# US 12,180,526 B2 · Subterranean microalgae for production of microbial biomass, substances, and compositions

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- Number: US 12,180,526 B2
- Document ID: US12180526B2
- Status: Granted
- Kind: B2
- Application number: 17/810,522
- Filed: 2022-07-01
- Published: 2023-09-14
- Granted: 2024-12-31
- Earliest claimed: 2019-01-16
- Assignees: Kuehnle AgroSystems, Inc.
- Applicants: Kuehnle AgroSystems, Inc.
- Inventors: Adelheid R. Kuehnle; Robert J. Schurr; Norie Anne B. Nolasco; Jacob F. Wallace
- Web page: https://patentgrep.com/patent/US12180526B2

## CPC

- C12P 19/04: Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
- A23K 10/12: by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
- A23K 10/16: Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions (A23K10/12 takes precedence)
- A23K 20/179: Colouring agents, e.g. pigmenting or dyeing agents
- A23K 40/10: by agglomeration; by granulation, e.g. making powders
- A23K 50/80: for aquatic animals, e.g. fish, crustaceans or molluscs
- A23L 17/60: Edible seaweed
- A61K 8/11: Encapsulated compositions
- A61K 8/31: Hydrocarbons
- A61K 8/34: Alcohols
- A61K 8/345: {containing more than one hydroxy group}
- A61K 8/4913: {having five membered rings, e.g. pyrrolidone carboxylic acid}
- A61K 8/73: Polysaccharides
- A61K 8/9722: Chlorophycota or Chlorophyta [green algae], e.g. Chlorella
- A61K 9/0014: {Skin, i.e. galenical aspects of topical compositions (non-active ingredients are additionally classified in A61K47/00; A61K9/0009, A61K9/0021, A61K9/7015, A61K9/7023 take precedence; cosmetic preparations A61K8/00, A61Q; preparations for wound dressings or bandages A61L26/00)}
- A61K 9/06: Ointments; Bases therefor; {Other semi-solid forms, e.g. creams, sticks, gels (composition of ointments, creams or gels A61K47/00)}
- A61K 31/01: Hydrocarbons
- A61K 31/045: Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
- A61K 31/409: having four such rings, e.g. porphine derivatives, bilirubin, biliverdine (hemin, hematin A61K31/555)
- A61K 36/05: Chlorophycota or chlorophyta (green algae), e.g. Chlorella
- A61K 47/10: Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
- A61K 47/26: Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
- A61Q 17/04: Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
- A61Q 19/00: Preparations for care of the skin
- A61Q 19/007: {Preparations for dry skin}
- A61Q 19/08: Anti-ageing preparations
- C12N 1/12: Unicellular algae; Culture media therefor (as new plants A01H13/00)
- C12N 1/125: Unicellular algae isolates
- C12P 5/007: {containing one or more isoprene units, i.e. terpenes (carotenes C12P23/00)}
- C12P 5/026: {Unsaturated compounds, i.e. alkenes, alkynes or allenes}
- C12P 7/02: containing a hydroxy group
- C12P 17/165: {Heterorings having nitrogen atoms as the only ring heteroatoms}
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C12P 23/00: Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes (containing heterorings C12P17/00)
- A23V 2002/00: Food compositions, function of food ingredients or processes for food or foodstuffs
- C12R 2001/89: Algae {; Processes using algae}
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- A23K: FODDER
- A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61Q: SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A23V: INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS

## Abstract

The invention pertains to a method for synthesizing a product of interest by culturing a microalgal cell obtained from a subterranean habitat for producing the product of interest. The microalgal cell obtained from a subterranean habitat can be cultured in the dark, in light, in low nutrition, or nutrient rich conditions for at least a portion of production cycle. A combination of these conditions can be used to specifically manipulate a microalgal cell culture to produce a product of interest. The product of interest can be a water-soluble carotenoid, for example, a water-soluble carotenoid produced by culturing an algae belonging to the genus Haematococcus or a capsular exopolysaccharide produced by culturing an algae belonging to the genus Parachlorella. Compositions containing the water-soluble carotenoid, for example, as sunscreen and compositions containing the exopolysaccharide, for example, as moisturizing cream are also described.

## Claims

### Claim 1

A composition comprising:
a proteoglycan, wherein the proteoglycan comprises a microalgal exopolysaccharide covalently linked to at least one protein, wherein the microalgal exopolysaccharide comprises rhamnose, xylose, 3-O-methyl rhamnose, glucuronic acid, galactose, glucose, and mannose, or
a microalgal water-soluble carotenoid complex, wherein the microalgal water-soluble carotenoid complex comprises astaxanthin, lutein, zeaxanthin, and canthaxanthin; and
a carrier or excipient wherein the proteoglycan or the carotenoid complex is extracted from microalgae cultured in a purposeful change to a condition of nitrogen depletion.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the microalgal exopolysaccharide is from a Parachlorella species cell.

### Claim 3 (depends on 2)

The composition of claim 2, wherein the Parachlorella species is Parachlorella kessleri.

### Claim 4 (depends on 1)

The composition of claim 1, wherein the microalgal water-soluble carotenoid complex is from a Haematococcus species cell.

### Claim 5 (depends on 4)

The composition of claim 4, wherein the Haematococcus species is Haematococcus pluvialis.

### Claim 6 (depends on 1)

The composition of claim 1, wherein the composition is present in a microalgal biomass comprising a plurality of microalgal cells, a secretion of a plurality of microalgal cells, or an extract of a plurality of microalgal cells.

### Claim 7 (depends on 1)

The composition of claim 1, wherein the composition further comprises additional pigment, protein, amino acid, carbohydrate, colorant, UV ray protectant or absorbent, antioxidant, metal chelator, blue-light attenuator, whitener, moisturizer, humectant, protein stabilizer, anti-frizz agent, anti-pollution agent, enzyme inhibitor, ophthalmic agent, a precursor or derivative thereof, or any combination of distinct agents thereof.

### Claim 8 (depends on 7)

The composition of claim 7, wherein the pigment is an isoprenoid, fucoxanthin, lycopene, carotene, phytofluene, phytoene, a chlorophyll, a precursor or a derivative thereof or any combination of distinct pigments thereof.

### Claim 9 (depends on 1)

The composition of claim 1, wherein the composition is an animal feed, a food, an ingredient used in personal care products, or a consumer product selected from the group consisting of a gel, oil, lotion, spray, cream, emulsion, lip care product, hair care product, and ointment.

### Claim 10 (depends on 1)

The composition of claim 1, wherein the microalgal water-soluble carotenoid complex or exopolysaccharide is from a cell selected from the group consisting of KAS1543, KAS1544, KAS1545, KAS1562A, KAS1562B, KAS1537, KAS1565, KAS1566, KAS1535, KAS1708, KAS1601, KAS1561, KAS1562C, and KAS1533.

### Claim 11 (depends on 9)

The composition of claim 9, wherein the composition comprises a cosmetically acceptable vehicle and the water-soluble carotenoid complex is from a Haematococcus species.

### Claim 12 (depends on 11)

The composition of claim 11, wherein the Haematococcus species is Haematococcus pluvialis.

### Claim 13 (depends on 9)

The composition of claim 9, wherein the composition comprises a cosmetically acceptable vehicle and the exopolysaccharide from a Parachlorella species.

### Claim 14 (depends on 13)

The composition of claim 13, wherein the Parachlorella species is Parachlorella kessleri.

### Claim 15 (depends on 1)

The composition of claim 1, wherein the microalgal water-soluble carotenoid complex or exopolysaccharide is from a cell from a taxon selected from Cyanoprokaryota, Chlorophyta, Bacillariophyta, Xanthophyta, or Charophyta.

### Claim 16 (depends on 1)

The composition of claim 1, wherein the microalgal water-soluble carotenoid complex or exopolysaccharide is from a cell from a genus selected from Anabaena, Parachlorella, Desmodesmus, Scenedesmus, Monoraphidium, Coelastrella, Chlorella, Coccomyxa, Pseudococcomyxa, Klebsormidium, Haematococcus, Hantzschia, Aulacoseira, Nitzschia, Navicula, Humidophila, Diadesmis, Eolimna, Sellaphora, Chamaepinnularia, Chroococcus, Mychonastes, Nostoc, Oculatella, Leptolyngbya, Dolichospermum, Geissleria, or Navigolum.

### Claim 17 (depends on 1)

The composition of claim 1, wherein the proteoglycan comprises a microalgal exopolysaccharide covalently linked to at least one protein, resulting in added hydration and stability as compared to the protein in an unlinked state.

### Claim 18 (depends on 1)

The composition of claim 1, wherein the microalgal water-soluble carotenoid complex further comprises doradexanthin.