Methods for purifying phycocyanin

Abstract

The present invention relates to a novel method for purifying phycocyanin produced by microalgae fermentation, in particular produced by Galdieria sulphuraria, which method involves the enzymatic degradation of glycogen.

Description

[Technical Field]

[0001] The present invention relates to a novel method for purifying phycocyanin produced by microalgae fermentation, in particular produced by Galdieria sulphuraria, which method involves the enzymatic degradation of glycogen. [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), but is very difficult to apply on an industrial scale because it requires large amounts of ammonium sulfate and causes serious problems in reprocessing the ammonium sulfate and the supernatant.

[0003] Other disclosed purification methods to obtain certain purity levels, such as chromatographic methods, are very expensive to implement.

[0004] Phycocyanin extraction methods generally consist in precipitating the non-phycocyanin organic matter present in the aqueous crude extract from microalgae fermentation, which is filtered before precipitating phycocyanin, in order to retain phycocyanin in the supernatant. However, some organic compounds, particularly complex polysaccharides such as glycogen, remain insensitive to this precipitation.

[0005] In industrial phycocyanin purification processes, a filtration (ultrafiltration) step is used to remove water and concentrate phycocyanin, removing small molecules (proteins, ions, organic acids, etc.) smaller than the filter's cutoff threshold to obtain the purest phycocyanin possible. However, if the filter's cutoff threshold is smaller than the size of glycogen, the latter will not be removed, increasing the viscosity of the retentate and limiting the performance and maintenance of optimal parameters of the filtration. A concentration-dependent viscosity effect of glycogen has been demonstrated using purified glycogen from Galdieria sulphuraria (Martinez-Garcia et al., 2017).

[0006] Furthermore, the resulting purified phycocyanin retains high levels of these sugars, which can alter the properties of the purified product, particularly its coloring strength, requiring greater amounts of phycocyanin for the same visual effect. These residual polysaccharides act as fillers that increase the cost of phycocyanin production and may limit the commercial use of the resulting phycocyanin, for example, in the preparation of foods with a low sugar content. The presence of residual polysaccharides can limit the use of the product for the preparation of foods with a low sugar content, resulting in additional costs for the removal of these sugars.

[0007] Glycogen is a complex sugar that is difficult to remove if the goal is to protect phycocyanin from the normal conditions of glycolysis. Glycogen is a branched polyglucoside consisting of α(1-4) glucosidic chains branched by α(1-6) bonds.

[0008] The use of enzymes for cell lysis is known in methods for extracting phycocyanin from microbial cultures (CN106749633, CN102433015, and CN1117973). This cell lysis step, which disrupts the cell walls and releases phycocyanin, and the subsequent extraction of the released phycocyanin into the medium, has no significant effect on the glycogen released and extracted with the phycocyanin.

[0009] Enzymatic degradation of glycogen is possible. However, this polysaccharide is a polymer that is partially resistant to enzymes capable of degrading it. Due to the large number of α1-6 glycosidic branching bonds, the use of enzymes such as β-amylase (α1-4 glucosidase) is inappropriate, as shown by Martinez-Garcia et al. The authors demonstrated the relatively limited activity of pancreatic α-amylase (α1-4 glucosidase) on glycogen. The reducing sugar measurements, which indicate the level of digestion, remain low and rapidly saturate. The use of enzymes with α1-6 glucosidase activity (isoamylase, pullulanase) to debranch glycogen is possible, as shown by the studies of Martinez-Garcia et al. or Shimonaga et al. However, digestion is also incomplete, releasing glucose polymers after long digestion times (24–48 h).

[0010] These glycogen digestion experiments reported in the prior art have not comprehensively addressed the issue of phycocyanin protection, even though the enzymes used affect the integrity of phycocyanin and therefore alter its coloring and antioxidant properties.

[0011] The object of the present invention is to improve the process for purifying phycocyanin extracted from biomass, both from a qualitative point of view and from an industrial and economic point of view, by reducing the residual sugar content, in particular the residual glycogen content, in the final product while preserving the properties of phycocyanin. Summary of the Invention

[0012] The method according to the invention consists in carrying out an enzymatic treatment of the phycocyanin solution to reduce the glycogen content with suitable enzymes for degrading glycogen, i.e. enzymes active at a pH below 6 and at reaction temperatures below 40°C, such as glucoamylase, pectinase and pullulanase and mixtures thereof, under temperature and pH conditions which do not substantially degrade the phycocyanin present.

[0013] The method according to the invention is particularly suitable for purifying acid-resistant phycobiliproteins produced by Galdieria sulphuraria, the enzymatic reaction being carried out at a pH below 6, advantageously at a pH of about 4.

[0014] The present invention also relates to a phycocyanin extract 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. [Brief explanation of the drawings]

[0015] [Figure 1] Figure 1 shows the phycocyanin loss curves (%) over time for digestion at pH = 4 at different enzyme concentrations. [Figure 2] Figure 1 shows the phycocyanin loss curves (%) over time for digestion at pH = 7 at different enzyme concentrations. [Figure 3] 1 shows glucose release curves after glycogen digestion over time for digestion at pH=4 at different enzyme concentrations. [Figure 4] 1 shows glucose release curves after glycogen digestion over time for digestion at pH=7 at different enzyme concentrations. [Figure 5] Figure 1 shows the change in permeate flux as a function of time for filtration of phycocyanin extract (C-PC) with and without enzymatic digestion. [Figure 6] 4 shows the curves after glycogen digestion at pH=4 for different enzymes. [Figure 7]4 shows the curves after glycogen digestion at pH=7 for different enzymes. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention relates to a method for purifying phycocyanin from a solution containing phycocyanin and glycogen, comprising the steps of enzymatically degrading glycogen with an appropriate enzyme for degrading glycogen under temperature and pH conditions that do not substantially degrade any phycocyanin present, and separating the phycocyanin from the glycogen degradation products.

[0017] The method according to the invention is particularly suitable for purifying a phycocyanin solution extracted from a culture of a phycocyanin-producing microorganism that 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 the biomass.

[0018] This 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 containing more than 10% glycogen on a total dry matter basis.

[0019] Phycocyanin-producing microorganisms, particularly 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, including, 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, and Galdieria sulphuraria. Galdieria sulphuraria (also known as Cyanidium caldarium) strain UTEX 2919 is particularly well known.

[0020] Also included are known phycocyanin producers, such as 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 Arthrospira, Spirulina, Synechococcus, Cyanidioschyzon, Cyanidium and Galdieria, more particularly Galdieria sulphuraria.

[0022] Glycogen is in fact a polysaccharide widely present in various organisms (bacteria, yeast, animal cells, etc.). While the structure of a glucose polymer with α1-4 bonds branched by α1-6 bonds is common, the difference comes from the percentage and distribution of branches. In particular, "glycogen" is understood in the present invention to mean the glucose polymer present in the phycocyanin-producing organisms already mentioned, whose distinctive feature is the majority branch size of less than 10 glucose units, as exemplified by the study by Martinez-Garcia et al.

[0023] Industrial methods for cultivating phycocyanin-producing microorganisms are well known to those skilled in the art, including, inter alia, applications WO2017 / 093345, WO2017 / 050917 and WO2018 / 178334.

[0024] Recovery of phycocyanin from biomass is also known to those skilled in the art. Patent application WO 2018 / 178334 is cited in particular. This typically requires a step of either mechanical or enzymatic cell lysis to release the phycocyanin produced in the cellular compartment of the microorganism. This cell lysis generally produces a phycocyanin solution containing organic matter in suspension (called a crude suspension), which can be separated by conventional separation methods, in particular filtration, particularly microfiltration, or centrifugation followed by filtration, more specifically microfiltration. 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. Tangential filtration on ceramic or organic membranes, such as polyethersulfone or polysulfone hollow fibers, particularly those proposed by Repligen, is particularly relevant. The threshold values ​​of these filters can be selected to separate molecules with molecular weights higher or lower than that of the target phycocyanin.

[0025] The resulting phycocyanin can then be purified, in particular by a diafiltration step, to remove as many low molecular weight organic residues as possible.

[0026] The enzymatic treatment according to the present invention can be carried out on both crude suspensions and crude solutions.

[0027] The method according to the invention is particularly suitable for purifying solutions of acid-resistant phycocyanins, in particular the phycocyanins described in application WO2017 / 050918.

[0028] In particular, the method according to the invention is used to purify acid-resistant phycocyanins produced by Galdieria sulphuraria, more particularly in industrial processes for producing these phycocyanins by fermenter culture of Galdieria sulphuraria.

[0029] Preferred conditions for carrying out the enzymatic reaction are a pH below 7 and a reaction temperature below 60°C, preferably below 50°C, and even more preferentially below 30°C.

[0030] Advantageously, the enzymatic dissolution of glycogen is carried out at a pH below 5, preferably at about 4.5.

[0031] Preferentially, the enzymatic reaction is carried out at room temperature, which corresponds to the definition of use in the temperate zone or in a room having a temperature in the temperate zone, i.e. in the range of 18-28°C, more generally in the range of 20-25°C.

[0032] These temperature and pH conditions are particularly suitable for protecting phycocyanin during the enzymatic reaction.

[0033] Enzymes active under acidic pH conditions and at room temperature are known to those skilled in the art, but conditions for digesting glycogen to protect phycocyanin and promote its production are unknown.

[0034] Surprisingly, it has been found that an enzyme known to have α1-4 galactosiduronic activity also has α1-4 glucosidase (or alpha-glucosidase) activity under pH and temperature conditions compatible with phycocyanin purification.

[0035] This is particularly true for pectinases known to degrade pectin, in particular pectinases extracted from filamentous fungi such as Aspergillus, more particularly pectinases extracted from Aspergillus aculeatus, such as the enzyme sold under the name Pectinex® by Novozymes.

[0036] The action of these enzymes reduces the size of the glycosidic chains which can then be removed by ultrafiltration under conditions which allow the phycocyanin to be retained while the glycogen fragments pass through.

[0037] The inventors have found that these enzymatic dissolution conditions are particularly suitable for releasing polyglucoside chains and a small number of glucose monomers, thus avoiding contamination with other microorganisms, especially organisms pathogenic to humans or animals, which is essential if the resulting phycocyanin is to be used as a food colorant.

[0038] According to certain embodiments, enzymatic lysis of glycogen can be achieved using α1-6 glucosidase activity in addition to α1-4 glucosidase or polygalacturonase activity.

[0039] The enzymes used in the method of the invention may then be a mixture of enzymes, ie a first enzyme with α1-4 glucosidase activity or a second enzyme with polygalacturonase and α1-6 glucosidase activity.

[0040] α1-6 glucosidases active under the above-mentioned pH and temperature conditions are also known to those skilled in the art, in particular pullulanases, which are known to hydrolyze the α1-6 glycosidic bonds of pullulan, in particular to remove starch branches.

[0041] These are generally enzymes extracted from bacteria, particularly 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. It should be noted that pullulanase / alpha-amylase mixtures have been described in the prior art, particularly for producing glucose syrup from starch (US 2017 / 159090).

[0042] According to another preferred embodiment of the invention, the enzyme has both α1-4 glucosidase activity and α1-6 glucosidase activity.

[0043] This is especially true for glucoamylases. These are also enzymes extracted from microorganisms, especially yeasts or fungi, such as S. diastaticus or A. niger. Many glucoamylases are known from the prior art and are described in the literature, in particular in patent applications such as WO2019 / 036721. They are generally used in fermentation methods for the production of alcohol for consumption (beer, spirits) or for the fermentation of biomass for the production of bioethanol. They are also used as baking additives or food supplements. Glucoamylases are known to be commercially available, inter alia, under the names "Amylase AG XXL" (Novozymes) or "Panzym® AG XXL" (Eaton).

[0044] Advantageously, the enzymes used in the method according to the invention are enzymes approved for use in the food industry.

[0045] The optimal enzyme content to be used in the glycogenolysis step can be determined by those skilled in the art according to the activity of the enzyme used under the above temperature and pH conditions.

[0046] The enzyme concentration is generally between 0.0001% and 5%, preferentially between 0.0025% and 1%, more preferentially between 0.005% and 0.5%, and even more preferentially between 0.01% and 0.25%, where the percentages are expressed as the volume of enzyme solution relative to the total volume of the crude suspension or crude suspension.

[0047] Enzyme solutions generally have enzyme concentrations ranging from 100 to 20,000 units / mL, and enzyme activity is generally attributed to these enzymes as identified by the manufacturer.

[0048] The use of α1-6 glucosidase reduces the amount of α1-4 glucosidase or polygalacturonase used. The total enzyme concentration (α1-4 glucuronidase + α1-6 glucosidase) is generally between 0.0001% and 5%, preferentially between 0.0025% and 1%, more preferentially between 0.005% and 0.5%, and even more preferentially between 0.01% and 0.25%, where the percentages are expressed as the volume of enzyme solution relative to the total volume of the crude suspension or crude suspension.

[0049] When α1-4 glucosidase or polygalacturonase is used alone or in combination with α1-6 glucosidase or with an enzyme having α1-4 glucosidase activity and α1-6 glucosidase activity, the reaction is advantageously carried out for less than 48 hours, preferably less than 24 hours, more preferentially between 5 and 12 hours.

[0050] For the method according to the invention, and more particularly for the step of isolation by tangential filtration to isolate phycocyanin, it is not necessary to obtain complete digestion of glycogen to glucose monomers. Partial digestion of the polysaccharide and reduction to oligomers of a size below the filtration cut-off threshold is sufficient to remove glycogen from the suspension or phycocyanin solution.

[0051] Those skilled in the art will know how to determine the appropriate time to best reduce the amount of glycogen as a function of the initial glycogen content, the amount of enzyme used, and the desired purity of the phycocyanin produced.

[0052] The implementation of glycogen reduction by enzymatic digestion can be associated with or replaced by the use of microorganisms capable of degrading this polysaccharide. Those skilled in the art will know how to take advantage of the ability of these microorganisms to produce and secrete enzymes capable of digesting glycogen in crude extracts, more particularly the enzymes already described. Those skilled in the art will know how to select and take advantage of the ability of these microorganisms to metabolize glycogen or the products resulting from the degradation of polysaccharides. Advantageously, those skilled in the art will know how to take advantage of the ability of these microorganisms to limit the growth of undesirable or pathogenic microorganisms, in particular by synthesizing substances with antimicrobial activity.

[0053] Preferred conditions for carrying out glycogen degradation ex vivo or in vivo are a pH below 7 and a reaction temperature below 50°C, preferably below 40°C, even more preferentially below 37°C.

[0054] Advantageously, the ex vivo or in vivo degradation of glycogen is carried out at a pH below 5, preferably at about 4.5 or 4.

[0055] Due to their unique characteristics of growing and degrading polysaccharides, lactic acid bacteria are considered to be particularly suitable, among which bacteria belonging to the genera Lactobacillus, Pediococcus, Tetragenococcus, Carnobacterium, Vagococcus, Leuconostoc, Weissella, Oenococcus, Atopobium, Streptococcus, Enterococcus, Lactococcus, Aerococcus, Alloiococcus, Melissococcus or Bifidobacterium can be mentioned.

[0056] The present invention also relates to a phycocyanin extract 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.

[0057] According to a first embodiment, this phycocyanin extract is a crude phycocyanin suspension obtained after enzymatic lysis.

[0058] This treated crude suspension, also called "enzymatically treated crude suspension", contains in the suspension insoluble matter resulting from cell lysis, in particular phycocyanin released after cell lysis, glucose oligomers which are products of enzymatic lysis of glycogen, and residual glycogen.

[0059] According to a second embodiment of the invention, the phycocyanin extract is a crude phycocyanin solution obtained after isolation of a crude suspension and enzymatic lysis of glycogen, the lysis being carried out before or after isolation of the crude suspension, or before and after isolation (isolation of an enzymatically treated crude suspension and / or carrying out an enzymatic reaction on the crude solution).

[0060] This crude solution contains, among other things, phycocyanin released after cell lysis, glucose oligomers that are the product of enzymatic lysis of glycogen, and residual glycogen. This treated crude solution, also called "enzymatically treated phycocyanin crude solution," generally contains 0.1 to 10 g / L of phycocyanin, more preferentially 1 to 5 g / L.

[0061] The dry weight ratio of glycogen to phycocyanin is advantageously less than 3, preferably less than 2.5.

[0062] The enzymatically treated crude solution according to the invention may optionally be concentrated by removing a portion of the water according to conventional methods in the art, carried out under conditions that substantially respect the integrity of the phycocyanin, in which case the phycocyanin content of the concentrated enzymatically treated crude solution is advantageously between 10 and 50 g / L.

[0063] According to another embodiment, the phycocyanin extract is phycocyanin isolated after extraction from a crude solution enzymatically treated according to the method described above.

[0064] For isolated phycocyanin, the dry weight ratio of glycogen to phycocyanin is advantageously less than 2, preferably less than 1.

[0065] According to another embodiment, the phycocyanin extract is a purified phycocyanin obtained after purification of the isolated extract according to the above-described method, in particular by diafiltration.

[0066] For purified phycocyanin, the dry weight ratio of glycogen to phycocyanin is advantageously less than 1, preferably less than 0.1.

[0067] Both isolated and purified phycocyanin may still contain trace amounts of glucose oligomers, which are products of the enzymatic dissolution of glycogen.

[0068] The phycocyanin obtained has an E10 tinting strength of between 90 and 400, preferentially of at least 120, more preferentially of at least 150.

[0069] For enzymatically treated crude solutions, the E10 tint strength is advantageously between 90 and 110.

[0070] For isolated phycocyanins, the E10 tinting strength is advantageously between 150 and 210.

[0071] For purified phycocyanin, the color strength is advantageously between 210 and 400.

[0072] The present invention also provides a method for producing phycocyanin of microbial origin, comprising the steps of: (a) culturing a phycocyanin-producing microorganism under culture conditions to produce a fermented must containing greater than 30 g / L dry matter and at least 4% phycocyanin on a dry matter basis; (b) lysing the cells to release the phycocyanin and glycogen produced, to obtain a crude suspension as defined above; (c) separating the crude suspension and recovering a crude solution containing phycocyanin and glycogen, and then optionally (d) isolating the phycocyanin from the crude solution, and then optionally (e) Purifying the isolated phycocyanin wherein the step of enzymatic lysis of glycogen is carried out under the conditions defined above with the enzymes defined above or by microbial degradation, said enzymatic lysis being carried out on a crude suspension and / or a crude solution.

[0073] Advantageously, the phycocyanin obtained is a phycocyanin containing less than 50% glycogen.

[0074] Cultivation methods are well known to those skilled in the art and are described in particular in patent applications WO2017 / 050917, WO2017 / 093345 and WO2018 / 178334.

[0075] These make it possible to obtain a fermented must of more than 30 g / L of dry matter, which can amount to more than 100 g / L of dry matter.

[0076] The phycocyanin content is at least 4%, but can reach more than 10%, depending on the fermentation conditions and the strain cultivated.

[0077] Those skilled in the art will know how to determine the culture conditions according to their own industrial phycocyanin production purposes.

[0078] Separation step (c), in particular by conventional filtration methods, such as microfiltration, or centrifugation followed by filtration, in particular microfiltration, is also known and described in the prior art.

[0079] 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 solid or liquid foods, in particular beverages, containing the phycocyanin with low glycogen content according to the present invention.

[0080] The phycocyanin used as a colorant may be in the form of enzymatically treated crude solution, isolated phycocyanin or purified phycocyanin, as defined above. [Example]

[0081] Example 1 - Monitoring C-PC concentration before and after enzymatic lysis The phycocyanin concentration of the crude extract was monitored at pH 4 and pH 7 using different amounts of the enzyme "Pectinex." Crude phycocyanin extract from Galdieria sulphuraria was produced according to the method described in application WO 2018 / 178334. For this monitoring, the enzyme and crude phycocyanin extract were filtered through a 0.22 μm filter. Digestion was carried out at room temperature. For each kinetic point, absorbance readings useful for determining the phycocyanin concentration were measured in parallel with glucose measurements after enzyme denaturation (95°C, 5 minutes) using a YSI 2700 Biochemical Analyzer.

[0082] The results are shown in Figures 1 to 4.

[0083] These results indicate that the amount of digested glycogen varies between different conditions of pH and enzyme concentration. Excessive enzyme may result in the degradation of phycocyanin.

[0084] However, the inventors have been able to see that after less than 24 hours of digestion at pH 4 and 0.05% "Pectinex", significant glycogenolysis is obtained while substantially limiting phycocyanin degradation.

[0085] Example 2 - Monitoring the rate of glycogen digestion in crude phycocyanin solutions Monitoring of the rate of glycogen digestion in crude solution is carried out at pH 4 and pH 7 using different enzymes: alpha amylase (Novozymes Ban 480L), polygalacturonase (Novozymes Pectinex Ultra SP-L) and glucoamylase (Novozymes Amylase AG XXL).

[0086] A crude phycocyanin solution from Galdieria sulphuraria is produced according to the method described in application WO2018 / 178334. For this monitoring, the enzyme and crude phycocyanin solution are filtered through a 0.22 μm filter. Digestion is carried out at room temperature. For each kinetic point, glucose measurements are performed after enzyme denaturation (95°C, 5 minutes) using a YSI 2700 Biochemistry Analyzer. The percentage of glycogen digestion is the ratio of the glucose concentration to the glucose concentration after complete hydrolysis of the polysaccharide.

[0087] The results are shown in Figure 6 (pH=4) and Figure 7 (pH=7).

[0088] Example 3 - glycogen content in purified products with or without enzymatic lysis The crude phycocyanin solution, either untreated or digested with 0.25% (v / v) α1-6 glucosidase for 12 hours and then with 0.1% (v / v) α1-4 polygalacturonase for 2 hours, is filtered through a hollow fiber membrane with a porosity of 70 kDa in a final diafiltration step.

[0089] Various measurements carried out at the end of each filtration and / or filtration step show that the concentration of glycogen increases significantly in the retentate until it reaches a concentration that is not negligible compared to PC. It is therefore necessary to remove all or part of this glycogen to avoid diluting the color strength of the final product, the E10 color strength of which was between 90 and 400.

[0090] The E10 color value (10% E618nm) indicates the color strength measured at 618nm after dissolving the powder in an aqueous solution.

[0091] Protocol: 0.25 grams of sample is measured and dissolved in 100 mL of citrate buffer solution adjusted to pH 6.0. This solution is then also diluted 10 times with citrate buffer, and the absorbance at 618 nm is measured using a 1 cm thick cuvette. E10 color value (10% E618 nm) = absorbance (618 nm) x 100 / 0.25 grams.

[0092] [Table 1]

[0093] Example 4 - Transmembrane pressure with or without enzymatic lysis Crude phycocyanin extract from Galdieria sulphuraria, produced according to the method described in patent application WO2018 / 178334, was clarified on a 0.05 μm PES hollow fiber membrane. The results below show filtration parameter monitoring of the same volume of 250 mL of crude extract, with and without digestion with "Pectinex" (0.05%, 5.5 hours, room temperature, pH = 4).

[0094] The results are shown in Figure 5. They demonstrate the effect of glycogen digestion on the microfiltration of crude extracts. It can be seen that to filter a given volume, the time required for the digested sample is approximately half that required for the undigested sample due to the increase in transmembrane flow.

[0095] 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., Korean Journal of Chemical Engineering (2014) 31, 490-495 ·Shimonaga et al., Marine Biotechnology (2007) 9, 192-202. ·Shimonaga et al., Plant and Cell Physiology (2008) 49, 103-116. ·CN 106749633, CN102433015 and CN1117973 ·US 6,074,854, US 5,817,498, US 2017 / 159090 ·WO 2009 / 07568, WO 20, WO 2017 / 050918, WO 2017 / 093345, WO 2018 / 178334, WO 2019 / 036721

Claims

1. A method for purifying phycocyanin from a solution containing phycocyanin and glycogen, comprising the steps of (i) enzymatically hydrolyzing glycogen at a pH below 6 and a reaction temperature below 40°C using an enzyme that degrades glycogen, and (ii) separating phycocyanin from glycogen degradation products, wherein the phycocyanin contained in the solution is produced by a microorganism of the order Cyanidiales, and the enzyme is pectinase, glucoamylase, or a mixture of pectinase and pullulanase.

2. 2. The method of claim 1, wherein the temperature is below 30° C. and / or the pH is below 5.

3. 3. The method according to claim 1 or 2, characterized in that the solution containing phycocyanin and glycogen is a crude suspension obtained after cell lysis of a phycocyanin-producing microbial biomass.

4. 3. The method according to claim 1 or 2, characterized in that the solution containing phycocyanin and glycogen is a crude solution obtained after filtration of the crude suspension itself obtained after cell lysis of the phycocyanin-producing microbial biomass.

5. A method for producing phycocyanin of microbial origin, comprising: (a) culturing a phycocyanin-producing microorganism of the order Cyanidiales under culture conditions to produce a fermentation must containing more than 30 g / L dry matter and at least 4% by weight phycocyanin on a dry matter basis; (b) lysing the cells to release the produced phycocyanin and glycogen and obtaining a crude suspension; and (c) separating the crude suspension and recovering a crude solution containing phycocyanin and glycogen. The method further comprises the additional step of purifying phycocyanin from the crude suspension obtained in (b) and / or the crude solution obtained in (c), the additional steps comprising the substeps of (i) enzymatically hydrolyzing glycogen at a pH below 6 and a reaction temperature below 40°C using a glycogen-degrading enzyme, and (ii) separating phycocyanin from the glycogen degradation products, wherein the enzyme is pectinase, glucoamylase, or a mixture of pectinase and pullulanase.

6. 6. The method of claim 5, further comprising the step of (d) isolating phycocyanin from the crude solution.

7. 7. The method of claim 6, further comprising the step of (e) purifying the isolated phycocyanin.

8. 8. The method according to any one of claims 5 to 7, characterized in that the additional step is carried out on the crude solution obtained in (c).

9. 9. The method according to claim 1, wherein the phycocyanin is of microbial origin, produced by a microorganism selected from species of the genera Cyanidioschyzon, Cyanidium and Galdieria.

10. 10. The method of claim 9, wherein the microorganism is Galdieria sulphuraria.

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