FR2789399A1 - Preparing extracts from photosynthetic microorganisms, useful in nutritional, cosmetic and therapeutic compositions, comprises lysing cells grown on induction medium - Google Patents

Abstract

Production of an extract (A) of a photosynthetic microorganism comprises: (i) culturing the microorganism; (ii) transferring the cells to induction medium to increase production of metabolites; (iii) lysing the cells, in the cold; (iv) mixing the lysate with a separation medium; and (v) separating soluble and insoluble fractions of the lysate by tangential filtration, in the cold, to produce a crude extract. An Independent claim is also included for (A) produced using the new method and to which has been added at least one of honey, royal jelly and propolis.

Description

Method for manufacturing extracts of microorganisms

  photosynthetics such as in particular spirulina.

  The present invention relates to methods of manufacturing extracts of photosynthetic microorganisms (purple bacteria, cyanobacteria and microalgae). Such extracts can in particular be used for food, cosmetic and / or therapeutic purposes. In addition, these extracts constitute a basis for the purification of active ingredients or

  metabolites of economic interest.

  By way of example of a microorganism to which the method according to the invention can be applied, mention may be made of spirulina (a filamentous cyanobacterium comprising two species, Arthrospira platensis and Spirulina maxima) known for its nutritional qualities (richness in proteins in fatty acids essential, in sulfated polysaccharides and vitamins) and producing biologically active compounds (antioxidants, anticancer, antiviral, immunostimulants, bifidogen). It will however be understood that the invention may of course be applied to other photosynthetic microorganisms. Currently, spirulina is sold in the form of dry powder which is the preferred form of preservation of cyanobacteria and microalgae. Drying uses a thermal process, i.e. natural drying

  in the sun for a very long time, either forced drying at 60-70 C or more.

  This process if it allows the conservation of a food quality of spirulina, leads to many biochemical degradations and therefore different activities of spirulina. In addition, this form of conservation tends to reinforce the solidity of the wall of these microorganisms and therefore to reduce the availability of proteins and active molecules, the majority

  cells remaining intact during their adsorption or during their use.

  The main objective of the present invention is therefore to provide a microorganism powder allowing total availability of

  active molecules, and cellular content.

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  In particular, an objective of the present invention is to propose a new type of extract of microorganism and in particular of spirulina, of which

  the organoleptic and / or biochemical qualities are not degraded.

  Also one of the objectives of the present invention is to describe such a process which does not implement a temperature rise and which

  allows to preserve all the intrinsic biological qualities of micro-

  organisms and in particular Spirulina.

  These various objectives, as well as others which will appear subsequently, are achieved thanks to the invention which relates to a process for the manufacture of an extract of at least one photosynthetic microorganism characterized in that it comprises the steps of : culture of said microorganism in a liquid culture medium adapted to the nutritional needs of said microorganism, physiological induction consisting in placing said culture in a specific liquid induction medium in order to cause said microorganism to produce an increased amount of metabolites; cold grinding of said culture so as to obtain a ground product thereof; mixing of said ground material in a separation medium and separation of the insoluble part from the soluble part of said ground material by cold tangential filtration

  allowing the obtaining of a crude extract.

  The invention therefore provides a process which can be carried out without heating and without chemical treatment. All biological qualities

  of the treated microorganism can thus be preserved.

  According to a preferred variant of the invention, said culture medium, as well as where appropriate the induction and extraction media, have controlled contents of heavy metals. Indeed, photosynthetic microorganisms have the distinction of accumulating heavy metals in their polysaccharide wall. Some of these metals, such as mercury,

  lead or chromium are toxic. As a result, the powders of these micro-

  organisms can be unfit for human consumption and also for the therapeutic use that can be made of them. The use of a medium

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  culture with controlled heavy metal contents opposes

  extensive cropping systems commonly used around the world.

  Similarly, some pesticides have an action on the human and animal organism, without having any on the growth of micro-algae, they can accumulate or even degrade in a more toxic form when these cultures are carried out in an environment. natural or in an uncontrolled culture medium. This is why the culture step is preferably carried out in a photobioreactor isolated from the outside, the parameters of which

physico-chemical are controlled.

  The second stage of the process (physiological induction stage) consists in directing the metabolism of microorganisms towards the production of products useful for the chosen purposes. If the culture medium of the first stage is optimized for the production of biomass, it is not for the production of active molecules and / or of economic interest. The biomass of the first stage is recovered and placed in a second controlled medium allowing the overexpression of molecules of interest compared to others. For example in the case of spirulina, the simple fact of putting it in the presence of sodium chloride at 0.5 M is sufficient to cause an overexpression of phycocyanin. The physiological induction medium is subjected to the same control conditions as the culture medium. This physiological induction also has the objective of promoting the synthesis of proteins and metabolites specific to stress and which have the property of promoting the conservation of the extracts. Physiological induction for products intended for food and cosmetic purposes will preferably be carried out "in seawater sterilized by ultrafiltration. Water

  allows the overexpression of phycobiliproteins and the intake of oligo-

  elements. This seawater must correspond to the criteria defined above, that is to say, have a concentration of toxic heavy metals

  slightly lower than the admissible concentration on the final product.

  The physiological induction medium is adapted to the purpose of the product, we

  could, for example enrich the culture with nitrate, sulfate or even oligo-

4.

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  elements to induce the expression of certain metabolic pathways

  specific, and / or accumulate trace elements.

  The third step in the process consists of grinding the cells. This step is preferably carried out under controlled pH. After culture and physiological induction, the cells are separated from the medium by mechanical means, washed with sterile water and suspended in an aqueous medium at a known concentration and ground. This grinding is carried out cold (at 4 C for

  spirulina) by mechanical means. Sterile water is the preferred solvent.

  In this case, the biomass is taken up in 2 to 3 times its volume of sterile water.

  The objective is to break down the walls so as to release the cellular content and its active products such as proteins, water-soluble vitamins, phycobiliproteins, carbohydrates and free polysaccharides in soluble form. This lysis of the cell makes it possible to have these nutrients in a form immediately assimilable by the body, unlike whole cells which can be poorly digested. To avoid the rush of

  certain proteins, the pH is controlled at this stage.

  The fourth step consists in separating the soluble part from the insoluble part. This step makes it possible to obtain a clear lysate free of unbroken cells, fragments of membranes and also nucleic acids of very high molecular weight. This separation is done by tangential filtration, preferably by tangential microfiltration. The filtrate

  obtained is the raw extract. This can then be valued in several ways.

  For short-term use, this raw extract will be filtered by frontal filtration

  or tangential ultra-filtration at 0.2 micrometers and sterile conditioned.

  For long-term use an authorized preservative may be added. The

  retentate and / or the extract may be lyophilized.

  It will be noted that all the fractionation stages take place cold, which allows the conservation of all the heat-sensitive molecules (vitamins, proteins, etc.). This raw extract also allows mixing with other natural products or extracts, for use as a food supplement, for example we can add to it bee products (honey, jelly

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  royal, propolis) in which case we can do without preservatives. The upgrading of the filtrate can involve purification processes in order to isolate a particularly advantageous fraction. For example, nanofiltration or reverse osmosis can be used to isolate oligosaccharides, antioxidant proteins, vitamins, peptides, hormones or growth factors. The purification can be continued by liquid chromatography. The retentate containing the membranes can be upgraded by additional purification steps. This fraction includes liposoluble molecules such as vitamins, lipoproteins, polysaccharides (anti-virals, anti-inflammatories, immunostimulants), rare essential fatty acids such as gamma-linoleic acid (compulsory for the development of the animal but not synthesized by him), carotenes (antioxidants) and hormones (precursors of prostaglandins and steroids). In addition to their nutritional richness, these molecules constitute the basis of dietary formulations. In addition, these molecules have important therapeutic activities, applications for which it is necessary to obtain

  pure molecules to allow the dosage.

  The method according to the invention, as well as the various advantages it

  present will be more easily understood thanks to the nonlimiting description

  which will follow from a nonlimiting example of implementation thereof

  relating to the manufacture of a liquid extract of Spirulina.

  Spirulina cells are cultivated in a photobioreactor constituted by a transparent cylindrical column letting pass the light rays necessary for photosynthesis. The culture medium used was Zarouk's medium, the composition of which is given below: NaHCO3: 16.8 g / l K2 HPO4: 0.5 g / l Na NO3: 2.5 g / MgSO4.7 H2O: 0.2 g / l K2SO4: 1 g / 1

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NaCI: 1 g / l.

  The cells are removed at the end of the exponential phase (1.5 to 2.5 units of optical density at 750 nm) by filtration on a 120 micron sieve and re-cultured in the physiological induction medium, which in this case is sea water sterilized by 0.2 micron filtration. An addition of carbonate is necessary to correct the pH as well as a nitrogen source favoring the synthesis of pigments. This stage lasts 12 hours. A variant of the process consists in adjusting the trace elements of seawater by adding some of them. In this context, zinc, copper and manganese were measured before and after adjustment. The results are given below Before: - Zn: 30 mg / Kg - Cu: 10 mg / Kg - Mn: 30 mg / Kg After: - Zn: 810 mg / Kg - Cu: 1540 mg / Kg - Mn: 1540 mg / Kg The spirulina cells are then harvested by sieving at 120 microns

  washed three times with demineralized water and frozen at -40 C in the absence of air.

  The frozen dough is then crushed and dissolved in twice its volume of sterile water at 4 ° C. and homogenized. The solution is then subjected to mechanical grinding or sonication until the cells have completely burst. This operation is carried out at a temperature of +2 C in order to avoid denaturation of the biological material. A pH control is necessary to avoid the precipitation of certain proteins, in particular phycocyanin. The ground material is then clarified by tangential microfiltration at 0.5 micron. the filtrate is sterilized by frontal filtration or by 0.2 micron ultrafiltration. This fraction is sterile packaged in ampoules or vials with or without preservatives for end use. The retentate is

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  lyophilized and packaged in sachets or capsules for later use. This embodiment is not intended to reduce the scope of the invention. Many modifications can therefore be made without departing from its scope. In particular, it should be noted that this process could be transposed to other micro-algae, in particular other cyanobacteria as well

  than any other suitable microorganism.

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Claims (12)

  1. Method for manufacturing an extract of at least one photosynthetic microorganism characterized in that it comprises the steps of: - culture of said microorganism in a liquid culture medium adapted to the nutritional needs of said microorganism, - physiological induction consisting in placing said culture in a particular liquid induction medium in order to cause said microorganisms to produce an increased quantity of metabolites; - cold grinding of said culture so as to obtain a ground product thereof; mixing of said ground material in a separation medium and separation of the insoluble part from the soluble part of said ground material by cold tangential filtration   allowing the obtaining of a crude extract.   2. Method according to claim 1 characterized in that all of said culture, induction and extraction media have contents tested in heavy metals.   3. Method according to any one of claims 1 or 2 characterized   in that said culture step is carried out in an isolated photobioreactor   from the outside whose physico-chemical parameters are controlled.   4. Method according to any one of claims 1 to 3 characterized   in that said induction medium comprises sea water.   5. Method according to any one of claims 1 to 4 characterized   in that it comprises an additional step of taking up the biomass originating from the first step in two or three times its volume of sterile water.   6. Method according to any one of claims I to 5 characterized   in that it comprises a step consisting in controlling the pH of the medium during   said grinding step to avoid precipitation of certain proteins.   7. Method according to any one of claims I to 6 characterized   in that said separation step is carried out by microfiltration tangential. 9 2789399   8. Method according to any one of claims 1 to 7 characterized   in that it comprises a final additional stage of sterilization of said extract by frontal filtration or ultra-filtration tangential to 0.2 micrometers of said raw extract.   9. Method according to any one of claims 1 to 8 characterized   in that it comprises a step of lyophilization of the crude extract, and / or retentate.   10. Method according to any one of claims 1 to 9 characterized   in that it comprises a step of purifying the filtrate by nano-   filtration or reverse osmosis to isolate a fraction   particularly interesting of it.   11. Method according to any one of claims I to 10 characterized   in that said microorganism is Arthrospira platensis or Spirulina maxima.   12. Extract of photosynthetic microorganism obtained according to the process   according to one of claims I to 11 1 characterized in that it   add at least one compound chosen from the group consisting of   honey, royal jelly, propolis.