, in order to extract the phycobiliprotein from the biomass.

One non-limiting example of biomass that may be used is seaweed (also known as macroalgae). Seaweed includes a variety of macroscopic, multicellular, marine algae. One example of macroalgae include those belonging to the genus Asparagopsis, also known as the red macroalgae. Many such macroalgae are edible. Non-limiting examples of seaweed within the red macroalgae genus include Asparagopsis taxiformis or Asparagopsis armata. Various cultivars have been developed, including the Icarus variety described in US Plant Patent No. PP 34,510, incorporated herein by reference.

Other examples of macroalgae that may be enhanced include, but are not limited to, macroalgae in the following: Acinetosporaceae, Agaraceae, Ahnfelitaceae, Alariaceae, Arthrospira, Bangiaceae, Bonnemaisoniaceae, Caulerpacaea, Chlorella, Chordariaceae, Cladophoraceae, Codiaceae, Compsopogonales, Cyanidioschyzonaceae, Cyanidiaceae, Cylindrospermum, Delesseriaceae, Desmarestiaceae, Dictyotaceae, Dumontiaceae, Erythropeltales, Fucaceae, Furcellariaceae, Gelidiellacaeae, Gigartinaceae, Gracilariaceae, Halimedacaea, Haylmedacaea, Faminariaceae, Naccariaceae, Nannochloropsis, Palmariaceae, Phaeophyceae, Phyllophoraceae, Phragmonemataceae, Porphyridiaceae, Pterocladiaceae, Rhodomelaceae, Rhodochaetales, Rhizophyllidacaeae, Rhodomelaceae, Rufusiaceae, Sargassacaea, Sphacelariaeceae, Stylonemataceae, or Ulvaceae. Other examples include other types of red seaweed, such as those in Kappaphycus . Yet other examples include, but are not limited to, Halymenia hawaiiana, or other members of Halymenia. In addition, in some cases, more than one type of macroalgae may be present in an aquaculture setting.

In some cases, the seaweed or macroalgae may be cultivated, or “farmed,” e.g., in an aquaculture setting for a variety of uses, such as food, animal feed, biofuel, raw materials for chemical production, or the like. In some cases, the aquaculture may be present in an artificially constructed system. Examples include but are not limited to, tanks, aquariums, artificial ponds, canals, etc. In addition, in some embodiments, the aquaculture may be present in an ocean-based system (e.g., in shallow waters of the ocean, enclosed sections of ocean water, etc.). For example, the macroalgae may be cultivated attached or unattached to a substrate, e.g., in enclosed sections of open ocean, farms in littoral waters, in artificially constructed tanks, aquariums, ponds, canals, etc. that contain ocean water, or the like.

As noted above, the biomass may be frozen, which facilitates at least partial breakdown of cell walls within the biomass. However, unlike some conventional techniques such as mechanical disruption, enzymatic treatment, and/or solvent extraction, freezing preserves some compounds (e.g., bromoform) while allowing the phycobiliproteins to exit the biomass. Freezing can be accomplished using conventional techniques, such as commercial freezers and/or temperature baths of an appropriate temperature (e.g., approximately 0 °C, approximately -20 °C). In some embodiments, the biomass may be contained within a confined volume before freezing. For example, the biomass may be placed in a container, e.g., containing water, which can then be frozen as discussed herein. For example, the container may be positioned within a commercial freezer, a temperature bath, or the like. The container may, for example, be relatively rigid (e.g., a plastic box) or soft (e.g., a plastic bag). In some cases, the container may be relatively impermeable to the media, e.g., to prevent media from interacting with the biomass. For example, biomass may be placed in the container and the container sealed to prevent media from interacting with the biomass during the freezing process.

In some cases, the container may be contained within a freeze -retarding (or freeze- slowing) media. Such media may be used to control the rate of freezing, or in some cases, the uniformity of freezing (e.g., for a duration greater than or equal to 1 hour, greater than or equal to 2 hours, greater than or equal to 4 hours, etc.). Nonlimiting examples of freeze retarding media include buffers such as any of those described herein, water, aqueous solutions, solutions that exhibit freezing points below 0 °C, for example, antifreeze solutions (e.g., comprising ethylene glycol, propylene glycol, glycerol, or the like, e.g., in water), seawater, or the like. Without wishing to be bound by any theory, it is believed that such systems may be helpful to control the rate of freezing of the biomass. A non-limiting example of such a system is schematically shown in Fig. 2, wherein biomass is contained within a container, which is contained within a freeze-retarding media.

In some embodiments, a rate of freezing is monitored and/or controlled. In some such embodiments, a freezing rate is greater than or equal to 0 °C/hour, greater than or equal to 0.1 °C/hour, greater than or equal to 0.2 °C/hour, greater than or equal to 0.3 °C/hour, greater than or equal to 0.4 °C/hour, or greater than or equal to 0.5 °C/hour. In some embodiments, the freezing rate is less than or equal to 0.5 °C/hour, less than or equal to 0.4 °C/hour, less than or equal to 0.3 °C/hour, less than or equal to 0.2 °C/hour, or less than or equal to 0.1 °C/hour. Combinations of the above-referenced ranges are also possible (e.g., greater than or equal to 0 °C/hour and less than or equal to 0.5 °C/hour). Other ranges are possible.

After freezing, the biomass may be thawed. Thawing may occur, for example, by exposing the frozen biomass to ambient conditions and/or may be facilitated by exposing the frozen biomass to an appropriate temperature bath (e.g., set at approximately 20 °C or approximately 25 °C) to facilitate warming of the frozen biomass to form thawed biomass. For example, the temperature bath may contain water, saline, seawater, or another fluid, which is used to heat the biomass.

Other non-limiting examples of thawing methods include exposure to heat lamps, electrically-based heating methods, exposure to sunlight (e.g., direct sunlight), exothermic chemical reactions, radiative heat (e.g., IR lamps), or the like.

In some embodiments, the rate of heating may be monitored and/or controlled. In some embodiments, a thawing rate is greater than or equal to 0.3 °C/hour, greater than or equal to 0.5 °C/hour, greater than or equal to 0.6 °C/hour, greater than or equal to 0.7°C/hour, greater than or equal to 1 °C/hour, greater than or equal to 1.3 °C/hour, greater than or equal to 1.5 °C/hour, greater than or equal to 1.7 °C/hour, greater than or equal to 2 °C/hour, greater than or equal to 2.3 °C/hour, greater than or equal to 2.5 °C/hour, greater than or equal to 2.7 °C/hour, or greater than or equal to 3 °C/hour. In some embodiments, the thawing rate is less than or equal to 3 °C/hour, less than or equal to 2.7 °C/hour, less than or equal to 2.5 °C/hour, less than or equal to 2.3 °C/hour, less than or equal to 2 °C/hour, less than or equal to 1.7 °C/hour, less than or equal to 1.5 °C/hour, less than or equal to 1.3 °C/hour, less than or equal to 1 °C/hour, less than or equal to 0.7 °C/hour, less than or equal to 0.6 °C/hour, less than or equal to 0.5 °C/hour, or less than or equal to 0.3 °C/hour.

Due in part to the photosensitivity of certain phycobiliproteins, in some embodiments, freezing and/or thawing (or other steps in the method) are performed in the absence of light (e.g., in a dark room).

In some embodiments, extraction of the phycobiliprotein from the biomass is facilitated by an extraction solution. The extraction solution may comprise a buffer (e.g., an acid and its conjugate base; a base and its conjugate acid). Many know buffers are suitable. Non-limiting examples of buffers include phosphate buffers, such as Na2HPO4 KH2PO4, acetate buffers, or the like. Other buffers are possible, for example, phosphate buffered saline. Other examples of extraction solution include, but are not limited to, ethylenediamine tetra-acetic acid (EDTA), water, or the like. In addition, other methods of extracting phycobiliprotein from the biomass can be used, e.g., instead of or in addition to solution-based extraction techniques. Without wishing to be bound by any theory, it is believed that certain phycobiliproteins, such as phycoerythrin, are relatively water-soluble, and can be extracted from biomass upon exposure to an extraction solution, such as an aqueous solution.

In some embodiments, the biomass extract is filtered to separate the solution (e.g., the extraction solution) from the remaining solid biomass. For example, the solution may be passed through a 0.1 micrometer filter or a 0.2 micrometer filter.

In some embodiments, this solution is further concentrated using techniques known to those of ordinary skill in the art (e.g., solvent evaporation, lyophilization, centrifugation, ultrafiltration, reverse osmosis, crystallization, or the like).

Once one or more phycobiliproteins have been extracted from the biomass, the remaining biomass (e.g., extracted biomass) may retain useful compounds (e.g., bromoform). By contrast with conventional extraction methods (e.g., mechanical disruption, enzymatic treatment, and/or solvent extraction), the methods described herein may retain useful compounds at concentrations similar to a concentration of useful compounds prior to extracting the phycobiliproteins from the biomass.

Phycobiliproteins (e.g., extracted phycobiliproteins) find a variety of uses, such as dyes and coloring agents. For example, phycoerythrin is a photopigment found in red algae and cyanobacteria and it imparts a distinctive red color to these organisms and plays a vital role in photosynthesis by absorbing light in the blue and green spectrum. Beyond its natural role, phycoerythrin is also used in anthropogenic applications, such as fluorescence labeling in biomedical research and other scientific and technological fields.

In some cases, the biomass may be prepared for uses such as for food, animal feed, biofuel, raw materials for chemical production, or the like. In one set of embodiments, for example, the biomass may be dried and/or pressed to form a solid material, for example, tablets or pellets, e.g., for use as animal feed (e.g., for cattle).

In some cases, the solid material may have a concentration of bromoform in the biomass of at least 0.001 mg/g, at least 0.002 mg/g, at least 0.003 mg/g, at least 0.005 mg/g, at least 0.01 mg/g, at least 0.02 mg/g, at least 0.03 mg/g, at least 0.05 mg/g, at least 0.1 mg/g, at least 0.2 mg/g, at least 0.3 mg/g, at least 0.5 mg/g, at least 1 mg/g, etc.

U.S. Provisional Patent Application Serial No. 63/549,964, filed February 5, 2024, entitled “Methods and Systems for Extracting Photopigments,” is incorporated herein by reference in its entirety.

The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention. EXAMPLE 1

The following example describes extracting phycoerythrin (PE) from seaweed of the genus Asparagopsis. By carefully manipulating the freeze-thaw parameters such as PE was either (1) extracted into saline buffer through freeze-thaw or (2) fractionated during oil emulsification of the seaweed. In either case, there was an aqueous buffer solution used to extract the PE from the seaweed.

Extraction methods

(1) Extraction into saline buffer through freeze-thaw:

• Phosphate buffered saline (PBS) solution: o Prepared in DI and/or RODI/MilliQ

■ 0.137 M NaCl

■ 2.68 mM KC1

■ 10.1 mM Na₂HPO₄

■ 1.80 mM KH₂PO₄

■ pH adjusted to 8.20 with NaOH

■ Salinity 17 ppt

The seaweed was placed in PBS pH 8.20 and salinity 17 ppt, vacuum sealed, frozen for 16 hours, submerged in deep sea water (a freeze-retarding agent) at temperatures of -20 °C. The seaweed was then thawed for 8 hours at 4 °C. Freezing and thawing procedures were completed in the dark to reduce photodegradation of the PE.

Thawed seaweed was either (i) placed into a mesh bag and squeezed into a collection vessel containing saline buffer or (ii) placed into a mesh bag and pressed over a dry collection vessel.

(2) Fractionation during oil emulsification:

The seaweed was spun and frozen. Thawed seaweed was incubated in oil at temperatures 36 °C, 46 °C, and 60 °C for 30 to 1440 minutes. Fractionated products were obtained by squeezing the infused seaweed through a mesh bag. PE is water soluble and extracted into the water fraction. The bottom water layer containing PE was collected.

Processing and Purification methods of extracted PE Extracted PE was filtered through a 0.2 micrometer filter to remove debris or bits of the seaweed. The remaining PE was concentrated using a centrifuge spin filter of 30 kDa, scalable using tangential flow filtration.

To streamline purification to 1 step, purification used ion-exchange chromatography and buffer exchange for the final formulation of the PE. Low purification fractions were saved and formulated for food and textile uses.

While several embodiments of the present disclosure have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present disclosure is/are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and/or methods, if such features, systems, articles, materials, and/or methods are not mutually inconsistent, is included within the scope of the present disclosure.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and/or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. Some embodiments may be embodied as a method, of which various examples have been described. The acts performed as part of the methods may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include different (e.g., more or less) acts than those that are described, and/or that may involve performing some acts simultaneously, even though the acts are shown as being performed sequentially in the embodiments specifically described above.

Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

CLAIMS What is claimed is:

1. A method for extracting phycoerythrin from seaweed, the method comprising: freezing seaweed in a confined volume; thawing the seaweed; and extracting phycoerythrin from the thawed seaweed.

2. The method of claim 1, wherein the thawed seaweed has a bromoform concentration of greater than or equal to 4 mg per gram of the seaweed.

3. The method of any one of the preceding claims, wherein a rate of freezing is controlled to retard the total freezing process.

4. The method of any one of the preceding claims, further comprising concentrating extracted phycoerythrin.

5. The method of any one of the preceding claims, further comprising removing a solvent from extracted phycoerythrin.

6. The method of any one of the preceding claims, wherein the freezing, thawing, and/or extracting steps occur in the absence of light.

7. The method of any one of the preceding claims, wherein the extracting step comprises extracting phycoerythrin into a buffered solution.

8. The method of any one of the preceding claims, wherein extracted phycoerythrin is filtered.

9. The method of any one of the preceding claims, further comprising forming the thawed seaweed into pellets.

10. A method for extracting a phycobiliprotein from biomass, the method comprising: freezing the biomass in a confined volume; thawing the biomass; and extracting phycobiliprotein from the thawed biomass.

11. The method of claim 10, wherein the phycobiliprotein comprises phycoerythrin.

12. The method of any one of claims 10 or 11, wherein the rate of thawing is greater than or equal to 0.3 °C/hour and less than or equal to 3 °C/hour.

13. The method of any one of claims 10-12, wherein the thawed biomass has a bromoform concentration of greater than or equal to 4 mg per gram of the biomass.

14. The method of any one of claims 10-13, further comprising concentrating extracted phycobiliprotein.

15. The method of any one of claims 10-14, further comprising removing a solvent from extracted phycobiliprotein.

16. The method of any one of claims 10-15, wherein the freezing, thawing, and/or extracting steps occur in the absence of light.

17. The method of any one of claims 10-16, wherein the extracting step comprises extracting phycobiliprotein into a buffered solution.

18. The method of any one of claims 10-17, wherein extracted phycobiliprotein is filtered.

19. The method of any one of claims 10-18, further comprising forming the thawed biomass into a solid mass.