TW201107478A - Photosynthetic microorganisms enriched in selenium using selenohydroxy acid compounds, uses thereof in nutrition, cosmetics and pharmacy - Google Patents

Abstract

The invention relates to the enrichment of photosynthetic microorganisms in organic selenium using selenohydroxy acid compounds, in particular 2-hydroxy-4-methylselenobutanoic acid, in D or L form, or an enantiomer, salt or ester or amide derivative of these compounds, and also to the use of the microorganisms thus enriched in animal or human nutrition, in cosmetics or in pharmacy.

Description

201107478 VI. INSTRUCTIONS OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to the use of a selenic acid compound of the D or L type, in particular 2 hydroxy-4-methyl seletonic acid or the enantiomers, salts or The use of vinegar or _amine derivatives to enrich photosynthetic microorganisms with organic selenium, and the use of such photosynthetic microorganisms in animal or human nutrition, cosmetics or pharmaceuticals to thereby increase nutritional value. 〇 [Prior Art] Selenium is especially a necessary micronutrient for humans and mammals (Wend el 5 A .; Phosphorus, Sul fur Silicon Rel at E1 em. 3 1 992, 67, 1 -4, 404-41 5 ). In particular, he participates in selenium in the form of L(+)-selenocysteine or L(+)-selenomethionine (Muller, S. et al., Arch. Microbiol, 1, 997, 168, 421). Bio-synthesis of proteins such as glutathione peroxidase, thioredoxin reductase, and selenoprotein P. Selenium deficiency has been reported in humans, especially in patients who have been receiving parenteral feeding for a long time (Von Stockhausen, HB, Biol. Trace Elem. Res., 1 98 8, 1 5:1 47-1 5 5 ). Daily supplementation of 200 micrograms Selenium is considered safe and suitable for adults with average body weight (Schrauzer, G.N., J. Am. Col. Nutr., 200 1, 20:1 -1 4 ). Selenium is found in nature in two forms (organic and inorganic). Inorganic compounds are most commonly salts such as sodium selenite or selenate. These compounds are extremely toxic to humans and most animals. -5- 201107478 Organic compounds (organic selenium compounds) in living tissues, especially amino acid L( + )-selenyl methionine, L( + )-methyl selenocysteine and L( + )- Selenium cysteine is representative. L( + )_selen methionine is the main source of organic selenium in humans and animals. However, humans and animals are not self-supporting for this kind of amino acid, but can only be obtained through diet. Therefore, selenium should ideally be incorporated into food supplements for the treatment or prevention of selenium deficiency in this organic form. Therefore, it has been shown that the supplementation of L(+)-selen methionine is much less toxic than the intake of sodium selenite and provides better bioavailability (Mony, MC et al., J. of Trace) Elem. Exp. Med., 2000, 1 3 :367-380)= Nowadays, in addition to the use of inorganic selenium (mainly in the form of sodium selenite) and selenium methionine as matrix, the metabolism of selenium absorption by living tissue The route is unknown. A suitable supply of organic selenium can be found in higher plants (especially wheat, corn, soybeans), where more than 80% of the selenium is composed of L(+)-selen thiomethionine (Schrauzer, GN, J. Am) Coll. Nutrit., 200 1, 20(1): 1-4). However, the concentration of selenium in these plants is insufficient, so that food additives cannot be easily and inexpensively manufactured. One of the methods developed for obtaining a composition rich in selenium methionine consists of using inorganic selenium to make certain microorganisms rich in organic selenium. Once enriched with organic selenium, these microorganisms can be used as a raw material for the preparation of food or cosmetic products. -6 - 201107478 Many publications describe the preparation of, for example, selenium-rich yeast and more particularly brewer's yeast (Oh Tae-Kwang et al., 0 6 · 6.1 9 9 5 KR9 5 0 0 0 6 9 5 patent) For the purpose of using itself or for the purpose of incorporating it into a food composition (Moesgaard S. et. al., DK 200200408 patent of 09.16.2003); or for obtaining a selenium-rich derivative product such as selenium-rich Bread (Wang Boaquan, CN 817143 patent of 0 8 · 1 6 · 2 0 0 6), milk (Jeng Chang-Yi, TW 5 6 5 4 3 2 patent of 1 2 1 1 · 2 0 0 3), egg (Cui O Li et al., CN1 302723C patent of 03.07.2007), chocolate (In Gyeong Suk et al., 1 1.0 8.2 0 04 KR2 0 0 4 0 1 0 1 1 4 5 patent) or beer (Jakovleva LG) Et al., 0 7.2 7 · 2 0 0 3 RU 2 2 0 9 2 3 7 Patent). In healthy foods, preparations containing selenium-rich yeast have also been proposed for use by pregnant women (Wang Weiyi, CN 1 778 1 99 of 05.3 1.2006), or for improving the microenvironment of the intestines of patients with hypoglycemia (Li Tao Zhao, CN1810161 patent of 08.02.2006). In the field of dermatological cosmetics, compositions containing selenium-rich yeast have been developed for the purpose of reducing hair damage (Kasik Heinz, DE 1 98 5 8670 of 06.2 1.2000) or preventing photoaging (Kawai Norihisa el al., JP07300409 patent of 11.14.1995). Medicinal preparations containing selenium-enriched yeast have been used to prevent and treat inflammatory conditions such as diabetic retinopathy (Crary Ely J., US Patent No. 5,623, 482 to 6.1 997), or cardiovascular inflammatory conditions (Nagy PL el al. , 09.28.1 992 of the HUT060436 patent). Bacteria and more particularly probiotics themselves are the subject of selenium enrichment (Calomme M. et al., Biol. Trace Elem. Res., 1 995, 47, 3 79-201107478 3 83 ). Lactobacillus acidophilus, reuteri lactic acid bacteria, ferintoshensis lactobacillus, buchn/parabuchneri lactobacillus (Andreoni V_al., US 02 58 964 patent) have been described as rich Selenium-containing food supplements. A mixture of probiotics from the yeast and lactobacilli groups (Huang Kehe Qin, 1 1.08.2006 CN 1 283 1 7 1 C patent) which has been prepared for the purpose of strengthening the immune system and disease resistance. However, in all of these formulations, the selenium-rich microorganisms were prepared only from machine selenium. Therefore, the most commonly used source of selenium consists of sodium selenite or sodium selenate which is stabilized in a microbial medium. Although a satisfactory amount of organic selenium which can be digested and absorbed by the human body has been synthesized, the selenium-rich micro-organism often has a high residual concentration of unconverted inorganic selenium, which has been confirmed to be dangerous to an individual who consumes. In the prior application disclosed in WO 2006/008 1 90, novel organic compounds of the hydroxy acid type have been described as precursors for the formation of L(+)-selenomethionine in humans and animals. Surprisingly, the Applicant has noted that selenoic acid type organic compounds, such as those described in the WO 2006/008 1 90 application, can be incorporated into a culture medium in which the polyphotosynthetic microorganism is enriched in organic selenium. The results obtained have revealed that these compounds are extremely efficient in making such microorganisms particularly rich in L(+)-selen thioglycolic acid, and the yield is equal to or even higher than that obtained using the inorganic compound generally used. Therefore, it has been shown that the use of selenic acid-type organic compounds to photosynthetically enriched organic selenium can produce organic selenium that does not contain inorganic selenium, and can also be produced by ΪΓ et 出生 出生 出生 彼 硒 硒 硒-8 - 201107478 Determine the toxicity issues associated with prior art methods. The selenium-rich photosynthetic microorganism can be directly used in food transactions for preventing or treating selenium deficiency, and is particularly suitable for the manufacture of pharmaceutical, nutraceutical or cosmetic products and compositions. SUMMARY OF THE INVENTION The present application relates to the acquisition of photosynthetic microorganisms, i.e., microorganisms grown in accordance with the source of light energy. The term "microorganism" attempts to identify any living single-cell organism that belongs to one of the following categories: monospores, unicellular organisms, molds, or protozoa, which have microscopic or ultramicroscopic eukaryotic or prokaryotic cellular structures. And he has metabolic and replication potential. These single cell microorganisms may be associated with the formation of fibrils or biofilms. Preferably, the photosynthetic microorganism according to the present invention is a eukaryotic microalgae, more preferably a Chlorella of the genus Chlorella; or a prokaryotic microalgae, such as a cyanobacteria, preferably a genus of the genus Aspergillus or Arthrospira ( Spirulina). The latter is customary for use as a food supplement for those skilled in the art, especially in developing countries. The term "organic selenium" is intended to indicate a collection of molecules containing a compound having at least one selenium atom in its chemical structure and capable of being produced by living tissue, particularly such as selenium methionine, methyl selenium cysteamine. An amino acid such as an acid or cysteine, or a peptide or protein containing the same. The selenium-rich photosynthetic microorganism itself can be used as a food additive. The latter may, for example, be dehydrated to form a stable powder which can be incorporated into the composition as a substrate for the preparation of the conversion product, but may also be used as a probiotic in the food conversion method by the method of -9 - 201107478. For the purpose of obtaining, for example, fermented milk or fake products. The subject matter of the present invention is therefore a novel method for enriching photosynthetic microorganisms with selenium methionine and/or selenocysteine, characterized in that the photosynthetic microorganism is cultured in a culture medium containing a selenoic acid type compound. Preferably, the selenoic acid type compound is a compound of the formula (I), or a precursor, a salt or an ester thereof or a guanamine derivative:

In the formula: η is equal to 0, 1 or 2; 1^ is 011, OCOR3, OP03H2, 0P03R4R5 or OR6 group; R2 is OH, R3, NHR7, S-cysteamine thiol or S-glutathione Base group; it should be understood that when n=l and ruler 2 is 011, R! cannot be OH; R3 is alkoxy group, neuropterin 1, neural guanamine 2, neural guanamine 3, neural guanamine 4, Ceramide 5, ceramide 6a and 6b, S-cysteinyl thiol or S-glutathionyl group, or a group selected from the group consisting of: -10- 201107478

Preferably, r3 is an alkoxy group, an S-cysteinyl sulfhydryl group or an S-glutathione group; OR4 is a (C^-Cm) alkoxy group, a ceramide, a ceramide 2. Neuropterin 3, neuropterin 4, neuropterin 5, neuropterin 6a and 6b, or a group selected from the group consisting of:

OK

-11 - 201107478 Preferably, '0Κ·4 is a (C1-C26) alkoxy group; 〇R5 is a (CVC26) alkoxy group, a ceramide 1 'neoxime 2, a ceramide 3 Ceramide 4, neuropterin 5, neuropterin 6a and 6b

Preferably *OR5 is a (C1-C26) alkoxy group; 〇R6 is propionate, lactate, citrate, fumarate's maleate, butanthate, palmitate, hard a fatty acid, palm oleate, oleate or linoleate group, a natural fatty acid group or a 13-cis-retinoate group; Κ·7 is a ruthenium or (C1-C26) aristocratic group, A natural amino acid or natural moon female in the above formula (I): - the term "hospital base" is intended to mean a straight or cyclic, arbitrary branch, any fluorinated or polyfluorinated one containing from 1 to 26 carbon atoms Any group optionally comprising one or more -12-201107478 carbon-carbon double bonds, such as methyl, ethyl, isopropyl, trifluoromethyl, linoleyl, linoleyl, or palmitic; _" The term "alkoxy" is intended to indicate that a straight chain or a ring, any branch, any fluorination or polyfluorination is derived from a group containing from 1 to 26 carbon atoms and optionally containing one or more carbon-carbon double bonds. a group of a secondary or tertiary alcohol such as methoxy, ethoxy, isopropoxy, trifluoromethoxy, linoleyloxy, linoleyloxy, or granule The structure of a thiol group; a structure of a neurosteroid type is described in particular in "Cosmetics Lipids and the Skin Barrier", Thomas Forster Ed. 2002, M arce 1 D ekker, I n c., Figure 2 on page 2 - The term "natural" is intended to indicate any related compound found in the metabolism of tissues in the plant and animal kingdoms and also in human metabolism (Steglich W., Rompp Encyclopedia Natural Products, G. Thieme ed.); - " "Oligomer" is intended to indicate any compound formed by the association of 2 to 15 monomers interconnected by an ester type linkage; a "polymer" is intended to indicate more than 15 single sheets interconnected by an ester type linkage Any compound formed by the association of the body. According to the present invention, the compound of the formula (I) is preferably used in the form of a calcium salt, a zinc salt or a magnesium salt, which forms a better solubility in a culture medium and a better digestion and absorption by a photosynthetic microorganism. In a preferred embodiment of the invention, the photosynthetic microorganism is selected from the group consisting of cyanobacteria and green algae. Thus, the photosynthetic microorganism is advantageously selected from the group consisting of cyanobacteria or green algae, preferably selected from the group consisting of the green algae of the genus Chlorella and the cyanobacteria of the genus Algae or Arthrospira. The invention more particularly relates to the use of a compound of formula (I) selected below: -L-2-hydroxy-4-methylselenobutyric acid, a D-2-hydroxy-4-methylselenidine Acid, -DL-2-hydroxy-4-methylselenobutyric acid, or a salt of these compounds. These compounds are described in the WO 2006/008 1 90 application. The subject matter of the invention is also a photosynthetic microorganism enriched with selenium which can be obtained in accordance with the method of the invention. Such microorganisms typically have an organic selenium content of greater than 500 ppm, preferably greater than 1000 ppm, more preferably greater than 2 00 ppm, based on selenium equivalents, and less than 0.5% by dry weight of the microorganism, preferably less than 0. %, and more preferably less than 0.1% inorganic selenium content. Preferably, the invention relates to the case where the photosynthetic microorganism comprises less than 1.5%, preferably less than 0.5%, more preferably less than 0.1% of inorganic selenium relative to total selenium. In other words, the selenium residue in the inorganic form present in the photosynthetic microorganism enriched in selenium in accordance with the process of the present invention is typically less than 1.5% of the total selenium present in the microorganism, which typically represents less than 0.5%. The total dry biomass (dry weight) of the microorganism. The invention particularly relates to a photosynthetic microorganism enriched in organic selenium, characterized in that the selenium content of the selenium methionine form of the microorganism is more than 50 relative to the total selenium present in the photosynthetic microorganism. The mass%, preferably more than 70% by mass, more preferably more than 80% by mass, and even more preferably more than 90% by mass of selenium. The ratio of such selenium methionine represents a significant and particularly advantageous improvement in terms of the amount of organic selenium present in the microorganism and the quality of -14-201107478 as compared to those obtained in the prior art. In particular, the present invention relates to the case where the microorganism is characterized in that it is a selenium-rich green algae microalgae, preferably a green algae, and is characterized in that the microorganism contains usually more than 50 micrograms based on the dry weight of the microorganism. Selenium equivalent / gram (AgSe / g), preferably greater than 7 〇 AgSe / g, and more preferably greater than 100 〆 gSe / g 〇 ^ organic molecular type fixed in the microorganism (selen methionine, selenium The amount of selenium in the cysteine and the like or the inorganic molecular type (selenium salt) is expressed in terms of selenium mass/gram (y gSe/g) of the dry weight of the microorganism. In other words, the selenium content of the photosynthetic microorganism is established by calculating the quality of the selenium present in these organic or inorganic molecules and retrospectively correlates it with the total dry biomass of the microorganism. In addition, the mass ratio of selenium in organic and inorganic forms is also established and expressed as a percentage relative to the total selenium mass. The total selenium of the photosynthetic microorganism according to the present invention and the selenium content of the selenium methionine type selenium can be determined by mineralization and enzymatic digestion after centrifugation and lyophilization of the microorganism, for example, by following Lobinsky el al· The method described in Mester, Z. et al. (2006) Annal. Bioanal. Chem. 3 85: 1 68- 1 80. The results obtained according to the present invention as illustrated in the examples of the present application show that photosynthetic microorganisms (more particularly green algae and cyanobacteria microalgae) accumulate selenium in the form of selenium methionine at a dry weight of these microalgae. Typically greater than 1 〇〇 microgram selenium equivalent per gram (#gSe/g), preferably greater than 200//g Se/g, more preferably greater than 500/zgSe/g, even more preferably greater than 1000/zgSe/g, -15- 201107478 and even greater than 1400 // gSe/g. The invention therefore more particularly relates to organic selenium-rich green algae or cyanobacteria characterized by a selenium methionine form having an organic selenium content generally greater than 100 micrograms of selenium equivalent per gram (ygSe/g) on a dry weight basis. Preferably, it is greater than 200 //gSe/g, more preferably greater than 500; zgSe/g, and even more preferably greater than 1 〇〇〇//gSe/g. Such organic selenium-rich green algae and cyanobacteria are generally characterized in that the selenium methionine form has an organic selenium content of more than 50% of the total selenium contained therein, preferably more than 7%, preferably more preferably More than 8 〇%, even more preferably more than 90% 'and even more than 95%, and is characterized by a residual inorganic selenium content of less than 1.5% of the total selenium contained therein. The good land is less than 〇. 5 %, more preferably less than 1%. Usually, the residual inorganic selenium content thereof is less than 1% by weight of the total biomass of the green algae, preferably less than 〇·5%, more preferably less than 0.2%, and even less than 〇. %. The invention also relates to the manufacture of food, cosmetic or pharmaceutical products from selenium-rich photosynthetic microorganisms in accordance with the method of the invention. Such fabrication uses techniques known to those skilled in the art. The photosynthetic microorganisms according to the invention can also be used in animal nutrition, in particular for the purpose of obtaining secondary derivatives rich in organic selenium, such as fish, milk or eggs. The resulting derivatives and molecules are therefore used in a variety of applications, including those enumerated above, particularly as cosmetic, pharmaceutical or nutraceutical agents. The subject matter of the invention is also the use of a selenium-rich photosynthetic microorganism according to the invention as a cosmetic, pharmaceutical (or therapeutic) or nutritional product (agent). -16- 201107478 The invention also relates to a composition comprising the photosynthetic microorganism, usually a makeup, pharmaceutical or nutritional composition. The invention also relates to a culture medium for photosynthetic microorganisms characterized in that it comprises one or more compounds of formula (I) as defined above. Such a culture medium has the use of a method according to the present invention for enriching selenium by photosynthetic microorganisms. In particular, the present invention relates to a solid or liquid culture medium comprising at least one compound of the formula (I), preferably 2-hydroxy-4-methylselenobutyric acid or a salt thereof, at a concentration of 0.5 and 2000 mg/ Between 1 and preferably between 1 and 1 〇〇〇 mg/1, more preferably between 2 and 500 mg/1, that is, between about 0.2 and 800 mg/1 on a selenium equivalent basis. The compound, preferably between 0.4 and 400 mg/1 on a selenium equivalent basis, more preferably between 〇·8 and 200 mg/1 on a selenium equivalent basis. For microalgae derived from seawater, the compound of formula (I) can be diluted in sterile filtered seawater or in synthetic seawater (for example, manufactured by Aquarium O Systems Inc., the company's "Reef Crystal" media). Minimum necessary culture medium. The method for preparing the microalgae according to the invention may in particular comprise one or more of the following steps: preparing a culture medium containing all the chemical elements required for the growth of the microalgae, preferably a minimum medium; I) a compound, preferably 2-hydroxy-4-methylselenobutyric acid, is introduced into the culture medium as a source of organic selenium; and the pH of the mixture is adjusted to be between 6 and 10; -17-201107478 - The inoculum of the pre-culture of the microalgae cultured in the mixture thus formed is placed at a temperature between 12 and 45 ° C, and may be contained in a gas containing 0 to 20% oxygen and 0.3% to 20% carbon dioxide. Preferably, it is 24 to 120 hours and orbital shaking is performed at 100 to 500 rpm; - the mixture is centrifuged at 4000 and 10 rpm for several minutes, or the mixture is filtered through a 0.2 micron filter and treated with physiological saline. The filter was washed; _ cells were immersed in physiological saline; - centrifuged again at 4000 and 1000 rpm for several minutes; - wet cells containing the selenium-rich microalgae were recovered. The moist cells can be lyophilized or air dried. Other features and advantages of the invention are set forth in the examples which follow. The following examples are provided to illustrate and not to limit the scope of the invention in any way. [Examples] Example 1: Production of selenium-rich Chlorella sp. microalgae in a medium containing 2-hydroxy-4-methylselenobutyric acid (THD-177) under self-operating conditions The strain used is Chlorella sp. SAG211-11B: a parasitic-free strain collected from the SAG of the University of G6ttingen (SAG: Sammlung von Algenkulturen der Universitat Gottingen [Collection of Alga Cultures of the -18-201107478

University of Gottingen]). This strain was cultured in a BG-11 medium (blue-green medium) as described in [Stanley RY et al_1971 Bacteriol. Rev. 35:17, 205], and the composition of the BG-1 1 medium was as follows (per liter):

O o (1 ) NaN03 : 1.5 g ( 2 ) K 2 HP 〇 4 : 0.04 g ( 3 ) MgS04 . 7H20 : 0.075 g ( 4 ) CaCl 2 · 2H20 : 0.036 g (5 ) citric acid : 0.006 g (6 ) ferric citrate Ammonium: 0.006 g (7) EDTA-Na2: 0.02 g (8) Na2C03: 0.02 g (9) 1.0 L of distilled water (10) Solution of trace elements: 1 ml / liter of H3B 〇 3 : 2.86 g

MnCI〗 4H2〇 : 1.81 g ZnS04 · 7H20 : 0.222 g Na2Mo04 . 2H2 : 0.39 g CuS〇4 · 5H2〇 : 0.08 g

Co(N03)2 . 6H20 : 0.05 g The pH was adjusted to 7.1 and the medium was autoclaved at 121 ° C for 15 minutes. This strain was cultured at 25 °C for 24 〇 0 + /-200 Lux under light self-operating conditions for 2 to 7 days and orbital (80 rpm), ODinit66Gnm = 〇.〇5. The strain D66Qnni reached 0.5 in 48 hours. -19- 201107478 The source of organic selenium in culture conditions, namely 2-hydroxy-4-methylselenobutyric acid (THD-177, Tetrahedron S AS, France, CAS: 873660-49-2) is 0.5 based on selenium equivalent. Concentrations between mg/L and 100 ml/L were administered, i.e., 1.25 mg/L and 250 mg/L 2-hydroxy-4-methylselenobutyric acid, respectively. The selenium-containing compound is added only once at the beginning of the culture (ie, between 0.125 mg and 25 mg per 100 ml culture), or several times at regular intervals, at intervals of 6 The culture has been maintained for 2 to 7 weeks between 24 hours. Example 2: In a medium containing 2-hydroxy-4-methylselenobutyric acid (THD-177) (in accordance with an example of the present invention) or in a medium containing sodium selenite (comparative example), in a mixed camp Production of selenium-rich Chlorella microalgae under conditions (light and carbohydrate-glucose in medium). In these tests, the strain used was Chlorella sp. SAG21 1-11 B strain: one from the University of Gdttngen Parasitic-free strain collected by SAG (SAG: Sammlung von Algenkulturen der

Universitat Gottingen[Collection of Alga Cultures of th

University of Gottingen]) , Quality Culture: Yeast Extract... Beef Extract... Tryptose . . . F 6 S 0 4........ It is under the mixed conditions of 0.33 g 0.33 g 0.66 g 0.66 mg-20- 201107478 glucose........ 3.3 g distilled water........ Add to 1 · Soaring pH is adjusted to 7.2 and the medium is at 121. Press under the pressure for 15 minutes. The source of organic selenium, ie 2-hydroxy-4-methylselenobutyric acid (THD-177 'Tetrahedron SAS, France, CAS: 873660-49-2) was cast at a concentration of 20 mg/l on a selenium equivalent basis. To this, 50 mg/L of 2-hydroxy-4-methylselenobutyric acid was administered. The source of inorganic selenium (NaSe, sodium selenite) was administered at a concentration of 20 mg/liter on the basis of selenium equivalent, that is, 43.9 mg/liter of sodium selenite was administered. The selenium-containing organic compound was added only once in the exponential growth phase of the Chlorella strain (i.e., 3 days after inoculation). Preparation of sample for analysis After 7 days of inoculation, the medium was filtered through a 0.2 μm Nalgene film (Ref a-PES, diameter 90 mm), and the cell retentate was washed with physiological saline. Wet cell mass was lyophilized for analysis of selenium components (total selenium, selenium methionine and selenosine). Analysis of Selenium Composition of Chlorella After the mineralization of the sample, total selenium was analyzed by coupling to mass detection ICP. After enzymatic digestion of the sample according to the method described by Lobinsky el al., Mester, Z. et al. (2006) Annal. B io an al · C h em. 3 8 5 : 1 6 8 - 1 80 Selenium species formation is achieved by coupling to the tandem mass detection of the high-21 - 201107478 efficiency liquid chromatography. Results Table 1 below indicates the average enthalpy obtained by repeating the vaccination time three times on the basis of the selenium equivalent. Table 1 Selenium component analysis of Chlorella sp. microalgae Total selenium mg selenium / kg Biomass selenium methionine mg selenium / kg biomass selenium (IV) mg selenium / kg biomass added THD177 20 mg selenium / l 1293 ± 23 1274±109 (98.5% of total selenium) 6±1 (0.4% of total selenium) Add NaSe 20 mg selenium/liter 144+5 29+2 (20% of total selenium) 4.1±0.4 (2.7% of total selenium) For the same dose of selenium (in this case 20 mg Se/Ι) added in the form of THD177 or NaSe, the results obtained show that: the addition in the form of THF177 is 9 times more than the addition in the form of NaSe. Selenium was detected; - the concentration of selenium accumulated in the cell in the form of selenium methionine (obtained by the addition of THD177) is 44 times higher than that obtained by the addition of the NaSe form; - if the addition is THD 1 In the form of 77, the concentration of selenium accumulated in the cell in the form of selenium methionine is substantially 100% (98.5%) of the intracellular selenium compound type: in comparison with the concentration of 20% using NaSe; If the addition is THD 177, only 0.4% of the total selenium is detected in S e -22- 201107478 (IV); In the case of NaSe detected 2.7% of Se (IV) in the total selenium. Example 3: Under self-operating conditions in a medium containing 2-hydroxy-4-methylselenobutyric acid (THD1 77) (in accordance with an example of the invention) or in a medium containing sodium selenite (comparative example) Production of selenium-rich Spirulina platensis microalgae under the self-operating conditions in the following medium to culture S. serrata (FLAMANT VERT®, 18000 Bourges, the standard strain of the blunt-tip snail bath)

France - Similar results were obtained using other strains 3054-E0001): Yeast extracts... 0.33 grams of beef extract... 0.33 grams of colored sugar (tryptose) · . . . 0_66 grams of F e S Ο 4......... 0.66 mg of distilled water........ The pH was adjusted to 7.2 to 1.0 liter and the medium was autoclaved at 121 ° C for 15 minutes. The source of organic selenium, ie 2 -transmethyl-4 -methylselenobutyric acid (THD-177, Tetrahedron S AS, France, CAS : 8 73 660-49-2 ) is 25 mg / liter based on selenium equivalent The concentration was administered, i.e., 62. 5 mg/L of 2-hydroxy-4-methylselenobutyric acid was administered. The source of inorganic selenium (NaSe, sodium selenite) was administered at a concentration of 25 mg/liter on the basis of selenium equivalent, that is, 54.4 mg/liter of sodium selenite was administered. The selenium-containing organic compound was added immediately after inoculation with S. sinensis strain (also -23-201107478, ie, τ = ο). Preparation of sample for analysis After 10 days of inoculation, nine cells of the cells were filtered through a 0.2 μm Nalgene membrane, and the cell retentate was washed with physiological saline. The wet cell mass was lyophilized for analysis of the selenium components (total sputum, methionine and sodium selenite). Selenium Component Analysis of Spirulina platensis After the mineralization of the sample, total selenium was analyzed by coupling to mass detection ICP. After enzymatic digestion of the sample according to the method described by Lobinsky el al., Mester, Z. et al. (2006) Annal. Bioanal. Chem. 385: 168-180, selenium species formation by coupling to tandem Mass detection is performed by high efficiency liquid chromatography. Results Table 2 below indicates the average enthalpy obtained by repeating the inoculation time of 1 day on the basis of selenium equivalents three times. Table 2 Selenium component analysis of Spirulina platensis microalgae Total selenium mg selenium/kg biomass selenium methionine mg selenium/kg biomass selenium (IV) mg selenium/kg biomass added THD177 25 mg selenium/liter 1431 ±68 1402147 (98% of total selenium) 17.2±0.7 (1.2% of total selenium) Add NaSe 25 mg selenium/liter 177+2 13±3 (7% of total selenium) 5.1 soil 0.3 (2.9% of total selenium) -24 - 201107478 For the same dose of selenium added in the form of THD177 or NaSe (in this case 2 5 mg S e /1 ), the results obtained show that: - the addition in the form of THF 177 is carried out in the form of NaSe The addition of 8 times more total selenium was detected; - the concentration of selenium accumulated in the cell in the form of selenium methionine (the one obtained by the addition of THD177) was 108 times higher than that obtained by the addition of the NaSe form; If the addition is carried out in the form of THD 177, the concentration of selenium accumulated in the cell in the form of selenium methionine reaches 98% of the compound type of selenium in the cell; compared with 7% in the case of NaSe Concentration; - If the addition is THD177, only 1.2% of Se (IV ) in total selenium is detected: but NaS is added In the case of e, 2.9% of Se (IV) was detected in total selenium. Example 4: Production of selenium-rich Spirulina platensis microalgae in a medium containing 2-hydroxy-4-methylselenobutyric acid (THD177) (in accordance with an example of the invention). In these tests, the strain used was Spirulina platensis strain (FLAMANT VERT®, 1 8000 Bourges, France). Comparing the results between the previous examples (Example 3) and the new experiments: - During the example 3, the addition of the selenium-containing THD-177 compound according to the invention was carried out once after inoculation with the S. serrata strain; In the new experiment, the addition of the selenium-containing THD-177 compound according to the present invention was carried out as in Example 2 in the exponential phase of the cultivation of S. serrata. The results are as follows. Table 3 below indicates on the basis of selenium equivalents. The average enthalpy obtained was repeated three times for the 10 day vaccination time. Table 3 Selenium component analysis of Spirulina platensis microalgae Selenium component analysis of total algae selenium/kg biomass selenium methionine/kg biomass THD177 mg selenium/kg biomass selenium IV) mg selenium/kg biomass THD177 25 mg selenium/liter 1431±68 1402±47 (98% of total selenium) 5±1 (0.35% of total selenium) 17.2±0.7 (1.2% of total selenium) In the exponential phase, add THD177 25 mg selenium / liter 1274 ± 16 1078 ± 89 (85% of total selenium) 11 ± 2 (0.86% of total selenium) 14 ± 2 (1.1% of total selenium) Results show: During the "additional period of growth" test (more specifically during the expiration period), in the "addition at vaccination" test, get 12% more total selenium and 30% more selenium methionine type selenium. This difference may be the result of a longer contact time between the biomass and the THD-177 in the "add at inoculation" test compared to the one in the other test ("added in the exponential phase"). In both cases, the concentration of selenium (IV) in the cells remained at a low concentration of 1% of total selenium. -26-

Claims (1)

201107478 VII. Patent application scope: 1. A method for enriching photosynthetic microorganisms with organic selenium, characterized in that the photosynthetic microorganism is cultured in a medium containing a selenohydroxy acid type compound, and the selenoic acid type compound grows in the microorganism The medium is introduced into the culture medium during the process. 2. The method of claim 1, wherein the cultivation is carried out under self-operating conditions. 3. The method of claim 2, wherein the photosynthetic microorganism is selected from the group consisting of cyanobacteria and green algae, preferably selected from the group consisting of Chlorella spp. and Spirulina or Arthrospira. The ethnic group formed by cyanobacteria. 4. The method of claim 1, wherein the cultivation is carried out under mixedotrophic conditions. 5. The method of claim 4, wherein the photosynthetic microorganism is selected from the group consisting of green algae, preferably a group formed by green algae of the genus Chlorella. 〇 ^ 6. The method of claim 1, wherein the cultivation is carried out under heterotrophic conditions. 7. The method of claim 1, wherein the selenoic acid type compound is of the formula (I) as defined below, or a precursor, a salt or an ester or a guanamine derivative of the compound: Wherein: -27- 201107478 η shows 0, 1 or 2; RJOH, OCOR3, 0Ρ03Η2, 0P03R4R5 or OR6 group; R2 is OH, R3, NHR7, S-cysteamine sulfhydryl or S-glutathione group It is understood that when n=l and R2S〇H, I cannot be OH; R3 is an alkoxy group, an S-cysteinyl sulfhydryl group or an S-glutathione group, or a group selected from the group consisting of group: OR4 is a (C^-Cn) alkoxy group, or a group selected from the group consisting of: -28- 201107478 〇r6 is propionate, lactate, citrate, fumarate, methacrylate, nitrite, palmitate, stearate, palmitolate, oleate or linoleate, natural Fatty acid group or 13-cis-retinylate (reti η 〇ate ) group; -29- 201107478 R7 is hydrazine or ((^-(:26)alkyl group, natural amino acid or natural amine 〇 8. The method of claim 7, wherein the compound of formula (I) is selected from the group consisting of: L-2-hydroxy-4-methylselenobutyric acid, _D-2-hydroxy-4-methyl selenium Butyric acid, DL-2-hydroxy-4-methylselenobutyric acid, or a salt of these compounds. 9. The method of claim 8, wherein the compound of formula (I) is a calcium salt, a zinc salt or The method of any one of claims 1 to 9, wherein the photosynthetic microorganism is selected from the group consisting of cyanobacteria and green algae, preferably selected from the group consisting of Chlorella spp. a group formed by cyanobacteria or cyanobacteria of the genus Arthrospira 11. A photosynthetic microorganism rich in organic selenium, which can be patented as before A method of any of the scopes is obtained. 12. The photosynthetic microorganism characterized by the selenium methionine form of the microorganism, as compared to the photosynthetic organism in the selenium methionine form of the microorganism, as claimed in claim 11 The total selenium present in the microorganism is more than 50% by mass, preferably more than 7% by mass, more preferably more than 80% and even more preferably more than 90% by mass of selenium. A green algae containing selenium, characterized in that the selenium methionine form has an organic selenium content of more than 5%, more preferably more than 70%, more preferably more than 70%, more preferably 80% and even more preferably more than 90%. -30- 201107478 14. A green algae rich in organic selenium, characterized in that the content of organic selenium in the form of selenium methionine is greater than 200# by dry weight gSe/g' is preferably greater than 500 y gSe/g, and more preferably greater than 1000//gSe/g. 15. The organic selenium-rich green algae according to claim 13 or 14 of the patent application, The residual inorganic selenium content is less than 2% by mass, preferably less than 1.5% by mass, and more preferably less than the total selenium contained therein. 1% by mass. 16. The organic selenium-rich green algae according to claim 13 or 14 of the patent, the residual inorganic selenium content of which is less than 0.5 mass by dry weight relative to the total biomass of the green algae %, preferably less than 0.2% by mass, more preferably less than 0.1% by mass of inorganic selenium. 17. An organic selenium-rich cyanobacterium characterized by selenium methionine in the form of organic selenium The content is more than 70%, more preferably more than 80%, and even more preferably more than 90% of the total mass of selenium contained therein. It is characterized in that the selenium methionine form has an organic selenium content of more than 200 / z gSe / g ', preferably more than 500 / z gSe / g, and more preferably more than 1000 / z gSe / on a dry weight basis g. 19. The residual inorganic selenium content of the organic selenium-rich cyanobacteria as claimed in claim 17 or 18 is less than 2% by mass, preferably less than 1.25, relative to the total selenium contained therein. % by mass, more preferably less than 1% by mass. 2 0. If the organic selenium-rich green algae of claim No. 17 or 18 of the patent application, the residual inorganic selenium content of the cyanobacteria is less than 〇.5 by dry weight. %, preferably less than 2% by mass, more preferably less than 0.1% by mass of inorganic selenium. -31 - 201107478 2 1. A cyanobacteria as claimed in claim 17 or 18, belonging to the genus Spirulina or Arthrospira. 22. The use of an organic selenium-rich photosynthetic microorganism as claimed in any one of claims 11 to 21 as a cosmetic, pharmaceutical and nutraceutical. A composition comprising at least one organic selenium-rich photosynthetic microorganism according to any one of claims 11 to 21. 2 4 . The composition of claim 23, wherein the composition is a cosmetic, pharmaceutical or nutritional product. -32- 201107478 IV. Designated representative map: (1) The representative representative of the case is: None. (2) A brief description of the symbol of the representative figure: None ❹ 201107478 V. If there is a chemical formula in this case, please disclose the chemical formula that best shows the characteristics of the invention: none -4-