Marine microalga as producer of beta-carotene, lutein and other carotenoids

Abstract

The invention relates to the field of microbiology and biotechnology, and more particularly to a novel strain of the microalga Desmodesmus subspicatus, Dia 5, which is a producer of carotenoids, especially lutein and beta-carotene. The substances produced can be used in biologically active additives and food supplements for human consumption, as well as in various feed supplements, primarily for use in aquaculture. The novel strain has the ability to accumulate a high level of both lutein and β-carotene, and the strain biomass additionally has good organoleptic properties. The strain does not require complex culture conditions, inter alia, it does not require a high concentration of NaCl in the cultivation medium.

Description

[0001] MARINE MICROALGAE - PRODUCER OF BETA-CAROTENE, LUTEIN AND OTHER CAROTENOIDS

[0002] Field of technology

[0003] The invention relates to the field of microbiology and biotechnology and can be used to obtain biologically active substances, primarily carotenoids, especially lutein and beta-carotene, for use in biologically active additives (BAA) and food additives for humans, as well as in various feed additives, primarily for aquaculture.

[0004] Prior art

[0005] Carotenoids are essential for plants to regulate photosynthesis and are used primarily as antioxidants. In animals, carotenoids most often act as provitamins, primarily as precursors to retinoids (a group of structurally similar compounds, including vitamin A). However, modern data show that the spectrum of carotenoid biological activity in animals is significantly broader, as exemplified by lutein, from which retinoids cannot be derived. Modern research demonstrates the colossal role of carotenoids in human and animal health throughout life, and their use can significantly improve the quality of life for the end consumer.

[0006] Carotenoids are widely used in aquaculture and as feed additives for animals and poultry. Demand for carotenoids has grown significantly recently. Chemically synthesized carotenoids are characterized by extremely low solubility in aqueous media and the ability to form large aggregates. This significantly reduces their bioavailability for both humans and animals. Furthermore, synthetic carotenoids often form various spatial E- and Z-isomers with a distorted structure, which become unrecognizable to human enzymes, making it difficult to eliminate them from the body when present in excess. This can lead to their accumulation in the form of aggregates and even lead to visual impairment. Direct comparisons of synthetic carotenoids with natural carotenoids in their native environment (e.g., crab meal) show that natural carotenoids stimulate the growth of fish fry significantly better than synthetic ones (Goda A.A. et al., Evaluation of Natural and Synthetic Carotenoid Supplementation on Growth, Survival, Total Carotenoid Content, Fatty Acids Profile and Stress Resistance of European Seabass, Dicentrarchus labrax / / Aquaculture Studies 2018, 18(1), 27-39. https: / / doi.org / 10.4194 / 2618-6381-v18_1_04). Therefore, "native" carotenoids, obtained, for example, in the form of lyophilized microalgae biomass, are the most suitable for food and feed additives. Many eukaryotic microalgae are capable of accumulating hundreds and sometimes even thousands of times more carotenoids than any known higher plants, which is associated with their sometimes extreme living conditions. It is this property to synthesize a gigantic amount of carotenoids that began to be used to create biologically active food and feed additives. Industrial cultivation of microalgae is now used in many countries.

[0007] Various microalgae are known to produce various carotenoids, for example, the Mallomonas kalinae strain SX-1 VKPM AI-23 is a fucoxanthin producer (RU2644260, published 02 / 08 / 2018), the Coelastrella sp. K1 (IPPAS C-2028) strain is a producer of a mixture of astaxanthin and β-carotene (RU2703420, published 10 / 16 / 2019). Some representatives of the Desmodesmus genus of microalgae are also known as possible carotenoid producers. For example, the strains Desmodesmus sp. (2-6) and Desmodesmus sp. (RUC-2), isolated from freshwater bodies of Sweden, capable of producing lipids and various carotenoids (Mehariya et al. Improving the content of high value compounds in Nordic Desmodesmus microalgal strains / / Bioresour. Technol., 2022, 359, 127445. https: / / doi.Org / 10.1016 / j.biortech.2022.127445).

[0008] Despite the wide diversity of microorganisms capable of producing carotenoids, they vary in their ability to produce them. The carotenoids themselves exhibit enormous diversity (approximately 600 different carotenoids have been described). Furthermore, microorganisms have different requirements for cultivation conditions (in many cases, unsuitable for industrial use due to environmental regulations and / or requiring close proximity to the sea). Furthermore, not all strains are commercially available. Therefore, the search for new carotenoid-producing strains is a pressing issue.

[0009] Disclosure of invention

[0010] The technical problem and task that the invention aims to solve is to obtain a new strain of microalgae with a high level of accumulation of lutein and p-carotene (beta-carotene).

[0011] The problem is solved by obtaining a new strain of Desmodesmus subspicartus (Chodat) E / Hegewald & A / W / F / Schmidt 2000 (Dia 5), ​​a carotenoid producer with predominant accumulation of lutein and β-carotene. The strain is deposited in the Microalgae Culture Collection of the K. A. Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences (hereinafter referred to as the IPPS Microalgae Collection RAS, IPPAS) under the number IPPAS C-2072, and also deposited in the All-Russian Collection of Microorganisms (VKM) according to the international depository system in accordance with the Budapest Treaty under the number VKM AI-522D (short strain name: Desmodesmus subspicartus Dia 5). Another aspect of the invention is the production of carotenoids, primarily lutein and p-carotene, during the cultivation of the Desmodesmus subspicartus Dia 5 strain.

[0012] Another aspect of the invention is a method for producing carotenoids, primarily lutein and p-carotene, by cultivating the Desmodesmus subspicartus Dia 5 strain.

[0013] The proposed strain Desmodesmus subspicartus Dia 5 has a number of advantages that provide the technical result of the present invention, which consists of the following:

[0014] - a new strain of microalgae Desmodesmus subspicartus Dia 5 expands the arsenal of sources of carotenoids, with a predominant content of lutein and β-carotene;

[0015] - the microalgae strain Desmodesmus subspicartus Dia 5 has the ability to produce a mixture of carotenoids, including lutein, a-carotene, β-carotene, neoxanthin, violaxanthin and neolutein B, with a high level of their accumulation (at least 12.2 mg / g biomass), with about 64% of this being lutein and about 22% being β-carotene;

[0016] - the biomass of the microalgae Desmodesmus subspicartus strain Dia 5 has good organoleptic properties (pleasant marine smell and pleasant taste), which is an important indicator and increases the prospects and advantages of its use as a source of carotenoids in the production of food additives, medicine and feed production;

[0017] - the Desmodesmus subspicartus Dia 5 microalgae strain is undemanding to cultivation conditions: cultivation is carried out in a single stage, and does not require a high NaCl content in the cultivation medium (a content at the level of a saline solution is sufficient), which allows the strain to be grown far from the sea coast and does not have a high impact on the environment when recycling the spent medium.

[0018] The Desmodesmus subspicartus Dia 5 strain is a potential industrial producer of biologically active substances – carotenoids, which can be used in industry, healthcare, agriculture, fish farming and other sectors.

[0019] Brief description of the drawings

[0020] Fig. 1 - cells of microalgae Desmodesmus subspicartus strain Dia 5 (bright-field microscopy, Leica Flexacam C3 camera).

[0021] Terms and definitions

[0022] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as understood by those skilled in the art. References to techniques used in describing this invention refer to well-known techniques, including variations of these techniques and their replacement with equivalent techniques known to those skilled in the art.

[0023] In the documents of this invention, the terms "includes," "including," and the like, as well as "contains," "comprising," and the like, are interpreted to mean "includes, among other things" (or "contains, among other things"). These terms are not intended to be construed as meaning "consists solely of."

[0024] The term "and / or" means one, more than one, or all of the listed elements.

[0025] Also here, listing numeric ranges by endpoints includes all numbers within that range.

[0026] The term "optional" or "optional" or "optionally" as used herein means that the subsequently described event or circumstance may, but does not necessarily, occur, and that the description includes instances in which the event or circumstance occurs and instances in which it does not occur.

[0027] Desmodesmus subspicartus Dia 5 refers to the Desmodesmus subspicartus Dia 5 strain, which was deposited in the Microalgae Collection of the Institute of Physiology and Microbiology of the Russian Academy of Sciences under accession number IPPAS C-2072 and deposited in the VKM collection under accession number VKM AI-522D. Desmodesmus subspicartus Dia 5 also refers to a mutant strain obtained using the deposited strain as starting material and retaining all the properties of the original strain, i.e., possessing all the defining (identifying) characteristics of Desmodesmus subspicartus Dia 5. This includes, at a minimum, its suitability for use in the production of carotenoids, primarily lutein and β-carotene.In particular, the Desmodesmus subspicartus Dia 5 strain has the following identifiable features, including 1) the ability to produce carotenoids, primarily lutein and β-carotene; 2) the presence of essential genes with greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity with the essential genes of Desmodesmus subspicartus Dia 5, which can be used to confirm that the analyzed variant is Desmodesmus subspicartus Dia 5 (the genetic sequence of the strain is registered in GenBank under the number OR682197).

[0028] Unless otherwise defined, technical and scientific terms in this application have the standard meanings generally accepted in the scientific and technical literature.

[0029] Detailed description of the invention

[0030] The strain Desmodesmus subspicartus Dia 5 was isolated from the coastal waters of the Sea of ​​Azov in the area of ​​the village of Dolzhanskaya in the Krasnodar Territory.

[0031] The full name of the strain is Desmodesmus subspicartus (Chodat) E / Hegewald & A / W / F / Schmidt 2000 (Dia 5). The short name is Desmodesmus subspicartus Dia 5. The strain Desmodesmus subspicartus Dia 5 was deposited on December 25, 2023, in the Microalgae Culture Collection at the Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences under the number IPPAS C-2072. The Desmodesmus subspicartus Dia 5 strain was also deposited on July 15, 2025, in the All-Russian Collection of Microorganisms (VKM) through the international deposition system in accordance with the Budapest Treaty under the number VKM AI-522D as part of the international patent deposition (the international deposition document is an appendix to this application).

[0032] The genetic sequence of the microalga Desmodesmus subspicartus Dia 5 has been registered in the international GenBank database.

[0033]

[0034] (htip: / / vv^^ ) under number OR682197.

[0035] Morphological features: cells are spherical or broadly oval, the cell wall is rigid, and contains short (up to 1 µm long) spines on its surface. A pyrenoid is present, the cell size is 4.5-8.5 µm in diameter. The chromatophore is single, parietal, and the flagellum is absent. Grains containing reserve nutrients are present in the cytoplasm. Reproduction is by autospores, predominantly 4 autospores of equal size. Growth pattern in storage medium: green colonies on solid medium. A bright-field microscopy image reflecting the morphology of the microalga Desmodesmus subspicartus Dia 5 is shown in Fig. 1.

[0036] Degree of purity: algologically pure culture.

[0037] Physiological properties of the strain. Optimal cultivation conditions: any sterile deionized water can be used, in particular, tap water passed through a reverse osmosis unit without remineralization. The classic F / 2 medium is used, but without biotin, and KH2PO4 is used instead of NahkPC. Additional salt solutions: 200 g / l (or 20%) NaCl (stock 20x solution), 0.5 mol / l (NN4)28O4 (stock 2000x solution), 150 g / l NaNOs (stock 2000x solution). Minimal medium: F / 2 + 1% NaCl, while the pH at the end of cultivation may increase. Can also grow on standard BG-11 medium. Composition of the gas-air mixture (GAM): atmospheric air (bubbling with atmospheric air passed through the medium). Temperature: 20-30 ° C. Lighting: day / night or 24 / 7.

[0038] Strain characteristics: Desmodesmus subspicartus strain Dia 5 produces carotenoids (lutein, α-carotene, β-carotene, neoxanthin, violaxanthin, and neolutein B). The yield of useful product is at least 12.2 mg per g of dry biomass (of which lutein is approximately 64% and β-carotene is approximately 22%). It also produces small amounts of polyunsaturated fatty acids, including linolenic acid (up to 33.7% of all fatty acids). Strain identification was performed morphologically using microscopy. Molecular genetic analysis was performed using PCR of the ITS1-5.8S-ITS2 region. As the analysis showed, the Dia 5 strain is a representative of the species Desmodesmus subspicatus (Chlorophyceae, Chlorophyta), despite the absence of coenobium typical for the genus in the culture.Verification was carried out, among other things, with the involvement of the ACSSI Algological Collection of the Institute of Physicochemical and Biological Problems of Soil Science of the Russian Academy of Sciences - a separate division of the Federal State Budgetary Scientific Institution "Putin Scientific Center for Biological Research of the Russian Academy of Sciences".

[0039] A preliminary taxonomic diagnosis was made based on morphological analysis performed using microscopic examination of the prepared specimen. The specimen was prepared on a glass slide with a drop of water in which a sample of the microalgae strain was placed. Light microscopy was performed using a Leica DM750 laboratory microscope (Germany). The observations were documented with color digital photography using a Leica Flexacam C3 camera (Germany) (see Fig. 1).

[0040] The cells of strain Dia 5 are solitary, broadly oval or spherical, with one parietal chloroplast and one pyrenoid, 4.5-8.5 µm in size, and contain reserve nutrients in the cytoplasm. The cell wall is rigid, with short (up to 1 µm long) spines on its surface. Reproduction occurs by (2)-4-(8) autospores. The culture contains "giant" cells of irregular shape with several pyrenoids, which most likely represent fused autospores. Remnants of the maternal membranes persist in the culture for some time, becoming mucilaginous.

[0041] Desmodesmus (Chlorophyceae, Chlorophyta) was isolated relatively recently based on analysis of 18S rRNA and the internal transcribed spacer ITS2 (An et al., 1999). Morphologically, Desmodesmus representatives are close to Scenedesmus representatives. Traditionally, they have elongated cells of varying shapes, which are usually arranged in flat coenobia of 2 to 32 cells in one or two rows (Komarek and Fott, 1983). However, sometimes in different populations of the same species isolated from the same ecotope, the ratio of single cells to coenobia can vary greatly. Thus, populations represented almost entirely by unicellular organisms can coexist in the same body of water with predominantly coenobic populations (Hegewald et al., 2001). Also, another characteristic feature of this genus is the presence of a four-layered cell wall with submicroscopic structures that are often visible under a light microscope as granulations or ribs.In addition, most Desmodesmus species have one or more spines (Hegewald, 1978; An et al., 1999). Representatives of this genus are widely found in fresh and brackish waters worldwide (Fawley et al., 2013; Nguyen et al., 2023; Hegewald et al., 2001). The studied strain Dia 5 clustered with representatives of Desmodesmus subspicatus UTEX 2532 and SAG 54.80 with high statistical support (BP - 97%). Although an authentic strain has not been assigned for Desmodesmus subspicatus, it was the strain UTEX 2532 that was used by Hegewald et al. (2001) to analyze relationships within the genus Desmodesmus based on the analysis of the 18S rRNA gene. The genetic distances between the Dia 5 strain and its sister strains ranged from 1.4-1.9%, and with other representatives of the genus, from 2.9% to 32.4%. The genetic distances between Desmodesmus protuberans and Desmodesmus pseudoprotuberans are 1%, but this is the only exception.In other cases, interspecific differences are higher, for example, the genetic distance between Desmodesmus shmidtii and Desmodesmus protuberans is 2.4%.

[0042] Due to the ability of the Desmodesmus subspicartus Dia 5 strain to produce carotenoids with high levels of lutein and β-carotene, the strain can be used as a source of these biologically active substances. Carotenoids act as antioxidants in humans and animals. Carotenoids are also metabolic precursors of vitamin A; β-carotene is the most important. In addition to their provitamin and antioxidant activity, carotenoids can stimulate immune cell function and modulate immune activity.

[0043] The Desmodesmus subspicartus Dia 5 strain can be used as freeze-dried biomass to create encapsulated dietary supplements (DS) or in powder form (including pressed tablets), as a source of carotenoids, either as a single supplement or in combination with other biologically active substances. It can also be used as an animal feed additive. Due to their antioxidant properties, carotenoids are of particular interest in the prevention of chronic diseases such as cardiovascular disease, diabetes, eye diseases, arthritis, kidney failure, osteoporosis, and even cognitive dysfunction. Vitamin A is a component of the visual pigment rhodopsin, which explains the important role of carotenoids, especially β-carotene. Lutein is the main protective component of the macular pigment spot and prevents the development of oxidative stress caused by excess blue light (Cristaldi M.et al. Comparative Efficiency of Lutein and Astaxanthin in the Protection of Human Corneal Epithelial Cells in Vitro from Blue-Violet Light Photo-Oxidative Damage / / Applied Sciences 2022, 12(3):1268 https / / Qs.Qrg: I(1339g / p&pl2g3 268). As a dietary supplement, the Desmodesmus subspicartus Dia 5 strain can be used to create dishes with the ideology of the Okinawan longevity diet, that is, "treatment through nutrition" (Sho H. History and characteristics of Okinawan longevity food / / Asia Race. J. Clin. Nutr. 2001 , 10, 159-64. https: / / doi.org / 10-1111 / j.1440-6047.2001 ,00235.x). Carotenoids are also used in food technology as food colorings, and recently also increasingly for the purpose of creating functional food products enriched with carotenoids (Achmiz A.D. et al. Physiological role of carotenoids and their application in food technology and New technologies, 2023, 19(1):14-25).

[0044] Desmodesmus subspicartus Dia 5 strain's use as a carotenoid source for aquaculture of finfish (such as rainbow trout, tilapia, and grouper) and crustaceans (especially shrimp) is best described as "provitamins"—substances that improve feed conversion, which promotes growth and development, strengthens their immune system, and ultimately improves their survival. For the aquaculture of mollusc larvae (scallops, mussels, and oysters) and sea cucumbers, Desmodesmus subspicartus Dia 5 strain can be used as a primary feed and, in some cases, as a superior feed compared to other feeds.

[0045] Carotenoids are also natural pigments, contributing to the coloration of feathers and skin in animals and enhancing the coloration of aquaculture species. Therefore, the use of the Desmodesmus subspicartus Dia 5 strain in feed additives can be used not only as a source of biologically active substances but also to enhance coloration to enhance the consumer value of products (egg yolk coloring, pigmentation of other poultry products, as well as livestock and aquaculture products).

[0046] The possibility of objectively demonstrating the technical result when using the invention is confirmed by reliable data given in examples containing experimental information obtained in the process of conducting research using methods accepted in this field.

[0047] It should be understood that the examples given in the application materials are not limiting and are given only to illustrate the present invention.

[0048] Example 1.

[0049] To cultivate the Desmodesmus subspicartus Dia 5 strain, we used the classic F / 2 medium, but without biotin, and KH2PO4 instead of NahkPC, to which additional salt solutions were added: 200 g / L (or 20%) NaCl (stock 20x solution), 0.5 mol / L (NN4)28O4 (stock 2000x solution), 150 g / L NaNOs (stock 2000x solution). To create salinity, 1% NaCl (final concentration) was used. At the beginning, cultivation was started on 1x or 2x medium; after reaching near-maximum optical density, additional F / 2 medium (up to 4-5x) and NaNOs were added and cultivation was continued. The pH at the beginning of cultivation was set at 7.0. pH was adjusted using 2M HCl or 2M NaOH.

[0050] Cultivation was carried out in 20- and 250-liter vessels at temperatures ranging from 20 to 30°C, using atmospheric air bubbling through the medium at a rate of 0.5-1 liter per liter of medium per minute. Fluorescent lamps were used for illumination, with constant illumination (24 hours a day) and an illumination intensity of 3.25-3.5 μmol / sec per liter (the lamps were placed inside the solution).

[0051] Studies have shown that the strain's biomass productivity (dry weight) reached 0.5 g / L, with an average daily growth rate (p) of up to 0.56. In terms of cell count, the observed increase ranged from approximately 2x at the beginning to 0.2x per day at the end of the cultivation cycle. The culture density at the end of cultivation was 50-70 million cells / mL.

[0052] The pigment composition of the cells was analyzed as follows. Pigments were extracted with 100% acetone. 500 μl of 100% acetone was added to the precipitated cells. To completely extract the pigments, the cells were disrupted using glass beads on a vortex mixer and an ultrasonic bath. Next, centrifugation was performed at 10,000 rpm. The supernatant was poured into a clean tube, and another 500 μl of 100% acetone was added to the sediment. The procedure was repeated five times, collecting the supernatants of each sample in a single tube. Pigment concentration was determined spectrophotometrically. Pigment analysis of the samples was performed chromatographically on an Agilent Zorbax SB-C18 5 μm 4.6x250 mm reversed-phase column. To separate the pigment mixture, a mixture of 23% ethyl acetate and 77% solution C was used as solution A, pure ethyl acetate as solution B, and a mixture of 90% acetonitrile and 10% water as solution C. The feed rate of the solvents was 1 ml / min.The HPLC system consisted of a Shimadzu LC-10ADVP pump with an FCV-10ALVP module to create a solvent gradient on the low-pressure side, an SPD-M20A detector, and a STO-20AC thermostat.

[0053] The composition of photosynthetic pigments (mg / g dry mass) at the plateau stage is presented in Table 1:

[0054] Sample

[0055] Dia5, 250L Dia5, 20L

[0056] Chlorophyll a

[0057] 36.40 38.55

[0058] Chlorophyll b

[0059] 12.541 13.42

[0060] Chlorophyll (a+b) 48.941 51.97

[0061] Carotenoids

[0062]

[0063] 12.21 12.62

[0064] As can be seen from Table 1, the yield of the useful product, carotenoids, was approximately 12.21-12.62 mg per g of dry biomass.

[0065] The analysis of the carotenoid composition of the algae showed that the Desmodesmus subspicartus Dia 5 strain produces various carotenoids, such as lutein, α-carotene, β-carotene, neoxanthin, violaxanthin, and neolutein B (the MOHO-Z isomer of (αH-E)-lutein, consisting of (9Z)- or (E^)-lutein or a mixture thereof). Among the carotenoids produced by the strain, the highest accumulation levels are for lutein (about 64%) and β-carotene (about 22%). A more detailed analysis of the carotenoid composition of the Desmodesmus subspicartus Dia 5 microalgae is given in Table 2:

[0066] Carotenoid Sample

[0067] Dia5, 250L Dia5, 20L

[0068] Neoxanthin 4.6 4.2

[0069] Violaxanthin 4.6 3.9

[0070] Lutein 63.7 64.9

[0071] Zeaxanthin 0.0 0.0

[0072] Neolutein B 2.0 2.5

[0073] a-carotene 3.0 2.9

[0074] β-carotene 22.1 21.6

[0075]

[0076] Thus, the study showed that the microalgae strain Desmodesmus subspicartus Dia 5 has a high level of accumulation of a mixture of natural carotenoids (about 4.5 mg per g of dry biomass), mainly lutein (about 64%) and p-carotene (more than 50% at the growth stage, but decreasing to 22% at the plateau stage).

[0077] As is known, cultivation conditions (such as lighting, composition and pH of the medium, etc.) can have a significant impact on the productivity of microalgae (see, for example, Lichtenthaler H.K. et al. Chlorophylls and Carotenoids: Measurement and Characterization by UV-VIS Spectroscopy / / Current Protocols in Food Analytical Chemistry, 2001, F4.3.1- F4.3.8), so it is obvious that creating optimal cultivation conditions for the Desmodesmus subspicartus Dia 5 strain will ensure a higher level of carotenoid production, including their individual fractions.

[0078] Example 2.

[0079] A study of the fatty acid profile produced by the microalgae strain Desmodesmus subspicartus Dia 5 was also conducted. Fatty acid composition was analyzed using the following method. Frozen samples (weighing 100 mg) in 2 ml Eppendorf tubes were homogenized in 1 ml of a chloroform / methanol solution (1:2) with the addition of ionol. To improve extraction efficiency, the samples were mixed on a vortex mixer with the addition of glass beads. The sample was centrifuged at 3000 rpm for 5 min, and the supernatant was transferred to a glass tube with a tight lid. 1 ml of a chloroform / methanol solution (1:2) and 270 μl of 1% KCl were added to the sediment, and the mixture was shaken well (30 s) with glass beads. An ultrasonic bath was used for more complete cell disruption. The mixture was centrifuged at 3000 rpm for 5 minutes, and the supernatant was transferred to the glass vial containing the supernatant obtained in the previous step. 670 µl of chloroform and 0.01 were added to the collected supernatant.4 ml of 1% KCl were mixed well and centrifuged at 3000 rpm for 5 min. The lower phase was transferred to a clean glass vial and dried under a stream of nitrogen. Fatty acid methyl esters were prepared by adding 1 ml of 8% (w / v) sulfuric acid in methanol. The samples were kept at 90°C for 90 min and cooled to room temperature. 1 ml of 10% sodium chloride in water and 250 μl of hexane were added to the vial, mixed, left on the workbench for 10 min, and the upper phase containing fatty acid methyl esters was transferred to a gas chromatography-mass spectrometry vial. The obtained fatty acid esters were analyzed by gas chromatography and mass spectrometry. Identification of peaks in the total ion chromatograms was performed by comparison with the Wiley Mass Spectral Data Register and the chromatographic profile of the sample with the profile of Supelco 37 Component FAME Mix fatty acid standards (Sigma-Aldrich).Peak areas were determined by computerized integration.

[0080] The fatty acid composition of the cells of the microalgae strain Desmodesmus subspicartus Dia 5 based on the results of the analysis is shown in Table 3:

[0081] Fatty acid content (mol % of total) 16:0 18.6 ± 0.5

[0082] 16:1D 7 5.3 ± 0.3

[0083] 16:2D 7 ' 10 2.2 ± 0.1

[0084] 16:30 7 ' 10 ' 13 2.6 ± 0.1

[0085] 16:4D 4 ' 7 ' 10 ' 13 18.7 ± 1.1

[0086] 18:0 0.8 ± 0.6

[0087] 18:1 A 9 2.1 ± 0.3

[0088] 18:2D 912 8.4 ± 0.3

[0089] 18:30 9 ' 12 ' 15 33.7 ± 0.8

[0090] 18:30 6 ' 9 ' 12 1.6 ± 0.1

[0091] 18:4D 6 ' 9' 12 ' 15 4.0 ± 0.2

[0092] 20:0 - Others 2.0 ± 0.1

[0093] Index

[0094] 2.46

[0095]

[0096] unsaturation

[0097] As the analysis showed, despite the diverse fatty acid composition, the total fatty acid content of the dry microalgae biomass is only approximately 20%, making it impractical to consider the Desmodesmus subspicartus Dia 5 strain as a significant source of fatty acids for the food industry. Thus, after studying the potential of the new Desmodesmus subspicartus Dia 5 microalgae strain, it was found that it exhibits a high capacity for carotenoid production, primarily accumulating lutein and β-carotene. Its fatty acid production is significantly less significant. These properties of the Desmodesmus subspicartus Dia 5 microalgae strain allow it to be considered primarily as a source of beta-carotene and lutein.

[0098] Although the invention has been described with reference to the disclosed embodiments, it will be apparent to those skilled in the art that the specific, detailed examples are provided merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the invention in any way. It should be understood that various modifications are possible without departing from the spirit of the present invention.

Claims

Invention formula The microalgae strain Desmodesmus subspicartus (Chodat) E / Hegewald & A / W / F / Schmidt 2000 (Dia 5), ​​deposited in the All-Russian Collection of Microorganisms (VKM) according to the international deposition system in accordance with the Budapest Treaty under the number VKM AI-522D, is a producer of carotenoids, with a predominant accumulation of lutein and β-carotene.

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