WO2023035088A1 - Integrated mixotrophic induction bioprocess for astaxanthin accumulation in strains of the green microalga haematococcus lacustris - Google Patents

Integrated mixotrophic induction bioprocess for astaxanthin accumulation in strains of the green microalga haematococcus lacustris Download PDF

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WO2023035088A1
WO2023035088A1 PCT/CL2021/050083 CL2021050083W WO2023035088A1 WO 2023035088 A1 WO2023035088 A1 WO 2023035088A1 CL 2021050083 W CL2021050083 W CL 2021050083W WO 2023035088 A1 WO2023035088 A1 WO 2023035088A1
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atx
accumulation
autotrophic
astaxanthin
induction
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PCT/CL2021/050083
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Spanish (es)
French (fr)
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Cristian AGURTO MUÑOZ
Chris LANDAHUR ESCALONA
Juan GALLARDO RODRIGUEZ
Sergio SAN MARTÍN PARRAGUEZ
Cristina PINTO FIGUEROA
Mónica LATORRE CASTAÑEDA
Adolfo HENRIQUEZ POBLETE
Andrea DONOSO YOULTON
Jessy PAVON PEREZ
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Universidad de Concepción
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Priority to ES202490017A priority Critical patent/ES2968529A1/en
Priority to MX2024003034A priority patent/MX2024003034A/en
Publication of WO2023035088A1 publication Critical patent/WO2023035088A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes

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  • the present invention is related to the industrial area, more specifically with microalgae cultures of interest in the aquaculture and food industry.
  • the red carotenoid pigment astaxanthin (ATX) is in high demand due to its important characteristics.
  • Synthetic ATX is used as a pigment in aquaculture and food industry, to achieve orange or reddish color in salmon and trout kept in captivity, which can only obtain it from their diet (Liu, 2010).
  • it is essential for the normal development and reproduction of salmonids, accelerating sexual maturity and increasing fertilization, egg maturation and defense against oxidative stress (Nakano et al.1995).
  • these beneficial characteristics are less in synthetic ATX compared to natural ATX.
  • Natural ATX has 500 times more antioxidant capacity than o-tocopherol (vitamin E), showing the highest free radical absorbance capacity (ORAC) of all carotenoids (Nguyen, 2013), arousing great interest for its use as a supplement. or functional ingredient in human food.
  • the natural pigment has applications in the pharmaceutical and nutraceutical industry (Lorenz and Cysewsky, 2000), in the case of human consumption, it has demonstrated capacity for the prevention of degenerative diseases (Iwamoto et al. 2000; Guerin et al. 2003), cancer prevention (McCarty, 2012), immune response enhancement (Kobayashi et al.
  • H. piuviaiis the fungus Xanthophyiiomyces dendrorhous and the green microalgae Chioreiia zofingiensis and Haematococcus iacustr ⁇ s
  • H. iacustris is the most productive organism, accumulating large amounts of astaxanthin upon encystment, reaching up to 4% of the compound based on dry weight under laboratory conditions (Ambati et al. 2014; Boussiba, 2000). .
  • Wild strains of the microalga achieve a maximum production reported in the literature of 4% under ideal laboratory conditions in low culture volumes. However, the contents obtained in larger-scale processes at an industrial level rarely exceed 2%. To improve productivity, random mutagenesis has been used, achieving increases between 2.5 - 2.6% (Chen et al., 2003; Gomez et al. 2013), but with reversion problems, low biomass and accumulation of other non-binding compounds. desired (An et al. 1989). The generation of transgenics can be successful (Liu, 2010; Teng, 2002; Steinbrenner and Sandmann, 2006; Kathiresan et al. 2009) but their use at the national level or for human consumption is not allowed, emphasizing the need for natural sources (Nguyen , 2013).
  • Figure 1 Selected stressors to induce ATX production in H. lacustris strains.
  • Figure 2 Three-dimensional graph of the response surface for the productivity of ATX induction by the mixotrophic pathway in strain 024.
  • Figure 3 ATX chromatograms in cultures of strain P3 with autotrophic induction. Signals: Left corresponds to sample without hydrolyzing; right corresponds to hydrolyzed sample.
  • Figure 4 ATX chromatograms in P3 strain cultures with mixotrophic induction. Signals: left corresponds to a hydrolyzed sample and right corresponds to a sample without hydrolyzate.
  • Figure 7 10 pg/mL synthetic ATX standard together with a sample of strain 024 NaCI treatment.
  • Figure 11 Assays of antioxidant capacity (%) of ATX from strain 024 (left) and P3 (right).
  • Figure 12 ATX antioxidant capacity assays from strain 024 (left) and P3 (right).
  • Figure 13 ATX concentration and biomass obtained from strain 024 during the induction process.
  • Figure 14 Productivity of the optimized mixotrophic induction of ATX in FBR 3 L, 5 experiences.
  • Figure 15 Productivity of the optimized mixotrophic induction of ATX in 200 L FBR, 3 experiences.
  • a new non-autotrophic integrated bioprocess of astaxanthin (ATX) accumulation via mixotrophic induction in Haematococcus lacustris for the nutraceutical industry is presented.
  • the bioprocess comprises at least the following stages: a) Growth phase of autotrophic culture under standard conditions: an autotrophic culture is prepared under standard conditions in a period of 10-20 days in bioreactors between 5 and 800 L at 23°C, with constant bubbling and a light intensity of 50 pE m -2 s -1 ; b) ATX accumulation induction phase: In a photobioreactor, the culture is subjected to the following conditions: Carbon source, sodium acetate-glycerol mixture at 10-50 mM; temperature 25-28°C; irradiance at 500-700 pE m“ 2 s -1 ; pH between 7 - 8; stirring at 100 - 250 rpm; duration 6 to 12 days; c) Biomass harvesting and drying phase, under standard conditions; and d) ATX extraction phase, under standard conditions.
  • the integrated bioprocess allows further accumulation of the ATX carotenoid pigment in at least two H. lacustris strains, representing industrial strains and polyploid strains. Since there were no significant differences between both strains studied under these optimal induction conditions, we can indicate that this process can be used for any strain of H. lacustris. Under these conditions, for the industrial strain, ATX productivities of 3.27 g nr 3 d 1 were obtained and for the polyploid strain, ATX productivities of 2.61 g nr 3 d' 1 were obtained. Which means increasing between 15 and 18 times the productivity of ATX compared to the obtained by the industry under traditional conditions in the industry of northern Chile 0.18g nr 3 d' 1 .
  • Another of the competitive characteristics of the bioprocess is the reduction of induction time, achieving high productivities between 6-12 days of induction, while, with the traditional method of the industrial process, induction is achieved in periods between 15-45 days.
  • the photobioreactor used can have a capacity from 250ml to 200L, without having to modify the operating conditions and without affecting the quality of the ATX obtained.
  • This bioprocess can be implemented without limitations, sanitary, seasonal, climatic or geographical.
  • the advantages and competitive potential of the developed non-autotrophic bioprocess are:
  • the polyploid strain (P3) did not see its carotenoid composition affected, either by its polyploidization treatment or by the non-autotrophic inductive treatments.
  • the industrial strain (024) was also not affected by the non-autotrophic inductive treatments regarding the carotenoid profile and the isomeric composition of the ATX molecule (3S,3'S), The effect that could be verified is the increase in the concentration of ATX for both strains in the non-autotrophic induction treatments, especially of the mixotrophic type.
  • Example 1 Evaluation of stressors that induce the accumulation of astaxanthin by mixotrophic pathway.
  • the P3 strain corresponds to a polyploid from the GIBMAR UdeC laboratory.
  • the P3 strain is deposited in the Spanish Algae Bank (BEA) under the access code BEA IDA 0064B, the deposit was made on November 21, 2017.
  • carbon sources sodium acetate, glycerol and a mixture of them, in a concentration range between 5-100 mM;
  • UV-C radiation at a range between 10-100 mJcnr 2 .
  • Irradiance range between 2.31 ⁇ 0.09 1.35 ⁇ 0.28
  • Example 2 Optimized mixotrophic astaxanthin induction bioprocess.
  • Example 1 The optimization of the mixotrophic induction parameters selected in Example 1 was performed, which generated a greater accumulation of the ATX carotenoid pigment in the two strains of H. lacustris.
  • the stressors selected to evaluate in this optimization process were: acetate carbon source, acetate-glycerol carbon source, irradiance and temperature.
  • the factorial design of experiments method with 3 factors and three levels was used.
  • the selected factors correspond to the concentration of sodium acetate added to the culture, the temperature of the reactor where the cultures are kept between 6 and 12 days, and its irradiance.
  • the evaluated response was the productivity of the ATX induction for each of the strains. Twenty one experiments were carried out in each case. In total, 4 response surfaces were obtained, evaluating the concentration of sodium acetate, the mixture of sodium acetate with glycerol, for each strain (024 and P3).
  • Cultures grown in 5 L bottles were used, which were kept for 10 days at 23°C, with constant bubbling and a light intensity of 50 pE m -2 s -1 . After this growth phase, accumulation induction treatments were carried out.
  • the 6 blocks were evaluated with 3 replicates in 250 mL Erlenmayer flasks inoculated with 100 mL of culture, which were subjected to the established conditions to induce encystment of the H. lacustris strains (024 and P3).
  • an industrial control was added, diluting the culture and adding NaCI for 4 consecutive days, to induce encystment. To simulate these conditions, 0.25 gL 1 was added the first 4 days until 1 gL 1 of NaCI was obtained in 100 mL of the culture.
  • Figure 2 shows the model optimized to increase ATX productivity in strain 024 with the sodium acetate stressor.
  • the temperature and irradiance values are optimal at 27.5 °C and 637 pE m“ 2 s -1 at a concentration of 50 mM (Table 2), the latter being the most significant factor in the model, since an increase in the sodium acetate concentration could increase the productivity values of ATX of 2.29 gnr 3 d' 1 .
  • Table 2 Selected stressors optimized to increase ATX productivity, through optimized mixotrophic induction
  • Example 3 H. lacustris strains validated based on the ATX produced
  • the industrial strain and the polyploid strain were validated for the best induction process compared to the ATX of the base strain obtained. by autotrophic induction.
  • Free ATX was quantified by means of high performance liquid chromatography (HPLC), by means of enzymatic hydrolysis of carotenoid esters and the corresponding stereoisomers were identified.
  • the prepared ATX standards were filtered and injected in triplicate. These standards correspond to concentrations of 0.75; 1.5; 3.0; 4.5; 6.0; and 7.5 pg/mL ATX.
  • the HPLC equipment was conditioned to be able to determine the signal corresponding to free ATX at a retention time of 7.92 min.
  • the analysis conditions were determined at room temperature, with a flow of 1 mL/min and using a column (Luna 3pm Silica) in normal phase under isocratic conditions of hexane/acetone 82: 18 v/v with detection at 474 nm. Aliquots of 20 pl were injected with a total run time of 15 min. Regarding the evaluation of some validation parameters, the method was linear in the concentration ranges evaluated and both the detection limit and the quantification limit were well below the first point of the calibration curve. This reflects the ability of the method to determine ATX concentrations at low concentrations.
  • Figures 3-6 show free ATX together with the sample without hydrolyzation for autotrophic and mixotrophic induction in H. lacustris strain P3 and 024. It is important to highlight that regardless of the strain and the treatment to which the samples were subjected, the results indicate that there is no variation in the carotenoid profile between the samples analyzed and only an increase in ATX is observed as in the rest of the pigments. detected, indicating an induction of the entire ATX metabolic pathway in H. lacustris. Stereoisomers of astaxanthin produced in cultures (D3 V 024) of H. acustris.
  • Figure 7 shows the stereoisomer profile of synthetic astaxanthin, which comprises the three optical isomers that this molecule has in a 1:2:1 ratio (3S,3'S):(3S,3R):(3R, 3'R).
  • Example 4 Mixotrophic inductive bioprocess optimized to obtain ATX at laboratory scale (3L) and at pilot scale (200L).
  • the inductive bioprocess was validated under laboratory conditions in stirred Infers bioreactors of 3 and 200L.
  • Table 4 Induction parameters used in the ATX induction process in 3 and 200L.
  • Figure 13 shows the results of the final concentration of ATX expressed in % w/w of biomass samples harvested at the end of each experience and lyophilized. In addition, the final concentration of biomass produced is indicated in g L -1 . With the 5 experiences of mixotrophic induction, it was determined that the average of the productivities obtained was 3.27 g nr 3 d -1 , which is equivalent to 18.2 times the average annual productivity of the industry (0.18 g nr 3 d -1 ).
  • Figure 14 shows the 5 experiences carried out in the 3L photobioreactor and Figure 15 indicates the productivity of the mixotrophic induction bioprocess in 3 experiences in 200L stirred bioreactors.
  • ATX productivities of 2.68 g nr 3 d 1 which is equivalent to 15 times the annual average productivity of the industry (0.18 g nr 3 d -1 ).

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Abstract

The present invention relates to an integrated bioprocess for the mixotrophic induction of astaxanthin (ATX) accumulation in Haematococcus lacustris, comprising, at least, the following steps: autotrophic culture growth under standard conditions; mixotrophic induction of ATX accumulation in photobioreactor; biomass harvesting and drying under standard conditions; and ATX extraction under standard conditions. The use of this integrated bioprocess enables cultivation without sanitary, seasonal, climatic or geographical constraints.

Description

Bioproceso integrado de inducción mixotrófica para la acumulación de astaxantina en cepas de la microalga verde en Haematococcus lacustrís Integrated bioprocess of mixotrophic induction for the accumulation of astaxanthin in strains of the green microalgae in Haematococcus lacustris
Sector Técnico Technical Sector
La presente invención está relacionada con el área industrial, más específicamente con los cultivos de microalgas de interés en la industria acuicultura e industria alimentaria. The present invention is related to the industrial area, more specifically with microalgae cultures of interest in the aquaculture and food industry.
Estado del Arte State of the Art
Dentro de los llamados químicos finos producidos por microalgas, el pigmento carotenoide rojo astaxantina (ATX) posee una alta demanda gracias a sus importantes características. Se utiliza ATX sintética como pigmento en acuicultura e industria alimentaria, para lograr el color anaranjado o rojizo en salmones y truchas mantenidas en cautiverio, las que solo pueden obtenerlo desde su alimentación (Liu, 2010). Además, es indispensable para el desarrollo normal y reproducción de salmónidos, acelerando la madurez sexual e incrementando la fertilización, maduración de los huevos y defensa frente a estrés oxidativo (Nakano y cois.1995). Sin embargo, estas características beneficiosas son menores en la ATX sintética comparado con la ATX natural. La ATX natural posee 500 veces más capacidad antioxidante que el o-tocoferol (vitamina E), mostrando la capacidad de absorción de radicales libres (ORAC) más alta de todos los carotenoides (Nguyen, 2013), despertando gran interés para su uso como suplemento o ingrediente funcional en la alimentación humana. Adicionalmente el pigmento natural posee aplicaciones en la industria farmacéutica y nutracéutica (Lorenz y Cysewsky, 2000), en el caso del consumo humano, ha demostrado capacidad para la prevención de enfermedades degenerativas (Iwamoto y cois. 2000; Guerin y cois. 2003), prevención del cáncer (McCarty, 2012), mejoramiento de la respuesta inmune (Kobayashi y cois. 1997; Lorenz y Cysewsky, 2000; Lignell y Bottiger, 2001), prevención de enfermedades oculares (Cort y cois. 2008), cuidado de la piel (Lorenz, 2002; Tominaga y cois. 2012), agente antiinflamatorio (Speranza y cois. 2012), e inhibición del colesterol LDL (Choi y cois. 2011). Estas sobresalientes cualidades hacen que la ATX natural sea considerada el "rey de los carotenoides", con un renovado interés para el consumo humano (Nguyen, 2013). Esta gran variedad de efectos benéficos ha permitido el ingreso de la astaxantina microalgal al mercado de productos nutracéuticos, con una enorme variedad de productos con propiedades antioxidantes que se comercializan en tiendas de alimentos naturales (McCoy, 1999) y en diversas cadenas de farmacias. En general, la evidencia acumulada ha demostrado que la ATX de la microalga H. /acustrís conocida antiguamente como H. piuviaiis (Nakada & Ota, 2016; Guiry, 2018), es beneficiosa para la salud humana por la prevención de diversas enfermedades Among the so-called fine chemicals produced by microalgae, the red carotenoid pigment astaxanthin (ATX) is in high demand due to its important characteristics. Synthetic ATX is used as a pigment in aquaculture and food industry, to achieve orange or reddish color in salmon and trout kept in captivity, which can only obtain it from their diet (Liu, 2010). In addition, it is essential for the normal development and reproduction of salmonids, accelerating sexual maturity and increasing fertilization, egg maturation and defense against oxidative stress (Nakano et al.1995). However, these beneficial characteristics are less in synthetic ATX compared to natural ATX. Natural ATX has 500 times more antioxidant capacity than o-tocopherol (vitamin E), showing the highest free radical absorbance capacity (ORAC) of all carotenoids (Nguyen, 2013), arousing great interest for its use as a supplement. or functional ingredient in human food. Additionally, the natural pigment has applications in the pharmaceutical and nutraceutical industry (Lorenz and Cysewsky, 2000), in the case of human consumption, it has demonstrated capacity for the prevention of degenerative diseases (Iwamoto et al. 2000; Guerin et al. 2003), cancer prevention (McCarty, 2012), immune response enhancement (Kobayashi et al. 1997; Lorenz and Cysewsky, 2000; Lignell and Bottiger, 2001), eye disease prevention (Cort et al. 2008), skin care (Lorenz, 2002; Tominaga et al. 2012), anti-inflammatory agent (Speranza et al. 2012), and LDL cholesterol inhibition (Choi et al. 2011). These outstanding qualities make natural ATX considered the "king of carotenoids", with renewed interest for human consumption (Nguyen, 2013). This wide variety of beneficial effects has allowed microalgal astaxanthin to enter the nutraceutical market, with a huge variety of products with antioxidant properties being sold in health food stores (McCoy, 1999) and in various drugstore chains. In general, the accumulated evidence has shown that ATX from the microalga H. /acustris, formerly known as H. piuviaiis (Nakada & Ota, 2016; Guiry, 2018), is beneficial for human health by preventing various diseases.
Sus excepcionales características han creado un importante mercado para su producción, que se estima alcanzó los US$ 200 millones el 2015 (Frost y Sullivan 2008) manteniendo un significativo crecimiento año a año. Incluso cuando la mayor parte del pigmento que se utiliza en la industria de la acuicultura es de origen sintético, el mercado para consumo humano está en activo crecimiento y se estima que alcanzó los US$ 60 millones el 2008 (Frost y Sullivan 2008), principalmente en forma de encapsulados o en formulación de cosméticos, bebidas y alimentos funcionales. Its exceptional characteristics have created an important market for its production, which is estimated to have reached US$ 200 million in 2015 (Frost and Sullivan 2008), maintaining significant year-on-year growth. Even though most of the pigment used in the aquaculture industry is of synthetic origin, the market for human consumption is actively growing and is estimated to have reached US$60 million in 2008 (Frost and Sullivan 2008), mainly in the form of encapsulations or in the formulation of cosmetics, beverages and functional foods.
De forma natural el hongo Xanthophyiiomyces dendrorhous y las microalgas verdes Chioreiia zofingiensis y Haematococcus iacustrís (ex H. piuviaiis; Nakada & Ota, 2016; Guiry, 2018), son los únicos organismos con potencialidad para competir con la ATX sintética (Tinoi y cois. 2006). H. iacustrís (ex H. piuviaiis) es el organismo más productivo, acumulando grandes cantidades de astaxantina al enquistarse, alcanzando hasOta un 4% del compuesto en base a peso seco en condiciones de laboratorio (Ambati y cois. 2014; Boussiba, 2000). No obstante, se requieren 56 kg de biomasa para obtener un kilo de pigmento a un valor de US$12.000. Pese a que la producción de ATX derivada de esta microalga es más costosa comparada con la artificial y de levaduras, es la de mejor calidad en cuanto a su índice de capacidad antioxidante ORAC, y tiene aprobación FDA para uso en consumo humano (Nguyen, 2013). Adicionalmente la ATX natural está asociada a otros compuestos que estabilizan el pigmento, evitando su oxidación e incrementando su duración en almacenamiento (Holtin y cois. 2009; Nguyen, 2013), sin embargo, la ATX natural representa solo un 30% de la producción actual (Frost y Sullivan 2008), existiendo una baja productividad en los cultivos y procesos inductivos de acumulación de ATX (Lorenz & Cysewski, 2000), los cuales son tradicionalmente de tipo autotróficos, necesitando grandes extensiones de terreno, mucho tiempo y elevados costos de producción (Nguyen, 2013), lo que indica una clara necesidad de nuevas fuentes naturales de ATX y por sobre todo, de nuevas formas de cultivar y particularmente, de inducir la acumulación de ATX con mayores productividades, que permitan una producción comercial sustentadle para cubrir su creciente mercado. Incluso, debe considerarse que los costos de producción no están demasiado alejados de la ATX sintética, por lo que mejoras en el proceso de inducción de ATX podrían hacer económicamente más viable la producción de ATX natural en un futuro próximo (L¡ y col., 2011). Naturally, the fungus Xanthophyiiomyces dendrorhous and the green microalgae Chioreiia zofingiensis and Haematococcus iacustrís (ex H. piuviaiis; Nakada & Ota, 2016; Guiry, 2018), are the only organisms with the potential to compete with synthetic ATX (Tinoi et al. 2006). H. iacustris (ex H. piuviaiis) is the most productive organism, accumulating large amounts of astaxanthin upon encystment, reaching up to 4% of the compound based on dry weight under laboratory conditions (Ambati et al. 2014; Boussiba, 2000). . However, 56 kg of biomass is required to obtain one kilo of pigment at a value of US$12,000. Although the production of ATX derived from this microalgae is more expensive compared to artificial and yeast production, it is the best quality in terms of its ORAC antioxidant capacity index, and it has FDA approval for use in human consumption (Nguyen, 2013). ). Additionally, natural ATX is associated with other compounds that stabilize the pigment, preventing its oxidation and increasing its storage life (Holtin et al. 2009; Nguyen, 2013), however, natural ATX represents only 30% of current production. (Frost and Sullivan 2008), with low productivity in crops and inductive processes of ATX accumulation (Lorenz & Cysewski, 2000), which are traditionally autotrophic, requiring large tracts of land, a long time and high production costs. (Nguyen, 2013), which indicates a clear need for new natural sources of ATX and above all, for new ways of cultivating and particularly, to induce the accumulation of ATX with higher productivities, which allow sustainable commercial production to cover their growing market. It should even be considered that production costs are not too far from synthetic ATX, so improvements in the ATX induction process could make the production of natural ATX more economically viable in the near future (L¡ et al., 2011).
La producción de ATX a partir de microorganismos ha sido un tema de intensa investigación en los últimos años (Chen et al, 1997; Ip & Chen, 2005), en comparación con otros microorganismos productores, las microalgas verdes, tales como H. /acustrísy C. zofíngiensis, tienen la capacidad de acumular grandes cantidades de ATX (Rise et al, 1994; Boussiba et al, 1999, Borowitzka, 1999; Curtain, 2000). En la actualidad la producción de ATX natural se realiza mayoritariamente, a través del cultivo e inducción de H. /acustrís bajo condiciones autotróficas, es decir, utilizando nutrientes inorgánicos, luz y CO2 como fuente de carbono (carbono inorgánico), requiriendo grandes extensiones de terreno, prolongados tiempos (21 a 27 días) para su cultivo e inducción de acumulación de ATX y elevados costos en operación. En este escenario, los existentes cultivos y procesos de inducción de acumulación de ATX autotróficos de H. lucustrís no satisfacen la demanda mundial de ATX natural, considerando que las condiciones actuales de cultivo e inducción de ATX presentan bajas productividades de 0,18 g ATX nr3 d -1con un 1,8% (porcentaje en masa de ATX respecto a la biomasa seca) promedio anual en un amplio terreno de 4000 m2 para raceways de cultivo y 25000 m2 para raceways de inducción autotróficas (Datos Planta industrial de Pigmentos Naturales S.A.). The production of ATX from microorganisms has been a subject of intense research in recent years (Chen et al, 1997; Ip & Chen, 2005), in comparison with other producing microorganisms, the green microalgae, such as H. /acustrísy C. zofingiensis, have the capacity to accumulate large amounts of TXA (Rise et al, 1994; Boussiba et al, 1999, Borowitzka, 1999; Curtain, 2000). At present, the production of natural ATX is carried out mainly through the cultivation and induction of H. /acustris under autotrophic conditions, that is, using inorganic nutrients, light and CO2 as a carbon source (inorganic carbon), requiring large areas of field, long times (21 to 27 days) for its cultivation and induction of ATX accumulation and high operating costs. In this scenario, the existing cultures and processes for induction of autotrophic TXA accumulation of H. lucustris do not satisfy the world demand for natural TXA, considering that the current culture and TXA induction conditions present low productivities of 0.18 g TXA nr 3 d -1 with 1.8% (mass percentage of ATX with respect to dry biomass) annual average in a large field of 4000 m 2 for culture raceways and 25000 m 2 for autotrophic induction raceways (Data Industrial Plant of Natural Pigments SA).
Las cepas silvestres de la microalga logran una producción máxima reportada en bibliografía de 4% en condiciones ideales de laboratorio en bajos volúmenes de cultivo. Sin embargo, los contenidos obtenidos en procesos de mayor escala a nivel industrial raramente superan el 2%. Para mejorar la productividad se ha utilizado mutagénesis al azar, logrando incrementos entre 2,5 - 2,6% (Chen y cois, 2003; Gomez y cois. 2013), pero con problemas de reversión, baja biomasa y acumulación de otros compuestos no deseados (An y cois. 1989). La generación de transgénicos puede ser exitosa (Liu, 2010; Teng, 2002; Steinbrenner y Sandmann, 2006; Kathiresan y cois. 2009) pero no está permitido su uso a nivel nacional ni en alimentación humana, enfatizando la necesidad de fuentes naturales (Nguyen, 2013). Wild strains of the microalga achieve a maximum production reported in the literature of 4% under ideal laboratory conditions in low culture volumes. However, the contents obtained in larger-scale processes at an industrial level rarely exceed 2%. To improve productivity, random mutagenesis has been used, achieving increases between 2.5 - 2.6% (Chen et al., 2003; Gomez et al. 2013), but with reversion problems, low biomass and accumulation of other non-binding compounds. desired (An et al. 1989). The generation of transgenics can be successful (Liu, 2010; Teng, 2002; Steinbrenner and Sandmann, 2006; Kathiresan et al. 2009) but their use at the national level or for human consumption is not allowed, emphasizing the need for natural sources (Nguyen , 2013).
Con estos antecedentes expuestos, es altamente necesario desarrollar un nuevo bioproceso de acumulación de astaxantina vía inducción mixo o heterotrófica en H. lacustris como una herramienta validada, con un gran potencial económico para obtener incrementos significativos de astaxantina, libre de las restricciones sanitarias, geográficas o climáticas. With these exposed background, it is highly necessary to develop a new bioprocess of astaxanthin accumulation via mixo or heterotrophic induction in H. lacustris as a validated tool, with great economic potential to obtain significant increases in astaxanthin, free of sanitary, geographical or climatic restrictions.
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• Nguyen, K. (2013) Astaxanthin: A Comparative Case of Synthetic VS. Natural Production. Chemical and Biomolecular Engineering Publications and Other Works. Http://trace.tennessee.edu/utk_chembiopubs/94. Official Journal of the European Communities L106, DIRECTIVE 2001/18/EC OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 12 March 2001, on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC. BREVE DESCRIPCIÓN DE LAS FIGURAS • Nguyen, K. (2013) Astaxanthin: A Comparative Case of Synthetic VS. Natural Production. Chemical and Biomolecular Engineering Publications and Other Works. http://trace.tennessee.edu/utk_chembiopubs/94. Official Journal of the European Communities L106, DIRECTIVE 2001/18/EC OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 12 March 2001, on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC. BRIEF DESCRIPTION OF THE FIGURES
Figura 1: Estresores seleccionados para inducir la producción de ATX en cepas de H. lacustrís. Figure 1: Selected stressors to induce ATX production in H. lacustris strains.
Figura 2: Gráfica tridimensional de la superficie de respuesta para la productividad de la inducción de ATX por vía mixotrófica en la cepa 024. Figure 2: Three-dimensional graph of the response surface for the productivity of ATX induction by the mixotrophic pathway in strain 024.
Figura 3: Cromatogramas de ATX en cultivos de cepa P3 con inducción autotrófica. Señales: Izquierda corresponde a muestra sin hidrol ¡zar; derecha corresponde a muestra hidrolizada. Figure 3: ATX chromatograms in cultures of strain P3 with autotrophic induction. Signals: Left corresponds to sample without hydrolyzing; right corresponds to hydrolyzed sample.
Figura 4. Cromatogramas de ATX en cultivos de cepa P3 con inducción mixotrófica. Señales: izquierda corresponde a muestra hidrolizada y derecha corresponde a muestra sin hidrolizada. Figure 4. ATX chromatograms in P3 strain cultures with mixotrophic induction. Signals: left corresponds to a hydrolyzed sample and right corresponds to a sample without hydrolyzate.
Figura 5. Cromatogramas de ATX en cultivos de cepa 024 con inducción autotrófica. Señales: Izquierda corresponde a muestra hidrolizada y derecha corresponde a muestra sin hidrolizar. Figure 5. ATX chromatograms in cultures of strain 024 with autotrophic induction. Signals: Left corresponds to hydrolyzed sample and right corresponds to non-hydrolyzed sample.
Figura 6. Cromatogramas de ATX en cultivos de cepa 024 con inducción mixotrófica. Señales: izquierda corresponde a muestra hidrolizada y derecha corresponde a muestra sin hidrolizar. Figure 6. ATX chromatograms in cultures of strain 024 with mixotrophic induction. Signals: left corresponds to a hydrolyzed sample and right corresponds to a non-hydrolyzed sample.
Figura 7: Estándar de ATX sintética 10 pg/mL junto a muestra de cepa 024 tratamiento NaCI. Figure 7: 10 pg/mL synthetic ATX standard together with a sample of strain 024 NaCI treatment.
Figura 8: Determinación estereoisómeros en ATX de cepa P3 con inducción autotróficaFigure 8: Determination of stereoisomers in ATX of strain P3 with autotrophic induction
Figura 9: Determinación estereoisómeros en ATX de cepa P3 con inducción mixotróficaFigure 9: Determination of stereoisomers in ATX of strain P3 with mixotrophic induction
Figura 10: Determinación estereoisómeros en ATX de cepa 024 con inducción mixotrófica Figure 10: Determination of stereoisomers in ATX of strain 024 with mixotrophic induction
Figura 11: Ensayos de capacidad antioxidante (%) de ATX proveniente de la cepa 024 (izquierda) y P3 (derecha). Figure 11: Assays of antioxidant capacity (%) of ATX from strain 024 (left) and P3 (right).
Figura 12: Ensayos de capacidad antioxidante de ATX proveniente de la cepa 024 (izquierda) y P3 (derecha). Figure 12: ATX antioxidant capacity assays from strain 024 (left) and P3 (right).
Figura 13: Concentración de ATX y biomasa obtenida de la cepa 024 durante el proceso de inducción. Figure 13: ATX concentration and biomass obtained from strain 024 during the induction process.
Figura 14: Productividad de la inducción mixotrófica optimizada de ATX en FBR 3 L, 5 experiencias. Figura 15: Productividad de la inducción mixotrófica optimizada de ATX en FBR de 200 L, 3 experiencias. Figure 14: Productivity of the optimized mixotrophic induction of ATX in FBR 3 L, 5 experiences. Figure 15: Productivity of the optimized mixotrophic induction of ATX in 200 L FBR, 3 experiences.
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
Se presenta un nuevo bioproceso integrado no autotrófico de acumulación de astaxantina (ATX), vía inducción mixotrófica en Haematococcus lacustrís para la industria nutracéutica. A new non-autotrophic integrated bioprocess of astaxanthin (ATX) accumulation via mixotrophic induction in Haematococcus lacustris for the nutraceutical industry is presented.
Durante el desarrollo de esta tecnología se utilizaron diferentes estresores los que fueron seleccionados según su capacidad de inducción de ATX. Se utilizaron diferentes fuentes de carbono: acetato, glicerol y acetato-glicerol; irradiancia y temperatura. During the development of this technology, different stressors were used, which were selected according to their ATX induction capacity. Different carbon sources were used: acetate, glycerol and acetate-glycerol; irradiance and temperature.
El bioproceso comprende al menos las siguientes etapas: a) Fase de crecimiento de cultivo autotrófico en condiciones estándar: se prepara un cultivo autotrófico en condiciones estándar en un periodo de 10-20 días en bioreactores entre 5 y 800 L a 23°C, con burbujeo constante y a una intensidad lumínica de 50 pE m-2 s -1; b) Fase de Inducción de acumulación de ATX: En un fotobioreactor se somete el cultivo a las siguientes condiciones: Fuente de carbono mezcla de acetato de sodio-glicerol a 10-50 mM; temperatura de 25-28°C; irradiancia a 500-700 pE m“2s-1; pH entre 7 - 8; agitación a 100 - 250 rpm; duración 6 a 12 días; c) Fase de cosecha y secado de biomasa, bajo condiciones estándar; y d) Fase de extracción de ATX, bajo condiciones estándar. The bioprocess comprises at least the following stages: a) Growth phase of autotrophic culture under standard conditions: an autotrophic culture is prepared under standard conditions in a period of 10-20 days in bioreactors between 5 and 800 L at 23°C, with constant bubbling and a light intensity of 50 pE m -2 s -1 ; b) ATX accumulation induction phase: In a photobioreactor, the culture is subjected to the following conditions: Carbon source, sodium acetate-glycerol mixture at 10-50 mM; temperature 25-28°C; irradiance at 500-700 pE m“ 2 s -1 ; pH between 7 - 8; stirring at 100 - 250 rpm; duration 6 to 12 days; c) Biomass harvesting and drying phase, under standard conditions; and d) ATX extraction phase, under standard conditions.
El bioproceso integrado permite una mayor acumulación del pigmento carotenoide ATX en al menos dos cepas de H. lacustrís, que representan cepas industriales y cepas poliploides. Dado que no existieron diferencias significativas entre ambas cepas estudiadas bajo estas condiciones óptimas de inducción, podemos indicar que este proceso puede servir para cualquier cepa de H. lacustrís. En estas condiciones para la cepa industrial se obtuvieron productividades de ATX 3,27 g nr3 d 1 y para la cepa poliploide se obtienen productividades de ATX 2,61 g nr3 d’1. Lo que significa incrementar entre 15 y 18 veces más la productividad de ATX en comparación a la obtenida por la industria bajo condiciones tradicionales en la industria del norte de Chile 0,18g nr3 d'1. The integrated bioprocess allows further accumulation of the ATX carotenoid pigment in at least two H. lacustris strains, representing industrial strains and polyploid strains. Since there were no significant differences between both strains studied under these optimal induction conditions, we can indicate that this process can be used for any strain of H. lacustris. Under these conditions, for the industrial strain, ATX productivities of 3.27 g nr 3 d 1 were obtained and for the polyploid strain, ATX productivities of 2.61 g nr 3 d' 1 were obtained. Which means increasing between 15 and 18 times the productivity of ATX compared to the obtained by the industry under traditional conditions in the industry of northern Chile 0.18g nr 3 d' 1 .
Otra de las características competitivas del bioproceso es la disminución del tiempo de inducción, logrando obtener altas productividades entre 6-12 días de inducción, mientras que, con el método tradicional del proceso industrial, la inducción se logra en períodos entre 15-45 días. Another of the competitive characteristics of the bioprocess is the reduction of induction time, achieving high productivities between 6-12 days of induction, while, with the traditional method of the industrial process, induction is achieved in periods between 15-45 days.
El fotobioreactor utilizado puede tener una capacidad desde los 250ml hasta 200L, sin tener que modificar las condiciones de operación y sin afectar la calidad de la ATX obtenida. The photobioreactor used can have a capacity from 250ml to 200L, without having to modify the operating conditions and without affecting the quality of the ATX obtained.
Este bioproceso puede ser implementado sin limitaciones, sanitarias, estacionales, climáticas o geográficas. Las ventajas y potencialidades competitivas del bioproceso no autotrófico desarrollado son: This bioprocess can be implemented without limitations, sanitary, seasonal, climatic or geographical. The advantages and competitive potential of the developed non-autotrophic bioprocess are:
- Mayor productividad de ATX debido a la optimización de dos factores: i) incremento en la producción de ATX y ¡i) menor tiempo de proceso de inducción. - Higher ATX productivity due to the optimization of two factors: i) increase in ATX production and ii) shorter induction process time.
- Menor costo de operación debido a: i) reducción del consumo de agua, ¡i) reducción de costos energéticos y iii) menor mano de obra. - Lower operating cost due to: i) reduced water consumption, ii) reduced energy costs and iii) less labor.
- Aplicable a cualquier zona geográfica por la utilización de sistemas cerrados. - Applicable to any geographical area due to the use of closed systems.
- Menor costo de inversión, dado el menor requerimiento de terreno para su implementación. - Lower investment cost, given the lower land requirement for its implementation.
- No depende de la variabilidad estacional. - Does not depend on seasonal variability.
- Mayor control de las variables críticas del proceso (por ejemplo, temperatura). - Greater control of critical process variables (for example, temperature).
- Disminuye la contaminación de la producción por el uso de sistemas cerrados. - Decreases production contamination due to the use of closed systems.
En la caracterización química e isomérica de la molécula de ATX obtenida en este bioproceso con respecto a la inducción autotrófica, no se encontraron diferencias en los perfiles de carotenoides ni en la composición isomérica de la molécula de ATX. In the chemical and isomeric characterization of the ATX molecule obtained in this bioprocess with respect to autotrophic induction, no differences were found in the carotenoid profiles or in the isomeric composition of the ATX molecule.
La cepa poliploide (P3) no vio afectada su composición de carotenoides, ya sea por su tratamiento de poliploidización como por los tratamientos inductivos no autotróficos. The polyploid strain (P3) did not see its carotenoid composition affected, either by its polyploidization treatment or by the non-autotrophic inductive treatments.
La cepa industrial (024) tampoco se vio afectada por los tratamientos inductivos no autotróficos en lo referente al perfil de carotenoides como en la composición isomérica de la molécula de ATX (3S,3'S), El efecto que se pudo comprobar es el aumento de la concentración de ATX para ambas cepas en los tratamientos de inducción no autotrófica, especialmente del tipo mixotrófico. The industrial strain (024) was also not affected by the non-autotrophic inductive treatments regarding the carotenoid profile and the isomeric composition of the ATX molecule (3S,3'S), The effect that could be verified is the increase in the concentration of ATX for both strains in the non-autotrophic induction treatments, especially of the mixotrophic type.
En los ensayos de capacidad antioxidante (CA), se logró comprobar que esta propiedad es relativamente afectada por la concentración de ATX en cada ensayo. No obstante, la naturaleza diferente de cada ensayo antioxidante no siempre demuestra que existe una relación directamente proporcional entre la concentración de ATX con la capacidad antioxidante (CA). In the antioxidant capacity (AC) trials, it was possible to verify that this property is relatively affected by the ATX concentration in each trial. However, the different nature of each antioxidant assay does not always demonstrate that there is a directly proportional relationship between the ATX concentration and the antioxidant capacity (CA).
En todos los ensayos antioxidantes realizados, a partir de los tratamientos de inducción mixotrófica, existió un aumento de la CA con respecto a la inducción autotrófica (NaCI), aunque el ensayo de CA por DPPH claramente indico que no existen diferencias significativas entre los tratamientos de inducción. Sin embargo, en el resto de los ensayos hubo un efecto importante en el aumento de la capacidad antioxidante de la molécula de ATX en las inducciones mixotróficas. In all the antioxidant tests carried out, from the mixotrophic induction treatments, there was an increase in CA with respect to autotrophic induction (NaCI), although the CA test by DPPH clearly indicated that there are no significant differences between the mixotrophic induction treatments. induction. However, in the rest of the trials there was a significant effect in increasing the antioxidant capacity of the ATX molecule on mixotrophic inductions.
EJEMPLOS DE APLICACIÓN APPLICATION EXAMPLES
Ejemplo 1: Evaluación de estresores que inducen la acumulación de astaxantina por vía mixotrófica. Example 1: Evaluation of stressors that induce the accumulation of astaxanthin by mixotrophic pathway.
Se evaluaron los estresores en dos cepas de H. lacustrís, la primera cepa denominada 024, corresponde a la cepa industrial de la empresa Astax Chile SpA, y la cepa P3 corresponde a un poliploide del laboratorio GIBMAR UdeC. La cepa P3 se encuentra depositada en el Banco Español de Algas (BEA) bajo el código de acceso BEA IDA 0064B, el deposito fue realizado con fecha 21 de noviembre de 2017. Stressors were evaluated in two strains of H. lacustrís, the first strain called 024, corresponds to the industrial strain of the Astax Chile SpA company, and the P3 strain corresponds to a polyploid from the GIBMAR UdeC laboratory. The P3 strain is deposited in the Spanish Algae Bank (BEA) under the access code BEA IDA 0064B, the deposit was made on November 21, 2017.
Los 4 estresores evaluados fueron: The 4 stressors evaluated were:
• fuentes de carbono: acetato de sodio, glicerol y mezcla de ellos, en rango de concentración entre 5-100 mM; • carbon sources: sodium acetate, glycerol and a mixture of them, in a concentration range between 5-100 mM;
• temperatura a un rango entre 25-30°C; • temperature in a range between 25-30°C;
• irradiancia a un rango entre 400 y 700 pE m-2 s-1; y • irradiance in a range between 400 and 700 pE m -2 s -1 ; and
• radiación UV-C a un rango entre 10-100 mJcnr2. • UV-C radiation at a range between 10-100 mJcnr 2 .
Después de la inducción, se mantuvieron en un shaker JSOS-500, JSR con una agitación de 120 rpm a 25, 30 y 35°C entre 6 y 12 días. Para la evaluación de cada estresor se utilizaron cultivos crecidos en botellones entre 2- 5 L que fueron mantenidos por 10 días a 23°C, con burbujeo constante y a una intensidad lumínica de 50 pmol m-2 s-1. Tras esta fase de crecimiento, se procedió con los tratamientos de inducción de acumulación. Estos se llevaron a cabo en matraces erlenmayer de 250 mL que fueron inoculados con 100 mL de cultivo y, posteriormente, cada uno de ellos fueron sometidos a diferentes tratamientos para inducir el enquistamiento de las cepas de H. lacustrís (024 y P3 . El control se dejó sin ningún estresor, y se mantuvo en las mismas condiciones que los tratamientos restantes. Adicionalmente NaCI fue utilizado como un control industrial (en la industria se diluye el cultivo y durante 4 días consecutivos se le añade NaCI para inducir el enquistamiento), para simular estas condiciones se añadieron 0,25 gL 1 los 4 primeros días hasta obtener 1 gL 1 de NaCI en los 100 mL del cultivo. Los estresores fueron analizados en las cepas 024 y P3 dos veces de forma independiente, incluyendo 3 réplicas para cada uno. After induction, they were kept in a JSOS-500, JSR shaker with a shaker of 120 rpm at 25, 30 and 35°C for 6 to 12 days. For the evaluation of each stressor, cultures grown in bottles between 2-5 L were used, which were kept for 10 days at 23°C, with constant bubbling and a light intensity of 50 pmol m -2 s -1 . After this growth phase, we proceeded with the accumulation induction treatments. These were carried out in 250 mL Erlenmayer flasks that were inoculated with 100 mL of culture and, subsequently, each one of them was subjected to different treatments to induce the encystment of the H. lacustrís strains (024 and P3 . The control it was left without any stressor, and it was maintained under the same conditions as the remaining treatments.Additionally, NaCI was used as an industrial control (in the industry the culture is diluted and NaCI is added for 4 consecutive days to induce encystment), to To simulate these conditions, 0.25 gL 1 was added the first 4 days until 1 gL 1 of NaCI was obtained in 100 mL of the culture.The stressors were analyzed in strains 024 and P3 twice independently, including 3 replicates for each one. .
En cada caso se determinó la productividad de ATX de los estresores evaluados. En la Tabla 1 se presentan las mejores condiciones seleccionadas. In each case, the ATX productivity of the evaluated stressors was determined. Table 1 presents the best selected conditions.
Tabla 1: Condiciones seleccionadas para la optimización. Table 1: Conditions selected for optimization.
Productividad de ATX Productividad de ATX Estresor seleccionado (gnr3 d _1) cepa 024 (gnr3 d -1) cepa P3ATX productivity ATX productivity Selected stressor (gnr 3 d _1 ) strain 024 (gnr 3 d -1 ) strain P3
Irradiancia: rango entre 2,31 ± 0,09 1,35 ± 0,28Irradiance: range between 2.31 ± 0.09 1.35 ± 0.28
500- 700 pE rrr2 s 1 500- 700 pE rrr 2 s 1
Temperatura: rango entre Temperature: range between
25-28°C con Acetato de 2,3 ± 0,16 1,09 ± 0,03 sodio 25-28°C with 2.3 ± 0.16 1.09 ± 0.03 sodium acetate
Rango entre 10-50 mM de Range between 10-50 mM of
1,9 ± 0,16 2,07 ± 0,18 acetato de sodio y glicerol 1.9 ± 0.16 2.07 ± 0.18 sodium acetate and glycerol
Mezcla de glicerol con 1,78 ± 0,06 1,69 ± 0,3Glycerol mixture with 1.78 ± 0.06 1.69 ± 0.3
Acetato de sodio sodium acetate
Se seleccionaron 4 estresores de la figura 1 para ambas cepas, obteniendo una productividad de ATX hasta 13 veces más que el promedio anual de la industria en el norte de Chile (0,18 gnr3 d 1), estos estresores fueron utilizados para la etapa de optimización. Es importante destacar que en ambas cepas coincide que las mayores productividades se obtienen con los mismos estresores. Ejemplo 2: Bioproceso de inducción de astaxantina mixotrófico optimizado. Four stressors from figure 1 were selected for both strains, obtaining an ATX productivity up to 13 times more than the annual average of the industry in northern Chile (0.18 gnr 3 d 1 ), these stressors were used for the stage optimization. It is important to highlight that in both strains it coincides that the highest productivities are obtained with the same stressors. Example 2: Optimized mixotrophic astaxanthin induction bioprocess.
Se realizó la optimización de los parámetros de inducción mixotróficos seleccionados en el Ejemplo 1, los que generaron una mayor acumulación del pigmento carotenoide ATX en las dos cepas de H. lacustrís. Los estresores seleccionados para evaluar en este proceso de optimización fueron: fuente de carbono acetato, fuente de carbono acetato- glicerol, i rradiancia y temperatura. The optimization of the mixotrophic induction parameters selected in Example 1 was performed, which generated a greater accumulation of the ATX carotenoid pigment in the two strains of H. lacustris. The stressors selected to evaluate in this optimization process were: acetate carbon source, acetate-glycerol carbon source, irradiance and temperature.
Estas condiciones fueron evaluadas mediante proceso de optimización multivariable para lo cual se organizaron en 6 bloques experimentales triplicados (n = 3 réplicas por 6 tratamientos; N=18). These conditions were evaluated through a multivariate optimization process, for which they were organized into 6 triplicate experimental blocks (n = 3 replicates for 6 treatments; N=18).
Para evaluar la interacción entre los factores y optimizar el bioproceso, se utilizó el método de diseño de experimentos factorial con 3 factores y tres niveles. Los factores seleccionados corresponden a la concentración de acetato de sodio añadido al cultivo, la temperatura del reactor en donde se mantienen los cultivos entre 6 y 12 días y la ¡rradiancia del mismo. La respuesta evaluada fue la productividad de la inducción de ATX para cada una de las cepas. Veintiún experimentos se llevaron a cabo en cada caso. En total se obtuvieron 4 superficies de respuestas, evaluando la concentración de acetato de sodio, la mezcla de acetato de sodio con glicerol, para cada cepa (024 y P3). To evaluate the interaction between the factors and optimize the bioprocess, the factorial design of experiments method with 3 factors and three levels was used. The selected factors correspond to the concentration of sodium acetate added to the culture, the temperature of the reactor where the cultures are kept between 6 and 12 days, and its irradiance. The evaluated response was the productivity of the ATX induction for each of the strains. Twenty one experiments were carried out in each case. In total, 4 response surfaces were obtained, evaluating the concentration of sodium acetate, the mixture of sodium acetate with glycerol, for each strain (024 and P3).
Las superficies de respuesta fueron evaluadas mediante el análisis de varianza (ANOVA) utilizando el software de análisis de datos MODDE 12.1 (Umetrics, Umea, Sweden) para ajustar los datos experimentales a la ecuación polinómica de segundo orden mediante una técnica de regresión lineal. Response surfaces were evaluated by analysis of variance (ANOVA) using MODDE 12.1 data analysis software (Umetrics, Umea, Sweden) to fit the experimental data to the second-order polynomial equation using a linear regression technique.
Se utilizaron cultivos crecidos en botellones de 5 L que fueron mantenidos por 10 días a 23°C, con burbujeo constante y a una intensidad lumínica de 50 pE m-2 s-1. Tras esta fase de crecimiento se procedió con los tratamientos de inducción de acumulación. Se evaluaron los 6 bloques con 3 réplicas en matraces erlenmayer de 250 mL inoculados con 100 mL de cultivo los que fueron sometidos a las condiciones establecidas para inducir el enquistamiento de las cepas de H. lacustrís (024 y P3). Además, se añadió un control industrial, diluyendo el cultivo y añadiendo NaCI durante 4 días consecutivos, para inducir el enquistamiento. Para simular estas condiciones se añadieron 0,25 gL 1 los 4 primeros días hasta obtener 1 gL 1 de NaCI en los 100 mL del cultivo. Cultures grown in 5 L bottles were used, which were kept for 10 days at 23°C, with constant bubbling and a light intensity of 50 pE m -2 s -1 . After this growth phase, accumulation induction treatments were carried out. The 6 blocks were evaluated with 3 replicates in 250 mL Erlenmayer flasks inoculated with 100 mL of culture, which were subjected to the established conditions to induce encystment of the H. lacustris strains (024 and P3). In addition, an industrial control was added, diluting the culture and adding NaCI for 4 consecutive days, to induce encystment. To simulate these conditions, 0.25 gL 1 was added the first 4 days until 1 gL 1 of NaCI was obtained in 100 mL of the culture.
Se tomaron muestras de todos los tratamientos en cada bloque entre el día 6 y 12 de cultivo con la finalidad de determinar la densidad celular, peso seco y cantidad de ATX. A modo de ejemplo, se muestra en la figura 2 el modelo optimizado para aumentar la productividad de ATX en la cepa 024 con el estresor acetato de sodio. Los valores de temperatura e irradiancia son óptimos a 27,5 °C y 637 pE m“2s-1 a una concentración de 50 mM (Tabla 2), siendo esta última el factor más significativo del modelo, puesto que un aumento en la concentración de acetato de sodio podría incrementar los valores de productividad de ATX de 2,29 gnr3 d’1. Samples were taken from all treatments in each block between day 6 and 12 of culture in order to determine cell density, dry weight and amount of ATX. As an example, Figure 2 shows the model optimized to increase ATX productivity in strain 024 with the sodium acetate stressor. The temperature and irradiance values are optimal at 27.5 °C and 637 pE m“ 2 s -1 at a concentration of 50 mM (Table 2), the latter being the most significant factor in the model, since an increase in the sodium acetate concentration could increase the productivity values of ATX of 2.29 gnr 3 d' 1 .
Tabla 2: Estresores seleccionados optimizados para aumentar la productividad de ATX, mediante una inducción mixotrófica optimizada Table 2: Selected stressors optimized to increase ATX productivity, through optimized mixotrophic induction
Estresor Valor optimizado Stressor Optimized value
Acetato adaptado (mM) 50 ± 0,12 Adapted acetate (mM) 50 ± 0.12
Temperatura (°C) 27,5 ± 0,12 Temperature (°C) 27.5 ± 0.12
Irradiancia pEnr2s 1 637 ± 0,10 Irradiance pEnr 2 s 1 637 ± 0.10
Ejemplo 3: Cepas de H. lacustris validada en base a la ATX producidaExample 3: H. lacustris strains validated based on the ATX produced
Mediante técnicas analíticas y funcionales que permiten determinar la calidad de la ATX producida (composición isomérica y capacidad antioxidante) por vía mixotrófica, se validó la cepa industrial y la cepa poliploide para el mejor proceso de inducción en comparación con la ATX de la cepa base obtenida por inducción autotrófica. Using analytical and functional techniques that allow determining the quality of the ATX produced (isomeric composition and antioxidant capacity) by mixotrophic pathway, the industrial strain and the polyploid strain were validated for the best induction process compared to the ATX of the base strain obtained. by autotrophic induction.
Se utilizaron cultivos crecidos en botellones de 2- 5 L que fueron mantenidos por 10 días a 23°C, con burbujeo constante y a una intensidad lumínica de 50 pE m“2s-1. Tras esta fase de crecimiento se procedió con los tratamientos de inducción de acumulación. Se evaluaron los rangos de las condiciones optimizadas descritas en la Tabla 3, para inducir el enquistamiento de las cepas de H. lacustris (024 y P3), cada una con 4 réplicas en matraces Erlenmeyer de 250 mL inoculados con 100 mL de cultivo. Además, se añadió un control industrial de inducción descrito anteriormente. Cultures grown in 2-5 L bottles were used, which were kept for 10 days at 23°C, with constant bubbling and a light intensity of 50 pE m“ 2 s -1 . After this growth phase, accumulation induction treatments were carried out. The ranges of the optimized conditions described in Table 3 were evaluated to induce encystment of the H. lacustris strains (024 and P3), each with 4 replicates in 250 mL Erlenmeyer flasks inoculated with 100 mL of culture. In addition, an industrial induction control described above was added.
Se tomaron muestras de todos los tratamientos entre el día 6 y 12 de cultivo con la finalidad de determinar peso seco y cantidad de ATX. Tabla 3: Estresores optimizados. Samples of all treatments were taken between day 6 and 12 of culture in order to determine dry weight and amount of ATX. Table 3: Optimized stressors.
Estresor optimizado Ejemplo 3 Rango de valoresOptimized stressor Example 3 Range of values
Irradiancia 500-700 pE m-2s-lIrradiance 500-700 pE m-2s-l
Temperatura 25-28 °C Temperature 25-28°C
Acetato de sodio 10-50 mM Sodium acetate 10-50mM
Glicerol con Acetato de sodio Mezcla Glycerol with Sodium Acetate Mix
Se cuantificó ATX libre por medio de cromatografía líquida de alta eficiencia (HPLC), por medio de la hidrólisis enzimática de los ásteres de carotenoides y se identificaron los estereoisómeros correspondientes. Free ATX was quantified by means of high performance liquid chromatography (HPLC), by means of enzymatic hydrolysis of carotenoid esters and the corresponding stereoisomers were identified.
Cuantifícación de estereoisómeros de ATX por cromatografía líquida de alto eficiencia HPLC. Quantification of ATX stereoisomers by HPLC high performance liquid chromatography.
Los estándares de ATX preparados fueron filtrados e inyectados por triplicado. Estos estándares corresponden a concentraciones de 0,75; 1,5; 3,0; 4,5; 6,0; y 7,5 pg/mL de ATX. El equipo de HPLC fue acondicionado para poder determinar la señal correspondiente a la ATX libre a un tiempo de retención de 7,92 min. The prepared ATX standards were filtered and injected in triplicate. These standards correspond to concentrations of 0.75; 1.5; 3.0; 4.5; 6.0; and 7.5 pg/mL ATX. The HPLC equipment was conditioned to be able to determine the signal corresponding to free ATX at a retention time of 7.92 min.
Las condiciones del análisis fueron determinadas a temperatura ambiente, con un flujo de 1 mL/min y utilizando una columna (Luna 3pm Sílice) en fase normal en condiciones ¡socráticas de hexano/acetona 82: 18 v/v con detección de 474 nm. Se inyectaron alícuotas de 20 pl con tiempo de corrida total de 15 min. En cuanto a la evaluación de algunos parámetros de validación, el método resulto lineal en los rangos de concentraciones evaluados y tanto el límite de detección como el de cuantifícación estuvieron muy por debajo del primer punto de la curva de calibrado. Lo cual refleja la capacidad del método para determinar concentraciones de ATX en concentraciones bajas. The analysis conditions were determined at room temperature, with a flow of 1 mL/min and using a column (Luna 3pm Silica) in normal phase under isocratic conditions of hexane/acetone 82: 18 v/v with detection at 474 nm. Aliquots of 20 pl were injected with a total run time of 15 min. Regarding the evaluation of some validation parameters, the method was linear in the concentration ranges evaluated and both the detection limit and the quantification limit were well below the first point of the calibration curve. This reflects the ability of the method to determine ATX concentrations at low concentrations.
Hidrólisis enzimática de los ásteres de carotenoides: Enzymatic hydrolysis of carotenoid esters:
En las figuras 3-6 se observa la ATX libre junto a la muestra sin hidrol ¡zar para para la inducción autotrófica y mixotrófoca en la cepa P3 y 024 de H. lacustrís. Es importante destacar que independiente de la cepa y del tratamiento a los cuales fueron sometidas las muestras, los resultados indican que no hay una variación en el perfil de carotenoides entre las muestras analizadas y sólo se observa un incremento de ATX como del resto de los pigmentos detectados, indicando una inducción de toda la ruta metabólica de ATX en H. lacustrís. Estereoisómeros de la astaxantina producida en los cultivos (D3 V 024) de H. ¡acustrís. Figures 3-6 show free ATX together with the sample without hydrolyzation for autotrophic and mixotrophic induction in H. lacustris strain P3 and 024. It is important to highlight that regardless of the strain and the treatment to which the samples were subjected, the results indicate that there is no variation in the carotenoid profile between the samples analyzed and only an increase in ATX is observed as in the rest of the pigments. detected, indicating an induction of the entire ATX metabolic pathway in H. lacustris. Stereoisomers of astaxanthin produced in cultures (D3 V 024) of H. acustris.
En la figura 7, se puede observar el perfil de estereoisómeros de astaxantina sintética la cual comprende a los tres isómeros ópticos que tiene esta molécula en una proporción 1:2: 1 (3S,3'S):(3S,3R):(3R,3'R). Figure 7 shows the stereoisomer profile of synthetic astaxanthin, which comprises the three optical isomers that this molecule has in a 1:2:1 ratio (3S,3'S):(3S,3R):(3R, 3'R).
Esta conformación de isómeros nos permite diferenciar la molécula de astaxantina sintética de la natural, en este caso de H. lacustrís la cual corresponde a su totalidad al isómero (3S,3'S). This conformation of isomers allows us to differentiate the synthetic astaxanthin molecule from the natural one, in this case from H. lacustris which corresponds entirely to the (3S,3'S) isomer.
En las figuras 7 a 10, se puede observar y confirmar que el proceso de poliploidización, así como los tratamientos de inducción mixotróficas no afectan la composición isomérica de la molécula de ATX, manteniendo la estructura (3S,3'S), lo cual valida la obtención ATX para su utilización como nutraceútico en humanos. In figures 7 to 10, it can be observed and confirmed that the polyploidization process, as well as the mixotrophic induction treatments do not affect the isomeric composition of the ATX molecule, maintaining the structure (3S,3'S), which validates the obtaining ATX for use as a nutraceutical in humans.
Por diferentes ensayos se determinó la relación del porcentaje de capacidad antioxidante (CA) de la ATX producida por inducción mixotrófica, con respecto a CA de ATX obtenida de cepa base por inducción autotrófica. The ratio of the percentage of antioxidant capacity (CA) of the ATX produced by mixotrophic induction, with respect to the CA of ATX obtained from the base strain by autotrophic induction, was determined by different assays.
• Resonancia paramagnética de espín (EPR) • Spin Paramagnetic Resonance (EPR)
• 2,2-difenil-l-picrylhydrazyl (DPPH) • 2,2-diphenyl-l-picrylhydrazyl (DPPH)
• Ácido 2,2'-azino-bis-(3-etillbenzotiazolin-6-sulfonico) (ABTS) • 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)
• Actividad reductora del hierro férrico/poder antioxidante (FRAP) • Ferric iron reducing activity/antioxidant power (FRAP)
• Capacidad de absorción de radicales de oxígeno (ORAC). • Oxygen Radical Absorbance Capacity (ORAC).
En los ensayos de capacidad antioxidante (CA), se logró comprobar que esta propiedad es relativamente afectada por la concentración de ATX en cada ensayo. No obstante, la naturaleza diferente de cada ensayo antioxidante no siempre demuestra que existe una relación directamente proporcional entre la concentración de ATX con la capacidad antioxidante (CA), figuras 11 y 12. In the antioxidant capacity (AC) trials, it was possible to verify that this property is relatively affected by the ATX concentration in each trial. However, the different nature of each antioxidant assay does not always show that there is a directly proportional relationship between the ATX concentration and the antioxidant capacity (CA), figures 11 and 12.
En todos los ensayos realizados, hubo un aumento de la CA con respecto a la inducción autotrófica (NaCI), aunque el ensayo de CA por DPPH claramente indico que no existen diferencias significativas entre los tratamientos de inducción mixotrófica. Sin embargo, en el resto de los ensayos demostró un efecto importante en el aumento de la capacidad antioxidante de la molécula de ATX en las inducciones mixotrófica (acetato y mezcla de acetato de sodio y glicerol). La CA fue determinada por diferentes métodos antioxidantes, puesto que definen diferentes mecanismos de reacción, tales como la transferencia de átomos de hidrógeno (como es en el caso del ensayo ORAC), transferencia de electrones (como el FRAP), mezcla entre los mecanismos de transferencia de átomos de hidrógeno y transferencia de electrones (como el DPPH y el ABST) o de manera inversa por capacidad proxidante (EPR). In all the tests carried out, there was an increase in CA with respect to autotrophic induction (NaCI), although the CA test by DPPH clearly indicated that there are no significant differences between the mixotrophic induction treatments. However, in the rest of the tests, it demonstrated an important effect in increasing the antioxidant capacity of the ATX molecule in mixotrophic inductions (acetate and a mixture of sodium acetate and glycerol). The CA was determined by different antioxidant methods, since they define different reaction mechanisms, such as the transfer of hydrogen atoms (as in the case of the ORAC assay), electron transfer (such as FRAP), mixture between the mechanisms of transfer of hydrogen atoms and transfer of electrons (such as DPPH and ABST) or inversely by proxidant capacity (EPR).
Ejemplo 4: Bioproceso inductivo mixotrófico optimizado para obtención de ATX a escala de laboratorio (3L) y a escala piloto (200L). Example 4: Mixotrophic inductive bioprocess optimized to obtain ATX at laboratory scale (3L) and at pilot scale (200L).
Se validó el bioproceso inductivo en condiciones de laboratorio en biorreactores Infers agitados de 3 y 200L. The inductive bioprocess was validated under laboratory conditions in stirred Infers bioreactors of 3 and 200L.
Se utilizaron cultivos crecidos en botellones de 5 y 20 L que fueron mantenidos por 10 días a 23°C, con burbujeo constante y a una intensidad lumínica de 50 pE m-2 s -1. Tras esta fase de crecimiento se procedió con el tratamiento de inducción de acumulación de ATX, bajo las condiciones descritas en la tabla 4. Posteriormente se llevó a cabo la inducción, tanto en un fotobiorreactor de 3L como en biorreactores agitados de 200 L. Cultures grown in 5 and 20 L bottles were used, which were kept for 10 days at 23°C, with constant bubbling and a light intensity of 50 pE m -2 s -1 . After this growth phase, the ATX accumulation induction treatment was carried out, under the conditions described in Table 4. Subsequently, the induction was carried out, both in a 3L photobioreactor and in 200L stirred bioreactors.
Tabla 4: Parámetros de inducción utilizados en el proceso de inducción de ATX en 3 y 200L. Table 4: Induction parameters used in the ATX induction process in 3 and 200L.
Condición Rango Condition Range
Inductor de inducer of
Mezcla de acetato de sodio y glicerol enquistamiento Mixture of sodium acetate and glycerol encystment
Temperatura 25-28°C Temperature 25-28°C
Irradiancia 500-700 pE nr2s 1 pH 7,6 Irradiance 500-700 pE nr 2 s 1 pH 7.6
Agitación 100-250 rpm Stirring 100-250rpm
Para evaluar la replicabilidad del método, en el caso del fotobiorreactor de 3L se llevaron a cabo cinco experiencias independientes bajo las mismas condiciones operacionales (réplicas). En la figura 13 se muestran los resultados de la concentración final de ATX expresada en % p/p de muestras de la biomasa cosechada al final de cada experiencia y liofilizada. Además, se indican la concentración final de biomasa producida en g L-1. Con las 5 experiencias de inducción mixotrófica, se determinó que el promedio de las productividades obtenidas fue de 3,27 g nr3 d -1, lo cual es equivalente a 18,2 veces la productividad promedio anual de la industria (0,18 g nr3 d -1). To evaluate the replicability of the method, in the case of the 3L photobioreactor, five independent experiences were carried out under the same operational conditions (replicates). Figure 13 shows the results of the final concentration of ATX expressed in % w/w of biomass samples harvested at the end of each experience and lyophilized. In addition, the final concentration of biomass produced is indicated in g L -1 . With the 5 experiences of mixotrophic induction, it was determined that the average of the productivities obtained was 3.27 g nr 3 d -1 , which is equivalent to 18.2 times the average annual productivity of the industry (0.18 g nr 3 d -1 ).
En la figura 14, se pueden observar las 5 experiencias realizadas en el fotobiorreactor de 3L y en la figura 15 se indica la productividad del bioproceso de inducción mixotrófica en 3 experiencias en biorreactores agitados de 200L, En este caso, se obtuvieron productividades de ATX de 2,68 g nr3 d 1 lo cual es equivalente a 15 veces la productividad promedio anual de la industria (0,18 g nr3 d -1). Figure 14 shows the 5 experiences carried out in the 3L photobioreactor and Figure 15 indicates the productivity of the mixotrophic induction bioprocess in 3 experiences in 200L stirred bioreactors. In this case, ATX productivities of 2.68 g nr 3 d 1 which is equivalent to 15 times the annual average productivity of the industry (0.18 g nr 3 d -1 ).

Claims

REIVINDICACIONES Un bioproceso integrado no autotrófico de acumulación de astaxantina (ATX) en Haematococcus /aovs/r/s CARACTERIZADO porque la acumulación de ATX es vía inducción mixotrófica y comprende a lo menos las siguientes etapas: a) Fase de crecimiento de cultivo autotrófico en condiciones estándar; b) Fase de Inducción de acumulación de ATX en un fotobioreactor se somete el cultivo a los siguientes estresores: fuente de carbono; temperatura; e irradiancia; c) Fase de cosecha y secado de biomasa, bajo condiciones estándar; y d) Fase de extracción de ATX, bajo condiciones estándar. El bioproceso integrado no autotrófico de acumulación de astaxantina (ATX) en Haematococcus /acustrís, según reivindicación 1, CARACTERIZADO porque el cultivo autotrófico de la fase de crecimiento comprende un periodo de 10-20 días en bioreactores entre 5 y 800 L a 23°C, con burbujeo constante y a una intensidad lumínica entre 50 - 100 pE m-2 s -1. El bioproceso integrado no autotrófico de acumulación de astaxantina (ATX) en Haematococcus /acustrís, según reivindicación 1, CARACTERIZADO porque en la fase de inducción de acumulación de ATX la fuente de carbono es una mezcla de acetato de sodio-glicerol. El bioproceso integrado no autotrófico de acumulación de astaxantina (ATX) en Haematococcus /acustrís, según reivindicación 3, CARACTERIZADO porque la mezcla de acetato de sodio-glicerol está a una concentración entre 10-50 mM. El bioproceso integrado no autotrófico de acumulación de astaxantina (ATX) en Haematococcus /acustrís, según reivindicación 1, CARACTERIZADO porque en la fase de inducción de acumulación de ATX la temperatura es de 25-28°C. El bioproceso integrado no autotrófico de acumulación de astaxantina (ATX) en Haematococcus /acustrís, según reivindicación 1, CARACTERIZADO porque en la fase de inducción de acumulación de ATX la irradiancia es de 500-700 pE m“2s-1. El bioproceso integrado no autotrófico de acumulación de astaxantina (ATX) en Haematococcus /acustrís, según reivindicación 1, CARACTERIZADO porque en la fase de inducción de acumulación de ATX el cultivo se somete a pH entre 7 - 8 y a una agitación de 100 - 250 rpm. El bioproceso integrado no autotrófico de acumulación de astaxantina (ATX) en Haematococcus /acustris, según reivindicación 1, CARACTERIZADO porque la fase de inducción de acumulación de ATX tiene una duración de 6 a 12 días. El bioproceso integrado no autotrófico de acumulación de astaxantina (ATX) en Haematococcus /acustris, según reivindicación 1, CARACTERIZADO porque en la fase de inducción de acumulación de ATX el fotobioreactor utilizado tiene una capacidad de 250ml hasta 200L. Uso del bioproceso integrado no autotrófico de acumulación de astaxantina (ATX) en Haematococcus iacustrís, según reivindicación 1, CARACTERIZADO porque su utilización permite obtener entre 15 y 18 veces más productividad de ATX en comparación a la obtenida por la industria bajo condiciones tradicionales. Uso del bioproceso integrado no autotrófico de acumulación de astaxantina (ATX) en Haematococcus iacustrís, según reivindicación 1, CARACTERIZADO porque su utilización permite un cultivo sin limitaciones, sanitarias, estacionales, climáticas o geográficas. CLAIMS A non-autotrophic integrated bioprocess of astaxanthin (ATX) accumulation in Haematococcus /aovs/r/s CHARACTERIZED in that ATX accumulation is via mixotrophic induction and comprises at least the following stages: a) Growth phase of autotrophic culture under conditions standard; b) ATX accumulation induction phase in a photobioreactor, the culture is subjected to the following stressors: carbon source; temperature; and irradiance; c) Biomass harvesting and drying phase, under standard conditions; and d) ATX extraction phase, under standard conditions. The non-autotrophic integrated bioprocess of astaxanthin (ATX) accumulation in Haematococcus /acustris, according to claim 1, CHARACTERIZED in that the autotrophic culture of the growth phase comprises a period of 10-20 days in bioreactors between 5 and 800 L at 23°C , with constant bubbling and a light intensity between 50-100 pE m -2 s -1 . The non-autotrophic integrated bioprocess of astaxanthin (ATX) accumulation in Haematococcus / acustris, according to claim 1, CHARACTERIZED in that in the ATX accumulation induction phase the carbon source is a mixture of sodium acetate-glycerol. The non-autotrophic integrated bioprocess of accumulation of astaxanthin (ATX) in Haematococcus / acustris, according to claim 3, CHARACTERIZED in that the sodium acetate-glycerol mixture is at a concentration between 10-50 mM. The non-autotrophic integrated bioprocess of astaxanthin (ATX) accumulation in Haematococcus / acustris, according to claim 1, CHARACTERIZED because in the ATX accumulation induction phase the temperature is 25-28°C. The non-autotrophic integrated bioprocess of astaxanthin (ATX) accumulation in Haematococcus /acustris, according to claim 1, CHARACTERIZED because in the ATX accumulation induction phase the irradiance is 500-700 pE m“ 2 s -1 . The non-autotrophic integrated bioprocess of astaxanthin (ATX) accumulation in Haematococcus / acustris, according to claim 1, CHARACTERIZED because in the ATX accumulation induction phase the culture is subjected to a pH between 7 - 8 and an agitation of 100 - 250 rpm . The non-autotrophic integrated bioprocess of astaxanthin (ATX) accumulation in Haematococcus /acustris, according to claim 1, CHARACTERIZED in that the ATX accumulation induction phase lasts from 6 to 12 days. The non-autotrophic integrated bioprocess of astaxanthin (ATX) accumulation in Haematococcus /acustris, according to claim 1, CHARACTERIZED because in the ATX accumulation induction phase the photobioreactor used has a capacity of 250ml to 200L. Use of the non-autotrophic integrated bioprocess of astaxanthin (ATX) accumulation in Haematococcus iacustris, according to claim 1, CHARACTERIZED because its use allows obtaining between 15 and 18 times more ATX productivity compared to that obtained by the industry under traditional conditions. Use of the non-autotrophic integrated bioprocess of astaxanthin accumulation (ATX) in Haematococcus iacustris, according to claim 1, CHARACTERIZED in that its use allows cultivation without sanitary, seasonal, climatic or geographical limitations.
PCT/CL2021/050083 2021-09-09 2021-09-10 Integrated mixotrophic induction bioprocess for astaxanthin accumulation in strains of the green microalga haematococcus lacustris WO2023035088A1 (en)

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