METHOD FOR OBTAINING FAT-SOLUBLE AND WATER-SOLUBLE COMPOUNDS FROM MICROALGAE BY MODULATING THE POLARITY OF VEGETABLE OR ANIMAL OILS

Abstract

The present invention relates to the field of converting algal biomass. The present invention relates to a method for obtaining fat-soluble and water-soluble compounds from a biomass of eukaryotic or prokaryotic microalgae (cyanobacteria), and the oil obtained by said method and the uses thereof, particularly in the food and food supplement sectors.

Claims

1. Method for obtaining fat-soluble and water-soluble compounds from a biomass of eukaryotic or prokaryotic microalgae (cyanobacteria), characterized in that said method comprises: 1. a step of mixing said biomass with oil, preferably vegetable, said oil comprising between 0.25% and 10% by mass of at least one amphiphilic additive, preferably being a monoglyceride, a diglyceride, or a phospholipid, allowing for modulation of the polarity of said oil, and 2. a step of macerating said biomass with said oil, allowing for the mixture to be homogenized, and/or 3. a step of extracting said fat-soluble and water-soluble compounds by means of ultrasound treatment with an application of ultrasonic power (Pus) of between 1 and 1000 W/L, applied to said biomass mixed with said oil obtained in step 1) at a temperature (T) of between 15 and 70 C., and/or 4. a step of extracting said fat-soluble and water-soluble compounds by means of treatment by electromagnetic microwaves with an application of power (W) of between 1 and 1000 W/L, applied to said biomass mixed with said oil obtained in step 1) at a temperature (T) of between 15 and 70 C.

2. Method according to the preceding claim, characterized in that the biomass of eukaryotic or prokaryotic microalgae (cyanobacteria) is in dry, moist, or pre-extracted form.

3. Method according to either of the preceding claims, characterized in that the duration of step 3) and/or of step 4) is in each case between 30 seconds and 1 hour.

4. Method according to any of the preceding claims, characterized in that the fat-soluble compounds are selected from the short-chain saturated fatty acids, such as butyric acid, caproic acid, or caprylic acid, the long-chain saturated fatty acids such as capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, or stearic acid, very long-chain saturated fatty acids such as arachidic acid, docosanoic acid, tricosanoic acid, or tetracosanoic acid, monounsaturated fatty acids such as undecanoic acid, lauroleic acid, pentadecanoic acid, palmitoleic acid, heptadecanoic acid, oleic acid, eicosenoic acid, docosaenoic acid or tetracosenoic acid, polyunsaturated fatty acids such as hexadecadienoic acid, linoleic acid (precursor of omegas 3 and 6), alpha-linolenic acid (ALA, precursor of omegas 3 and 6), gamma-linolenic acid (GLA), eicosadienoic acid, dihomolinoleic acid, eicosapentaenoic acid (EPA), fat-soluble vitamins such as -tocopherol (vitamin E), or the fat-soluble pigments such as chlorophyll, or carotenoids such as -carotene (vitamin A precursor), lutein, asthaxanthin, zeaxanthin, cryptoxanthin (vitamin A precursor), lycopene, sterols, alkaloids, phenolic compounds such as flavonoids, or terpenes such as phytol (precursor of vitamin E).

5. Method according to any of the preceding claims, characterized in that the eukaryotic microalgae are selected from Chlorella, Nannocloropsis, Duanaliella and Euglena, and in that the cyanobacteria are selected from spirulina (Arthrospira platensis or Spirulina maxima) and AFA (Aphanizomenon Flos-aquae).

6. Method according to any of the preceding claims, characterized in that it comprises an additional step 5) of solid/liquid separation, preferably by means of centrifugation, of the extract obtained after step 2), 3) or 4).

7. Method according to the preceding claim, characterized in that it comprises an additional step 6) of filtering the liquid part obtained by separation of the extract obtained after step 5).

8. Method according to any of the preceding claims, characterized in that the microalgae or cyanobacteria/oil ratio is between .sup.th and 1/50.sup.th of the volume, preferably 1/20.sup.th of the volume.

9. Use of the method according to any of the preceding claims, where, in the case of the method comprising a step 3) of ultrasound treatment, the ultrasound power and the temperature are modulated in order to vary the type and the quantity of compounds extracted from said biomass.

10. Use of the method according to claims 1 to 8, where, in the case of the method comprising a step 4) of electromagnetic microwave treatment, the power, the exposure number and the temperature are modulated in order to vary the type and the quantity of compounds extracted from said biomass.

11. Use of the method according to claims 1 to 8, where the form of the original biomass, the type and quantity of the oil, and the type and quantity of said at least one amphiphilic additive, and the step 3) of ultrasound treatment and/or 4) of electromagnetic microwave treatment are modulated in order to vary the fat-soluble and water-soluble compounds extracted from said eukaryotic microalgae or cyanobacteria.

12. Oil enriched in fat-soluble and water-soluble compounds of eukaryotic or prokaryotic microalgae (cyanobacteria), such as obtained by the method according to claims 1 to 8.

13. Use of the enriched oil according to the preceding claim in chemical, food, cosmetic or pharmaceutical, preferably food, compositions.

Description

DESCRIPTION OF THE FIGURES

[0054] FIG. 1 Shows absorption spectra of the oil alone (A), the oil enriched with spirulina compounds (B) after the biomass of Arthrospira platensis has been mixed into the extraction solvent (sunflower vegetable oil at 1% Tween 80) according to the ratio 1/20 (0.5 g of spirulina in 10 g of oil), and the mixture has been introduced into a stirred ball mill having ceramic balls and subjected to rotations of a speed of 4000 rotations/minute for 1 hour at ambient temperature, and then centrifuged for 10 minutes at 9000 rpm.

[0055] FIG. 2 Shows a quantitative analysis carried out by means of spectrophotometric measurements in order to evaluate the chlorophyll a and carotenoid content depending on the biomass type (moist, dry or pre-extracted) and the amphiphilic additive type (none, Tween 80, or Span 80), after having introduced the mixture into a stirred ball mill for 1 hour at 4000 rpm at ambient temperature. The first line shows a control value. The following 3 lines show the data after using moist biomass. The following 3 lines show the data after using dry biomass. The following 3 lines show the data after using pre-extracted biomass, in this case a lyophilized pellet.

[0056] FIG. 3: Shows a quantitative analysis of neutral lipids, carried out by means of high-performance thin-layer chromatography (HPTLC). 20 L of the sample, at 1 mg/mL, were deposited in chloroform. The presence of monoglycerides (MAG), free fatty acids (FFA), triglycerides (TAG), diglycerides (DAG) and sterols (STE) is studied.

[0057] FIG. 4: Shows a quantitative analysis carried out by means of spectrophotometric measurements in order to evaluate the chlorophyll a and carotenoid content depending on the extraction technique (stirred ball mill, maceration, microwaves or ultrasound) and the amphiphilic additive type (none, Tween 80, or Span 80), using a moist biomass (80% moisture). The first line shows a control value. The following 3 lines show the data after maceration for 24 hours. The following 3 lines show the data after treatment with electromagnetic microwaves, 4 times a minute. The following 3 lines show the data after ultrasound treatment with application of ultrasonic power for 30 minutes. The last 3 lines show the data after treatment using a stirred ball mill for 1 hour.

[0058] FIG. 5: Shows a schematic diagram of the microwave application cycles. The rising curves represent the points of microwave treatment.

[0059] FIG. 6: Shows photographs which characterize the extractions achieved by means of microwave treatment at powers of 300, 600, 850 and 1000 W, and according to the amphiphilic additive type (none, Tween 80, or Span 80).

[0060] FIG. 7: Shows a quantitative analysis carried out by means of spectrophotometric measurements in order to evaluate the chlorophyll a and carotenoid content depending on the power of the microwave treatment (300, 600, 850 and 1000 W). The first 3 lines show the data in the case of treatment at 1000 W. The following 3 lines show the data in the case of treatment at 850 W. The 3 lines show the data in the case of treatment at 600 W. The last 3 lines show the data in the case of treatment at 300 W.

EXAMPLES

Example 1

Study of the Modularity of the State of the Biomass and of the Addition of Amphiphilic Additive by Enrichment of Sunflower Oil Using a Stirred Ball Mill

[0061] Moist, dry or pre-extracted biomass (Arthrospira platensis) is mixed with the extraction solvent (sunflower vegetable oi, sunflower vegetable oil at 1% Tween 80, and sunflower vegetable oil at 1% Span 80) according to the ratio 1/20; i.e. 1 g of biomass for 20 g of solvent. In this case, 0.5 g of spirulina is introduced into 10 g of oil. The mixtures are then introduced into a stirred ball mill having ceramic balls, and subjected to rotations of a speed of 4000 rotations/minute for 1 hour, at ambient temperature. The extracts are then centrifuged for 10 minutes at 9000 rpm in order to achieve a clear oil.

[0062] The oil collected after these extractions is of a green color which is different from the initial color of the sunflower oil at the outset (light yellow). Comparing the absorption spectra of the oil alone (FIG. 1A) and the sunflower vegetable oil at 1% Tween 80 enriched in spirulina compounds (FIG. 1B) reveals the presence of peaks characteristic of vegetable pigments: carotenoids (.sub.max=416 nm) and chlorophyll (.sub.max=668 nm). Thus, according to these preliminary qualitative observations, the sunflower oil appears to have been enriched with spirulina pigments by the method according to the invention.

[0063] A quantitative analysis was then carried out by means of spectrophotometric measurements in order to evaluate the content of each of the pigments thereof, depending on the biomass and amphiphilic additive type. The results obtained (FIG. 2) demonstrate that the biomass type modulates the pigment content. Indeed, the maximum pigment contents are achieved in the case of a pre-extracted biomass (lyophilized pellet, having already undergone extraction). Modulating the polarity by adding amphiphilic additive into the oil also affects the pigment yields. Thus, in the case of the surfactant Tween 80, the extract contains 238.61 g of chlorophyll a per g of oil, and 61.46 g of carotenoids per g of oil.

[0064] Furthermore, focusing on the lipids classes, the enrichment of vegetable oil with neutral lipids of spirulina following extraction is also evident. A qualitative analysis of the neutral lipids has been carried out (FIG. 3) by means of high-performance thin-layer chromatography (HPTLC), where 20 L of the sample, at 1 mg/mL, was deposited in chloroform. The results obtained show the presence of monoglycerides (MAG) in extracts 1 to 19 but not in the control, and the presence of free fatty acids (FFA) in extracts 12 to 19 but not in the control. These results highlight the presence of neutral lipids such as free fatty acids (FFA) and monoglycerides (MAG) in vegetable oil following extraction. The vegetable oil, which did not contain these compounds in the initial state, has therefore been enriched.

Example 2

Study of the Modularity of the Extraction TechniqueEnrichment of Sunflower Oil Using Different Extraction Methods

[0065] For a given biomass (in this case moist biomass (80% moisture) of Arthrospira platensis), different extraction methods have been used in order to enrich the sunflower oil:

Stirred ball mill: 4000 rpm for 1 hour, using 20 g of ceramic balls
Ultrasound: 25 kHz, 150 W, for 30 min
Microwaves: test using different powers of from 300 to 1000 W, by means of treatment by 1-minute cycles

Maceration.

[0066] The spectrophotometric measurements carried out (FIG. 4) show that the yields of chlorophyll a and carotenoids are modulated by the extraction technique selected. Indeed, the maximum yields are achieved in the case of ultrasound extraction: 209.92 g of chlorophyll a per g of oil, and 35.60 g of carotenoids per g of oil (in the case of oil at 1% Span 80). In this case, adding Span 80 to the oil modulates the polarity in favor of the extraction of chlorophyll and carotenoid pigments.

Example 3

Study of the Modularity of the Extraction ParametersEnrichment of Sunflower Oil Using Microwaves

[0067] In order to highlight the modularity of the extraction technique, various parameters have been tested. The biomass selected for these extractions is a spirulina paste having 79.87% moisture. The biomass/oil mixture is made at a dry ratio of 1/20.sup.th. A plurality of cycles of exposure to microwaves, of a duration of 1 minute, were applied to the solvent/biomass mixture, interspersed with cooling in an ice bath (shown in FIG. 5).

[0068] Extractions have been carried out by microwave treatment at powers of 300, 600, 850 and 1000 W. FIG. 6 shows that the oil becomes darker and darker as the microwave power increases, showing that the pigment extraction is thus favored by an increased microwave power. Furthermore, modulating the polarity also plays a significant role in the extraction of pigments, since it is possible to identify a color difference depending on the amphiphilic additive used, for the same power. Thus, at 600 W, oil comprising 1% Tween 80 has a more intense color than the extracts achieved using oil alone and using oil with added Span 80, at 1%.

[0069] The results shown in FIG. 7 confirm the observations of the above paragraph. The greater the microwave power, the greater the pigment yield. In the same way, adding Tween 80 to the oil allows for a greater yield than that achieved using oil alone, or using oil with Span 80 added: 11.37 g of chlorophyll a per g of oil, and 9.76 g of carotenoids per g of oil in the case of oil with Tween 80 added at 1%, at 850 and 1000 W, respectively.

Conclusions

[0070] The results obtained in these three examples demonstrate that the modularity of the biomass type affects the quantity of fat-soluble and water-soluble compounds extracted. Indeed, using a pre-extracted biomass will allow for better extraction of fat-soluble and water-soluble compounds than in the case of a dry biomass, although said dry biomass makes it possible to achieve better extraction of fat-soluble and water-soluble compounds than in the case of extraction from moist biomass.

[0071] The results obtained in these three examples also demonstrate that the extraction technique used modulates the yields for extraction of fat-soluble and water-soluble compounds. Indeed, using ultrasound makes it possible to achieve greater yields of carotenoid and chlorophyll a extraction, compared with maceration, microwave treatment, or using a stirred ball mill.

[0072] The results obtained in these three examples also demonstrate that the modulation of the technique based on microwaves influences the yields for extraction of fat-soluble and water-soluble compounds. Indeed, a maximum quantity of carotenoids is extracted at the microwave power of 1000 W, whereas a maximum of chlorophyll a is extracted at 850 W.

[0073] Indeed, the results obtained in these three examples demonstrate that adding amphiphilic additive affects the yields for extraction of fat-soluble and water-soluble compounds, whatever the technique used. Adding for example Tween 80 or Span 80 makes it possible to increase the quantity of compounds extracted, in the case of ultrasound treatment or in the case of microwave treatment. Treatment by maceration has also made it possible to identify that adding Span 80 makes it possible to increase the yield of extraction of carotenoids and of chlorophyll a, whereas adding Tween 80 makes it possible to increase only the yield of extraction of chlorophyll a, the yield of extraction of carotenoids reducing.