A method for the purification of mycosporine-like amino acids from organisms for cosmetics and sunscreens

Abstract

The present disclosure describes methods to extract, separate and purify, from a microbial culture, or naturally occurring sources of MAA producing organisms, mycosporine like amino acids that are ultraviolet protective compounds, as well as antioxidants for use in cosmetics and sunscreens. The purification methods include, centrifugation, cell lysis, chemical extraction, as well as column chromatography that includes affinity chromatography using aptamers as the solid-phase and ion exchange or size exclusion chromatography.

Description

A METHOD FOR THE PURIFICATION OF MYCOSPORINE-LIKE AMINO ACIDS FROM ORGANISMS FOR COSMETICS AND SUNSCREENSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 559,368 entitled “A METHOD FOR THE PURIFICATION OF MYCOSPORINE-LIKE AMINO ACIDS FROM ORGANISMS FOR COSMETICS AND SUNSCREENS,” filed February 29, 2024, the disclosure of which is incorporated herein in its entirety by reference.BACKGROUND

[0002] Mycosporines and mycosporine-like amino acids (MAAs) are UV-absorbing metabolites initially isolated from fungi.SUMMARY OF THE DISCLOSURE

[0003] The present disclosure provides a method and process for purifying mycosporine like amino acids that absorb UV radiation and serve as an antioxidant at commercial / industrial scale for use in cosmetics, sunscreens and other therapeutic applications (e.g., anti-inflammatory agents, wound healing, anti-cancer treatments, etc.). The present disclosure recites a process of purification of MAAs using centrifugation to separate the biomass from extracellular material that may include MAAs excreted from the cell, cell lysis to obtain intracellular MAAs, and column chromatography for further purification at the industrial scale.

[0004] As mentioned above, mycosporines and mycosporine-like amino acids (MAAs) are UV-absorbing metabolites initially isolated from fungi. Beginning in the 1960’ s they have been increasingly identified in a broad range of organisms including cyanobacteria, algae, lichens, corals, dinoflagellates and sea stars. They have long been recognized as having commercial value for their UV protective properties by absorbing UV radiation in the harmful range from 309 to 362 nm and having a high molar absorptivity from 2.81 x 104to 5.00 x 104M^cm’1. They are also recognized as having anti-aging, anti-oxidant andanti-inflammatory properties. Given the range of organisms producing these compounds and the important cellular protection functions they provide, it is clear they are evolutionarily selected to protect cells from a variety of environmental and cellular stressors.

[0005] Mycosporine-like amino acids (MAAs) demonstrate significant UV-protective properties by absorbing UV radiation specifically in the harmful range from 309 to 362 nm, with a high molar absorptivity ranging from 2.81 x 104to 5.00 x 104M^cm’1. These compounds have been evolutionarily selected to protect cells from various environmental and cellular stressors, making them particularly effective natural UV-protective agents.

[0006] The MAAs' protective capabilities extend beyond direct UV absorption, as they also exhibit anti-aging, antioxidant and anti-inflammatory properties. This multi-functional protective mechanism has been demonstrated through their effectiveness in vivo, suggesting potential synergistic effects when combined with other protective compounds in cosmetic formulations.

[0007] The formulations can incorporate MAAs alongside traditional UV filters, including both mineral filters (such as zinc oxide and titanium dioxide) and organic UV filters, creating a comprehensive approach to UV protection. The combination of these different protective mechanisms - MAAs' UV absorption properties, mineral UV reflection / scattering, and organic UV absorption - provides broad-spectrum protection against harmful UV radiation.

[0008] The biochemistry and metabolic function of MAAs is well documented in the literature. Additionally, their commercial value has been reported in both the scientific literature and via the filing of numerous patents. Despite their effectiveness, in vivo commercial applications have seen limited success. This is due in large part because recovery of sufficient quantities via an economically viable process has remained elusive. Recovery for analytical work based on chemical extraction is efficient for laboratory scale recovery, but not commercial scale. A variety of approaches to increasing industrial access to MAAs include engineering yeast to produce specific MAAs, an integrated multi- trophic aquaculture system to increase production potential, and development of synthetic analogs with improved stability and processes. Gadusol Laboratories Inc, and Oregon State University have filed a patent application on a syntheticgene cluster, expression vectors, genetically engineered microorganisms and methods for the production of derivatives of the MAA gadusol.

[0009] Our method of MAA purification demonstrates broad applicability across any number of cultivation or natural harvesting of MAAs from the biomass. Examples of MAAs include, without limitation, asterina-330, euhalothece-362, mycosporine-2-glycine, mycosporine-glycine, mycosporine- glycine-valine, mycosporine-glutamic acid-glycine, mycosporine-methylamine- serine, mycosporine-methylamine-threonine, mycosporine-taurine, palythenic acid, palythene, palythine, palythine-serine, palythine-serine-sulfate, palythinol, porphyra-334, shinorine, usujirene, M-343, palythene, palythine-threonine- sulfate, 4-dexoygadusol, and dehydroxyl-usjirene.

[0010] One aspect of the instant disclosure relates to purification using aptamers. Aptamers, a sub-set of ligands, include nucleic acid sequences capable of binding to a variety of molecular targets. Contrary to the actual genetic material, their specificity and characteristics are not directly determined by their primary sequence, but rather by their secondary and / or tertiary structures. Aptamers are typically characterized by binding to their target molecules via non-Watson-Crick (i.e., non-hybridization) mechanisms, such as by intermolecular forces resulting from the secondary or tertiary structure of the aptamer. This is especially true of non-nucleic acid target molecules where Watson-Crick mechanisms typically do not apply.

[0011] Aptamers are commonly identified by an in vitro method of selection sometimes referred to as Systematic Evolution of Ligands by Exponential enrichment or “SELEX”. The basic SELEX protocol and aptamers are described in U.S. Patent No. 5,270,163, entitled “Methods for identifying nucleic acid ligands”. SELEX typically begins with a very large pool of randomized polynucleotides which is generally narrowed to one aptamer ligand per molecular target. Once multiple rounds (typically 10-15) of SELEX are completed, the nucleic acid sequences are identified by conventional cloning and sequencing. After identification of a nucleic acid sequence that binds to the target molecule, the aptamer is manufactured as with any other oligonucleotide, in standard commercial nucleic acid synthesis (e.g. oligonucleotide synthesis byphosphoramidite method, etc.), or the sequence may be utilized with biological synthesis methods, such polymerase chain reaction (PCR).

[0012] Herein we describe a process of purification of MAAs using centrifugation, cell lysis if the MAAs are intracellular, and column chromatography that utilizes aptamers and ion exchange chromatography. The disclosed method demonstrates exceptional versatility in handling MAA purification across concentration ranges from micrograms per liter to several grams per liter

[0013] There is an extensive literature detailing the biochemistry and metabolic function of MAAs. Additionally, their commercial value has been reported in both the scientific literature and via the filing of numerous patents. Despite their effectiveness in vivo commercial applications have seen limited success. This is due in large part because recovery of sufficient quantities via an economically viable process has remained elusive. Recovery for analytical work based on chemical extraction is efficient for laboratory scale recovery, but not commercial scale. A variety of approaches to increasing industrial access to MAAs include engineering yeast to produce specific MAAs, integrating a multi- trophic aquaculture system to increase production potential, and development of synthetic analogs with improved stability and processes. Gadusol Laboratories Inc, and Oregon State University have filed a patent application on a synthetic gene cluster, expression vectors, genetically engineered microorganisms, and methods for the production of derivatives of the MAA gadusol. In contrast HelioBioSys’s innovation avoids genetic modification and relies on natural production of a suite of MAAs. HelioBioSys’s robust cultivation process produces large volumes of MAAs. Our method also demonstrates broad applicability across any number of cultivation systems or natural harvesting of MAA containing biomass.BRIEF DESCRIPTION OF THE FIGURES

[0014] FIG. 1 provides a general scheme for the Isolation & purification of MAAs for commercial applications including cosmetics & sunscreens.DETAILED DESCRIPTION OF THE DISCLOSUREBiomass Separation

[0015] Centrifugation is typically used to separate the cell culture (feed) from the biomass. However, biomass from some systems may be harvested using sedimentation (gravity settling), filtration, flocculation, or flotation. Centrifugation may be performed by disc stack centrifugation (DSC), tubular centrifugation, or other commercial centrifugation systems. This includes any means where centrifugal force is used to separate solid / liquid mixtures in continuous, semi-continuous and batch configurations based on density differences between the phases present in the feed. This results in a light phase (supernatant) and a heavy phase (solids or biomass).Intracellular MAAs - Cell Lysis

[0016] For intracellular MAAs the biomass (heavy phase from the centrifugation) may be diluted from 0 to 100 or more fold. Cell lysis may be performed by re-centrifuging through a DSC, sonication, homogenization, or pulsed electric field. The MAAs are further separated from the heavy phase lysed cells using all, or any combination thereof, membrane filtration separation, chemical extraction, flocculation, and column chromatography.Extracellular MAAs

[0017] If the MAAs are extracellular they will be in the light phase after centrifugation or in the filtrate after filtration, or in the liquid phase after settling or flocculation. The MAAs are further separated from the light phase using, chemical extraction, flocculation and membrane filtration.

[0018] MAA Extraction For purifying MAAs from either a cell lysate, whole cells or the extracellular light phase, extraction with either ethanol or acid is used to separate the lysed cells particulate matter from water soluble cell contents that include MAAs. This is followed by column chromatography.Column Chromatography

[0019] Column chromatography can refer to liquid chromatography performed on a three-dimensional stationary phase packed inside of a glass, plastic, or metal column and can be used for both preparative and analytical purposes. Based on the characteristics of the compounds to be separated, and thecompositions of the mobile and stationary phases, different compounds will move through the column at different speeds as the mobile phase flows through it, enabling the collection of discrete fractions at the outlet containing one or more components of the original mixture. Column chromatography techniques encompass a broad range of applications based on diverse solid and liquid phase chemistries and column configurations, enabling precise analyses and the resolution of complex mixtures that would be difficult or impossible to separate by other means. Column chromatography can be performed using gravity flow or with the aid of various types of pumps and specialized instrumentation to control the flow of the fluid phase through the column. There are different categories of column chromatography, including ion exchange, size exclusion, and affinity chromatography.

[0020] Ion exchange chromatography is a technique for separating compounds based on their net charge. Ion exchange chromatography media contain negatively or positively charged functional groups covalently bound to a solid support, yielding either a cation or anion exchanger, respectively. Charged compounds are adsorbed and retained by an ion exchanger having the opposite charge, whereas compounds that are neutral or have the same charge as the media pass through the void volume and are eluted from the column. The binding of the charged compounds is reversible, and adsorbed compounds are commonly eluted with a salt or pH gradient. Ion exchange media are available in various particle sizes, ionic forms, and purity ranges.

[0021] Size exclusion chromatography (SEC) separates molecules based on their size by filtration through a gel. The gel consists of spherical beads containing pores of a specific size distribution. Separation occurs when molecules of different sizes are included or excluded from the pores within the matrix. Small molecules diffuse into the pores and their flow through the column is retarded according to their size, while large molecules do not enter the pores and are eluted in the column's void volume. Consequently, molecules separate based on their size as they pass through the column and are eluted in order of decreasing molecular weight (MW). Operating conditions and gel selection depend on the application and the desired resolution. Two common types of separations performed by SEC are fractionation and desalting (or buffer exchange.)Desalting — A common use of SEC is for desalting protein or nucleic acid samples. The molecule of interest is eluted in the void volume, while smaller molecules are retained in the gel pores. To obtain the desired separation, the gel should have an exclusion limit significantly smaller than the molecule of interest. Fractionation — Molecules of varying molecular weights are separated within the gel matrix. With this separation method, the molecules of interest should fall within the fractionation range of the gel.

[0022] Affinity chromatography is a separation method based on a specific binding interaction between an immobilized ligand and its binding partner. Examples include antibody / antigen, enzyme / substrate, and enzyme / inhibitor interactions. The degree of purification can be high depending on the specificity of the interaction and, consequently, it is generally the first step, if not the only step, in a purification strategy. Affinity chromatography offers high selectivity, resolution, and capacity in most protein purification schemes. It has the advantage of utilizing a protein's biological structure or function for purification. As a result, purifications that would otherwise be time consuming and complicated, can often be easily achieved with affinity chromatography. Here we use a nucleotide (RNA or DNA) as the ligand (aptamer) immobilized in the column. The aptamer binds the MAAs as they pass through the column. When all of the cell lysate has passed through the column the MAAs are released from the aptamer and eluted from the column with a buffer. The aptamer-based affinity chromatography step provides highly selective MAA purification through specific molecular recognition. The aptamers can be conjugated to various chromatographic supports and demonstrate stability under the range of pH and ionic strength conditions required for efficient binding and elution. The small size of aptamers enables higher density on columns compared to traditional antibody-based approaches, potentially increasing column capacity and throughput.

[0023] Aptamers offer several unique characteristics that are desirable for affinity chromatography. Aptamers are easily conjugated to chromatographic supports. Aptamer stability is important in harsh elution conditions. Small aptamer size enables higher density on columns and increased capacity.Aptamers can be selected under pH, ionic strength, and temperature conditionsfavorable for affinity chromatography. High aptamer purity and low-cost chemical production are also very important when a large quantity of material is required to build columns to purify very precious material. Aptamer affinity chromatography has been used to purify small molecules, proteins, and even for cell separation. The use of aptamer affinity columns to remove environmental contaminants has also been demonstrated. Modifications to produce aptamers with slower off-rates combined with new support materials are making aptamers even better choices for affinity chromatography.

[0024] The instantly disclosed method demonstrates broad applicability across any number of cultivation or natural harvesting of MAAs from the biomass. Examples of MAAs include, without limitation, asterina-330 (2-[[5- hy droxy-3-(2 -hydroxy ethylamino)-5-(hydroxymethyl)-2-methoxycy cl ohex-2-en- l-ylidene]amino]acetic acid), euhalothece-362 (25)-2-[[(5A)-3-[[(E)-2,3- dihydroxyprop-l-enyl]amino]-5-hydroxy-5-(hydroxymethyl)-2- methoxycyclohex-2-en-l-ylidene]amino]propanoic acid), mycosporine-2- glycine (2-[[3-(carboxymethylimino)-5-hydroxy-5-(hydroxymethyl)-2- methoxycyclohexen-l-yl]amino]acetic acid), mycosporine-glycine (2-[[(55)-5- hydroxy-5-(hydroxymethyl)-2-methoxy-3-oxocyclohexen-l-yl]amino]acetic acid), mycosporine-glycine-valine (2-[[(5A)-3-(carboxymethylimino)-5- hydroxy-5-(hydroxymethyl)-2-methoxycyclohexen-l-yl]amino]-3- methylbutanoic acid), mycosporine-glutamic acid-glycine, mycosporine- methylamine-serine, mycosporine-methylamine-threonine, mycosporine-taurine, palythenic acid, palythine-serine, palythine-serine-sulfate, palythinol, porphyra- 334 (2-[[3-(carboxymethylimino)-5-hydroxy-5-(hydroxymethyl)-2- methoxycyclohexen-l-yl]amino]-3-hydroxybutanoic acid), shinorine (2-[[3- (carboxymethylimino)-5-hydroxy-5-(hydroxymethyl)-2-methoxycyclohexen-l- yl]amino]-3-hydroxypropanoic acid), usujirene-M343palythine threonine sulfate, 4-dexoygadusol (3,5-dihydroxy-5-(hydroxymethyl)-2-methoxycyclohex-2-en-l- one), and dehydroxyl-usjirene.

[0025] The method begins with a microbial culture separated from the cell biomass by centrifugation followed by cell lysis. Alternatively, biomass can be harvested from a natural or managed system depending upon the MAA producing organism.

[0026] For intracellular MAAs the biomass from any method of separation, including but not limited to flocculation, centrifugation, settling or filtration, may be diluted from 0 to 100 or more fold, resulting in the cells within the biomass being lysed. Cell lysis may be performed by re-centrifuging through a DSC, sonication, homogenization, or pulsed electric field. The cell may or may not be genetically modified to produce specific MAAs. These organisms may or may not be sourced from nature, including but not limited to fungi, cyanobacteria, bacteria, algae, dinoflagellates, etc.

[0027] The MAAs in the lysed cells are further separated from non- MAA cellular material using either ethanol extraction in the range of 50% to 100%, 90% to 100%, less than, equal to, or greater than about 50%, 60, 70, 80, 90, or 100%, or acid extraction where the pH ranges from 2.0 to 6.9, about 2.5 to about 3.9, less than, equal to, or greater than about 2.0, 2.5, 3. 3.5, 4. 4.5, 5. 5.5, 6, or about 6.9. The concentration of MAAs can range from a few ug / L to a few g / L.

[0028] The MAAs that have been partially purified using chemical extraction can be further purified using commercial scale ion exchange chromatography. This reduces the amount of small molecular weight non-MAA components resulting in a more purified MAA stream. Ion exchange chromatography can be used in the purification of a broad variety of compounds, importantly, it has not been used in the purification of MAAs.

[0029] The MAAs that have been partially purified using chemical extraction can be further purified using commercial scale affinity chromatography using aptamers, or ligands (synthetic single stranded DNA) that bind to individual MAAs with a high specificity. The process of using aptamers in resin-based column chromatography will yield highly purified MAAs. We have preliminary data showing that aptamers can bind and remove MAAs from a cell lysate using magnetic beads at the lab scale. The use of resin-bound aptamers on a column used to purify MAAs is unique and provide a purity of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The purification process demonstrates broad applicability across cultivation systems while avoiding genetic modification approaches. The method's scalability is supported by the use of industrial-standard equipmentincluding disc stack centrifugation operating at 9,600 x g for initial biomass separation, followed by extraction and chromatography systems capable of handling industrial-scale volumes. The process can accommodate biomass dilution ratios from 0 to 100-fold or greater, providing flexibility in handling various input concentrations.

[0030] The industrial-scale purification process employs a systematic approach beginning with biomass separation through disc stack centrifugation operating at 9,600 x g. The process demonstrates flexibility in handling varying biomass concentrations through dilution ratios ranging from 0 to 100-fold or greater, allowing optimization based on equipment capacity and downstream processing requirements.

[0031] For chemical extraction scale-up, the process utilizes industrial standard systems for ethanol and acidified extraction.

[0032] The commercial-scale chromatography process leverages both ion exchange and aptamer-based affinity methods. The aptamer affinity columns offer several advantages for industrial scale operations, including high-density ligand packing due to small aptamer size, stability under various elution conditions, and cost-effective chemical production when large quantities of material are required for column preparation. These characteristics make aptamer-based separation particularly suitable for precious material purification at commercial scale.

[0033] The process demonstrates broad applicability across cultivation systems while avoiding genetic modification approaches. Recovery of sufficient quantities through an economically viable process has historically been challenging, but this method addresses those limitations through the integration of standard industrial equipment and scalable separation techniques. The process can handle MAA concentrations ranging from micrograms per liter to several grams per liter, making it suitable for commercial production volumes.

[0034] It is possible to create many different cosmetic compositions including MAAs according to the instant disclosure. Those cosmetic compositions can include a sun cream, sunscreen, a moisturizer, a foundation, an anti-wrinkle claim, or the like. Examples of such compositions are below.

[0035] Sun cream: Oil phase components (4-14 wt%), UV filters (0-10 wt%), Emollients (0-10 wt%), Mineral UV filters (0-10 wt%), UV absorbers (0- 8 wt%), Natural oils (0-8 wt%), Natural butters (0-7 wt%), Thickening agents (0-6.4 wt%), Emulsifiers (0-6.2 wt%), Mycosporine-like amino acids (0-5.5 wt%) (in liposomes), Antioxidants (0-5.005 wt%) (in liposomes), Vitamins (0- 5.001 wt%) (in liposomes), Aqueous phase components and Preservative system to 100%.

[0036] Sun cream: Oil phase components (4-14 wt%), Organic UV filters (3-13 wt%), Emollients (1-11 wt%), UV absorbers (0-8 wt%), Natural oils (0-8 wt%), Natural butters (0-7 wt%), UV stabilizers (0-6.5 wt%), Thickening agents (0-6.4 wt%), Emulsifiers (0-6.2 wt%), Mycosporine-like amino acids (0-5.1 wt%), Skin conditioning agents (0-5.1 wt%), Aqueous phase components and Preservative system to 100%.

[0037] Sunscreen: Mineral UV filters (18.5-28.5 wt%), Humectants (3- 13 wt%), Aqueous phase components, Natural oils (0-8 wt%), Plant oils (0-8 wt%), Natural extracts (0-10 wt%), Natural waxes (0-7 wt%), Emulsifiers (0-6 wt%), Natural oils (0-7 wt%), Antioxidants (0-7 wt%), Vitamins (0-7 wt%), Natural starches (0-8 wt%), Thickening agents (0-8 wt%), Mycosporine-like amino acids (0-6 wt%), Preservative system and Aqueous phase components to 100%.

[0038] Moisturizer: Emollients (0-10 wt%), Botanical extracts (0-10 wt%), Emulsifiers (0-9 wt%), Mineral oils (0-7 wt%), Humectants (0-7 wt%), Fatty alcohols (0-6.5 wt%), Fatty alcohols (0-6.5 wt%), Fatty acids (0-6 wt%), Synthetic emollients (0-5.5 wt%), Thickening agents (0-5.15 wt%), pH adjusters (0-5.12 wt%), Plant-derived compounds (0-5.1 wt%), Chelating agents (0-5.1 wt%), Skin conditioning agents (0-5.001 wt%), Mycosporine-like amino acids (0-5.0005 wt%), Aqueous phase components and Preservative system to 100%.

[0039] Sun cream: Mineral Oil (4-14 wt%), PPG- 15 Stearyl Ether (0-10 wt%), Titanium Dioxide (0-10 wt%), Zinc Oxide (0-10 wt%), Octyl Salicylate (0-8 wt%), Jojoba Oil (0-8 wt%), Cocoa Butter (0-7 wt%), Hydrogenated Castor Oil (0-6.4 wt%), Sorbitan Stearates (0-6.2 wt%), Mycosporine-like Amino Acids (MAAs) (0-5.5 wt%) (in liposomes), Soy Isoflavones (0-5.005 wt%) (inliposomes), Tocopherol (0-5.001 wt%) (in liposomes), Aqua, Perfume and Preservatives to 100%.

[0040] Sun cream: Mineral Oil (4-14 wt%), EthylhexylMethoxy cinnamate (3-13 wt%), PPG- 15 Stearyl Ether (1-11 wt%), Octyl Salicylate (0-8 wt%), Jojoba Oil (0-8 wt%), Cocoa Butter (0-7 wt%), Butyl Methoxy dibenzoylmethane (0-6.5 wt%), Hydrogenated Castor Oil (0-6.4 wt%), Sorbitan Stearates (0-6.2 wt%), My cosporine-like Amino Acids (MAAs) (0-5.1 wt%), Sodium Carboxymethyl Beta-Glucan (0-5.1 wt%), Aqua, Perfume and Preservatives to 100%.

[0041] Sunscreen: Zinc Oxide (18.5-28.5 wt%), Vegetable Glycerine (3- 13 wt%), Distilled Water, Simmondsia Chinesis (Jojoba) Oil (0-8 wt%), Olea Europaea Olive (0-8 wt%), Aloe (0-10 wt%), Cera Flava Citrus Aurantium Dulces (Orange) Peel Wax (0-7 wt%), Phosphatidyl Choline / Cetearyl Glucoside (0-6 wt%), Refined Coconut Oil Extract (0-7 wt%), Tocopherol (0-7 wt%), Ascorbic Acid (0-7 wt%), Zea Mays (Corn) Starch (0-8 wt%), Xanthan Gum (0- 8 wt%), Mycosporine-Like Amino Acids (0-6 wt%), Preservatives and Distilled Water to 100%.

[0042] Sunscreen: Mycosporine-like Amino Acids (0-7 wt%), Vegetable Glycerine (3-13 wt%), Distilled Water, Simmondsia Chinesis (Jojoba) Oil (0-8 wt%), Olea Europaea Olive (0-8 wt%), Aloe (0-10 wt%), Cera Flava Citrus Aurantium Dulces (Orange) Peel Wax (0-7 wt%), Phosphatidyl Choline / Cetearyl Glucoside (0-6 wt%), Refined Coconut Oil Extract (0-7 wt%), Tocopherol (0-7 wt%), Ascorbic Acid (0-7 wt%), Zea Mays (Com) Starch (0-8 wt%), Xanthan Gum (0-8 wt%), Mycosporine-Like Amino Acids (0-6 wt%), Preservatives and Distilled Water to 100%.

[0043] Moisturizer: Caprylic / Capric Triglycerides (0-10 wt%), Vitis Vinifera (Grape) Seed Extract (0-10 wt%), Glyceryl Stearate (0-9 wt%), Paraffinum Liquidum (0-7 wt%), Glycerin (0-7 wt%), Stearyl Alcohol (0-6.5 wt%), Cetyl Alcohol (0-6.5 wt%), Palmitic / Stearic Acid (0-6 wt%), Hydrogenated Polyisobutenes (0-5.5 wt%), Carbomer (0-5.15 wt%), Sodium Hydroxides (0-5.12 wt%), Ghicosylrutin (0-5.1 wt%), Disodium EDTA (0-5.1 wt%), Sodium Carboxymethyl Beta-Glucan (0-5.001 wt%), Mycosporine-likeAmino Acids (MAAs) (0-5.0005 wt%), Aqua, Perfume and Preservatives to 100%.Examples

[0044] Various aspects of the present disclosure can be better understood by reference to the following examples which are offered by way of illustration. The present disclosure is not limited to the examples given herein.

[0045] Purification of the MAAs, specifically, porphyra-334, shinorine, asterine, palythine, mycosporine glycine, palythinol, and palythene: Separation of a microbial culture into two phases, the biomass and the extracellular material using a disc stack centrifuge (DSC) such as the Alfa Laval Clara 20 operating at 9600 x g. For MAAs in the cell biomass the biomass is diluted 1-100 fold and the cells are lysed using either pulsed electric field, homogenization, sonication or re-centrifuging in a DSC. The MAAs are then further separated from the cell debris and particulate matter by chemical extraction with either ethanol at concentrations ranging from 50% to 100%, acid extraction using a variety of acids (e.g., HC1) to reduce the pH of the system from less than 3.0 to a maximum of 6.9. The extract is then purified using affinity chromatography, using aptamers specifically designed to bind to MAAs as the agent immobilized in the column. The MAAs bind to the aptamers and stick to the resin within the column. The MAAs are then eluted from the column with a solution that changes the binding affinity by increasing the pH or decreasing the pH. The purified MAAs are then formulated into skin care products such as but not limited to; face creams, sun creams, sunscreens, and other therapeutic agents.

[0046] Sun cream. Mineral Oil 9.0% PPG- 15 stearyl ether 6.0% Titanium Dioxide 5.0% Zinc Oxide 5.0% Octyl, salicylate 3.0% Jojoba Oil 3.0% Cocoa butter 2.0% Hydrogenated Castor Oil 1.4% Sorbitan stearates 1.2% Mycosporine-like amino acids (MAAs) 0.5% (in liposomes) Soy Isogavones 0.005% (in liposomes) tocopherol 0.001% (in liposomes) Aqua, perfume and preservatives to 100%

[0047] Sun cream. Mineral Oil 9.0% Ethylhexyl methoxy cinnamate, 8.0% PPG-15 stearyl ether 6.0% Octyl salicylate 3.0% Jojoba Oil 3.0% Cocoa butter 2.0% Butyl methoxydibenzoylmethane 1.5% Hydrogenated Castor Oil1.4% Sorbitan, stearates 1.2% Mycosporine-like amino acids (MAAs) 0.1% Sodium carboxymethyl beta-glucan 0.1% Aqua, perfume and preservatives to 100%

[0048] Sun cream. Mineral Oil 9.0% Ethylhexyl methoxy cinnamate, 8.0% PPG-15 stearyl ether 6.0% Octyl salicylate 3.0%, Jojoba Oil 3.0% Cocoa butter 2.0% Butyl methoxydibenzoylmethane 1.5% Hydrogenated Castor Oil 1.4% Sorbitan, stearates 1.2% Mycosporine-like amino acids (MAAs) 0.1% Sodium carboxymethyl beta-glucan 0.1% Aqua, perfume and preservatives to 100%

[0049] Sunscreen. Zinc Oxide 23.5%, vegetable glycerine 8%, distilled water, Simmondsia chinesis (jojoba) oil 3%, Olea europaea olive, 3% Aloe 5%, Cera flava Citrus aurantium dulces (orange) peel wax 2%, phosphatidyl choline / cetearyl glucoside 1%, refined coconut oil extract 2%, tocopherol 2%, ascorbic acid 2%, Zea mays (corn) starch 3%, , xanthan gum 3%, Mycosporing Like Amiono Acids 1%, preservatives and distilled water to 100%

[0050] Sunscreen, mysosporine like amino acids 2%, vegetable glycerine 8%, distilled water, Simmondsia chinesis (jojoba) oil 3%, Olea europaea olive, 3% Aloe 5%, Cera flava Citrus aurantium dulces (orange) peel wax 2%, phosphatidyl choline / cetearyl glucoside 1%, refined coconut oil extract 2%, tocopherol 2%, ascorbic acid 2%, Zea mays (com) starch 3%, , xanthan gum 3%, Mycosporing Like Amiono Acids 1%, preservatives and distilled water to 100%

[0051] Moisturizer. Caprylic / Capric triglycerides 5.0% Vitis Vinifera (Grape) Seed Extract 5.0% Glyceryl stearate 4.0% ParaWnum Liquidum 2.0% glycerin, 2.0% Stearyl alcohol 1.5% Cetyl alcohol 1.5% Palmitic / Stearic Acid 1.0% Hydrogenated polyisobutenes 0.5% Carbomer 0.15% Sodium hydroxides 0.12% glucosylrutin, 0.1% Disodium EDTA 0.1% Sodium carboxymethyl betaglucan 0.001% Mycosporine-like amino acids (MAAs) 0.0005% Aqua, perfume and preservatives to 100%

[0052] The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the aspects of the present disclosure. Thus, itshould be understood that although the present disclosure has been specifically disclosed by specific aspects and optional features, modification and variation of the concepts herein disclosed may be resorted to by those of ordinary skill in the art, and that such modifications and variations are considered to be within the scope of aspects of the present disclosure.Exemplary Aspects.

[0053] The following exemplary embodiments are provided, the numbering of which is not to be construed as designating levels of importance:

[0054] Aspect 1 provides a method for separating intracellularly produced and extracellularly produced mycosporine like amino acids from a cell culture or biomass, the method comprising: using centrifugation to separate the biomass from the extracellular mycosporine-like amino acids; and incorporating the mycosporine-like amino acid into a cosmetic product.

[0055] Aspect 2 provides the method of Aspect 1, wherein the cosmetic product comprises a sunscreen, a sun cream, a therapeutic product, or a combination thereof.

[0056] Aspect 3 provides the method of any of Aspects 1 or 2, wherein centrifugation comprises use of a disc stack centrifugation.

[0057] Aspect 4 provides the method of any of Aspects 1 or 2, wherein the method of centrifugation comprises the use of tubular centrifugation.

[0058] Aspect 5 provides a method of lysing cells in the biomass that mycosporine like amino acids for use in cosmetics, sunscreens, sun creams and therapeutic products, the method comprising: homogenization; sonication; pulsed electric field processing; repeated disc stack centrifugation.

[0059] Aspect 6 provides a method for separating the mycosporine like amino acids in either a cell lysate or extracellular material non- mycosporine like amino acids material, the method comprising using chemical extraction.

[0060] Aspect 7 provides the method of Aspect 6, wherein the extraction is performed with ethanol with concentrations ranging from 50% to 100%.

[0061] Aspect 8 provides the method of Aspect 6, wherein the extraction is performed with a strong acid such as HC1 to reduce the pH of the system in the range of 6.9 to 3.0.

[0062] Aspect 9 provides a method for purifying mycosporine like amino acids from a cell lysate and an extracted cell lysate using affinity column chromatography, wherein the immobile phase is comprised, or partially comprised, of aptamers or nucleic acid ligands that bind to the mycosporine like amino acids.

[0063] Aspect 10 provides the method of Aspect 9, wherein the aptamers or nucleic acid ligands are resin-bound to a column.

[0064] Aspect 11 provides a method of for purifying mycosporine like amino acids from a cell lysate, filtered cell lysate and filtered extracellular material using ion exchange or size exclusion column chromatography.

[0065] Aspect 12 provides a method for separating intracellular mycosporine like amino acids from a cell culture or biomass from any source using centrifugation to separate the mycosporine like amino acids containing biomass from the extracellular material.

[0066] Aspect 13 provides a method for formulating mycosporine like amino acids purified by column chromatography into cosmetics, sun creams and sunscreens and other therapeutic agents, the method comprising incorporating the mycosporine like amino acids into the cosmetic, sun cream and sunscreen and other therapeutic agents, such as anti-aging creams (anti-oxidant properties).

[0067] Aspect 14 provides a cosmetic composition, sun cream composition, sunscreen composition, or therapeutic composition comprising the mycosporine like amino acid of any of Aspects 1-13.

[0068] Aspect 15 provides a method for separating intracellularly produced and extracellularly produced mycosporine like amino acids from a cell culture or biomass using centrifugation to separate the biomass from the extracellular material to produce cosmetics, sunscreens, sun creams and therapeutic products.

[0069] Aspect 16 provides the method of Aspect 15 wherein the method of centrifugation is disc stack centrifugation.

[0070] Aspect 17 provides the method of any of Aspects 16 or 17, wherein the method of centrifugation is tubular centrifugation.

[0071] Aspect 18 provides a method for lysing cells in the biomass that contain MAAs for use in cosmetics, sunscreens, sun creams and therapeutic products.

[0072] Aspect 19 provides the method of Aspect 18, wherein the method of cell lysis of the biomass is homogenization

[0073] Aspect 20 provides the method of Aspect 18 wherein the method of cell lysis of the biomass is sonication

[0074] Aspect 21 provides the method of Aspect 18, wherein the method of cell lysis of the biomass is pulsed electric field processing.

[0075] Aspect 22 provides the method of Aspect 18, wherein the method of cell lysis of the biomass is repeated disc stack centrifugation.

[0076] Aspect 23 provides a method for separating the MAAs in either a cell lysate or extracellular material non-MAA material using chemical extraction.

[0077] Aspect 24 provides the method of Aspect 24, wherein the extraction is performed with ethanol.

[0078] Aspect 25 provides the method of Aspect 24, wherein the extraction is performed with an acid (e.g., HC1).

[0079] Aspect 26 provides a method for purifying MAAs from a cell lysate and a filtered cell lysate using affinity column chromatography wherein the immobile phase is comprised, or partially comprised, of aptamers or nucleic acid ligands that bind specifically to MAAs.

[0080] Aspect 27 provides a method of for purifying MAAs from a cell lysate, filtered cell lysate and filtered extracellular material using ion exchange or size exclusion column chromatography.

[0081] Aspect 28 provides a method for separating intracellular mycosporine like amino acids from a cell culture or biomass from any source using centrifugation to separate the MAA containing biomass from the extracellular material.

[0082] Aspect 29 provides a method for formulating MAAs purified by column chromatography into cosmetics, sun creams and sunscreens and other therapeutic agents.

Claims

CLAIMSWhat is claimed is:

1. A method for separating intracellularly produced and extracellularly produced mycosporine like amino acids from a cell culture or biomass using centrifugation to separate the biomass from the extracellular material to produce cosmetics, sunscreens, sun creams and therapeutic products.

2. The method of claim 1 wherein the method of centrifugation is disc stack centrifugation.

3. The method of claim 1 wherein the method of centrifugation is tubular centrifugation.

4. A method for lysing cells in the biomass that contain MAAs for use in cosmetics, sunscreens, sun creams and therapeutic products.

5. The method of claim 4 wherein the method of cell lysis of the biomass is homogenization.

6. The method of claim 4 wherein the method of cell lysis of the biomass is sonication.

7. The method of claim 4 wherein the method of cell lysis of the biomass is pulsed electric field processing.

8. The method of claim 4 wherein the method of cell lysis of the biomass is repeated disc stack centrifugation.

9. A method for separating the MAAs in either a cell lysate or extracellular non-MAA material using chemical extraction with different solvents.

10. The method of claim 9 wherein the solvent is ethanol.

11. The method of claim 9 wherein the solvent is an acid.

12. A method for purifying MAAs from a cell lysate and a filtered cell lysate using affinity column chromatography wherein the immobile phase is comprised, or partially comprised, of aptamers or nucleic acid ligands that bind specifically to MAAs.

13. A method of for purifying MAAs from a cell lysate, filtered cell lysate and filtered extracellular material using ion exchange or size exclusion column chromatography.

14. A method for separating intracellular mycosporine like amino acids from a cell culture or biomass from any source using centrifugation to separate the MAA containing biomass from the extracellular material.

15. A method for formulating MAAs purified by column chromatography into cosmetics, sun creams and sunscreens and other therapeutic agents.

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