• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    The present invention relates to a culture for the production of dha comprising at least one dha-producing algae of the laberintulide class, and comprising in specific proportions at least one carbon source, at least one yeast extract and one or more secondary sources of nitrogen. The present invention is also directed to a fermentation process using said culture and to the obtained fermentation products. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2617081A1
    申请号:ES201531814
    申请日:2015-12-15
    公开日:2017-06-15
    发明作者:María Teresa PELLICER MOYA；Sergi ABAD I SANCHEZ；Antoni Planas Sauter；Xavier TURON CASALPRIM
    申请人:Institut Quimic de Sarria CETS Fundacio Privada；Interquim SA；
    IPC主号:
    专利说明:

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    • Aeration should be high enough to achieve good biomass production without yet causing excessive DHA production. Typical values are between 1 and 5 L / min, preferably between 2 and 4 L / min.
    • Reactor volume: The volume of the first reactor may be between
    5 5% and 25%, preferably between 10% and 20% of the total system volume.
    • The dilution rate should be as high as possible, to establish a high growth rate. This will impact the final productivity of DHA. You can work between: 0.0211 to 0.070 h-1. The optimum would be between 0.050 and 0.060 h-1.
    10 Therefore, all conditions are aimed at maximizing biomass production, and the person skilled in the art can apply different variations depending on the circumstances of each case. In the second phase the maximum production of DHA is sought, increasing its content in the cells that have occurred in the first phase. For this, a time of
    15 adequate residence to accumulate significant amounts of DHA. This can be achieved, for example, by reducing the dilution rate and thus increasing the volume in the second phase, which can be done in a single tank if it is large enough, or in several tanks. This causes a greater residence of the cells in this phase. The dilution in this second phase is preferably set between 0.0001h-1 and 0.0100h-1,
    20 preferably between 0.001h-1 and 0.010h-1. The total volume in the tank (s) of this second phase is therefore preferably between 75% and 95%, preferably between 80% and 90%, of the total system volume. Conditions that typically stimulate the production of DHA are one or more of the following:
    • Modulation of the concentration of the carbon source. The inventors have
    25 found that a lower glycerol concentration than in the first stage, for example between 0.1 and 8 g / L, surprisingly stimulates the accumulation of fatty acids, an effect that until now had not been observed in the literature . Thus, for example, it was found that at the end of a culture, the addition of a small amount of glycerol (up to 5g / l) increased the yield by 30%.
    30 • This reduction in the concentration of the carbon source may be accompanied by a lower temperature than that used in the first phase, for example between 1020 ° C. In WO 2004/083442 a two-stage fermentation process is described in which the second stage comprises lowering the temperature to 10 ° C, but the effect of reducing the concentration of the carbon source is not described.
    35 • Less agitation, for example, between 200 rpm and 500 rpm, or greater than 350 rpm to ensure good oxygen availability.
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    • Less aeration, for example between 0.1 and 1 g / min to stimulate the production of DHA almost exclusively.
    • Zero or negligible concentration of the nitrogen source.
    • At the same time, the conditions of this second phase may be such that they cause the necessary stress to cause pigment generation in the presence of adequate amounts of glutamate. These conditions generally involve low oxygen availability and low temperatures.
    Therefore, researchers have found that the combination of a higher first temperature in the first phase with a lower temperature in the second one, increases the efficiency in DHA production in a surprising way. In the prior art it had already been observed that a lower temperature favors the production of DHA
    (Y. Taoka et al., Effects of cold shock treatment on total lipid content and fatty acid composition of Aurantiochytrium limacinum strain mh0186., J. Oleo Sci. 60, 217–20 (2011)). However, biomass growth proved to be so low that the final productivity made the process unfeasible for the industrial scale. On the other hand, a thermal shock was attempted, that is, storing the biomass at low temperatures after fermentation at a higher temperature, without obtaining an increase in the amount of DHA produced. On the other hand, the presence in low concentrations of the carbon source in the second phase (either because it is added or because it comes from the first phase) allows DHA yields to increase around 5% to 15%. The process of the invention may comprise a third phase in which the culture is stored at temperatures below 10 ° C, preferably below 7 ° C, in the presence of glycerol. As with temperature, in the case of oxygen the conditions for a better production of DHA and biomass do not converge. Many studies have identified that when the availability of oxygen is lowered, the concentration of DHA increases. For example, in the first phase the concentration of dissolved oxygen can be as high as possible, for example it can be between 2 and 70%, preferably more than 20%, while it can fall to less than 2% during The second phase It should be added that if temperatures of 15 ° C or lower are combined, as well as low oxygen availability, for example between 0.5 and 20%, preferably between 1 and 10%, pigments are generated with antioxidant capacity mentioned above. This phenomenon is important, as it protects the DHA at the time of extraction. Therefore, the process of the invention can be a continuous process where the first phase takes place at a higher temperature and oxygen concentration than in the second phase, and the second phase takes place in the presence of a glutamate concentration greater than 10 mM . Alternatively, the first phase takes place at a temperature between 20 ° C and 35 ° C,
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    of culture is between 0.007 and 0.6; at a temperature greater than 20 ° C and an oxygen concentration greater than 20%; Y
    (ii) during a second phase at a temperature less than 20 ° C, in the presence of a lower oxygen concentration of 20% and a glutamate concentration greater than 10 mM. Preferably, the fermentation product has a concentration of said pigments, such as Astaxanthin, greater than 20 µg of pigment per g of biomass, preferably greater than 30 µg of pigment per g of biomass. Typically, the pigment concentration is between 20 and 300 µg of pigment per g of biomass. These high concentrations of pigment are obtained directly from the fermentation process without the need for external addition, and whose antioxidant properties during the fermentation process and subsequent manipulation, allow to obtain a higher purity DHA more easily. These compositions comprising DHA and at least one pigment, preferably a tetraterpene, in concentrations greater than 20 µg of pigment per g of biomass also constitute an aspect of the present invention, as is a composition comprising DHA and at least one pigment. , preferably a tetraterpene, wherein said composition is obtainable by a process comprising cultivating a culture comprising at least one DHA-producing algae of the Labyrinthulide class:
    (i) during a first phase in the presence of at least one carbon source, at least one yeast extract and one or more secondary sources of nitrogen selected from the group consisting of tryptone, fermented corn liquor, peptone and casamino acid, characterized in that the proportion in the culture between the concentration of the sum of secondary sources of nitrogen in grams per liter of culture and the concentration of the source of carbon in grams per liter of culture is between 0.005 and 0.4, and the ratio between the concentration of the extract of yeast in grams per liter of culture and the concentration of the carbon source in grams per liter of culture is between 0.008 and 1, and where the ratio between the concentration of the sum of secondary sources of nitrogen in grams per liter of culture and the concentration of yeast extract in grams per liter of culture is between 0.007 and 0.6; at a temperature greater than 20 ° C and an oxygen concentration greater than 20%;
    (ii) during a second phase at a temperature less than 20 ° C, in the presence of an oxygen concentration less than 20% and a glutamate concentration greater than 10 mM;
    (iii) extract from the resulting biomass at least the resulting lipids and pigments.


    On the other hand, the invention is not limited to the specific embodiments that have been described but also covers, for example, the variants that can be made by the average person skilled in the art (for example, in terms of the choice of materials, dimensions , components, configuration, etc.), within what follows from the
    5 claims
    Examples
    The following examples show the efficacy and improvements of a crop according to the present invention against a crop prepared according to the state of the art
    10 (Standard medium). The cultures were prepared according to the compositions indicated below in Table 1.
    Component Concentration ComponentConcentration
    KH2PO4 0.85 g / LNa2EDTA2H2O0.20 mM
    NaOH 0.14 g / LH3BO31 mM
    NaCl 18 g / LMnSO4H2O0.097 mM
    CaCl2 0.19 g / LZnSO47H2O7 µM
    MgSO4 0.1 g / LFeSO47H2O0.83 mg / L
    Yeast extract 2.3 (C / 10)Medium of the present invention
    Tryptone 0.4 (C / 10)
    Carbon source C value between 10 g / L and 100 g / L
    Component Concentration ComponentConcentration
    Tris 1 g / LNa2EDTA2H2O0.20 mM
    CH3COONH4 1 g / LH3BO31 mM
    NaCl 18 g / LMnSO4H2O0.097 mM
    MgSO47H2O 2.5 g / LZnSO47H2O7 µM
    CaCl2 0.3 g / LCoCl26H2O2 µm
    KCl 0.6 g / LFeSO47H2O0.83 mg / L
    NaNO3 1 g / LFeCl36H2O0.018 mM
    NH4Cl 0.03 g / LStandard medium
    KH2PO4 0.05 g / L
    Yeast extract 1 g / L
    Peptone 1 g / L
    B12 vitamin 0.15 µg / L
    Carbon source C value between 10 g / L and 100 g / L
    Table 1: crop composition
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    Table 2
    * Indicates approximate calculation of productivities in batch, to be comparable to the continuum (described in the following paragraphs)
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    Comparing cultivation strategies, it can be seen that the medium according to the present invention has a positive effect on productivity as well when a continuous system is used, providing the necessary nutrients to maintain sustained growth, even in situations of high biomass concentration. On the other hand, with the multi-tank system, pigments and DHA can be produced simultaneously and sustainably. In addition, the cost per liter of medium is reduced by 30%.
    Aspects and embodiments of the invention can be found in the following numbered clauses: Clause 1: Cultivation for the production of DHA comprising at least one DHA producing algae of the Labyrinthides class, at least one carbon source, at least one extract of yeast and one or more secondary sources of nitrogen selected from the group consisting of tryptone, fermented corn liquor, peptone and casamino acid, characterized in that the ratio between the concentration of the sum of secondary sources of nitrogen in grams per liter of culture and the concentration of the carbon source in grams per liter of culture is between 0.005 and 0.4, and the ratio between the concentration of the yeast extract in grams per liter of culture and the concentration of the carbon source in grams per liter of crop is between 0.008 and 1, and where the ratio between the concentration of the sum of secondary sources of nitrog In grams per liter of culture and the concentration of yeast extract in grams per liter of culture is between 0.007 and 0.6. Clause 2: Culture for the production of DHA comprising at least one algae of the Labyrinthide class in the presence of at least one carbon source and at least one nitrogen source, characterized in that it comprises MgSO4 in a concentration of less than 1 g / L. Clause 3: Culture for the production of DHA comprising at least one algae of the Labyrinthide class in the presence of at least one carbon source and at least one nitrogen source, characterized in that it comprises CaCl2 in a concentration less than 0, 25 g / L Clause 4: Cultivation for the production of DHA comprising at least one algae of the Labyrinthide class in the presence of at least one carbon source and at least one nitrogen source, characterized in that it does not contain one or more of the compounds that they are selected from the group consisting of KCl, NaNO3 and vitamin B12.
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    of culture is between 0.007 and 0.6; at a temperature greater than 20 ° C and an oxygen concentration greater than 20%;
    (ii) during a second phase at a temperature less than 20 ° C, in the presence of a concentration less than 20% and a glutamate concentration greater than 10 mM;
    (iii) extract from the resulting biomass at least the resulting lipids and pigments. Clause 33: The composition according to any one of clauses 31 or 32 wherein the concentration of said pigment is greater than 20 µg of pigment per g of biomass. Clause 34: Procedure for the preparation of the culture of clause 1 comprising mixing in an aqueous medium at least one DHA-producing algae of the Labyrinthides class, at least one carbon source, at least one yeast extract and one or more secondary sources of nitrogen selected from the group consisting of tryptone, fermented corn liquor, peptone and casamino acid so that the ratio between the concentration of the sum of secondary sources of nitrogen in grams per liter of culture and the concentration of the source of Carbon in grams per liter of culture is between 0.005 and 0.4, and the ratio between the concentration of the yeast extract in grams per liter of culture and the concentration of the carbon source in grams per liter of culture is between 0.008 and 1, and where the ratio between the concentration of the sum of secondary sources of nitrogen in grams per liter of culture and the concentration The yeast extract in grams per liter of culture is between 0.007 and 0.6. Clause 35: Procedure for the preparation of a crop according to clause 2 which comprises mixing in an aqueous medium at least one algae of the Labyrinthide class, at least one carbon source, at least one nitrogen source, and MgSO4 in one concentration less than 1 g / L. Clause 36: Procedure for the preparation of a crop according to clause 3 which comprises mixing in an aqueous medium at least one algae of the Labyrinthides class, at least one carbon source, at least one nitrogen source, and CaCl2 in a concentration less than 0.25 g / L. Clause 37: Procedure for the preparation of a crop according to clause 4 which comprises mixing in an aqueous medium at least one algae of the Labyrinthide class, at least one carbon source, at least one nitrogen source, in the absence of one or more of the compounds that are selected from the group consisting of KCl, NaNO3 and vitamin B12. Clause 38: Use of the crop according to any one of clauses 1-19 for the production of DHA.
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    权利要求:
    Claims (1)
    [1]
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    同族专利:
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    引用文献:
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    CN103614427A|2013-11-13|2014-03-05|福建师范大学|Method for producing docosahexaenoic acid through fermenting straw hydrolyzate|
    WO2015150716A2|2014-04-03|2015-10-08|Fermentalg|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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