Marine microalga as producer of 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 Parachlorella sp., Nanno 1, which is a producer of carotenoids, especially lutein. 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 carotenoids, primarily lutein, which constitutes approximately 2 / 3 of the carotenoids accumulated, and the strain biomass additionally has good organoleptic properties. The strain exhibits a high growth rate with rapid pigment accumulation, and has a simple culture protocol which can be easily scaled up.

Description

[0001] MARINE MICROALGAE - LUTEIN PRODUCER

[0002] AND OTHER CAROTENOIDS

[0003] Field of technology

[0004] The invention relates to the field of microbiology and biotechnology and can be used to obtain biologically active substances, primarily carotenoids, especially lutein, for use in biologically active additives (BAA) and food additives for humans, as well as in various feed additives, primarily for aquaculture.

[0005] Prior art

[0006] 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 biological activity of carotenoids in animals is significantly broader, as exemplified by lutein, from which retinoids cannot be derived. Lutein, known as macular pigment, is known to support visual function and plays a key role in alleviating age-related macular degeneration and proliferative diabetic retinopathy—diseases associated with excess levels of vascular endothelial growth factor (VEGF) in ocular tissue. Lutein has been shown to accumulate in ocular tissue and is a key protector against the development of these diseases by suppressing the VEGF signal (Gurunathan S. et al.Lutein downregulates retinal vascular endothelial growth factor, possibly via hypoxia inducible factor 1 alpha and X-box binding protein 1 expression in streptozotocin-induced diabetic rats / / Journal of Functional Foods, 2017, 31:97-103). 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 of the end consumer.

[0007] Carotenoids are widely used in aquaculture and as feed additives for animals and poultry. Recently, the demand for "native" (natural) carotenoids has grown significantly. However, chemically synthesized carotenoids are characterized by extremely low solubility in aquatic environments and the ability to form large aggregates, which significantly reduces their bioavailability for both humans and animals. Furthermore, synthetic carotenoids often form various E- and Z-isomers with distorted structures, which become unrecognizable to human enzymes, making it difficult to eliminate them from the body 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 fish fry growth 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 htfes: / / do L rgdL0.4 .942618 ;6381;;y18 I 04). Therefore, native carotenoids, obtained, for example, in the form of lyophilized microalgae biomass, are the most suitable for both food and feed additives.

[0008] Many eukaryotic microalgae are capable of accumulating hundreds and sometimes even thousands of times more carotenoids than any known higher plants, due to their often extreme living conditions. It is this ability to synthesize enormous quantities of carotenoids that has recently been exploited to create biologically active food and feed additives. Industrial microalgae cultivation is now used in many countries.

[0009] 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 08.02.2018), and the Coelastrella sp. K1 (IPPAS C-2028) strain is a producer of a mixture of astaxanthin and β-carotene (RU2703420, published 16.10.2019). Representatives of the genus Chlorella, as well as representatives of some other genera from the Chlorella clades (Parachlorella, Auxenochlorella), are also known to produce carotenoids. A number of them are known as lutein producers: for example, the Parachlorella kessleri HY1 strain is known, deposited at the Institute of Botany of the Czech Republic, Třeboň (CZ2019776A3, published on 27.01.2021), the Chlorella sorokiniana strain (SAG 211-32), deposited in the culture collection of the University of Göttingen (Germany) (EP21571676 published on 28.09.2011), the Chlorella minutissima MCC-27 strain, deposited in the CCUBGA collection (India) (Dineshkumar R. et al.Development of an optimal light-feeding strategy coupled with semi-continuous reactor operation for simultaneous improvement of microalgal photosynthetic efficiency, lutein production and CO2 sequestration / / Biochemical Engineering Journal, 2016, 113: 47-56).

[0010] Despite the wide variety of microorganisms capable of producing carotenoids, there is a tremendous diversity among the carotenoids themselves (approximately 600 different carotenoids have been described). Furthermore, microbial strains have different cultivation requirements, often 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. Disclosure of the invention

[0011] The technical problem and task that the invention aims to solve is to obtain a new strain of microalgae with a high level of lutein accumulation.

[0012] This problem is being solved by obtaining a new strain of Parachlorella sp. Nanno 1, a producer of carotenoids, primarily lutein. The strain is deposited in the Microalgae Culture Collection at the K. A. Timiryazev Institute of Plant Physiology, Russian Academy of Sciences (hereinafter referred to as the TPPS Microalgae Collection, IPPAS) under accession number IPPAS C-2073 and deposited in the All-Russian Collection of Microorganisms (VKM) under the international deposition system in accordance with the Budapest Treaty under accession number VKM AI-521D.

[0013] Another aspect of the invention is the production of carotenoids with a high lutein content during the cultivation of the Parachlorella sp. Nanno 1 strain.

[0014] Another aspect of the invention is a method for obtaining carotenoids, primarily lutein, by culturing the Parachlorella sp. Nanno 1 strain.

[0015] The proposed strain Parachlorella sp. Nanno 1 has a number of advantages that ensure the technical result of the present invention, which are as follows:

[0016] - a new strain of microalgae Parachlorella sp. Nanno 1 expands the arsenal of sources of carotenoids, primarily lutein;

[0017] - the Parachlorella sp. Nanno 1 strain has high productivity in terms of biomass, as well as carotenoids (about 1.4% of dry matter);

[0018] - the microalgae strain Parachlorella sp. Nanno 1 has the ability to produce a mixture of carotenoids, including lutein, a-carotene, p-carotene neoxanthin, violaxanthin, zeaxanthin and neolutein B, in high content; lutein accounts for approximately 2 / 3 of all carotenoids produced;

[0019] - Parachlorella sp. Nanno 1 strain has a high growth rate with rapid pigment accumulation, a simple cultivation protocol with easy scaling;

[0020] - the biomass of the microalgae Parachlorella sp. strain Nanno 1 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.

[0021] The Parachlorella sp. Nanno 1 strain is a potential industrial producer of biologically active substances—carotenoids—which can be used in industry, healthcare, agriculture, fish farming, and other sectors.

[0022] Brief description of the figures Fig. 1 - cells of microalgae Parachlorella sp. strain Nanno 1 (bright-field microscopy, Leica Flexacam C3 camera).

[0023] Terms and definitions

[0024] 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.

[0025] 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."

[0026] The term "and / or" means one, more than one, or all of the listed elements.

[0027] Also here, listing numeric ranges by endpoints includes all numbers within that range.

[0028] 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.

[0029] By strain Parachlorella sp. Nanno 1 is meant the strain Parachlorella sp. Nanno 1, which was deposited in the Microalgae Collection of the Institute of Physiology of the Russian Academy of Sciences under the number IPPAS C-2073, and deposited in the VKM collection under the number VKM AI-521D. By strain Parachlorella sp. Nanno 1 is also meant a mutant strain obtained using the deposited strain as a starting material and retaining all the properties of the original strain, i.e. possessing all the defining (identifying) characteristics of the strain Parachlorella sp. Nanno 1. Including, at least, the ability to use in the production of carotenoids, primarily lutein. In particular, the strain Parachlorella sp. Nanno 1 has the following identifiable features, including 1) the ability to produce carotenoids, primarily lutein; 2) the presence of essential genes with identity of more than 95%, more than 96%, more than 97%, more than 98% or more than 99% with the essential genes of Parachlorella sp.Nanno 1, which can be used to confirm that the analyzed variant is Parachlorella sp. Nanno 1 (the genetic sequence of the strain is registered in GenBank under the number OR682196).

[0030] Unless otherwise defined, technical and scientific terms in this application have the standard meanings generally accepted in the scientific and technical literature. Detailed Description of the Invention

[0031] The strain Parachlorella sp. Nanno 1 was isolated from a mixed culture of microalgae belonging to several different genera.

[0032] The Parachlorella sp. Nanno 1 strain was deposited on December 25, 2023, in the Microalgae Culture Collection of the K. A. Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences under the number IPPAS C-2073. The Parachlorella sp. Nanno 1 strain was also deposited on July 15, 2025, in the All-Russian Collection of Microorganisms (VKM) via the international deposit system in accordance with the Budapest Treaty under the number VKM AI-521D as part of the international patent deposit (the international deposit document is an appendix to this application).

[0033] The strain was identified using morphological and molecular genetic analysis, including whole-genome sequencing. The genetic sequence of the Parachlorella sp. Nanno 1 microalgae is registered in the international GenBank database (h it : / www . p ; n rn.njh.goy / gen bank / ) under accession number OR682196.

[0034] Morphological features: solitary cells, surrounded by common mucus, spherical, 5-10 µm in diameter, with one parietal chloroplast and one pyrenoid (rarely two). The cytoplasm is vacuolated, the cell wall is smooth. Reproduction is by 2-4 autospores. A bright-field microscopy image showing the morphology of the microalga Parachlorella sp. Nanno 1 is shown in Fig. 1.

[0035] Degree of purity: algologically pure culture.

[0036] Physiological properties of the strain. Any sterile deionized water can be used for cultivation, in particular, tap water passed through a reverse osmosis unit without remineralization. Optimal cultivation medium: classic F / 2 medium, but instead of NaH2PO4, KH2PO4 + 2% NaCI + 75 mg / L NaNOs + 0.25 mmol / L (NN4)28O4 is used. The multiplicity of the F / 2 medium can be increased up to 6x, as well as the addition of NaNOs up to 6x (it is not necessary to increase ammonium sulfate). Minimum medium: F / 2 + 2% NaCI + 0.25 mmol / L (NN4)28O4. Composition of the gas-air mixture (GAM): atmospheric air (bubbling with atmospheric air passed through the medium). Temperature: 25°C. Lighting: day / night during cultivation or around the clock for optimal growth.

[0037] Strain characteristics: Parachlorella sp. Nanno 1 produces a mixture of carotenoids (lutein, a-carotene, p-carotene, neoxanthin, violaxanthin, zeaxanthin and neolutein B) with a high content of about 14 mg / g dry matter, with lutein accounting for about 2 / 3 of the carotenoids produced.

[0038] The strain was identified morphologically using microscopy. Molecular genetic analysis was performed using PCR of the ITS1-5.8S-ITS2 region. Furthermore, since PCR was unable to provide unambiguous identification, a whole-genome analysis was also performed. As a result of these studies, the Nanno 1 strain was identified as a candidate for a new, previously undescribed genus with Chlorella-like morphology from the Parachlorella family (Trebouxiophyceae, Chlorophyta). Verification was conducted, in part, with the ACSSI Algological Collection of the Institute of Physicochemical and Biological Problems of Soil Science, Russian Academy of Sciences, a separate division of the Putin Research Center for Biological Research, Russian Academy of Sciences.

[0039] A preliminary taxonomic diagnosis was established based on a morphological analysis performed using microscopic examination of the prepared preparation. The preparation 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 observation results were documented by color digital photography using a Leica Flexacam C3 camera (Germany) (see Fig. 1). It was established that the cells of the Nanno 1 strain are solitary, surrounded by common mucus, spherical, 5-10 µm in diameter, with one parietal chloroplast and one pyrenoid (rarely two). The cytoplasm is vacuolated, the cell membrane is smooth. Reproduction is by 2-4 autospores. Remnants of the maternal membranes are preserved in the mucus for some time. The Nanno 1 strain was initially identified as Chlorella sp. based on morphological characteristics, but further molecular genetic analysis showed that this was incorrect.

[0040] Strain DNA was isolated for PCR analysis using DNeasy (QIAGEN, USA). Analysis of the strain's 18S rRNA gene in 1% agarose gel revealed consistently low-quality amplicons, despite various protocol modifications, preventing definitive identification. Therefore, a different DNA barcode—internal transcribed spacer 2 (ITS2)—was used for identification. Standard primers were used to amplify 18S rRNA and ITS2.

[0041] Based on molecular genetic identification, the studied microalgae strain Nanno 1 was included in the Parachlorella clade (Trebouxiophyceae, Chlorophyta). This clade, sister to the Chlorella clade, was isolated in 2004, and this division was subsequently confirmed by Yamamoto et al. (2005) based on an analysis of the cell wall ultrastructure of autospores. Representatives of this clade are widespread in freshwater and saltwater bodies and can also be found in biofilms and soil (Krivina et al., 2021; Song et al., 2018).

[0042] Representatives of the Parachlorella clade can be divided into four types based on their morphology, and the Nanno 1 strain is quite similar morphologically to Chlorella-like species, but united by a common mucus. The analyzed Nanno 1 strain clustered with Auxenochlorella pyrenoidosa strain 820 and Auxenochlorella pyrenoidosa isolate N with maximum statistical support. This cluster formed an independent phylogenetic lineage within the Parachlorella clade. However, these strains did not cluster with representatives of the Auxenochlorella genus, including the authentic strain of the type species Auxenochlorella protothecoides CCAP 211 / 7A. Genetic distances between the Nanno 1 strain and its sister strains ranged from 1.1% to 2.8% (intraspecific level). For comparison, within the genus Parachlorella, the level of genetic differences ranged from 3.6% to 6.6%. Genetic distances between strains Nanno 1, strain 820, isolate N and other representatives of the clade ranged from 16.9% to 35.5%.These values ​​correspond to the interspecific and intergeneric levels. For example, the genetic differences between the genera Dictyosphaerium and Kalenjinia are 13.8-15.2%. Thus, strain Nanno and its sister strains, strain 820 and isolate N, represent a new, undescribed species and, likely, a genus within the Parachlorella family.

[0043] To further confirm the results of the molecular genetic analysis, whole-genome sequencing of the Nanno 1 strain was performed on an Illumina Novaseq 6000 sequencer. Genome coverage was assessed using Jellyfish V2.2.10. Analysis of the k-mer distribution (at k = 23) showed an average genome coverage for the x60 sample. These indicators indicate sufficient read depth, good coverage, and, consequently, high sequencing quality. The quality of the raw data was assessed using FastQC. Read analysis demonstrates excellent quality: indicators such as average quality for each nucleotide, read length, adapter content, presence of highly represented sequences, etc., meet normal standards. The SPAdes v.3.15.4 assembler was used for de novo assembly. The quality of the genome assembly was assessed using QUAST and BUSCO.The analysis result showed that the assembly contains more than 85% of single-stranded orthologs of the taxonomic group Chlorophyta, which also confirms the high quality of the assembly. To assemble the 18S-ITS1-5.8S-ITS2-28S region, which was used for phylogenetic analysis, an array of more than 3000 homologous rRNA-coding regions for taxon 3041 from the NCBI database was used. The method of targeted region assembly was used using reads homologous to the sequences from the array. The final region has a length of 4712 nucleotide pairs (bp), of which 18S - 1798 bp, ITS1 -267 bp, 5.8S - 154 bp, ITS2 - 285 bp, 28S (partial) - 2206 bp. Sequence alignment was performed using the ClustalW algorithm in the BioEdit program. The rooted phylogenetic tree was constructed using the IQ-TREE web server with the following parameters: TIM2+I+G model, gamma categories = 4, bootstrap alignments = 1000, and the remaining settings were default.The analysis revealed that the analyzed sequence occupies a unique phylogenetic position, corresponding to the genus level of the Parachlorella clade. The closest genus is Parachlorella. Two species identical to the rRNA region of the studied organism and forming a single stable clade with it were found in the NCBI database: EU038290 and KJ868082. Their taxonomic affiliation is described as Chlorella (or Auxenochlorella) pyrenoidosa, which is likely incorrect.

[0044] Analysis of the biotechnological characteristics of the Parachlorella sp. Nanno 1 strain revealed that it has a high growth rate with rapid pigment accumulation, visible even to the naked eye (upon reaching the plateau stage, the microalgae acquires a dark green, or even almost black, color). It also produces a mixture of carotenoids with a high content of approximately 14 mg / g dry matter, with lutein accounting for approximately two-thirds of the carotenoids produced. The total carotenoid content of the Parachlorella sp. strain is 14 mg / g dry matter. Nanno 1 can account for up to 1.4% of dry weight, which can be compared with such “champions” in carotenoid production as Phaeodactylum trcornutum (1.02%), Isochrysis galbana (1.76%) (Di Lena et al., 2019) and Haematococcus pluvialis (2%) (Liu et al., 2016).

[0045] Due to the ability of the Parachlorella sp. Nanno 1 strain to produce carotenoids with high levels of lutein accumulation, it can be used as a source of these biologically active substances. Carotenoids function as antioxidants in humans and animals, and some carotenoids are also metabolic precursors of vitamin A.

[0046] The Parachlorella sp. Nanno 1 strain can be used as a 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 fight against 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. 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.

[0047]

[0048] 2031268). As a dietary supplement, the Parachlorella sp. Nanno 1 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 and Asia Pacific. J. Clin. Nutr. 2001, 10, 159-64. https: / / doi.org / 10-1111 / j.1440-6047.2001.00235.x).

[0049] The best definition of the use of Parachlorella sp. Nanno 1 as a carotenoid source for aquaculture of fish (such as rainbow trout, tilapia, and grouper) and crustaceans (especially shrimp) is "provitamins"—that is, substances that improve feed conversion, which promotes better growth and development, strengthens their immune system, and, consequently, improves their survival. For the aquaculture of mollusc larvae (scallops, mussels, and oysters) and sea cucumbers, the use of Parachlorella sp. Nanno 1 can be the primary feed and the best feed compared to other feeds.

[0050] Carotenoids, especially lutein, are also natural pigments that contribute to the coloration of feathers and skin in animals and enhance the coloration of aquaculture species. Therefore, the use of Parachlorella sp. Nanno 1 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). Furthermore, lutein can be used as a food coloring and to color pharmaceuticals and cosmetics.

[0051] 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.

[0052] It should be understood that the examples given in the application materials are not limiting and are given only to illustrate the present invention.

[0053] Example 1.

[0054] For cultivation of the Parachlorella sp. Nanno 1 strain, classical F / 2 medium was used, but without biotin, and KH2PO4 was used 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), vitamins B1 and B12. The pH at the beginning of cultivation was set at 7.0. pH was adjusted using 2M HCl or 2M NaOH.

[0055] Cultivation was carried out in 20- and 250-liter vessels at 25°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 a 24-hour light cycle during rearing, maintaining the light intensity at 200-240 lumens per liter (submersible lamps were used).

[0056] According to the conducted studies, the strain's productivity in terms of biomass accumulation (dry weight) amounted to 0.5 g / L, with an average daily growth rate (p) of up to 0.69. 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 50-90 million cells / ml. Because the strain produces a large amount of pigments (chlorophyll and various carotenoids), the culture turned dark green, almost black, at the end of the cultivation cycle.

[0057] 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.

[0058] The composition of photosynthetic pigments (mg / g dry mass) at the plateau stage is presented in Table 1:

[0059] Sample

[0060] Nannol, 250L Nannol, 20L

[0061] Chlorophyll a 41.84 35.85

[0062] Chlorophyll b 14.584 12.58

[0063] Chlorophyll (a+b) 56.424 48.43

[0064] Carotenoids

[0065]

[0066] 14.07 10.83

[0067] As can be seen from Table 1, the yield of the useful product, carotenoids, amounted to more than 14 mg per g of dry biomass per cultivation cycle.

[0068] The analysis of the carotenoid composition of the algae showed that the strain Parachlorella sp. Nanno 1 is a producer of various carotenoids, such as: lutein, α-carotene, β-carotene, neoxanthin, violaxanthin, zeaxanthin, and neolutein-B (a mono-β-isomer of (αH-E)-lutein, consisting of (9Z)- or (E^)-lutein or a mixture thereof). Moreover, among the carotenoids produced by the strain, lutein has the highest accumulation level (more than 60%). A more detailed analysis of the carotenoid composition of the microalgae strain Parachlorella sp. Nanno 1 is given in Table 2:

[0069] Carotenoid Sample

[0070] Nannol, 250L Nannol, 20L

[0071] Neoxanthin 5.1 2.9

[0072] Violaxanthin 6.3 2.2

[0073] Lutein 67.1 63.5

[0074] Zeaxanthin 0.1 0.0

[0075] Neolutein B 1.9 0.9

[0076] a-carotene 2.1 3.1

[0077] β-carotene 17.4 27.4

[0078]

[0079] Thus, the study showed that the microalgae strain Parachlorella sp. Nanno 1 has a high level of accumulation of a mixture of natural carotenoids (14.07 mg per g of dry biomass), primarily lutein (more than 67%, i.e., about 2 / 3 of all carotenoids produced), which significantly exceeds the productivity of many known carotenoid producers.

[0080] 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 Parachlorella sp. Nanno 1 strain will ensure a higher level of carotenoid production, including their individual fractions. Thus, cultivation of the Parachlorella sp. Nanno 1 strain in a 250-liter vessel and illumination of about 48,000 lumens showed a carotenoid content at the plateau stage of 14.07 mg / g dry weight with a lutein content of 67.1%. Further optimization of cultivation conditions will contribute to even greater productivity of the strain.

[0081] Example 2.

[0082] A study of the fatty acid profile produced by the microalgae strain Parachlorella sp. Nanno 1 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.

[0083] The fatty acid composition of the cells of the microalgae strain Parachlorella sp. Nanno 1 at the growth stage, based on the results of the analysis, is shown in Table 3:

[0084] Fatty acid content (mol % of total) 16:0 17.2 ± 0.3

[0085] 16:1D 7 5.0 ± 0.7

[0086] 16:2D 7 ' 10 15.7 ± 0.8

[0087] 16:30 7 ' 10 ' 13 14.2 ± 0.1

[0088] 16:4D 4 ' 7 ' 10 ' 13 - 18:0 1.3 ± 0.03

[0089] 18:1 A 9 - 18:2D 9 ' 12 20.0 ± 1.5

[0090] 18:30 9 ' 12 ' 15 22.8 ± 0.1

[0091] 18:30 6 ' 9 ' 12 - 18:4D 6 ' 9 ' 12 ' 15 - 20:0 2.5 ± 0.2

[0092] Others 1.3 ± 0.4

[0093] Index

[0094] 2.05

[0095]

[0096] unsaturation

[0097] As the analysis showed, despite the diverse fatty acid composition, the total amount of fatty acids in the dry microalgae biomass is low, making it impractical to consider the Parachlorella sp. Nanno 1 strain as a significant source of fatty acids for the food industry.

[0098] Thus, after studying the potential of the new microalgae strain Parachlorella sp. Nanno 1, it was found that the strain exhibits a high capacity for carotenoid production, primarily accumulating lutein; fatty acid production by the strain is significantly less significant. These properties of the microalgae strain Parachlorella sp. Nanno 1 allow it to be considered primarily as a source of lutein.

[0099] 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 Parachlorella sp. Nanno 1, deposited in the All-Russian Collection of Microorganisms (VKM) under the international deposition system in accordance with the Budapest Treaty under the number VKM AI-521D, is a carotenoid producer with predominant accumulation of lutein.

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