METHOD FOR EXTRACTING SOLUBLE PROTEINS FROM MICROALGAL BIOMASS

Abstract

Method for preparing a protein isolate of the biomass of microalgae of the genus Chlorella, includes: supplying a microalgal biomass produced by fermentation; optionally washing the biomass so as to eliminate soluble interstitial compounds, thermal permeabilization of the biomass at a temperature between 50 and 150 C., for between 10 seconds and 5 minutes; eliminating the thus permeabilized biomass by a solid-liquid separation technique selected from the group consisting of frontal or tangential filtration, flocculation and centrifuging, more specifically multi-stage centrifuging, in order to obtain a soluble fraction; optionally recovering and clarifying the soluble fraction thus obtained by microfiltration in such a way as to remove residual insoluble elements therefrom; ultrafiltration of the soluble fraction on a membrane with a cut-off threshold lower than 5 kDa, preferably between 1 and 5 kDa, in order to produce a 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 the following steps: providing a microalgal biomass produced by fermentation, optionally, washing the biomass so as to eliminate the interstitial soluble compounds, thermal permeabilization of the biomass at a temperature of between 50 and 150 C., for a duration of between 10 seconds and 5 minutes, elimination of the biomass permeabilized in this way by a technique of solid-liquid separation chosen from the group consisting of frontal or tangential filtration, flocculation and centrifugation, more particularly multistage centrifugation, to obtain a soluble fraction, optionally, recovery and clarification of the soluble fraction obtained in this way by microfiltration so as to remove residual insoluble substances therefrom, ultrafiltration of the soluble fraction on a membrane with a cut-off threshold of less than 5 kDa, so as to obtain a soluble protein isolate, then evaporation, pasteurization and atomization of said protein isolate.

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

3. The method as claimed in claim 1, wherein the thermal permeabilization of the biomass is carried out at a temperature of between 100 and 150 C., for a duration of between 10 seconds and 1 minute.

4. The method as claimed in claim 1, wherein the ultrafiltration of the soluble fraction is carried out on a membrane with a cut-off threshold of between 1 and 5 kDa.

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

Description

EXAMPLES

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

[0093] The strain used is Chlorella protothecoides UTEX 250.

[0094] Preculture: [0095] 500 ml of medium in a 2 l conical flask; [0096] 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 0.1 (antifoam) 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

[0097] Incubation is carried out under the following conditions: duration: 72 h; temperature: 28 C.; agitation: 110 rpm (Infors Multitron Incubator).

[0098] The preculture is then transferred to a 30 l Sartorius type fermenter.

[0099] Culture for Biomass Production:

[0100] 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 0.3 (antifoam) Microelements and CaCl.sub.22H.sub.2O 40 Vitamins (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

[0101] 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.

[0102] 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 agitation) Agitation Minimum 300 rpm Air flow rate 15 l/min

[0103] 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.

[0104] Results

[0105] In 40 h, 80 g/l of biomass containing 52% of proteins are obtained.

Example 2. Thermal Permeabilization of the Chlorella protothecoides Biomass and Recovery of the Soluble Fraction

[0106] The biomass obtained according to example 1 is: [0107] centrifuged and washed 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), then [0108] thermally treated by UHT for approximately ten seconds at 135 C.

[0109] The partially solubilized biomass obtained in this way has of the order of 50% peptides and proteins (expressed as total amino acids), 20% sugars and 15% lipids, corresponding to a degree of solubilization of between 20% and 50% relative to the total initial biomass.

[0110] It is then separated from the soluble fraction by centrifugal separation.

[0111] The raw soluble substances thus contain between 60% and 75% peptides and proteins (expressed as total amino acids, of which 90% arginine and glutamic acid), between 10% and 25% sugars and less than 1% lipids.

[0112] The depleted biomass, in the centrifugation pellet, still has between 20% and 35% peptides and proteins (expressed as total amino acids), 25% to 35% sugars and most of all between 20% to 25% lipids.

[0113] The microfiltration permeate P1 having a dry matter content of 5% and a titer between 60% and 75% of peptides and proteins (expressed as total amino acids) is then ultrafiltered on a membrane with a <5 kDa cut-off threshold.

[0114] The ultrafiltration retentate R2 obtained in this way has 10% (5% to 20%) dry matter, and contains more than 90% of peptides having a molecular weight of greater than or equal to 5 kDa.

[0115] The permeate P2 having a dry matter content of less than 3% contains peptides having a molecular weight less than 5 kDa and oligosaccharides having a DP less than or equal to 2.

[0116] 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: [0117] a retentate R3 having more than 10% dry matter, containing peptides having a molecular weight less than 5 kDa and oligosaccharides of DP 2, such as sucrose; [0118] a permeate R3 having 0.1% dry matter, containing oligosaccharides of DP 1, salts, free amino acids and organic acids.

[0119] The protein isolate R2 is then: [0120] neutralized to pH 7 with potassium hydroxide, [0121] concentrated by evaporation to 35% dry matter (DM), [0122] pasteurized and [0123] atomized.