Patent Application
20230136215
The present invention relates to Dunaliella algae, and extracts thereof, comprising increased levels of 9-cis β-carotene and/or increased levels of colourless carotenoids and/or increased levels of α-carotene, to processes for producing such Dunaliella algae, and to uses thereof.
Dunaliella is a green alga which is known to produce high concentrations of β-carotene, a naturally occurring pigment which has a variety of uses, including as a food colourant, an additive for cosmetics, and a nutritional or health supplement for veterinary and human use.
Other natural sources of β-carotene include carrots and palm oil, however, these produce a significantly lower β-carotene content compared with Dunaliella algae. D. salina is considered the best commercial source of natural β-carotene in the world (Borowitzka M.; J. Appl. Phycol. 1995; 7:3-15). β-Carotene exists in the all-trans, and in the 9-cis forms with the known natural sources producing β-carotene predominantly as the all-trans isomer. Synthetic methods for the production of β-carotene provide exclusively the all-trans isomer and there is no known method of converting all-trans-β-carotene to 9-cis-β-carotene. OsD27, a 9-cis/all-trans β-carotene isomerase, catalyses the reversible isomerization between 9-cis- and all-trans β-carotene but conversion of 9-cis into all-trans β-carotene is the preferred reaction (Bruno, M. & Al-Babili, S., 2016, Planta, 243(6), pp. 1429-1440).
Chemical Structures of A) 9-cis-β-carotene and B) all-trans-β-carotene
Therapeutic uses of Dunaliella salina bardawil have been proposed: US 2010/0221348 A1 discusses the use of Dunaliella salina bardawil powder in the treatment of atherosclerosis and/or diabetes mellitus. Shaish et al (The alga Dunaliella: physiology, genomics and biotechnology, ISBN 1578085454) hypothesize that the beneficial effects of Dunaliella salina bardawil on atherosclerosis is due to its high content of 9-cis-β-carotene. A clinical trial to test the effect of Dunaliella salina bardawil on psoriasis is discussed in Greenberger et al (J. Am. Coll. Nutr., 2012, October, 31(5), 320-326). Trials investigating the effect of Dunaliella salina bardawil on retinal dystrophy and retinitis pigmentosa are discussed in Rotenstreich et al (Br. J. Opthalmol., 2010, May, 94(5), 616-621 and JAMA Opthalmol., 2013, August, 131(8), 985-92).
The major all-trans isomer has low solubility in aqueous solvent systems and tends to form crystals or precipitate, requiring formulation in oil based systems or emulsions, and thus limiting the industrial and clinical utility of all-trans β-carotene. The minor 9-cis β-carotene has been found to dissolve crystalline all-trans β-carotene and to reduce the tendency of the all-trans form to precipitate. It would therefore be an advantage to produce β-carotene comprising predominantly the 9-cis isomer, which β-carotene can be more easily formulated. However, extraction of natural β-carotene from Dunaliella followed by purification to increase the ratio of 9-cis: all-trans β-carotene is currently the only known commercial method for producing preparations with a high 9-cis β-carotene content, as discussed in U.S. Pat. No. 5,612,485 and European Patent Application No. EP0933359. A recent paper Sher et al, 2018 (Scientific Reports (2018) 8:6130) discusses a synthetic method for the preparation of 9-cis-beta-carotene.
Dunaliella algae are also known to produce significant concentrations of the colourless carotenoids phytoene (IUPAC name (6E,10E,14E,16Z,18E,22E,26E)-2,6,10,14,19,23,27,31-octamethyldotriaconta-2,6,10,14,16,18,22,26,30-nonaene) and phytofluene (IUPAC name (6E,10E,12E,14E,16E,18E,22E,26E)-2,6,10,14,19,23,27,31-octamethyldotriaconta-2,6,10,12,14,16,18,22,26,30-decaene), precursors in the biosynthesis of all carotenoids. Phytoene and phytofluene are rarities among carotenoids due to their lower number of conjugated double bonds, as a result of which they absorb maximally in the UV region, with phytoene absorbing maximally in the UVB region and phytofluene in the UVA region. The compounds, which may be ingested or topically applied, are of great interest in the nutricosmetic field for their skin health and aesthetic benefits. Meléndez-Martinez et al 2018 (Journal of Food Composition and Analysis, 67, 91-103) discusses health and cosmetic benefits of phytoene and phytofluene. A review by Meléndez-Martinez et al 2015 (Archives of Biochemistry and Biophysics, 2015, 572, 188-200) discussed the possible beneficial effect of phytoene and phytofluene, concluding that these compounds may provide antioxidant activity, anticarcinogenic activity, anti-inflammatory activity, or protection against UVR-induced damage.
Chemical Structures of (A) Phytoene and (B) Phytofluene:
Ben-Amotz et al (J Phycol (2987) 23:176-181) reported an increase in the phytoene content, and corresponding decrease in the β-carotene content, of Dunaliella bardawil treated with the herbicide norflurazon, a phytoene desaturase inhibitor.
Dunaliella algae are also known to produce significant concentrations of α-carotene (IUPAC name 1,3,3-trimethyl-2-[(1E,3E,5E,7E,9E,11E,13E,15E,17E)-3,7,12,16-tetramethyl-18-(2,6,6-trimethylcyclohex-2-en-1-yl)octadeca-1,3,5,7,9,11,13,15,17-nonaenyl]cyclohexene): α-carotene has proven anti-metastatic action, which is not associated with provitamin A activity (Liu et al.; J Nutr Biochem. 2015 June; 26(6):607-15). The structure of α-carotene is shown below:
The invention provides a Dunaliella alga, or extract thereof, comprising
when compared to a Dunaliella alga, or extract thereof, which is grown or cultivated under natural light or white light conditions.
The invention further provides a powdered Dunaliella alga, or extract thereof, comprising:
when compared to a Dunaliella alga, or extract thereof, which is grown or cultivated under natural light or white light conditions.
The invention further provides Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; comprising a 9-cis β-carotene content of 60 wt % of total carotenoids or greater.
The invention further provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; comprising a colourless carotenoid content of 10 wt % of total carotenoids or greater.
The invention further provides a process for the preparation of a Dunaliella alga comprising exposing the Dunaliella alga to light of wavelength 500-1000 nm; and/or eliminating light of wavelength less than 500 nm (blue light).
The invention further provides the use of a Dunaliella alga or extract thereof, or a powdered Dunaliella alga or extract thereof, as a food colourant or food ingredient; or as a health supplement; or in a cosmetic composition, wherein the Dunaliella alga, or extract thereof, comprises
when compared to a Dunaliella alga, or extract thereof, which is grown or cultivated under natural light or white light conditions.
The invention further provides a Dunaliella alga or extract thereof, or a powdered Dunaliella alga or extract thereof, for use in therapy, wherein the Dunaliella alga, or extract thereof, comprises
when compared to a Dunaliella alga, or extract thereof, which is grown or cultivated under natural light or white light conditions.
The invention further provides a composition comprising: a) a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof and b) a pharmaceutically acceptable excipient, wherein the Dunaliella alga, or extract thereof, comprises
when compared to a Dunaliella alga, or extract thereof, which is grown or cultivated under natural light or white light conditions.
The invention further provides a process for the preparation of a Dunaliella alga comprising treating the Dunaliella alga by application of a herbicide selected from the group consisting of amino acid synthesis inhibitors, growth regulators, nitrogen metabolism inhibitor, pigment inhibitors, seedling root growth inhibitors, seedling shoot growth inhibitors, cell wall synthesis inhibitors, mitosis microtubule organisation inhibitors, and combinations thereof.
The inventors have surprisingly found that when exposed to red light (light of wavelength approximately 500 to 700 nm), eliminating blue light (light of wavelength less than 500 nm), green Dunaliella alga produces an increased content of all carotenoids, including phytoene, α-carotene and β-carotene, compared with the content produced by Dunaliella algae cultivated under normal white light (for example natural sun light). Alternatively, the Dunaliella alga may be exposed to red light of approximately 500 nm-700 nm and/or far-red light, and/or infrared light of wavelength approximately 700-1000 nm, preferably of wavelength approximately 500 nm to less than 830 nm. In particular, the ratio of 9-cis: all-trans-β-carotene is increased, therefore providing an improved yield of β-carotene product which has the additional advantage of being easier to formulate and administer due to the higher 9-cis: all-trans-β-carotene ratio. The relative increase in ratio of 9-cis: all-trans β-carotene on exposure to red light compared to white light is even greater using early-orange phase algae and even greater still when Dunaliella algae are cultivated during red light exposure under cool temperatures (for example 15° C. compared to 25° C.). Light filters that blocked out blue light wavelengths (400 nm-500 nm) from white light were also found to be effective in increasing the amount of 9-cis β-carotene and the ratio of 9-cis: all-trans β-carotene. In contrast, exposure to blue light decreased the amount of 9-cis β-carotene and the ratio of 9-cis: all-trans β-carotene produced by the Dunaliella alga. Furthermore, when cultivated under natural light, the properties of the Dunaliella alga vary seasonally, for example in content of carotenoids and/or colour. Such seasonal variation is reduced or eliminated when the Dunaliella alga is exposed to red light.
In embodiment 1, the invention provides a Dunaliella alga, or extract thereof, comprising
when compared to a Dunaliella alga, or extract thereof, which is grown or cultivated under natural light or white light conditions.
In embodiment 2, the invention provides a powdered Dunaliella alga, or extract thereof, comprising:
when compared to a Dunaliella alga, or extract thereof, which is grown or cultivated under natural light or white light conditions.
In embodiment 3, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; comprising a 9-cis β-carotene content of 60 wt % of total carotenoids or greater.
In embodiment 4, the invention provides a Dunaliella alga, or extract thereof according to embodiment 1; or a powdered Dunaliella alga, or extract thereof according to embodiment 2; wherein the 9-cis β-carotene content is 60 wt % of total carotenoids or greater.
In embodiment 5, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; according to any preceding embodiment, wherein the 9-cis β-carotene content is 65 wt % of total carotenoids or greater.
In embodiment 6, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; according to any preceding embodiment, wherein the 9-cis β-carotene content is 70 wt % of total carotenoids or greater
In embodiment 7, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; according to any preceding embodiment, wherein the 9-cis β-carotene content is 75 wt % of total carotenoids or greater.
In embodiment 8, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; according to any preceding embodiment, wherein the β-carotene has a 9-cis: all-trans ratio of 1.2 or greater.
In embodiment 9, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; according to any preceding embodiment, wherein the β-carotene has a 9-cis: all-trans ratio of 1.5 or greater.
In embodiment 10, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; according to any preceding embodiment, wherein the β-carotene has a 9-cis: all-trans ratio 2.0 or greater.
In embodiment 11, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; according to any preceding embodiment, wherein the β-carotene has a 9-cis: all-trans ratio 3.0 or greater.
In embodiment 12, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; comprising a colourless carotenoid content of 10 wt % of total carotenoids or greater.
In embodiment 13, the invention a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; according to any preceding embodiment, wherein the colourless carotenoid content is 11 wt % or greater.
In embodiment 14, the invention a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; according to any preceding embodiment, wherein the colourless carotenoid content is 12 wt % or greater.
In embodiment 15, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; according to any preceding embodiment, wherein the 9-cis β-carotene content is 60 wt % of total carotenoids or greater and the colourless carotenoid content is 10 wt % or greater of total carotenoids.
In embodiment 16, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; according to any preceding embodiment, wherein the 9-cis β-carotene content is 60 wt % of total carotenoids or greater and the colourless carotenoid content is 11 wt % or greater of total carotenoids.
In embodiment 17, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; wherein the 9-cis β-carotene content is 30 wt % of total carotenoids or greater and the colourless carotenoid content is 40 wt % or greater of total carotenoids.
In embodiment 18, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; wherein the 9-cis β-carotene content is 60 wt % of total carotenoids or greater and the colourless carotenoid content is 4 wt % or greater of total carotenoids.
In embodiment 19, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; wherein the 9-cis β-carotene content is 35 wt % of total carotenoids or greater and the colourless carotenoid content is 45 wt % or greater of total carotenoids.
In embodiment 20, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; according to any preceding embodiment, wherein the colourless carotenoid content is the combined content of phytoene and phytofluene.
In embodiment 21, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; according to any preceding embodiment, comprising a phytoene content of 10 wt % of total carotenoids or greater.
In embodiment 22, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; according to any preceding embodiment, comprising a phytoene content of 11 wt % of total carotenoids or greater.
In embodiment 23, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; according to any preceding embodiment, comprising a phytoene content of 12 wt % of total carotenoids or greater.
In embodiment 24, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; optionally according to any preceding embodiment, comprising a phytoene content of 15 wt % of total carotenoids or greater.
In embodiment 25, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; optionally according to any preceding embodiment, comprising a phytoene content of 20 wt % of total carotenoids or greater.
In embodiment 26, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; optionally according to any preceding embodiment, comprising a phytoene content of 25 wt % of total carotenoids or greater.
In embodiment 27, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; optionally according to any preceding embodiment, comprising a phytoene content of 30 wt % of total carotenoids or greater.
In embodiment 28, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; optionally according to any preceding embodiment; comprising a phytoene content of 40 wt % of total carotenoids or greater.
In embodiment 29, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; according to any preceding embodiment, comprising a phytoene content of 45 wt % of total carotenoids or greater.
For the avoidance of doubt, the content of total carotenoids will always total 100 wt %. In embodiment 30, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; according to any preceding embodiment, wherein the Dunaliella alga is selected from Dunaliella salina salina, Dunaliella salina bardawil and Dunaliella salina rubeus (accession number CCAP 19/41).
In embodiment 31, the invention provides a composition comprising: a) a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; according to any preceding embodiment; and b) a pharmaceutically acceptable excipient.
In embodiment 32, the invention provides a process for the preparation of a Dunaliella alga comprising exposing the Dunaliella alga to light of wavelength 500-1000 nm or 500-700 nm or 700-1000 nm; and/or eliminating light of wavelength less than 500 nm (blue light). The process preferably produces a Dunaliella alga which has increased 9-cis p-carotene content; and/or an increased colourless carotenoid content, particularly an increased phytoene content; and/or an increased α-carotene content.
In embodiment 33, the invention provides a process for the preparation of a Dunaliella alga comprising the steps:
In embodiment 34, the invention provides a process according to embodiment 32 or 33, wherein the step of exposing the Dunaliella alga to light of wavelength 500-1000 nm or 500-700 nm or 700-1000 nm; and/or eliminating light of wavelengths less than 500 nm (blue light); has a duration sufficient to achieve an increase in the 9-cis: all trans ration of 20% or greater; preferably 100% or greater; more preferably 150% or greater.
In embodiment 35, the invention provides a process according to embodiments 32 to 34, wherein the step of exposing the Dunaliella alga to light of wavelength 500-1000 nm or 500-700 nm or 700-1000 nm, preferably comprises the use of light of wavelength from greater than or equal to 500 to less than 830 nm.
In embodiment 36, the invention provides a process according to embodiments 32 to 35, wherein the step of exposing the Dunaliella alga to light of wavelength 500-1000 nm or 500-700 nm or 700-1000 nm, preferably comprises the use of light of wavelength 550-800 nm.
In embodiment 37, the invention provides a process according to embodiments 32 to 36, wherein the step of exposing the Dunaliella alga to light of wavelength 500-1000 nm or 500-700 nm or 700-1000 nm, preferably comprises the use of light of wavelength 600-750 nm.
In embodiment 38, the invention provides a process according to embodiments 32 to 37, wherein the step of exposing the Dunaliella alga to light of wavelength 500-1000 nm or 500-700 nm or 700-1000 nm, preferably comprises the use of light of wavelength 650-750 nm.
In embodiment 39, the invention provides a process according to embodiments 32 to 36, wherein the step of exposing the Dunaliella alga to light of wavelength 500-1000 nm or 500-700 nm or 700-1000 nm, preferably comprises the use of light of wavelength 600-700 nm or 650-700 nm.
The process according to embodiments 32 to 39 may be used for the cultivation of any strains of Dunaliella that produce carotenoids; preferably the Dunaliella alga is selected from Dunaliella salina salina, Dunaliella salina bardawil and Dunaliella salina rubeus (accession number CCAP 19/41).
In embodiment 40, the invention provides a process according to any one of embodiments 32 to 39, wherein the step of exposing the Dunaliella alga to light of wavelength 500-1000 nm, or 500-700 nm or 700-1000 nm, preferably from greater than or equal to 500 to less than 830 nm, preferably 550-800 nm, more preferably 600-750 nm, more preferably 650-750 nm, and more preferably 600-700 or 650-700 nm; and/or eliminating light of wavelength less than 500 nm (blue light); has a duration at least 4 hours.
In embodiment 41, the invention provides a process according to any one of embodiments 32 to 40, wherein the step of exposing the Dunaliella alga to light of wavelength 500-1000 nm, or 500-700 nm or 700-1000 nm, preferably from greater than or equal to 500 to less than 830 nm, preferably 550-800 nm, more preferably 600-750 nm, more preferably 650-750 nm, and more preferably 600-700 or 650-700 nm; and/or eliminating light of wavelength less than 500 nm (blue light); has a duration at least 12 hours.
In embodiment 42, the invention provides a process according to any one of embodiments 32 to 41, wherein the step of exposing the Dunaliella alga to light of wavelength 500-1000 nm, or 500-700 nm or 700-1000 nm, preferably from greater than or equal to 500 to less than 830 nm, preferably 550-800 nm, more preferably 600-750 nm, more preferably 650-750 nm, and more preferably 600-700 or 650-700 nm; and/or eliminating light of wavelength less than 500 nm (blue light); has a duration at least 24 hours.
In embodiment 43, the invention provides a process according to any one of embodiments 32 to 42, wherein the step of exposing the Dunaliella alga to light of wavelength 500-1000 nm, or 500-700 nm or 700-1000 nm, preferably from greater than or equal to 500 to less than 830 nm, preferably 550-800 nm, more preferably 600-750 nm, more preferably 650-750 nm, and more preferably 600-700 or 650-700 nm; and/or eliminating light of wavelength less than 500 nm (blue light); has a duration at least 48 hours.
The cultivation step a) of embodiments 33 to 43 may comprise cultivating the Dunaliella alga using any suitable method, such as in open pond systems, including cascade raceways and conventional raceways; and in closed cultivation systems, including tubular, flat-panel, green wall and thin-layer photobioreactors (PBRs). The cultivation step a) of embodiments 33 to 43 may take place outdoors or indoors, including in greenhouses.
The white light used in step a) of embodiments 33 to 43 may be any suitable source of white light, including natural light and white LED light.
The light used in step b) of embodiments 32 to 43 may be any suitable source of light of the desired wavelength, such as use of a red LED light, far-red light, or infrared light; or the use of a red filter such as the commercially available filters 26 Bright red, 27 Medium Red and 787 Marius Red (available from LEE Filters); or use of a filter that eliminates blue light, such as the commercially available filters 105 Orange, 101 Yellow, or 010 Medium Yellow. Far red light sources are known in the horticultural field.
In embodiment 44, the invention provides a process according to any one of embodiments 33 to 43, wherein step a) comprises cultivating the Dunaliella alga under natural light for a period from the beginning of cultivation to at least the log growth phase; preferably to the early orange phase.
In embodiment 45, the invention provides a process according to any one of embodiments 33 to 44, wherein in step b) the light has a wavelength in the range of from 650 nm to 700 nm and has an intensity of at least 10 μmol m−2s−1.
In embodiment 46, the invention provides a process according to any one of embodiments 33 to 45 wherein in step b) the light of the desired wavelength is applied using a red filter; or using a red LED light, far-red light or infrared light; or using an orange or yellow filter which eliminates light of wavelength less than 500 nm.
In embodiment 47, the invention provides a process according to any one of embodiments 32 to 46, wherein the step of exposing the Dunaliella alga to light of wavelength 500-1000 nm is carried out at a temperature of 20° C. or less.
In embodiment 48, the invention provides a process according to any one of embodiments 32 to 47, wherein the step of exposing the Dunaliella alga to light of wavelength 500-1000 nm is carried out at a temperature of 15° C. or less.
In embodiment 49, the invention provides a process according to any one of embodiments 32 to 48, wherein the step of exposing the Dunaliella alga to light of wavelength 500-1000 nm is carried out at a temperature of 12° C. or less.
In embodiment 50, the invention provides a process according to any one of embodiments 33 to 49, which comprises the additional steps:
In step c) of embodiment 50, the Dunaliella alga may be harvested by any suitable method; preferably using a centrifuge or membrane micro/ultrafiltration.
In step d) of embodiment 50, the carotenoids may be extracted by any suitable process known to a person skilled in the art, such as extraction into a suitable organic solvent, or using supercritical CO2, or any of the methods described in MAki-Arvela, et al (J. Chem. Technol. Biotechnol., 2014; 89: 1607-1626) or in Saini et al (Food Chemistry, 2018, 240, 90-103).
In embodiment 51, the invention provides a process according to any one of embodiments 31 to 50, wherein the Dunaliella alga is selected from any Dunaliella strain that produces carotenoids; preferably the Dunaliella alga is selected from Dunaliella salina salina, Dunaliella salina bardawil and Dunaliella salina rubeus (accession number CCAP 19/41).
In embodiment 52, the invention provides a process according to any one of embodiments 32 to 51, wherein in step a) the ambient temperature is in the range of from 4° C. to 45° C. The skilled person will understand that the temperature may vary during the cultivation step a) within the range of summer day time temperatures of up to 45° C. and winter night time temperatures down to 4° C.
The inventors have further surprisingly found that production of the colourless carotenoids phytoene and phytofluene by a Dunaliella alga or extract thereof is increased through the application of a herbicide. Thus, in embodiment 53, the invention provides a process according to any one of embodiments 32 to 52, which process comprises the steps:
For the avoidance of doubt, the term ‘a herbicide’ as used herein refers to a singular herbicide and to combinations of herbicides. When combinations of herbicides are used in the present invention, the herbicides may be applied simultaneously or sequentially.
Phytoene desaturase inhibitors, such as norflurazon, diflufenican and picolinafen, are known to have an effect on the accumulation of phytoene in Dunaliella alga. By inhibiting the activity of the phytoene desaturase (PDS), the carotenoid pathway is interrupted and the transformation of phytoene into other carotenoids is reduced. The inventors have now surprisingly found that phytoene and phytofluene content in Dunaliella alga can also be increased by treating the Dunaliella alga by application of a herbicide which is a cell division and phytochrome inhibitor, such as Chlorpropham, and postulate that such herbicides act by modulation of phytoene synthase, that is, by increasing the production of phytoene and phytofluene in the carotenoid pathway rather than by reducing the transformation of phytoene as has been seen with the application of a PDS inhibitor herbicide.
In addition to phytoene desaturase inhibitors, suitable herbicides for use in the present invention include those listed in the table below.
The active herbicidal ingredients listed above may be used as a free acid or base, or as a suitable salt. Where the compound possesses a chiral centre, the racemic form or a specific diastereoisomer or enantiomer may be used.
Particular suitable herbicides include:
Norflurazon [4-chloro-5-methylamino-2-(3-trifluoromethylphenyl)-pyridazin-3(2H)one] is a pyridazinone bleaching herbicide which inhibits carotene biosynthesis in photosynthetic organisms including D. salina, by binding reversibly in a non-competitive manner with its target enzyme phytoene desaturase. In Dunaliella sp it causes the accumulation of phytoene (Ben-Amotz A, Gressel J, Avron M (1987) Massive accumulation of phytoene induced by norflurazon in Dunaliella bardawil (Chlorophyceae) prevents recovery from photoinhibition. J Phycol 23:176-181), but not phytofluene (Ben-Amotz A, Lers A, Avron M (1988) Stereoisomers of beta carotene and phytoene in the alga Dunaliella bardawil. Plant Physiol 86:1286-1291). Other known phytoene desaturase (PDS) inhibitor herbicides, such as diflufenican and picolinafen, will also therefore permit phytoene accumulation and are suitable for use in the present invention.
Chlorpropham (isopropyl N-(3-chlorophenyl) carbamate (CIPC) (commercial names: Bud Nip, Taterpex, Preventol, Elbanil, Metoxon, Nexoval, Stickman Pistols, Preweed, Furloe, Stopgerme-S, Sprout Nip, Mirvale, Bygran, ChlorIPC, CHLOROPROPHAM, Spud-Nic, Spud-Nie, Chloro-IFK, Chloro-IPC, Keim-stop, Triherbicide CIPC) is a carbamate herbicide and plant growth regulator used for pre-emergence control of grass weeds in alfalfa, lima and snap beans, blueberries, cranberries, carrots, cranberries, ladino clover, garlic, seed grass, onions, spinach, sugar beets, tomatoes, safflower, soybeans, gladioli and woody nursery stock. In the post-harvest treatment of potatoes during storage and transport, it is also used as a sprout suppressant and for sucker control in tobacco. It is considered to be a phytochrome inhibitor (Mann et al 1967 Nature 213, 420-421), and in wheat, has been shown to disorganize cell microtubules and microtubule organizing centres to prevent cell division (Eleftheriou, E. & Bekiari, E. Plant and Soil (2000) 226: 11. Ultrastructural effects of the herbicide chlorpropham (CIPC) in root tip cells of wheat).
Aminopyralid (4-amino-3, 6-dichloropyridine-2-carboxylic acid) is a post-emergent, auxin-type herbicide that inhibits cell division and has been widely used for weed control. It is a member of the pyridine carboxylic acid family and induces an auxin-type response in susceptible plant species, causing epinastic bending and twisting of the stems that result in growth inhibition. (Li, W., et al (2018), Ecotoxicology and Environmental Safety, 155, 17-25).
Carbetamide ((R)-1-(ethylcarbamoyl)ethyl carbanilate) is a pre- and post-emergence herbicide which targets microtubule organizing centres and disrupts mitosis and cytokinesis in proliferating plant tissues, inhibiting cell division (Giménez-Abidn, M. I., Panzera, F., López-Sáez, J. F. et al. Protoplasma (1998) 204: 119).
Chlorsulfuron is a sulfonylurea herbicide which inhibits plant acetohydroxyacid synthase, the first enzyme in the branched-chain amino acid biosynthesis pathway and is closely associated with an inhibition of plant cell division.
Glyphosate acts as a transition state inhibitor of 5-enolpyruvylshikimate-3-phosphate synthase which is responsible for facilitating the assembly of shikimate-3-phosphate and phosphoenolpyruvate in the shikimate pathway and is a critical biosynthetic pathway in plant cellular plastids. (d'Avignon, Ge, (2018) J. Magnetic Resonance, 292, 59-72). It is also linked to phytochrome inhibition (Duke et al (1979), Effects of Glyphosate on Metabolism of Phenolic Compounds. Physiologia Plantarum, 46: 307-317).
In embodiment 54, the invention provides a process according to embodiment 53, wherein the herbicide is selected from amino acid synthesis inhibitors, growth regulators, nitrogen metabolism inhibitors, pigment inhibitors, seedling root growth inhibitors, seedling shoot growth inhibitors, cell wall synthesis inhibitors, mitosis microtubule organisation inhibitors, and combinations thereof.
In embodiment 55, the invention provides a process according to embodiment 53 or 54, wherein the herbicide is selected from acetolactate synthase (ALS) inhibitors, 5-enolpyruvyl-shikimate3-phosphate (EPSP) synthase inhibitors, transport inhibitor response (TIR) 1 auxin receptors (synthetic auxins), auxin transport inhibitors, glutamine synthetase inhibitors, phytoene desaturase inhibitors, bleaching 4-Hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors, carotenoid biosynthesis inhibitors (unknown target), microtubule inhibitors, long-chain fatty acid inhibitors (cell division inhibitors), cell wall synthesis inhibitors, mitosis microtubule organization inhibitors, and combinations therefore.
In embodiment 56, the invention provides a process according to any one of embodiments 53 to 55, wherein the herbicide is selected from amidosulfuron, azimsulfuron, bensulfuron-methyl, chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfuron-methyl, ethoxysulfuron, flazasulfuron, flupyrsulfuron-methyl-sodium, foramsulfuron, halosulfuron-methyl, imazosulfuron, iodosulfuron, mesosulfuron, metsulfuron-methyl, nicosulfuron, oxasulfuron, primisulfuron-methyl, prosulfuron, pyrazosulfuron-ethyl, rimsulfuron, sulfometuron-methyl, sulfosulfuron, thifensulfuron-methyl, triasulfuron, tribenuron-methyl, trifloxysulfuron, triflusulfuron-methyl, tritosulfuron, imazapic, imazamethabenz-methyl, imazamox, imazapyr, imazaquin, imazethapyr, cloransulam-methyl, diclosulam, florasulam, flumetsulam, metosulam, penoxsulam, bispyribac-sodium, pyribenzoxim, pyriftalid, pyrithiobac-sodium, pyriminobac-methyl, flucarbazone-sodium, propoxycarbazone-sodium, glyphosate, sulfosate, clomeprop, 2,4-D, 2,4-DB, dichlorprop (2,4-DP), MCPA, MCPB, mecoprop (MCPP or CMPP), chloramben, dicamba, TBA, clopyralid, fluroxypyr, picloram, triclopyr, quinclorac, Quinmerac, benazolin-ethyl, naptalam, diflufenzopyr-sodium, glufosinate-ammonium, bialaphos (bilanaphos), Norflurazon, diflufenican, picolinafen, beflubutamid, fluridone, flurochloridone, flurtamone, mesotrione, sulcotrione, isoxachlortole, isoxaflutole, benzofenap, pyrazolynate, pyrazoxyfen, Benzobicyclon, bromobutide, (chloro)-flurenol, Cinmethylin, Cumyluron, Dazomet, dymron (daimuron), methyl-dymron (methyl-dimuron), etobenzanid, fosamine, indanofan, metam, oxaziclomefone, oleic acid, pelargonic acid, pyributicarb, amitrole, benefin (benfluralin), butralin, dinitramine, ethalfluralin, oryzalin, pendimethalin, trifluralin, amiprophos-methyl, butamiphos, dithiopyr, thiazopyr, propyzamide (pronamide), tebutam, propyzamide (pronamide), tebutam, chlorthal-dimethyl (DCPA), acetochlor, alachlor, butachlor, dimethachlor, dimethenamid, metazachlor, metolachlor, pethoxamid, pretilachlor, propachlor, propisochlor, thenylchlor, diphenamid, napropamide, naproanilide, flufenacet, mefenacet, fentrazamide, anilofos, cafenstrole, piperophos, dichlobenil, chlorthiamide, chlorpropham, propham, carbetamide, and combinations thereof.
In embodiment 57, the invention provides a process according to any one of embodiments 53 to 56, wherein the herbicide is selected from amidosulfuron, azimsulfuron, bensulfuron-methyl, chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfuron-methyl, ethoxysulfuron, flazasulfuron, flupyrsulfuron-methyl-sodium, foramsulfuron, halosulfuron-methyl, imazosulfuron, iodosulfuron, mesosulfuron, metsulfuron-methyl, nicosulfuron, oxasulfuron, primisulfuron-methyl, prosulfuron, pyrazosulfuron-ethyl, rimsulfuron, sulfometuron-methyl, sulfosulfuron, thifensulfuron-methyl, triasulfuron, tribenuron-methyl, trifloxysulfuron, triflusulfuron-methyl, tritosulfuron, imazapic, imazamethabenz-methyl, imazamox, imazapyr, imazaquin, imazethapyr, cloransulam-methyl, diclosulam, florasulam, flumetsulam, metosulam, penoxsulam, bispyribac-sodium, pyribenzoxim, pyriftalid, pyrithiobac-sodium, pyriminobac-methyl, flucarbazone-sodium, propoxycarbazone-sodium, glyphosate, sulfosate, benazolin-ethyl, glufosinate-ammonium, bialaphos (bilanaphos), norflurazon, diflufenican, picolinafen, beflubutamid, fluridone, flurochloridone, flurtamone, mesotrione, sulcotrione, isoxachlortole, isoxaflutole, benzofenap, pyrazolynate, pyrazoxyfen, Benzobicyclon, bromobutide, (chloro)-flurenol, Cinmethylin, Cumyluron, Dazomet, dymron (daimuron), methyl-dymron (methyl-dimuron), etobenzanid, fosamine, indanofan, metam, oxaziclomefone, oleic acid, pelargonic acid, pyributicarb, benefin (benfluralin), butralin, dinitramine, ethalfluralin, oryzalin, pendimethalin, trifluralin, amiprophos-methyl, butamiphos, dithiopyr, thiazopyr, propyzamide (pronamide), tebutam, propyzamide (pronamide), tebutam, chlorthal-dimethyl (DCPA), chlorpropham, propham, carbetamide, and combinations thereof.
In embodiment 58, the invention provides a process according to any one of embodiments 53 to 57, wherein the herbicide is selected from norflurazon, diflufenican, picolinafen, beflubutamid, fluridone, flurochloridone, flurtamone, chlorpropham, propham, carbetamide, and combinations thereof; most preferably chlorpropham.
In embodiment 59, the invention provides a process for the preparation of a Dunaliella alga, comprising treating the Dunaliella alga by applying a herbicide selected from the group consisting of amino acid synthesis inhibitors, growth regulators, nitrogen metabolism inhibitor, pigment inhibitors (excluding phytoene desaturase inhibitors), seedling root growth inhibitors, seedling shoot growth inhibitors, cell wall synthesis inhibitors, mitosis microtubule organisation inhibitors, and combinations thereof.
In embodiment 60, the invention provides a process according to embodiment 59, wherein the herbicide is selected from acetolactate synthase (ALS) inhibitors, 5-enolpyruvyl-shikimate3-phosphate (EPSP) synthase inhibitors, transport inhibitor response (TIR) 1 auxin receptors (synthetic auxins), auxin transport inhibitors, glutamine synthetase inhibitors, bleaching 4-Hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors, carotenoid biosynthesis inhibitors (unknown target), microtubule inhibitors, long-chain fatty acid inhibitors (cell division inhibitors), cell wall synthesis inhibitors, mitosis microtubule organization inhibitors, and combinations therefore.
In embodiment 61, the invention provides a process according to embodiment 59 or 60, wherein the herbicide is selected from amidosulfuron, azimsulfuron, bensulfuron-methyl, chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfuron-methyl, ethoxysulfuron, flazasulfuron, flupyrsulfuron-methyl-sodium, foramsulfuron, halosulfuron-methyl, imazosulfuron, iodosulfuron, mesosulfuron, metsulfuron-methyl, nicosulfuron, oxasulfuron, primisulfuron-methyl, prosulfuron, pyrazosulfuron-ethyl, rimsulfuron, sulfometuron-methyl, sulfosulfuron, thifensulfuron-methyl, triasulfuron, tribenuron-methyl, trifloxysulfuron, triflusulfuron-methyl, tritosulfuron, imazapic, imazamethabenz-methyl, imazamox, imazapyr, imazaquin, imazethapyr, cloransulam-methyl, diclosulam, florasulam, flumetsulam, metosulam, penoxsulam, bispyribac-sodium, pyribenzoxim, pyriftalid, pyrithiobac-sodium, pyriminobac-methyl, flucarbazone-sodium, propoxycarbazone-sodium, glyphosate, sulfosate, clomeprop, 2,4-D, 2,4-DB, dichlorprop (2,4-DP), MCPA, MCPB, mecoprop (MCPP or CMPP), chloramben, dicamba, TBA, clopyralid, fluroxypyr, picloram, triclopyr, quinclorac, Quinmerac, benazolin-ethyl, naptalam, diflufenzopyr-sodium, glufosinate-ammonium, bialaphos (bilanaphos), mesotrione, sulcotrione, isoxachlortole, isoxaflutole, benzofenap, pyrazolynate, pyrazoxyfen, Benzobicyclon, bromobutide, (chloro)-flurenol, Cinmethylin, Cunyluron, Dazomet, dymron (daimuron), methyl-dymron (methyl-dimuron), etobenzanid, fosamine, indanofan, metam, oxaziclomefone, oleic acid, pelargonic acid, pyributicarb, amitrole, benefin (benfluralin), butralin, dinitramine, ethalfluralin, oryzalin, pendimethalin, trifluralin, amiprophos-methyl, butamiphos, dithiopyr, thiazopyr, propyzamide (pronamide), tebutam, propyzamide (pronamide), tebutam, chlorthal-dimethyl (DCPA), acetochlor, alachlor, butachlor, dimethachlor, dimethenamid, metazachlor, metolachlor, pethoxamid, pretilachlor, propachlor, propisochlor, thenylchlor, diphenamid, napropamide, naproanilide, flufenacet, mefenacet, fentrazamide, anilofos, cafenstrole, piperophos, dichlobenil, chlorthiamide, chlorpropham, propham, carbetamide, and combinations thereof.
In embodiment 62, the invention provides a process according to any one of embodiments 59 to 61, wherein the herbicide is selected from amidosulfuron, azimsulfuron, bensulfuron-methyl, chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfuron-methyl, ethoxysulfuron, flazasulfuron, flupyrsulfuron-methyl-sodium, foramsulfuron, halosulfuron-methyl, imazosulfuron, iodosulfuron, mesosulfuron, metsulfuron-methyl, nicosulfuron, oxasulfuron, primisulfuron-methyl, prosulfuron, pyrazosulfuron-ethyl, rimsulfuron, sulfometuron-methyl, sulfosulfuron, thifensulfuron-methyl, triasulfuron, tribenuron-methyl, trifloxysulfuron, triflusulfuron-methyl, tritosulfuron, imazapic, imazamethabenz-methyl, imazamox, imazapyr, imazaquin, imazethapyr, cloransulam-methyl, diclosulam, florasulam, flumetsulam, metosulam, penoxsulam, bispyribac-sodium, pyribenzoxim, pyriftalid, pyrithiobac-sodium, pyriminobac-methyl, flucarbazone-sodium, propoxycarbazone-sodium, glyphosate, sulfosate, benazolin-ethyl, glufosinate-ammonium, bialaphos (bilanaphos), mesotrione, sulcotrione, isoxachlortole, isoxaflutole, benzofenap, pyrazolynate, pyrazoxyfen, Benzobicyclon, bromobutide, (chloro)-flurenol, Cinmethylin, Cumyluron, Dazomet, dymron (daimuron), methyl-dymron (methyl-dimuron), etobenzanid, fosamine, indanofan, metam, oxaziclomefone, oleic acid, pelargonic acid, pyributicarb, benefin (benfluralin), butralin, dinitramine, ethalfluralin, oryzalin, pendimethalin, trifluralin, amiprophos-methyl, butamiphos, dithiopyr, thiazopyr, propyzamide (pronamide), tebutam, propyzamide (pronamide), tebutam, chlorthal-dimethyl (DCPA), chlorpropham, propham, carbetamide, and combinations thereof.
In embodiment 63, the invention provides a process according to any one of embodiments 59 to 62, wherein the herbicide is selected from chlorpropham, propham, carbetamide, and combinations thereof; most preferably chlorpropham.
In embodiment 64, the invention provides a process for preparing a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; according to any one of embodiments 24 to 30, wherein the process is as defined in any one of embodiments 53 to 63.
In embodiment 65, the invention provides the use of a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; as defined in any one of embodiments 1 to 30; as a food colourant or food ingredient; or as a health supplement.
In embodiment 66, the invention provides the use of a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; as defined in any one of embodiments 1 to 30; in a cosmetic composition.
In embodiment 67, the invention provides a Dunaliella alga, or extract thereof; or a powdered Dunaliella alga, or extract thereof; as defined in any one of embodiments 1 to 30; or a composition as defined in embodiment 31, for use in therapy.
The term ‘Dunaliella alga’ as used herein refers to the multiple strains of Dunaliella that produce carotenoids. Nomenclature of these strains has not historically been consistent.
For example, Dunaliella bardawil is considered by some references to be a strain of Dunaliella salina, but is considered by others to be a different strain.
The term ‘grown or cultivated under natural light or white light conditions’ as used herein, refers to Dunaliella algae growing, or specifically cultivated, in ponds, lakes, lagoons, raceways or closed vessels under natural light or under artificial white light.
The term ‘raceway’ as used herein, refers to a shallow pond that uses sunlight as the light source and paddlewheels to provide the flow to circulate algae, water and nutrients keeping the algae suspended in the water, and circulating them back to the surface on a regular frequency. The ponds are operated continuously with carbon dioxide or flue gas containing CO2 and nutrients are fed constantly or by batch to the ponds.
The term ‘cascade raceway’ as used herein, refers to a raceway which uses gravity instead of a paddlewheel to promote the mixing of the culture as it flows on the surface of inclined surfaces. After each cycle it is necessary to reposition the culture on the top part of the cascade through a pump or another device thus ensuring the flow cycle is closed.
The term ‘photobioreactor’ (PBR) as used herein, refers to a closed vessel or bioreactor, which incorporates some type of light source for photo- or mixo-trophic cultivation of algae. The light source is usually sunlight, but can also include artificial lighting. All essential nutrients must be introduced into the system to allow algae to grow and be cultivated. A photobioreactor can be operated in “batch mode” but it is also possible to introduce one or multiple continuous streams of process water containing nutrients, air and carbon dioxide. Temperature control (heating and cooling) are easily achievable. Many photobioreactor designs have been created and include vertical Green-wall flat panels (Green-walls, GW) comprising a thin layer of liquid (5-10 cm) contained in an aerated transparent plastic bag supported by a metal framework, and tubular photobioreactors, which consist of vertical or horizontally displayed transparent tubes, which can be stacked in groups to yield parallel fence-like vertical sets, and connected through piping accessories to a tank/degassing column, where most of the automation equipment is located, as well as the inlets and outlets for all the utilities. Culture mixing is ensured by pumping (in some cases also compressed air).
The term ‘increased content of’ as used herein, refers to an increase in the content of the carotenoid relative to the content found in Dunaliella algae which is grown or cultivated under natural conditions, i.e. under natural light or white light conditions and without herbicide treatment.
The term ‘early orange phase’ as used herein, refers to the growth phase that typifies the start of carotenogenesis, and is usually associated with the onset of stress related to deficiency in nitrate, sulfate, and phosphate in the culture media as well as high light intensity and high sodium chloride concentration. The carotenoid:chlorophyll ratio in cells is typically 3 or more.
The term ‘log growth phase’ as used herein refers to the period of algal growth characterized by cell doubling (also known as the logarithmic phase or exponential phase). The carotenoid:chlorophyll ratio in cells is typically around 1.
The term ‘powdered Dunaliella alga’ as used herein refers to a powdered product of Dunaliella alga which may be obtained by spray-drying or freeze-drying or any other method of dehydration.
The term ‘light of wavelength’ or ‘wavelength in the range of’ as used herein, refers to light having a wavelength of light emittance in the specified range by the source. For the avoidance of doubt the wavelength of light range includes either a single wavelength of light emittance within the specified range or any number of single wavelengths of light emittance within the specified range.
The term ‘herbicide’ as used herein refers to a composition which controls, suppresses or destroys plant growth. The herbicide may be defined by the mechanism of action, including phytoene desaturase inhibitors, phytochrome inhibitors, auxin-type (synthetic auxin) herbicides), cell division inhibitors, enolpyruvylshikimate 3-phosphate synthase enzyme (EPSPS) inhibitors, acetyl coenzyme A carboxylase (ACCase) inhibitors, acetolactate synthase (ALS) inhibitors, photostem II inhibitors, photostem I inhibitors, and 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors.
The invention is illustrated by the following examples.
Dunaliella algae were cultured in the laboratory in an ALGEM Environmental Modeling Labscale Photobioreactor (Algenuity, UK), at 25° C. Approximately 5×107 cells were inoculated in 500 ml Modified Johnsons Medium (Borowitzka, Algal growth media and sources of cultures, in Microalgal Biotecnology. Borowitzka, L. J. (Eds.), 1988, pp. 456-465) containing 1.5 M NaCl and placed under a cycle of 12 h/12 h Light/Dark conditions. Cells were grown under 200 μmol photons·m−2·s−1 of white LED light. In one set of experiments cells were cultivated to a cell density of ˜0.5×106 cells mL−1 and then the cultures were diluted with fresh medium to a cell density of ˜0.2×106 cells mL−1. Under these conditions cells were in the early orange phase of growth but not placed under nutrient stress. The cultures were then exposed to either white LED light, red LED light, or blue LED light at the same light intensity of 1000 μmol m−2 s−1, or white LED light of 1000 μmol m−2 s−1 covered with one of three different red filters (filter 26 Bright red, 27 Medium Red and 787 Marius Red supplied by LEE Filters) for 48 hours. Each light condition was set up in at least triplicate. Dunaliella algae used in these experiments were the following strains:
PLY DF15, classified as D salina rubeus (and held by the Marine Biological Association Culture Collection, origin Israel) and also classified as CCAP 19/41 and held by the Culture Collection of Algae and Protozoa (CCAP).
PLY DF17, classified as D. salina salina (held by the Marine Biological Association Culture Collection, origin Israel)
PLY DF40, classified as D. salina bardawil (held by the Marine Biological Association Culture Collection, origin Spain) and also classified as D. salina CCAP 19/40 and held by the Culture Collection of Algae and Protozoa (CCAP).
UTEX 2538, classified as D. salina bardawil (Culture Collection of Algae and Protozoa (CCAP))
In a second set of experiments cells were cultivated to the log phase of growth and then kept in the dark for 36 hours for dark adaption. After dark adaption, the cultures were exposed to continuous blue or red LED light at different light intensities of 200, 500, and 1000 μmol photons·m−2·s−1 for 48 hours. Each growth condition was set up in at least triplicate.
In a third set of experiments, Dunaliella algae were cultivated at 25° C. under 200 μmol photons·m−2·s−1 of white LED light to log phase and then kept in the dark for 36 hours for dark adaption. Then the cultures were exposed to continuous blue or red LED light at the light intensity of 1000 μmol photons·m−2·s−1 at 15° C. compared to 25° C. for 48 hours.
Cell concentration: Cell concentration was determined by counting the number of cells in culture broth using a haemocytometer, after fixing with 2% formalin. Samples were taken at 0, 24 and 48 hours to determine the cellular contents of carotenoids and chlorophyll and the composition of the carotenoids.
Pigment analysis: 1 ml culture broth was centrifuged at 3,000 g in a bench-top centrifuge for 5 min. to harvest the algal biomass and pigments were extracted from the biomass using 1 ml of 80% (v/v) acetone. After clarification at the centrifuge, the absorbance of the acetone extract was measured at 480 nm in a spectrophotometer. The content of total carotenoids was calculated according to Strickland & Parsons (Strickland, J. & Parsons, T. R., 1972. A practical handbook of seawater analysis 2nd ed., Fish Res Board Can Bull.): Total Carotenoids (μg·ml−1)=4.0*Abs480nm, where Abs480nm is the absorbance of 80% acetone extract measured at 480 nm.
Chlorophyll a, b and total Chlorophyll were evaluated by measuring the absorbance of the acetone extract at 664 nm and 647 nm and calculated according to Porra, R. J., Thompson, W. A. & Kriedemann, P. E., 1989. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 975(3), pp. 384-394:
Chl a (μg·ml−1)=(12.25*Abs664nm)−(2.55*Abs647nm);
Chl b (μg·ml−1)=(20.31*Abs647nm)−(4.91*Abs664nm),
Total Chl (μg·ml−1)=Chl a (μg·ml−1)+Chl b (μg·ml−1),
where Abs647nm and Abs664nm refer to the absorbance of the 80% acetone extract measured at 664 nm and 647 nm respectively.
The compositions of pigments were analysed using an HPLC with fitted with diode-array detection (DAD). 15 ml culture broth was centrifuged at 3,000 g in a bench-top centrifuge for 5 min. to harvest the algal biomass as before. Algal biomass was extracted with 10 ml MTBE-MeOH (20:80), after sonication for 20 s. Each sample was clarified by centrifugation at 3,000 g for 10 min then filtered through a 0.45 μm filter into amber HPLC vials. The samples were analysed using a YMC30 250×4.9 mm I.D S-5μ HPLC column with DAD at 25° C., and isocratic elution with 80% methanol: 20% MTBE, flow rate of 1 mL min−1, pressure of 90 bar. The quantities of β-carotene in the biomass were estimated using a β-carotene standard curve prepared with synthetic all-trans β-carotene from Sigma, and the quantities of phytoene and α-carotene, with reference to standards of each from Sigma. Each experiment was carried out in at least triplicate.
Treatment of D. salina cultures with red light included in the cultivation cycle was observed to increase both the ratio of 9-cis to all-trans β-carotene and the amount of carotenoid compared to cultivation under a white:dark light cycle, with the greatest increases occurring with continuous red light, whether applied with red LED or with red filters. Compensation for the intensity of light emitted by LED lights may be required when red filters are applied as covers to LED lights. The results are presented in
Treatment of D. salina cultures with far-red light of 730 nm was found to be as effective in increasing β-carotene production and the 9-cis/all-trans ratio as red light transmitted by Lee Filter 027 (600-700 nm). The carotenoids, 9-cis/all-trans ratio and chlorophyll content of cultures under far red and red light were identical. Both far red light and red light increased the 9-cis/all-trans ratio from ˜1.5 to ˜2.0 compared to white light alone. By contrast with LED light of wavelength 830 nm applied for 3 days, the cells did not divide, as was also found for cells placed in the dark for 3 days. The 9-cis/all-trans β-carotene ratio decreased for cells placed in the dark or treated with 830 nm light compared to untreated cells and the yield of carotenoids and β-carotene also slightly decreased. The results are presented in
Treatment of D. salina cultures with red light-dark cycles of increasing red light cycle duration was found to increase cell density, total carotenoids and 9-cis: all-trans ratio, with the greatest effect being meted with continuous red light. 9-cis-β-Carotene content was found to continuously increase with continuous red light for 140 h, whereas total carotenoid content showed no further increase after 72 h, which may reflect decreasing cellular synthetic capacity, since total chlorophyll content declines continuously in continuous red light for 140 h. Results are presented in
Treatment of D. salina cultures cultivated under red light with phytoene desaturase inhibitor herbicides was found to result in a significantly higher amount of phytoene when compared to cultivation under white light. Results are presented in
D. salina cultures were treated with herbicides which inhibit cell division, such as chlorpropham (CIPC), aminopyralid, carbetamide and chlorsulfuron, or with phytochrome inhibitors such as glyphosate. The content of both phytoene and phytofluene as well as the content of coloured carotenoids were found to have increased when D. salina is cultured in the presence of the herbicides, and cultivation under red light was found to magnify the effects. Results are presented in Table 4 and
D. salina cultures were treated with chlorpropham. The cellular content of colorless carotenoids was found to increase by more than 30-fold and the yield of the colorless carotenoids was found to increase more than 10-fold compared to untreated cultures. The optimal concentration range of chlorpropham added to the cultures was determined to be 10-50 μM. Cell density stopped increasing once chlorpropham was added, and carotenoids in particular phytoene and phytofluene started to accumulate. Chlorpropham is preferably added to the cultures when a high cell density is achieved.
Cells were cultured in light:dark 12 h:12 h in incubators with white light to early orange phase (cell density of ˜0.5×106 cells mL−1; carotenoid:chlorophyll ratio ˜3) and then cultures were diluted with fresh medium to a cell density of ˜0.2×106 cells mL−1 (no nutrient stress). The cultures were then exposed to either white LED light, red LED light, or blue LED light at the same light intensity of 1000 μmol m−2 s−1, or white LED light of 1000 μmol m−2 s−1 covered with one of three different red filters (Lee filter 26 Bright red, 27 Medium Red and 787 Marius Red, see
These data show that the cellular content of chlorophyll and in turn phytoene and α-carotene may vary to compensate for reduced light availability using filters (see
The total carotenoid content increased from 8.58±1.09 pg cell−1 (dark-adapted cells) to 22.47±2.34 pg cell−1 after treatment with red light for 48 h (2.6-fold increase) (see
The total carotenoid content increased from 8.58±1.09 pg cell−1 (dark-adapted cells) to 22.47±2.34 pg cell−1 after treatment with red light for 48 h (2.6-fold increase). The chlorophyll content decreased under these conditions such that the ratio of carotenoids:chlorophyll increased from 2 in white light on exposure to red light for 24 h, to 5.5.
These data show that red LED light specifically enhances production of 9-cis-β-carotene relative to all-trans-β-carotene. Furthermore, the effect on 9-cis-β-carotene and on all-trans-β-carotene is independent of light intensity.
Cultures of D. salina were grown to mid-log phase and then exposed to different 24 h cycles of light treatment applied for 3 days. Biomass was harvested at mid-day on the 3rd day for analysis by HPLC. The cycles were as follows:
Cultures of D. salina were grown to a cell density of ˜0.2 million cells ml−1 under white LED light and then transferred for either 48 h growth (
The carotenoids, 9-cis/all-trans ratio and chlorophyll content of cultures under far red and red light were identical (
Cultures of D. salina were grown to a cell density of ˜0.2 million cells ml−1 under white LED light and then transferred into red LED light growth cycles of different duration, which were maintained for 6 days. The light intensity of red LED light was set at 500 μmol m−2 s−1. The cycles were as follows:
(
(
(
(
Chlorpropham stock solution of 1 M was added to cultures of D. salina to different final concentrations (0, 0.1, 1, 10, 20, 50 and 100 μM) and cultures were maintained in an incubator at 25° C. Carotenoids profile was analysed for each culture by HPLC. For (
The optimal concentration of chlorpropham for phytoene production was between 10-50 μM.
After 6-days cultivation in white LED light in the presence of 20 μM chlorpropham, the phytoene content in cells increased ca. 50-fold compared to that in untreated cells (untreated cells: 0.55±0.01 pg cell−1, treated cells 25.76±1.58 pg cell−1) whilst the final phytoene concentration in the cultures increased 10-fold (untreated cultures 0.35±0.01 mg L−1; treated 3.55±0.11 mg L−1). With increasing light intensity of applied white light, phytoene content per cell and yield increased: after just 4 days' cultivation, the phytoene content reached above 30 pg cell−1 under 1500 μmol m−2 s−1, giving a yield of 8.2 mg L−1. Under red light, cultures had higher phytoene contents than cultures maintained under white light with the same concentration of chlorpropham treatment.
(
(
Cultures were maintained at 25° C. under continuous white or red LED light at ˜200 μmol m−2 s−1 and carotenoid contents determined daily by HPLC as before.
Samples were extracted using absolute ethanol and extracts were analysed using a YMC30 250×4.9 mm I.D S-5μ HPLC column with DAD at 25° C., and isocratic elution with 80% methanol: 20% MTBE, flow rate of 1 mL min−1, pressure of 90 bar. Alternatively they were analysed using a Waters Acquity UPCC (Waters, UK) instrument fitted with a Diode Array Detector and connected to a Synapt G2 HDMS (Waters, UK). The Synapt G2 was fitted with an electrospray source, and operated in positive ion mode over a mass range of 50-800 m/z units. Wavelength-dependent absorption was measured using the DAD, and operating in the wavelength range 200-700 nm. Phytoene was separated using an Acquity UPLC HSS C18 SB, 3.0×100 mm, 1.8 μm particle size, inlet conditions: scCO2 (
It can be seen from the data presented in Tables 1 to 3, and
Table 4 provides a comparison of the contents of carotenoids for cultures of D. salina cultivated for 48 h under either white LED light (A) or red LED light (B) in the presence of either Norflurazon or chlorpropham (C1PO compared to cultivation without herbicide Both white and red LED lights were applied at 200 pmol m−2 s−1 continuously for 48 h after adding the herbicides Red light more than doubled the vield of phv toene in either CIPC or norflurazon-treated cultures increased the content of total carotenoids, and increased the ratio of 9-cts all-trans β-carotene
Table 5 shows the contents of carotenoids produced for cultures of D. salina cultivated for 96 h and 144 h under red light of varying intensities and different illumination time, with the application of chlorpropham. Data for diflufenican are also shown.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1718822.8 | Nov 2017 | GB | national |
| 1719440.8 | Nov 2017 | GB | national |
| 1813776.0 | Aug 2018 | GB | national |
The present application is a divisional of U.S. patent application Ser. No. 16/763,919, filed May 13, 2020, the entire contents of which are hereby incorporated herein by reference; U.S. patent application Ser. No. 16/763,919 is a National Stage of International Application No.: PCT/GB2018/053278 that was filed Nov. 13, 2018; the entire contents of which are hereby incorporated by reference; International Application No. PCT/GB2018/053278 claims priority to GB patent application No. 1718822.8 that was filed Nov. 14, 2017, GB patent application No. 1719440.8 that was filed Nov. 23, 2017, and GB patent application No. 1813776.0 that was filed Aug. 23, 2018 the entire contents of all three of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16763919 | May 2020 | US |
| Child | 17964312 | US |
