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    • 专利摘要:
    The invention relates to a method for culturing a protist of the genus Aurantiochytrium mangrovei. The genus is characterized genetically and by its lipid profile. The method makes it possible to obtain a high yield of biomass and an enrichment of the protists thus cultivated with lipids and more particularly with docosahexaenoic acid (DHA). The invention relates to the development of a culture medium for the high-density cell production of a protist of the genus Aurantiochytrium mangrovei rich in DHA. The medium is chemically defined with reduced sodium (Na +) and chloride (Cl-) content.
    公开号:FR3019559A1
    申请号:FR1452960
    申请日:2014-04-03
    公开日:2015-10-09
    发明作者:Pierre Calleja；Julien Pagliardini；Olivier Cagnac；Francois Godart
    申请人:Fermentalg SA；
    IPC主号:
    专利说明:

    [0001] The invention relates to a method for culturing Thraustochytrid cells of the genus Aurantiochytrium mangrovei. The method makes it possible to obtain a high yield of biomass and an enrichment of the protists thus cultivated with lipids and more particularly with docosahexaenoic acid (DHA). The invention relates to the development of a culture method for the high-density production of Thraustochytrides rich in DHA on chemically defined medium reduced sodium (Na +) and chloride (CI-). The invention relates only to Thraustochytrids belonging to the genus Aurantiochytrium. This genus is delimited by a genetic, metabolic and physiological characterization. The cultivation process, by which the cells are cultured without significant addition of sodium ions (Na +) or chloride (CI-), allows high cell density production - about 125 to 140 g / L of dry matter - of Aurantiochytrium mangrovei. This biomass is rich in DHA, with a level of 15 to 20 g / l of culture, preferably 20 to 25 g / l of culture, or even 25 to 30 g / l of culture. Very small amounts of sodium chloride are present in the culture medium required for this process, specifically, very small amounts of chloride ions (CI -) and sodium (Na +). Thus, in the culture medium, there is less than 1 g / l, preferably less than 0.5 g / l, more preferably less than 0.2 g / l of chloride ions, and less than 100 mg / L, preferably less than 50 mg / L and more preferably less than 6 mg / L of sodium ions (Na +). This makes it possible to overcome the additional investment costs required for the equipment in contact with the environment and to substantially reduce the costs of effluent treatment, to overcome the disadvantages associated with the presence of sodium salt or chloride in the process. biomass and reduce the cost of the environment by decreasing inputs (products added to the crop to improve yield). Preamble The protists are currently the subject of many industrial projects because some species are able to accumulate or secrete significant amounts of lipids, including polyunsaturated fatty acids. Among the polyunsaturated fatty acids, some highly unsaturated (AGNI) omega-3 series (PUFA-w3), particularly eicosapentaenoic acid (EPA or C20: 5 w3) and docosahexaenoic acid (DHA or C22: 6 w3), and of the omega-6 series (PUFA-w6), in particular, arachidonic acid (ARA or AA or eicosatetraenoic acid C20: 4 w6), have a recognized nutritional importance and have high potential in term therapeutic applications. Considered an essential nutrient, DHA is necessary for the normal and functional development of cells, and plays a crucial role in various biochemical processes and functions. It is essential for the development of the central nervous system and retinal function, by incorporation into cell membranes, and plays a vital role in the satisfactory acquisition and maintenance of the mechanisms of vision and memory. Thraustochytrids, in particular Aurantiochytrium, are known to produce DHA when grown in heterotrophy [W.K. Hong et al. (2011); Production of lipids containing high levels of docosahexaenoic acid by a newly isolated microalga, Aurantiochytrium sp. KRS101. Appl. Biochem. Biotechnol .: 164 (8): 1468-80]. Aurantiochytrium is also known to produce carotenoids, such as astaxanthin, zeaxanthin, canthaxanthin, echinenone, beta-carotene and phoenicoxanthin [Yokoyama, R, Honda, D. (2007) Taxonomic rearrangement of the genus Schizochytrium sensu lato based on morphology, chemotaxonomic characteristics, and 18S rRNA gene phylogeny (Thraustochytriaceae, Labyrinthulomycetes): emendation for Schizochytrium and erection of Aurantiochytrium and Oblongichytrium gen. Nov.; Mycoscience, Vol. 48, pp. 199-211]. To implement the production of fatty acids by protists on an industrial scale, several factors must be taken into account to make production profitable. These factors include: - the costs of raw materials and equipment (their purchase or lease and maintenance), as well as labor; - the technical requirements of the production: for example, the number and the technical difficulty of the pre-culture and cultivation stages, the online monitoring of the crops, and the stages of treatment of the biomass resulting from the culture to valorize the product ; - the treatment of effluents from the culture. Culture media currently used to grow protists of the family Thraustochytrides heterotrophy or mixotrophy, contain significant amounts of salt, especially sodium chloride. By way of example, mention may be made of the ATCC medium medium No. 790 (11 g / L Na + and 19 g / L). The use of sodium chloride (NaCl) for the cultivation of marine protists of the family Thraustochytrids in oil production processes and / or other molecules of interest entails significant additional costs in terms of investment, treatment of effluents and limits the recovery of co-products, since chloride ions cause degradation of the steel stainless steel, a material used for the production of fermenters, tools for the preparation and sterilization of the culture medium and other equipment for the cultivation of microalgae and the treatment of the resulting biomass.One of the consequences of this phenomenon is the early degradation of the tools of production and processing of biomass ("DownStream Processing" or "DSP" in English) .To avoid this problem of equipment degradation, it is possible to use alloys of particular alloys more resistant to chloride ions. These more salt-resistant materials are more expensive. In this case, the investment costs for production are substantially increased. Furthermore, the use of sodium chloride (or other sodium salts, for example sodium sulfate, sodium carbonate) causes significant additional costs in terms of effluent treatment, in particular the desalination of water . Finally, the presence of sodium salts in the oil-cake-type co-products constituted by the remaining biomass after extraction of the oil hampers their recovery, especially for animal nutrition, aquaculture, or as an ingredient for cosmetics or in the pharmaceutical industry. No. 5,518,918 describes the replacement of sodium chloride by other types of sodium salts (sodium sulphate, sodium carbonate, etc.). Even if this makes it possible to overcome the early wear of stainless steel equipment, the addition of sodium salts to the medium does not make it possible to overcome the additional cost of effluent treatment or problems of recovery of co-products. indicated above. In addition, the replacement of NaCI with another sodium salt entails an additional cost linked to the purchase of the substitute salt. In the article "Optimization of docosahexaenoic acid production by Schizochytrium limacinum SR21" of Yokochi et al. [(1998) 301 9 5 5 9 Appl. Microbiol. Flight. 49, pp. 72-76], the tolerance of the strain Schizochytrium limacinum SR21 for salt conditions was studied. The strain had a high tolerance for high salt concentrations, the concentration being between 50% and 200% of that of seawater. The growth of the strain in the salt-free culture was half as great as in the The culture medium used in this study also included 3% glucose and 1% yeast extract. However, the addition of yeast extract represents an additional cost for the medium, and also a disadvantage compared to the quality of the final biomass for its use as a food or pharmaceutical product. Yeast extracts are not standardized products and therefore lots of yeast extracts are not similar to each other. This has an impact on the standardization of end products derived from the biomass of a culture medium containing yeast extracts. Shabala et al. ["Osmotic adjustment and requirement for sodium in marine protist thraustochytrid" (2009) Environmental Microbiology Vol. 11 (7), pp. 1835-1843] demonstrated that Thraustochytrium can grow in reduced-sodium (1 mM) culture medium, provided that the medium is supplemented with a compound such as mannitol or sucrose, which allows the osmotic adjustment of this culture medium without sodium. However, the presence of these compounds leads to additional costs associated with the cost of these materials. Moreover, despite the addition of compounds that adjust the osmolarity of the culture medium, biomass yields (200,000 cells per milliliter) obtained without salt and with mannitol remain insufficient for industrial production of DHA. In a recent article by Shabala et al. ["Thraustochytrids can be grown in low-salt media without PUFA production", Marine Biotechnology (2013) 15: 437-444], a mutant was obtained by random UV mutagenesis of Schizochytrium limacinum SR21 - named OUC88 and tested to determine environmental factors that induce a change in the composition of fatty acids. Figure 2 of the article shows that as soon as the salt concentration drops below 0.9%, the biomass and the amount of lipids decrease sharply (less than 20 g / L in biomass and less than 10 g / L in lipids). It is desirable to be able to cultivate Thraustochytrids under optimal conditions to increase the yield of the fatty acid (s) to be produced, while avoiding the problems related to the wear of steel equipment, by reducing the costs of production for fermentation, as well as for the treatment of the resulting biomass. In particular, it is desirable to provide Aurantiochytrium culture methods which make it possible to reduce, or even substantially eliminate, the sodium and chloride ions of the culture media, without the addition of other (s) component (s), which can lead to additional costs related to the treatment of effluents, as well as additional costs related to additional stages of PSD and problems of recovery of end products. In the context of the present invention it is desirable to obtain a yield of biomass and lipids sufficient for industrial production of DHA. It is therefore desirable to obtain concentrations of, for example, greater than 100 g / l of dry matter, preferably greater than 130 g / l of dry matter, more preferably still greater than 150 g / l of dry matter. It is therefore desirable to obtain, for example, more than 40% or even 50% of fatty acids relative to the total weight of the dry matter. It is also desirable to obtain, for example, more than 30% or even 40% or even 50% of DHA in relation to total lipids. Thus, the applicant has succeeded in identifying strains of Aurantiochytrium protists capable of growing in a chemically defined culture medium, substantially free of sodium (Na +) and chloride (CI-), without an organic source such as yeast extract, without osmotic agents such as mannitol or sucrose, as defined in the articles of Shabala et al. (2013) (or other osmotic agents such as sorbitol, polyethylene glycol PEG). These strains, cultured under the conditions of the present invention, make it possible to obtain a high-yield production of biomass (greater than 110 g / l, preferably 120 g / l) and of polyunsaturated fatty acids (greater than 15 g / l). preferably 20 g / L), especially DHA. A strain (FCC 1324), concerned with the invention, representative of the strains of Aurantiochytrium thus isolated and selected, was deposited with the CCAP (Culture Collection of Algae and Protozoa, Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Oban , Argyll PA371QA, Scotland, United Kingdom) on June 21, 2013, according to the provisions of the Budapest Treaty, accession number CCAP 4062/1. Figure 1: Phylogenetic analysis showing the relationships between DNA sequences encoding the small subunit of ribosomal RNA. The sequences were aligned with ClustalW of Mega 5.1. The analysis was conducted using the Maximum Likelihood method. The strains of Thraustochytrids used in this study belong to the genera: Aurantiochytrium mangrovei, Schizochytrium sp, and Schizochytrium aggregatum, Ulkenia visurgensis, Ulkenia sp, Ulkenia profunda, Botryochytrium sp., Botryochytriumradiatum, Parieticytrium sp., Parieticytrium sarkarianum, Aplanochtytrium kerguelense Aplanochtytrium stocchinoi, Oblongichytrium multirudimentale, Oblongichytrium sp. and Phytophthora infestans. Bootstrap values are considered significant if they are greater than 75%. 2: Growth tests in Erlen with FCC-M medium with a reduced content of Na + and Cl "Comparison of the growth of the strains concerned by the invention (in black) and not concerned by the invention (in gray and strokes ) in a culture medium according to one embodiment of the invention The length of the column for each strain represents the optical density (Example 1) Figure 3: Fatty acid profiles of the different strains of Thrautochytrides grown in Erlen (Example 2). (A) and (B): A comparison of the fatty acid profiles between the genus according to the invention, exemplified by Aurantiochytrium mangrovei, of which Aurantiochytrium limacinum are part (first three lines of the panels), and the kind of Schizochytrium sp., Which groups the ATCC strains 20888, as well as strains Aurantiochytrium sp SEK 217 and SEK 209 (last three lines of the panels) .The culture conditions are described in Example 2. FIG. differen Thrautochytrid strains cultured in a bioreactor (Examples 3 and 4). (A) and (B): Lipid profiles of the strains according to the invention. (A) Polyunsaturated fatty acids (PUFAs) expressed as percentages to total PUFAs. (B) Saturated fatty acids expressed as percentages of total saturated fatty acids. Cultures whose pH has been regulated by KOH or NH4OH have been identified by the initials (KOH) or (NH4OH), respectively. The culture conditions are described in Examples 3 and 4. Detailed Description By "strain" is meant not only the natural strains of the genus Aurantiochytrium defined according to the invention, but also the mutants of said natural strains. By "chemically defined culture medium" is meant culture medium in which the content of each element is known, that is to say in the absence of yeast extracts or other complex sources of proteins or other organic materials such as peptone or other complex growth agent having no fixed concentration of each of these components. By "osmotic regulating agent" is meant an agent present in a culture medium that maintains the osmotic pressure in the medium. By "genetic identity" is meant an identity between two DNA sequences, as performed by BLAST-type software. By "co-product" is meant products (other than DHA) that can be recovered in this cultivation process. These co-products include, for example, the biomass cake recovered from the extraction of the oil, or the fraction of the oil remaining after purification of the DHA. The subject of the present invention is therefore a process for culturing certain protists of the type Aurantiochytrium mangrovei and Aurantiochytrium limacinum in heterotrophy in a substantially free sodium (Na +) and chloride (Cl-) organic medium. This culture method makes it possible to obtain high yields of biomass, lipids, and specifically DHA. The strains concerned by the invention have the capacity to grow at high density in chemically defined culture media, without the addition of significant amounts of sodium ions or chloride, and without the addition of osmotic regulatory agents. such as mannitol, sorbitol, sucrose and polyethylene glycol. They are, according to recent phylogenetic classifications, strains of Thraustochytrides of the type Aurantiochytrium mangrovei and Aurantiochytrium limacinum, known to be producing DHA [Yokoyama R, et al. (2007). Taxonomic rearrangement of the genus Ulkenia sensu lato based on morphology, chemotaxonomical characteristics, and 18S rRNA gene phylogeny (Thraustochytriaceae, Labyrinthulomycetes): emendation for Ulkenia and Erection of Botryochytrium, Parietichytrium. Mycoscience. 48 (6) p. 329-341; Yokoyama, R., Honda, D. (2007) Taxonomic rearrangement of the genus Schizochytrium sensu lato based on morphology, chemotaxonomical characteristics and 18S rRNA gene phylogeny (Thraustochytriaceae, Labyrinthulomycetes, stramenopiles): emendation for Schizochytrium and erection of Aurantiochytrium and Oblongichytrium gen. Nov. Mycoscience 48, 199-211; Tsui CK, et al. (2009) Labyrinthulomycetes phylogeny and its implications for the evolutionary loss of chloroplasts and gain of ectoplasmic gliding. Mol Phylogenet Evol. 50 (1): p. 129-40]. It is recalled that in a conventional manner, heterotrophic cultures of these kinds of microalgae were carried out with a seawater-based culture medium, such as that used by the American Type Culture Collection, the ATCC 790 By + medium ( yeast extract 1.0 g, peptone 1.0 g D (+), glucose 5.0 g and seawater 1 liter). The strains are genetically characterized, as well as by their lipid profile. The strains concerned by the invention are characterized by the genetic identity of four of their genes, 18s, actin, tubulin and EF1-alpha, with genes of a strain representative of the strains of the invention, the FCC 1324 strain. strain FCC 1324 is representative of the new strains of Aurantiochytrium thus isolated and selected, and has been deposited with the CCAP (Culture Collection of Algae and Protozoa, Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Oban, Argyll PA371QA, Scotland, Kingdom United Kingdom) on 21 June 2013, according to the provisions of the Budapest Treaty, accession number CCAP 4062/1. Table 1 (a) is a comparison of the sequences of the four genes between the genus Aurantiochytrium mangrovei and the genetically closest genus Schizochytrium sp. as well as other genetically related strains. All strains having sequences having between 91 ° and 100% identity with the CCAP 4062-1 genes, depending on the genes compared, can be considered to be of the genus Aurantiochytrium. A genetic identity of at least 92% to the 18s gene of the CCAP 4062-1 strain characterizes the strains according to the invention. 18s Strains of Schizochytrium sp. Length sequence% identity compared with (1) / 0) SCC Aurantiochytrium_sp_SEK209_AB290574 4062-1 99% 91% 100% 91% Thraustochytriidae_sp_MBIC11093_AB183664 Aurantiochytrium_sp_AB073308 94% 91% 93% 91% Aurantiochytrium_sp_ATCC_PRA276_DQ836628 Schizochytrium_sp_ATCC20888_DQ367050 100% 91% 100% 91% Thraustochytriidae_sp_BURABG162_DQ100295 In rantiochytrium_sp_AB052555 100% 91% Aurantiochytrium_sp_SEK217_AB290572 100% 91% Au rantiochytrium_sp_SEK_218_AB290573 99% 90% Aurantiochytrium strains mangrovei Sequence length ° A) Identity with compared (%) CCAP 4062-1 At rantiochytrium_limacinum_AB022107 77% 96% At rantiochytrium_limacinum_H MO42909 100% 98% Aurantiochytrium_limacinum_SL1101 JN986842 100% 98% Aurantiochytrium_mangrovei DQ367649 100% 99% Aurantiochytrium_ p_BL10 '1821477 100% 98% Aurantiochytrium sp_LY20T2 JX847370 100% 98% Schizochytrium limacinum Oi-JC166 HM042907 100% 99% Thraustochytriiiae_sp_N I CTS1_AY70-5-769 100% 99% Thraustochytriidae_sp_LY2012 JX 847378 83% 98% Schizochytrium_sp_KGS2 KCi97137 77% 98% Aurantiochytrium_sp_LY_1012 PKU M n5 _JX847361 100% 98% Aurantiochytrium_limacinum JR1986J42 100% 98% Schizochytrium_ p_SKA10 UQ248009 99% 98% Aurantiochytrium_sp_SD1 i 100% 99% Actin Strains Sequence length% identity with compared (%) CCAP 4062-1 Aurantiochytrium mangrovei DQ356659 97% 99% Schizochytrium sp. ATCC 20888 DQ356660 98% 95% Thraustochytrium aureum DQ356666 98% 90% Japanochytrium marinum DQ356668 96% 89% Thraustochytriidae sp. # 32 DQ356663 96% 89% Thraustochytrium aggregatum DQ356662 96% 89% Schizochytrium aggregatum DQ356661 86% 92% Thraustochytrium striatum DQ356665 95% 88% Thraustochytriidae sp. RT49 DQ356669 91% 89% EF1 Strains Sequence Length% Identity with Compared (%) CCAP 4062-1 Aurantiochytrium mangrovei 100% 99% Schizochytrium sp. ATCC 20888 100% 94% Schizochytrium aggregatum 100% 87% Thraustochytrium striatum 99% 86% Thraustochytriidae sp. TheI2 91% 87% Thraustochytriidae sp. RT49 100% 84% Tubulin Strains Sequence Length% Identity with Compared (%) CCAP 4062-1 Aurantiochytrium mangrovei DQ323157 100% 97% Schizochytrium sp. ATCC 20111 DQ323158 * 100% 90% Schizochytrium aggregatum DQ323159 100% 87% Japanochytrium marinum DQ323166 99% 87% Thraustochytriidae sp. RT49 DQ323167 99% 87% Thraustochytrium kinnei DQ323165 99% 87% Thraustochytrium striatum DQ323163 99% 86% Thraustochytriidae sp. TheI2 DQ323162 99% 86% Thraustochytriidae sp. # 32 DQ323161 99% 85% * Schizochytrium sp. ATCC 20111 DQ323158 = ATCC 20888 Table 1 (a) Strains having a genetic identity of at least 92% to the 18s gene (SEQ ID NO: 1) are concerned by the invention, and are therefore capable of growing in a medium reduced by sodium and chloride. Applicants have also found that these strains thus defined by their genetic identity for the 18s gene, have a genetic identity of at least 96% for the actin gene (SEQ ID No. 2), of at least 91% for the tubulin gene (SEQ ID NO: 3) and at least 95% for the EF1-alpha gene (SEQ ID NO: 4). These percentages of identity are shown in Table 1 (b). 18s Actin Tubulin EF1-a Percent genetic identity: 92% 96% 91% 95% The strains of the invention have an identity with the genes of strain CCAP 4062-1 of at least: Table 1 (b) The Figure 1 shows the phylogenetic analysis that led to this definition of the strains concerned by the invention.
    [0002] The strains of Thraustochytrids used in this study belong to the genus Aurantiochytrium mangrovei, Schizochytrium sp and Schizochytrium aggregatum. The sequences have been aligned with CLUSTAL W of Mega 5.1. In Figure 1, the numbers at the branch lines are Bootstrap values. A Bootstrap value of 70% is considered as the limit below which it is not necessary to descend so that the branch between two groups remains significant. According to Hillis D.M. and Bull J.J. in their article "An empirical test of bootstrapping and a method for determining confidence in phylogenetic analysis" [(1993) Systematic Biology Vol. 42, pp. 182-192], Bootstrap proportions of more than 70% generally correspond to a probability of at least 95% that the corresponding clade (groups) is real. In Figure 1, this means that the difference between A.mangrovei and Schizochytrium sp. is significant because the node separating the two groups has a value of 100%, and so we are in the presence of two different kinds. These two groups are themselves far removed from the group Schizochytrium aggregatum, as already demonstrated by Yokoyama and Honda [(2007), Taxonomic rearrangement of the genus Schizochytrium sensu lato based on morphology, chemotaxonomic characteristics, and 18S rRNA gene phylogeny (Thraustochytriaceae, Labyrinthulomycetes): emendation for Schizochytrium and erection of Aurantiochytrium and Oblongichytrium gen. Nov.; Mycoscience Vol. 48, pp. 99-211]. In Figure 1, the strains that are concerned by the invention are those of the first group at the top of the Figure and have the name Aurantiochytrium mangrovei.
    [0003] Examples of the strains that are concerned by the invention are the strains Aurantiochytrium sp. SD116 (JX863672), Aurantiochytrium limacinum (AB022107), Aurantiochytrium mangrovei (DQ367049), Aurantiochytrium limacinum SL1101 (JN986842), Aurantiochytrium limacinum (JN986842), Aurantiochytrium sp. LY2012 (JX847370), Aurantiochytrium limacinum (HM042909) and Aurantiochytrium sp. BL10 (FJ821477), in the phylogenetic class classified as identified by the inventors. The numbers in parentheses are accession numbers.
    [0004] Indeed, each of these strains has a percentage identity with the sequences of the strain CCAP 4062/1 respectively of at least 92%, 96%, 91% and 94% to the sequences SEQ NO. 1, SEQ NO. 2, SEQ NO. 3 and SEQ NO. 4.
    [0005] For example, strains of the genus Aurantiochytrium having a genetic identity of 92%, 93%, 94%, 95%, 96%, 97%, 980, 99% or 100% in the sequence SEQ NO. 1 are concerned by the invention. These strains also have a 96%, 97%, 980, 99% or 100% genetic identity to the sequence SEQ NO. 2, a genetic identity of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% to the sequence SEQ NO. 3 and a 95%, 96%, 97%, 98%, 99% or 100% genetic identity to the sequence SEQ NO. 4. Table 1 (a) shows in detail the comparison of the genetic identities of the strains concerned, or not, by the invention with the CCAP 4062/1 strain. It is noted that neither the strain Schizochytrium sp ATCC 20888 (DQ367050) nor the strain Aurantiochytrium sp. SEK 209 (AB290574) do not belong to the strains concerned by the invention. Identity values in SEQ NO. 1 for these strains are 91%. These strains have the ability to grow at high density in a chemically defined culture medium (i.e. in the absence of yeast extract or other protein extracts) having very small amounts of sodium chloride. This medium is characterized in that it comprises less than 3.5 g / l, preferably less than 1 g / l, more preferably less than 10 mg / l of sodium ions and less than 1 g / l. L, preferably less than 0.5 g / l, more preferably less than 0.2 g / l of chloride ions. In Example 1, the applicants carried out Erlen growth tests with strains according to the invention, as well as comparison strains not concerned by the invention, in such a culture medium (see Table 2 (a). ) for the main culture medium). The cultures were carried out in the presence of mineral nitrogen (NH4) 2SO4, and glucose as carbon source, and without addition of organic nitrogen. Thus, Figure 2 illustrates results from his experiences. The results show that all strains of Aurantiochytrium mangrovei have the ability to grow on this medium, unlike the other genera of Thraustochytrids tested, Schizochytrium sp. and Schizochytrium aggregatum. It is found that the strains according to the invention have a genetic identity for the 18s, actin, tubulin and EF1-alpha genes, respectively of at least 92%, 96%, 91% and 95% of the FCC 1324 strain. strains concerned by the invention are also characterized by their lipid profile. The strain CCAP 4062/1 is taken as an example and is representative of the strains according to the invention. Figures 3 (A) and 3 (B) show a comparison of fatty acid profiles between strains of two different genera. The genus according to the invention, illustrated by Aurantiochytrium mangrovei, of which Aurantiochytrium limacinum are part (first three lines of each panel) and the genus Schizochytrium sp. which groups the ATCC strains 20888, as well as the strains Aurantiochytrium sp SEK 217 and SEK 209 (last three lines of each panel). The lipid profile of the genus Aurantiochytrium mangrovei has a majority of DHA (greater than 80% of the total PUFAs) under culture conditions according to one embodiment of the invention, described in Example 2. In Figure 3 (A) it can be seen that the ATCC 20888, Aurantiochytrium sp SEK 217 and SEK 209 strains have a different profile, with more EPA and DPA (n-6) than the strains according to the invention. For the strains according to the invention, the DPA (n-6) represents approximately 20% of the total PUFAs with minor amounts of AA and EPA.
    [0006] Figure 3 (B) shows that ATCC strains 20888, Aurantiochytrium sp SEK 217 and SEK 209 have a different saturated fatty acid profile compared to the strains according to the invention, with less palmitic acid and a larger amount of odd fatty acid C15: 0 and C17: 0. Figure 4 (A) shows the polyunsaturated fatty acids (PUFAs) expressed as percentages relative to the total PUFAs. Figure 4 (B) shows saturated fatty acids expressed as percentages of total saturated fatty acids. Cultures whose pH has been regulated by KOH or NH4OH have been identified by the initials (KOH) or (NH4OH), respectively. The profile remains almost unchanged according to the growing conditions. DHA is the major polyunsaturated fatty acid, at almost 80% of the total unsaturated acids. C16: 0 remains the majority saturated fatty acid, more than 90% of the total saturated acids, under the conditions of culture according to embodiments of the invention, described in Examples 3 and 4. Thus, the inventors have defined the strains concerned by the invention. These strains have the ability to grow at a high density in a chemically defined culture medium without the addition of significant amounts of sodium or chloride.
    [0007] The culture of these strains is generally carried out in heterotrophic mode. An example of a culture medium according to one embodiment of the invention is defined as in Table 2 (a) below. This medium, called FCC-M, contains 0.00038 g / l of sodium (Na +) and 0.437 g / l of chloride (CI-). The medium does not have NaCl.
    [0008] Ingredients Main solution Concentration g / L KCl 0.36 H3B03 0.175 MgSO4.7H20 6.750 CaCl2, 2H2O 0.55 KNO3 0.04667 KH2PQ4.7H2O 0.30940 Na2EDTA, 2H2O 0.003094 ZnSO4.7H2O 0.000073 CoCl2.6H2O 0, 000016 MnC12.4H2O 0.00054 Na2MoO4, 2H2O 0.00000148 Na2SeO3 0.00000173 NiSO4.6H2O 0.00000298 CuSO4.5H2O 0.0000098 EDTA-Fe 0.03 Carbon g / L Glucose 55 Nitrogen g / L (NH4) 2SO4 7 Post Autoclave Vitamins g / L Thiamine 0.008 Vitamin B12 0.00013 Panthothenate 0.0027 Table 2 (a): Culture medium FCC-M.
    [0009] Conventionally, during the culture of the strains according to the invention in bioreactor or fermenter, in heterotrophic conditions, the basic culture medium is supplemented with an addition solution to maintain the growth of microalgae. The carbon substrate, for example glucose, can also be added (addition solution 2). An example of such add-on solutions is given in Table 2 (b). Concentration Addition Solution 1 g / L K2SO4 31.9 MgSO4, 7H2O 25.8 KH2PO4.7H2O 61.38 FeSO4, 7H20 0.61 (NI-14) 2SO4 * 138.24 MnCl2 4H20 0.165 ZnSO4.7H20 0.165 CoCl2. 6H20 0.0016 Na2MoO4, 2H20 0.0016 CuSO4, 5H20 0.11 NiSO4, 6H20 0.086 Na2EDTA, 2H20 1.81 Thiamine 0.49 Vitamin B12 0.008 Panthothenate 0.1656 Addition solution 2 g / L Glucose 750 KH2PO4 6, 4 (NI-14) 2SO4 * 34 Table 2 (b): Example of addition solutions 1 and 2 for fermenter or bioreactor culture.
    [0010] According to one embodiment of the invention, when the culture is carried out in a fermentor or a bioreactor, typically after the addition solutions have been added to maintain the growth of the cells, the final concentration of Na + is approximately 10 mg / ml. L, preferably less than 6 mg / L, and the final concentration of Cl- is about 250 mg / L, preferably less than 200 mg / L. According to another embodiment of the invention, when the culture is carried out in Erlen, the concentration of Na + is less than 5 mg / L, preferably less than 2 mg / L and more preferably less than 1 mg / L and the concentration of Cl- is about 1 g / L, preferably less than 0.750 g / L and more preferably less than 0.5 g / L. In general, during cultivation, additions of an organic carbon substrate are made (see, for example, the addition solution 2 Table 2 (b)), in order to allow the cells to accumulate a high concentration of lipids. Additional substrate (addition solution 2) is added to the culture medium during the culture process to maintain a sufficient concentration. This organic carbon substrate preferably comprises, in pure form or in a mixture: glucose, cellulose derivatives, sucrose and / or glycerol. The concentration of organic substrate is generally between 200 mM and 500 mM. The organic carbon substrate contained in the culture medium may consist of complex molecules or a mixture of substrates. Products resulting from the biotransformation of starch, for example from corn, wheat or potato, in particular starch hydrolysates, which consist of small molecules, constitute, for example, substrates organic carbon adapted to the heterotrophic culture of the protists according to the invention.
    [0011] During cultivation, the pH is between 4 and 8, the temperature between 20 and 30 ° C and the concentration of dissolved oxygen is typically regulated between 5% and 30%. This process has the advantage of increasing the yield of biomass obtained from the culture. It also has the advantage of enriching the protists thus cultivated in polyunsaturated fatty acids, more particularly in docosahexaenoic acid (DHA). According to one embodiment of the invention, a preculture is carried out in a culture medium having a small amount of NaCl, such as FCC-M medium (Table 2 (a)) containing, for example, yeast as a source of nitrogen, and glucose, for example, as a source of carbon. After an incubation period, for example, 48 hours, the cells are centrifuged and the cell pellet rinsed in a culture medium having a small amount of NaCl, such as FCC-M, for example, containing no extract yeast, or any other source of mineral or organic nitrogen. This operation is intended to avoid any Na + intake in the main culture via the presence of yeast extract in the preculture, generally corresponding to 1/100 (v / v) of the culture volume of the main solution.
    [0012] The process according to the invention also comprises the following steps: a) the cultivation, under heterotrophic conditions, of one or more Labyrinthutomycete strains, in particular of the genus Aurantiochytrium having a genetic identity of at least 92% with the sequence SEQ NO. 1 in a chemically defined culture medium having less than 1 g / L, preferably less than 10 mg / L sodium (Na +) and less than 1 g / L, preferably less than 200 mg / L of chloride (CI -), b) a step of maintaining said culture over several generations, c) a step of recovering the biomass thus cultivated.
    [0013] The method may also comprise the additional steps: d) a lipid recovery step of the strains, and optionally, e) the extraction of DHA (docosahexaenoic acid) recovered lipids. The isolated Aurantiochytrium strains according to the invention make it possible to produce significant amounts of biomass as well as lipids, the lipids being rich in DHA. Indeed, the method of the invention under heterotrophic conditions makes it possible to obtain at the end of step b) a biomass concentration greater than 100 g / l, preferably greater than 120 g / l. This biomass generally has 50% to 60% of lipids relative to the weight of the dry matter. DHA generally represents more than 30%, preferably more than 40%, or more preferably more than 50% of the total fatty acids contained in the protists. The protists, according to one embodiment of the invention, can thus have a productivity (quantity of product of interest produced, per liter of culture, per hour) of DHA of at least 0.1 g / L / h, preferably at least 0.2 g / L / h, and more preferably at least 0.3 g / L / h.
    [0014] The strains of step (a) may also have a genetic identity of at least 96% to the sequence SEQ NO. 2, and / or a genetic identity of at least 91% to the sequence SEQ NO. 3 and / or a genetic identity of at least 95% to the sequence SEQ NO. 4. The culture method according to the invention makes it possible to carry out the cultures of these strains of Aurantiochytrium in culture media with a reduced sodium and chloride, without losing productivity and high yields of biomass, lipids and in particular DHA. Thus, the degradation of fermentors and other stainless steel equipment used for culturing cells and the treatment of the resulting biomass, as well as the early degradation by corrosion of biomass production and processing tools ("Down-stream") is avoided. Processing "or" DSP "in English). The invention also relates to the development of a culture medium for the high cell density production of strains according to the invention rich in DHA. The medium is chemically defined with reduced sodium (Na +) and chloride (Cl-) content. The concentration of Na + is generally less than 100 mg / l, preferably less than 50 mg / l and more preferably less than 6 mg / l, and the concentration of Cl- is preferably less than 0.5 g / l. and more preferably less than 200 mg / L.
    [0015] According to one embodiment of the invention, the culture of the strains concerned by the invention is carried out in a culture medium having less than 6 mg / l of sodium ions and less than 200 mg / l of chloride ions. . According to one embodiment, the culture medium is FCC-M (Table 2 (a)). If the culture is carried out in a fermentor or bioreactor, addition solutions will be desirable, as described above (see for example Table 2 (b)). Since the culture media according to the invention are chemically defined, they contain no growth agents, such as yeast extracts or peptones which also contain amounts of sodium chloride or osmotic regulatory agents, such as mannitol or sorbitol. Thus, in the absence of these agents, the biomass and lipids derived from the culture can be used for food products (or pharmaceuticals) without the many steps of DSP necessary to either characterize the content of the final products or eliminate these undesired added agents in end products. This avoids additional costs associated with these additional steps. Similarly, co-products obtained after extraction of the oil can be used for animal feed in the form of cakes, for example.
    [0016] Another advantage of the process of the invention and the medium of the invention is that the effluents from the cultures do not contain agents requiring additional processing steps which entail additional costs and thus reduce the profitability of the production.
    [0017] The method and the medium of the invention make it possible not only to optimize the production of the biomass obtained from the culture, while avoiding the use of the sodium and chloride ions and the problems of associated additional costs, but also to enrich the organisms thus cultivated in polyunsaturated fatty acids. Preferably, the strains are cultured according to the methods referred to above, and then recovered to extract the lipid content, in particular lipids including DHA. Selective lipid extraction methods, including DHA, are known to those skilled in the art and are, for example, described by [Bligh, EG and Dyer, WJ (1959) [A rapid method of total lipid extraction and purification. , Can. J. Biochem. Physiol., 37: 911-917]. Strains according to one embodiment of the invention can thus have a DHA productivity of at least 0.1 g / L / h, preferably at least 0.2 g / L / h, and more preferably at least 0.3 g / L / h. The biomass and / or co-products obtained from the process according to the invention can be used as a product or ingredient in an agri-food or aquaculture product. Example 1 Erlen Growth Tests: The strains listed in Figure 2 were previously cultured in a medium containing reconstituted sea salt, Instant Ocean® at 15 g / l for two days, then centrifuged and washed once with the FCC-M solution (Table 2 (a)), before being inoculated (1/1000 v / v) into Erlens containing 50 ml of FCC-M medium. The optical densities of the cell cultures were measured after 3 days of incubation at 26 ° C. and with stirring (220 rpm). Example 2 Ciras Acid Profiles of Erlen-grown Cells: The strains listed in FIG. 2 were previously cultured in a medium containing reconstituted sea salt, Instant Ocean® at 15 g / l for two days, then centrifuged and washed once with the FCC-M solution (Table 2 (a)), before being inoculated (1/1000 v / v) in the Erlens containing 50 ml of FCC-M medium, in which ammonium sulfate was substituted with yeast extract (4 g / L). The fatty acid profile (methyl esters of fatty acids or "FAME" in English) was determined from cell cultures incubated for 3 days at 26 ° C. and with stirring (220 rpm).
    [0018] Example 3 Growth tests and production of DHA in bioreactor: The Aurantiochytrium cultures were made in fermentors (bioreactors) of 1 to 2 L useful with dedicated automata and supervision by computer station. The system was regulated in pH via addition of base (NaOH, or KOH) and / or acid (sulfuric acid solution). The culture temperature was set at 26 ° C. Stirring was carried out by means of 3 stirring rods placed on the shaft according to the Rushton configuration (three-bladed pumping propellers). The dissolved oxygen pressure was regulated in the medium throughout the culture, the stirring speed (250 - 1200 rpm), the air flow (0.25 - 1 vvm), or even the flow rate of oxygen (0.1 0.5 vvm). The control parameters, integrated in the supervisory automaton, made it possible to maintain a constant p02 between 5% and 30%. The culture time was between 50 and 200 hours, preferably between 65 and 96 hours, for example 72 hours. Preculture was carried out on a stirring table (140 rpm) in a thermostatically controlled enclosure (26 ° C), in FCC-M medium, containing 4 g of yeast extract as a source of nitrogen and 30 g of glucose. as a source of carbon. After 48 hours of incubation, the cells were centrifuged for 5 minutes at 3000 g and the cell pellet was rinsed with FCC-M medium containing no yeast extract or any other mineral or organic nitrogen source. During culture, 3 additions of addition solution 1 were made, as well as additions of solution 2 in order to maintain glucose concentrations of between 200 mM and 500 mM. Crop monitoring: The total biomass concentration was monitored by measuring the dry mass (filter filtration GF / F, Whatman, then drying in an oven at 105 ° C, for 24 hours minimum before weighing). Total lipid content assays and FAMEs were performed according to the methods conventionally described in the literature [Folch J, et al., A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem.
    [0019] 1957 May; 226 (1): 497-509]. Example 4 Fermenter Culture: The cultures of Aurantiochytrium were carried out in fermentors, in a manner similar to that described in Example 3. The modification of the procedure was carried out on the pH regulation mode by addition of ammonia (NH4OH) to avoid the important contribution of Na + or e, related to the regulation of the pH by NaOH or KOH, which could have been embarrassing for the valorization of co-products for animal feed. Part of the nitrogen required for cell culture is provided via pH regulation with ammonia (NH4OH), so it was no longer necessary to include (NH4) 2SO4 in the composition of the solution. addition 1. Table 3 shows the results of this example: Dry Mass Productivity MG / MS% DHA / MG% DHA g / L 8 / I in MS (g / L / h) CCAP 4062-1 FCC-M (KOH) 1.83 40.75 31.92 CCAP 4062-1 FCC-M 127`_! 1.67 50 32.6 20.05 CCAP 4062-1 FCC-M (N1-1.101-1) 17fl 1.566 35.09 38.01 16.01 CCAP 4062-1 Instant Oceanre 13.1.5 1.85 23.00 5.09 10
    权利要求:
    Claims (15)
    [0001]
    REVENDICATIONS1. A process for producing DHA comprising the following step: a) culturing, under heterotrophic conditions, one or more strains of the genus Aurantiochytrium having a genetic identity of at least 92% to the sequence SEQ NO. 1, in a chemically defined culture medium having less than 3.5 g / l, preferably less than 1 g / l, more preferably less than 10 mg / l of sodium ions and less than 1 g / l, of preferably less than 500 mg / L, more preferably 200 mg / L of chloride ions.
    [0002]
    2. Method according to claim 1, characterized in that the strain or strains of the genus Aurantiochytrium has or also has a genetic identity of at least 96% to the sequence SEQ NO. 2, and / or at least 91% to the sequence SEQ NO. 3 and / or at least 95% to the sequence SEQ NO. 4.
    [0003]
    3. Method according to claim 1 or 2, characterized in that the culture medium has less than 6 mg / l of sodium ions and less than 200 mg / l of chloride ions.
    [0004]
    4. Method according to any one of claims 1 to 3, characterized in that the medium does not include an osmotic pressure regulating agent, such as mannitol, sorbitol, polyethylene glycol and sucrose.
    [0005]
    5. Method according to any one of claims 1 to 4, further comprising the steps of: b) maintaining said culture over several generations, c) recovering the biomass thus cultivated, d) recovering the lipids of the strains, and possibly e) extraction of DHA (docosahexaenoic acid).
    [0006]
    6. Method according to claim 5, characterized in that the biomass from step b) is at least 100 g / l, preferably 115 g / l, more preferably 120 g / l dry matter.
    [0007]
    7. The method of claim 5 or 6, characterized in that the concentration of DHA at the end of step b) is at least 15 g / L, preferably 20 g / L.
    [0008]
    8. Method according to any one of claims 5 to 7, characterized in that the DHA contained in the biomass at the end of step b) represents more than 30%, or preferably more than 40%, or more preferentially, more than 50% of the total lipids.
    [0009]
    9. Process according to any one of claims 1 to 8, characterized in that the process has a DHA productivity of at least 0.1 g / Uh, preferably at least 0.2 g / L / h. and more preferably at least 0.3 g / Uh.
    [0010]
    10. Process according to any one of claims 1 to 9, characterized in that the culture medium consists of: Ingredients Concentration (g / L) KCl 0.36 Fi3B03 0.175 MgSO4, 7H20 6.750 CaCl2, 2H20 0.55 KNO3 0 , 04667 KH2PO4, 7H20 0.30940 Na2EDTA, 2H2O 0.003094 ZnSO4.7H2O 0.000073 CoC12.6H2O 0.000016 MnC12.4H2O 0.00054 Na2MoO4, 2H2O 0.00000148 Na2SeO3 0.00000173 NiSO4.6H2O 0.00000298 CuSO4. 5H20 0.0000098 EDTA-Fe 0.03 Glucose 55 (NH4) 2SO4 7 Thiamine 0.008 Vitamin B12 0.00013 Panthothenate 0.0027
    [0011]
    11. Process according to any one of claims 1 to 10, characterized in that said organism of the genus Aurantiochytrium corresponds to the strain FCC 1324, deposited with the CCAP (Culture Collection of Algae and Protozoa), under accession number CCAP 4062/1.
    [0012]
    12. Use of all or part of the biomass and / or co-products obtained from the process according to any one of claims 1 to 11 as a product, or ingredient in an agri-food or aquaculture product.
    [0013]
    13. Culture medium characterized in that it consists of: Ingredients Concentration (g / L) KCl 0.36 H3B03 0.175 MgSO4, 7H20 6.750 CaCl2, 2H20 0.55 KNO3 0.04667 KH2PO4.7H20 0.30940 Na2EDTA, 2H20 0.003094 ZnSO4.7H20 0.000073 CoC12.6H20 0.000016 MnCl2.4H2O 0.00054 Na2MoO4, 2H2O 0.00000148 Na2SeO3 0.00000173 NiSO4.6H2O 0.00000298 CuSO4.5H2O 0.0000098 EDTA-Fe 0.03 Glucose 55 (NH4) 2SO4 7 Thiamine 0.008 Vitamin B12 0.00013 Panthothenate 0.0027
    [0014]
    14. Use of the culture medium of claim 13 for the cultivation of protists for the production of lipids and pigments.
    [0015]
    15. Use of the culture medium of claim 13 for carrying out the method according to any one of claims 1 to 12.
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    US10100340B2|2018-10-16|
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    优先权:
    申请号 | 申请日 | 专利标题
    FR1452960|2014-04-03|
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    US15/301,091| US10100340B2|2014-04-03|2015-04-03|Method for culturing microalgae of the aurantiochytrium genus in a culture medium without chloride and without sodium for the production of DHA|
    MYPI2016703593A| MY178364A|2014-04-03|2015-04-03|Method for culturing microalgae of the genus aurantiochytrium in a culture medium without chloride and without sodium for the production of dha|
    AU2015242493A| AU2015242493A1|2014-04-03|2015-04-03|Method for culturing microalgae of the Aurantiochytrium genus in a culture medium without chloride and without sodium, for the production of DHA|
    UAA201609969A| UA124963C2|2014-04-03|2015-04-03|Method for culturing microalgae of the aurantiochytrium genus in a culture medium without chloride and without sodium for the production of dha|
    AU2019200857A| AU2019200857B2|2014-04-03|2019-02-07|Method for culturing microalgae of the Aurantiochytrium genus in a culture medium without chloride and without sodium, for the production of DHA|
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