法国专利FR3038914A1 THRAUSTOCHYTRIDE BIOMASS, CULTURE METHOD AND USES

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专利摘要:
The invention is in the field of microalgae cultures, particularly Thraustochytrids. It relates to a biomass of Thraustochytrides rich in protein and containing polyunsaturated fatty acids such as DHA, its method of production and its uses.
公开号:FR3038914A1
申请号:FR1556792
申请日:2015-07-17
公开日:2017-01-20
发明作者:Olivier Cagnac；Julien Pagliardini；Pierre Calleja；Adeline Lapendrie
申请人:Fermentalg SA；
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专利说明:

FIELD OF THE INVENTION The invention is in the field of microalgae cultures, particularly Thraustochytrids. It relates to a biomass of Thraustochytrides rich in proteins and lipids, and possibly including carotenoids, its production process and its uses in food.
STATE OF THE ART
Several sources of proteins and sources of plant-derived lipids are known for their use in food or feed, directly or as food supplements, to provide animals and humans with the amino acids necessary for their metabolism. By "protein sources" is meant sources of amino acids available to animals or humans once the food has been ingested. Lipids, in particular polyunsaturated fatty acids (PUFAs), have an interest as a source of energy, as well as for their therapeutic properties. Among the polyunsaturated fatty acids, some highly unsaturated (omega-3) (PUFA-oo3), in particular, eicosapentaenoic acid (EPA or C20: 5 ω3) and docosahexaenoic acid (DFIA or C22: 6 ω3), and of the omega-6 series (PUFA-ωβ), in particular, arachidonic acid (ARA or AA or eicosatetraenoic acid C20: 4 ω6) have a recognized nutritional importance and have high potential in term therapeutic applications. It would therefore be desirable to be able to provide new sources of proteins and PUFAs for their use in animal and human nutrition. Sources which contain proteins as well as lipids, in particular PUFAs, would have considerable advantages.
As sources of protein, fish meal is known. This has the disadvantage of being a source of animal origin whose availability is reduced. This raw material comes from fishing, and its manufacturing processes are polluting and expensive because of applied heat treatment and waste treatment (wastewater, contaminants). Fishmeal is relatively rich in protein but, on the other hand, it is low in lipids (we can mention levels of about 8% in dry matter), since the oils are extracted beforehand for marketing in human and animal nutrition. Lipid profile varies depending on its origin. Fishmeal is now used in animal feed, especially in aquaculture
The best known source of vegetable protein used in animal feed is soy, usually used in the form of cake, solid residue remaining after extraction of the oil. However, the use of soybean meal has several disadvantages associated with their origin. The cakes are generally imported from countries that practice intensive soybean cultivation to the detriment of other biodiversity-producing plants. In addition, many countries promote the cultivation of genetically modified (GMO) soybean varieties, which are mixed with non-GMO soybeans in cakes, which does not meet the growing demand for food products of origin vegetable without GMO. Moreover, soy is not a source of lipids such as PUFAs.
Oilseed by-products from other oilseeds are also sources of protein commonly used in animal feed (rapeseed cake, sunflower, cotton, peanut, etc.) as well as co-products of other agro-industries, particularly starch and starch milling. (maize protein, wheat, peas, potato, rice, etc.). These protein sources are generally low in fat. In addition, the fatty acids constituents contain very little PUFA mainly omega 6 fatty acids whose overconsumption by animals impact the quality of lipids in their products.
These sources, often unfit for human consumption for nutritional reasons (anti nutritional factors) or sensory (taste) can be reworked so that they can be offered as human food in the form of isolates, concentrates, flours, gluten or hydrolysates or peptides. These often highly purified products are very expensive and provide only a few nutrients of interest next to the protein.
There are also new sources of protein from less common seeds (lupine, flax, hemp, chia ...). They remain very confidential because of their low availability and / or high price.
Other sources of vegetable proteins are known, such as spirulina or chlorella, used as dietary supplements in humans.
Spirulina, like chlorella, however, have the disadvantage of a low productivity of protein that does not allow the transition to a high-yielding fermentor culture. Their cultivation does not make it possible to meet the objective of a larger, economically viable industrial production of a food protein source whose qualities will enable it to replace the usual sources such as soya in the diet of animals and humans. . In addition, chlorella and spirulina have a very low content of PUFAs.
Like proteins and lipids, carotenoids are also of interest in animal and human nutrition. Carotenoids are rather orange and yellow, fat-soluble pigments. They are synthesized by all algae, all green plants and by many fungi and bacteria (including cyanobacteria). They are absorbed by animals and humans in their food. Carotenoids have two main absorption peaks around 440 and 475 nm.
Carotenoids are generally used as pigments, but they also have an important role for human and animal health as antioxidants. Finally, they have the ability to stimulate the immune system and prevent certain eye diseases, especially age-related macular degeneration (AMD). Particularly useful molecules of interest are, in particular, pigments, such as lutein, fucoxanthin, astaxanthin, zeaxanthin, canthaxanthin, echinenone, beta-carotene and phoenicoxanthin.
Currently, the pigments mentioned above are produced from vegetable sources. Often, the production of these pigments on a large scale requires intensive labor and large areas of production. For example, lutein is obtained from calendula petals, after a process of extraction and purification, concentration and recrystallization in a crystalline form. This form is difficult to handle and is sold in suspension in corn or sunflower oil. Synthetic chemical production of lutein is much more expensive than the extraction of calendulas. Other existing sources of lutein (crustaceans, egg yolks) have limited availability or relatively low content (corn) to make these sources profitable for lutein production on an industrial scale.
Moreover, today, none of the aforementioned sources of protein or fatty acids contain significant amounts of carotenoids.
New sources of carotenoids, including lutein, fucoxanthin, astaxanthin, zeaxanthin, cantaxanthin, echinenone, beta-carotenes and phoenicoxanthin should therefore be sought in order to meet the growing market demand for these molecules. .
Protists known to meet an industrial production capacity in fermenters, have long been used for the production of fats rich in polyunsaturated fatty acids such as DHA or ΓΕΡΑ, for example WO 97/37032. Some protists are able, under certain conditions, to produce carotenoids. However, the biomasses obtained so far, including after fat extraction, did not contain sufficient levels of protein to allow their use as a source of protein in the diet, at least without additional steps of protein enrichment economically expensive. One of the aims of the invention is to provide a new source of proteins and lipids, in particular PUFAs for animal feed or human feed for a broad, economically viable industrial production objective. This source of proteins and PUFAs can also comprise carotenoids, in particular carotenes (α, β, ε, γ, δ or ζ-carotene, lycopene and phytoene) and xanthophylls (astaxanthin, antheraxanthine, citranaxanthin, cryptoxanthin, canthaxanthin). , diadinoxanthine, diatoxanthin, flavoxanthin, fucoxanthin, lutein, neoxanthine, phoenicoxanthine, rhodoxanthine, rubixanthine, siphonaxanthin, violaxanthin, zeaxanthin). The invention shows that under certain culture conditions, Thraustochytrides, known for their use in the production of oils with high levels of polyunsaturated fatty acids (DHA and EPA, in particular) are microorganisms capable of producing a large quantity of proteins, which can make it a source of food protein substitutable for fishmeal, for animal feed and a high value protein source for human nutrition. The inventors, after numerous experiments, have been able to determine culture conditions which make it possible to obtain a high protein content while keeping a lipid content of at least 20% (relative to the dry matter), of which 25 -60%, preferably 40-60% of PUFAs. They were also able to determine culture conditions to direct cells to the production of carotenoids in addition to proteins and PUFAs.
SUMMARY OF THE INVENTION
Thus, the subject of the invention is primarily a biomass of Thraustochytrides which may comprise, by weight relative to the weight of the dry matter, at least 20% of proteins, preferably at least 30% of proteins, which may be up to more than 40% by weight. % protein, or even more than 60% protein by weight relative to the weight of the dry matter. The biomass may contain, in general, between 35% and 65% of proteins, in particular 45% to 55% of proteins relative to the weight of the dry matter.
The percentages by weight of proteins may be expressed both with respect to the proteins themselves and with respect to the amino acids contained in said proteins. The percentages given here are calculated by the sum of the total amino acids contained in the biomass (ISO 13903: 2005) and not from the total nitrogen. For example, an amino acid analyzer can be used to measure total amino acids. Tryptophan is dosed separately, its value is then added to the sum of the other amino acids to obtain the sum of the total amino acids.
In addition, said biomass may comprise, by weight relative to the weight of the dry matter, at least 20% fat, preferably at least 30% fat. This fat may contain between 25% and 60%, preferably between 40% and 60% of PUFAs, such as TARA, DFIA and / or ΓΕΡΑ. According to one embodiment of the invention, the DFIA may be the preferred PUFA.
According to some embodiments of the invention, said biomass may also comprise between 5 and 250 ppm (ng / mg of dry matter), preferably between 150 and 200 ppm of carotenoids. The invention also relates to a method for producing a biomass as defined above and below, characterized in that it comprises: a. a first step of cultivating Thraustochytrides under conditions of heterotrophy or mixotrophy in a suitable culture medium, at a temperature of between about 24 ° C. and 35 ° C. and under conditions that can promote the production of proteins with a content of less than 35% protein by weight relative to the weight of the dry matter; b. a second step of culture under heterotrophic or mixotrophic conditions at a temperature of between about 18 ° C and 25 ° C and lower than that of step a) under conditions that can promote the accumulation of fat up to at least 20%, preferably at least 30%, and may promote the accumulation of DHA within the fat to at least 25%, preferably at least 35% of the fat to obtaining a culture density of at least 40 g / l in dry matter, preferably at least 60 g / l, more preferably at least 80 g / l; vs. a third step of recovering the biomass obtained in the second step by separating said biomass from the culture medium (the harvest); this step may be followed by homogenisation and, where appropriate, d. a fourth step of drying the biomass recovered in the third step.
The cultivation time may be, generally, between 40 and 100 hours, preferably between 65 and 96 hours, for example, 70, 84 or 96 hours. The culture method which is the subject of the invention can generally make it possible to attain a biomass concentration of greater than 100 g / l or even 120 g / l.
In the first step a), the cells can be cultured for about 18-30 hours, preferably for 24 hours. After this first culture step, for example, from about 24 hours of culture, the temperature can be lowered to about 18-24 ° C (to start step b)). This drop in temperature can promote the production of fatty acids and possibly carotenoids. The low temperature culture can generally be continued up to between about 50 and 70 hours of cultivation, and in any case preferably until the end of the cultivation.
Carotenoid production may be induced during the cultivation, preferably during step b). For this purpose, mixotrophic conditions with a light, preferably blue, at about 435-475 nm, preferably 455 nm, can be applied. Carotenoids such as astaxanthin, canthaxanthin, beta-carotene and phoenicoxanthin can be produced with these wavelengths. The light can be generally applied for at least 18 hours, preferably for at least 24 hours.
According to a preferred embodiment of the invention, the light can be applied to the culture from 24 to 50 hours, more preferably from 45 hours of culture and until the end of the culture. The invention also relates to the use of biomass as above or hereafter, in the fields of human or animal nutrition, especially in aquaculture. The invention also relates to the use of biomass as above or hereafter, for improving the performance of animals. Said performance can be evaluated by measuring the consumption index, the weight gain or the "Feed Conversion Ratio", all known to those skilled in the art. The invention also relates to a food comprising such a biomass that allows both to increase its value and its nutritional density. The invention also relates to the biomass according to the invention for its use in therapy, particularly in the treatment and in the prevention of malnutrition.
It also relates to cosmetic or pharmaceutical compositions for humans or animals and foods, or food compositions for humans or animals, which comprise a biomass according to the invention.
DETAILED DESCRIPTION OF THE INVENTION
By "mutants" is meant an organism derived from the original strain whose genetic heritage has been modified either by a natural process or by physico-chemical methods known to those skilled in the art that can generate random mutations (UV etc.), or by genetic engineering methods.
By "mixotrophic conditions" or "in mixotrophic mode" is meant a culture with a light supply and a contribution of organic carbon substrate and in the dark.
By "predominantly heterotrophic mixotrophy conditions" are meant illumination conditions in which the bulk of the energy for cell growth is provided by a source of organic carbon, but in which light has an impact on the growth and / or cell composition. This light can be provided either continuously, with or without intensity variations, or discontinuously with periods of darkness between the illumination periods, as there is an effect on the metabolism of cells exposed to light. said illumination. The periodicity of the variation of the intensity or discontinuities can be regular over time, or can be modified with the different phases of cell growth. The predominantly heterotrophic mixotrophy makes it possible to produce molecules of interest by coupling the advantages of autotrophy and heterotrophy at the same time. Under these conditions, as in heterotrophy, the organic substrate feeds the microalgae to produce large amounts of biomass, but the chloroplast and other organelles light energy sensors of the cell are activated. Light acts as a signal that can induce a response of the metabolism, for example the synthesis of pigments.
The term "carotenoid" includes carotenes (α, β, ε, γ, δ or ζ-carotene, lycopene and phytoene) and xanthophylls (astaxanthin, antheraxanthine, citranaxanthin, cryptoxanthin, canthaxanthin, diadinoxanthine, diatoxanthin, flavoxanthin, fucoxanthin, lutein, neoxanthine, rhodoxanthine, rubixanthine, siphonaxanthin, violaxanthin, zeaxanthin).
The term "biomass" according to the invention is advantageously understood to mean a set of Thraustochytrid cells produced by the cultivation of said protists, and having the protein, fatty acid and possibly carotenoid contents described in the present text, which cells may to have preserved their physical integrity or not.
It is therefore understood that said biomass may comprise an amount of degraded Thraustochytrid cells ranging from 0% to 100%. By "degraded" is meant that said Thraustochytrid cells may have had their structure and / or composition modified. For example, they may have undergone a drying step or a step of harvesting their oil, the important thing being that the biomass comprising these cells has the protein, fatty acid and possibly carotenoid contents described herein. text.
According to a preferred embodiment of the invention, the biomass may not have undergone treatments that modify its amino acid composition during or after harvest, ie the treatments that this biomass can undergo after harvesting do not occur. do not alter the amino acid composition. In particular, the biomass may not undergo a protein enrichment step and amino acids.
According to a more preferred embodiment of the invention, the biomass has not undergone treatments which modify its composition in amino acids and in fat, and where appropriate in carotenoids, that is to say that the treatments that this biomass undergoes after harvest do not alter the composition of amino acids and fat, and possibly carotenoids. In particular, the relative composition of the amino acids with respect to the fat remains substantially constant.
It has been observed in some cases that non-degraded Thraustochytrides in the biomass according to the invention may have better preservation and digestibility properties than degraded Thraustochytrides. One of the preferred forms of the invention is a biomass comprising a substantially majority of Thraustochytrides which are not degraded.
By "degraded" is meant according to the invention Thraustochytrides whose structural integrity and / or chemical may have been altered such as lysed Thraustochytrides, resulting for example from a homogenization process.
It is nevertheless obvious that once produced, said biomass may be used raw, dried or not, or may undergo any treatment necessary for its use, including homogenized.
According to the invention, said biomass may have, by weight relative to the weight of the dry matter, a moisture content of 1% to 95%.
Preferably according to one embodiment of the invention, said biomass may have, by weight relative to the weight of the dry matter, a moisture content of 70% to 90%, preferably 80% to 85%.
Preferably also according to a second embodiment of the invention, said biomass may have, by weight relative to the weight of the dry matter, a moisture content of 1% to 10%, preferably 2% to 7%.
According to the invention, said thraustochytrids may be of the order of Thraustochytriales, preferably of the subclass of Thraustochytriaceae, even more preferably of a genus that may be chosen from the group comprising the genera Aurantiochytrium, Aplanochytrium, Botryochytrium,
Japonochytrium, Oblongichytrium, Parietichytrium, Schizochytrium, Sicyoidochytrium, Thraustochytrium and Ulkenia.
Thraustochytrids are non-genetically modified microorganisms or genetically modified microorganisms. In the event that genetically modified Thraustochytrids are used, they do not contain genes coding for one or more enzymes that degrade or digest biomass for use as a food.
Advantageously, said Thraustochytrides may be chosen from the species Aplanochytrium kerguelense; Aplanochytrium minuta; Aplanochytrium stocchinoi; Aplanochytrium sp. PR24-1; Aurantiochytrium limacinum;
Aurantiochytrium limacinum AB022107; Aurantiochytrium limacinum HM042909; Aurantiochytrium limacinum JN986842; Aurantiochytrium limacinum SL1101 JN986842; Aurantiochytrium mangrovei; Aurantiochytrium mangrovei DQ323157; Aurantiochytrium mangrovei DQ356659; Aurantiochytrium mangrovei DQ367049; Aurantiochytrium mangrovei CCAP 4062/2; Aurantiochytrium mangrovei CCAP 4062/3; Aurantiochytrium mangrovei CCAP 4062/4; Aurantiochytrium mangrovei CCAP 4062/5; Aurantiochytrium mangrovei CCAP 4062/6; Aurantiochytrium mangrovei CCAP 4062/1; Aurantiochytrium sp. AB052555; Aurantiochytrium sp. AB073308; Aurantiochytrium sp. ATCC PRA276 DQ836628; Aurantiochytrium sp. BL10 FJ821477; Aurantiochytrium sp. LY 2012 PKU Mn5 JX847361; Aurantiochytrium sp. LY2012 JX847370; Aurantiochytrium sp. N1-27; Aurantiochytrium sp. SD116; Aurantiochytrium sp. SEK209 AB290574; Aurantiochytrium sp. SEK217 AB290572; Aurantiochytrium sp. SEK 218 AB290573; Aurantiochytrium sp. 18W-13a; Botryochytrium radiatum Botryochytrium radiatum Raghukumar 16; Botryochytrium radiatum SEK353; Botryochytrium sp. ; Botryochytrium sp. BUTRBC 143; Botryochytrium sp. Raghukumar 29; Oblongichytrium minutum; Oblongichytrium multirudimentalis; Oblongichytrium sp. ; Oblongichytrium sp. SEK347; Parieticytrium sarkarianum; Parieticytrium sarkarianum SEK351; Parieticytrium sarkarianum SEK364; Parieticytrium sp. ; Parieticytrium sp. F3-1; Parieticytrium sp. H1-14; Parieticytrium sp. N B RC 102984; Phytophthora infestans; Schizochytrium aggregatum DQ323159; Schizochytrium aggregatum DQ356661; Schizochytrium aggregatum; Schizochytrium limacinum; Schizochytrium limacinum OUC166 HM042907; Schizochytrium mangrovei; Schizochytrium mangrovei FB1; Schizochytrium mangrovei FB3; Schizochytrium mangrovei FB5; Schizochytrium minutum; Schizochytrium sp. ATCC20888 DQ367050; Schizochytrium sp. KGS2 KC297137; Schizochytrium sp. SKA10 JQ248009; Schizochytrium sp. ATCC 20111; Schizochytrium sp. ATCC 20888; Schizochytrium sp. ATCC 20111 DQ323158 *; Schizochytrium sp. ATCC 20888 DQ356660; Schizochytrium sp. ATCC 20889; Schizochytrium sp. ATCC 26185; Schizochytrium sp. BR2.1.2; Schizochytrium sp. BUCAAA 032; Schizochytrium sp. BUCAAA 093;
Schizochytrium sp. B U CAC D 152; Schizochytrium sp. BUCARA 021;
Schizochytrium sp. BUCFIAO 113; Schizochytrium sp. BURABQ 133;
Schizochytrium sp. BURARM 801; Schizochytrium sp. BURARM 802;
Schizochytrium sp. CCAP 4087/3; Schizochytrium sp. CCAP 4087/1;
Schizochytrium sp. CCAP 4087/4; Schizochytrium sp. CCAP 4087/5, Schizochytrium sp. FJU-512; Schizochytrium sp. KFI105; Schizochytrium sp. KK17-3; Schizochytrium sp. KR-5; Schizochytrium sp. PJ10.4; Schizochytrium sp. SE K 210; Schizochytrium sp. SEK 345; Schizochytrium sp. SEK 346; Schizochytrium sp. SR21; Schizochytrium sp. TIO01; Sicyoidochytrium minutum SEK354; Sicyoidochytrium minutum NBRC 102975 Sicyoidochytrium minutum N B RC 102979; Thraustochytriidae sp. BURABG162 DQ100295;
Thraustochytriidae sp. CG9; Thraustochytriidae sp. LY2012 JX847378; Thraustochytriidae sp. MBIC11093 AB 183664; Thraustochytriidae sp. NIOS1 AY705769; Thraustochytriidae sp. # 32 DQ323161; Thraustochytriidae sp. # 32 DQ356663; Thraustochytriidae sp. RT49 DQ323167; Thraustochytriidae sp. RT49 DQ356669; Thraustochytriidae sp. RT49; Thraustochytriidae sp. Thel2 DQ323162; Thraustochytriidae sp. Thel2; Thraustochytrium aggregatum; Thraustochytrium aggregatum DQ356662; Thraustochytrium aureum;
Thraustochytrium aureum DQ356666; Thraustochytrium gaertnerium;
Thraustochytrium kinnei; Thraustochytrium kinnei DQ323165; Thraustochytrium motivum; Multirudimental thraustochytrium; Thraustochytrium pachydermum; Thraustochytrium roseum; Thraustochytrium sp. 13A4.1; Thraustochytrium sp. ATCC 26185; Thraustochytrium sp. BL13; Thraustochytrium sp. BL14; Thraustochytrium sp. BL2; Thraustochytrium sp. BL3; Thraustochytrium sp. BL4; Thraustochytrium sp. BL5; Thraustochytrium sp. BL6; Thraustochytrium sp. BL7;
Thraustochytrium sp. BL8; Thraustochytrium sp. BL9; Thraustochytrium sp. BP3.2.2; Thraustochytrium sp. BP3.3.3; Thraustochytrium sp. caudivorum; Thraustochytrium sp. CHN-1; Thraustochytrium sp. FJN-10; Thraustochytrium sp. HK1; Thraustochytrium sp. HK10; Thraustochytrium sp. HK5; Thraustochytrium sp. HK8; Thraustochytrium sp. HK8a; Thraustochytrium sp. KK17-3; Thraustochytrium sp. KL1; Thraustochytrium sp. KL2; Thraustochytrium sp. KL2a; Thraustochytrium sp. ONC-T18; Thraustochytrium sp. PJA10.2;
Thraustochytrium sp. TR1.4; Thraustochytrium sp. TRR2; Thraustochytrium striatum; Thraustochytrium striatum ATCC24473; Thraustochytrium striatum DQ323163; Thraustochytrium striatum DQ356665; Thraustochytrium visurgense; Uikenia amoeboidea SEK 214; Uikenia profunda; Uikenia profunda BUTRBG 111; Uikenia sp; Uikenia sp. ATCC 28207; Uikenia visurgensis; Uikenia visurgensis BURAAA 141; Uikenia visurgensis ATCC 28208.
Preferentially according to the invention, the Thraustochytrides may be chosen from the genera Aurantiochytrium and Schizochytrium, preferably from the species Aurantiochytrium mangrovei CCAP 4062/2; Aurantiochytrium mangrovei CCAP 4062/3; Aurantiochytrium mangrovei CCAP 4062/4; Aurantiochytrium mangrovei CCAP 4062/5; Aurantiochytrium mangrovei CCAP 4062/6; Aurantiochytrium CCAP 4062/1; Schizochytrium sp. CCAP 4087/3;
Schizochytrium sp. CCAP 4087/1; Schizochytrium sp. CCAP 4087/4; Schizochytrium sp. CCAP 4087/5.
According to preferred embodiments of the invention, the biomass may have a protein content, by weight relative to the weight of the dry matter, of at least 30%, preferably of at least 40%, very preferably of 35% to 55%. According to preferred embodiments of the invention the biomass may have a protein content, by weight relative to the weight of the dry matter of between 35% and 65%.
According to preferred embodiments of the invention, the biomass may have a fat content, by weight relative to the weight of the dry matter, of at least 20%, preferably at least 30%, of which 25% at 60%, preferably 40% to 60% PUFAs. In particular, the ratio DFIA: proteins (in weight relative to the weight of the dry matter) can be between 1: 1.5 and 1: 9, preferably between 1: 2 and 1: 7, more preferably between 1: 5 and 1: 6.
According to some of the preferred embodiments of the invention, the biomass may optionally have a carotenoid content of between 5 and 250 ppm relative to the weight of the dry matter, preferably between 150 and 200 ppm. The carotenoid content may depend on the lighting conditions but also on the culture medium and the conduct of the process. Therefore, the same strain of microalgae can produce different amounts of pigments depending on the culture conditions.
According to preferred embodiments of the invention, said biomass may be a biomass: Thraustochytrides may be chosen from the genera Aurantiochytrium and Schizochytrium, preferentially from the species Aurantiochytrium mangrovei CCAP 4062/2; Aurantiochytrium mangrovei CCAP 4062/3; Aurantiochytrium mangrovei CCAP 4062/4; Aurantiochytrium mangrovei CCAP 4062/5; Aurantiochytrium mangrovei CCAP 4062/6; Aurantiochytrium mangrovei CCAP 4062/1; Schizochytrium sp. CCAP 4087/3; Schizochytrium sp. CCAP 4087/1; Schizochytrium sp. CCAP 4087/4; Schizochytrium sp. CCAP 4087/5; and which may have: a protein content, by weight relative to the weight of the dry matter, of at least 30%, preferably of at least 40%, very preferably of 35% to 55%, or 45% to 55%; a fat content, by weight relative to the weight of the dry matter, of at least 20%, preferably of at least 30%, of which 25 to 60%, preferably 40 to 60% of PUFAs; optionally a carotenoid content of between 5 and 250 ppm relative to the weight of the dry matter, preferably between 150 and 200 ppm; and a moisture content before drying of between 70% and 90%, preferably between 80% and 85% or a moisture content after drying of between 1% and 10%, preferably between 2% and 7%.
The levels and types of carotenoids obtained can be controlled by the choice of the wavelength of the incident light, as well as by the illumination conditions. Carotenoids such as lutein, fucoxanthin, astaxanthin, zeaxanthin, canthaxanthin, echinenone, beta-carotene and phoenicoxanthin may be produced according to embodiments of the invention. To induce the production of the carotenoids selected from astaxanthin, canthaxanthin, beta-carotene and phoenicoxanthin, the culture is exposed to a blue light (about 435-475 nm, preferably 455 nm). According to one embodiment, the carotenoids may have an astaxanthin fat content of between 5% and 30%, a phoenicoxanthin content of between 15% and 30%, and a beta-carotene content between 0% and 30%. . The invention also relates to a method for producing a biomass as described above which comprises: a. a first step of culture in heterotrophy or mixotrophy Thraustochytrides in a suitable culture medium, at a temperature between about 24 ° C and 35 ° C and under conditions that can promote the production of protein at a content of at least 35 % of proteins by weight relative to the weight of the dry matter under conditions; b. a second step of heterotrophic or mixotrophic culture at a temperature between about 18 ° C and 25 ° C and lower than that of step a) under conditions that can promote the accumulation of fat up to at least 20% , preferably at least 30%, and which can promote the accumulation of polyunsaturated fatty acids (PUFAs), preferably DHA, and / or ΓΕΡΑ, and / or TARA, within the fat up to at least 25%, preferably at least 35% of the fat, and optionally promoting the production of carotenoids, until a culture density of at least 40 g / L in dry matter is obtained, preferably at least 60 g / L, more preferably at least 80 g / L; vs. a third step of recovering the biomass obtained in the second step by separating said biomass from the culture medium (the harvest); and, where appropriate, d. a fourth step of drying the biomass recovered in the third step.
Preferably, step a) of Thraustochytrid culture can be carried out in a culture medium and under appropriate conditions to promote the production of proteins.
In general, said appropriate culture medium may be a culture medium known to those skilled in the art for the cultivation of thraustochytrids, adapting it for cells which are not deficient in nitrogen during step a. ). As examples, examples may be made of marine salt-based media supplemented with a carbon source. Mention may be made, for example, of the modified Verduyn type medium (marine salts 15 g / L, (NH 4) 2 SO 4 3 g / L, KH 2 PO 4 1 g / L, MgSO 4 7H 2 O, 0.5 g / L, Na 2 EDTA 24 mg / L, ZnSO 4 -7H20 3 mg / L, MnCl 2-2H 2 O 3 mg / L, Na 2 MoO 4-2H 2 O 0.04 mg / L, FeSO 4 - 7H 2 O 10 mg / L, Pantothenate 3.2 mg / L, Thiamine hydrochloride 9.5 mg / L, vitamin B12 0.15 mg / L). It is also possible to mention culture media of the ATCC790 MB2216 or F / 2 type.
According to one embodiment of the invention, said suitable culture medium may preferably be a chemically defined culture medium which may comprise a carbon source, a nitrogen source, a phosphorus source and salts.
By "chemically defined culture medium" is meant according to the invention a culture medium in which the content of each element is known. Specifically, the invention is directed to a medium that may not contain rich or complex organic materials. Rich or complex organic materials are non-purified organic materials, in the form of mixtures for which the exact composition and concentrations of the various components of the mixture are not known exactly, not controlled, and may exhibit variability. significant from one batch to another. As an example of rich or complex organic material, there may be mentioned yeast extracts or peptones which are products of a protein hydrolysis reaction or rich mineral materials such as, for example, marine mineral salts or other growth agents. complex, having no fixed concentration of each of their components.
According to the invention, said defined medium may comprise salts chosen from calcium, cobalt, manganese, magnesium, zinc, nickel, copper, potassium, iron and sodium salts, and mixtures thereof.
Advantageously, said salts may be chosen from calcium chloride, cobalt chloride, manganese chloride, magnesium sulphate, zinc sulphate, nickel sulphate, copper sulphate, potassium sulphate and sulphate. ferrous, potassium sulphate, sodium molybdate, sodium selenite, sodium chloride, and mixtures thereof.
According to one embodiment of the invention and depending on the strains used, the medium may also comprise sodium chloride (NaCl), in particular for certain strains of marine origin.
According to this embodiment, mention may be made, by way of example, of marine strains capable of admitting a culture medium which may comprise sodium chloride, the strains of Schizochytrium sp., In particular Schizochytrium sp. CCAP 4062/3 and CCAP4087 / 4.
According to another embodiment of the invention and depending on the strains used, the medium may not comprise sodium chloride (NaCl), at least comprise a very small amount of sodium chloride, 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, preferably less than 500 mg / l, more preferably 200 mg / l d chloride ions.
According to this embodiment, mention may be made, by way of example, of strains capable of admitting a culture medium which may not comprise sodium chloride (NaCl), at least to include a very small amount of sodium chloride, the strains of Aurantiochytrium mangrovei, in particular the Aurantiochytrium mangrovei CCAP 4062/4 and CCAP 4062/1 strains.
According to one embodiment of the invention, the carbon source of said defined medium may be a carbohydrate (s), acetate (s), alcohol (s) or complex molecule (s). or any mixture, in any proportion of at least two of these sources.
By any mixture, in any proportion of at least two of these sources, is meant at least one of the following mixtures, in any proportion: a carbohydrate (s) and an acetate (s) , carbohydrate (s) and alcohol (s), carbohydrate (s) and complex molecule (s), (s) acetate (s) and one or more alcohol (s), acetate (s) and complex molecule (s) and alcohol (s) and complex molecule (s), carbohydrate (s) and acetate (s) and alcohol (s), carbohydrate (s) ( s) and one or more acetate (s) and one or more complex molecule (s), carbohydrate (s) and acetate (s) and / or alcohol) and a complex molecule (s).
According to the invention, said nitrogen source of said defined medium may be chosen from nitrate salt (s), glutamate salt (s), salt (s) and / or salt (s). ) ammonium, urea, ammonia or any mixture, in any proportion of at least two of these sources.
By any mixture, in any proportion of at least two of these sources, at least one of the following mixtures is to be understood, in any proportion: a nitrate salt (s) and a salt (or salts) ( s) glutamate and ammonium salt (s) and urea and ammonia; nitrate salt (s) and glutamate salt (s) and ammonium salt (s) and urea; nitrate salt (s) and glutamate salt (s) and ammonium salt (s) and ammonia; nitrate salt (s) and glutamate salt (s) and urea and ammonia; nitrate salt (s) and ammonium salt (s) and urea and ammonia; nitrate salt (s) and glutamate salt (s) and ammonium salt (s); nitrate salt (s) and glutamate salt (s) and urea; nitrate salt (s) and glutamate salt (s) and ammonia; nitrate salt (s) and ammonium salt (s) and urea; nitrate salt (s) and ammonium salt (s) and ammonia; nitrate salt (s) and urea and ammonia; glutamate salt (s) and ammonium salt (s) and urea and ammonia; glutamate salt (s) and ammonium salt (s) and urea; glutamate salt (s) and ammonium salt (s) and ammonia; glutamate salt (s) and urea and ammonia; ammonium salt (s) and urea and ammonia; nitrate salt (s) and glutamate salt (s); nitrate salt (s) and ammonium salt (s); nitrate salt (s) and urea;
According to one embodiment of the invention, the phosphorus source of said defined medium may be chosen from phosphoric acid, phosphate salts, advantageously sodium hydrogen phosphate (Na 2 HPO 4), or sodium dihydrogen phosphate (NaH 2 PO 4) or potassium dihydrogenphosphate (KH2PO4), or potassium hydrogen phosphate (K2HPO4), or any mixture, in any proportion of at least two of these sources.
By any mixture, in any proportion of at least two of these sources, is meant at least one of the following mixtures, in any proportion of phosphoric acid and phosphate salt (s), advantageously one of the following mixtures, in any proportion: phosphoric acid and sodium hydrogenphosphate and sodium dihydrogenphosphate and potassium dihydrogenphosphate and potassium hydrogenphosphate; phosphoric acid and sodium hydrogenphosphate and sodium dihydrogenphosphate and potassium dihydrogenphosphate; phosphoric acid and sodium hydrogen phosphate and sodium dihydrogenphosphate and potassium hydrogenphosphate; phosphoric acid and sodium hydrogenphosphate and potassium dihydrogenphosphate and potassium hydrogenphosphate; phosphoric acid and sodium dihydrogenphosphate and potassium dihydrogenphosphate and potassium hydrogenphosphate; sodium hydrogen phosphate and sodium dihydrogenphosphate and potassium dihydrogenphosphate and potassium hydrogenphosphate; phosphoric acid and sodium hydrogen phosphate and sodium dihydrogenphosphate; phosphoric acid and sodium hydrogenphosphate and potassium dihydrogenphosphate; phosphoric acid and sodium hydrogen phosphate and potassium hydrogen phosphate; phosphoric acid and sodium dihydrogenphosphate and potassium dihydrogenphosphate; phosphoric acid and sodium dihydrogenphosphate and potassium hydrogen phosphate; phosphoric acid and potassium dihydrogen phosphate and potassium hydrogen phosphate; sodium hydrogen phosphate and sodium dihydrogenphosphate and potassium dihydrogenphosphate; sodium hydrogen phosphate and sodium dihydrogenphosphate and potassium hydrogen phosphate; sodium hydrogen phosphate and potassium dihydrogen phosphate and potassium hydrogen phosphate; sodium dihydrogenphosphate and potassium dihydrogenphosphate and potassium hydrogenphosphate; phosphoric acid and sodium hydrogen phosphate; phosphoric acid and sodium dihydrogenphosphate; phosphoric acid and potassium dihydrogen phosphate; phosphoric acid and potassium hydrogen phosphate; sodium hydrogen phosphate and sodium dihydrogenphosphate; sodium hydrogen phosphate and potassium dihydrogenphosphate; sodium hydrogen phosphate and potassium hydrogen phosphate; sodium dihydrogenphosphate and potassium dihydrogenphosphate; sodium dihydrogen phosphate and potassium hydrogen phosphate; potassium dihydrogen phosphate and potassium hydrogen phosphate.
According to one embodiment of the invention, said culture medium may comprise magnesium chloride, advantageously in the form of tetrahydrate (MgCl 2 4H 2 O); calcium chloride, advantageously in the dihydrate form (CaCl 2 2H 2 O); cobalt chloride hexahydrate (CoCl2 6H2O); manganese (II) chloride tetrahydrate (MnCl 24 H 2 O); magnesium sulfate heptahydrate (MgSO4, 7H2O); zinc sulfate heptahydrate (ZnSO4, 7H2O); nickel sulphate hexahydrate (NiSO 4 6H 2 O); copper sulfate pentahydrate (CuSO 4 5H 2 O); potassium sulphate (K2SO4); ferrous sulfate heptahydrate (FeSO4, 7H2O); boric acid (H3BO3); ethylene diamine tetraacetic acid in disodium form dihydrate (Na 2 EDTA, 2H 2 O); sodium molybdate dihydrate, (Na2MoO4, 2H2O); sodium selenite (Na2SeOs); as a vitamin of thiamine, cobalamin or vitamin B12, panthotenate or vitamin B5; a source of carbon; a nitrogen source; a source of phosphorus.
According to a preferred form of the invention, in said culture medium, the magnesium chloride may be at a concentration of between 0.008 and 0.012 g / l, advantageously between 0.009 and 0.011 g / l; the calcium chloride may be at a concentration of between 0.40 and 0.70 g / L, advantageously between 0.50 and 0.60 g / L; the cobalt chloride hexahydrate may be at a concentration of between 0.00008 and 0.00013 g / L, preferably between 0.00009 and 0.00012 g / L; the manganese (II) chloride tetrahydrate can be at a concentration of between 0.008 and 0.013, advantageously between 0.009 and 0.012 g / L; the magnesium sulphate heptahydrate may be at a concentration of between 6 and 10 g / l, advantageously between 7 and 9 g / l; the zinc sulphate heptahydrate may be at a concentration of between 0.008 and 0.013, advantageously 0.009 and 0.012 g / L; the nickel sulfate hexahydrate may be at a concentration of between 0.004 and 0.007 g / L, advantageously 0.005 and 0.006 g / L; the copper sulfate pentahydrate may be at a concentration of between 0.005 and 0.009 g / L, advantageously between 0.006 and 0.008 g / L; the potassium sulphate may be at a concentration of between 0.5 and 3.5 g / l, advantageously between 1 and 3 g / l; the ferrous sulphate heptahydrate may be at a concentration of between 0.03 and 0.05 g / l, advantageously between 0.035 and 0.045 g / l; the boric acid may be at a concentration of between 0.0155 and 0.0195 g / L, preferably between 0.0165 and 0.0185 g / L; the ethylene diamine tetraacetic acid in disodium form dihydrate may be at a concentration of between 0.10 and 0.14 g / L, advantageously between 0.11 and 0.13 g / L; the sodium molybdate dihydrate may be at a concentration of from 0.00001 to 0.0003 g / L, preferably from 0.00005 to 0.0002 g / L; the sodium selenite may be at a concentration of between 0.00000015 and 0.000019 g / L, preferably between 0.00000016 and 0.00000018 g / L; the thiamine may be at a concentration of between 0.015 and 0.05 g / L, advantageously between 0.025 and 0.04 g / L; cobalamin or vitamin B12 may be at a concentration of between 0.0004 and 0.00065 g / L, preferably between 0.00045 and 0.00060 g / L; the panthotenate or vitamin B5 may be at a concentration of between 0.008 and 0.013, advantageously between 0.009 and 0.012 g / L; the carbon source may be at a concentration of between 45 and 65 g / l, advantageously between 50 and 60 g / l; the nitrogen source may be at a concentration of between 7 and 11 g / l, advantageously between 8 and 10 g / l; the phosphorus source may be at a concentration of between 2 and 6 g / l, advantageously between 3 and 5 g / l.
Very preferably according to the invention, in said culture medium the magnesium chloride is at a concentration of 0.0108 g / L, the calcium chloride is at a concentration of 0.55 g / L; cobalt chloride hexahydrate (CoCl 2 6H 2 O) is at a concentration of 0.000108 g / L; the manganese (II) chloride tetrahydrate is at a concentration of 0.0108 g / L; the magnesium sulfate heptahydrate is at a concentration of 8.01 g / L; the zinc sulfate heptahydrate is at a concentration of 0.0108 g / L; nickel sulfate hexahydrate is at a concentration of 0.0056 g / L; copper sulfate pentahydrate is at a concentration of 0.0072 g / L; potassium sulfate is at a concentration of 2.09 g / L; the ferrous sulphate heptahydrate is at a concentration of 0.04 g: I; the boric acid is at a concentration of between 0.0155 and 0.0195 g / L of 0.0175g / L; ethylene diamine tetraacetic acid in disodium form dihydrate is at a concentration of 0.12 g / L; sodium molybdate dihydrate is at a concentration of 0.000108 g / L; sodium selenite, is at a concentration of 0.000000173 g / L; the thiamine is at a concentration of 0.032 g / L; cobalamin or vitamin B12 is at a concentration of 0.00052 g / L; Panthotenate or vitamin B5 is at a concentration of 0.0108 g / L; the carbon source is at a concentration of 55 g / L; the nitrogen source is at a concentration of 9 g / L; the phosphorus source is at a concentration of 4 g / L.
According to the invention, the first step a) of culturing the process can be carried out in discontinuous mode called "batch", in semi-continuous mode called "fed batch" or in continuous feed mode.
According to one embodiment of the invention, when the method is implemented in "fed batch" mode, it may comprise a first step divided into two substeps, a first substep a1) of growth in the medium of suitable culture until a culture density of at least 20 g / l, preferably at least 40 g / l, more preferably at least 60 g / l, more preferably at least 80 g / l, is obtained, followed by a second sub-step a2) of protein production in which one or more carbon source enrichment solution (s), a nitrogen source and a nitrogen source can be added to the culture medium simultaneously or successively. source of phosphorus, so as to maintain in the culture medium a carbon source content of between 5 and 200 g / l, preferably between 10 and 50 g / l, a nitrogen source content of between 0.5 and 5 g / L, preferably between 0.5 and 2 g / L and a content of phosphorus source of between 0.5 and 5 g / l, preferably 0.5 and 2 g / l. In general, the sub-steps a1) and a2) are carried out at a temperature between 24 and 35 ° C, preferably between 25 and 30 ° C.
The second step of process b) generally starts from about 24 hours after the beginning of the culture. It is generally carried out at a temperature between 18 and 25 ° C. This drop in temperature directs production towards polyunsaturated fatty acids of long chains (PUFAs), in particular of DHA, ARA, and EPA, instead of proteins. The profile of fatty acid (s) produced (s) depends, generally, the strain cultured.
During step b) generally, as is done for step a2), one or more carbon source enrichment solution (s) may be added to the culture medium, simultaneously or successively, and / or as a source of nitrogen and / or as a source of phosphorus, so as to maintain a carbon source content in the culture medium of between 5 and 200 g / l, preferably between 10 and 50 g / l, a source content nitrogen content between 0.5 and 5 g / l, preferably between 0.5 and 2 g / l and a phosphorus source content of between 0.5 and 5 g / l, preferably 0.5 and 2 g / L.
The culture is stopped, generally, between 50 and 70 hours after the beginning of the culture.
Carotenoid production may be induced during the cultivation, preferably during step b), but it can be done for the duration of the culture as well. To induce the production of carotenoids, mixotrophic conditions with light, preferably blue, at about 435-475 nm, preferably 455 nm, are applied. The culture is thus in mixotrophic mode. Carotenoids such as astaxanthin, canthaxanthin, beta-carotenes and phoenicoxanthin are produced with these wavelengths. The light is generally applied for at least 18 hours, preferably for at least 24 hours. According to a preferred embodiment of the invention, the light is applied to the culture from 24 to 50 hours, more preferably from 45 hours of culture. This illumination is generally continued, preferably until the end of the culture. It is also possible to stop the illumination shortly before the end of the crop (for example, a quarter of an hour, half an hour, three quarters hours or an hour before the end).
According to some embodiments of the invention the culture is exposed to light, in general, for at least 18 hours, preferably at least 24 hours before the end of the culture. According to one embodiment of the invention, the culture is illuminated from the outset, that is to say during the steps a) and b) of culture. According to a preferred embodiment of the invention, the culture is illuminated from the moment when step b) begins. According to a preferred embodiment of the invention, the culture is illuminated for at least the last 18 hours of culture, preferably for at least the last 24 hours of culture.
White light can be used, but the inventors have found higher carotenoid levels using blue light at a wavelength of about 435-475 nm, preferably 455 nm. In general, the culture is exposed to light from 40 to 50 hours of culture (i.e., from 40 to 50 hours after the start of cultivation), preferably from 45 hours of culture and until the end of the cultivation (between 50 and 70 hours). Under these conditions, a carotenoid content of about 150-250 ppm (ng / mg), preferably 180-200 ppm (dry matter) is obtained. In one embodiment, carotenoids have an astaxanthin content of between 5 and 30%. In one embodiment, carotenoids have a phoenicoxanthin content of between 15 and 30%. According to one embodiment, carotenoids have a beta-carotene content between 0 and 30%.
In general, steps a) and b) of the process can be carried out under heterotrophic conditions and / or under mixotrophic conditions. To obtain carotenoids, in step b) the culture is carried out in a mixotrophy process for at least 24 hours before the end of the cultivation.
According to one embodiment of the invention, for example when no carotenoids are produced, the culture is carried out entirely under heterotrophic conditions. In general, the use of the conditions of mixotrophy during the cultivation has a positive effect on the production of lipids, in particular PUFAs, as well as on the production of carotenoids, and makes it possible to increase the yield in biomass, in fatty acids, especially in PUFAs, in particular in DFIA and carotenoids.
Steps a1), a2) and b) can be performed independently under heterotrophic conditions or under mixotrophic conditions. According to one embodiment of the invention, steps a1) and a2) are carried out under heterotrophic conditions, and then step b), wholly or in part, under mixotrophic conditions. According to a preferred embodiment of the invention, step a1) is conducted under heterotrophic conditions and step a2) under mixotrophic conditions.
According to another embodiment of the invention, during step b) the culture is exposed, under mixotrophic conditions, to a white light. According to another embodiment of the invention, during step b) the culture is exposed, under the conditions of mixotrophy, to a white light, up to 40 to 50, preferably 45 hours of culture, then the culture is exposed to a blue light with a wavelength of about 435-475 nm, preferably 455 nm for at least 24 hours.
According to one embodiment of the invention, in the implementation of a culture in mixotrophic mode, the illumination is variable and / or discontinuous. An illumination in the form of flashes is particularly favorable on the development of protists and increases the productivity of these, particularly with regard to their production of lipids and carotenoids.
"Discontinuous illumination" means an illumination punctuated by periods of darkness. The periods of darkness may occupy more than a quarter of the time, preferably half or more of the time, during which the algae are grown.
According to a preferred aspect of the invention, the illumination is discontinuous and more preferably in the form of flashes. A flash, within the meaning of the invention, is a short period of illumination, that is to say less than 30 minutes. The duration may be less than 15 minutes, preferably less than 5 minutes or more preferably less than 1 minute. According to some embodiments of the invention, the flash duration may be less than one second. For example, the flash duration can be 1/10 of a second, or 2/10 of a second, or 3/10 of a second, or 4/10 of a second, or 5/10 of a second, or 6/10 of a second, or 7/10 of a second, or 8/10 of a second, or 9/10 of a second. The illuminance, or flash, is usually longer than 15 seconds. It is generally between 5 seconds and 10 minutes, preferably between 10 seconds and 2 minutes, more preferably between 20 seconds and 1 minute. These flash times are suitable for "low frequency" light inputs.
According to this last embodiment where the illumination regime (the contribution of light) is "low frequency", the number of flashes can be between about 2 and 3600 per hour. It can be, for example, between 100 and 3600 flashes per hour. It can also be between 120 and 3000, or between 400 and 2500, or between 600 and 2000, or between 800 and 1500 flashes per hour. It may also be between 2 and 200, preferably between 10 and 150, more preferably between 15 and 100, and even more preferably between 20 and 50 per hour.
According to one embodiment, a flash may have a duration between 1/150000 seconds and 1/1000 seconds. These flash times are suitable for "high frequency" illumination regimes; that is with flash rates of 150kHz to 1kHz respectively.
According to this embodiment where the illumination regime (the contribution of light) is "high frequency", the flashes can take place between 3.6 x105 and 5.4 x 109 times per hour. In this case, the variation of the light has a frequency between 1 kHz and 150 kHz, that is to say between 1000 and 150 000 flashes per second. The light input according to this last embodiment of the invention is called "high frequency".
The number of flashes per hour can be chosen according to the intensity and duration of the flashes (see below). In general, the intensity of the light provided in the form of flashes may be between 5 and 1000 μmol.m -2 s -1, preferably between 5 and 500 pmol, m 2 s -1, or 50 and 400 pmol. . m'2 s "1, and more preferably between 150 and 300 pmol. m'2. s" 1. 1 pmol. m'2. s'1 corresponds to 1pE m'2. s'1 (Einstein), unit often used in the literature.
According to another embodiment of the invention, the illumination may be variable, which means that the illumination is not interrupted by dark phases, but that the light intensity varies over time. This variation in light intensity can be regular and can be periodic or cyclic. According to the invention, it is also possible to carry out a light supply combining continuous and discontinuous illumination phases.
According to a preferred embodiment of the invention, predominantly heterotrophic mixotrophy conditions are used. Thraustochytrids do not have chloropastes. As a result, most of the energy used for growth comes from the carbon source of the culture medium. In this case, the light induces certain metabolic pathways in the cell but does not make it possible to fix the carbon by photosynthesis.
According to a preferred embodiment, the illumination of the cultures can be provided by an illumination device internal to the fermenter. Thus, the efficiency of the illumination is better compared to a configuration where the light enters through portholes from sources disposed outside. The illumination devices can be arranged on the rotating assembly provided with blades or on tubular parts immersed in the mass to be treated, or at the bottom or the ceiling of the tank. According to a preferred embodiment of the invention, the illumination device can be located on the counter-blades inside the fermenter. Such a device is described in French Patent Application No. 1353641. The fermenter can thus be provided with a plurality of illumination sources carried by counter-blades, the latter having the function of preventing the formation of a vortex within the biomass under the action of the rotating assembly. brewing. These sources of illumination are preferably encapsulated, partially or completely in at least a portion of these against blades, in a material compatible with the biomass and in a thickness for diffusing said light to the inside of the tank.
According to the invention, the culture can be carried out by any known culture technique, for example in flasks or in a reactor, but also in fermenters or in any container suitable for the growth of protists, particularly Thraustochytrides, such as, for example, basins of the "raceway" type, provided that said technique makes it possible to implement the required culture conditions.
Preferably, the culture can be carried out in fermenters according to the known methods for culturing protists in fermenters.
According to the invention, the third stage c) of the process allowing the recovery of said biomass can be carried out under appropriate conditions to obtain a biomass that can have the desired moisture content.
Said recovery of the protists can be carried out by any technique allowing the recovery of biomass, including filtration methods, gravimetric or under reduced pressure, centrifugation, decantation, or else precipitation methods followed by gravimetric filtration. The subject of the invention is also a biomass capable of being obtained by the process according to the invention as described above in all its variants. The invention also relates to the use of biomass as described above in the fields of cosmetics, pharmaceuticals, food (human or animal food).
In animal feed, we can distinguish between the feeding of farmed animals, especially industrial livestock and domestic animals, or pets or so-called "leisure" animals, such as aquarium fish or aviary birds or caged.
"Farmed animal" includes grazing animals (especially cattle raised for meat, milk, cheese and leather, sheep raised for meat, wool and cheese, goats), pigs, rabbits, poultry (chickens, chickens, turkeys, ducks, geese and others), equines (ponies, horses, foals), intended to support human activities (transport, leisure) or their feeding aquatic animals (eg fish, shrimp, oysters and mussels). However, it will be possible to distinguish the feeding of fish to the fry stage, and that of the raised fish, including the food and food compositions intended for them.
They will be distinguished from domestic animals, pets or recreational animals. They also include mammals, ruminants or not, birds or fish. They include especially dogs and cats. The invention also relates to a food, or food composition, for humans or animals, which may comprise a biomass according to the invention as described above. By "food" is meant any composition that can be used for food of humans or animals, especially farm animals.
According to one embodiment of the invention, the food may consist of the biomass, dried or not, transformed or not, or biomass, dried or not, transformed or not, mixed with any other additive, vehicle or support , used in the field of food or feed. Examples that may be mentioned as additives include food preservatives, colorants, flavor enhancers, pH regulators, fillers, thickeners, texturizers or binders. It may also include vitamins, probiotics and prebiotics, or pharmaceutical additives such as growth hormones, antibiotics.
According to one embodiment of the invention, the biomass, whether or not dried, whether or not transformed, may itself be used as a carrier or carrier, additive, food preservative, dye, flavor enhancer, filler, thickening agent, texturing agent or binding agent in a product intended for human or animal consumption.
The present invention relates in particular to animal feeds and more particularly to livestock. These foods can usually be in the form of simple mixtures, flours, granules, soup or fodder (fresh fodder, silage, hay ...) in which the biomass according to the invention can be incorporated. These foods can be made by the breeder himself or made from a professional animal feed. By "food" is meant anything that can be used to feed animals.
For intensive animal husbandry, foods may include, in addition to algal biomass, a nutritional basis and nutritional additives. Most of the animal's food intake can be constituted by the "nutritional base" and the algal biomass. This base can be constituted as an example by a mixture of cereals, proteins and fats of animal and / or vegetable origin.
The nutritional bases for animals are adapted to the diet of these animals and are well known to those skilled in the art. In the context of the present invention, these nutritional bases may comprise, for example, fishmeal, corn derivatives, wheat, peas and soya and other seeds. These nutritional bases can be adapted to the needs of the different animal species for which they are intended. These nutritional bases may already contain nutritional additives such as vitamins, mineral salts and amino acids.
Additives used in animal feed can be added to the nutritional base or the algal biomass. These additives can be added to improve certain characteristics of foods, for example to enhance their taste, to make animal feed raw materials more digestible, to preserve food more efficiently or to protect animals. They are frequently used in intensive intensive breeding.
The additives that can be used in animal feed are available, in particular, in the following subcategories (source EFSA): - technological additives: for example, preservatives, antioxidants, emulsifiers, stabilizers, acidity regulators and silage additives; - sensory additives: for example, flavorings, colorants, - nutritional additives: for example, vitamins, amino acids, trace elements; - zootechnical additives: for example, digestibility enhancers, intestinal flora stabilizers, such as probiotics and prebiotics; - coccidiostatic and histomonostatic (antiparasitic).
In one embodiment, the invention relates to feedstuffs for livestock which may comprise between 1% and 60%, preferably between 1 and 20%, and most preferably between 3% and 8% of a biomass. dried or obtained by the process according to the invention.
In another embodiment, the invention relates to animal feeds which may comprise between 1% and 40%, preferably between 5% and 10%, of an undried biomass obtained by the method of the invention.
According to a particular embodiment of the invention, the food may be intended for farm animals, in particular cattle, sheep, pigs, rabbits, poultry and equines.
According to another particular embodiment of the invention, the food may be intended for aquatic animals, in particular fish, at least up to the fry stage, or even including farmed fish. The use of a biomass rich in proteins and PUFAs and including pigments at the end of the growth phase makes it possible to improve the quality of the flesh (thanks to the presence of PUFAs and pigments).
According to another particular embodiment of the invention, the food may be intended for domestic animals, pets and / or recreational animals.
Finally, according to another embodiment of the invention, the food composition can be intended for humans, in particular, the composition can be adapted to children, adolescents, adults or the elderly. The invention also relates to a cosmetic or pharmaceutical composition for humans or animals that may comprise a biomass according to the invention as described above.
According to the invention, the cosmetic or pharmaceutical composition may comprise only biomass, dried or not, transformed or otherwise, or biomass, dried or not, transformed or otherwise, mixed with any other additive, vehicle or carrier, used in the field of cosmetics or pharmacy such as preservatives, dyes, pH regulators. The invention also relates to the use of biomass as described above in therapy, for example in the prevention and treatment of malnutrition. It is known that in the treatment of malnutrition, protein deficiency is the most detrimental and expensive to treat. Biomass, according to one embodiment of the invention, can be a complete solution of choice for preventing and / or treating malnutrition, especially children and seniors. It can be a balanced and digestible protein source (protein deficiency is the most detrimental and expensive). Its protein fraction can be associated with a source of high nutritional quality lipids necessary for healthy development of children. The biomass according to the invention may also be adapted for use by the elderly for the prevention and / or treatment of malnutrition, and other diseases related to aging. In this context, the carotenoid intake of the biomass according to the invention can also be very beneficial for the health of these populations, the elderly being susceptible to diseases such as AMD.
Biomass can therefore be incorporated in a complete food (in wet form or powder for rehydration) a snack food such as cereal bars, rusks, biscuits or not, confectionery, cooking products such as creams, mix powders, dairy products, cereals or drinks. Biomass can be present in food supplements, for example in the form of powder, spread or sauces to add to the daily diet.
EXAMPLES Other aspects and features of the invention may appear on reading the examples which follow.
EXAMPLE 1 Cultivation Method at 25 ° C.
The cultures of Aurantiochytrium CCAP4062 / 1 were carried out in fermenters (bioreactors) of 1 to 2 L useful with dedicated automata and supervision by computer station. The system was regulated in pH via the addition of base (NH 4 OH). The culture temperature was set at 25 ° C and then lowered to 18 ° C at 24 hours of culture. The dissolved oxygen pressure was regulated in the medium throughout the culture, by the stirring speed (220 - 1200 rpm), the air flow (0.6 - 2 vvm), or even the oxygen flow (0-2 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 72 hours.
The composition of the culture medium used during the culture is given in the table:
Glucose additions have been made in the form of enrichment solutions having a molar ratio of carbon to phosphorus (C: P) of 530: 1.
Precultures were performed on a stirring table (140 rpm) in a controlled temperature chamber (26 ° C), a first 24 hours and then 70 hours.
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). The appearance of the biomass is light beige.
The total lipid content and fatty acid analyzes were carried out 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. 1957 May; 226 (1): 497-509]. Results:
Example 2: Culture method with step a) at 30 ° C.
The cultures of Aurantiochytrium CCAP4062 / 4 were carried out in fermenters (bioreactors) of 1 to 2 L useful with dedicated automata and supervision by computer station. The system was regulated at pH 6 via the addition of base (NH4OH). The culture temperature was set at 30 ° C and then lowered to 18 ° C at 24 hours of culture. The dissolved oxygen pressure was regulated in the medium throughout the culture, by the stirring speed (220 - 1200 rpm), the air flow (0.3 -1 vvm), or even the flow rate of oxygen (0 - 1 vvm). The control parameters, integrated in the supervisory automaton, made it possible to maintain a constant pO2 between 5% and 30%. The culture time was between 40 and 100 hours (test A: 69 hours, test B: 51 hours)
The culture medium is the same as that of Example 1.
Glucose additions in the form of an enrichment solution having a molar ratio of carbon: nitrogen: phosphorus (CNP) of 190.5: 7.3: 1 were made for test A. For test B the enrichment solution and had a CNP molar ratio of 95.3: 3.2: 1
Preculture chaining was carried out on a stirring table (140 rpm) in a controlled temperature chamber (26 ° C), a first 24h and then 70 hours.
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). The appearance of the biomass is light beige.
The total lipid content and fatty acid analyzes were carried out 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. 1957 May; 226 (1): 497-509].
Example 3: Cultivation method with illumination in blue light during the last 25 hours of step b)
The cultures of Aurantiochytrium CCAP4062 / 4 were carried out in fermenters (bioreactors) of 5 L useful with dedicated automata and supervision by computer station. The system was regulated at pH 5 via the addition of base (NaOH). The culture temperature was set at 30 ° C and then lowered to 18 ° C at 24 hours of culture. The control parameters, integrated in the supervisory automaton, made it possible to maintain a constant pO2 between 5% and 30%. The culture time was 70 hours. The culture was exposed to blue light (wavelength 455 nm) from 45h of culture. The light sources are in the form of LEDs (or LEDs in French, for light-emitting diode) were mounted on two counter-blades in the bioreactor.
The culture medium is the same as that of Example 1.
Glucose additions in the form of an enrichment solution with a CNP molar ratio of 37: 4: 1 were made.
Preculture chaining was carried out on a stirring table (140 rpm) in a controlled temperature chamber (26 ° C), a first 24h and then 70 hours. This was followed by a 24h fermenter preculture.
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 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. 1957 May; 226 (1): 497-509].
The appearance of the biomass is yellow / orange. The difference in color from Examples 1 and 2 which gave a white / light cream biomass is due to the presence of carotenoids (mainly beta-carotenes, cantaxanthin, phoenicoxanthin and astaxanthin) in the left vial.
Carotenoids measured in Run A: 199 ppm.
* Ppm = ng / mg of MS.

权利要求:
Claims (24)
[1" id="c-fr-0001]
1. Biomass of Thraustochytrides comprising, by weight relative to the weight of the dry matter, at least 35%, preferably at least 45% protein, up to more than 60% protein, in particular 45 to 55% protein, and at least 20%, preferably at least 30% of fat, and optionally between 5 and 250 ppm, preferably between 150 and 200 ppm of carotenoids.
[2" id="c-fr-0002]
2. Biomass according to claim 1, characterized in that carotenoids have a content relative to the fat astaxanthin between 5 and 30%, a phoenicoxanthine content between 15 and 30% and a beta-carotene content between 0 and 30%.
[3" id="c-fr-0003]
3. Biomass according to claim 1 or 2, characterized in that the fat has a content of 25 to 60%, preferably 40 to 60% of polyunsaturated fatty acids selected from AA, DHA and EPA.
[4" id="c-fr-0004]
4. Biomass according to any one of claims 1 to 3, characterized in that said Thraustochytrides are of the order of Thraustochytriales, preferably of the subclass of Thraustochytriaceae, more preferably of a genus selected from the group comprising Aurantiochytrium. , Aplanochytrium, Botryochytrium, Japonochytrium, Oblongichytrium, Parietichytrium, Schizochytrium, Sicyoidochytrium, Thraustochytrium and Ulkenia.
[5" id="c-fr-0005]
5. Biomass according to any one of claims 1 to 4, characterized in that said Thraustochytrides are selected from the genera Aurantiochytrium and Schizochytrium, preferentially among the species Aurantiochytrium mangrovei CCAP 4062/2; Aurantiochytrium mangrovei CCAP 4062/3; Aurantiochytrium mangrovei CCAP 4062/4; Aurantiochytrium mangrovei, CCAP 4062/5; Aurantiochytrium mangrovei CCAP 4062/6; Aurantiochytrium mangrovei CCAP 4062/1; Schizochytrium sp. 4087/3; Schizochytrium sp. CCAP 4087/1; Schizochytrium sp. CCAP 4087/4; Schizochytrium sp. CCAP 4087/5.
[6" id="c-fr-0006]
6. Process for producing a biomass as defined in one of claims 1 to 5, characterized in that it comprises: a. a first step of cultivating Thraustochytrides under conditions of heterotrophy or mixotrophy in a suitable culture medium, at a temperature of between about 24 ° C. and 35 ° C. and under conditions that can promote the production of proteins with a content of less than 35% protein by weight relative to the weight of the dry matter; b. a second step of culture under heterotrophic or mixotrophic conditions at a temperature between about 18 ° C and 25 ° C and lower than that of step a) under conditions that can promote fat accumulation up to less than 20%, preferably at least 30%, and promoting the accumulation of DHA, EPA and / or AA within the fat up to between 25% and 60%, preferably between 40 and 60% of the material fat and until a culture density of at least 40 g / L in dry matter is obtained, preferably at least 60 g / l, more preferably at least 80 g / l; vs. a third step of recovering the biomass obtained in the second step by separating said biomass from the culture medium (the crop), optionally followed by homogenization; and, where appropriate, d. a fourth step of drying the biomass recovered in the third step.
[7" id="c-fr-0007]
7. A method according to claim 6, characterized in that the culture is illuminated, preferably, with light of wavelength between 435 and 475 nm, for at least 18 hours, preferably at least 24 hours.
[8" id="c-fr-0008]
8. Method according to claim 7, characterized in that the culture is illuminated for at least the last 18 hours of culture, preferably for at least the last 24 hours of culture.
[9" id="c-fr-0009]
9. The method of claim 8, characterized in that the culture is illuminated from about 24-50 hours of culture, preferably from 45 hours of culture and until the end of the culture.
[10" id="c-fr-0010]
10. The method of claim 6 to 9, characterized in that said suitable culture medium is a defined medium comprising a carbon source, a nitrogen source, a phosphorus source and salts.
[11" id="c-fr-0011]
11. Method according to any one of claims 6 to 10, characterized in that said defined medium comprises salts selected from calcium salts, cobalt, manganese, magnesium, zinc, nickel, copper, potassium, iron, sodium, and mixtures thereof, advantageously chosen from calcium chloride, cobalt chloride, manganese chloride, magnesium sulphate, zinc sulphate, nickel sulphate, copper sulphate, potassium sulphate, ferrous sulphate, potassium sulphate, sodium molybdate, sodium selenite, sodium chloride and mixtures thereof.
[12" id="c-fr-0012]
12. Method according to one of claims 6 to 11, characterized in that the first step a) of culture is carried out in said mode "batch" mode said "fed batch" or continuous mode.
[13" id="c-fr-0013]
13. Method according to claim 12, characterized in that the "fed batch" culture comprises a first step divided into two sub-steps, a first substep a1) of growth in the appropriate culture medium up to obtaining a culture density of at least 20 g / l, preferably at least 40 g / l, more preferably at least 60 g / l, even more preferably at least 80 g / l, followed by a second sub-component. production step a2) in which one or more carbon source enrichment solution (s), nitrogen source and phosphorus source are added to the culture medium simultaneously or successively.
[14" id="c-fr-0014]
14. The method of claim 13, characterized in that, in step a2), the carbon source content is maintained between 5 and 200 g / l, preferably between 10 and 50 g / l, the source content of nitrogen is maintained between 0.5 and 5 g / l, preferably between 0.5 and 2 g / l and the phosphorus source content is maintained between 0.5 and 5 g / l, preferably 0.5 and 2 g. / L.
[15" id="c-fr-0015]
15. Method according to any one of claims 7 to 14, characterized in that the illumination is variable and / or discontinuous, preferably in the form of flashes.
[16" id="c-fr-0016]
16. Biomass obtainable by a process according to any one of claims 6 to 15.
[17" id="c-fr-0017]
17. Non-therapeutic use of a biomass as described in any one of claims 1 to 5 or 16 in the cosmetic field and in food or feed.
[18" id="c-fr-0018]
18. Non-therapeutic use of a Thraustochytrid biomass as described in any one of Claims 1 to 5 or 16 for the improvement of the performance of the animals, evaluated by measuring the consumption index, the gain in weight or "Feed Conversion Ratio".
[19" id="c-fr-0019]
19. A cosmetic or pharmaceutical composition for humans or animals comprising a biomass as described in any one of claims 1 to 5 or 16.
[20" id="c-fr-0020]
20. Food characterized in that it comprises a biomass as described in any one of claims 1 to 5 or 16.
[21" id="c-fr-0021]
21. Food according to claim 20 characterized in that it is intended for human food, in particular, a food suitable for children, adolescents, adults or elderly,
[22" id="c-fr-0022]
22. Feed for farm animals, characterized in that it comprises between 1% and 60%, preferably between 1% and 20%, more preferably between 3% and 8% of a biomass as described according to US Pat. one of claims 1 to 5, or 16.
[23" id="c-fr-0023]
23. Biomass according to any one of claims 1 to 5 or 16 for its use in therapy.
[24" id="c-fr-0024]
24. Feed for aquaculture, characterized in that it comprises between 1% and 60%, preferably between 1% and 20%, more preferably between 3% and 8% of a biomass as described according to one of the claims. 1 to 5, or 16.

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同族专利:

公开号 | 公开日

CN109072169A|2018-12-21|

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FR3038914B1|2020-03-13|

PH12018500129A1|2018-07-23|

JP2018521652A|2018-08-09|

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EP3325606A1|2018-05-30|

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WO2012175027A1|2011-06-23|2012-12-27|Roquette Freres|Methods of mutagenesis of schizochytrium sp and variant strains produced thereof|

WO2015004403A2|2013-07-12|2015-01-15|Fermentalg|Uncoupled cell culture method|

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WO2015079182A1|2013-11-29|2015-06-04|Roquette Freres|Process for enrichment of microalgal biomass with carotenoids and with proteins|

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2017-01-20| PLSC| Publication of the preliminary search report|Effective date: 20170120 |

2017-01-20| EXTE| Extension to a french territory|Extension state: PF |

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2021-07-29| PLFP| Fee payment|Year of fee payment: 7 |

优先权:

申请号 | 申请日 | 专利标题

FR1556792A|FR3038914B1|2015-07-17|2015-07-17|THRAUSTOCHYTRIDE BIOMASS, CULTURE METHOD AND USES|

FR1556792|2015-07-17|FR1556792A| FR3038914B1|2015-07-17|2015-07-17|THRAUSTOCHYTRIDE BIOMASS, CULTURE METHOD AND USES|

PCT/EP2016/066594| WO2017012933A1|2015-07-17|2016-07-13|Thraustochytrid biomass enriched with oils and proteins, culturing method, and uses|

EP16754404.8A| EP3325606A1|2015-07-17|2016-07-13|Thraustochytrid biomass enriched with oils and proteins, culturing method, and uses|

JP2018502069A| JP6976926B2|2015-07-17|2016-07-13|Oil- and protein-rich Thrust Kitrid Biomass, Culture Methods and Uses|

CN201680054170.5A| CN109072169A|2015-07-17|2016-07-13|Thraustochytriale biomass, cultural method and purposes rich in oil and protein|

CA2992149A| CA2992149A1|2015-07-17|2016-07-13|Thraustochytrid biomass enriched with oils and proteins, culturing method, and uses|

PH12018500129A| PH12018500129A1|2015-07-17|2018-01-16|Thraustochytrid biomass enriched with oils and proteins, culturing method, and uses|

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