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EP3908652A1 - Process for extracting phycocyanins - Google Patents

Process for extracting phycocyanins

Info

Publication number
EP3908652A1
EP3908652A1 EP20700136.3A EP20700136A EP3908652A1 EP 3908652 A1 EP3908652 A1 EP 3908652A1 EP 20700136 A EP20700136 A EP 20700136A EP 3908652 A1 EP3908652 A1 EP 3908652A1
Authority
EP
European Patent Office
Prior art keywords
phycocyanin
phycocyanins
solution
concentration
precipitation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20700136.3A
Other languages
German (de)
French (fr)
Inventor
Olivier CAGNAC
Axel ATHANE
Julien DEMOL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fermentalg SA
Original Assignee
Fermentalg SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fermentalg SA filed Critical Fermentalg SA
Publication of EP3908652A1 publication Critical patent/EP3908652A1/en
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/40—Colouring or decolouring of foods
    • A23L5/42—Addition of dyes or pigments, e.g. in combination with optical brighteners
    • A23L5/46—Addition of dyes or pigments, e.g. in combination with optical brighteners using dyes or pigments of microbial or algal origin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/405—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from algae
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/89—Algae ; Processes using algae

Definitions

  • the present invention relates to a new process for extracting and purifying phycocyanins produced by fermentation of microalgae, in particular produced by Galdieria sulphuraria, by selective precipitation.
  • Phycocyanin extraction processes generally consist in precipitating organic matter other than phycocyanins present in an aqueous crude extract resulting from a fermentation of microalgae to preserve the phycocyanins in the supernatant which will be filtered before precipitating the phycocyanins (JP 2004359638).
  • certain organic compounds, in particular complex polysaccharides such as glycogen remain soluble under the same conditions as phycocyanins.
  • a filtration step (ultrafiltration) can be used to remove the water in order to concentrate the phycocyanin and remove small molecules (proteins, ions, organic acid, etc.) whose size is smaller. at the cutoff threshold of the filter used, to obtain the purest phycocyanin possible.
  • the cutoff threshold of the filter being lower than the size of the glycogen, this is not eliminated and increases the viscosity of the retentate, reducing the filtration rates.
  • the viscosifying effect of glycogen as a function of its concentration has been demonstrated using purified glycogen from Galdieria sulphuraria (Martinez-Garcia et al., 2017).
  • the purified phycocyanins obtained retain high levels of these sugars which can alter the qualities of the phycocyanins, in particular their coloring power, requiring the production and / or use of larger quantities of phycocyanins for the same effect.
  • These residual polysaccharides behave like a filler which increases the costs of manufacturing phycocyanin and can limit the commercial uses of the phycocyanin obtained, for example for the preparation of foods which have a low sugar content.
  • the method according to the invention consists in performing a selective precipitation of the phycocyanins directly from the crude extract which contains them under conditions which respect the integrity of the phycobilliproteins and which allow the main impurities, in particular the polysaccharides, to be kept in solution. including glycogen.
  • This selective precipitation results from a combined action on two factors, simultaneously or in sequence in an indifferent order, on the one hand the pH of the solution and on the other hand the phycocyanin concentration.
  • the process according to the invention is particularly suitable for the purification of phycocyanins resistant to acidic pH produced by Galdieria sulphuraria.
  • the invention relates to a process for extracting phycocyanins from a solution comprising the phycocyanin (s), also called initial phycocyanin solution, comprising a selective precipitation step which consists firstly in adjusting the pH of the initial solution at a value chosen in a range of pH values in which the phycocyanins are less soluble (also called instability range) and on the other hand to concentrate the phycocyanins in the solution to promote their precipitation, then a step of recovering the precipitated phycocyanin.
  • a selective precipitation step which consists firstly in adjusting the pH of the initial solution at a value chosen in a range of pH values in which the phycocyanins are less soluble (also called instability range) and on the other hand to concentrate the phycocyanins in the solution to promote their precipitation, then a step of recovering the precipitated phycocyanin.
  • the two pH adjustment and concentration actions can be implemented simultaneously or in sequence, either by adjusting the pH of the initial solution before concentrating, or by concentrating the initial solution before adjusting the pH.
  • the conditions of differential concentration mean that only the phycocyanins precipitate, the other products which can be described as impurities, especially the polysaccharides, remain in solution.
  • the process according to the invention is particularly suitable for the purification of a phycocyanin solution extracted from a culture of phycocyanin-producing microorganism which also produces glycogen, in particular within the framework of an industrial process for the production of phycocyanin which comprises culturing the microorganisms, then recovering the biomass produced to extract phycocyanin, and recovering phycocyanin from this biomass.
  • the process is particularly suitable for phycocyanins produced by microorganisms which produce high glycogen contents, in particular for the extraction and purification of phycocyanins from a biomass which comprises more than 10% glycogen relative to the total dry matter.
  • Phycocyanin-producing microorganisms are well known, in particular algae (or microalgae) of the orders of Cyanidiales.
  • the order of Cyanidiales includes the families of Cyanidiaceae or Galdieriaceae, themselves subdivided into the genera Cyanidioschyzon, Cyanidium or Galdieria, to which belong among others the species Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarum, Cyanidium maximum, Cyanidium , Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita or even Galdieria sulphuraria. Mention will in particular be made of the strain Galdieria sulphuraria (also called Cyanidium caldarium (UTEX 2919).
  • microorganisms which produce phycocyanin with a high glycogen content are more particularly identified among the microorganisms mentioned above, in particular the species of the genera Cyanidioschyzon, Cyanidium or Galdieria, more particularly Galdieria sulfuraria.
  • the recovery of phycocyanin from biomass is also known from the skilled person. Mention may in particular be made of patent application WO 2018/178334. It generally requires a cell, mechanical or enzymatic lysis step in order to release the phycocyanin produced in the cellular compartments of the microorganisms. This lysis is advantageously carried out at a pH favorable to the solubilization of phycocyanins. This cell lysis will generally generate a phycocyanin solution which comprises organic matter in suspension (called crude suspension) which can be separated by usual filtration methods. A crude phycocyanin solution is then obtained which can be further purified so as to remove low molecular weight organic residues by usual ultrafiltration methods to obtain a refined solution from which phycocyanin will be obtained.
  • crude suspension organic matter in suspension
  • a crude phycocyanin solution is then obtained which can be further purified so as to remove low molecular weight organic residues by usual ultrafiltration methods to obtain a refined solution from which phycocyanin will be obtained.
  • the process according to the invention is particularly suitable for the purification of a solution of phycocyanins resistant to acidic pH, in particular the phycocyanins described in application WO 2017/050918.
  • the process according to the invention is implemented for the purification of phycocyanins resistant to acid pH produced by Galdieria sulphuraria, more particularly in a process for the industrial production of these phycocyanins by culture in a fermenter of Galdieria sulphuraria.
  • the method is advantageously implemented for extracting phycocyanin from the raw juice obtained from a biomass of microorganisms producing phycocyanin.
  • the initial solution of phycocyanin in particular the raw juice, comprises from 0.1 to 10 g / L of phycocyanin.
  • the concentration consists in eliminating the water so as to obtain a phycocyanin content of at least 15 g / L, preferably at least 20 g / L, more preferably at least 30 g / L, or even at least 40 g / L.
  • This concentration can be defined in% of volume loss depending on the content of phycocyanins in the initial solution.
  • the raw juice will advantageously comprise at least 1 g / L of phycocyanins.
  • the concentration will consist in eliminating at least 93% of the initial volume of liquid.
  • the concentration is done by any known method to allow the elimination of water under conditions which preserve the integrity of the phycocyanins. Mention may be made of the methods of evaporation of water, in particular under reduced pressure to promote this evaporation under temperature conditions which respect the integrity of phycocyanins, without affecting their coloring power. We can also cite methods which make it possible to eliminate a liquid, such as tangential filtration with pore sizes which allow the passage of water and small molecules in solution but retain the proteins.
  • pore filters from 50 kD to 100 kD will advantageously be chosen, in particular polyethersulfone or polysulfone filters.
  • Adjusting the pH involves adding an acid or base to the initial solution or to the concentrated solution so as to reach a pH value within the instability range.
  • the range of instability will depend on the phycocyanins to be purified, including the microorganism that produced it. In general, this instability range is from 4.5 to 5.5, in particular for phycocyanins resistant to acidic pHs as described above.
  • cell lysis is carried out at an acidic pH, preferably less than 4.5, generally around 4, or even up to 3.
  • Adjusting the pH then involves adding a base to reach the pH in the instability range.
  • the method first consists in concentrating the initial juice.
  • the concentration is carried out at a pH favorable for the solubilization of phycocyanins, that is to say outside the instability range.
  • these pHs favorable to the solubilization of phycocyanins will advantageously be less than 4 or more than 5.
  • the method consists first of adjusting the pH to the instability range and then concentrating the solution until the phycocyanins precipitate.
  • This selective precipitation step is advantageously carried out at room temperature.
  • those skilled in the art can modify the temperature so as to promote precipitation, for example by lowering the temperature to implement the second part of the step (concentration or adjustment of the pH) during which the precipitation has location.
  • polysaccharides in solution in particular glycogen
  • glycogen can then be recovered by usual methods of precipitation of polysaccharides, for example by adding ethanol (Martinez-Garcia et al., 2016), these polysaccharides can also then be purified.
  • the polysaccharides contained in the initial solution with the phycocyanins are the subject of an enzymatic lysis which promotes their maintenance in solution.
  • the traces of these polysaccharides liable to be entrained with the precipitation of the already weak phycocyanins are even more reduced when the polysaccharides are lysed into even more soluble low molecular weight polysaccharides.
  • the concentration step is carried out by tangential filtration, the low molecular weight polysaccharides are eliminated with the other small molecules in solution, which favors obtaining a phycocyanin solution with an even higher content.
  • the enzymatic lysis of glycogen is carried out at a pH less than or equal to 5, preferably about 4.5, at room temperature.
  • the enzymes active under acidic pH conditions and at room temperature are chosen from enzymes known for glucuronidase a1-4, glucosidase a1-4 (or alpha-glucosidase) activity. Mention will in particular be made of pectinases known to degrade pectin and in particular pectinases extracted from filamentous fungi such as Aspergillus, more particularly from pectinases extracted from Aspegillus aculeatus, such as the enzymes sold under the name Pectinex® by the company Novozymes.
  • the enzymatic lysis of glycogen can also be carried out with a glucosidase a1-6 in addition to glucuronidase a1-4 or glucosidase a1-4.
  • Glucosidases a1-6 active under the pH and temperature conditions set out above are also known to those skilled in the art. These are in particular the pullulanases known to hydrolyze the glucosidic bonds a1-6 of the pullulan, in particular known to suppress the ramifications of starch.
  • pullulanase / alpha-amylase mixtures are described in the state of the art, but in particular for producing glucose syrup from starch (US 2017/159090). Those skilled in the art will know how to determine the appropriate reaction conditions to best reduce the amounts of glycogen as a function of the initial glycogen content in the solution to be treated, the amount of enzymes used and the purity sought for the phycocyanin produced.
  • the recovery of solid precipitated phycocyanins is carried out by any method known to a person skilled in the art, such as filtration or centrifugation.
  • a person skilled in the art may consider any method of recovering solids so as to reduce the volume to be treated by filtration or centrifugation.
  • This recovery can be done discontinuously, in batches, or even continuously, the addition of initial solution to compensate for the recovery of solid phycocyanins.
  • This continuous recovery step will advantageously be carried out with a concentration by tangential filtration on a solution of unstable pH, the skilled person being able to adjust the respective flow rates of water removal and supply of solution of unstable pH for favor the precipitation of phycocyanins.
  • Such a continuous process will be particularly suitable for treating initial solutions in which the polysaccharides and in particular the glycogen have undergone an enzymatic lysis which promotes their elimination by tangential filtration with water and the other small soluble molecules.
  • the invention also relates to a method for producing phycocyanins by fermentation of microorganisms, said method comprising the following steps of (i) culturing the microorganisms to obtain a biomass rich in phycocyanin, (ii) recovering the biomass and cell lysis to dissolve the phycocyanins released in a suspension of cellular particles, (iii) clarification of the suspension previously obtained to obtain a crude solution of phycocyanin and (iv) recovery of phycocyanin from the crude solution previously obtained, characterized in that the recovery of the phycocyanin comprises a selective precipitation step as defined above.
  • the solid recovered can then be dried by any suitable method, and if necessary ground.
  • the recovered solid comprising phycocyanin can also be subjected to purification by methods known to those skilled in the art, such as diafiltration.
  • the selective precipitation implemented according to the invention makes it possible to globally reduce the energy necessary for the production of powdered phycocyanin from of an initial solution, in particular from a raw juice, both on the quantity of material to be handled and on the energy necessary for the drying of the solid phycocyanin and its grinding.
  • the phycocyanin obtained by this process has a purity index of at least 2, preferably at least 3, or even greater than 4.
  • This purity index is measured by absorbance measurement with the method described by Moon & al. (2014).
  • the phycocyanin obtained is a phycocyanin which has a glycogen / phycocyanin ratio (by dry weight) of less than 6, advantageously less than 4, preferably less than 3, more preferably less than 2.5, even more preferably less than 1.
  • the invention also relates to the use of the phycocyanins obtained as coloring agents, in particular as food coloring agents. It also relates to foodstuffs, solid or liquid, in particular drinks which comprise a phycocyanin with a low glycogen content according to the invention.
  • Figure 1 shows the mass of the precipitate obtained at different concentrations of phycocyanin and different pH in the initial solution.
  • FIG. 2 represents the concentration of phycocyanin in the supernatant after recovery of the precipitate as a function of the pH for different concentrations of phycocyanin.
  • Galdieria sulphuraria also called Cyanidim caldarium
  • the biomass is obtained by fermentation in Fedbatch mode using the conditions described in patent WO2017050918A1.
  • PC Phycocyanin
  • the crude PC extract is recovered after separation of the cellular debris by centrifugation at 10,000 g for 10 min at room temperature.
  • the crude extract is concentrated by tangential filtration with a ceramic membrane or an organic membrane with a cut-off threshold allowing phycocyanin to be retained.
  • the samples are then centrifuged in order to separate the precipitate from the supernatant.
  • the mass of the base is measured using a balance of precision.
  • the pellet is re-suspended in an aqueous solution of pH 7 allowing its resolubilization, this in order to be able to quantify the precipitated phycocyanin.
  • a crude phycocyanin solution with an initial concentration of 1 g / l of PC and an initial purity of 1.6 is concentrated by tangential filtration at pH 4 until a retentate with a concentration of 20 g / l is obtained, then 30 g / l then 45 g / l.
  • a retentate with a concentration of 20 g / l is obtained, then 30 g / l then 45 g / l.
  • this purity does not exceed the value of 2 despite the degree of concentration of the product.
  • the precipitation of phycocyanin is weak at pH 4 and increases slightly while making evolve the pH towards higher values (Fig. 1).
  • Table 1 reports the measurement of the purity of phycocyanin after re-solubilization of the phycocyanin precipitate for precipitation at pH 7.5.
  • Example 2 Effect of the concentration and of the pH on the precipitation and the purification of the PC on an enzymatically digested sample.
  • the crude solution is subjected to an enzymatic digestion in order to degrade the glycogen present.
  • precipitation is important for a pH range between 4.5 and 5.5, and the level of purity in this case reaches values greater than 3.8 during the resolubilization of the precipitated phycocyanin.
  • Enzymatic digestion of glycogen does not affect precipitation purification.
  • Table 2 below gives the values of the purity index of phycocyanin after precipitation by adjustment to the instability pH but also after re-solubilization of the phycocyanin precipitate.

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Abstract

The invention relates to a new process for extracting and purifying phycocyanins produced by fermenting microalgae, in particular produced by Galdieria sulphuraria, by means of selective precipitation.

Description

PROCÉDÉ D’EXTRACTION DE PHYCOCYANINES PHYCOCYANIN EXTRACTION PROCESS
DOMAINE DE L'INVENTION FIELD OF THE INVENTION
La présente invention concerne un nouveau procédé d’extraction et de purification de phycocyanines produites par fermentation de microalgues, en particulier produites par Galdieria sulphuraria, par précipitation sélective. The present invention relates to a new process for extracting and purifying phycocyanins produced by fermentation of microalgae, in particular produced by Galdieria sulphuraria, by selective precipitation.
ETAT DE LA TECHNIQUE STATE OF THE ART
La purification de phycobiliprotéines extraites de Galdieria sulphuraria et de Spiruline par précipitation au sulfate d’ammonium a déjà été décrite dans la littérature (Moon et al., 2015 ; Cruz de Jesûs et al., 2006, CN 106190853) mais elle est très difficilement applicable à l’échelle industrielle car elle demande beaucoup de sulfate d’ammonium, ce qui pose de gros problèmes de retraitement du sulfate d’ammonium et du surnageant. The purification of phycobiliproteins extracted from Galdieria sulphuraria and Spirulina by precipitation with ammonium sulphate has already been described in the literature (Moon et al., 2015; Cruz de Jesûs et al., 2006, CN 106190853) but it is very difficult applicable on an industrial scale because it requires a lot of ammonium sulfate, which poses major problems of reprocessing of ammonium sulfate and the supernatant.
Les autres méthodes de purification décrites permettant d’obtenir un taux de pureté élevé, telles que des méthodes de chromatographie ou de purification sur résine échangeuses d’ions (JP 2004359638) mais sont très coûteuses à mettre en œuvre. The other purification methods described making it possible to obtain a high level of purity, such as chromatography or purification methods on ion exchange resin (JP 2004359638) but are very expensive to implement.
La précipitation de la phycocyanine de Spiruline a été décrite avec l’ajout d’acide à la solution brute de phycocyanine (TN 2009000406, JP 2004359638, JP06271783, CN 106190853). Toutefois compte tenu du point isoelectrique de la phycocyanine de Spiruline, autour de 4,5 et proche du point isoelectrique d’un grand nombre d’autres protéines, cette méthode ne permet pas de précipiter sélectivement la phycocyanine et donc ne permet pas de la purifier. De même, certains décrivent l’utilisation de l’acide salicylique pour précipiter la phycocyanine de Spiruline (WO 2016/030643). L’utilisation de cet acide génère une précipitation non sélective de la PC et le précipité obtenu est particulièrement difficile à re-solubiliser. Un résultat identique a été obtenu avec la PC de Galdieria. The precipitation of phycocyanin from Spirulina has been described with the addition of acid to the crude phycocyanin solution (TN 2009000406, JP 2004359638, JP06271783, CN 106190853). However, given the isoelectric point of spirulina phycocyanin, around 4.5 and close to the isoelectric point of a large number of other proteins, this method does not make it possible to selectively precipitate phycocyanin and therefore does not allow it to be purified . Similarly, some describe the use of salicylic acid to precipitate phycocyanin from Spirulina (WO 2016/030643). The use of this acid generates non-selective precipitation of the PC and the precipitate obtained is particularly difficult to re-dissolve. An identical result was obtained with the PC of Galdieria.
Les procédés d’extraction des phycocyanines consistent généralement à faire précipiter les matières organiques autres que les phycocyanines présentes dans un extrait brut aqueux issu d’une fermentation de microalgues pour conserver les phycocyanines dans le surnageant qui sera filtré avant de faire précipiter les phycocyanines (JP 2004359638). Toutefois, certains composés organiques, notamment des polysaccharides complexes comme le glycogène, restent solubles dans les mêmes conditions que les phycocyanines. Phycocyanin extraction processes generally consist in precipitating organic matter other than phycocyanins present in an aqueous crude extract resulting from a fermentation of microalgae to preserve the phycocyanins in the supernatant which will be filtered before precipitating the phycocyanins (JP 2004359638). However, certain organic compounds, in particular complex polysaccharides such as glycogen, remain soluble under the same conditions as phycocyanins.
Dans un procédé industriel de purification de phycocyanine, on peut employer une étape par filtration (ultrafiltration) pour éliminer l'eau afin de concentrer la phycocyanine et éliminer des petites molécules (protéines, ions, acide organique, etc.) dont la taille est inférieure au seuil de coupure du filtre utilisé, pour obtenir une phycocyanine la plus pure possible. Toutefois, le seuil de coupure du filtre étant inférieur à la taille du glycogène celui- ci n’est pas éliminé et augmente la viscosité du rétentat, diminuant les débits de filtration. L’effet viscosant du glycogène en fonction de sa concentration a été démontré à partir de glycogène purifié de Galdieria sulphuraria (Martinez-Garcia ét al., 2017). In an industrial process for purifying phycocyanin, a filtration step (ultrafiltration) can be used to remove the water in order to concentrate the phycocyanin and remove small molecules (proteins, ions, organic acid, etc.) whose size is smaller. at the cutoff threshold of the filter used, to obtain the purest phycocyanin possible. However, the cutoff threshold of the filter being lower than the size of the glycogen, this is not eliminated and increases the viscosity of the retentate, reducing the filtration rates. The viscosifying effect of glycogen as a function of its concentration has been demonstrated using purified glycogen from Galdieria sulphuraria (Martinez-Garcia et al., 2017).
De plus les phycocyanines purifiées obtenues conservent des taux élevés de ces sucres qui peuvent altérer les qualités des phycocyanines, notamment leur pouvoir colorant, nécessitant la production et/ou l’utilisation de plus grandes quantités de phycocyanines pour un même effet. Ces polysaccharides résiduels se comportent comme une charge qui vient renchérir les coûts de fabrication de la phycocyanine et peut limiter les usages commerciaux de la phycocyanine obtenue, par exemple pour la préparation d’aliments qui ont une faible teneur en sucres. In addition, the purified phycocyanins obtained retain high levels of these sugars which can alter the qualities of the phycocyanins, in particular their coloring power, requiring the production and / or use of larger quantities of phycocyanins for the same effect. These residual polysaccharides behave like a filler which increases the costs of manufacturing phycocyanin and can limit the commercial uses of the phycocyanin obtained, for example for the preparation of foods which have a low sugar content.
On cherche donc à améliorer les procédés d’extraction et de purification des phycocyanines extraites d’une biomasse, tant d’un point de vue qualitatif que d’un point de vue industriel et économique et notamment en diminuant la teneur résiduelle en sucres dans le produit final, en particulier la teneur résiduelle en glycogènes. We therefore seek to improve the extraction and purification processes of phycocyanins extracted from a biomass, both from a qualitative point of view and from an industrial and economic point of view and in particular by reducing the residual sugar content in the final product, in particular the residual glycogen content.
EXPOSE DE L'INVENTION STATEMENT OF THE INVENTION
Le procédé selon l’invention consiste à effectuer une précipitation sélective des phycocyanines directement à partir de l’extrait brut qui les contiennent dans des conditions respectueuses de l’intégrité des phycobilliprotéines et qui permettent le maintien en solution des principales impuretés, en particulier les polysaccharides dont le glycogène. The method according to the invention consists in performing a selective precipitation of the phycocyanins directly from the crude extract which contains them under conditions which respect the integrity of the phycobilliproteins and which allow the main impurities, in particular the polysaccharides, to be kept in solution. including glycogen.
Cette précipitation sélective résulte d’une action combinée sur deux facteurs, simultanément ou en séquence dans un ordre indifférent, d’une part le pH de la solution et d’autre part la concentration en phycocyanine. This selective precipitation results from a combined action on two factors, simultaneously or in sequence in an indifferent order, on the one hand the pH of the solution and on the other hand the phycocyanin concentration.
Le procédé selon l’invention est particulièrement adapté pour la purification de phycocyanines résistantes aux pH acides produites par Galdieria sulphuraria. The process according to the invention is particularly suitable for the purification of phycocyanins resistant to acidic pH produced by Galdieria sulphuraria.
DESCRIPTION DETAILLEE DE L'INVENTION DETAILED DESCRIPTION OF THE INVENTION
L’invention concerne un procédé d’extraction de phycocyanines à partir d’une solution comprenant la ou les phycocyanines, également appelée solution initiale de phycocyanine, comprenant une étape de précipitation sélective qui consiste d’une part à ajuster le pH de la solution initiale à une valeur choisie dans une plage de valeurs de pH dans laquelle les phycocyanines sont moins solubles (également appelée plage d’instabilité) et d’autre part à concentrer les phycocyanines dans la solution pour favoriser leur précipitation, puis une étape de récupération de la phycocyanine précipitée. The invention relates to a process for extracting phycocyanins from a solution comprising the phycocyanin (s), also called initial phycocyanin solution, comprising a selective precipitation step which consists firstly in adjusting the pH of the initial solution at a value chosen in a range of pH values in which the phycocyanins are less soluble (also called instability range) and on the other hand to concentrate the phycocyanins in the solution to promote their precipitation, then a step of recovering the precipitated phycocyanin.
Les deux actions d’ajustement du pH et de concentration peuvent être mises en œuvres simultanément ou en séquence, soit en ajustant le pH de la solution initiale avant de concentrer, soit en concentrant la solution initiale avant d’ajuster le pH. The two pH adjustment and concentration actions can be implemented simultaneously or in sequence, either by adjusting the pH of the initial solution before concentrating, or by concentrating the initial solution before adjusting the pH.
De manière surprenante, les conditions de concentration différentielle font que seules les phycocyanines précipitent, les autres produits que l’on peut qualifier d’impuretés, en particulier les polysaccharides, restent en solution. Surprisingly, the conditions of differential concentration mean that only the phycocyanins precipitate, the other products which can be described as impurities, especially the polysaccharides, remain in solution.
Ainsi il est possible de récupérer les phycocyanines précipitées en les séparant de la solution, et le cas échéant de les sécher pour obtenir une poudre de phycocyanines purifiées. Thus it is possible to recover the precipitated phycocyanins by separating them from the solution, and if necessary to dry them to obtain a powder of purified phycocyanins.
Non seulement le procédé selon l’invention permet d’extraire les phycocyanines de la solution, mais permet en une même étape de les purifier, les phycocyanines obtenues étant particulièrement pures avec de faibles teneurs en sucres résiduels. Not only does the process according to the invention make it possible to extract the phycocyanins from the solution, but allows them to be purified in the same step, the phycocyanins obtained being particularly pure with low contents of residual sugars.
Le procédé selon l’invention est particulièrement adapté pour la purification d’une solution de phycocyanine extraite d’une culture de microorganisme producteur de phycocyanines qui produit également du glycogène, en particulier dans le cadre d’un procédé industriel de production de phycocyanine qui comprend la culture des microorganismes, puis la récupération de la biomasse produite pour extraire la phycocyanine, et la récupération de la phycocyanine à partir de cette biomasse. The process according to the invention is particularly suitable for the purification of a phycocyanin solution extracted from a culture of phycocyanin-producing microorganism which also produces glycogen, in particular within the framework of an industrial process for the production of phycocyanin which comprises culturing the microorganisms, then recovering the biomass produced to extract phycocyanin, and recovering phycocyanin from this biomass.
Le procédé est particulièrement adapté pour les phycocyanines produites par des microorganismes qui produisent de fortes teneurs de glycogène, en particulier pour l’extraction et la purification des phycocyanines à partir d’une biomasse qui comprend plus de 10% de glycogène par rapport à la totalité de la matière sèche. The process is particularly suitable for phycocyanins produced by microorganisms which produce high glycogen contents, in particular for the extraction and purification of phycocyanins from a biomass which comprises more than 10% glycogen relative to the total dry matter.
Des microorganismes producteurs de phycocyanine sont bien connus, notamment les algues (ou microalgues) des ordres des Cyanidiales. L'ordre des Cyanidiales, englobe les familles des Cyanidiaceae ou des Galdieriaceae, elles-mêmes subdivisées en les genres Cyanidioschyzon, Cyanidium ou Galdieria, auxquelles appartiennent entre autres les espèces Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarum, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita ou encore Galdieria sulphuraria. On citera en particulier la souche Galdieria sulphuraria (aussi appelée Cyanidium caldarium (UTEX 2919). Phycocyanin-producing microorganisms are well known, in particular algae (or microalgae) of the orders of Cyanidiales. The order of Cyanidiales, includes the families of Cyanidiaceae or Galdieriaceae, themselves subdivided into the genera Cyanidioschyzon, Cyanidium or Galdieria, to which belong among others the species Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarum, Cyanidium maximum, Cyanidium , Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita or even Galdieria sulphuraria. Mention will in particular be made of the strain Galdieria sulphuraria (also called Cyanidium caldarium (UTEX 2919).
On citera aussi des producteurs connus de phycocyanines comme les cyanobactéries filamenteuses du genre Arthrospira, cultivées industriellement sous le nom commun de spiruline. Mention will also be made of known producers of phycocyanins such as the filamentous cyanobacteria of the genus Arthrospira, cultivated industrially under the common name of spirulina.
Les microorganismes qui produisent de la phycocyanine avec une teneur élevée en glycogènes sont plus particulièrement identifiés parmi les microorganismes cités précédemment, en particulier les espèces des genres Cyanidioschyzon, Cyanidium ou Galdieria, plus particulièrement Galdieria sulfuraria. The microorganisms which produce phycocyanin with a high glycogen content are more particularly identified among the microorganisms mentioned above, in particular the species of the genera Cyanidioschyzon, Cyanidium or Galdieria, more particularly Galdieria sulfuraria.
Les procédés industriels de culture de microorganismes producteurs de phycocyanine sont bien connus de l’homme du métier. On citera en particulier les demandes de brevet WO 2017/093345, WO 2017/050917. Industrial methods for growing phycocyanin-producing microorganisms are well known to those skilled in the art. Mention will be made in particular of patent applications WO 2017/093345, WO 2017/050917.
La récupération de la phycocyanine à partir de la biomasse est également connue de l’homme du métier. On citera notamment la demande de brevet WO 2018/178334. Elle requiert généralement une étape de lyse cellulaire, mécanique ou enzymatique de manière à libérer la phycocyanine produite dans les compartiments cellulaires des microorganismes. Cette lyse est avantageusement effectuée à un pH favorable à la solubilisation des phycocyanines. Cette lyse cellulaire va généralement générer une solution de phycocyanine qui comprend des matières organiques en suspensions (appelée suspension brute) qui peuvent être séparées par des méthodes usuelles de filtration. On obtient alors une solution brute de phycocyanine qui peut faire l’objet d’une nouvelle purification de manière à éliminer des résidus organiques de faible poids moléculaire par des méthodes usuelles d’ultrafiltration pour obtenir une solution raffinée à partir de laquelle on obtiendra la phycocyanine par des méthodes usuelles de précipitation et séchage. On citera notamment la filtration tangentielle sur membranes céramiques ou membranes organiques tel que les fibres creuses en polyéthersulfone. Les seuils de ces filtres peuvent être choisis pour séparer des molécules de poids moléculaire supérieur ou inférieur aux phycobiliprotéines ciblées. The recovery of phycocyanin from biomass is also known from the skilled person. Mention may in particular be made of patent application WO 2018/178334. It generally requires a cell, mechanical or enzymatic lysis step in order to release the phycocyanin produced in the cellular compartments of the microorganisms. This lysis is advantageously carried out at a pH favorable to the solubilization of phycocyanins. This cell lysis will generally generate a phycocyanin solution which comprises organic matter in suspension (called crude suspension) which can be separated by usual filtration methods. A crude phycocyanin solution is then obtained which can be further purified so as to remove low molecular weight organic residues by usual ultrafiltration methods to obtain a refined solution from which phycocyanin will be obtained. by usual methods of precipitation and drying. These include tangential filtration on ceramic membranes or organic membranes such as hollow polyethersulfone fibers. The thresholds of these filters can be chosen to separate molecules of molecular weight higher or lower than the targeted phycobiliproteins.
Le procédé selon l’invention est particulièrement adapté pour la purification de solution de phycocyanines résistantes à des pH acides, en particulier les phycocyanines décrites dans la demande WO 2017/050918. The process according to the invention is particularly suitable for the purification of a solution of phycocyanins resistant to acidic pH, in particular the phycocyanins described in application WO 2017/050918.
En particulier, le procédé selon l’invention est mis en œuvre pour la purification de phycocyanines résistantes aux pH acides produites par Galdieria sulphuraria, plus particulièrement dans un procédé de production industrielle de ces phycocyanines par culture en fermenteur de Galdieria sulphuraria. In particular, the process according to the invention is implemented for the purification of phycocyanins resistant to acid pH produced by Galdieria sulphuraria, more particularly in a process for the industrial production of these phycocyanins by culture in a fermenter of Galdieria sulphuraria.
Le procédé est avantageusement mis en œuvre pour extraire la phycocyanine du jus brut obtenu à partir d’une biomasse de microorganismes producteurs de phycocyanine. The method is advantageously implemented for extracting phycocyanin from the raw juice obtained from a biomass of microorganisms producing phycocyanin.
Avantageusement, la solution initiale de phycocyanine, en particulier le jus brut, comprend de 0, 1 à 10 g/L de phycocyanine. Advantageously, the initial solution of phycocyanin, in particular the raw juice, comprises from 0.1 to 10 g / L of phycocyanin.
La concentration consiste à éliminer l’eau de manière à obtenir une teneur en phycocyanine d’au moins 15 g/L, de préférence d’au moins 20 g/L, plus préférentiellement d’au moins 30 g/L, voire au moins 40 g/L. The concentration consists in eliminating the water so as to obtain a phycocyanin content of at least 15 g / L, preferably at least 20 g / L, more preferably at least 30 g / L, or even at least 40 g / L.
Cette concentration peut être définie en % de perte de volume en fonction de la teneur en phycocyanines dans la solution initiale. This concentration can be defined in% of volume loss depending on the content of phycocyanins in the initial solution.
Dans un procédé industriel de production de phycocyanines, le jus brut comprendra avantageusement au moins 1 g/L de phycocyanines. Dans ce cas, la concentration consistera à éliminer au moins 93% de volume initial de liquide. In an industrial process for the production of phycocyanins, the raw juice will advantageously comprise at least 1 g / L of phycocyanins. In this case, the concentration will consist in eliminating at least 93% of the initial volume of liquid.
La concentration se fait par toute méthode connue pour permettre l’élimination de l’eau dans des conditions qui préservent l’intégrité des phycocyanines. On peut citer les méthodes d’évaporation de l’eau, notamment sous pression réduite pour favoriser cette évaporation dans des conditions de température respectueuses de l’intégrité de phycocyanines, sans affecter leur pouvoir colorant. On peut également citer des méthodes qui permettent d’éliminer un liquide, comme la filtration tangentielle avec des tailles de pores qui permettent le passage de l’eau et des petites molécules en solution mais retiennent les protéines. The concentration is done by any known method to allow the elimination of water under conditions which preserve the integrity of the phycocyanins. Mention may be made of the methods of evaporation of water, in particular under reduced pressure to promote this evaporation under temperature conditions which respect the integrity of phycocyanins, without affecting their coloring power. We can also cite methods which make it possible to eliminate a liquid, such as tangential filtration with pore sizes which allow the passage of water and small molecules in solution but retain the proteins.
Ces méthodes de filtration et les appareils qui permettent leur mise en œuvre sont bien connues de l’homme du métier, notamment les systèmes « TFF Spectrum Lab » de Repligen. Pour les phycocyanines, on choisira avantageusement des filtres des pores de 50kD à 100kD, en particulier des filtres de polyéthersulfone ou de polysulfone. These filtration methods and the devices that allow their implementation are well known to those skilled in the art, in particular Repligen's "TFF Spectrum Lab" systems. For phycocyanins, pore filters from 50 kD to 100 kD will advantageously be chosen, in particular polyethersulfone or polysulfone filters.
L’ajustement du pH consiste à ajouter un acide ou une base à la solution initiale ou à la solution concentrée de manière à atteindre une valeur de pH dans la plage d’instabilité. La plage d’instabilité dépendra des phycocyanines à purifier, et notamment du microorganisme qui l’a produite. De manière générale, cette plage d’instabilité va de 4,5 à 5,5, en particulier pour des phycocyanines résistantes aux pH acides telles que décrites ci- dessus. Adjusting the pH involves adding an acid or base to the initial solution or to the concentrated solution so as to reach a pH value within the instability range. The range of instability will depend on the phycocyanins to be purified, including the microorganism that produced it. In general, this instability range is from 4.5 to 5.5, in particular for phycocyanins resistant to acidic pHs as described above.
Pour ces phycocyanines résistantes aux pH acides, la lyse cellulaire est faite à pH acide, de préférence inférieur à 4,5, généralement d’environ 4, voire jusqu’à 3. For these phycocyanins resistant to acidic pH, cell lysis is carried out at an acidic pH, preferably less than 4.5, generally around 4, or even up to 3.
L’ajustement du pH consiste alors à ajouter une base pour atteindre le pH dans la plage d’instabilité. Adjusting the pH then involves adding a base to reach the pH in the instability range.
Selon un premier mode de réalisation de l’invention, le procédé consiste d’abord à concentrer le jus initial. Dans ce cas, la concentration est mise en œuvre à un pH favorable à la solubilisation des phycocyanines, c’est à dire hors de la plage d’instabilité. Pour les phycocyanines résistantes aux pH acides décrite précédemment, ces pH favorables à la solubilisation des phycocyanines sera avantageusement inférieur à 4 ou supérieur à 5. According to a first embodiment of the invention, the method first consists in concentrating the initial juice. In this case, the concentration is carried out at a pH favorable for the solubilization of phycocyanins, that is to say outside the instability range. For phycocyanins resistant to the acidic pH described above, these pHs favorable to the solubilization of phycocyanins will advantageously be less than 4 or more than 5.
Selon un autre mode préféré de réalisation de l’invention, le procédé consiste d’abord à ajuster le pH à la plage d’instabilité puis à concentrer la solution jusqu’à la précipitation des phycocyanines. According to another preferred embodiment of the invention, the method consists first of adjusting the pH to the instability range and then concentrating the solution until the phycocyanins precipitate.
On peut alors décrire le procédé comme l’obtention d’une solution dite de pH instable à partir d’une solution initiale, puis de concentration de la solution de pH instable pour entraîner la précipitation des phycocyanines. Le pourcentage de réduction de volume sera atteint dès lors que l’on observe la précipitation des phycocyanines. We can then describe the process as obtaining a solution called pH unstable from an initial solution, then concentrating the solution of pH unstable to cause the precipitation of phycocyanins. The percentage reduction in volume will be achieved as soon as the precipitation of phycocyanins is observed.
Cette étape de précipitation sélective est avantageusement mise en œuvre à température ambiante. Bien entendu, l’homme du métier pourra modifier la température de manière à favoriser la précipitation, par exemple en diminuant la température pour mettre en œuvre la deuxième partie de l’étape (concentration ou ajustement du pH) au cours de laquelle la précipitation a lieu. This selective precipitation step is advantageously carried out at room temperature. Of course, those skilled in the art can modify the temperature so as to promote precipitation, for example by lowering the temperature to implement the second part of the step (concentration or adjustment of the pH) during which the precipitation has location.
Les polysaccharides en solution, en particulier le glycogène, pourront être ensuite récupérés par des méthodes usuelles de précipitation des polysaccharides, par exemple par ajout d’éthanol (Martinez-Garcia ét al., 2016), ces polysaccharides pouvant également ensuite être purifiés. The polysaccharides in solution, in particular glycogen, can then be recovered by usual methods of precipitation of polysaccharides, for example by adding ethanol (Martinez-Garcia et al., 2016), these polysaccharides can also then be purified.
Selon un mode particulier de réalisation de l’invention, les polysaccharides contenus dans la solution initiale avec les phycocyanines font l’objet d’une lyse enzymatique qui favorise leur maintien en solution. Les traces de ces polysaccharides susceptibles d’être entraînés avec la précipitation des phycocyanines, déjà faibles, sont encore plus réduites lorsque les polysaccharides sont lysés en polyosides de faibles poids moléculaires encore plus solubles. Au surplus, lorsque l’étape de concentration est réalisée par filtration tangentielle, les polyosides de faibles poids moléculaires sont éliminés avec les autres petites molécules en solution, ce qui favorise l’obtention de solution en phycocyanines à plus grande teneur encore. According to a particular embodiment of the invention, the polysaccharides contained in the initial solution with the phycocyanins are the subject of an enzymatic lysis which promotes their maintenance in solution. The traces of these polysaccharides liable to be entrained with the precipitation of the already weak phycocyanins are even more reduced when the polysaccharides are lysed into even more soluble low molecular weight polysaccharides. In addition, when the concentration step is carried out by tangential filtration, the low molecular weight polysaccharides are eliminated with the other small molecules in solution, which favors obtaining a phycocyanin solution with an even higher content.
En particulier, la lyse enzymatique du glycogène est mise en œuvre à un pH inférieure ou égal à 5, de préférence d’environ 4,5, à température ambiante. In particular, the enzymatic lysis of glycogen is carried out at a pH less than or equal to 5, preferably about 4.5, at room temperature.
Ces conditions de température et de pH sont particulièrement adaptées pour préserver la phycocyanine au cours de la réaction enzymatique. These temperature and pH conditions are particularly suitable for preserving phycocyanin during the enzymatic reaction.
Les enzymes actives en conditions de pH acide et à température ambiante sont choisies parmi des enzymes connues pour une activité glucuronidase a1-4 , glucosidase a1-4 (ou alpha-glucosidase). On citera en particulier des pectinases connues pour dégrader la pectine et en particulier des pectinases extraites de champignons filamenteux comme Aspergillus, plus particulièrement de pectinases extraites d’Aspegillus aculeatus, comme les enzymes commercialisées sous la dénomination Pectinex® par la société Novozymes. The enzymes active under acidic pH conditions and at room temperature are chosen from enzymes known for glucuronidase a1-4, glucosidase a1-4 (or alpha-glucosidase) activity. Mention will in particular be made of pectinases known to degrade pectin and in particular pectinases extracted from filamentous fungi such as Aspergillus, more particularly from pectinases extracted from Aspegillus aculeatus, such as the enzymes sold under the name Pectinex® by the company Novozymes.
La lyse enzymatique du glycogène pourra également être réalisée avec une glucosidase a1-6 en plus de la glucuronidase a1-4 ou glucosidase a1-4. Des glucosidases a1-6 actives dans les conditions de pH et de température exposées ci-dessus sont également connues de l’homme du métier. Il s’agit en particulier des pullulanases connues pour hydrolyser les liaisons glucosidiques a1-6 de la pullulane, notamment connues pour supprimer les ramifications de l’amidon. The enzymatic lysis of glycogen can also be carried out with a glucosidase a1-6 in addition to glucuronidase a1-4 or glucosidase a1-4. Glucosidases a1-6 active under the pH and temperature conditions set out above are also known to those skilled in the art. These are in particular the pullulanases known to hydrolyze the glucosidic bonds a1-6 of the pullulan, in particular known to suppress the ramifications of starch.
Il s’agit généralement d’enzymes extraites de bactéries, notamment des genres Bacillus. US 6,074,854, US 5,817,498 et WO 2009/075682 décrivent de telles pullulanases extraites de Bacillus deramificans ou de Bacillus acidopullulyticus. On connaît aussi des pullulanases disponibles dans le commerce, notamment sous les dénominations « Promozyme D2 » (Novozymes), « Novozym 26062 » (Novozymes) et « Optimax L 1000 » (DuPont-Genencor). These are generally enzymes extracted from bacteria, including the genera Bacillus. US 6,074,854, US 5,817,498 and WO 2009/075682 describe such pullulanases extracted from Bacillus deramificans or Bacillus acidopullulyticus. There are also known commercially available pullulanases, in particular under the names "Promozyme D2" (Novozymes), "Novozym 26062" (Novozymes) and "Optimax L 1000" (DuPont-Genencor).
On notera que des mélanges pullulanases/alpha-amylases sont décrits dans l’état de la technique, mais en particulier pour produire du sirop de glucose à partir de l’amidon (US 2017/159090). L’homme du métier saura déterminer les conditions réactionnelles appropriés pour réduire au mieux les quantités de glycogène en fonction de la teneur initiale en glycogène dans la solution à traiter, la quantité d’enzymes employée et la pureté recherchée pour la phycocyanine produite. It will be noted that pullulanase / alpha-amylase mixtures are described in the state of the art, but in particular for producing glucose syrup from starch (US 2017/159090). Those skilled in the art will know how to determine the appropriate reaction conditions to best reduce the amounts of glycogen as a function of the initial glycogen content in the solution to be treated, the amount of enzymes used and the purity sought for the phycocyanin produced.
La récupération des phycocyanines précipitées solides se fait par toutes méthodes connues de l’homme du métier, comme la filtration ou la centrifugation. The recovery of solid precipitated phycocyanins is carried out by any method known to a person skilled in the art, such as filtration or centrifugation.
L’homme du métier pourra envisager toute méthode de récupération des solides de manière à diminuer le volume à traiter par filtration ou centrifugation. A person skilled in the art may consider any method of recovering solids so as to reduce the volume to be treated by filtration or centrifugation.
Cette récupération pourra se faire de manière discontinue, par lots, ou encore de manière continue, l’ajout de solution initiale venant compenser la récupération des phycocyanines solides. This recovery can be done discontinuously, in batches, or even continuously, the addition of initial solution to compensate for the recovery of solid phycocyanins.
Cette étape de récupération en continu sera avantageusement mise en œuvre avec une concentration par filtration tangentielle sur une solution de pH instable, l’homme du métier pouvant ajuster les débits respectifs d’élimination d’eau et d’alimentation en solution de pH instable pour favoriser la précipitation des phycocyanines. This continuous recovery step will advantageously be carried out with a concentration by tangential filtration on a solution of unstable pH, the skilled person being able to adjust the respective flow rates of water removal and supply of solution of unstable pH for favor the precipitation of phycocyanins.
Un tel procédé continu sera particulièrement adapté pour traiter des solutions initiales dans lesquelles les polysaccharides et en particulier le glycogène auront fait l’objet d’une lyse enzymatique qui favorise leur élimination par filtration tangentielle avec l’eau et les autres petites molécules solubles. Such a continuous process will be particularly suitable for treating initial solutions in which the polysaccharides and in particular the glycogen have undergone an enzymatic lysis which promotes their elimination by tangential filtration with water and the other small soluble molecules.
L’invention concerne aussi un procédé de production de phycocyanines par fermentation de microorganismes, ledit procédé comprenant les étapes suivantes de (i) culture des microorganismes pour obtenir une biomasse riche en phycocyanine, (ii) récupération de la biomasse et lyse cellulaire pour solubiliser les phycocyanines libérées dans une suspension de particules cellulaires, (iii) clarification de la suspension préalablement obtenue pour obtenir une solution brute de phycocyanine et (iv) récupération de la phycocyanine à partir de la solution brute préalablement obtenue, caractérisé en ce que la récupération de la phycocyanine comprend une étape de précipitation sélective telle que définie précédemment. The invention also relates to a method for producing phycocyanins by fermentation of microorganisms, said method comprising the following steps of (i) culturing the microorganisms to obtain a biomass rich in phycocyanin, (ii) recovering the biomass and cell lysis to dissolve the phycocyanins released in a suspension of cellular particles, (iii) clarification of the suspension previously obtained to obtain a crude solution of phycocyanin and (iv) recovery of phycocyanin from the crude solution previously obtained, characterized in that the recovery of the phycocyanin comprises a selective precipitation step as defined above.
Le solide récupéré peut ensuite être séché par toute méthode appropriée, et le cas échéant broyé. The solid recovered can then be dried by any suitable method, and if necessary ground.
Le solide récupéré comprenant la phycocyanine peut également faire l’objet d’une purification par des méthodes connues de l’homme du métier, comme la diafiltration. The recovered solid comprising phycocyanin can also be subjected to purification by methods known to those skilled in the art, such as diafiltration.
Les méthodes de culture par fermentation, de récupération de la biomasse, de lyse et de clarification sont bien connues de l’homme du métier, notamment décrites dans les demandes de brevet WO 2017/050917, WO 2017/093345 et WO 2018/178334. The methods of culture by fermentation, of biomass recovery, of lysis and of clarification are well known to those skilled in the art, in particular described in patent applications WO 2017/050917, WO 2017/093345 and WO 2018/178334.
La précipitation sélective mise en œuvre selon l’invention permet de diminuer de manière globale l’énergie nécessaire à la production de phycocyanine en poudre à partir d’une solution initiale, en particulier à partir d’un jus brut, tant sur la quantité de matière à manipuler que de l’énergie nécessaire au séchage de la phycocyanine solide et son broyage. The selective precipitation implemented according to the invention makes it possible to globally reduce the energy necessary for the production of powdered phycocyanin from of an initial solution, in particular from a raw juice, both on the quantity of material to be handled and on the energy necessary for the drying of the solid phycocyanin and its grinding.
La phycocyanine obtenue par ce procédé a un indice de pureté d’au moins 2, de préférence d’au moins 3, voire supérieur à 4. The phycocyanin obtained by this process has a purity index of at least 2, preferably at least 3, or even greater than 4.
Cet indice de pureté est mesuré par mesure d’absorbance avec la méthode décrite par Moon & al. (2014). This purity index is measured by absorbance measurement with the method described by Moon & al. (2014).
De manière avantageuse, la phycocyanine obtenue est une phycocyanine qui a un rapport glycogène/phycocyanines (en poids sec) inférieur à 6, avantageusement inférieur à 4, de préférence inférieur à 3, plus préférentiellement inférieur à 2,5, encore plus préférentiellement inférieur à 1. Advantageously, the phycocyanin obtained is a phycocyanin which has a glycogen / phycocyanin ratio (by dry weight) of less than 6, advantageously less than 4, preferably less than 3, more preferably less than 2.5, even more preferably less than 1.
L’invention concerne également l’utilisation des phycocyanines obtenues comme colorants, en particulier comme colorants alimentaires. Elle concerne aussi des aliments, solides ou liquides, en particulier des boissons qui comprennent une phycocyanine à faible teneur en glycogène selon l’invention. The invention also relates to the use of the phycocyanins obtained as coloring agents, in particular as food coloring agents. It also relates to foodstuffs, solid or liquid, in particular drinks which comprise a phycocyanin with a low glycogen content according to the invention.
DESCRIPTION DES FIGURES DESCRIPTION OF THE FIGURES
La Figure 1 représente la masse du précipité obtenu à différentes concentrations en phycocyanine et différents pH dans la solution initiale. Figure 1 shows the mass of the precipitate obtained at different concentrations of phycocyanin and different pH in the initial solution.
La Figure 2 représente la concentration en phycocyanine dans le surnageant après récupération du précipité en fonction du pH pour différentes concentrations en phycocyanine. FIG. 2 represents the concentration of phycocyanin in the supernatant after recovery of the precipitate as a function of the pH for different concentrations of phycocyanin.
EXEMPLES EXAMPLES
Matériel et méthodes Material and methods
Souche : Galdieria sulphuraria (aussi appelée Cyanidim caldarium) UTEX#2919. Strain: Galdieria sulphuraria (also called Cyanidim caldarium) UTEX # 2919.
Conditions de culture : Culture conditions:
La biomasse est obtenue par fermentation en mode Fedbatch en utilisant les conditions décrites dans le brevet WO2017050918A1. The biomass is obtained by fermentation in Fedbatch mode using the conditions described in patent WO2017050918A1.
Conditions d’extraction : Extraction conditions:
Les cellules sont broyées mécaniquement, à l’aide d’un broyeur à billes de type DYNO®-MILL KD (Willy A. Bachofen AG Maschinenfabrik). La phycocyanine (PC) étant une molécule hydrophile, son extraction se fait à l’eau en ajustant le pH à la valeur souhaitée avec une base (NaOH, KOH, NhUOH... ) ou un acide (H2SO4, acide citrique... .). L’extrait brut de PC est récupéré après séparation des débris cellulaires par centrifugation à 10000g pendant 10 min à température ambiante. L’extrait brut est concentré par filtration tangentielle avec membrane céramique ou membrane organique avec un seuil de coupure permettant de retenir la phycocyanine. Les échantillons sont par la suite centrifugés afin de séparer le précipité du surnageant. La masse du culot est mesurée grâce à une balance de précision. Le culot est re-suspendu dans une solution aqueuse de pH 7 permettant sa resolubilisation, ceci afin de pouvoir quantifier la phycocyanine précipitée. The cells were mechanically ground by means of a type of ball mill DYNO ® -MILL KD (Willy A. Bachofen AG Maschinenfabrik). Phycocyanin (PC) being a hydrophilic molecule, its extraction is done with water by adjusting the pH to the desired value with a base (NaOH, KOH, NhUOH ...) or an acid (H2SO4, citric acid ... .). The crude PC extract is recovered after separation of the cellular debris by centrifugation at 10,000 g for 10 min at room temperature. The crude extract is concentrated by tangential filtration with a ceramic membrane or an organic membrane with a cut-off threshold allowing phycocyanin to be retained. The samples are then centrifuged in order to separate the precipitate from the supernatant. The mass of the base is measured using a balance of precision. The pellet is re-suspended in an aqueous solution of pH 7 allowing its resolubilization, this in order to be able to quantify the precipitated phycocyanin.
Dosage de la PC : Dosage of the PC:
L’estimation de la teneur en phycocyanine et de l’indice de pureté ont été réalisés par mesure d’absorbance grâce à la méthode décrite par Moon & al (Moon et al., Korean J. Chem. Eng., 2014, 1-6). The estimation of the phycocyanin content and of the purity index were carried out by measurement of absorbance using the method described by Moon & al (Moon et al., Korean J. Chem. Eng., 2014, 1- 6).
Exemple 1 : Effet de la concentration et du pH sur la précipitation et la purification de la phycocyanine (PC) Example 1 Effect of the Concentration and of the pH on the Precipitation and the Purification of Phycocyanin (PC)
Une solution brute de phycocyanine de concentration initiale de 1 g/l de PC et de pureté initiale de 1 ,6 est concentrée par filtration tangentielle à pH 4 jusqu’à obtenir un rétentat d’une concentration de 20 g/l, puis 30 g/l puis 45 g/l. Au cours de la filtration une augmentation de la pureté du produit peut être observée, toutefois cette pureté ne dépasse pas la valeur de 2 malgré le degré de concentration du produit. Dans la figure 1 nous pouvons voir que pour une concentration de 20 g/l, la précipitation de la phycocyanine est faible à pH 4 et augmente légèrement en faisant évoluer le pH vers des valeurs plus élevées (Fig. 1). Parallèlement la mesure de la concentration en phycocyanine soluble dans le surnageant lors de cette remontée du pH montre une diminution relativement faible. Pour une concentration de PC à 30 g/l ce phénomène de précipitation par changement de pH est beaucoup plus marqué et parait être maximale pour les valeurs de 4,5 et 5,5 (Fig. 1 et Fig. 2). Sur la poursuite de la filtration et la concentration de la phycocyanine jusqu’à la valeur de 40g/L soluble, la formation d’un précipité important est observée durant la filtration avant même modification du pH (Fig. 1). Comme précédemment le changement de pH accroît le phénomène de précipitation de la phycocyanine. A crude phycocyanin solution with an initial concentration of 1 g / l of PC and an initial purity of 1.6 is concentrated by tangential filtration at pH 4 until a retentate with a concentration of 20 g / l is obtained, then 30 g / l then 45 g / l. During filtration an increase in the purity of the product can be observed, however this purity does not exceed the value of 2 despite the degree of concentration of the product. In figure 1 we can see that for a concentration of 20 g / l, the precipitation of phycocyanin is weak at pH 4 and increases slightly while making evolve the pH towards higher values (Fig. 1). In parallel, the measurement of the concentration of soluble phycocyanin in the supernatant during this rise in pH shows a relatively small decrease. For a concentration of PC at 30 g / l, this precipitation phenomenon by change of pH is much more marked and appears to be maximum for the values of 4.5 and 5.5 (Fig. 1 and Fig. 2). On continuing filtration and the concentration of phycocyanin up to the value of 40g / L soluble, the formation of a large precipitate is observed during filtration before even changing the pH (Fig. 1). As before, the change in pH increases the phenomenon of precipitation of phycocyanin.
Par filtration tangentielle plus la mesure de pureté de phycocyanine diminue et inversement concernant la pureté du précipité collecté et resolubilisé à pH7. Ceci indiquant une précipitation préférentielle de la phycocyanine, qui peut être resolubilisable dans des conditions de pH plus favorable. By tangential filtration, the measurement of purity of phycocyanin decreases and vice versa concerning the purity of the precipitate collected and resolubilized at pH 7. This indicates a preferential precipitation of phycocyanin, which can be resolubilizable under more favorable pH conditions.
Le Tableau 1 rapporte la mesure de la pureté de la phycocyanine après re solubilisation du précipité de phycocyanine pour une précipitation à pH de 7,5. Table 1 reports the measurement of the purity of phycocyanin after re-solubilization of the phycocyanin precipitate for precipitation at pH 7.5.
Tableau 1 Table 1
Exemple 2: Effet de la concentration et du pH sur la précipitation et la purification de la PC sur un échantillon digéré enzymatiquement. Example 2: Effect of the concentration and of the pH on the precipitation and the purification of the PC on an enzymatically digested sample.
Dans cet exemple, la solution brute est soumise à une digestion enzymatique afin de dégrader le glycogène présent. La dégradation enzymatique est faite à température ambiante et pH = 4 avec les enzymes glucoronidase alpha 1-4 (« pectinex Ultra SP-L ») et glucosidase alpha 1-6 (« Novozyme 26062 »). In this example, the crude solution is subjected to an enzymatic digestion in order to degrade the glycogen present. The enzymatic degradation is carried out at room temperature and pH = 4 with the enzymes glucoronidase alpha 1-4 ("pectinex Ultra SP-L") and glucosidase alpha 1-6 ("Novozyme 26062").
Un enrichissement par filtration tangentielle est réalisé pour atteindre une concentration en phycocyanine de plusieurs dizaines de g/L puis un ajustement pH est réalisé, provoquant une précipitation. Des prélèvements de pH aux valeurs de 4.5 ; 5 et 5.5 sont réalisés avec une mesure de la phycocyanine soluble résiduelle et une mesure de la phycocyanine précipitée après collecte et resolubilisation du culot avec une solution tampon à pH 7.5. An enrichment by tangential filtration is carried out to reach a phycocyanin concentration of several tens of g / L then a pH adjustment is carried out, causing precipitation. PH samples at values of 4.5; 5 and 5.5 are carried out with a measurement of the residual soluble phycocyanin and a measurement of the precipitated phycocyanin after collection and resolubilization of the pellet with a buffer solution at pH 7.5.
De façon similaire à l’exemple précédent, la précipitation est importante pour une gamme de pH comprise entre 4,5 et 5,5, et le niveau de pureté atteint dans ce cas des valeurs supérieures à 3,8 lors de la resolubilisation de la phycocyanine précipitée. La digestion enzymatique du glycogène n’affecte pas la purification par précipitation. Le Tableau 2 ci-après donne les valeurs d’indice de pureté de la phycocyanine après précipitation par ajustement aux pH d’instabilité mais aussi après re-solubilisation du précipité de phycocyanine. Similar to the previous example, precipitation is important for a pH range between 4.5 and 5.5, and the level of purity in this case reaches values greater than 3.8 during the resolubilization of the precipitated phycocyanin. Enzymatic digestion of glycogen does not affect precipitation purification. Table 2 below gives the values of the purity index of phycocyanin after precipitation by adjustment to the instability pH but also after re-solubilization of the phycocyanin precipitate.
Tableau 2 Table 2
REFERENCES REFERENCES
Cruz de Jésus ét al., Int J Food Nutr Sci (2016) 3(3): 1-0 Cruz de Jésus et al., Int J Food Nutr Sci (2016) 3 (3): 1-0
Martinez-Garcia ét al., Int J Biol Macromol. (2016) 89:12-8 Martinez-Garcia et al., Int J Biol Macromol. (2016) 89: 12-8
Martinez-Garcia ét al., Carbohydrate Polymers (2017) 169: 75-82 Martinez-Garcia et al., Carbohydrate Polymers (2017) 169: 75-82
- Moon et al., 2014 Korean J. Chem. Eng., 2014, 1-6 - Moon et al., 2014 Korean J. Chem. Eng., 2014, 1-6
- TN 2009000406, US 6,074,854, US 5,817,498, US 2017/159090, WO 2009/075682, WO 2016/030643, WO 2017/050917, WO 2017/050918, WO 2017/093345, WO 2018/178334 - TN 2009000406, US 6,074,854, US 5,817,498, US 2017/159090, WO 2009/075682, WO 2016/030643, WO 2017/050917, WO 2017/050918, WO 2017/093345, WO 2018/178334

Claims

REVENDICATIONS
1. Procédé d’extraction de phycocyanines à partir d’une solution initiale de phycocyanine, caractérisé en ce qu’il comprend les étapes suivantes : 1. Process for extracting phycocyanins from an initial phycocyanin solution, characterized in that it comprises the following steps:
a) précipitation sélective qui consiste à ajuster le pH de la solution initiale à une valeur choisie dans une plage de valeurs de pH dans laquelle les phycocyanines sont moins solubles (également appelée plage d’instabilité) et à concentrer les phycocyanines dans la solution pour favoriser leur précipitation, puis a) selective precipitation which consists in adjusting the pH of the initial solution to a value chosen within a range of pH values in which the phycocyanins are less soluble (also called range of instability) and in concentrating the phycocyanins in the solution to favor their precipitation and then
b) de récupération de la phycocyanine précipitée. b) recovery of the precipitated phycocyanin.
2. Procédé selon la revendication 1 , caractérisée en les deux actions d’ajustement du pH et de concentration sont mises en œuvre simultanément ou en séquence, soit en ajustant le pH de la solution initiale avant de concentrer, soit en concentrant la solution initiale avant d’ajuster le pH. 2. Method according to claim 1, characterized in that the two pH adjustment and concentration actions are implemented simultaneously or in sequence, either by adjusting the pH of the initial solution before concentrating, or by concentrating the initial solution before to adjust the pH.
3. Procédé selon l’une des revendications 1 ou 2, caractérisé en ce que la solution initiale est une solution brute provenant d’une lyse d’une biomasse de microorganismes cultivés pour produire de la phycocyanine. 3. Method according to one of claims 1 or 2, characterized in that the initial solution is a crude solution from a lysis of a biomass of microorganisms cultivated to produce phycocyanin.
4. Procédé selon l’une des revendications 1 à 3, caractérisé en ce que la phycocyanine est une phycocyanine stable à pH acide. 4. Method according to one of claims 1 to 3, characterized in that the phycocyanin is a stable phycocyanin at acidic pH.
5. Procédé selon l’une des revendications 1 à 4, caractérisé en ce que la phycocyanine est une phycocyanine d’origine microbienne, produite par un microorganisme choisi parmi les espèces des genres Cyanidioschyzon, Cyanidium ou Galdieria. 5. Method according to one of claims 1 to 4, characterized in that the phycocyanin is a phycocyanin of microbial origin, produced by a microorganism chosen from the species of the genera Cyanidioschyzon, Cyanidium or Galdieria.
6. Procédé selon l’une des revendications 1 à 4, caractérisé en ce que le pH de la plage d’instabilité va de 4,5 à 5,5. 6. Method according to one of claims 1 to 4, characterized in that the pH of the instability range is from 4.5 to 5.5.
7. Procédé selon l’une des revendications 1 à 6, caractérisé en ce que la concentration consiste à éliminer l’eau de manière à obtenir une teneur en phycocyanine d’au moins 15 g/L, de préférence d’au moins 20 g/L, plus préférentiellement d’au moins 30 g/L, voire au moins 40 g/L. 7. Method according to one of claims 1 to 6, characterized in that the concentration consists in eliminating the water so as to obtain a phycocyanin content of at least 15 g / L, preferably at least 20 g / L, more preferably at least 30 g / L, or even at least 40 g / L.
8. Procédé selon l’une des revendications 1 à 7, caractérisé en ce que la concentration est réalisée par filtration tangentielle avec un seuil de coupure permettant de retenir la phycocyanine. 8. Method according to one of claims 1 to 7, characterized in that the concentration is achieved by tangential filtration with a cutoff threshold for retaining phycocyanin.
9. Procédé selon l’une des revendications 1 à 8, caractérisé en ce que la phycocyanine récupérée est séchée et éventuellement broyée. 9. Method according to one of claims 1 to 8, characterized in that the recovered phycocyanin is dried and optionally ground.
10. Procédé selon l’une des revendications 1 à 8, caractérisé en ce que la phycocyanine récupérée a un indice de pureté d’au moins 2, de préférence d’au moins 3. 10. Method according to one of claims 1 to 8, characterized in that the recovered phycocyanin has a purity index of at least 2, preferably at least 3.
1 1. Procédé selon la revendication 10, caractérisé en ce que la phycocyanine récupérée à un indice de pureté supérieur à 4. 1 1. Process according to claim 10, characterized in that the phycocyanin recovered has a purity index greater than 4.
12. Phycocyanine purifiée obtenue par le procédé selon l’une des revendications 1 à 11. 12. Purified phycocyanin obtained by the process according to one of claims 1 to 11.
13. Utilisation d’une phycocyanine purifiée selon la revendication 12 comme colorant alimentaire. 13. Use of a purified phycocyanin according to claim 12 as a food coloring.
14. Aliment, caractérisé en ce qu’il comprend une phycocyanine purifiée selon la revendication 12. 14. Food, characterized in that it comprises a purified phycocyanin according to claim 12.
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