METHOD FOR THERMAL PERMEABILIZATION OF A MICROALGAE BIOMASS

Abstract

Described are methods for thermal permeabilization of the biomass of microalgae of the Chlorella genus. The methods are useful in recovering the soluble fractions, including polypeptides and saccharide, of the microalgae.

Claims

1.-10. (canceled)

11. A process for thermal permeabilization of biomass of microalgae of the Chlorella genus, which comprises the following steps: Provision of a microalal biomass; heat treatment in steps at a temperature of between 60 and 130 C., for 1 to 5 minutes, cooling to a temperature of between 0 and 10 C., and recovery, concentration and enrichment of the soluble fractions from which the microalgal cells have been removed, thereby recovering from said microalgae the soluble fractions which are enriched in particular with proteins and with oligosaccharides.

12. The thermal permeabilization process of claim 11, which comprises the following steps: (1) culturing of the microalgae by fermentation under heterotrophic conditions and in the absence of light, (2) collection of the biomass, (3) washing of the biomass in order to remove the residues of the interstitial fluid that result from the fermentation, (4) heat treatment in steps at a temperature of between 60 and 130 C., for 1 to 5 minutes, (5) cooling to ambient temperature and maintaining at this temperature for 30 minutes to 3 hours, so as to allow the intracellular components to diffuse into the reaction medium, (6) cooling to a temperature between 0 C. and 10 C., (7) elimination of the residual biomass by means of a solid-liquid separation technique, (8) recovery, concentration and enrichment of the soluble fractions from which the microalgal cells have been removed.

13. The process of claim 11, wherein the microalgae of the Chlorella genus are chosen from the group consisting of Chlorella vulgaris, Chlorella sorokiniana and Chlorella protothecoides.

14. The process of claim 11, wherein the biomass is collected by solid-liquid separation, by frontal or tangential filtration or by centrifugation.

15. The process of claim 11, wherein the heat treatment comprises several steps of increasing temperatures.

16. The process of claim 11, wherein the heat treatment comprises a first step making it possible to bring the biomass to a temperature of approximately 60-70 C., one or more steps making it possible to reach a maximum temperature applied of approximately 90 to 130 C.

17. The process of claim 11, wherein the heat treatment comprises the following phases: increase in temperature from ambient temperature to 60 C. for 30 seconds, increase in temperature from 60 C. to 90 C. for a further 30 seconds, maintenance of the temperature at 90 C. for 3 minutes.

18. The process of claim 11, wherein the heat treatment comprises the following phases: raising of the temperature from 28 C. to 60 C. for 30 seconds, raising of the temperature from 60 C. to 90 C. for 30 seconds, maintenance of the temperature at 90 C. for 1 minute, cooling from 90 to 60 C. for 30 seconds, cooling form 60 C. to 4 C.

19. A process for preparing compositions which are enriched with soluble peptides and polypeptides and with oligosaccharides from microalgae of the Chlorella genus, wherein the soluble fractions obtained by carrying out the process of claim 11 are filtered on a membrane separation system chosen from the group consisting of microfiltration, ultrafiltration, nanofiltration and diafiltration, taken alone or in combination.

20. A process for fractionating the compositions which are enriched with soluble peptides and polypeptides and with oligosaccharides obtained as claimed in claim 19, wherein membranes of reverse osmosis type are used.

21. The process of claim 11, wherein the temperature of the heat treatment step is between 60 and 90 C.

22. The process of claim 12, wherein the heat treatment temperature is between 60 and 90 C., and the cooling step is performed at a temperature of about 4 C.

23. The process of claim 15, wherein the heat treatment further comprises several steps of decreasing temperatures, each step being from 10 to 40 C.

24. The process of claim 16, wherein the heat treatment further comprises one or more steps making it possible to reduce the temperature.

25. The process of claim 18, wherein the cooling from 60 C. to 4 C. is performed for 1 minute.

Description

EXAMPLES

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

[0100] The strain used is Chlorella protothecoides UTEX 250

[0101] Preculture: [0102] 500 ml of medium in a 2 l Erlenmeyer flask; [0103] 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.47H2O 0.25 (NH.sub.4).sub.2SO.sub.4 1 Citric acid 1 clerol FBA 3107 (antifoam) 0.1 Microelements CaCl.sub.22H2O 30 and Vitamins FeSO47H2O 1 (mg/l) MnSO41H2O 8 CoSO47H2O 0.1 CuSO45H2O 0.2 ZnSO47H2O 0.5 H3BO3 0.1 Na2MoO42H2O 0.4 Thiamine HCl 1 Biotin 0.015 B12 0.01 Calcium pantothenate 0.03 p-aminobenzoic acid 0.06

[0104] Incubation is carried out under the following conditions: duration: 72 h; temperature: 28 C.; shaking: 110 rpm (Infors Multitron incubator).

[0105] The preculture is then transferred to a 30 l Sartorius type fermenter.

[0106] Culture for Biomass Production:

[0107] The medium is as follows:

TABLE-US-00002 TABLE 2 Macro Glucose 40 elements (g/l) K.sub.2HPO.sub.4 1.8 NaH.sub.2PO.sub.4 1.4 MgSO.sub.47H2O 3.4 (NH.sub.4).sub.2SO.sub.4 0.2 clerol FBA 3107 (antifoam) 0.3 Microelements CaCl.sub.22H2O 40 and Vitamins FeSO47H2O 12 (mg/l) MnSO41H2O 40 CoSO47H2O 0.1 CuSO45H2O 0.5 ZnSO47H2O 50 H3BO3 15 Na2MoO42H2O 2 Thiamine HCl 6 Biotin 0.1 B12 0.06 Calcium pantothenate 0.2 p-aminobenzoic acid 0.2

[0108] 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.

[0109] The parameters for carrying out 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

[0110] 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.

[0111] Results

[0112] 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

[0113] The biomass obtained according to example 1 is: [0114] centrifuged and washed so as to be brought to a dry matter content of 150 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 [0115] heat treated in the following way: [0116] raising of the temperature from 28 C. to 60 C. for 30 seconds, [0117] raising of the temperature from 60 C. to 90 C. for 30 seconds, [0118] maintenance of the temperature at 90 C. for 1 minute, [0119] cooling from 90 to 60 C. for 30 seconds, [0120] cooling from 60 C. to 4 C., preferably for 1 minute.

[0121] The biomass thus obtained is separated from the soluble fraction by centrifugal separation. Said soluble fraction is then microfiltered on a 0.14 m ceramic membrane at 60 C.

[0122] The transmembrane pressure is fixed at a value of between 0.2 and 0.6 bar and the microfiltration is carried out so as to obtain a volume concentration factor of 2.5 (100 l of this microfiltered soluble fraction thus generate 40 liters of retentate R1 and 60 liters of permeate P1).

[0123] This microfiltration permeate P1 has a dry matter content of 4% and a titer between 60% and 80% of soluble proteins, expressed as N6.25.

Example 3. Fractionation of the Protein and Saccharide Components of the Intracellular Content of the Permeabilized Chlorella protothecoides Biomass

[0124] In order to obtain the fractions which are rich in soluble proteins and in saccharides, the microfiltration permeate P1 obtained at the end of example 2 having a dry matter content of 4% is in particular ultrafiltered on a membrane with a cut-off threshold of 10 kDa, so as to obtain: [0125] a retentate R2 having a dry matter content of 10%, containing peptides having a molecular weight greater than or equal to 5 kDa and oligosaccharides having high DPs; [0126] a permeate P2 having a dry matter content of 1%, containing peptides having a molecular weight less than 5 kDa and oligosaccharides having a DP less than or equal to 2.

[0127] This permeate P2 can then be in particular filtered on a reverse osmosis membrane (having a degree of NaCl rejection of 93%), so as to obtain: [0128] a retentate R3 having a dry matter content of 10%, containing peptides having a molecular weight less than 5 kDa and oligosaccharides of DP 2, such as sucrose; [0129] a permeate R3 having a dry matter content of 0.1%, containing oligosaccharides of DP 1, salts, free amino acids and organic acids.