METHOD FOR EXTRACTING SOLUBLE PROTEINS FROM MICROALGAL BIOMASS

Abstract

The invention relates to a method for preparing a protein isolate of the biomass of microalgae of the genus Chlorella, characterised in that it comprises the following steps: supplying a microalgal biomass produced by fermentation, washing the biomass so as to eliminate the soluble interstitial compounds and concentrating the biomass, mechanically grinding the washed and concentrated biomass in a horizontal ball grinder-type system in order to produce an emulsion, destructuring the emulsion thus produced, triple-phase separation so as to separate the soluble fraction from the fractions containing the lipids and the cell debris, recovery of the soluble fraction thus produced in order to produce the soluble protein isolate, then evaporation, pasteurisation and atomisation of said protein isolate.

Claims

1-7. (canceled)

8. A method for preparing a protein isolate from the biomass of microalgae of the Chlorella genus, comprising: providing a microalgal biomass produced by fermentation of microalgae of the Chlorella genus; washing the microalgal biomass so as to eliminate interstitial soluble compounds and concentrate the microalgal biomass; mechanical milling of the washed and concentrated microalgal biomass to obtain an emulsion; destructuring the obtained emulsion by (i) or (ii), thus resulting in a destructured emulsion: (i) by enzymatic predigestion, by treatment with polar solvent, and/or by controlled alkaline treatment targeting the protein fraction of the emulsion; or (ii) by adjusting the pH and the temperature, by treatment with a polar solvent, and/or by enzymatic digestion targeting the interface with the lipid fraction of the emulsion; triphase separation so as to separate the soluble fraction from the fractions containing lipids and cell debris; recovering the soluble fraction obtained in this way in order to obtain the soluble protein isolate; then evaporation, pasteurization and atomization of the protein isolate.

9. The method as claimed in claim 8, wherein the microalgae of the Chlorella genus are selected from the group consisting of Chlorella vulgaris, Chlorella sorokiniana, and Chlorella protothecoides.

10. The method as claimed in claim 8, wherein the triphase separation of the destructured emulsion is carried out by centrifugation.

11. The method as claimed in claim 8, wherein the soluble protein isolate is obtained from the soluble fraction by: clarifying the soluble fraction by microfiltration so as to remove residual insoluble substances therefrom; ultrafiltration of the clarified soluble fraction on a membrane with a cut-off threshold of less than 5 kDa; and optionally, neutralizing at a pH of between 6 and 8.

12. The method as claimed in claim 8, wherein the soluble protein isolate is obtained from the soluble fraction by: precipitating the proteins at their pI by adjusting the pH of the medium to a value of between 4 and 5; centrifugation or microfiltration in order to recover the precipitated proteins; and dissolving in water at a pH of between 6 and 8.

13. The method as claimed in claim 9, wherein the microalgae of the Chlorella genus is Chlorella protothecoides.

14. The method as claimed in claim 9, wherein the triphase separation of the destructured emulsion is carried out by centrifugation.

15. The method as claimed in claim 2, wherein the soluble protein isolate is obtained from the soluble fraction by: clarifying the soluble fraction by microfiltration so as to remove residual insoluble substances therefrom; ultrafiltration of the clarified soluble fraction on a membrane with a cut-off threshold of less than 5 kDa; and optionally, neutralizing at a pH of between 6 and 8.

16. The method as claimed in claim 9, wherein the soluble protein isolate is obtained from the soluble fraction by: precipitating the proteins at their pI by adjusting the pH of the medium to a value of between 4 and 5; centrifugation or microfiltration in order to recover the precipitated proteins; and dissolving in water at a pH of between 6 and 8.

17. The method as claimed in claim 12, wherein the dissolving in water is carried out at a pH of 7.

Description

DETAILED DESCRIPTION OF THE INVENTION

Choice of the Microalgal Biomass

[0055] Preferably, the microalgae of the Chlorella genus are chosen from the group consisting of Chlorella vulgaris, Chlorella sorokiniana and Chlorella protothecoides, and are more particularly Chlorella protothecoides.

[0056] In one particular embodiment, the strain is Chlorella protothecoides (strain UTEX 250The Culture Collection of Algae at the University of Texas at AustinUSA).

[0057] In another particular embodiment, the strain is Chlorella sorokiniana (strain UTEX 1663The Culture Collection of Algae at the University of Texas at AustinUSA).

[0058] The culturing under heterotrophic conditions and in the absence of light conventionally results in the production of a chlorella biomass having a protein content (evaluated by measuring the nitrogen content N6.25) of 45% to 70% by weight of dry cells.

[0059] As will be exemplified hereinafter, this culturing is carried out in two steps: [0060] preculturing in a medium containing glucose and yeast extract for 72 h at 28 C. with agitation, then [0061] culturing for production of the biomass per se in glucose and yeast extract for more than 36 h at 28 C., with agitation and at pH 6.5 adjusted with aqueous ammonia, which results in approximately 80 g/l of biomass with a protein content (evaluated by N6.25) of the order of 52% by weight of dry cells.

[0062] The biomass is then collected by solid-liquid separation, by frontal or tangential filtration or by any means known, moreover, to those skilled in the art.

[0063] Advantageously, the Applicant company then recommends washing and concentrating the biomass so as to eliminate the interstitial soluble compounds by a succession of concentration (by centrifugation)/dilution of the biomass.

[0064] On the industrial scale, in-line dilution and separation by centrifugation in one or two stages is advantageously chosen.

[0065] For the purposes of the invention, the term interstitial soluble compounds is intended to mean all the soluble organic contaminants of the fermentation medium, for example the hydrosoluble compounds such as the salts, the residual glucose, the oligosaccharides with a degree of polymerization (or DP) of 2 or 3, or the peptides.

[0066] This biomass purified in this way of its interstitial soluble compounds is then preferentially adjusted to a dry matter of between 15% and 30% by weight, preferably to a dry matter of between 20% and 30%.

[0067] For the remainder of the method of the invention, the biomass obtained in this way may be used as is, or thermally permeabilized (by a high-temperature short-time or HTST methodalso developed by the Applicant company and protected in one of its as yet unpublished applications) so as to release the content of soluble peptides therefrom.

[0068] The residual proteins of this biomass may be extracted by the subsequent following steps.

Biomass Milling

[0069] The Applicant company recommends using (horizontal) bead mill technology.

[0070] More particularly, the milling may advantageously be carried out according to a method which the Applicant company has developed and protected in one of its as yet unexamined applications, in which: [0071] the zirconium silicate beads have an apparent density of between 2 and 3.5 kg/l, and [0072] the filling rate of the milling chamber is greater than or equal to 80%.

[0073] The milling is carried out in continuous mode, for example by successive passes in series.

[0074] The density of the microalgae to be milled is chosen at a level of less than 250 g/l.

[0075] At the end of milling, an emulsion is obtained.

Destructuring of the Emulsion and Separation of its Components

[0076] The separation of the components of the emulsion in order to extract the peptide or polypeptide fraction of interest therefrom requires destructuring/destabilization of the emulsion resulting from the cell milling (complex mixture of lipids, proteinspeptides and polypeptidesand cell debris).

[0077] This destructuring/destabilization of the emulsion may be facilitated: [0078] either by enzymatic predigestion, especially by specific proteases, by treatment with polar solvent and/or by controlled alkaline treatment targeting the protein fraction of the emulsion, [0079] or by adjusting the pH and the temperature, by treatment with a polar solvent and/or by enzymatic digestion, especially of cellulase type, targeting the interface with the lipid fraction of the emulsion.

[0080] Thus, the milled cell material is conditioned in a stirred reactor fitted with a low shear stirring module, so as to limit emulsification while enabling homogeneous mixing promoting the specific treatment chosen (setting a pH, action of the lytic enzyme, etc.).

[0081] For example, in the case of a treatment which aims to destabilize the emulsion by treating the protein fraction in mixture via the enzymatic route, for example by a basic protease, the temperature and the pH of the emulsion are adjusted to the reaction conditions for said protease: [0082] the temperature is adjusted to a value of greater than 30 C., preferably of the order of 60 C., and [0083] the pH is adjusted to a value of greater than 7, preferably of the order of 8 (or even optionally of the order of 10 if only the action of pH is being utilized).

[0084] The duration of the reaction is between 2 and 8 h.

[0085] At the end of the lysis, ethanol at more than 5% (v/v) may be added to the reaction mixture as destabilizing agent for the emulsion (in the case of an oil in water emulsion).

[0086] The emulsion destabilized in this way may be (partially) split up by triphase separation, for example by centrifugation.

[0087] Thus, 3 phases are obtained: [0088] an upper lipid cream, [0089] an aqueous/intermediate (=raw soluble substances) soluble compounds (and residual insoluble substances) phase, and [0090] a pellet concentrating the cell debris.

[0091] The soluble fraction is essentially composed of a predominant protein fraction, soluble sugars, salts and residual lipid globules.

Membrane Separation

[0092] To release peptides and polypeptides, the method of the invention next leads to the isolation of the proteins of interest, preferably by membrane fractionation.

[0093] The Applicant company thus recommends carrying out the process in three steps: [0094] recovery and clarification of the soluble fraction obtained in this way by microfiltration so as to remove residual insoluble substances therefrom, [0095] ultrafiltration of the clarified soluble fraction on a membrane with a cut-off threshold of less than 5 kDa, preferably of between 1 and 5 kDa, and [0096] optional neutralization at a pH of between 6 and 8, preferably at a value of 7.

[0097] Utilizing these pathways makes it possible to purify the soluble peptides and polypeptides of their residual salts and sugars.

Precipitation at the pI

[0098] Alternatively, to isolate the peptides and polypeptides of interest, the choice may be made to carry out the process in three steps: [0099] precipitating the proteins at their pI, by adjusting the pH of the medium to a value of between 4 and 5, [0100] centrifugation or microfiltration in order to recover the precipitated proteins, and [0101] dissolving in water at a pH of between 6 and 8, preferably 7.

[0102] It should be noted that although the latter two steps make it possible, according to the method of the invention, to obtain protein isolates having a protein content of more than 80%, preferably of more than 90% by weight, they lead, by their implementational methods, to compositions distinct in nature.

Obtaining the Isolate in Powder Form

[0103] The protein isolate in soluble form obtained in this way may be: [0104] concentrated by evaporation, [0105] pasteurized, and finally [0106] atomized.

[0107] The invention will be understood more clearly from the following examples which are intended to be illustrative and nonlimiting.

EXAMPLES

Example 1

Production of Chlorella protothecoides by Fed-Batch Fermentation

[0108] The strain used is Chlorella protothecoides UTEX 250

Preculture:

[0109] 500 ml of medium in a 21 conical flask; [0110] Composition of the medium (in g/l):

TABLE-US-00001 TABLE 1 Macro- Glucose 40 elements (g/l) K.sub.2HPO.sub.4 3 Na.sub.2HPO.sub.4 3 MgSO.sub.47H.sub.2O 0.25 (NH.sub.4).sub.2SO.sub.4 1 Citric acid 1 Clerol FBA 3107 (antifoam) 0.1 Microelements and Vitamins CaCl.sub.22H.sub.2O 30 (mg/l) FeSO.sub.47H.sub.2O 1 MnSO.sub.41H.sub.2O 8 CoSO.sub.47H.sub.2O 0.1 CuSO.sub.45H.sub.2O 0.2 ZnSO.sub.47H.sub.2O 0.5 H.sub.3BO.sub.3 0.1 Na.sub.2MoO.sub.42H.sub.2O 0.4 Thiamine HCl 1 Biotin 0.015 B12 0.01 Calcium pantothenate 0.03 p-Aminobenzoic acid 0.06

[0111] Incubation is carried out under the following conditions: duration: 72 h; temperature: 28 C.; agitation: 110 rpm (Infors Multitron incubator).

[0112] The preculture is then transferred to a 30 l Sartorius type fermenter.

Culture for Biomass Production:

[0113] The medium is as follows:

TABLE-US-00002 TABLE 2 Macro- Glucose 40 elements (g/l) KH.sub.2PO.sub.4 1.8 NaH.sub.2PO.sub.4 1.4 MgSO.sub.47H.sub.2O 3.4 (NH.sub.4).sub.2SO.sub.4 0.2 Clerol FBA 3107 (antifoam) 0.3 Microelements and Vitamins CaCl.sub.22H.sub.2O 40 (mg/l) FeSO.sub.47H.sub.2O 12 MnSO.sub.41H.sub.2O 40 CoSO.sub.47H.sub.2O 0.1 CuSO.sub.45H.sub.2O 0.5 ZnSO.sub.47H.sub.2O 50 H.sub.3BO.sub.3 15 Na.sub.2MoO.sub.42H.sub.2O 2 Thiamine HCl 6 Biotin 0.1 B12 0.06 Calcium pantothenate 0.2 p-Aminobenzoic acid 0.2

[0114] The initial volume (Vi) of the fermenter is adjusted to 17 l after inoculation. It is brought to a final volume of approximately 20-25 l.

[0115] The parameters for performing the fermentation are as follows:

TABLE-US-00003 TABLE 3 Temperature 28 C. pH 5.0-5.2 by 28% w/w NH.sub.3 pO.sub.2 20% 5% (maintained by shaking) Shaking Minimum 300 rpm Air flow rate 15 l/min

[0116] When the residual glucose concentration falls below 10 g/l, glucose in the form of a concentrated solution at approximately 800 g/l is introduced so as to maintain the glucose content between 0 and 20 g/l in the fermenter.

Results

[0117] In 40 h, 80 g/l of biomass containing 52% of proteins are obtained.

Example 2

Milling the Chlorella protothecoides Biomass and Recovery of the Soluble FractionDestructuring of the Emulsion by Treatment of the Peptide and Polypeptide Fraction

[0118] The biomass obtained according to example 1 is washed and concentrated by centrifugation so as to be brought to a dry matter content of 220 g/l and to a purity of more than 90% (purity defined by the ratio of the dry matter of the biomass to the total dry matter).

[0119] It is then milled by bead milling (horizontal bead mill) with zirconium silicate beads (0.6 mm diameter, apparent density 2.4).

[0120] The milled biomass is then agitated in a reactor fitted with a marine impeller and baffles. The temperature is adjusted to 60 C. and the pH to 8 with potassium hydroxide. A basic protease in combination with a cellulase are added, with these reaction conditions being maintained for a duration of 6 h.

[0121] The emulsion is then centrifuged on a triphase centrifuge which makes it possible to obtain 3 phases: an upper lipid cream, an aqueous/intermediate (=raw soluble substances) soluble compounds (and residual insoluble substances) phase, and a pellet concentrating the cell debris.

[0122] The fraction of raw soluble substances is clarified by microfiltration. The microfiltration permeate P1 has a titer between 55% and 70% of peptides and proteins (expressed as total amino acids) and is then ultrafiltered on a membrane with a <5 kDa cut-off threshold.

[0123] The ultrafiltration retentate R2 obtained in this way contains more than 80% of peptides having a molecular weight of greater than or equal to 5 kDa.

[0124] The permeate P2 contains peptides having a molecular weight of less than 5 kDa and oligosaccharides and residual salts.

[0125] This permeate P2 can then especially be filtered on a reverse osmosis membrane (having a degree of NaCl rejection of 93%), so as to obtain: [0126] a retentate R3, containing peptides having a molecular weight of less than 5 kDa and oligosaccharides of DP 2, such as sucrose; and [0127] a permeate R3, containing oligosaccharides of DP 1, salts, free amino acids and organic acids.

[0128] The protein isolate R2 is then: [0129] neutralized to pH 7 with potassium hydroxide, [0130] concentrated by evaporation to 35% dry matter (DM), [0131] pasteurized, then [0132] atomized.

Example 3

Milling the Chlorella protothecoides Biomass and Recovery of the Soluble FractionDestructuring of the Emulsion by Treatment of the Lipid Fraction

[0133] According to the same sequence as in example 2, the milled biomass is agitated in a reactor fitted with a marine impeller and baffles. The temperature is adjusted to 50 C. without adjusting the pH (naturally between 5 and 6).

[0134] A cellulase having optimum activity in this pH and temperature range is added, with these reaction conditions being maintained for a duration of 6 h.

[0135] At the end of the reaction, the pH is adjusted to 8 before the separation into 3 phases.

[0136] The remainder of the operations is described in example 2.