Method for extracting soluble proteins from microalgal biomass

Abstract

A method for preparing a protein isolate of the biomass of microalgae of the genus Chlorella, includes 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, pasteurization and atomization of the protein isolate.

Claims

1. 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, wherein the fermentation is carried out for more than 36 hours at 28 C. with agitation and at a pH of 6.5, 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, carried out in a horizontal bead mill type system, 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 for a duration of 2-8 hours at a temperature of more than 30 C. and a pH of greater than 7; or (ii) by treatment with a polar solvent, and/or by enzymatic digestion targeting the interface with the lipid fraction of the emulsion for a duration of 6 hours at a temperature of 50 C. and a pH of 5-6; 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.

2. The method as claimed in claim 1, wherein the microalgae of the Chlorella genus are selected from the group consisting of Chlorella vulgaris, Chlorella sorokiniana, and Chlorella protothecoides.

3. The method as claimed in claim 1, wherein the triphase separation of the destructured emulsion is carried out by centrifugation.

4. The method as claimed in claim 1, 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, optionally, neutralizing at a pH of between 6 and 8.

5. The method as claimed in claim 1, 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.

6. The method as claimed in claim 2, wherein the microalgae of the Chlorella genus is Chlorella protothecoides.

7. The method as claimed in claim 2, wherein the triphase separation of the destructured emulsion is carried out by centrifugation.

8. 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.

9. The method as claimed in claim 2, 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.

10. The method as claimed in claim 5, wherein the dissolving in water is carried out at a pH of 7.

Description

EXAMPLES

Example 1: Production of Chlorella protothecoides by Fed-Batch Fermentation

(1) The strain used is Chlorella protothecoides UTEX 250

(2) Preculture: 500 ml of medium in a 2 l conical flask; Composition of the medium (in g/l):

(3) TABLE-US-00001 TABLE 1 Macro- Glucose 40 elements K.sub.2HPO.sub.4 3 (g/l) 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 Micro- CaCl.sub.22H.sub.2O 30 elements FeSO.sub.47H.sub.2O 1 and MnSO.sub.41H.sub.2O 8 Vitamins CoSO.sub.47H.sub.2O 0.1 (mg/l) 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

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

(5) The preculture is then transferred to a 30 l Sartorius type fermenter.

(6) Culture for Biomass Production

(7) The medium is as follows:

(8) TABLE-US-00002 TABLE 2 Macro- Glucose 40 elements KH.sub.2PO.sub.4 1.8 (g/l) 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 Micro- CaCl.sub.22H.sub.2O 40 elements FeSO.sub.47H.sub.2O 12 and MnSO.sub.41H.sub.2O 40 Vitamins CoSO.sub.47H.sub.2O 0.1 (mg/l) 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

(9) 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.

(10) The parameters for performing the fermentation are as follows:

(11) 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

(12) 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.

(13) Results

(14) 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

(15) 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).

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

(17) 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.

(18) 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.

(19) 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.

(20) 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.

(21) The permeate P2 contains peptides having a molecular weight of less than 5 kDa and oligosaccharides and residual salts.

(22) 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: a retentate R3, containing peptides having a molecular weight of less than 5 kDa and oligosaccharides of DP 2, such as sucrose; and a permeate R3, containing oligosaccharides of DP 1, salts, free amino acids and organic acids.

(23) The protein isolate R2 is then: neutralized to pH 7 with potassium hydroxide, concentrated by evaporation to 35% dry matter (DM), pasteurized, then atomized.

Example 3. Milling the Chlorella protothecoides Biomass and Recovery of the Soluble FractionDestructuring of the Emulsion by Treatment of the Lipid Fraction

(24) 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).

(25) 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.

(26) At the end of the reaction, the pH is adjusted to 8 before the separation into 3 phases.

(27) The remainder of the operations is described in example 2.