Methods for extracting phycocyanin

Abstract

The present invention relates to a novel method for extracting and purifying phycocyanin produced by fermentation of microalgae, in particular produced by Galdieria sulphuraria, by selective precipitation.

Description

[Technical field]

[0001] The present invention relates to a novel method for the extraction and purification by selective precipitation of phycocyanin produced by fermentation of microalgae, in particular produced by Galdieria sulphuraria. [Background technology]

[0002] The purification of phycobiliproteins extracted from Galdieria sulphuraria and Spirulina by ammonium sulfate precipitation has already been described in the literature (Moon et al., 2015; Cruz de Jesus et al., 2006, CN106190853), but is very difficult to apply on an industrial scale, since it requires large amounts of ammonium sulfate and causes serious problems in reprocessing the ammonium sulfate and the supernatant.

[0003] Other disclosed purification methods for obtaining high purity levels, such as chromatography or ion exchange resin purification (JP2004359638), are very expensive to implement.

[0004] Precipitation of Spirulina phycocyanin using the addition of acid to crude phycocyanin solutions has been described (TN2009000406, JP2004359638, JP06271783, CN106190853). However, taking into account the isoelectric point of Spirulina phycocyanin, which is approximately 4.5 and close to that of many other proteins, this method does not allow selective precipitation and therefore does not allow purification of phycocyanin. Similarly, others have described the use of salicylic acid to precipitate phycocyanin from Spirulina (WO2016 / 030643). The use of this acid results in non-selective precipitation of PC, and the resulting precipitate is particularly difficult to resolubilize. The same results were obtained with Galdieria PC.

[0005] Phycocyanin extraction methods generally consist in precipitating organic matter other than phycocyanin present in the aqueous crude extract derived from microalgae fermentation in order to preserve phycocyanin in the supernatant, which organic matter is filtered before precipitating phycocyanin (JP2004359638). However, some organic compounds, especially complex polysaccharides such as glycogen, remain soluble under the same conditions as phycocyanin.

[0006] In industrial phycocyanin purification processes, a filtration (ultrafiltration) step may be used to remove small molecules (proteins, ions, organic acids, etc.) smaller than the cut-off threshold of the filter used to remove water and concentrate phycocyanin, obtaining the purest phycocyanin possible. However, if the cut-off threshold of the filter is smaller than the size of glycogen, it will not be removed, increasing the viscosity of the retentate and decreasing the filtration rate. A concentration-dependent viscosity effect of glycogen has been demonstrated using purified glycogen from Galdieria sulphuraria (Martinez-Garcia et al., 2017).

[0007] Also, the resulting purified phycocyanins retain high levels of these sugars, which may alter the quality of the phycocyanins, especially their coloring strength, and require the production and / or use of greater amounts of phycocyanins for the same effect. These residual polysaccharides act as fillers that increase the cost of phycocyanin production and may limit the commercial use of the resulting phycocyanins, for example, in the preparation of foodstuffs with low sugar content.

[0008] The object of the present invention is to improve the process for extracting and purifying phycocyanin extracted from biomass, both from a qualitative point of view and from an industrial and economic point of view, in particular by reducing the residual sugar content, in particular the residual glycogen content, in the final product. Summary of the Invention

[0009] The method according to the invention consists in carrying out the selective precipitation of phycocyanin directly from a crude extract containing it, under conditions which respect the integrity of the phycobiliproteins and which make it possible for the main impurities, in particular the polysaccharides, including glycogen, to be maintained in solution.

[0010] This selective precipitation results from the combined action of two factors, namely the pH of the solution on the one hand and the concentration of phycocyanin on the other hand, either simultaneously or successively in any order.

[0011] The method according to the invention is particularly suitable for purifying the acid pH-resistant phycocyanin produced by Galdieria sulphuraria. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention relates to a method for extracting phycocyanin from a solution containing phycocyanin, also called the initial phycocyanin solution, comprising the steps of: a selective precipitation step consisting, on the one hand, of adjusting the pH of the initial solution to a selected value within the range of pH values ​​at which phycocyanins are poorly soluble (also called the instability range), and, on the other hand, of concentrating the phycocyanins in the solution to favor their precipitation, and then Recovery process of precipitated phycocyanin The present invention relates to a method comprising the steps of:

[0013] The two acts of pH adjustment and concentration can be performed simultaneously or sequentially by adjusting the pH of the initial solution before concentrating, or by concentrating the initial solution before adjusting the pH.

[0014] Surprisingly, the characteristic concentration conditions mean that only phycocyanin precipitates, while other products, which may be described as impurities, especially polysaccharides, remain in solution.

[0015] It is therefore possible to recover the precipitated phycocyanins by separating them from the solution and, if necessary, drying them to obtain a purified phycocyanin powder.

[0016] The method according to the invention not only allows the extraction of phycocyanins from solution, but also makes it possible to purify them in the same step, the phycocyanins obtained being particularly pure with a low residual sugar content.

[0017] The method according to the invention is particularly suitable for purifying a phycocyanin solution extracted from a culture of a phycocyanin-producing microorganism which also produces glycogen, especially in the context of an industrial phycocyanin production process which comprises culturing the microorganism, then recovering the produced biomass, extracting the phycocyanin, and recovering the phycocyanin from this biomass.

[0018] The method is particularly suitable for phycocyanin produced by microorganisms that produce high levels of glycogen, and in particular for extracting and purifying phycocyanin from biomass that contains more than 10% glycogen on a total dry matter basis.

[0019] Phycocyanin-producing microorganisms, such as algae (or microalgae) of the order Cyanidiales, are well known. The order Cyanidiales includes the family Cyanidiaceae or Galdieriaceae, which itself is subclassified into the genera Cyanidioschyzon, Cyanidium or Galdieria, which include, among other species, Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita or Galdieria sulphuraria. In particular, Galdieria sulphuraria (also known as Cyanidium caldarium) strain UTEX 2919.

[0020] Mention may also be made of known phycocyanin producers, such as the filamentous cyanobacteria of the genus Arthrospira, which are cultivated industrially under the common name spirulina.

[0021] Microorganisms which produce phycocyanin with a high glycogen content have been identified in particular from among the above-mentioned microorganisms, in particular from species of the genera Cyanidioschyzon, Cyanidium and Galdieria, more particularly Galdieria sulphuraria.

[0022] Industrial methods for culturing phycocyanin-producing microorganisms are well known to those skilled in the art, in particular in patent applications WO2017 / 093345 and WO2017 / 050917.

[0023] The recovery of phycocyanin from biomass is also known to those skilled in the art. In particular, patent application WO2018 / 178334 is cited. This usually requires a step of mechanical or enzymatic cell lysis to release the phycocyanin produced in the cellular compartment of the microorganism. This lysis is advantageously carried out at a pH favorable for the solubilization of phycocyanin. This cell lysis generally produces a phycocyanin solution containing organic matter in suspension (called crude suspension) which can be separated by conventional filtration methods. A crude phycocyanin solution is then obtained which can be further purified to remove low molecular weight organic residues by conventional ultrafiltration methods to obtain a purified solution from which the phycocyanin can be obtained by conventional precipitation and drying methods. In particular, tangential filtration on ceramic or organic membranes, such as polyethersulfone hollow fibers, is cited. The threshold value of these filters can be selected to separate molecules with a higher or lower molecular weight than the target phycobiliprotein.

[0024] The method according to the invention is particularly suitable for purifying acid pH resistant phycocyanin solutions, in particular the phycocyanins described in application WO2017 / 050918.

[0025] In particular, the method according to the invention is used to purify acid pH resistant phycocyanins produced by Galdieria sulphuraria, more particularly in an industrial process for producing these phycocyanins by fermenter culture of Galdieria sulphuraria.

[0026] The method is advantageously carried out to extract phycocyanin from crude juice obtained from a phycocyanin-producing microbial biomass.

[0027] Advantageously, the initial phycocyanin solution, in particular the crude juice, contains between 0.1 and 10 g / L of phycocyanin.

[0028] The concentration consists in removing water so as to obtain a phycocyanin content of at least 15 g / L, preferably at least 20 g / L, more preferentially at least 30 g / L, or even more preferentially at least 40 g / L.

[0029] This concentration can be defined as the percentage (%) of volume loss based on the phycocyanin content in the initial solution.

[0030] In an industrial phycocyanin production process, the crude juice advantageously contains at least 1 g / L of phycocyanin, where concentration consists in removing at least 93% of the initial volume of liquid.

[0031] Concentration is carried out by any method known to allow the removal of water under conditions that protect the integrity of phycocyanin, including water evaporation methods, particularly under reduced pressure to facilitate this evaporation under temperature conditions that respect the integrity of phycocyanin without affecting its coloring power, and methods that allow the removal of liquid, such as tangential filtration using pore sizes that allow the passage of water and small molecules in solution but retain proteins.

[0032] These filtration methods and the devices for carrying them out, in particular the Spectrum Labs TFF systems from Repligen, are well known to those skilled in the art. For phycocyanin, it is advantageous to choose filters with pores between 50 kD and 100 kD, in particular polyethersulfone or polysulfone filters.

[0033] The pH adjustment consists in adding an acid or base to the initial or concentrated solution in order to reach a pH value within the unstable range. The unstable range depends on the phycocyanin to be purified, and in particular on the microorganism that produced the phycocyanin. In general, the unstable range is between 4.5 and 5.5, in particular for the acid pH-resistant phycocyanins mentioned above.

[0034] For these acid pH resistant phycocyanins, cell lysis is carried out at an acidic pH, preferably below 4.5, usually at a pH of about 4, or even lowered to 3.

[0035] As a result, pH adjustment consists of adding base to reach a pH within the unstable range.

[0036] According to a first embodiment of the invention, the method of the invention consists first in concentrating the initial juice, the concentration being carried out at a pH favourable for the solubilisation of phycocyanin, i.e. outside the unstable range. For the acid pH resistant phycocyanins, the pH favourable for the solubilisation of phycocyanin is advantageously below 4 or above 5.

[0037] According to another preferred embodiment of the invention, the method of the invention consists first in adjusting the pH to the unstable range and then concentrating the solution until phycocyanin precipitates.

[0038] As a result, the method may be described as obtaining an unstable pH solution from an initial solution, and then concentrating the unstable pH solution to cause precipitation of phycocyanin. A percentage of volume reduction is reached at which precipitation of phycocyanin is observed.

[0039] This selective precipitation step is advantageously carried out at room temperature. Of course, the skilled person can modify the temperature in a way that favors precipitation, for example by lowering the temperature to carry out the second part of the step (concentration or pH adjustment) where precipitation occurs.

[0040] The polysaccharides in solution, particularly glycogen, can then be recovered by conventional polysaccharide precipitation methods, for example by the addition of ethanol (Martinez-Garcia et al., 2016), and these polysaccharides can also be subsequently purified.

[0041] According to a particular embodiment of the invention, the polysaccharides contained in the initial solution together with the phycocyanin are subjected to an enzymatic dissolution which favors their retention in the solution. Traces of these polysaccharides are likely to be removed by phycocyanin precipitation, and are even further reduced if the amount of polysaccharides is already low and is dissolved into more soluble low molecular weight oligosaccharides. Furthermore, if the concentration step is carried out by tangential filtration, the low molecular weight oligosaccharides are removed together with other small molecules in the solution, which favors obtaining a solution with an even higher phycocyanin content.

[0042] In particular, the enzymatic lysis of glycogen is carried out at a pH below 5, preferably about 4.5, and at room temperature.

[0043] These temperature and pH conditions are particularly suitable for protecting phycocyanin during the enzymatic reaction.

[0044] Enzymes active under acidic pH conditions and at room temperature are selected from enzymes known to have α1-4 glucuronidase, α1-4 glucosidase (or alpha-glucosidase) activity, in particular pectinases known to degrade pectin, in particular pectinases extracted from filamentous fungi such as Aspergillus, and more particularly from Aspergillus aculeatus, such as the enzyme sold under the name Pectinex® by the company Novozymes.

[0045] Enzymatic dissolution of glycogen can be achieved using α1-4 glucuronidase or α1-4 glucosidase as well as α1-6 glucosidase. α1-6 glucosidases active under the above mentioned pH and temperature conditions are also known to those skilled in the art. In particular, these are pullulanases, which are known to hydrolyze the α1-6 glycosidic bonds of pullulan, in particular to remove starch branches.

[0046] These are generally enzymes extracted from bacteria, in particular from the genus Bacillus. US 6,074,854, US 5,817,498 and WO 2009 / 075682 describe pullulanases extracted from Bacillus deramificans or Bacillus acidopullulyticus. Commercially available pullulanases are also known under the names "Promozyme D2" (Novozymes), "Novozym 26062" (Novozymes) and "Optimax L 1000" (DuPont-Genencor), among others.

[0047] It should be noted that pullulanase / alpha-amylase mixtures have been described in the prior art, inter alia, for the production of glucose syrup from starch (US2017 / 159090).

[0048] The skilled artisan will know how to determine the appropriate reaction conditions that best reduce the amount of glycogen as a function of the initial glycogen content in the solution to be treated, the amount of enzyme used, and the desired purity of the phycocyanin produced.

[0049] Recovery of the precipitated solid phycocyanin is carried out by any method known to those skilled in the art, such as filtration or centrifugation.

[0050] One skilled in the art can envision any method of recovery of solids to reduce the volume processed by filtration or centrifugation.

[0051] This recovery can be carried out discontinuously, in batches, or continuously, with the addition of initial solution to supplement the recovery of solid phycocyanin.

[0052] This continuous recovery step is advantageously carried out using concentration by tangential filtration of the unstable pH solution, the skilled person being able to adjust the respective flow rates of water removal and of the unstable pH solution feed to promote phycocyanin precipitation.

[0053] Such a continuous method is particularly adapted for treating an initial solution in which polysaccharides, especially glycogen, undergo enzymatic dissolution favoring their removal by tangential filtration together with water and other soluble small molecules.

[0054] The present invention also relates to a method for producing phycocyanin by microbial fermentation, comprising the steps of: (i) cultivating a microorganism to obtain a phycocyanin-rich biomass; (ii) harvesting the biomass and lysing it to solubilize the released phycocyanin in a suspension of cell particles; (iii) clarifying the suspension previously obtained to obtain a crude phycocyanin solution; and (iv) recovering the phycocyanin from the crude solution previously obtained, characterized in that the recovery of the phycocyanin comprises a selective precipitation step as defined above.

[0055] The recovered solids may then be dried by any suitable method and, if desired, ground.

[0056] The recovered solids containing phycocyanin may be subjected to purification by methods known to those skilled in the art, for example, by diafiltration.

[0057] Methods of fermentation culture, biomass recovery, lysis and clarification, in particular those described in patent applications WO2017 / 050917, WO2017 / 093345 and WO2018 / 178334, are well known to those skilled in the art.

[0058] The selective precipitation practiced in the present invention reduces the overall energy required to produce phycocyanin powder from an initial solution, and in particular from crude juice, both in terms of the amount of material handled and the energy required to dry the solid phycocyanin and grind it.

[0059] The phycocyanin obtained by the method of the present invention has a purity index of at least 2, preferably at least 3, or even higher than 4.

[0060] This purity index is measured by absorbance measurements using the method described by Moon et al. (2014).

[0061] Advantageously, the phycocyanin obtained is a phycocyanin having a glycogen / phycocyanin ratio (by dry weight) of less than 6, advantageously less than 4, preferably less than 3, more preferentially less than 2.5 and even more preferentially less than 1.

[0062] The present invention also relates to the use of the obtained phycocyanin as a colorant, in particular as a food colorant.The present invention also relates to a solid or liquid food product, in particular a beverage, containing the glycogen-depleted phycocyanin according to the invention. [Brief description of the drawings]

[0063] [Figure 1] The mass of precipitate obtained at different phycocyanin concentrations and different pH in the initial solution is shown. [Diagram 2] FIG. 1 shows the phycocyanin concentration in the supernatant after precipitate recovery as a function of pH for different phycocyanin concentrations. EXAMPLES

[0064] material and method KK :Galdieria sulphuraria (also known as Cyanidium caldarium) UTEX#2919.

[0065] Culture conditions : Biomass is obtained by fed-batch fermentation using the conditions described in WO2017050918A1.

[0066] Extraction conditions : The cells are mechanically pulverized using a DYNO®-MILL KD ball mill (Willy A. Bachofen AG Maschinenfabrik). As phycocyanin (PC) is a hydrophilic molecule, it is extracted with water by adjusting the pH to the desired value with bases (NaOH, KOH, NH4OH, etc.) or acids (H2SO4, citric acid, etc.). The crude PC extract is recovered after separation of cell debris by centrifugation at 10000 g for 10 min at room temperature. The crude extract is concentrated by tangential filtration using ceramic or organic membranes with a cut-off threshold that allows the retention of phycocyanin. The sample is then centrifuged to separate the precipitate from the supernatant. The mass of the pellet is measured using a precision balance. To quantify the precipitated phycocyanin, the pellet is resuspended in an aqueous solution at pH 7 to allow its resolubilization.

[0067] PC measurement : The evaluation of phycocyanin content and purity index was carried out by absorbance measurements using the method described by Moon et al. (Moon et al., Korean J. Chem. Eng., 2014, 1-6).

[0068] Example 1: Effect of concentration and pH on precipitation and purification of phycocyanin (PC) A crude phycocyanin solution with an initial concentration of 1 g / L PC and an initial purity of 1.6 is concentrated by tangential filtration at pH 4 to obtain a retentate with a concentration of 20 g / L, then 30 g / L, and then 45 g / L. An increase in the purity of the product can be observed during the filtration, but this purity does not exceed a value of 2, despite the degree of concentration of the product. In Figure 1 it can be seen that for a concentration of 20 g / L, the precipitation of phycocyanin is low at pH 4 and increases slightly as the pH increases to higher values ​​(Figure 1). At the same time, the measurements of the soluble phycocyanin concentration in the supernatant during this pH increase show a relatively small decrease. For a PC concentration of 30 g / L, this phenomenon of precipitation due to the change in pH is even more pronounced and appears to be maximum for values ​​of 4.5 and 5.5 (Figures 1 and 2). Upon further filtration and concentration of phycocyanin up to soluble values ​​of 40 g / L, significant precipitate formation is observed during filtration even before pH modification (Figure 1). Again, changes in pH increase the phenomenon of phycocyanin precipitation.

[0069] Tangential filtration reduces the purity of phycocyanin, and the opposite is true for the purity of the precipitate collected and resolubilized at pH 7. This indicates preferential precipitation of phycocyanin that can be resolubilized under more favorable pH conditions.

[0070] Table 1 reports the measured phycocyanin purity after resolubilization of the phycocyanin precipitate for precipitation at pH 7.5.

[0071] [Table 1]

[0072] Example 2: Effect of concentration and pH on precipitation and purification of PC from enzymatically digested samples In this example, the crude solution is subjected to enzymatic digestion to degrade the glycogen present. The enzymatic digestion is carried out at room temperature and pH=4 using the enzymes alpha 1-4 glucuronidase ("Pectinex Ultra SP-L") and alpha 1-6 glucosidase ("Novozyme 26062").

[0073] Enrichment by tangential filtration is performed to reach a phycocyanin concentration of several tens of g / L, then pH adjustment is performed to induce precipitation. Measurements of the remaining soluble phycocyanin are made in pH samples at 4.5, 5 and 5.5, and of the precipitated phycocyanin after collection of the pellet and its resolubilization in a buffer solution at pH 7.5.

[0074] As in the previous examples, precipitation is evident for the pH range comprised between 4.5 and 5.5, with purity levels reaching values ​​above 3.8 upon resolubilization of the precipitated phycocyanin. Enzymatic digestion of glycogen does not affect purification by precipitation. Table 2 below gives the purity index values ​​of phycocyanin after precipitation by adjustment to an unstable pH, and also after resolubilization of the phycocyanin precipitate.

[0075] [Table 2]

[0076] References ·Cruz de Jesus et al., Int J Food Nutr Sci (2016) 3(3): 1-0 ·Martinez-Garcia et al., Int J Biol Macromol. (2016) 89:12-8 ·Martinez-Garcia et al., Carbohydrate Polymers (2017) 169: 75-82 ·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

Claims

1. 1. A method for extracting phycocyanin from an initial phycocyanin solution comprising: a) performing selective precipitation comprising adjusting the pH of the initial phycocyanin solution to a selected value within the range of pH values ​​4.5 to 5.5, and concentrating the phycocyanin in the solution to promote its precipitation; b) recovering the precipitated phycocyanin; Including, 1. A method according to claim 1, wherein the phycocyanin is an acid pH resistant phycocyanin that is stable at an acid pH of 4 or less.

2. 2. The method according to claim 1, characterized in that the two acts of pH adjustment and concentration are carried out simultaneously or successively by adjusting the pH of the initial phycocyanin solution before concentration or by concentrating the initial phycocyanin solution before pH adjustment.

3. 3. The method according to claim 1, characterized in that the initial phycocyanin solution is a crude solution from the lysis of a microbial biomass cultured to produce phycocyanin.

4. The method according to any one of claims 1 to 3, characterized in that the phycocyanin is of microbial origin, produced by a microorganism selected from species of the genera Cyanidioschyzon, Cyanidium or Galdieria.

5. 5. The method according to claim 1, wherein the concentration is characterized by removing water so as to obtain a phycocyanin content of at least 15 g / L.

6. 6. The method according to claim 1, wherein the concentration is carried out by tangential filtration having a cut-off threshold allowing the retention of phycocyanin.

7. The method according to any one of claims 1 to 6, characterized in that the recovered phycocyanin is dried.

8. The method according to any one of claims 1 to 6, characterized in that the recovered phycocyanin has a purity index of at least 2.

9. 9. The method according to claim 8, characterized in that the recovered phycocyanin has a purity index higher than 4.

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