New strain of the microalga haematococcus pluvialis with high astaxanthin yield

Abstract

The present invention relates to a strain of microalgae belonging to the genus Haematococcus with a high astaxanthin yield, and to a method for producing astaxanthin; in particular, the invention relates to a strain of the microalga Haematococcus pluvialis comprising at least 5% astaxanthin by weight relative to the total weight of Haematococcus pluvialis dry matter.

Description

[0001] NEW STRAIN OF HAEMATOCOCCUS PLUVIALIS MICROALGAE WITH HIGH ASTAXANTHIN YIELD

[0002] technical field

[0003] The invention relates to the technical field of microalgae. In particular, the invention relates to a specific strain of Haematococcus pluvialis with high astaxanthin yield and a process for producing astaxanthin using said strain.

[0004] State of the art

[0005] The growing demand for naturally sourced molecules is prompting manufacturers to explore innovative production methods.

[0006] Microalgae, unicellular photosynthetic organisms, are emerging as prime candidates to meet this need, offering a unique cellular environment conducive to the biosynthesis of compounds of interest.

[0007] In the context of natural molecule production, microalgae offer many advantages such as their ability to thrive in varied environmental conditions, their rapid growth rate, and their low environmental footprint, making them ideal candidates for sustainable and economically viable production processes.

[0008] However, despite the many advances in the field of biotechnology, microalgae sometimes have a production yield that is too low compared to market needs.

[0009] This is particularly true for the production of naturally sourced astaxanthin. Astaxanthin (3,3'-dihydroxy-3,P'-carotene-4,4'-dione) is a red carotenoid belonging to the xanthophyll family. Astaxanthin is known for its beneficial properties in humans and animals, such as:

[0010] *Its antioxidant effect: One of the most remarkable properties of astaxanthin is its exceptional antioxidant power. Astaxanthin has a capacity far superior to that of vitamin E, vitamin C, and many other carotenoids; and

[0011] *its anti-inflammatory effects.

[0012] Due to its exceptional properties, astaxanthin finds applications in many fields such as the food, pharmaceutical, cosmetic and aquaculture industries.

[0013] Hematococcus pluvialis, a freshwater microalga, is the main natural source of astaxanthin. However, despite its inherent capabilities, the natural production of this molecule by Hematococcus pluvialis remains limited, not fully meeting the growing market demand.

[0014] Despite the difficulties in producing naturally sourced astaxanthin, astaxanthin remains a compound of vital importance, motivating the search for innovative solutions to overcome the limitations related to its natural production.

[0015] There is therefore a need for new approaches aimed at substantially increasing the production capacity of astaxanthin from microalgae, particularly from Hematococcus pluvialis, and thus meeting the demand of industries that need it.

[0016] In particular, there is a need for microalgae strains capable of producing astaxanthin more efficiently, with increased biomass productivity and the ability to accumulate astaxanthin under less stressful culture conditions. Such strains would optimize industrial astaxanthin production, reducing energy costs and increasing yields.

[0017] Furthermore, there is a growing interest in more sustainable and economically viable astaxanthin production processes. This includes cultivation methods using moderate light intensities, which could reduce energy consumption and the costs associated with large-scale production.

[0018] Finally, there is a need for microalgae strains capable of producing high-quality astaxanthin under various culture conditions, including autotrophic and mixotrophic conditions, in order to adapt to different industrial production configurations.

[0019] These needs underline the importance of developing new strains of microalgae and innovative cultivation processes to improve the production of natural astaxanthin and meet the growing demands of industries.

[0020] Summary of the invention

[0021] To meet this need, the invention proposes a new strain of Hematococcus pluvialis microalgae comprising at least 5% astaxanthin by weight relative to the total weight of dry matter.

[0022] Surprisingly, the inventors succeeded in obtaining a new strain of interest from a wild strain of Haematococcus pluvialis. The invention addresses the needs of industry by offering a new microalgae strain with improved astaxanthin production compared to strains described in the prior art, particularly the wild strain UTEX 2505 referenced in the algae culture collection at the University of Texas at Austin. Indeed, the wild strain UTEX 2505, known for its high astaxanthin production capacity, contains less than 5% astaxanthin by weight relative to the total dry weight.

[0023] Preferably, the microalgae strain according to the invention is a particular microalgae strain belonging to the genus Hematococcus and the species pluvialis, also called Hematococcus pluvialis, chosen from the strain deposited with the Spanish algae bank (Banco Español de Algas) under number BEA_IDA_0084, BEA_IDA_0087 or BEA_IDA_0089.

[0024] These new, genetically stable strains exhibit higher astaxanthin production than the wild-type UTEX 2505 strain, which is known for its high astaxanthin production. The strain according to the invention is characterized by a high astaxanthin content, increased biomass productivity, and its ability to produce astaxanthin at moderate light intensities. Most preferably, said strain achieves an astaxanthin content of at least 6.2% of the dry weight of the biomass and a biomass productivity of 0.20 g / L / day. It allows for efficient pigment accumulation without requiring light stress exceeding 150 pE / m². 2 / s, which makes it particularly suited to industrial closed culture conditions.

[0025] According to a preferred object of the invention, the microalgae strain according to the invention is a strain derived from at least one strain chosen from the microalgae strain filed under number BEA_IDA_0084 or the microalgae strain filed under number BEA_IDA_0087 or the microalgae strain filed under number BEA_IDA_0089.

[0026] According to another particularly preferred object of the invention, the microalgae strain according to the invention is a strain comprising an 18S rDNA sequence having at least 97% identity with the SEQ ID NO: 1 18S rDNA sequence of the strain filed under number BEA_IDA_0084.

[0027] Advantageously, the strain filed under number BEA_IDA_0084 has an 18S rDNA sequence identical to the 18S rDNA sequence of the strain filed under number BEA_IDA_087 and to the 18S rDNA sequence of the strain filed under number BEA_IDA_0089.

[0028] Most preferably, the invention relates to a new strain of Haematococcus pluvialis microalgae filed under number BEA_IDA_0084 or under number BEA_IDA_0087 or under number BEA_IDA_0089.

[0029] According to another aspect, the invention relates to the use of at least one strain of Haematococcus pluvialis microalgae to produce astaxanthin, preferably said microalgae strain is chosen from the microalgae strain filed under number BEA_IDA_0084 or the microalgae strain filed under number BEA_IDA_0087 or the microalgae strain filed under number BEA_IDA_0089.

[0030] Finally, the invention relates, according to another aspect, to a process for the production of astaxanthin comprising the cultivation of at least one strain of microalgae of Hematococcus pluvialis comprising at least 5% astaxanthin by weight of dry matter, preferably the strain of microalgae of Hematococcus pluvialis is chosen from the strain of microalgae filed under number BEA_IDA_0084 or the strain of microalgae filed under number BEA_IDA_0087 or the strain of microalgae filed under number BEA_IDA_0089.

[0031] Other features and advantages will become apparent from the detailed description of the invention, the examples and figures being purely illustrative and in no way limiting of the scope of the invention.

[0032] Brief description of the figures

[0033] [Fig. 1] Figure 1 is a graphical representation of a comparative test of the amount of dry matter produced after 18 days of culture of a wild strain of Hematococcus pluvialis compared with a strain according to the invention.

[0034] [Fig. 2] Figure 2 is a graphical representation of a comparative test of the concentration of astaxanthin found in a sample of microalgae after 18 days of culture belonging to a wild strain of Hematococcus pluvialis compared with a strain according to the invention.

[0035] [Fig. 3] Figure 3 is a graphical representation of a comparative test of the mass concentration of astaxanthin found in the dry matter produced after 18 days of culture of a wild strain of Hematococcus pluvialis compared to a strain according to the invention.

[0036] [Fig. 4] Figure 4 is a graphical representation of a comparative trial of biomass productivity between the wild strain Utex 2505 and the strain BEA_IDA_0089 according to the invention, under different culture conditions.

[0037] [Fig. 5] Figure 5 is a graphical representation of a comparative trial of astaxanthin production between the wild strain Utex 2505 and the strain BEA_IDA_0089 according to the invention, under different culture conditions.

[0038] [Fig. 6] Figure 6 is a graphical representation of a comparative test of astaxanthin production between the wild strain Utex 2505 and the strain BEA_IDA_0089 according to the invention, under low light intensity conditions.

[0039] Detailed description of the invention

[0040] Definitions. For the purposes of this invention, "microalgae" refers to microorganisms that are largely photosynthetic and unicellular or multicellular. Microalgae are classified by family, genus, and species. Each species of microalgae comprises a diversity of microalgal strains. In the context of this invention, the microalgal strain belongs to the family Haematococcaceae, the genus Hematococcus, and the species pluvialis.

[0041] For the purposes of this invention, "microalgae strain" or "strain" means a specific microalgae strain but also all microalgae derived from the strain or obtained from the strain or corresponding to the microalgae strain and having the same metabolic functions, for example, at least one microalga taken from a colony derived from the strain.

[0042] For the purposes of this invention, "microalgae according to the invention" means a strain of microalgae according to the invention.

[0043] For the purposes of this invention, "derived microalgae strain" or "mutant strain" or "derived strain" means a microalgae strain having a high similarity to the microalgae strain filed under number BEA_IDA_0084 or the microalgae strain filed under number BEA_IDA_0087 or the microalgae strain filed under number BEA_IDA_0089, in particular by maintaining or improving the astaxanthin production capacities of the microalgae strain BEA_IDA_0084 or the microalgae strain BEA_IDA_0087 or the microalgae strain BEA_IDA_0089.Preferably, the strain comprises a nucleotide sequence having at least 99.90% ANI identity with the nucleotide sequence of the genome of microalgae strain BEA_IDA_0084 or microalgae strain BEA_IDA_0087 or microalgae strain BEA_IDA_0089, more preferably at least 99.91%, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, or at least 99.99% ANI identity.

[0044] For the purposes of this invention, "ANI" means the average percentage of nucleotide identity calculated from a pairwise comparison of all genome sequences shared between the two microalgae strains. According to the general knowledge well known to a person skilled in the art, from at least one strain of interest chosen from the microalgae strain deposited with the BEA under number BEA_IDA_0084 or the microalgae strain deposited with the BEA under number BEA_IDA_0087 or the microalgae strain deposited with the BEA under number BEA_IDA_0089, genomic DNA can be extracted from a pure microalgae culture from said strain of interest, followed by DNA sequencing according to various well-known methods, for example Sanger, Roche 454, Illumina, Oxford Nanopore, then the sequenced genome is assembled by bioinformatics and the sequences obtained are analyzed.Finally, the genomes of interest are compared two by two to calculate the ANI.

[0045] For the purposes of this invention, "wild strain" or "wild strain of Hematococcus pluvialis" refers to the UTEX 2505 wild strain referenced in the University of Texas at Austin algae culture collection, known for its ability to produce astaxanthin. This wild strain according to the invention was collected from a natural environment and has not undergone any selection or genetic modification.

[0046] For the purposes of this invention, "by weight of dry matter" refers to the quantity of astaxanthin present in the dry matter of Hematococcus pluvialis. This percentage can also be expressed in grams per 100 grams of dry matter.

[0047] For the purposes of this invention, "dry matter" refers to dried Hematococcus pluvialis biomass. Preferably, the dry matter is obtained after culturing the microalgae strain according to the invention under optimal conditions to induce astaxanthin production.

[0048] For the purposes of this invention, "microalgae culture" means the liquid culture medium suitable for the growth and reproduction of microalgae, as well as said microalgae.

[0049] By "liquid culture medium suitable for the growth and reproduction of microalgae" is meant a culture medium having at least one of the following parameters, namely: a temperature between 25 and 32°C, advantageously between 28 and 32°C; or a pH greater than 6.8, advantageously a pH between 7.5 and 9, very advantageously between 8.5 and 9.

[0050] Microalgae strain according to the invention

[0051] The present invention therefore relates to a strain of microalgae of Hematococcus pluvialis comprising at least 5% astaxanthin by weight relative to the total weight of the dry matter of Hematococcus pluvialis.

[0052] The inventors have thus succeeded in obtaining a strain of Hematococcus pluvialis with high astaxanthin yield, that is to say producing a quantity of astaxanthin greater than the quantity of astaxanthin produced by the wild strain of Hematococcus pluvialis such as the UTEX 2505 strain.

[0053] Thus, the microalgae strain according to the invention comprises at least 5% astaxanthin by weight relative to the total dry weight of Hematococcus pluvialis, preferably at least 5.1%, at least 5.2%, at least 5.3%, at least 5.4%, and more preferably at least 5.5% by weight of the dry weight of Hematococcus pluvialis. In a particular embodiment, the strain according to the invention can achieve an astaxanthin content of at least 6.2% of the dry weight of the biomass. According to a particular embodiment, the microalgae strain according to the invention is capable of growing in a liquid medium having at least one parameter selected from:

[0054] * a temperature between 25 and 32°C, advantageously between 28 and 32°C; and

[0055] *a pH greater than 7.5 advantageously a pH between 7.5 and 9, very advantageously between 8.5 and 9.

[0056] Thus, in a particularly advantageous manner, said strain according to the invention grows under culture conditions that do not allow the wild strain UTEX 2505 to develop and grow, namely:

[0057] * a temperature between 25 and 32°C, typically between 28 and 32°C; and

[0058] *a pH greater than 7.5, in particular a pH between 7.5 and 9, typically between 8.5 and 9.

[0059] According to another particular embodiment, the microalgae strain according to the invention is capable of growing in a liquid medium having at least one parameter selected from:

[0060] * a temperature between 25 and 32°C, advantageously between 28 and 32°C; and

[0061] *a pH greater than 6.8 advantageously a pH between 6.8 and 8, very advantageously between 7.1 and 7.3.

[0062] Thus, in a particularly advantageous manner, said strain according to the invention grows under culture conditions that do not allow the wild strain UTEX 2505 to develop and grow, namely:

[0063] * a temperature between 25 and 32°C, typically between 28 and 32°C; and

[0064] *a pH greater than 6.8, in particular a pH between 6.8 and 8, typically between 7.1 and 7.3.

[0065] In a particularly preferred manner, the microalgae strain according to the invention is a strain of Haematococcus pluvialis chosen from the strain filed with the BEA under number BEA_IDA_0084 or the strain filed with the BEA under number BEA_IDA_0087 or the strain filed with the BEA under number BEA_IDA_0089.

[0066] The said strains of Hematococcus Pluvialis microalgae have thus been deposited with the Spanish Algae Bank (Banco Español de Algas) under number BEA_IDA_0084, BEA_IDA_0087, or BEA_IDA_0089. The said strain deposited under number BEA_IDA_0084 has the 18S rDNA sequence SEQ ID NO: 1. Furthermore, a person skilled in the art, by virtue of their general knowledge, is able to determine the complete genome sequence (AN I) of the microalga according to the invention from the strain deposited and accessible at the BEA under number BEA_IDA_0084.

[0067] The scientific classification of the microalgae strain BEA_IDA_0084 is as follows: Domain: Eukaryota; Phylum: Chlorophyta; Class: Chlorophycea; Order: Chlamydomonadales; Family: Haematococcaceae; Genus: Hematococcus; Species: pluvialis.

[0068] When the strain according to the invention is cultivated under appropriate conditions, i.e., in a suitable culture medium, the size of its vegetative cells varies from 5 to 25 micrometers. These cells are oval-shaped, green in color, actively motile, with a narrow tip and a papillary structure. The cells also possess two isometric flagella that enable the movement of the microalgae.

[0069] Astaxanthin accumulates within granules that cluster around the nucleus of microalgae. During astaxanthin accumulation, the cells increase in volume and lose their flagella.

[0070] Thus, when the strain according to the invention is cultured under appropriate conditions, the majority of cells of said strain do not exhibit a flagellum.

[0071] According to a preferred embodiment, the microalgae strain according to the invention comprising at least 5% astaxanthin by weight relative to the total dry weight of Haematococcus pluvialis is a strain derived from at least one microalgae strain selected from the strain filed under number BEA_IDA_0084 or the strain filed under number BEA_IDA_0087 or the strain filed under number BEA_IDA_0089. Said strain maintains or improves the capabilities described in the present invention, namely an astaxanthin production greater than 5% by weight of the dry matter of Haematococcus pluvialis.

[0072] The derived strain can alternatively be produced naturally or intentionally, by mutagenesis methods known to those skilled in the art. For example, mutagenesis methods that can be implemented within the framework of the present invention include the growth of the original microorganism in the presence of mutagenic or stress-producing agents, or by genetic engineering aimed at modifying specific or non-specific genes, such as targeted mutagenesis or random mutagenesis. When the strain is obtained by targeted or random mutagenesis, the strain derived from Haematococcus pluvialis strain BEA_IDA_0084, BEA_IDA_0087, or BEA_IDA_0089 is preferably a genetically modified mutant.

[0073] According to another preferred embodiment, the microalgae strain according to the invention comprises at least 5% astaxanthin by weight relative to the total dry weight of Haematococcus pluvialis and has an 18S rDNA sequence having at least 97%, preferably at least 98%, more preferably at least 99% identity with the SEQ ID NO:1 18S rDNA sequence of the microalgae strain BEA_IDA_0084.

[0074] 18S ribosomal RNA (18S rRNA) is the ribosomal RNA that constitutes the 40S subunit of ribosomes in eukaryotes. The genes encoding this RNA are called 18S rDNA. The 18S rRNA or 18S rDNA sequence is regularly used in phylogenetic analyses to study the evolutionary relationships between different species of microalgae. This allows us to understand the genetic diversity and evolution of microalgae at a molecular level. Indeed, the 18S rRNA or 18S rDNA sequence is a highly conserved structure that allows for the identification and characterization of microalgae and is therefore particularly well-suited to the context of this invention.

[0075] The percentage of identity of the 18S rDNA sequence between two microalgal strains, more specifically two strains of Haematococcus pluvialis, can be determined by the BLAST method, a well-known heuristic search method. It allows for the identification of similar regions between two or more nucleotide or amino acid sequences and the alignment of these homologous regions.

[0076] In some cases, the 18S rDNA sequence does not always allow differentiation between two strains of the same species that nevertheless have different properties, for example, different astaxanthin production capacities. Therefore, a person skilled in the art, thanks to their general knowledge, is able to characterize a microalgal strain according to other parameters such as the calculation of phylogenetic distance based on the complete genome (ANI), genome size, the number of CDSs (Coding DNA Sequences), and also the identification of genes specific to the strain of interest.

[0077] According to one embodiment, the invention also relates to a microalgae strain comprising at least 5% astaxanthin by weight relative to the dry weight of Haematococcus pluvialis and having a nucleotide sequence having at least 99% ANI identity with the nucleotide sequence of the microalgae strain BEA_IDA_0084. Preferably, said strain according to the invention comprises a nucleotide sequence having at least 99.91%, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, at least 99.99% ANI identity with the nucleotide sequence of the microalgae strain BEA_IDA_0084.

[0078] Such a strain is therefore a strain derived from the microalgae strain BEA_IDA_0084 or the strain BEA_IDA_0087 or the strain BEA_IDA_0089 allowing to maintain or improve the production capacities of astaxanthin, namely at least 5% by weight relative to the total weight of the dry matter of Haematococcus pluvialis.

[0079] Advantageously, the strain according to the invention exhibits remarkable characteristics in terms of biomass productivity and ability to produce astaxanthin under various culture conditions.

[0080] A particularly advantageous aspect of the strain according to the invention, preferably strain BEA_IDA_0089, is its ability to efficiently produce astaxanthin at moderate light intensities. This characteristic makes it particularly well-suited to industrial closed culture conditions, allowing for efficient pigment accumulation without requiring light stress exceeding 150 pE / m². 2 / s. Thus, the strain according to the invention, preferably strain BEA_IDA_0089, makes it possible to produce astaxanthin with reduced energy consumption compared to known strains of the prior art.

[0081] This performance is achieved without requiring stressful conditions such as nitrogen deficiency or high light intensity, which represents a considerable advantage for industrial production.

[0082] According to an advantageous embodiment, the microalgae strain according to the invention is obtained by implementing a culture process comprising the following steps: a) a proliferation step of at least one microalgae strain according to any previously described embodiment in a suitable culture medium; and b) an induction step of astaxanthin production from the microalgae obtained from step a).

[0083] Preferably, the proliferation step a) is carried out in an autotrophic, heterotrophic or mixotrophic environment.

[0084] According to a much preferred embodiment, step a) is carried out in an autotrophic culture medium selected from Bolb basal modified medium (BBM), Blue green 11 medium (BG-11) and Kobayashi medium 1 (KM1).

[0085] According to a preferred embodiment, the suitable culture medium of step a) includes at least one carbon source and at least one nitrogen source.

[0086] When the medium includes at least one carbon source, this may be chosen from glucose, fructose, maltose, galactose, mannose, sucrose, arabinose, xylose, acetate, and glycerol, with acetate being the preferred carbon source. When the medium includes at least one nitrogen source, this may be i) an organic nitrogen source selected from the group consisting of yeast extract and tryptone, or ii) an inorganic nitrogen source selected from the group consisting of sodium nitrate and potassium nitrate.

[0087] According to one embodiment, the culture medium of step a) comprises at least one vitamin, preferably at least one vitamin selected from vitamins Bl, B12, H and their mixtures.

[0088] When it includes at least one vitamin, the culture medium in step a) preferentially includes:

[0089] *between 2.10-7M and 4.10-7M of vitamin B1;

[0090] *between 3 x 10⁻¹⁰ and 5 x 10⁻¹⁰M of vitamin B12; and

[0091] *between 1.10-9 and 3.10-9M of vitamin H;

[0092] Preferably, step a) of proliferation of at least one strain of microalgae includes exposure of said culture medium comprising the microalgae to a light intensity of between 5 and 100 pmol m-2 s-1, more preferably 20 pmol m-2 s-1.

[0093] Light exposure for the crop can be achieved in particular by means of suitable LEDs or fluorescent neon lights.

[0094] In the context of the invention, the measurement of light intensity is carried out by a photometer (li-cor LI-250A).

[0095] Thus, the culture according to step a) of Haematococcus pluvialis microalgae is carried out under optimal growth conditions.

[0096] According to another embodiment, step b) of inducing the production of astaxanthin includes exposing said microalgae culture from step a) to an intensity of between 110 and 500 pmol m-2 s-1, preferably between 110 and 250 pmol m-2 s-1, more preferably between 110 and 150 pmol m-2 s-1, even more preferably between 110 and 130 pmol m-2 s-1, and even more preferably between 125 pmol m-2 s-1.

[0097] According to another preferred embodiment, step b) of inducing astaxanthin production includes exposing said microalgae culture from step a) to a light intensity between 5 and 110 pmol m-2 s-1, more preferably less than 110 pmol m-2 s-1. Advantageously, the strain according to the invention, preferably strain BEA_IDA_0089, is capable of producing astaxanthin in industrially satisfactory quantities without requiring light exposure greater than 110 pmol m-2 s-1.

[0098] Preferably, step b) includes a reduction in the input of at least one element chosen from nitrogen, phosphorus or sulfur.

[0099] Preferably, step b) includes the application of salt stress, said salt stress includes a concentration of sodium chloride in the culture medium of at least 0.2% of the culture medium by weight of the total weight of the culture medium.

[0100] According to a preferred embodiment, step b) includes the addition of sodium acetate and / or ferrous ions.

[0101] Preferably, step b) includes the addition of at least 20mM sodium acetate and / or at least 400pM iron sulfate.

[0102] According to a particularly preferred embodiment, step b) comprises exposing the microalgae culture from the proliferation step a) to a light intensity greater than 100 pmol m⁻² s⁻¹ and / or adding sodium acetate and / or ferrous ions. Advantageously, at least 20 mM sodium acetate and / or at least 400 pM iron sulfate.

[0103] Thus, according to one embodiment, the invention relates to a strain of microalgae of Hematococcus Pluvialis comprising at least 5% astaxanthin by weight of the dry matter obtained after exposure of a culture of said microalgae to a light intensity greater than 100 pmol m-2 s-1 and / or addition of sodium acetate, preferably at least 20mM of sodium acetate and / or addition of ferrous ions, preferably at least 400pM of iron sulfate.

[0104] According to a particular embodiment of the invention, the microalgae strain according to the invention is obtained by implementing a culture process comprising the following steps: a) a proliferation step of at least one microalgae strain according to any of the previously described embodiments in a suitable culture medium, in which:

[0105] - the culture medium is an autotrophic culture medium; and

[0106] - the microalgae culture is exposed to a light intensity between 5 and 100 pmol m-2 s-1; and

[0107] - the culture medium comprises at least acetate and nitrate; and - optionally, the culture medium comprises at least one vitamin chosen from vitamin B1 and / or B12 and / or H; and b) an induction step of astaxanthin production from microalgae originating from proliferation step a), in which:

[0108] - the microalgae culture from step a) is exposed to a light intensity between 110 and 500 pmol m-2 s-1; and / or

[0109] - the culture medium including the microalgae from step a) is supplemented with sodium acetate and / or ferrous ions.

[0110] According to another particular embodiment of the invention, the microalgae strain according to the invention is obtained by implementing a culture process comprising the following steps: a) a proliferation step of at least one microalgae strain according to any of the previously described embodiments in a suitable culture medium, in which:

[0111] - the culture medium is an autotrophic culture medium; and

[0112] - the microalgae culture is exposed to a light intensity between 5 and 100 pmol m-2 s-1; and

[0113] - the culture medium comprises at least acetate and nitrate; and

[0114] - optionally, the culture medium includes at least one vitamin chosen from vitamin B1 and / or B12 and / or H; and b) an induction step for the production of astaxanthin from microalgae originating from proliferation step a), in which:

[0115] - the microalgae culture from step a) is exposed to a light intensity between 5 and 110 pmol m-2 s-1; and / or

[0116] - the culture medium including the microalgae from step a) is supplemented with sodium acetate and / or ferrous ions.

[0117] Advantageously, step b) allows for further optimization of astaxanthin production from the microalgae strain according to the invention.

[0118] According to another aspect, the invention relates to the use of a microalgae strain according to any one of the preceding embodiments for producing astaxanthin. According to another aspect, the invention relates to a Haematococcus pluvialis microalgae strain filed under number BEA_IDA_0084, BEA_IDA_0087, or BEA_IDA_0089.

[0119] Preferably, the Haematococcus pluvialis microalgae strain filed under number BEA_IDA_0084 or under number BEA_IDA_0087 or under number BEA_IDA_0089 exhibits increased astaxanthin production compared to the wild strain UTEX 2505.

[0120] Thus, the present invention also aims at the use of a microalgae strain of Haematococcus pluvialis filed under number BEA_IDA_0084 or under number BEA_IDA_0087 or under number BEA_IDA_0089 to increase the production of astaxanthin compared to the production of astaxanthin from a wild strain of Haematococcus pluvialis and preferably by reducing the energy consumption necessary in order to optimize the performance of industrial processes.

[0121] Astaxanthin production process

[0122] The invention also relates to a high-yield astaxanthin production process.

[0123] Thus, the invention relates to a process for producing astaxanthin comprising at least one step of cultivating at least one strain of microalgae according to any of the embodiments previously described.

[0124] According to one embodiment, the astaxanthin production process comprises the cultivation of at least one microalgal strain of Haematococcus pluvialis, said strain comprising at least 5%, preferably at least 5.1%, at least 5.2%, at least 5.3%, at least 5.4%, more preferably at least 5.5%, in particular at least 6.2% astaxanthin by weight of the dry matter of Haematococcus pluvialis. Preferably, the astaxanthin production process comprises at least one step of cultivating at least one microalgal strain of Haematococcus pluvialis registered under number BEA_IDA_0084, BEA_IDA_0087, or BEA_IDA_0089.

[0125] According to another preferred embodiment, the astaxanthin production process comprises carrying out the following steps: a) a step of proliferating at least one strain of microalgae according to any embodiment previously described in a suitable culture medium; b) a step of inducing astaxanthin production from the microalgae produced in step a); and c) a step of extracting astaxanthin from the microalgae produced in step b). Preferably, the culture medium for step a) is selected from an autotrophic, heterotrophic, or mixotrophic medium.

[0126] According to another preferred embodiment, the proliferation step a) is carried out in an autotrophic culture medium selected from BBM, BG-11 and KM1.

[0127] Preferably, the culture medium in step a) includes at least one carbon source and at least one nitrogen source.

[0128] When it includes at least one carbon source, said carbon source may be chosen from glucose, fructose, maltose, galactose, mannose, sucrose, arabinose, xylose, acetate and glycerol, preferably the carbon source is acetate.

[0129] When it includes at least one nitrogen source, said nitrogen source may be i) an organic nitrogen source selected from the group consisting of yeast extract and tryptone, or ii) an inorganic nitrogen source selected from the group consisting of sodium nitrate and potassium nitrate. Preferably, said at least one nitrogen source is a nitrate.

[0130] According to one embodiment, the culture medium of step a) comprises at least one vitamin, preferably at least one vitamin selected from vitamins Bl, B12, H and their mixtures.

[0131] Preferably, the culture medium in stage a) comprises:

[0132] *between 2 x 10⁻⁷M and 4 x 10⁻⁷M of vitamin B1; and

[0133] *between 3 x 10⁻¹⁰ and 5 x 10⁻¹⁰M of vitamin B12; and

[0134] *between 1.10-9 and 3.10-9M of vitamin H;

[0135] Preferably, step a) of proliferation of at least one strain of microalgae includes exposure of said culture medium comprising the microalgae to a light intensity of between 5 and 100 pmol m-2 s-1, more preferably 20 pmol m-2 s-1.

[0136] Light exposure for the crop can be achieved in particular by means of suitable LEDs or fluorescent neon lights.

[0137] In the context of the invention, the measurement of light intensity is carried out by a photometer (li-cor LI-250A).

[0138] Thus, the culture according to step a) of Haematococcus pluvialis microalgae is carried out under optimal growth conditions.

[0139] According to another embodiment, step b) of inducing the production of astaxanthin includes exposing said microalgae culture from step a) to a light intensity greater than 110 pmol m-2 s-1, preferably between 110 and 500 pmol m-2 s-1, in particular between 110 and 250 pmol m-2 s-1, more preferably between 110 and 150 pmol m-2 s-1, even more preferably between 110 and 130 pmol m-2 s-1, and even more preferably between 125 pmol m-2 s-1.

[0140] According to another preferred embodiment, step b) of inducing astaxanthin production includes exposing said microalgae culture from step a) to a light intensity between 5 and 110 pmol m-2 s-1, more preferably less than 110 pmol m-2 s-1.

[0141] Advantageously, the strain according to the invention, preferably strain BEA_IDA_0089, is capable of producing astaxanthin in industrially satisfactory quantities without requiring light exposure exceeding 110 pmol m-2 s-1. Preferably, step b) includes a reduction in the supply of at least one element selected from nitrogen, phosphorus, or sulfur.

[0142] Preferably, step b) includes the application of salt stress, said salt stress includes a concentration of sodium chloride in the culture medium of at least 0.2% of the culture medium by weight of the total weight of the culture medium.

[0143] According to a preferred embodiment, step b) includes the addition of sodium acetate and / or ferrous ions.

[0144] Preferably, step b) includes the addition of at least 20mM sodium acetate and / or at least 400pM iron sulfate.

[0145] According to a particularly preferred embodiment, step b) comprises exposing the microalgae culture from the proliferation step a) to a light intensity greater than 100 pmol m⁻² s⁻¹ and / or adding sodium acetate and / or ferrous ions. Advantageously, at least 20 mM sodium acetate and / or at least 400 pM iron sulfate.

[0146] According to a particular embodiment, the process according to the invention comprises carrying out the following steps: a) a proliferation step of at least one strain of microalgae according to any of the embodiments previously described in a suitable culture medium, in which:

[0147] - the culture medium is an autotrophic culture medium; and

[0148] - the microalgae culture is exposed to a light intensity between 5 and 100 pmol m-2 s-1; and

[0149] - the culture medium comprises at least acetate and nitrate; and - optionally, the culture medium comprises at least one vitamin chosen from vitamin B1 and / or B12 and / or H; and b) an induction step of astaxanthin production from microalgae originating from proliferation step a), in which:

[0150] - the microalgae culture from step a) is exposed to a light intensity between 110 and 500 pmol m-2 s-1; and / or

[0151] - the culture medium including the microalgae from step a) is supplemented with sodium acetate and / or ferrous ions.

[0152] According to another particular embodiment of the invention, the process according to the invention comprises carrying out the following steps: a) a proliferation step of at least one strain of microalgae according to any of the embodiments previously described in a suitable culture medium, in which:

[0153] - the culture medium is an autotrophic culture medium; and

[0154] - the microalgae culture is exposed to a light intensity between 5 and 100 pmol m-2 s-1; and

[0155] - the culture medium comprises at least acetate and nitrate; and

[0156] - optionally, the culture medium includes at least one vitamin chosen from vitamin B1 and / or B12 and / or H; and b) an induction step for the production of astaxanthin from microalgae originating from proliferation step a), in which:

[0157] - the microalgae culture from step a) is exposed to a light intensity between 5 and 110 pmol m-2 s-1; and / or

[0158] - The culture medium containing the microalgae obtained in step a) is supplemented with sodium acetate and / or ferrous ions. Advantageously, step b) further optimizes the production of astaxanthin by the microalgae strain according to the invention.

[0159] According to one embodiment, step c) of extraction is carried out using carbon dioxide in a supercritical state.

[0160] Advantageously, supercritical carbon dioxide extraction allows for the selective extraction of astaxanthin and thus optimizes its extraction.

[0161] According to a preferred embodiment, extraction step c) includes the implementation of the following steps: *drying of the microalgae biomass from step b);

[0162] *lysis of microalgal cell walls, preferably by means of at least one enzyme or at least one mechanical means; and

[0163] * extraction of astaxanthin using carbon dioxide in a supercritical state.

[0164] According to one embodiment, step c) of extraction includes a drying step, preferably using an oven at 95°C for 24 hours. of astaxanthin from a strain sa of Haematococcus pluvialis UTEX 2505 vs the strain according to the invention registered under number BEA IDA 0084.

[0165] Strains

[0166] The wild strain UTEX 2505 was obtained from the University of Texas at Austin (UTEX) algae culture collection.

[0167] The strain according to the invention was obtained in a surprising way, from a wild strain of Hematococcus pluvialis by combining Darwinian selection methods and a very high throughput screening process.

[0168] For the realization of this example, the strain according to the invention is the strain filed under number BEA_IDA_0084.

[0169] The two microalgae strains were cultured on a basal fatty medium (BBM) at 24°C under continuous light.

[0170] Culture

[0171] Cultures were carried out in 250 ml Erlenmeyer flasks containing 70 ml of BG-11 medium at 24°C, under orbital shaking at 140rpm and under continuous illumination (50 pmol m-2 s-1) for 7 days then 350 pmol m-2 s-1 for 10 days.

[0172] Contamination

[0173] The presence of contamination was verified by the detection of a drop of culture on the LB+agar medium at the end of each culture.

[0174] Tests

[0175] Determination of dry matter

[0176] After 18 days of culture, a sample from each culture was taken and centrifuged at 3500 g for 5 minutes at room temperature. The cell pellet was washed once with 2 ml of distilled water and placed on previously dried Eppendorf microtubes. The microtubes were then dried at 60°C for 24 hours. The results are illustrated in Figure 1 and demonstrate improved proliferation of the strain according to the invention compared to the wild-type strain. In other words, the strain according to the invention produces a greater microalgal biomass compared to the wild-type strain, and therefore a larger quantity of dry matter.

[0177] Astaxanthin dosage

[0178] The astaxanthin (AC) concentration was determined by the photometric method. Briefly, after 18 days of culture under the aforementioned conditions, microalgae cells were collected from a 1 ml sample by centrifugation (3000 rpm, 2 min) and then treated with 5% (w / v) KOH in 30% (v / v) methanol at 80 °C for 30 min. The remaining pellet was washed three times and extracted with 5 ml of DMSO / acetone (80 / 20) for 30 min in a sonication bath. The supernatant was collected after centrifugation (10000 rpm, 5 min). The extraction procedure was repeated several times until the pellet became colorless. Finally, the absorbance of the extract was measured at a wavelength of 492 nm.

[0179] Thus, the concentration of astaxanthin was calculated according to the formula: [Math 1]

[0180] AC=4.5xA492xVa / 102 where A492 is the absorbance of the extracts at 492 nm and Va is the volume of the extracts.

[0181] The results are illustrated in Figure 2 and demonstrate that the strain according to the invention has a superior astaxanthin production capacity compared to the wild strain.

[0182] In addition, the inventors measured the astaxanthin concentration in the dry matter resulting from culturing the two microalgae strains for 18 days. The results are shown in Figure 3 and demonstrate a concentration of at least 5% astaxanthin by weight relative to the total dry matter weight.

[0183] Example 2: Biomass productivity of strain BEA IDA 0089

[0184] This example illustrates the superior biomass productivity of strain BEA_IDA_0089 compared to the wild strain WT Utex 2505.

[0185] Materials and methods:

[0186] The strains BEA_IDA_0089 and WT Utex 2505 were cultured under different conditions for 15 days. The tested conditions included a BBM (Bold Basal Medium) for autotrophic culture, and a BBM supplemented with yeast extract (YE) and acetate (Ac) for mixotrophic culture.

[0187] The cultures were grown in triplicate at a temperature of 25°C and a light intensity of 150 pE / m 2 / s. Results:

[0188] As illustrated in Figure 4, strain BEA_IDA_0089 demonstrated significantly higher biomass productivity than strain WT Utex 2505 under all tested conditions.

[0189] Under autotrophic conditions (BBM), strain BEA_IDA_0089 reached a biomass concentration of approximately 2.5 g / L, corresponding to a productivity of approximately 0.17 g / L / day. Under mixotrophic conditions (BBM+YE+Ac), the performance of strain BEA_IDA_0089 was even more remarkable, reaching a biomass concentration of approximately 3.0 g / L, corresponding to an average productivity of 0.204 g / L / day.

[0190] These results demonstrate the superiority of strain BEA_IDA_0089 in terms of biomass productivity under various culture conditions, without requiring the addition of CO2 or pH regulation.

[0191] Example 3: Production of astaxanthin by the BEA IDA 0089 strain

[0192] This example illustrates the superior astaxanthin biosynthesis capacity of strain BEA_IDA_0089 compared to the wild strain.

[0193] Materials and methods:

[0194] Strains BEA_IDA_0089 and WT Utex 2505 were cultured under the same conditions as in Example 2 for 21 days. Astaxanthin content was measured spectrophotometrically after extraction.

[0195] Results :

[0196] As shown in Figure 5, strain BEA_IDA_0089 demonstrated a significantly higher astaxanthin production capacity than strain WT Utex 2505 under all tested conditions.

[0197] Under autotrophic conditions (BBM), strain BEA_IDA_0089 reached an astaxanthin content of approximately 6.2% of the dry weight of biomass, compared to approximately 4.2% for strain WT.

[0198] Under mixotrophic conditions (BBM+YE+Ac), although the astaxanthin content was generally lower, strain BEA_IDA_0089 maintained its superiority with approximately 4.2% compared to 2.5% for the WT strain.

[0199] These results demonstrate the superior ability of strain BEA_IDA_0089 to produce astaxanthin under various culture conditions, thus offering an optimized solution for applications requiring a high level of pigment concentration.

[0200] Example 4: Production of astaxanthin under low light intensity conditions This example demonstrates the ability of strain BEA_IDA_0089 to efficiently produce astaxanthin under low light intensity conditions.

[0201] Materials and methods:

[0202] The BEA_IDA_0089 and WT Utex 2505 strains were cultured for 21 days under continuous light at 100 pmol / m² -2 s -1 The astaxanthin content was measured as in the previous example.

[0203] Results :

[0204] Figure 6 clearly illustrates the superiority of strain BEA_IDA_0089 under low light intensity conditions. While the WT strain showed limited astaxanthin accumulation (2.27% after 21 days), strain BEA_IDA_0089 produced significantly more astaxanthin (5.78% over the same period), without requiring stressful conditions such as nitrogen deficiency or high light intensity.

[0205] These results demonstrate the ability of strain BEA_IDA_0089 to efficiently produce astaxanthin under low light conditions, enabling energy-efficient production compatible with low-energy LED closed photobioreactors. This characteristic paves the way for sustainable, high-yield astaxanthin production, particularly for the nutraceutical, cosmetic, and aquaculture sectors.

[0206]

[0207] Algas

[0208] BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSE OF DECLARING A PATENT PROCEDURE IN THE CASE OF AN ORIGINAL DEPOSIT

[0209] INTERNATIONAL FORM University of Banco Las Palmas de Espanol G ran Canaria deAlgas

[0210] BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICRO-

[0211] BODIES FOR THE PURPOSE OF DECLARING PATENT PROCEDURES IN THE CASE OF AN ORIGINAL FILING

[0212] INTERNATIONAL FORM

[0213] 1. Where rule 6.4(d) applies, this date is the date on which the status of international depositary authority was acquired. Form BP / 4 (single page) (24 / 0112023)

[0214] Scanned with CamScanner

Claims

DEMANDS

1. Haematococcus pluvialis microalgae strain comprising at least 5% astaxanthin by weight relative to the total dry weight of Haematococcus pluvialis.

2. Microalgae strain according to the preceding claim, characterized in that it is derived from at least one strain selected from the microalgae strain filed under number BEA_IDA_0084 or the microalgae strain filed under number BEA_IDA_0087 or the microalgae strain filed under number BEA_IDA_0089.

3. Microalgae strain according to any one of the preceding claims, characterized in that it comprises an 18S rDNA sequence having at least 97% identity with the SEQ ID NO: 1 18S rDNA sequence of the strain filed under number BEA_IDA_0084.

4. Microalgae strain according to any one of the preceding claims, characterized in that it is selected from the strain filed under number BEA_IDA_0084 or the strain filed under number BEA_IDA_0087 or the strain filed under number BEA_IDA_0089.

5. Use of a microalgae strain according to any one of the preceding claims, to produce astaxanthin.

6. A process for producing astaxanthin, characterized in that it comprises at least one step of cultivating at least one strain of microalgae according to any one of claims 1 to 4.

7. A method according to the preceding claim, characterized in that it comprises carrying out the following steps: a) a step of proliferation of at least one strain of microalgae according to any one of claims 1 to 4 in a suitable culture medium; b) a step of induction of the production of astaxanthin from the microalgae from step a); c) a step of extraction of astaxanthin from the microalgae from step b).

8. A method according to the preceding claim, characterized in that the culture medium of step a) is autotrophic, heterotrophic or mixotrophic.

9. A method according to any one of claims 7 or 8, characterized in that the culture medium of step a) comprises a carbon source and a nitrogen source.

10. A method according to the preceding claim, characterized in that the carbon source is selected from glucose, fructose, maltose, galactose, mannose, sucrose, arabinose, xylose, acetate and glycerol.

11. A process according to any one of claims 9 or 10, characterized in that the nitrogen source is: i) an organic nitrogen source selected from the group consisting of yeast extract, peptone, and tryptone; or ii) an inorganic nitrogen source selected from the group consisting of ammonium chloride, ammonium sulfate, sodium nitrate, urea, and monosodium glutamate (MSG).

12. A method according to any one of claims 7 to 11, characterized in that the culture medium of step a) also comprises at least one vitamin selected from vitamin Bl, vitamin B12, vitamin H and mixtures thereof.

13. A method according to any one of claims 7 to 12, characterized in that step a) of proliferation comprises exposing the microalgae culture to a light intensity of between 5 and 100 pmol nr 2 s 1 .

14. A method according to any one of claims 7 to 13, characterized in that step b) comprises exposing the microalgae culture from the proliferation step a) to a light intensity greater than 110 pmol nr 2 s 1 and / or to the addition of sodium acetate and / or ferrous ions.

15. A method according to any one of claims 8 to 14, characterized in that step c) is carried out using carbon dioxide in a supercritical state.

16. A method according to the preceding claim, characterized in that step c) comprises carrying out the following steps: *drying of the microalgae biomass from step b); *lysis of microalgal cell walls, preferably by means of at least one enzyme or at least one mechanical means; and *Extraction of astaxanthin using carbon dioxide in a supercritical state.

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