WO2017129914A1

WO2017129914A1 - Method for producing pigments

Info

Publication number: WO2017129914A1
Application number: PCT/FR2017/050188
Authority: WO
Inventors: Rémi PRADELLES; Antoine DELBRUT; Vincent USACHE
Original assignee: Microphyt
Priority date: 2016-01-29
Filing date: 2017-01-27
Publication date: 2017-08-03
Also published as: FR3047252B1; FR3047252A1

Abstract

The invention relates to a method for producing pigments by cells capable of photosynthesis, according to which said cells are cultivated in conditions suitable for the development thereof, in order to obtain a biomass, the method comprising a step of overproduction of said pigments according to which a pre-determined light intensity normalised in relation to dry biomass is applied to said cells, said light intensity, expressed in µmoles of photons per gram of said dry biomass and per second, being at least 2µmoles.g^-1.s^-1 and at most 200 µmoles.g^-1.s^-..

Description

PROCESS FOR PRODUCING PIGMENTS

The invention relates to the production of pigments by cells capable of photosynthesis.

These pigments include photosynthetic pigments capable of capturing light and photoprotective pigments which do not capture light or very little and which protect cells exposed to an excess of photons, or more broadly to oxidative stress.

Photosynthetic pigments are chemical compounds that allow the transformation of light energy into chemical energy in photosynthetic organisms. When a photon strikes a molecule of photosynthetic pigment, its energy excites an atom of this molecule, and makes it go to an excited state, of high energy level. The energy accumulated in the pigment molecule is released during the return to the ground state of the molecule by a release of heat, and by a fluorescence phenomenon, by the transmission of the excitation state to another molecule and / or by the conversion of energy.

The photosynthetic pigments are essentially divided into two types, the active pigments, capable of performing the three modes of release of the energy accumulated above, which are in particular able to transfer the light energy in the photosynthetic electron transport chain. where the light energy is converted into chemical energy, and of which chlorophyll a is the representative, and the accessory pigments incapable of directly effecting this conversion of energy and which include in particular phycobilins and some carotenoids such as neoxanthine, siphonoxanthin , diadinoxanthin, fucoxanthin, violaxanthin, peridinine and echinenone.

The photoprotective pigments include non-photosynthetic carotenoids and in particular carotenes including lycopene and beta-carotene, and xanthophylls such as antheraxanthin, zeaxanthin, neoxanthine, lutein, loroxanthin, astaxanthin, canthaxanthin, and diatoxanthin.

The ^invention relates more particularly to the production of accessories photosynthetic pigments as well as pigments phot.oprotect.eurs.

Beyond their coloring functions widely used in the food industry, many of these pigments have biological properties of interest, including antioxidant activity, thus finding various uses in pharmacy and cosmetics. For example, different studies on the fucoxanthin, a pigment of the family of xanthophylls, report that in addition to its applications as antioxidant, it has anti-inflammatory properties, anti-cancer, anti-obesity, anti-diabetes, anti-angiogenesis and can even to be used to treat malaria.

The future of these pigments is therefore promising and it is essential to have tools for their production on an industrial scale, in particular for accessory photosynthetic pigments for which no profitable route is yet available.

By way of example, fucoxanthin is only synthesized by certain macroalgae and certain microalgae.

The production of fucoxanthin by macroalgae is problematic because of the seasonal nature of such production, the contamination by elements present in the marine environment (chemical pollutants, heavy metals, iodine, etc.) and the low fucoxanthin content of obtained biomass averaging 0.09% (m: m) relative to dry biomass.

As for the production of fucoxanthin by microalgae, the concentrations reached within the dry biomass are on average 0.70% (m: m). Only one study reported a content of 1.98% (m: m) in the case of the microalga Isochrysis sp. (Crupi P. et al., Rapid Commun Mass Spectrom 2013, 27, 1027-1035).

At present, some experts suggest that the only possibility of overproducing these pigments would be molecular engineering (KJM M ulders et al., 2014, J. Phycol 2014, 50 (2), 229-242), and , despite the poor knowledge of the metabolic pathways and enzymes involved in their synthesis and the low degree of maturity of the molecular tools available for certain microorganisms.

In addition, it has been shown that in the case of natural light production, the quantity and concentration of accessory photosynthetic pigments of the production is dependent on the time of harvest of the system during the day because of the different variations. luminous intensities during the sunny phase and the value of the light intensities (Obata and Taguchi, 2012, Plankton Benthos Res 7 (3), 101-110). This makes industrial exploitation difficult to optimize and rationalize as part of a reproducibility of the characteristics of a production.

According to WQ2013 / 136025A1, there is known a process for producing docosahexaenoic acid and eicosapentaenoic acid by culturing a microalgae of the genus Nitzschia, subjected to discontinuous and / or variable illumination of light over time, this process allowing, also to produce fucoxanthin. The present illumination intensity variations, the amplitude of which is between 5 micromoles of photon.m ^-2 biomass exposée.s ^-1 and 1000 μmoles.m ^{- 2} .s ^-1 and speaker variations between 2 and 3600 times per hour, this method of culture allows to enrich the biomass fucoxanthin only 0.2% (m: m) in dry matter and therefore does not solve the problem of too low yields.

To date, there is a real need for a more efficient production process.

The ^invention provides a solution with a cost-effective process and a simple implementation for a production of pigments involved, directly or indirectly, in photosynthesis, especially photosynthetic accessory pigments and pigment photoprotective.

Surprisingly, the authors discovered that by subjecting a photosynthetic cell culture to normalized light power over dry biomass, pigment production was greatly increased.

Thus, the invention relates to a process for the production of pigments by cells capable of photosynthesis, according to which said cells are cultivated under conditions suitable for their development, in order to obtain a biomass, said process comprising a step of overproduction of said pigments according to which one applies to said cells a normalized luminous power with respect to the dry biomass, dependent on said pigments, said luminous power, expressed in μmoles of photons per gram of said dry biomass per second, being at least 2 μmoles.g ^_1 .s ^-1 and not more than 200 μmol

Before exposing the invention in more detail, certain terms used are defined.

According to the invention, biomass means the mass of the biological material present in the culture medium which consists essentially of cells. This biomass being dependent on its biotope, its quantity evolves during the process according to the conditions that are applied to it. This quantity, generally expressed in grams, at a given instant is determined in dry biomass by conventional techniques known to those skilled in the art. By way of example, one skilled in the art can refer to the document published by the Algae Biomass Organization (ABO), Industrial Algae Measurements, October 2013, Version 6.0, in particular but not only, available at

which is a compendium of standard methodologies used and endorsed by the main global players in the seaweed sector, and particular to its Annex A, on pages 7-8 where is described the protocol for measuring the dry biomass of a cell culture.

Normalized light power with respect to dry biomass means a light intensity expressed in μmoies of photons applied per gram of dry biomass per second. This value is said to be normalized because, because of its dependence on the pigment to be produced, this value is predetermined. Example 1 illustrates a method of predermination of this value for the production of fucoxanthin and for two species of microalgae, Tisochrysis lutea and Phaedactylum tricornutum.

The invention is more specifically described with reference to the accessory photosynthetic pigments as defined above, because to date, no production method on an industrial scale exists. But as said above, the method of the invention also applies to photoprotective pigments.

The invention is hereinafter described in more detail and its variants, especially preferred variants, presented.

The value of the normalized luminous power with respect to the dry biomass, to be applied to the culture, which will be designated by parameter in the remainder of the description, is dependent on the nature of the pigment to be produced.

The authors observed that it should be at least 2 μmoles.g ^{- 1} .s ^-1 and at most 200 μmoles.g ^{- 1} .s ^-1 . In this range, its value is advantageously higher for the production of photoprotective pigments compared to that of accessory photosynthetic pigments.

Thus, according to a preferred embodiment of the process of the invention applied to the production of photosynthetic pigments, the value of the parameter is at least 2, preferably at least 5, and at most 150, preferably of not more than 130 μιmoles e photons per gram of dry biomass per second. In this variant, the pigments are chosen from carotenoids and photosynthetic phycobilins. Advantageously, the process is applied to the production of photosynthetic xanthophils, such as fucoxanthin, and the value of the parameter is chosen from values of 5 to 30 μmoles.g ^-1 .s-. ¹

According to a preferred implementation of the process of the invention applied to the production of photoprotective pigments, the value of the parameter is at least 30, preferably at least 50, and at most 200, preferably at most 190 μmol of photons per gram of dry biomass per second. In this variant, the pigments are preferably chosen from photoprotective carotenoids, such as beta-carotene and asthaxanthin. Parameter management is carried out during the light phases or in a relevant way within a sufficient period of time before the harvest of the production. Preferably, the normalized light power relative to the dry biomass is applied during all or part of the exponential growth phase of the cell culture.

The parameter is characterized during production by the measurement of the average photon flux, expressed in μmoles of photon per m ² and per second, applied to the production system subtracted from the average photon flux absorbed by the production system and normalized by the amount of biomass contained in the production system at the time of measurement. The measurements of the normalized light power with respect to the dry biomass can be carried out by a quantum-meter type device. Dry biomass measurements can be carried out after filtration or centrifugation of the culture, rinsing and drying of the biomass thus recovered, or by on-line absorbance measurements at the appropriate wavelengths after determining the correlation between the absorbance at a given wavelength and dry biomass.

The light source applied to the production system is of natural or artificial origin or a combination of both.

The light source has continuous operation and / or operation in day / night mode according to naturally occurring photoperiods. The emission spectrum of the light source can be selected, modified, adapted to meet particular characteristics specific to the pigments to be produced.

The modification of the emission spectrum of the light source can occur during production.

The regulation of this parameter to maintain constant can be carried out by the variation of one or more factors:

- increase or decrease in the flow of photons applied to the system, or

- increase or decrease in dry biomass within the production system, or

- coupling of these actions

The increase or decrease in photon flux applied to the system can be effected by mechanized or manual moving net systems between the light source and the production system. The increase or decrease of the flux of photons applied to the system can be effected by direct action on the light source in the case of an artificial source.

The increase in dry biomass within the production system can be achieved through controlled additions of nutrients that will have a direct influence on the amount of biomass.

The decrease in dry biomass in the production system can be achieved by dilution of the production system.

The parameter described by the process, can be controlled and kept constant during all or part of the production via an automated device connected to light intensity sensors and sensors allowing the quantification of dry biomass, by acting on the light intensity applied to the crop, as previously described, and / or dry biomass within the production system.

The method described allows the production of pigments by cells capable of photosynthesis according to the batch, fed-batch, continuous, semi-continuous, turbidostat or chemostat type of culture control modes within a photobioreactor type culture system or any type of microalgae production system allowing a large and controlled mixing of the reaction medium to ensure homogeneity of the received photon flux per cell.

The cells capable of photosynthesis may be cells of prokaryotic or eukaryotic organisms such as algae, and in particular microalgae or macroalgae. According to a variant of the invention, said organisms are microalgae, such as those belonging to the classes Pinguiophyceae, Chrysophyceae, Bacillariophyceae, Mamiellophyceae, Prymnesiophyceae, Haptophyceae or Coccolithophyceae.

For the purposes of the production of fucoxanthin according to the invention, the cells are advantageously microalgae cells of the species Tisochrysis lutea of the class of Prymnesiophyceae or of the species Phaedactylum tricornutum of the class Bacillariophyceae and the normalized luminous power relative to the applied dry biomass is at least 2 μmoles.g ^{- 1} .s ^-1 and at most 30 μmoles.g ^{- 1} .s-. ¹ Such a process is illustrated in the following examples in support of the figure which represents a standard curve of fucoxanthin concentration produced in the biomass as a function of the parameter value.

In an interesting application of the process of the invention, the pigment is fucoxanthin, the cells are microalgae cells of the species Tisochrysis lutea or the species Phaedactylum tricornutum and the normalized luminous power relative to the applied dry biomass is at least 15 μmoles.g ^{- 1} .s ^-1 and at most 25 μmoles.g ^{- 1} .s ^-1 . Example 1 Determination of the Normalized Light Output with Respect to the Dry Biomass to be Applied to the Culture

As indicated previously, the parameter quantity of photons per unit of dry biomass in the production system and per unit of time is dependent on the pigment in question. To a lesser extent, it is also dependent on the organism whose cells are cultured.

This is why the determination of this parameter is carried out by experimentation, which however falls within the general skills of a person skilled in the art. A perfectly reproducible determination technique is hereinafter described.

Different values of this parameter, that is to say μιηοΐθ of photons per gram of dried biomass per second, are applied to pigment productions by cells capable of photosynthesis and the pigment contents thus produced are quantified.

Thus, such experiments are exemplified in the case of fucoxanthin production by a culture of T. lutea microalgae and illustrated by the curve shown in FIG.

It follows from this curve that the peak of production is obtained with a parameter value of the order of 20 μmol of photons per gram of dry biomass per second.

Example 2 Process for producing fucoxanthin from microalgae cultures

This example relates to the production of fucoxanthin from microalgae cultures of the species Tisochrysis lutea of the class Prymnesiophyceae and Phaedactylum tricornutum of the class Bacillariophyceae.

The cultures are carried out under the same conditions which are detailed below:

The culture medium used is mineral, conventionally used for the culture of marine microalgae species, f / 2 type (RRL Guillard and JH Ryther, 1962, Gran Can J. Microbiol 8, 229-239) sterilized by autoclaving. . The cultures are carried out in photobioreactors of 10 L capacity, previously sterilized.

Aeration and agitation of crops are provided by a bubbling system with a flow rate of 10 L / h ± 1 of air, 1.5% of this flow is C0 ₂ .

The culture temperature is 25 ° C ± 1 ° C.

The pH of the cultures is 7.5 ± 0.5.

Crop lighting is provided by neon lights and lighting is provided 24 hours a day.

The inoculation rates applied allow an initial cell density of cultures of 1 to 2.10 ⁶ cells / ml.

The monitoring of cultures are daily after sterile samples.

Quantified parameters are:

- The cell density by counting on Malassez's blade

Absorbance at 880 nm by spectrophotometry

- The concentration of dry biomass by centrifugation of a known volume, rinsing of the pellet and drying in an oven until stabilization of the residual mass

- The concentration of P0 ₄ ^{2 "} by colorimetric test

- The concentration in N0 ₃ ^" by colorimetric test

- The light intensity coming in and out of crops via a quantum meter.

Two cultures of the microalga P. tricornutum are carried out in an identical manner and without nutrient deficiency, with the exception of the mode of management of the applied light intensity: a culture being carried out in a conventional manner and a culture being carried out according to the method of the invention.

Six cultures of the T. lutea microalgae are carried out identically and without nutrient deficiency, with the exception of the applied light intensity management mode: three cultures are carried out in a conventional manner and three cultures are conducted according to the method of the invention.

The management of the so-called conventional light is characterized by the application of a constant light intensity of 100 μΐΎΐοΙ.ητ ² ^ ^"1 to the culture and therefore of a value of μιηοΐθ of photons per cell per second, which will decrease at during the culture because of the increase of the cell density.

The light management method of the invention involves the adaptation of the light intensity applied to the crop, by determining a normalized light power with respect to the dry biomass as a function of the concentration. cellular; the amount of photon per cell per second is thus kept constant. Since the pigment to be overproduced is a photosynthetic pigment, it is advantageously chosen in a range of between 2 and 30 μmoles of photons per gram of dry biomass per second. It emerges from example 1 that it is of the order of 20 μmol.g ^-1. s ^-1 . It is applied as early as the growth phase of primed culture.

For each crop, the biomass is harvested at the beginning of the stationary phase, the fucoxanthin is extracted and quantified by HPLC. The fucoxanthin concentrations of the dry biomasses from the cultures, expressed as a percentage (m: m), as well as the fucoxanthin productivity, expressed in mg per liter of culture medium and per day, are compared according to the management mode of the light tracking. The results are shown in the following Table 1.

Table 1

From Table 1 it can be seen that, compared to conventional cultures, the method of the invention allows:

- »An increase in fucoxanthin levels by a factor of 2 to 2.4 and -» An increase in fucoxanthin productivity by a factor of 2 to 2.4, due to a conservation of biomass productivities.

Example 3 Process for producing fucoxanthin from microalgae cultures on an industrial scale

The work focuses on the management of normalized light output compared to dry biomass in a microalgae culture (Tisochrysis lutea).

The culture medium used is mineral, of f / 2 type as in Example 2.

The culture systems are industrial photobioreactors (CAMARGUE) of 4730 L capacity, previously sterilized. The aeration of the cultures is ensured with a flow rate of 20L / min of air in cocurrent of the culture as described in the patent application WO2010 / 109108A1.

The culture temperature is 25 ° C ± 5 ° C.

The pH of the cultures is 7.5 ± 0.5. The pH of the cultures is automatically regulated by the injection of C0 ₂ .

The crop lighting is of natural origin (sun), the incoming light intensity is regulated by opening-closing protective curtains constituting the climatic greenhouse in which is implanted photobioreactor.

The monitoring of cultures are daily after sterile samples.

Quantified parameters are:

- The cell density by counting on Malassez's blade

Absorbance at 880 nm by spectrophotometry

- The concentration of P0 ₄ ^2- by colorimetric test

- The concentration in N0 ₃ - by colorimetric test

- The light intensity coming in and out of crops via a quantum meter

Two cultures of the T. lutea microalgae are carried out identically and without nutrient deficiency, with the exception of the mode of management of the applied light intensity: a culture being carried out in a conventional manner and a culture being carried out according to the method of the invention.

The so-called conventional light management is characterized by the application of an unregulated light intensity to the culture and therefore a value of μmole of photons per cell per second variable during the culture due to the increase of cell density.

On the other hand, the light management method involves the adaptation of the light intensity applied to the culture as a function of the cell concentration: the quantity of photon per cell and per second is thus kept constant, and this value is of the order of 20 μmol of photons per gram of dry biomass per second, from the primed culture growth phase.

For each crop, the biomass is harvested at the beginning of the stationary phase, the fucoxanthin is extracted and quantified by HPLC. Concentrations in fucoxanthin, dry biomass from the cultures, expressed as a percentage (m: m), as well as fucoxanthin productivity, expressed in mg per liter of culture medium and per day, are compared according to the modality of the management of the light. The results are shown in the following Table 2.

Surprisingly, it can be seen from Table 2 that, compared to cultures carried out according to a conventional method, the method of the invention makes it possible:

- »An increase in fucoxanthin levels by a factor of 1.8 and -» An increase in fucoxanthin productivity by a factor of 2.4.

Claims

1. A process for producing pigments by cells capable of photosynthesis, according to which said cells are cultivated under conditions suitable for their development, in order to obtain a biomass, characterized in that the process comprises a step of overproduction of said pigments according to which one applies said cells have a light power normalized with respect to the predetermined dry biomass, said light power, expressed in μmoles of photons per gram of said dry biomass per second, being at least 2 μmol.g ^-1. s ^-1 and at most 200 μmoles.g ^-1. s ^-1 .

2. Method according to claim 1, characterized in that the cells capable of photosynthesis are cells of prokaryotic or eukaryotic organisms.

3. Method according to claim 2, characterized in that said organisms are microalgae, such as those belonging to the classes of Pinguiophyceae, Chrysophyceae, Bacillariophyceae, Mamiellophyceae, Prymnesiophyceae, Haptophyceae or Coccolithophyceae.

4. Method according to any one of the preceding claims, characterized in that the pigments are chosen from photosynthetic carotenoids and phycobilins and the normalized luminous power with respect to the applied dry biomass is at least 2 μmol.g ^-1. s ^-1 and at most 150 μmoles.g ^-1. s ^-1

5. Method according to any one of the preceding claims, characterized in that the pigments are photosynthetic xanthophylls and the normalized light power relative to the applied dry biomass is at least 5 μmol.g ^-1. s ^-1 and at most 30 μmoles.g ^-1. s ^-1 .

6. Process according to any one of the preceding claims, characterized in that the pigment is fucoxanthin, the cells are microalgae cells of the species Tisochrysis lutea or of the species Phaedactylum tricornutum and the light power normalized with respect to the applied dry biomass is at least 15 μmol.g ^-1. s ^-1 and at most 25 μ μmoles.g ^-1. s ^-1

7. Method according to any one of the preceding claims, characterized in that the pigments are chosen from photoprotective carotenoids and the normalized luminous power with respect to the applied dry biomass is at least 30, preferably at least 50 μmoles. .g ^-1. s ^-1 .

8. Method according to any one of the preceding claims, characterized in that the normalized light power relative to the dry biomass is kept constant by variation of the photon flux applied to the crop or variation of dry biomass within the culture. .

9. Method according to any one of the preceding claims, characterized in that applying the normalized light power relative to the dry biomass during all or part of the exponential growth phase of the cell culture.