COMPOSITION

Abstract

The present invention is related to extracts rich in polar lipids obtained or obtainable from macroalgae, microalgae, photosynthetic bacteria and/or photosynthetic organ(s) and/or tissue(s) of a plant and combinations thereof as well as their uses as emulsifiers.

Claims

1. Extract rich in polar lipids obtained from microalgae, macroalgae, photosynthetic bacteria and/or photosynthetic organ(s) and/or tissue(s) of a plant and combinations thereof.

2. Extract according to claim 1, wherein the photosynthetic organ(s) and/or tissue(s) of a plant, macroalgae, microalgae and/or photosynthetic bacteria are recognized as food grade or recognized as GRAS (Generally Recognized as Safe).

3. Extract according to claim 1, wherein the photosynthetic organ(s) and/or tissue(s) is from one or more of the following plants: alfalfa, spinach, broccoli rabe, broccoli, red radish, guarana, rosemary, sage, thyme, mint, basil, Perilla frutescens, ajwain, angelica, anise, asafoetida, caraway, carrot, celery, chervil, coriander, cumin, dill, fennel, lovage, cow parsley, parsley, parsnip, sea holly, silphium oregano, lettuce, fenugreek, lentil, lupine, pea, garlic, scallion, leek, chive, and chinese onion, onions, green onions, beetroot, parsley, yerba mate, tea, endive, watercress, nettle, carrots, sweet potato, pak choi, water spinach.

4. Extract according to claim 1, wherein the microalgae is selected from one or more of Chlorella, Chysophyceae, Xantophyceae, Baccilariophyceae, Dinophyceae, Rodophyceae, Phaeophyceae, Chlorophyceae, Prasinophyceae, Cryptophyceae, Crypthecodinium, Cylindrothec, Botryococcus, Dunaliella (such as Dunaliella salina), Euglena gracilis, Isochrysis, Nannochloropsis, Neochloris, Nitzschia Scenedesmus, Chlorobotrys, Eustigmatos, Phaeodactylum, Porphyridium, Pseudostaurastrum, Schizochytrium, Tetraselmis, Vischeria, Monodopsis, Pseudocharaciopsis.

5. Extract according to claim 1, wherein the photosynthetic bacteria is selected from one or more of the following genera: Spirulina (Arthrospira, such as Arthrospira platensis, Arthrospira maxima), Limnospira (Limnospira platensis), Synechocystis, Nostoc, Cyanothece, Aphanizomenon (such as Aphanizomenon flosaquae).

6. Extract according to claim 1, wherein the macroalgae is selected from one or more of the following species: Ascophyllum nodosum, Fucus serratu, F. vesiculosus. Himanthalia elongata, Undaria pinnatifida, Laminaria digitata, L. saccharina, L. japonica, Alaria esculenta, Palmaria palmata (dulse), Porphyra umbilicalis, P. tenera, P. yezoensis, P. dioica, P. purpurea, P. laciniata, P. leucostica, Chondrus crispus, Gracilaria verrucosa, Lithothamnium calcareum, Enteromorpha spp., and Ulva spp.

7. Extract according to claim 1, wherein the extract is selected from methyltetrahydrofuran extracts or hydro methyltetrahydrofuran extracts, acetate extracts or hydro-acetate extracts, isopropanol or hydroisopropanolic extracts, acetone or hydroacetonic extracts, chloroform extracts, methanol or hydromethanolic extracts, ethanol or hydroethanolic extracts or mixtures thereof.

8. Extract according to claim 1, wherein the photosynthetic organ(s) and/or tissue(s) of a plant, macroalgae, microalgae and/or photosynthetic bacteria is a raw material and/or a spent material.

9. Extract according to claim 1 wherein the extract is selected from a Crude extract rich in polar lipids or a Purified extract rich polar lipids.

10. Crude Extract according to claim 9, wherein the extract comprises at least 5%, at least 10% of polar lipids, at least 20% of polar lipids, at least 25 wt %, at least 30%, at least 40%, at least 50%, such as at least 60% of polar lipids based on the total weight of the extract.

11. Purified extract rich in polar lipids according to claim 9, wherein the purified extract comprises at least 10% of polar lipids, at least 15 wt %, at least 20 wt %, at least 30 wt %, at least 35 wt %, at least 40 wt % at least 45 wt %, at least 50 wt %, at least 55 wt %, at least 60 wt %, at least 65 wt %, at least 70 wt %, at least 75 wt %, at least 80 wt %, at least 85 wt %, at least 90 wt %, at least 95 wt % or at least 99 wt % of polar lipids in based on the total weight of the purified extract.

12. Purified extract rich in polar lipids according to claim 9, wherein the purified extract was is obtained using activated carbon and/or carbon filter plate.

13. Purified extract rich in polar lipids according to claim 9 having improved organoleptic properties including such as a neutral odour, a light colour and/or absence of off-taste.

14. Purified extract rich in polar lipids according to claim 9 having a L1*, a1*, and b1* that corresponds to white or is near to white.

15. Extract according to claim 1, wherein the polar lipids phase comprises galactosyl acylglycerols, phospholipids, lysophospholipids, sulphur lipids, betain lipids and/or furan-based lipids or oxidation products thereof.

16. Extract according to claim 1, wherein the polar lipid phase comprises at least 5 wt %, at least 8 wt %, at least 10 wt %, at least 11 wt %, at least 12 wt %, at least 20%, or at least 30 wt % of galactosyl acylglycerols based on the total weight of the polar lipid phase.

17. Extract according to claim 1, wherein the polar lipid phase comprises at least 5 wt % of sulphur lipids, based on the total weight of the polar lipid fraction.

18. Purified extract rich in polar lipids according to claim 9, wherein the Purified extract contain less than 10% of sugars, proteins, peptides, chlorophylls and/or waxes.

19. (canceled)

20. (canceled)

21. Emulsion comprising at least one Extract according to claim 1 as an emulsifying agent, wherein the emulsion optionally does not comprise other emulsifying agents.

22. (canceled)

23. Emulsion according to claim 21, wherein the emulsifying agent is present in a concentration of from about 0.1 to about 10 wt %.

24. Emulsion according to claim 21, wherein the emulsion is selected from the group consisting of a water-in-oil emulsion and an oil-in-water emulsion.

25. Process for preparing an emulsion comprising: a) mixing ingredients of an aqueous phase; b) mixing ingredients of a lipid phase; c) dispersing one or more Extracts according to claim 1 in one or both of the aqueous phase or the lipid phase; and d) homogenizing the two phases to form an emulsion.

26. (canceled)

27. (canceled)

28. Emulsion according to claim 21, wherein the emulsion is free of synthetic or artificial emulsifiers and/or structuring agents.

29. Emulsion according to claim 21, wherein the emulsion has a pH from about 2 to 10, such as from about 3 to 7.

30. Emulsion according to claim 21, wherein the droplets size is comprised between 0.05 and 50 micrometres.

31. Emulsion according to claim 21, wherein the droplet size remains stable for at least one day of storage at ambient temperature (25° C.).

32. A food or beverage product for humans or animals, a nutritional supplement, a nutraceutical formulation, a fragrance or flavouring, a pharmaceutical or veterinary formulation, an oenological or cosmetic formulation comprising an emulsion according to claim 21.

33. A food or beverage product for humans or animals, a nutritional supplement, a nutraceutical formulation, a fragrance or flavouring, a pharmaceutical or veterinary formulation, an oenological or cosmetic formulation comprising at least one emulsifying extract according to claim 1.

34. Food or beverage according to claim 32 selected from sauces, mayonnaises, snacks, ice creams and desserts, dairy products, beverages, sausages and condiments, process products, meat analogues, coffee creamers, baked goods, spreads or margarines.

Description

FIGURES

[0214] FIG. 1. Mass yield (%) obtained for spinach leave extraction using various solvents. Independently duplicated result are indicated with the mention n=2.

[0215] FIG. 2. Mass yield (%) obtained for spinach leave extraction using a Soxhlet apparatus and various chloroform:methanol mixtures as extraction solvents.

[0216] FIG. 3. Polar lipid content (%) in crude extracts of spinach leaves obtained by a typical S/L extraction procedure or by a Soxhlet procedure (only for chloroform:methanol mixtures).

[0217] FIG. 4. Emulsifying activity of the spinach leaves crude extracts (1%) in oil-in-water emulsion at pH 3.5 as measured by the droplet size (D.sub.v98) and compared to the reference extracts (oat). Droplet size values for spinach and oat extracts are expressed as the average of two independent replicates ±the standard deviation.

[0218] FIG. 5. Emulsifying activity of the spinach leaves crude extracts (1%) in oil-in-water emulsion at pH 7 as measured by the droplet size (D.sub.v98) and compared to the reference extracts (oat). Droplet size values for spinach and oat extracts are expressed as the average of two independent replicates ±the standard deviation.

[0219] FIG. 6. Emulsifying activity of the spinach leaves crude extracts (5%) in water-in-oil emulsions at pH 3.5 or 7 as measured by the droplet size (D.sub.v98).

[0220] FIG. 7. Mass yield (%) obtained for spirulina extraction cake extraction using various solvents.

[0221] FIG. 8. Mass yield (%) obtained for spirulina extraction using a Soxhlet apparatus and various chloroform:methanol mixtures as extraction solvents.

[0222] FIG. 9. Polar lipid content (%) in crude extracts of spinach leaves obtained by a typical S/L extraction procedure or by a Soxhlet procedure (only for chloroform:methanol mixtures).

[0223] FIG. 10. Emulsifying activity of the spirulina crude extracts (1%) in oil-in-water emulsion at pH 3.5 as measured by the droplet size (D.sub.v98) and compared to the reference extracts (oat). Droplet size values for spinach and oat extracts are expressed as the average of two independent replicates ±the standard deviation.

[0224] FIG. 11. Emulsifying activity of the spirulina crude extracts (1%) in oil-in-water emulsion at pH 7 as measured by the droplet size (D.sub.v98) and compared to the reference extracts (oat). Droplet size values for spinach and oat extracts are expressed as the average of two independent replicates ±the standard deviation.

[0225] FIG. 12. Emulsifying activity of the spirulina crude extracts (5%) in oil-in-water emulsion at pH 3.5 as measured by the droplet size (D.sub.v98) and compared to the reference extracts (oat). Droplet size values for spinach and oat extracts are expressed as the average of two independent replicates ±the standard deviation.

[0226] FIG. 13. Emulsifying activity of the spirulina crude extracts (5%) in oil-in-water emulsion at pH 7 as measured by the droplet size (D.sub.v98) and compared to the reference extracts (oat). Droplet size values for spinach and oat extracts are expressed as the average of two independent replicates ±the standard deviation.

[0227] FIG. 14. Mass yield (%) obtained for Ulva spp. extraction using various solvents.

[0228] FIG. 15. Emulsifying activity of an ethanol:water 90:10 Ulva crude extracts (1%) in oil-in-water emulsion at pH 3.5 as measured by the droplet size (D.sub.v98) and compared to the reference extract (oat). Droplet size values for the Ulva extract are expressed as the average of two independent replicates ±the standard deviation.

[0229] FIG. 16. Emulsifying activity of an ethanol:water 90:10 Ulva crude extracts (1%) in oil-in-water emulsion at pH 7 as measured by the droplet size (D.sub.v98) and compared to the reference extract (oat). Droplet size values for the Ulva extract are expressed as the average of two independent replicates ±the standard deviation.

[0230] FIG. 17. Mass yield (%) obtained for Agarophyton chilensis extraction using various solvents.

[0231] FIG. 18. Polar lipid content (%) in crude extracts of Agarophyton chilensis.

[0232] FIG. 19. Decrease of AE between emulsions stabilized with oat oil (reference) or spinach extracts depending on the applied purification process. Color was measured in emulsions of type 2 (1% extract, pH 7). Averages and standard deviations were calculated from n=2 repetitions for crude extracts (Y06 and Y13), n=3 for the extracts decolorized with the powdered charcoal (Y09, Y10 and Y15), n=1 for the extract decolorized with the R55S filter sheet (Y37), and n=1 for the extract decolorized with the Filtrox filter sheet (Y41).

[0233] FIG. 20. Influence of the purification protocol on the droplet size of oil-in-water emulsions stabilized by 1 and 5% of ethanolic:water (90:10) extracts of spinach (crude or decolorized on charcoal, R55S or Filtrox) at pH 3.5 or 7. The standard deviation was calculated from the ES of different extracts. The number of repetitions (n) was n=2 for the crude extracts (Y13 and Y32), n=3 for the extracts decolorized with the charcoal powder (Y05, Y33 and Y39), n=1 for the extract decolorized with the R55S filter sheet (Y37), and n=1 for the extract decolorized with the Filtrox filter sheet (Y41).

[0234] FIG. 21. Influence of the purification protocol on the droplet size of oil-in-water emulsions stabilized by 5% of ethanolic:water (90:10) extracts of spirulina (crude or decolorized on charcoal) at pH 3.5 or 7.

[0235] FIG. 22: The D.sub.v98 of emulsions prepared with a commercially available soy lecithin (Topcithin, Cargill) is shown in comparison with three purified plant extracts from Spinach.

EXAMPLES

Material and Methods

[0236] 1. Biological Materials Dried Spinach leaves and alfalfa grass were purchased from Hungarian Food Ingredients Ltd., while extraction cakes of spirulina were obtained from a spirulina purchased from C.B.N Spirulina Bioengineering Co., Ltd and extracted with an aqueous solvent. Rosemary leaves were obtained from Naturex. Dried parsley leaves were purchased from VNK B.V. Biddinghuizen, dried black tea leaves (Lipton yellow) as well as fresh plant materials such as green peas, celery, carrot, broccoli rabe, spring onion and radish were purchased from a local supermarket and the green parts (leaves, pods, etc.) were manually sorted to provide the green samples.

[0237] Agarophyton chilensis and Ulva sp. were obtained from Kaiso Spa. Nannochloropsis sp. were obtained from Necton and Monzon Biotech, while Chlorella sorokiniana. Isochrysis and Tetraselmis were purchased from Necton. Dunaliella salina was purchased from Monzon.

2. Chemicals

[0238] For the extractions, ethanol (further referred sometime to as ‘EtOH’) at 99.9% purity was purchased from Christalco, hexane (C6 alcane>98%; n-hexane>45%) from Azelis, acetone (99.5%) from Univar, methanol (99.9%) from Honeyweel, isopropanol (>98%), ethyl acetate (>99%), and chloroform (>98%, stabilized with 0.6% ethanol) from VWR, 2-methyltetrahydrofuran (>99.5% stabilized with 150-400 ppm BHT) from Sigma-Aldrich.

[0239] For purification purpose, powdery supercritical water activated carbon (SCW) was purchased from Chemviron (France), while CarbofilCA and R55S activated carbon filter plates were provided by Filtrox (Switzerland) and 3M (United States), respectively.

[0240] For the analytics, digalactosyldiacylglycerol (DGDG, plant) was purchased from Avanti Polar Lipids Inc. (Alabaster, Ala., US). Acetonitrile (ACN), methanol (MeOH) and acetic acid (AA) were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France). Tetrahydrofuran (THF) was obtained from Biosolve Chime (Dieuze, France). Ultrapure water was obtained from a Milli-Q purification system (Millipore, Billerica, Mass., US).

[0241] For the preparation of emulsions, MCT (middle chain triglycerides, Mygliol 812, ex Oleo) was purchased from Oleon MV (Belgium), and deionised water was obtained from Adesco (Spain).

3. Solid/Liquid Extractions on Dried Biological Materials to Obtain Crude Extracts

[0242] Two hundred grams of dried biological material were mixed with 2 L of solvent (3 L for spinach leaves only) for two hours at reflux and mechanically stirred (175 rpm). The solid/liquid weight ratio was thus 1/10 for oat flour, spirulina extraction cakes, Agarophyton chilensis, Ulva spp., Nannochloropsis, Isochrysis, Tetraselmis, Chlorella vulgaris, Chlorella sorokiniana and Chlorella zofingensis, and 1/15 for spinach. The homogenate was filtered through an AF06 filter plate (retention rate: 15-35 μm) on a Büchner apparatus with a slight suction. Filtration is normally quite rapid and when the residue becomes dry, the solution is let to cool down for one hour approximately. When room temperature is reached, a new filtration is done in some cases with an AF31H filter plate (retention rate: 5-12 μm) on the same system to ensure the resulting extract is devoid of any potential solid particles coming from the biological material or from precipitates forming after the temperature reached 25° C. A rotary evaporator is used to remove the solvent from the extract. The solid extract is then freeze-dried with a dry matter typically >90%. For dry matter determination, the extract is placed in an oven at 125° C. We repeated the freeze-drying step whenever the dry matter was found <90%.

4. Direct Solid/Liquid Extractions on Fresh Biological Materials to Obtain Crude Extracts

[0243] One litre of ethanol was placed in a necked Erlenmeyer flask and heated to 70° C. One hundred grams of fresh biological material were weighted and properly mixed with a classic cooking blender (Moulinex, France) during a few min. The plant was then added to the hot solvent and magnetically stirred at 450 rpm for 2 hours at reflux. The solid/liquid weight ratio was thus 1/10 for vegetables, based on the fresh weight. The rest of the process is essentially the same as the one described above in section 3. The filtration cake is left under the fume hood for 16 hours. An estimation of the alcoholic degree of the recovered solvent was performed with a hydrometer immersed in 500 mL in a graduated cylinder. The dry matter percentage was determined on the fresh biological material before the extraction, and on the filtration cake to calculate the rate of the released water.

5. Indirect Solid/Liquid Extractions on Fresh Biological Materials to Obtain Crude Extracts

[0244] One litre of ethanol was placed in a necked Erlenmeyer flask and heated to 70° C. Two hundred and fifty grams of fresh biological material were juiced using a cooking juice-extractor device (Philips, Netherlands) at room temperature for 5 min. The residue was recovered and added to the hot solvent to proceed to the extraction, except for one attempt (extract Z69, fresh spinach leaves) where the juice was clarified through an AF31H filtration on a Buchner apparatus, and the resulting filtration cake was pooled with the recovered residue from the juice-extractor, and pressed together through a 1 μm bag (Filtration Group, The Netherlands) by a hydraulic press (SAM Outillage, France) at 15 bar (10 bar stabilised during 1 min) to remove as much residual juice as possible. The dry cake was then extracted according to the rest of the mixing process. The dry matter percentage was determined on the fresh biological material, before and after the juice extraction, and also on the filtration cake after the extraction, to determine both the loss of water and its release rate.

6. Soxhlet Extraction for Total Lipid Determination

[0245] Ten grams of biological material were mixed with 450 mL of solvent and extracted for eight hours in a Soxhlet apparatus. A rotary evaporator is used to remove the solvent from the extract. The solid extract is then freeze-dried with a dry matter typically >90%. For dry matter determination, the extract is placed in an oven at 125° C. We repeated the freeze-drying step whenever the dry matter was found <90%.

7. Purification with Powdery Activated Carbon to Obtain Purified Extracts

[0246] Right after the filtration step described above in section 3 (Solid/liquid extractions to obtain crude extracts), 10% of activated carbon, relative to the starting biological material amount, were added to the filtrate. The mixture was heated to 50° C. for 30 min under magnetic stirring at 300 rpm. A new filtration was performed through an AF31H filter plate on a Büchner apparatus with a slight suction. The rest of the process is essentially the one described above in section 3.

8. Purification with Activated Carbon Filter Plate to Obtain Purified Extracts

[0247] At the filtration step described above in section 3 (Solid/liquid extractions to obtain crude extracts), the conventional cellulosic filter plate was replaced by an activated carbon plate either a CarbofilCA plate (Filtrox, Switzerland) or a R55S plate (3M, United States). The filtration was performed as usual and the rest of the process is essentially the same as the one described above in section 3.

9. HPLC-ELSD Method to Measure the Polar Lipid Content

[0248] Quantification of the polar lipid was performed by reverse phase high performance liquid chromatography (RP-HPLC) (Agilent Technologies instrument, 1260 Infinity Series) using a 380-ELSD (Agilent Technologies) connected to the instrument. An Agilent Infinity Lab Poroshell 120 EC-C8 column 4 μm 3 mm inner diameter and 150 mm length (Agilent Technologies) was used as stationary phase. Separation of the lipids was carried out using an elution gradient analysis displayed in Table 1 with 0.8 mL/min flow rate. Mobile phase A consisted of a mixture of methanol-water-acid acetic (750:250:4; v/v/v), whereas mobile phase B consisted of mixture of acetonitrile-methanol-THF-acid acetic (500:375:125:4; v/v/v/v). Standard lipid solution of DGDG was dissolved in chloroform:methanol (1.5:1; v/v) prior to injection. The HPLC-ELSD settings were kept constant as follows: 4 μL injection volume, column temperature was maintained at 40° C., the ELSD Evaporator and Nebulizer temperature were set at 40° C. Nitrogen was used as a carrier gas with a gas flow rate at 1.2 SLM. Data rate was 80 Hz, Led intensity 90%, smoothing 3.0 seconds and PMT Gain at 8.0. Chromatograms were analyzed with Agilent OpenLab Rev. C.01.06 software.

TABLE-US-00001 TABLE 1 Gradient elution method for quantitative analysis of polar lipids % Eluent A % Eluent B Time MeOH-H.sub.2O-AA ACN-MeOH-TFH-AA (min) (750/250/4) (500/375/125/4) 0 90 10 10 60 40 30 40 60 45 30 70 60 10 90 65.1 90 10 75 90 10

[0249] Standard solutions were injected into the HPLC system prior to each measurement in order to establish the calibration curve in quadratic mode from five levels in the range 10 to 1000 ppm of DGDG. For quantification, all compounds were quantified as DGDG.

[0250] Extracts from Agarophyton chilensis, oat, spinach, spirulina, and Ulva were dissolved in chloroform:methanol (1.5:1 v/v) and filtered through a 0.45 μm PTFE filter prior to injection. Concentration of samples were 20 mg/mL for Agarophyton and oat, 10 mg/mL for spinach and 5 mg/mL for spirulina and Ulva.

10. Preparation of the Oil-In-Water Emulsions and Measurement of their Droplet Size

[0251] According to the present invention, the solubility of the crude or purified extract in oil and water was determined by adding 1% of extract to water or to a vegetable oil respectively (middle chain triglycerides (MCT) oil fraction, Mygliol 812). Depending on the sample in which the extract dissolved better, the crude or purified extract was classified as “oil soluble” or “water soluble”.

[0252] For oil soluble crude or purified extracts, a series of oil-in-water emulsions according to the present invention were obtained by performing, for each of them, the steps of: [0253] (i) Mixing a known amount of MCT with a known amount of oil soluble extract in a glass vessel (see Table 2). (For emulsions of type 5 the oil phase was prepared in excess to ensure accurate weighing of the plant extract. If the extract was not completely soluble and sediment resulted, only the supernatant was used in the next step); [0254] (ii) Adding a known amount of deionized water or deionized water adjusted to pH 3.5 with citric acid (see Table 2), in order to obtain an aqueous phase; [0255] (iii) Emulsifying the oil and the aqueous phases with a Branson Digital Sonifier 450, 102c using a 6.3 mm tip. The tip was immersed into—and positioned in the upper third of—the mixture, which was then emulsified for 3 min at 80% amplitude. The emulsification time was split over 5:50 min alternating 10 s pulses and 10 s pauses. The glass vessel was immersed into cold water (10° C.) to cool the emulsion.

[0256] For water-soluble crude or purified extracts, a series of oil-in-water emulsions according to the present invention were obtained by performing, for each of them, the steps of: [0257] (i) Mixing a known amount of deionized water or deionized water adjusted to pH 3.5 with citric acid and a known amount of water-soluble extract in a glass vessel (see Table 2) in order to obtain an aqueous phase. (For emulsions of type 5, the aqueous phase was prepared in excess to ensure accurate weighing of the plant extract. If the extract was not completely soluble and sediment resulted, only the supernatant was used in the next step, adding a known amount of MCT (see Table 2). [0258] (ii) Emulsifying the oil and the aqueous phases with a Branson Digital Sonifier 450, 102c (for Alfalfa a SONICS 500 Watt Ultrasonic Processors—VCX was used) using a 6.3 mm tip. The tip was immersed into—and positioned in the upper third of—the mixture, which was then emulsified for 3 min at 80% amplitude (75% for Alfalfa). The emulsification time was split over 5:50 min alternating 10 s pulses and 10 s pauses. The glass vessel was immersed into cold water (10° C.) to cool the emulsion.

[0259] All emulsions following the formulas shown in Table 2 were of oil-in-water type.

TABLE-US-00002 TABLE 2 Oil-in-water emulsion formulas. The batch size was 7 g for all plant extracts except for alfalfa where it was 20 g. Emulsion 1 Emulsion 2 Emulsion 3 Emulsion 4 Emulsion 5 Ingredients (%) (%) (%) (%) (%) Deionized water — 89 — 85 89.9 Deionized water adjusted 89 — 85 — to pH 3.5 with citric acid MCT oil 10 10 10 10 10 Crude or purified extract 1  1 5  5 0.1

[0260] The droplet size distribution of the oil droplets was measured by Static Light Scattering with a Malvern Mastersizer 3000 using laser diffraction particle size analysis and the Mie scattering theory. For the continuous phase, the refractive index of water, and for the dispersed phase, the refractive index of MCT oil, were used respectively.

[0261] The measurement cell was filled with degassed deionized water. The emulsions were diluted by adding the emulsion dropwise to the measurement cell following the obscuration measurement in the device. The D.sub.v98 of the droplet size distribution were then calculated using the software implemented in the measurement instrument. The D.sub.v98, is defined as the diameter where 98% of the population (in volume) lies below this value. All measurements were performed at room temperature. All emulsions were stored at 5° C. for 7 days and the droplet size distribution was measured after 1 day and after 7 days to check the stability of the sample.

11. Preparation of the Water-In-Oil Emulsions and Measurement of their Droplet Size

[0262] For oil soluble purified extracts, a series of water-in-oil emulsions according to the present invention were obtained by performing, for each of them, the steps of: [0263] (i) Mixing a known amount of MCT with a known amount of oil soluble extract in a glass vessel (Table 3). [0264] (ii) Adding a known amount of deionized water or deionized water adjusted to pH 3.5 with citric acid (see Table 3), to obtain an aqueous phase; [0265] (iii) Emulsifying the oil and the aqueous phases with a Branson Digital Sonifier 450, 102c using a 6.3 mm tip. The tip was immersed into the sample and positioned in its upper third. The mixture was emulsified for 3 min at 80% amplitude. The emulsification time was split over 5:50 min alternating 10 s pulses and 10 s pauses. The glass vessel was immersed into cold water (10° C.) to cool the emulsion.

[0266] For water-soluble purified extracts, a series of water-in-oil emulsions according to the present invention were obtained by performing, for each of them, the steps of: [0267] (i) Mixing a known amount of deionized water or deionized water adjusted to pH 3.5 with citric acid and a known amount of water soluble plant extract in a glass vessel (see Table 3) to obtain a polar phase; [0268] (ii) Adding a known amount of MCT (see Table 3). [0269] (iii) Emulsifying the oil and the aqueous phases with a Branson Digital Sonifier 450, 102c using a 6.3 mm tip. The tip was immersed into the sample and positioned in its upper third. The mixture was emulsified for 3 min at 80% amplitude. The emulsification time was split over 5:50 min alternating 10 s pulses and 10 s pauses. The glass vessel was immersed into cold water (10° C.) to cool the emulsion.

[0270] All emulsions following the formulas shown in Table 3 were of water-in-oil type.

TABLE-US-00003 TABLE 3 Water-in-oil emulsion formulas. The batch size was 30 g. Emulsion 6 Ingredients (%) Deionized water 10 MCT oil 89 purified extract or emulsifier 1

[0271] The droplet size distribution of the water droplets was measured by Static Light Scattering with Malvern Mastersizer 3000 using laser diffraction particle size analysis and the Mie scattering theory. For the continuous phase, the refractive index of MCT, and for the dispersed phase, the refractive index of water, were used respectively.

[0272] If unpurified samples with high chlorophyll concentrations were used it was not possible to get reliable measurements as the green colour is changing the adsorption index of the continuous phase which is falsifying the measurement. As this could not be adjusted via the measurement parameters only purified extracts were used for water-in-oil emulsions.

[0273] The measurement cell was filled with MCT oil. The emulsions were diluted by adding the emulsion dropwise to the measurement cell following the obscuration measurement in the device. The D.sub.v98 of the droplet size distribution were then calculated using the software implemented in the measurement instrument. The D.sub.v98, is defined as the diameter where 98% of the population (Volume) lies below this value. All measurements were performed at room temperature and all emulsions were stored at 5° C. for 1 day and then the droplet size distribution was measured again to check the stability of the sample.

12. Calculation of the Emulsifying Score (ES)

[0274] We captured the droplet size as well as the stability of the emulsions in one single index referred to as ‘emulsifying score’ or ES.

[0275] We first assigned each dataset (droplet size day 0, day 1 and day 7) a ‘stability index’ (SI) according to the rules described in Table 4.

TABLE-US-00004 TABLE 4 Algorithmic rules used for the calculation of the stability index (SI) Stability index Conditions 3 the increase of the droplet size after one day is larger than 100% or the droplet size at day 0 is higher than 40 μm 2.5 the increase of the droplet size after one day is between 30 and 100% 2 the increase of the droplet size after one day is smaller than 30% and the increase of the droplet size between day one and day seven is more than 100% 1.5 the increase of the droplet size after one day is smaller than 30% and the increase of the droplet size between day one and day seven is between 30 and100% 1 the increase of the droplet size after one day is smaller than 30% and the increase of the droplet size between day one and day seven is smaller than 30%

[0276] The ES was then calculated according to the following formula:

ES=10*log(SI.sup.3*D(98))

[0277] The D.sub.v98 is expressed in μm. The lower the ES, the better the emulsifier. Negative ES can even be obtained.

Example 1. Production of Crude Extracts from Dried Spinach Leaves by S/L Extraction

[0278] Two hundred grams of dried flakes of spinach leaves were mixed with 3 L of solvent (ethanol; ethanol:water, 90:10; ethanol:water, 80:20; ethanol:water, 70:30; ethanol:water, 60:40; ethanol:water, 50:50; acetone; acetone:water, 90:10; acetone:water, 80:20; acetone:water, 70:30; hexane, ethyl acetate, isopropanol, methyltetrahydrofuran, and methanol) for two hours at reflux and mechanically stirred (175 rpm). The solid/liquid weight ratio was thus 1/15. The extraction medium was then filtered through an AF06 filter plate (retention rate: 15-35 μm) on a Buchner apparatus with a slight suction to remove the plant residues. A rotary evaporator was used to remove the solvent from the extract by evaporation at reduced pressure. The solid extract was then freeze-dried with a dry matter typically reaching >90%. For dry matter determination, the extract was placed in an oven at 125° C. We repeated the freeze-drying step whenever the dry matter was found <90% until it overpassed this threshold.

[0279] FIG. 1 shows the mass yield obtained for the different tested solvents with maximal values for ethanol:water, 80:20 (21.2%), methanol (18.1%), and ethanol:water, 90:10 (17.0%). The minimal yield were obtained with the apolar solvent hexane (1.4%).

Example 2. Production of Crude Extracts from Dried Spinach Leaves by Soxhlet Extraction Using Chloroform:Methanol Mixtures as Extraction Solvents

[0280] Around 6 to 7 grams of dried flakes of spinach leaves were mixed with 450 mL of solvent and extracted for eight hours in a Soxhlet apparatus using chloroform:methanol mixtures as extraction solvents at different volume ratio (2:1, 1:1, or 1:2). The solid/liquid weight ratio was thus ranging from 1/74 to 1/65 depending on the initial amount of spinach. The extraction medium was then filtered through an AF06 filter plate (retention rate: 15-35 μm) on a Buchner apparatus with a slight suction to remove the plant residues. A rotary evaporator was used to remove the solvent from the extract by evaporation at reduced pressure. The solid extract was then freeze-dried with a dry matter typically reaching >90%. For dry matter determination, the extract was placed in an oven at 125° C. We repeated the freeze-drying step whenever the dry matter was found <90%, until it overpassed this threshold.

[0281] FIG. 2 shows the mass yield obtained for the different tested solvents with maximal values for chloroform:methanol 2:1 (19.2%).

Example 3. Polar Lipid Characterization of Crude Extracts of Spinach Leaves Obtained by a Typical S/L Extraction Procedure or by a Soxhlet Procedure (Only for Chloroform:Methanol Mixtures)

[0282] Quantification of the polar lipid was performed by reverse phase HPLC as described above in section 9 (HPLC-ELSD method to measure the polar lipid content). Results show that the highest polar lipid content (>50%) was reached for crude spinach extracts obtained using ethyl acetate, acetone, chloroform:methanol 2:1 (Soxhlet procedure), and ethanol as extraction solvents (FIG. 3). By contrast, crude extract obtained using ethanol:water mixtures with at least 40% of water resulted in low amounts of polar lipids (<10%).

Example 4. The Use of 1% of Crude Extracts of Spinach Leaves as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 3.5

[0283] One percent of the different spinach leave crude extracts was used to stabilize oil-in-water emulsions at pH 3.5 made with medium chain triglycerides as the oily phase. The emulsifying activity of these extracts was estimated by the droplet size (D.sub.v98) of the fresh emulsions and its variation over a day of storage (FIG. 4). The variation over a longer storage period (7 days) was also measured for a few selected extracts. Most of the droplet size values are expressed as the average of two independent replicates ±the standard deviation. The lower the droplet size, the higher the emulsifying activity. The higher the increase of the droplet size with time, the lower the stability.

[0284] The emulsifying activities of the extracts of the invention (crude spinach extracts) and the stabilities of the emulsions they formed were further compared with those of reference extracts obtained from dehulled oat kernels, a known source of emulsifiers. The extracts of the invention and the reference extracts were obtained using the same conditions of extraction and their emulsifying activities were measured with the exact same protocol. Interestingly, many spinach extracts exhibited a significant ability to form emulsions with smaller droplet sizes than the ones obtained for the reference oat extracts (FIG. 4). Such is the case for the crude spinach extracts obtained using ethyl acetate (D.sub.v98 fresh=2.8 μm), isopropanol (2.6 μm), ethanol:water 90:10 (2.5 and 1.6 μm; two independent extracts referred to as X14 and X65), ethanol (2.4 and 2.3 μm; two independent extracts), acetone (2.3 μm), methanol (1.9 μm), acetone:water 90:10 (1.5 μm), and acetone:water 80:20 (1.4 μm).

Example 5. The Use of 1% of Crude Extracts of Spinach Leaves as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 7

[0285] One percent of the different spinach leave crude extracts was used to stabilize oil-in-water emulsions at pH 7 made with medium chain triglycerides as the oily phase. The emulsifying activity of these extracts was estimated by the droplet size (D.sub.v98) of the fresh emulsions and its variation over a day of storage (FIG. 5). The variation over a longer storage period (7 days) was also measured for a few selected extracts. Most of the droplet size values are expressed as the average of two independent replicates ±the standard deviation. The lower the droplet size, the higher the emulsifying activity. The higher the increase of the droplet size with time, the lower the stability.

[0286] The emulsifying activities of the extracts of the invention (crude spinach extracts) and the stabilities of the emulsions they formed were further compared with those of reference extracts obtained from dehulled oat kernels, a known source of emulsifiers. The extracts of the invention and the reference extracts were obtained using the same conditions of extraction and their emulsifying activities were measured with the exact same protocol. Interestingly, many spinach extracts exhibited a significant ability to form emulsions with smaller droplet sizes than the ones obtained for the reference oat extracts (FIG. 5). Such is the case for the crude spinach extracts obtained using ethanol:water 60:40 (D.sub.v98 fresh=3.3 μm), methyl tetrahydrofuran (2.6 μm), ethanol (1.8 and 2.5 μm; two independent extracts referred to as X15 and X64), ethyl acetate (2.5 μm), hexane (2.2 μm), isopropanol (2.2 μm), methanol (2.2 μm), acetone:water 80:20 (2.1 μm), acetone (1.9 μm), ethanol:water 90:10 (1.9 μm), acetone:water, 90:10 (1.8 μm), and ethanol (1.8 μm). Among them, the stability of the crude spinach extracts obtained using ethanol:water 60:40 and methyl tetrahydrofuran was not satisfying after one and seven days, respectively.

Example 6. The Use of 5% of Crude Extracts of Spinach Leaves as Emulsifiers in a Water-In-Oil Emulsion with a Continuous Phase at pH 3.5 or 7

[0287] Five percents of spinach leave crude extracts obtained using ethanol:water 90:10, acetone:water 90:10, and methanol, were used to stabilize water-in-oil emulsions at pH 3.5 and 7 made with medium chain triglycerides as the oily phase. The emulsifying activity of these extracts was estimated by the droplet size (D.sub.v98) of the fresh emulsions and its variation over a day of storage (FIG. 6). The lower the droplet size, the higher the emulsifying activity. The higher the increase of the droplet size with time, the lower the stability.

[0288] The emulsifying activities of the extracts of the invention (crude spinach extracts) and the stabilities of the emulsions they formed were measured. Interestingly, the tested spinach extracts exhibited a significant ability to form reverse emulsions with relatively small droplet sizes for this type of emulsions (FIG. 6). The obtained D.sub.v98 values for the extract obtained using ethanol:water (90:10) were 9.2 vs. 8.6 μm, for the fresh emulsion and after one day of storage, respectively. They were 6.2 vs. 2.5 μm for the extract obtained using methanol, and 0.2 vs. 0.4 μm for the extract obtained using acetone:water 90:10.

Example 7. Production of Crude Extracts from Dried Spirulina Extraction Cakes by Sa Extraction

[0289] Two hundred grams of dried spirulina (cyanobacteria) extraction cakes were mixed with 2 L of solvent (ethanol; ethanol:water, 90:10; ethanol:water, 80:20; ethanol:water, 70:30; ethanol:water, 60:40; ethanol:water, 50:50; acetone; hexane, isopropanol, and methyltetrahydrofuran) for two hours at reflux and mechanically stirred (175 rpm). The solid/liquid weight ratio was thus 1/10. The extraction medium was then filtered through an AF06 filter plate (retention rate: 15-35 μm) on a Buchner apparatus with a slight suction to remove the plant residues. Filtration is normally quite rapid and when the residue becomes dry, the solution is let to cool down for one hour approximately. When room temperature is reached, a second filtration was done with an AF31H filter paper (retention rate: 5-12 μm) on the same system to ensure the resulting extract was devoid of any potential solid particles coming from the biological material. A rotary evaporator was then used to remove the solvent from the extract by evaporation at reduced pressure. The solid extract was freeze-dried with a dry matter typically reaching >90%. For dry matter determination, the extract was placed in an oven at 125° C. We repeated the freeze-drying step whenever the dry matter was found <90% until it overpassed this threshold.

[0290] FIG. 7 shows the mass yield obtained for the different tested solvents with maximal values for ethanol:water, 70:30 (13.7%), ethanol:water, 80:20 (13.2%), and ethanol:water, 90:10 (13.0%). The minimal yield were obtained with the apolar solvent hexane (1.4%).

Example 8. Production of Crude Extracts from Dried Spirulina Extraction Cakes by Soxhlet Extraction Using Chloroform:Methanol Mixtures as Extraction Solvents

[0291] Around 12 to 13 grams of a dried powder of spirulina (cyanobacteria) extraction cakes were mixed with 450 mL of solvent and extracted for eight hours in a Soxhlet apparatus using chloroform:methanol mixtures as extraction solvents at different volume ratios (2:1, 1:1, or 1:2). The solid/liquid weight ratio was thus ranging from 1/35 to 1/38 depending on the initial amount of spirulina. The extraction medium was then filtered through an AF06 filter plate (retention rate: 15-35 μm) on a Buchner apparatus with a slight suction to remove the cyanobacteria residues. Filtration is normally quite rapid and when the residue becomes dry, the solution is let to cool down for one hour approximately. When room temperature is reached, a second filtration was done with an AF31H filter paper (retention rate: 5-12 μm) on the same system to ensure the resulting extract was devoid of any potential solid particles coming from the biological material. A rotary evaporator was used to remove the solvent from the extract by evaporation at reduced pressure. The solid extract was then freeze-dried with a dry matter typically reaching >90%. For dry matter determination, the extract was placed in an oven at 125° C. We repeated the freeze-drying step whenever the dry matter was found <90%, until it overpassed this threshold.

[0292] FIG. 8 shows the mass yield obtained for the different tested solvents with maximal values for chloroform:methanol 2:1 (6.4%).

Example 9. Polar Lipid Characterization of Crude Extracts of Spirulina Extraction Cakes Obtained by a Typical S/L Extraction Procedure or by a Soxhlet Procedure (Only for Chloroform:Methanol Mixtures)

[0293] Quantification of the polar lipid was performed by reverse phase HPLC as described above in section 9 (HPLC-ELSD method to measure the polar lipid content). Results show that the highest polar lipid content (>50%) was reached for crude spirulina extraction cake extracts obtained using hexane, acetone, methyltetrahydrofuran (MeTHF), and isopropanol (iPrOH) as extraction solvents (FIG. 9). By contrast, crude extract obtained using ethanol:water mixtures with 50% of water resulted in low amounts of polar lipids (<10%).

Example 10. The Use of 1% of Crude Extracts of Spirulina Extraction Cakes as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 3.5

[0294] One percent of the different spirulina crude extracts was used to stabilize oil-in-water emulsions at pH 3.5 made with medium chain triglycerides as the oily phase. The emulsifying activity of these extracts was estimated by the droplet size (D.sub.v98) of the fresh emulsions and its variation over a day of storage (FIG. 10). The variation over a longer storage period (7 days) was also measured for selected extracts. Most of the droplet size values are expressed as the average of two independent replicates ±the standard deviation. The lower the droplet size, the higher the emulsifying activity. The higher the increase of the droplet size with time, the lower the stability.

[0295] The emulsifying activities of the extracts of the invention (crude spirulina extracts) and the stabilities of the emulsions they formed were further compared with those of reference extracts obtained from dehulled oat kernels, a known source of emulsifiers. The extracts of the invention and the reference extracts were obtained using the same conditions of extraction and their emulsifying activities were measured with the exact same protocol. Interestingly, two spirulina extracts exhibited a significant ability to form emulsions with smaller droplet sizes than the ones obtained for the reference oat extracts (FIG. 10). Such is the case for the crude spirulina extracts obtained using ethanol (D.sub.v98 fresh=1.8 μm) and ethanol:water 90:10 (1.7 μm). Both extracts formed satisfactorily stable emulsions for 7 days.

Example 11. The Use of 1% of Crude Extracts of Spirulina Extraction Cakes as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 7

[0296] One percent of the different crude extracts of spirulina extraction cakes was used to stabilize oil-in-water emulsions at pH 7 made with medium chain triglycerides as the oily phase. The emulsifying activity of these extracts was estimated by the droplet size (D.sub.v98) of the fresh emulsions and its variation over a day of storage (FIG. 11). The variation over a longer storage period (7 days) was also measured for selected extracts. Most of the droplet size values are expressed as the average of two independent replicates ±the standard deviation. The lower the droplet size, the higher the emulsifying activity. The higher the increase of the droplet size with time, the lower the stability.

[0297] The emulsifying activities of the extracts of the invention (crude spirulina extracts) and the stabilities of the emulsions they formed were further compared with those of reference extracts obtained from dehulled oat kernels, a known source of emulsifiers. The extracts of the invention and the reference extracts were obtained using the same conditions of extraction and their emulsifying activities were measured with the exact same protocol. Interestingly, several spirulina extracts exhibited a significant ability to form emulsions with smaller droplet sizes than the ones obtained for the reference oat extracts (FIG. 11). Such is the case for the crude spirulina extracts obtained using methyl tetrahydrofuran (D.sub.v98 fresh=2.2 μm), ethanol:water 90:10 (1.6 μm), ethanol (1.2 μm) and isopropanol (1.1 μm).

Example 12. The Use of 5% of Crude Extracts of Spirulina Extraction Cakes as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 3.5

[0298] Five percents of the different crude extracts of spirulina extraction cakes were used to stabilize oil-in-water emulsions at pH 3.5 made with medium chain triglycerides as the oily phase. The emulsifying activity of these extracts was estimated by the droplet size (D.sub.v98) of the fresh emulsions and its variation over a day of storage (FIG. 12). The variation over a longer storage period (7 days) was also measured for selected extracts. Most of the droplet size values are expressed as the average of two independent replicates ±the standard deviation. The lower the droplet size, the higher the emulsifying activity. The higher the increase of the droplet size with time, the lower the stability. The emulsifying activities of the extracts of the invention (crude spirulina extracts) and the stabilities of the emulsions they formed were further compared with those of reference extracts obtained from dehulled oat kernels, a known source of emulsifiers. The extracts of the invention and the reference extracts were obtained using the same conditions of extraction and their emulsifying activities were measured with the exact same protocol. Interestingly, several spirulina extracts exhibited a significant ability to form emulsions with smaller droplet sizes than the ones obtained for the reference oat extracts (FIG. 12). Such is the case for the crude spirulina extracts obtained using methyl tetrahydrofuran (D.sub.v98 fresh=1.7 μm), ethanol:water 90:10 (1.6 μm), ethanol (1.0 μm) and isopropanol (0.7 μm). All of them formed satisfactorily stable emulsions for 7 days.

Example 13. The Use of 5% of Crude Extracts of Spirulina Extraction Cakes as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 7

[0299] Five percents of the different crude extracts of spirulina extraction cakes were used to stabilize oil-in-water emulsions at pH 7 made with medium chain triglycerides as the oily phase. The emulsifying activity of these extracts was estimated by the droplet size (D.sub.v98) of the fresh emulsions and its variation over a day of storage (FIG. 13). The variation over a longer storage period (7 days) was also measured for selected extracts. Most of the droplet size values are expressed as the average of two independent replicates ±the standard deviation. The lower the droplet size, the higher the emulsifying activity. The higher the increase of the droplet size with time, the lower the stability.

[0300] The emulsifying activities of the extracts of the invention (crude spirulina extracts) and the stabilities of the emulsions they formed were further compared with those of reference extracts obtained from dehulled oat kernels, a known source of emulsifiers. The extracts of the invention and the reference extracts were obtained using the same conditions of extraction and their emulsifying activities were measured with the exact same protocol. Interestingly, several spirulina extracts exhibited a significant ability to form emulsions with smaller droplet sizes than the ones obtained for the reference oat extracts (FIG. 13). Such is the case for the crude spirulina extracts obtained using methyl tetrahydrofuran (D.sub.v98 fresh=1.9 μm), isopropanol (0.7 μm), and ethanol (0.7 μm). All of them formed satisfactorily stable emulsions for 7 days.

Example 14. Production of Crude Extracts from Dried Ulva Spp. Seaweed (Macroalgae) by S/L Extraction

[0301] Two hundred grams of dried Ulva spp. (sea lettuce) were mixed with 2 L of solvent (ethanol, ethanol:water, 90:10; isopropanol, acetone, and methyltetrahydrofuran) for two hours at reflux and mechanically stirred (175 rpm). The solid/liquid weight ratio was thus 1/10. The extraction medium was then filtered through an AF06 filter plate (retention rate: 15-35 μm) on a Buchner apparatus with a slight suction to remove the seaweed residues. When the residue becomes dry, the solution was let to cool down for one hour approximately. When room temperature was reached, a second filtration was done with an AF31H filter paper (retention rate: 5-12 μm) on the same system to ensure the resulting extract was devoid of any potential solid particles coming from the biological material. A rotary evaporator was then used to remove the solvent from the extract by evaporation at reduced pressure. The solid extract was freeze-dried with a dry matter typically reaching >90%. For dry matter determination, the extract was placed in an oven at 125° C. We repeated the freeze-drying step whenever the dry matter was found <90% until it overpassed this threshold.

[0302] FIG. 14 shows the mass yield obtained for the different tested solvents with maximal values for ethanol:water, 90:10 (3.0%). The minimal yield were obtained with acetone (0.3%) and ethanol (0.4%).

Example 15. Polar Lipid Characterization of Crude Extracts of Ulva Spp. Seaweed (Macroalgae)

[0303] Quantification of the polar lipid was performed by reverse phase HPLC as described above in section 9 (HPLC-ELSD method to measure the polar lipid content). Polar lipid contents of 33.9 and 45.5% were reached for crude Ulva sp. extracts obtained using methyl tetrahydrofuran and ethanol:water, 90:10 (respectively) as extraction solvent.

Example 16. The Use of 1% of Crude Extracts of Ulva Spp. Seaweed (Macroalgae) as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 3.5

[0304] One percent of an Ulva spp. (sea lettuce) crude extract was used to stabilize oil-in-water emulsions at pH 3.5 made with medium chain triglycerides as the oily phase. The emulsifying activity of this extract was estimated by the droplet size (D.sub.v98) of a fresh emulsion and its variation over a day of storage (FIG. 15). The droplet size values for the Ulva spp. extract are expressed as the average of two independent replicates ±the standard deviation. The lower the droplet size, the higher the emulsifying activity. Moreover, the higher the increase of the droplet size with time, the lower the stability.

[0305] The emulsifying activity of the extract of the invention (Ulva spp.) and the stability of the emulsion they formed were further compared with those of a reference extract obtained from dehulled oat kernels, a known source of emulsifiers. The extract of the invention and the reference extract were obtained using the same solvent (ethanol:water, 90:10) and conditions of extraction and their emulsifying activities were measured with the exact same protocol. Interestingly, the Ulva spp. extract exhibited a significant ability to form emulsions with smaller droplet sizes (D.sub.v98 fresh=2.6 μm) than the ones obtained for the reference oat extract (5.0 μm) (FIG. 15). Furthermore, the Ulva extract-stabilized emulsion was physically stable after one day of storage at room temperature (2.6 vs. 2.8 μm).

Example 17. The Use of 1% of Crude Extracts of Ulva Spp. Seaweed (Macroalgae) as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 7

[0306] One percent of an Ulva spp. (sea lettuce) crude extract was used to stabilize oil-in-water emulsions at pH 7 made with medium chain triglycerides as the oily phase. The emulsifying activity of this extract was estimated by the droplet size (D.sub.v98) of a fresh emulsion and its variation over a day of storage (FIG. 16). The droplet size values for the Ulva spp. extract are expressed as the average of two independent replicates ±the standard deviation. The lower the droplet size, the higher the emulsifying activity. Moreover, the higher the increase of the droplet size with time, the lower the stability.

[0307] The emulsifying activity of the extract of the invention (Ulva spp.) and the stability of the emulsion they formed were further compared with those of a reference extract obtained from dehulled oat kernels, a known source of emulsifiers. The extract of the invention and the reference extract were obtained using the same solvent (ethanol:water, 90:10) and conditions of extraction and their emulsifying activities were measured with the exact same protocol. The Ulva spp. extract exhibited a significant ability to form emulsions with smaller droplet sizes (D.sub.v98 fresh=2.5 μm) than the ones obtained for the reference oat extract (2.7 μm) (FIG. 16), even though the stabilities of the emulsion after one day of storage were equivalent between the extract of the invention and the oat reference (2.8 vs. 2.7 μm, respectively).

Example 18. Production of Crude Extracts from Dried Agarophyton chilensis by S/L Extraction

[0308] Two hundred grams of dried Agarophyton chilensis seaweed were mixed with 2 L of solvent (ethanol; ethanol:water, 90:10; ethanol:water, 80:20; ethanol:water, 70:30; ethanol:water, 60:40; and acetone) for two hours at reflux and mechanically stirred (175 rpm). The solid/liquid weight ratio was thus 1/10. The extraction medium was then filtered through an AF06 filter plate (retention rate: 15-35 μm) on a Buchner apparatus with a slight suction to remove the seaweed residues. When the residue becomes dry, the solution was let to cool down for one hour approximately. When room temperature was reached, a second filtration was done with an AF31H filter paper (retention rate: 5-12 μm) on the same system to ensure the resulting extract was devoid of any potential solid particles coming from the biological material. A rotary evaporator was then used to remove the solvent from the extract by evaporation at reduced pressure. The solid extract was freeze-dried with a dry matter typically reaching >90%. For dry matter determination, the extract was placed in an oven at 125° C. We repeated the freeze-drying step whenever the dry matter was found <90% until it overpassed this threshold.

[0309] FIG. 17 shows the mass yield obtained for the different tested solvents with maximal values for ethanol:water, 60:40 (10.2%), ethanol:water, 80:20 (9.7%), and ethanol:water, 70:30 (9.3%). The minimal yield were obtained with acetone (0.1%).

Example 19. Polar Lipid Characterization of Crude Extracts of Agarophyton chilensis

[0310] Quantification of the polar lipid was performed by reverse phase HPLC as described above in section 9 (HPLC-ELSD method to measure the polar lipid content). Results show that the highest polar lipid content (>50%) was reached for crude Agarophyton chilensis extracts obtained using ethanol as extraction solvent (FIG. 18). By contrast, crude extract obtained using ethanol:water mixtures with at least 10% of water resulted in low amounts of polar lipids (<10%).

Example 20. The Use of 5% of an Ethanolic:Water (80:20) Crude Extract of Agarophyton chilensis as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 7

[0311] Five percents of a crude extract of Agarophyton chilensis obtained in Example 18 using an ethanol:water (80:20) mixture as extraction solvent were used to stabilize an oil-in-water emulsion at pH 7 made with medium chain triglycerides as the oily phase. The droplet size (D.sub.v98) of this emulsion was 3.4, 3.6 and 3.6 μm, just after emulsification and after one and seven days of storage, respectively. The corresponding emulsifying score (ES)—which was calculated as described above in section 12 of the material and methods—was of 5.4, which demonstrates a very good emulsifying activity because extracts with an ES equal to, or lower than, 20 are considered as emulsifying agents.

Example 21. Production of an Ethanol:Water (80:20) Crude Extracts from Dried Alfalfa (Medicago sativa) by S/L Extraction and Use of 5% Thereof as Emulsifiers in 10% Oil-In-Water Emulsions at pH 3.5 and 7

[0312] Dried alfalfa was coarsely ground using a food machine. 200 or 150 g of each dried plant were weighed and extracted with 1200-1800 ml of solvent in a beaker. A mixture of ethanol and water (80:20 vol:vol) was used as extraction solvent. The extraction was done for 3 h at 60° C. in a glass beaker equipped with a stirrer running at 500 rpm. After filtration (Spectum EBEP-25-3-UK), the extract was filtered again using Whatman™ filter paper (CAT No 1003-110), and it was concentrated by rotary evaporator. Finally, the extracts were stored at 4° C. in a refrigerator until used. For each measurement as well as for emulsification, the extracts were adjusted to 20% solid content. A mass yield of 11.8% was obtained.

[0313] Table 5 shows the values of droplet size in alfalfa-stabilized emulsion for the ethanolic:water (80:20) alfalfa crude extract. Emulsions stable at pH 3.5 and 7 for at least 7 days could be obtained. The polar lipid content in this extract was 18%. The saponin content in this extract was of 23.6%.

TABLE-US-00005 TABLE 5 Droplet size obtained in emulsions prepared at pH 3.5 (emulsion type 3) and 7 (emulsion type 4) using an ethanolic:water (80:20) alfalfa extract at 5% in the emulsion adjusted at 20% of solid, in fresh samples and after 7 days Droplet size-Dv98[μm] Fresh 7 days pH 3.5 pH 7 pH 3.5 pH 7 5.11 ± 0.11 5.24 ± 0.08 5.68 ± 0.68 5.65 ± 0.62

Example 22. Production of an Ethanolic:Water (90:10) Crude Extract from Dried Alfalfa (Medicago sativa) by S/L Extraction and Use of 5% Thereof as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 7

[0314] Two hundred grams of dried alfalfa were mixed with 2 L of an ethanol:water (90:10) mixture for two hours at reflux and mechanically stirred (175 rpm). The extraction was performed as described above in section 3 of the material and methods. One percent of the resulting extract (C04) was then used to stabilize an oil-in-water emulsion at pH 7 made with medium chain triglycerides as the oily phase. The D.sub.v98 of this emulsion measured in duplicate was 2.3 and 2.2 just after emulsification, and 2.1 and 2.2 μm (resp.) after one day of storage. These results indicate that the crude extract had the ability to form a stable emulsion.

Example 23. Production of Crude Extracts from Dried Microalgae Biomass by S/L Extraction

[0315] Two hundred grams of dried microalgae biomass (Isochrysis, Nannochloropsis, Tetraselmis, Chlorella sorokiniana, Chlorella vulgaris, and Chlorella zofingensis) were mixed with 2 L of solvent (ethanol; ethanol:water, 90:10; ethanol:water, 80:20; acetone; ethyl acetate, isopropanol) for two hours at reflux and mechanically stirred (175 rpm). The solid/liquid weight ratio was thus 1/10. The extraction medium was then filtered on a Buchner apparatus with a slight suction to remove the plant residues. In some specific cases, we observed a precipitate in the filtrate when the extract was let to cool down. In these cases, a second filtration was performed. For all the extracts, a rotary evaporator was used to remove the solvent from the extract by evaporation at reduced pressure. The solid extract was then freeze-dried with a dry matter typically reaching >90%. For dry matter determination, the extract was placed in an oven at 125 20° C. We repeated the freeze-drying step whenever the dry matter was found <90% until it overpassed this threshold.

Example 24. The Use of 0.1% of Crude Extracts from Microalgae as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 7

[0316] 0.1 percent of the different crude extracts from microalgae obtained in Example 23 was used to stabilize oil-in-water emulsions at pH 7 made with medium chain triglycerides as the oily phase. The emulsifying activity of these extracts was estimated by the droplet size (D.sub.v98) of a fresh emulsion and its variation after one and seven days of storage (Table 6). For most extracts, the droplet size values are expressed as the average of two independent replicates. The lower the droplet size, the higher the emulsifying activity. Moreover, the higher the increase of the droplet size with time, the lower the stability. Table 6 also shows the emulsifying scores (ES) calculated as described above in section 12 of the material and methods. Crude extracts with an ES equal to, or lower than, 20 are considered as emulsifying agents and are shown below in Table 6. The solvent of extraction is mentioned in Table 6 for all extracts. Apart from extracts obtained from Chlorella vulgaris (Z54, Z55, Z56 and Z57) and Chlorella sorokiniana (Z25 and Z26), all crude extracts have been obtained from microalgae grown photo-autotrophically.

TABLE-US-00006 TABLE 6 Emulsifying activity of the microalgae crude extracts (0.1%) in oil-in-water emulsion at pH 7 as measured by the droplet size (D.sub.V98) and ES. Data are sorted in decreasing order of ES. In some cases, the polar lipid content (which have been determined as described in section 9 of material and methods) is given in % of dry matter. Droplet size (μm) Microalgae material Day 0 Day 1 Day 7 ES Lipid level (%) Chlorella vulgaris-HPD-Filt. AF31H-EtOH90-Z57 9.7 9.8 36.4 18.9 — Chlorella vulgaris-H-Filt. AF31H-NPD-EtOH90-Z56 17.7 14.7 28.3 17.8 — Nannochloropsis-EtOH90-Z09 7.4 7.2 41.1 17.7 — Tetraselmis-EtOH90-Z39 6.9 7.6 24.7 17.4 — Chlorella vulgaris-MNPD-EtOH90-Z54 15.2 12.1 21.3 17.1 — Chlorella vulgaris-MPD-EtOH90-Z55 16.0 13.2 16.3 12.0 52.7 Chlorella sorokiniana-M-EtOH90-Z25 7.5 7.6 5.5 8.8 — Chlorella sorokiniana-MBC-EtOH90-Z26 6.1 5.4 5.0 7.8 100   Nannochloropsis-EtOH90-Z12 4.1 4.3 5.0 6.2 —

Example 25. The Use of 1% of Crude Extracts from Microalgae as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 3.5

[0317] One percent of the different crude extracts from microalgae obtained in Example 23 was used to stabilize oil-in-water emulsions at pH 3.5 made with medium chain triglycerides as the oily phase. The emulsifying activity of these extracts was estimated by the droplet size (D.sub.v98) of the fresh emulsions and its variation over a day of storage (Table 7). The variation over a longer storage period (7 days) was also measured. Most droplet size values are expressed as the average of two independent replicates. The lower the droplet size, the higher the emulsifying activity. The higher the increase of the droplet size with time, the lower the stability. Table 7 also shows the emulsifying scores (ES) calculated as described above in section 12 of the material and methods. Crude extracts with an ES equal to, or lower than, 20 are considered as emulsifying agents and are shown below in Table 7. The solvent of extraction is mentioned in Table 7 for all extracts. Apart from extracts obtained from Chlorella vulgaris (Z54, Z55, Z56 and Z57), Chlorella sorokiniana (Z24, Z25 and Z26) and Chlorella zofingensis (Z27), all crude extracts have been obtained from microalgae grown photo-autotrophically.

TABLE-US-00007 TABLE 7 Emulsifying activity of the microalgae crude extracts (1%) in oil-in-water emulsion at pH 3.5 as measured by the droplet size (D.sub.V98) and ES. Data are sorted in decreasing order of ES. In some cases, the polar lipid content (which have been determined as described in section 9 of material and methods) is given in % of dry matter. Droplet size (μm) Microalgae material Day 0 Day 1 Day 7 Score Polar lipids (%) Isochrysis-Ethyl Acetate-Z04 3.7 32 184.8 20 — Nannochloropsis-Ethyl Acetate-Z06 3.1 15.7 96.4 19.3 — Chlorella sorokiniana-H-EtOH90-Z24 5.1 8.7 12.8 19 — Nannochloropsis-EtOH80-Z13 4.4 7.2 — 18.4 — Nannochloropsis-EtOH80-Z14 2.4 665.3 3.3 18.1 — Nannochloropsis-EtOH80-Z33 4 6.4 892.4 18 — Chlorella vulgaris-M-EtOH90-Z54 2.9 5.2 52.6 16.6 — Nannochloropsis-EtOH90-Z32 4.6 4.3 28.5 15.7 55.1 Chlorella vulgaris-EtOH80-Z21 4.6 4.8 89.7 15.7 — Nannochloropsis-EtOH80-Z34 3.9 4.1 15 15 — Nannochloropsis-Acetone-Z16 3.6 3 593.1 14.6 — Chlorella sorokiniana-M-EtOH90-Z25 3.3 3.3 694.5 14.2 — Isochrysis-Acetone-Y29 3.2 3.1 79.5 14.1 — Isochrysis-iPrOH-Z02 2.7 3.2 26.3 13.3 — Nannochloropsis-EtOH90-Z09 4.1 5.7 4.9 11.5 73.7 Tetraselmis-Ethyl Acetate-Z05 2.3 2.5 3.8 9 — Nannochloropsis-EtOH80-Y27 6.2 5.7 4.1 7.9 — Isochrysis-EtOH90-Z37 5.1 5.2 5 7 53.4 Isochrysis-EtOH90-Y23 4.6 5.5 5.8 6.6 29.3 Nannochloropsis-EtOH80-Z17 4.2 4.8 5.9 6.2 — Chlorella sorokiniana-MBC-EtOH90-Z26 4.2 4.6 4.5 6.2 100   Chlorella vulgaris-H-Filt. AF31H-EtOH90-Z56 4.1 3.4 3.4 6.2 — Isochrysis-EtOH100-Y20 4 4.5 5.3 6.1 — Nannochloropsis-EtOH90-Z12 3.8 4.3 4.4 5.7 — Chlorella vulgaris-EtOH100-Z19 3.6 3 3.6 5.6 — Chlorella zofingensis-M-EtOH90-Z27 3.4 3.6 3.3 5.3 54.8 Tetraselmis-Acetone-Y31 3.3 3.4 4 5.1 — Nannochloropsis-iPrOH-Z01 3.2 3.4 3.4 5.1 — Chlorella vulgaris-HPD-Filt. AF31H-EtOH90-Z57 2.8 2.8 2.8 4.5 — Nannochloropsis-EtOH90-Y24 2.8 3 3.1 4.4 — Nannochloropsis-iPrOH-Z29 2.8 2.7 2.9 4.4 — Nannochloropsis-EtOH100-Z08 2.6 2.7 3.2 4.2 — Nannochloropsis-iPrOH-Z30 2.6 2.8 3.1 4.1 — Chlorella vulgaris-MPD-EtOH90-Z55 2.6 2.5 2.6 4.1 52.7 Nannochloropsis-EtOH100-Z07 2.3 2.9 2.4 3.6 — Tetraselmis-EtOH80-Y28 2.2 2.2 2.1 3.5 — Tetraselmis-EtOH90-Z39 2.2 2 2.4 3.4 — Tetraselmis-EtOH100-Y22 1.9 1.9 1.8 2.8 — Tetraselmis-EtOH90-Y25 1.5 1.6 1.6 1.9 69.4 Nannochloropsis-EtOH100-Y21 1.3 1.4 1 1.2 Tetraselmis-iPrOH-Z03 1.2 1.2 1.2 0.8 98.4

Example 26. The Use of 1% of Crude Extracts from Microalgae as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 7

[0318] One percent of the different crude extracts from microalgae obtained in Example 23 was used to stabilize oil-in-water emulsions at pH 7 made with medium chain triglycerides as the oily phase. The emulsifying activity of these extracts was estimated by the droplet size (D.sub.v98) of the fresh emulsions and its variation over a day of storage (Table 8). The variation over a longer storage period (7 days) was also measured for selected extracts. Most droplet size values are expressed as the average of two independent replicates. The lower the droplet size, the higher the emulsifying activity. The higher the increase of the droplet size with time, the lower the stability. Table 8 also shows the emulsifying scores (ES) calculated as described above in section 12 of the material and methods. Crude extracts with an ES equal to, or lower than, 20 are considered as emulsifying agents and are shown below in Table 8. The solvent of extraction is mentioned in Table 8 for all extracts. Apart from extracts obtained from Chlorella vulgaris (Z19, Z21, Z55, Z56 and Z57), Chlorella sorokiniana (Z25 and Z26) and Chlorella zofingensis (Z27), all crude extracts have been obtained from microalgae grown photo-autotrophically.

TABLE-US-00008 TABLE 8 Emulsifying activity of the microalgae crude extracts (1%) in oil-in-water emulsion at pH 7 as measured by the droplet size (D.sub.V98) and ES. Data are sorted in decreasing order of ES. In some cases, the polar lipid content (which have been determined as described in section 9 of material and methods) is given in % of dry matter. Droplet size (μm) Microalgae material Day 0 Day 1 Day 7 Score Polar lipids (%) Chlorella sorokiniana-M-EtOH90-Z25 3.4 752.2 — 19.6 — Nannochloropsis-Acetone-Y30 3.3 13.5 — 19.5 — Nannochloropsis-EtOH80-Z14 3.1 923.8 — 19.2 — Nannochloropsis-Ethyl Acetate-Z40 3.0 327.3 28.0 19.1 — Chlorella vulgaris-HPD-Filt. AF31H-EtOH90-Z57 52.9 3.6 3.7 17.2 — Nannochloropsis-EtOH80-Z13 5.6 6.6 15.2 16.5 — Chlorella sorokiniana-MBC-EtOH90-Z26 4.0 4.0 686.2 15.0 100   Chlorella vulgaris-EtOH80-Z21 3.8 3.8 428.0 14.8 — Isochrysis-Ethyl Acetate-Z04 3.4 4.2 58.0 14.4 — Nannochloropsis-Acetone-Z15 2.9 2.6 24.7 13.7 — Isochrysis-Acetone-Y29 2.9 2.8 106.4 13.6 — Tetraselmis-Acetone-Y31 2.9 3.0 354.3 13.6 — Isochrysis-iPrOH-Z02 2.7 2.6 10.7 13.4 — Nannochloropsis-EtOH90-Z32 4.7 4.4 6.5 12.0 55.1 Nannochloropsis-EtOH100-Z07 1.8 1.7 705.0 11.5 — Isochrysis-EtOH90-Z37 6.0 5.9 6.6 7.8 53.4 Isochrysis-EtOH90-Y23 5.9 7.0 7.2 7.7 29.3 Nannochloropsis-EtOH90-Z31 4.1 4.4 4.5 6.1 — Isochrysis-EtOH100-Y20 4.0 4.3 4.6 6.0 — Nannochloropsis-EtOH90-Z09 3.7 4.2 4.1 5.7 73.7 Chlorella vulgaris-EtOH100-Z19 3.7 3.0 3.7 5.6 — Nannochloropsis-EtOH80-Z17 3.6 3.7 4.0 5.6 — Tetraselmis-Ethyl Acetate-Z05 3.5 4.2 4.1 5.4 — Chlorella vulgaris-H-filtered AF31H-EtOH90-Z56 3.5 3.5 3.4 5.4 — Nannochloropsis-EtOH100-Y21 3.4 1.0 0.9 5.3 — Chlorella zofingensis-M-EtOH90-Z27 3.4 3.4 3.3 5.3 54.8 Nannochloropsis-EtOH80-Z34 3.4 3.4 3.6 5.3 — Nannochloropsis-EtOH80-Z33 3.3 4.2 4.7 5.2 — Nannochloropsis-EtOH80-Z36 2.7 2.7 2.8 4.4 — Nannochloropsis-EtOH90-Z12 2.6 2.8 2.9 4.2 — Chlorella vulgaris-MPD-EtOH90-Z55 2.4 2.5 2.5 3.8 52.7 Tetraselmis-EtOH90-Z39 2.1 2.1 2.1 3.3 — Nannochloropsis-iPrOH-Z01 2.1 2.4 2.3 3.3 — Nannochloropsis-iPrOH-Z30 1.8 1.8 1.8 2.6 — Nannochloropsis-EtOH100-Z08 1.8 1.9 1.9 2.6 — Tetraselmis-EtOH90-Y25 1.4 1.5 1.5 1.4 69.4 Tetraselmis-iPrOH-Z03 1.0 1.0 1.0 0.2 98.4 Tetraselmis-EtOH100-Y22 0.9 1.0 1.0 −0.3 —

Example 27. The Use of 5% of Crude Extracts from Microalgae as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 3.5

[0319] Five percents of the different crude extracts from microalgae obtained in Example 23 were used to stabilize oil-in-water emulsions at pH 3.5 made with medium chain triglycerides as the oily phase. The emulsifying activity of these extracts was estimated by the droplet size (D.sub.v98) of the fresh emulsions and its variation over a day of storage (Table 9). The variation over a longer storage period (7 days) was also measured. Most droplet size values are expressed as the average of two independent replicates. The lower the droplet size, the higher the emulsifying activity. The higher the increase of the droplet size with time, the lower the stability. Table 9 also shows the emulsifying scores (ES) calculated as described above in section 12 of the material and methods. Crude extracts with an ES equal to, or lower than, 20 are considered as emulsifying agents and are shown below in Table 9. The solvent of extraction is mentioned in Table 9 for all extracts. Apart from extracts obtained from Chlorella vulgaris (Z55, Z56 and Z57) and Chlorella sorokiniana (Z24 and Z25), all crude extracts have been obtained from microalgae grown photo-autotrophically.

TABLE-US-00009 TABLE 9 Emulsifying activity of the microalgae crude extracts (5%) in oil-in-water emulsion at pH 3.5 as measured by the droplet size (D.sub.V98) and ES. Data are sorted in decreasing order of ES. In some cases, the polar lipid content (which have been determined as described in section 9 of material and methods) is given in % of dry matter. Droplet size (μm) Microalgae material Day 0 Day 1 Day 7 Score Lipid content (%) Nannochloropsis-Ethyl Acetate-Z06 3.62 11.77 27.78 19.9 — Nannochloropsis-Acetone-Z18 3.11 144.87 — 19.2 — Tetraselmis-Ethyl Acetate-Z05 2.57 53.12 1.04 18.4 — Nannochloropsis-Acetone-Y30 3.39 6.65 1.44 17.2 — Nannochloropsis-Acetone-Z15 2.67 2.22 24.02 13.3 — Isochrysis-Ethyl Acetate-Z04 1.87 2.08 16.04 11.7 — Chlorella sorokiniana-M-EtOH90-Z25 4.37 4.9 5.78 11.7 — Nannochloropsis-EtOH100-Y21 11 13.77 0.53 10.4 — Nannochloropsis-iPrOH-Z29 1.24 1.26 254.71 10.0 — Nannochloropsis-EtOH100-Z08 2.89 2.85 4.8 9.9 — Chlorella vulgaris-MPD-EtOH90-Z55 8.79 10.18 5.34 9.4 52.7 Chlorella vulgaris-HPD-Filt. AF31H-EtOH90- 8.46 8.36 8 9.3 — Tetraselmis-EtOH100-Y22 6.76 6.7 5.02 8.3 — Chlorella sorokiniana-H-EtOH90-Z24 6.29 6.15 1.86 8.0 — Isochrysis-EtOH80-Y26 5.86 5.82 5.36 7.7 — Chlorella vulgaris-H-Filt. AF31H-EtOH90-Z56 4.91 5.72 5.93 6.9 — Isochrysis-iPrOH-Z02 1.34 1.84 2.03 6.6 — Isochrysis-EtOH100-Y20 1.1 1.28 2.01 5.7 — Nannochloropsis-EtOH100-Z07 3.4 3.52 3.32 5.3 — Chlorella vulgaris-EtOH100-Z19 2.98 2.71 3.34 4.7 — Isochrysis-EtOH90-Y23 2.26 2.35 2.49 3.5 29.3 Isochrysis-EtOH80-Z38 1.83 1.95 1.95 2.6 — Nannochloropsis-iPrOH-Z01 1.69 1.77 1.01 2.3 — Tetraselmis-EtOH80-Y28 1.64 1.69 1.73 2.1 — Isochrysis-EtOH90-Z37 1.59 1.77 1.84 2.0 53.4 Isochrysis-Acetone-Y29 1.42 1.41 1.54 1.5 — Tetraselmis-EtOH90-Y25 1.12 0.96 0.96 0.5 69.4 Tetraselmis-EtOH90-Z39 1.07 1.12 1.29 0.3 — Nannochloropsis-iPrOH-Z30 0.88 0.89 0.86 −0.6 — Tetraselmis-iPrOH-Z03 0.81 0.73 0.71 −0.9 98.4

Example 28. The Use of 5% of Crude Extracts from Microalgae as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 7

[0320] Five percents of the different crude extracts from microalgae obtained in Example 23 were used to stabilize oil-in-water emulsions at pH 7 made with medium chain triglycerides as the oily phase. The emulsifying activity of these extracts was estimated by the droplet size (D.sub.v98) of the fresh emulsions and its variation over a day of storage (Table 10). The variation over a longer storage period (7 days) was also measured. Most droplet size values are expressed as the average of two independent replicates. The lower the droplet size, the higher the emulsifying activity. The higher the increase of the droplet size with time, the lower the stability. Table 10 also shows the emulsifying scores (ES) calculated as described above in section 12 of the material and methods. Crude extracts with an ES equal to, or lower than, 20 are considered as emulsifying agents and are shown below in Table 10. The solvent of extraction is mentioned in Table 10 for all extracts. Apart from extracts obtained from Chlorella vulgaris (Z54, Z55, Z56 and Z57) and Chlorella sorokiniana (Z24 and Z25), all crude extracts have been obtained from microalgae grown photo-autotrophically.

TABLE-US-00010 TABLE 10 Emulsifying activity of the microalgae crude extracts (5%) in oil-in-water emulsion at pH 7 as measured by the droplet size (D.sub.V98) and ES. Data are sorted in decreasing order of ES. In some cases, the polar lipid content (which have been determined as described in section 9 of material and methods) is given in % of dry matter. Droplet size Microalgae material Day 0 Day 1 Day 7 ES Lipid content (%) Chlorella sorokiniana-M-EtOH90-Z25 6.33 8.53 15.42 20.0 — Chlorella vulgaris-M-EtOH90-Z54 3.25 31.56 49.47 19.4 — Nannochloropsis-Ethyl Acetate-Z06 2.85 17.6 — 18.9 — Chlorella vulgaris-EtOH90-Z20 7.14 8.45 100.23 17.6 — Chlorella vulgaris-HPD-Filt. AF31H-EtOH90-Z57 13.96 14.19 18.89 16.7 — Tetraselmis-Acetone-Y31 40.16 4.9 0.88 16.0 — Isochrysis-Acetone-Y29 1.43 2.32 3.44 13.5 — Tetraselmis-Ethyl Acetate-Z05 2.37 2.32 23.7 12.8 — Isochrysis-iPrOH-Z02 1.83 2.14 4.05 11.7 — Nannochloropsis-Acetone-Z15 1.83 1.81 4.91 11.7 — Chlorella vulgaris-EtOH100-Z19 3.9 3.86 5.68 11.2 — Chlorella vulgaris-MPD-EtOH90-Z55 11.86 14.67 10.32 10.7 52.7 Nannochloropsis-Acetone-Y30 2.15 2.38 3.76 8.6 — Chlorella sorokiniana-H-EtOH90-Z24 6.59 6.43 2.77 8.2 — Chlorella vulgaris-H-Filt, AF31H-EtOH90-Z56 5.16 4.51 5.71 7.1 — Isochrysis-EtOH100-Y20 1.46 1.67 2.16 6.9 — Isochrysis-EtOH80-Y26 4.63 5.06 4.58 6.7 — Nannochloropsis-EtOH100-Z07 3.81 4.44 4.09 5.8 — Nannochloropsis-EtOH100-Z08 3.14 3.29 3 5.0 — Isochrysis-EtOH90-Y23 1.79 1.95 2.08 2.5 29.3 Isochrysis-EtOH90-Z37 1.75 1.9 1.94 2.4 53.4 Tetraselmis-EtOH80-Y28 1.71 1.75 1.84 2.3 — Isochrysis-EtOH80-Z38 1.63 1.68 1.74 2.1 — Tetraselmis-EtOH100-Y22 1.42 1.37 1.18 1.5 — Nannochloropsis-iPrOH-Z01 1.28 1.32 1.15 1.1 — Tetraselmis-EtOH90-Y25 1.04 0.96 0.95 0.2 69.4 Tetraselmis-EtOH90-Z39 1.04 1.11 1.28 0.2 — Nannochloropsis-iPrOH-Z29 0.9 0.89 0.88 −0.5 — Tetraselmis-iPrOH-Z03 0.84 0.76 0.71 −0.8 98.4 Nannochloropsis-iPrOH-Z30 0.78 0.77 0.78 −1.1 —

Example 29. Production of a Crude Extract from Dried Dunaliella salina Microalgae by S/L Extraction and Use of 1 and 5% Thereof as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 7

[0321] Two hundred grams of dried Dunaliella salina were mixed with 2 L of an ethanol:water (90:10) mixture for two hours at reflux and mechanically stirred (175 rpm). The extraction was performed as described above in section 3 of the material and methods. One percent of the resulting extract (C03) were then used to stabilize an oil-in-water emulsion at pH 7 made with medium chain triglycerides as the oily phase. The D.sub.v98 of this emulsion measured in duplicate was 2.4 and 2.4 μm just after emulsification, and still 2.4 and 2.4 μm after one day of storage, indicating that the crude extract had the ability to form a stable emulsion.

Example 30. Production of a Crude Extract from Dried Black Tea Leaves (Camellia sinensis) by S/L Extraction and Use of 1% Thereof as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 7

[0322] Two hundred grams of dried black tea leaves were mixed with 2 L of an ethanol:water (90:10) mixture for two hours at reflux and mechanically stirred (175 rpm). The extraction was performed as described above in section 3 of the material and methods. One percent of the resulting extract (Z58) was then used to stabilize an oil-in-water emulsion at pH 7 made with medium chain triglycerides as the oily phase. The D.sub.v98 of this emulsion was 11.4 μm just after emulsification, indicating that the crude extract had the ability to form an emulsion. A quantification of the polar lipid content has been achieved on the extract as described in section 9 and gave a value of 6.9%.

Example 31. Production of a Crude Extract from Dried Rosemary Leaves (Rosmarinus officinalis) by S/L Extraction and Use of 5% Thereof as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 7

[0323] Two hundred grams of dried rosemary leaves were mixed with 2 L of an ethanol:water (90:10) mixture for two hours at reflux and mechanically stirred (175 rpm). The extraction was performed as described above in section 3 of the material and methods. Five percents of the resulting extract (C02) was then used to stabilize an oil-in-water emulsion at pH 7 made with medium chain triglycerides as the oily phase. The D.sub.v98 of this emulsion was 5.11 and 3.9 μm just after emulsification and after one day of storage, respectively, indicating that the crude extract had the ability to form a stable emulsion.

Example 32. Production of a Crude Extract from Dried Green Pea Pods (Pisum sativum) by S/L Extraction and Use of 1% Thereof as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 7

[0324] Two hundred grams of dried green pea pods were mixed with 2 L of an ethanol:water (90:10) mixture for two hours at reflux and mechanically stirred (175 rpm). The extraction was performed as described above in section 3 of the material and methods. One percent of the resulting extract (Z67) was then used to stabilize an oil-in-water emulsion at pH 7 made with medium chain triglycerides as the oily phase. The D.sub.v98 of this emulsion was 5.7 μm just after emulsification, indicating that the crude extract had the ability to form an emulsion.

Example 33. Production of a Crude Extract from Fresh Spring Onion (Allium fistulosum) Biomass by a Direct S/L Extraction and Use of 1% Thereof as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 3.5

[0325] One litre of ethanol was heated to 70° C. One hundred grams of fresh spring onion were weighted and properly mixed with a classic cooking blender (Moulinex, France) during a few min. The plant was then added to the hot solvent and the extraction was performed as described above in section 4 (Direct S/L extraction on fresh materials).

[0326] One percent of the resulting extract (Z42 green) was then used to stabilize an oil-in-water emulsion at pH 3.5 made with medium chain triglycerides as the oily phase. The D.sub.v98 of this emulsion was 3.5, 3.1 and 164.9 μm, just after emulsification and after one and seven days of storage, respectively. The corresponding ES—which was calculated as described above in section 12 of the material and methods— was of 14.5, which demonstrates a notable emulsifying activity, at least after one day of storage, because extracts with an ES equal to, or lower than, 20 are considered as emulsifying agents. The droplet size increase after the seventh day of storage is likely to be solvable by a simple increase of the emulsion's viscosity.

Example 34. Production of a Crude Extract from Fresh Broccoli Rabe (Brassica ruvo) Biomass by an Indirect S/L Extraction and Use of 1% Thereof as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 3.5

[0327] One litre of ethanol was heated to 70° C. One hundred grams of fresh broccoli rabe were weighted and properly mixed with a classic cooking blender (Moulinex, France) during a few min. The plant was then added to the hot solvent and the extraction was performed as described above in section 5 (Indirect S/L extraction on fresh materials).

[0328] One percent of the resulting extract (Z49) was then used to stabilize an oil-in-water emulsion at pH 3.5 made with medium chain triglycerides as the oily phase. The D.sub.v98 of this emulsion was 3.6 μm just after emulsification, demonstrating that the extract was able to form an emulsion.

Example 35. Production of a Crude Extract from Fresh Carrot Leaves (Daucus carota) Biomass by an Indirect S/L Extraction and Use of 1% Thereof as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 7

[0329] One litre of ethanol was heated to 70° C. One hundred grams of fresh carrot leaves were weighted and properly mixed with a classic cooking blender (Moulinex, France) during a few min. The plant was then added to the hot solvent and the extraction was performed as described above in section 5 (Indirect S/L extraction on fresh materials).

[0330] One percent of the resulting extract (Z51) was then used to stabilize an oil-in-water emulsion at pH 7 made with medium chain triglycerides as the oily phase. The D.sub.v98 of this emulsion was 5.2 μm just after emulsification, demonstrating that the extract was able to form an emulsion.

Example 36. Production of Crude Extracts from Fresh Spinach Leaves by an Indirect S/L Extraction and Use of 1% Thereof as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 3.5

[0331] One litre of ethanol was heated to 70° C. The residue was recovered and added to the hot solvent to proceed to the extraction as described above in section 5 (Indirect S/L extraction on fresh materials). The resulting crude extract is further referred to as extract Z48.

[0332] In another extraction, 250 g of the same fresh spinach leaves were juiced using a cooking juice-extractor device at room temperature for 5 min, then the juice was clarified by filtration, and the resulting filtration cake was pooled with the recovered residue from the juice-extractor, and pressed using a hydraulic press at 15 bar to remove as much residual juice as possible. The dry cake was then extracted as described above in section 5 (Indirect S/L extraction on fresh materials). The resulting crude extract is further referred to as extract Z69.

[0333] One percent of both crude extracts was then used to stabilize an oil-in-water emulsion at pH 3.5 made with medium chain triglycerides as the oily phase.

[0334] For extract Z48, the D.sub.v98 of this emulsion was 4.6, 5.6 and 40.2 μm, just after emulsification and after one and seven days of storage, respectively. The corresponding ES—which was calculated as described above in section 12 of the material and methods— was of 14.5, which demonstrates a notable emulsifying activity, at least after one day of storage, because extracts with an ES equal to, or lower than, 20 are considered as emulsifying agents.

[0335] For extract Z69, the emulsifying performances were slightly better with a D.sub.v98 of the emulsion of 3.6, 3.4 and 21 μm, just after emulsification and after one and seven days of storage, respectively. The corresponding ES was of 14.6.

[0336] In both cases, the droplet size increase after the seventh day of storage is likely to be solvable by a simple increase of the emulsion's viscosity.

Example 37. Production of a Crude Extract from Fresh Spring Onion (Allium fistulosum) Biomass by a Direct S/L Extraction and Use of 1% Thereof as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 7

[0337] One litre of ethanol was heated to 70° C. One hundred grams of fresh spring onion were weighted and properly mixed with a classic cooking blender (Moulinex, France) during a few min. The plant was then added to the hot solvent and the extraction was performed as described above in section 4 (Direct S/L extraction on fresh materials).

[0338] One percent of the resulting extract (Z42 green) was then used to stabilize an oil-in-water emulsion at pH 7 made with medium chain triglycerides as the oily phase. The D.sub.v98 of this emulsion was 3.3, 3.2 and 3.0 μm, just after emulsification and after one and seven days of storage, respectively. The corresponding ES—which was calculated as described above in section 12 of the material and methods— was of 5.1, which demonstrates a very good emulsifying activity, because extracts with an ES equal to, or lower than, 20 are considered as emulsifying agents.

Example 38. Production of a Crude Extract from Fresh Celery Leaves (Apium graveolens) Biomass by a Direct S/L Extraction and Use of 1% Thereof as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 7

[0339] One litre of ethanol was heated to 70° C. One hundred grams of fresh celery leaves were weighted and properly mixed with a classic cooking blender (Moulinex, France) during a few min. The plant was then added to the hot solvent and the extraction was performed as described above in section 4 (Direct S/L extraction on fresh materials). One percent of the resulting extract (Z52) was then used to stabilize an oil-in-water emulsion at pH 7 made with medium chain triglycerides as the oily phase. The D.sub.v98 of this emulsion was 10.5 μm, just after emulsification, indicating that the extract was able to form an emulsion.

Example 39. Production of a Crude Extract from Fresh Broccoli Rabe (Brassica ruvo) Biomass by a Direct S/L Extraction and Use of 1% Thereof as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 7

[0340] One litre of ethanol was heated to 70° C. One hundred grams of fresh broccoli rabe were weighted and properly mixed with a classic cooking blender (Moulinex, France) during a few min. The plant was then added to the hot solvent and the extraction was performed as described above in section 4 (Direct S/L extraction on fresh materials).

[0341] One percent of the resulting extract (Z44) was then used to stabilize an oil-in-water emulsion at pH 7 made with medium chain triglycerides as the oily phase. The D.sub.v98 of this emulsion was 4.5 μm, just after emulsification, indicating that the extract was able to form an emulsion.

Example 40. Production of a Crude Extract from Fresh Radish Leaves (Raphanus sativus) Biomass by a Direct S/L Extraction and Use of 1% Thereof as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 7

[0342] One litre of ethanol was heated to 70° C. One hundred grams of fresh radish leaves were weighted and properly mixed with a classic cooking blender (Moulinex, France) during a few min. The plant was then added to the hot solvent and the extraction was performed as described above in section 4 (Direct S/L extraction on fresh materials).

[0343] One percent of the resulting extract (Z47) was then used to stabilize an oil-in-water emulsion at pH 7 made with medium chain triglycerides as the oily phase. The D.sub.v98 of this emulsion was 3.7 μm, just after emulsification, indicating that the extract was able to form an emulsion.

Example 41. Production of Crude Extracts from Fresh Spinach Leaves by an Indirect S/L Extraction and Use of 1% Thereof as Emulsifiers in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 7

[0344] One litre of ethanol was heated to 70° C. Two hundred and fifty grams of fresh spinach leaves were juiced using a cooking juice-extractor device at room temperature for 5 min. The juice was clarified by filtration, and the resulting filtration cake was pooled with the recovered residue from the juice-extractor, and pressed using a hydraulic press at 15 bar to remove as much residual juice as possible. The dry cake was then extracted as described above in section 5 (Indirect S/L extraction on fresh materials). The resulting crude extract is further referred to as extract Z69.

[0345] One percent of this extract was then used to stabilize an oil-in-water emulsion at pH 7 made with medium chain triglycerides as the oily phase. The D.sub.v98 of this emulsion was 4.0, 4.3 and 6.1 μm, just after emulsification and after one and seven days of storage, respectively. The corresponding ES—which was calculated as described above in section 12 of the material and methods— was of 11.3, which demonstrates a notable emulsifying activity, because extracts with an ES equal to, or lower than, 20 are considered as emulsifying agents.

Example 42. Production of Ethanolic:Water (90:10) Crude Extracts from Dried Spinach by S/L Extraction, Decolorization Thereof with Powdery Activated Carbon or Filter Plates Coated with Charcoal, and Use of 1 or 5% Thereof in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 3.5 or 7

[0346] For Y06, 200 g of dried spinach were mixed with 3 L of an ethanol:water (90:10) mixture for two hours at reflux and mechanically stirred (175 rpm). The extraction was performed as described above in section 3 of the material and methods (S/L ratio: 1:15). One and five percents of the resulting extract was then used to stabilize an oil-in-water emulsion at pH 3.5 and 7 made with medium chain triglycerides as the oily phase.

[0347] For Y13, the same process was applied except that before drying the filtrate of the first solvent pass, a second pass with a new ethanol:water (90:10) mixture was performed in the same conditions as the first pass (2 h at reflux under mechanically stirred at 175 rpm, S/L ratio: 1:15). One and five percents of the resulting extract was then used to stabilize the emulsion above-mentioned at pH 3.5 and 7.

[0348] For Y10 and Y15, the same one-pass extraction protocol as previously described for Y06, was applied, except that a decolorization step using powdery charcoal was performed at the end as described in section 7 of the material and methods.

[0349] For Y09, the same 2-pass extraction protocol as previously described for Y13 was applied, except that a decolorization step using powdery charcoal was performed at the end as described in section 7 of the material and methods.

[0350] For Y37, the same one-pass extraction protocol as previously described for Y06, was applied, except that a decolorization step using a R55S filter plate coated with charcoal was performed at the end as described in section 8 of the material and methods.

[0351] For Y41, the same one-pass extraction protocol as previously described for Y06, was applied, except that a decolorization step using a Filtrox filter plate coated with charcoal was performed at the end as described in section 8 of the material and methods. A quantification of the polar lipids (method described in section 9 of the material and methods) was also achieved on purified extract Y41 and found a content of 27.6%.

[0352] Color of 1% emulsions was measured by reflection in the CIELAB color space in a borosilicate tube cell with a spectrophotometer (Konica Minolta CM-5). The emulsions were given into the measurement cell at 10 mm height.

[0353] All spinach extracts were obtained from photosynthetically active parts of the plant (i.e. leaves) so that they are green. This color was still visible in the emulsions prepared with 1% extract. Emulsions prepared with Sweoat PL40 as reference (oat oil) showed no green color. Hence, we calculated the color difference ΔE* according to equation 1 with L.sub.1*, a.sub.1*, and b.sub.1* being measured in the emulsion with oat oil and L.sub.2*, a.sub.2*, and b.sub.2* being measured in the emulsion stabilized with an ethanolic:water (90:10) spinach.

ΔE*.sub.ab=√(L*.sub.2−L*.sub.1).sup.2+(a*.sub.2−a*.sub.1).sup.2+(b*.sub.2−b*.sub.1).sup.2  Equation 1:

[0354] FIG. 19 shows the AE decrease as a function of the applied decolorization (purification) protocol. The fact that AE decreases with the addition of growing quantities of charcoal and with the use of the R55S filter plate means that the resulting emulsions get closer to the whitish oat oil reference. There is still a visible difference between the emulsions even after purifying them with an activated carbon filter sheet R55S but the color changes from green to yellow and the L value increases distinctly (from 67 to 87), demonstrating that the sample is more white.

[0355] In FIG. 20, it can be seen that the emulsification performance is not negatively influenced by the purification within the reproducibility of the experiments. This means that no or only little amounts of emulsifying molecules adsorbed to the activated charcoal and that filter material of the right selectivity was chosen.

Example 43. Production of Ethanolic:Water (90:10) Crude Extracts from Dried Spirulina by S/L Extraction, Decolorization Thereof with Powdery Activated Carbon, and Use of 5% Thereof in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 3.5 or 7

[0356] For Y16, 200 g of dried spirulina were mixed with 2 L of an ethanol:water (90:10) mixture for two hours at reflux and mechanically stirred (175 rpm). The extraction was performed as described above in section 3 of the material and methods. Five percents of the resulting extract was then used to stabilize an oil-in-water emulsion at pH 3.5 and 7 made with medium chain triglycerides as the oily phase.

[0357] For Y18, the same process was applied except that before drying the filtrate of the first solvent pass, a second pass with a new ethanol:water (90:10) mixture was done in the same condition as the first pass (2 h at reflux under mechanically stirred at 175 rpm). Five percents of the resulting extract was then used to stabilize the emulsion above-mentioned at pH 3.5 and 7.

[0358] For Y17 and 19, the same protocol as previously described for Y16 and Y18, respectively, was applied, except that a decolorization step was performed as described in section 7 of the material and methods. Among these spirulina extracts, the polar lipid content of Y16 and 17 were measured as described in section 9 of the material and methods and gave values of 26.4 and 27.6%, respectively.

[0359] FIG. 21 shows that droplet size slightly increases with the purification. However, the droplet sizes achieved with the purified extract are still very small (about 1.5 μm) and the emulsions are very stable over time. Hence, the purified extracts are very good emulsifiers.

TABLE-US-00011 TABLE 11 Influence of the purification protocol on the colorimetric properties of spirulina extracts. The ΔE is calculated in comparison to a reference emulsion prepared with oat oil. Purification with 10% Extract powdered Number Description of sample charcoal L* a* b* ΔE Reference Sweoat PL15 — 94.3 −1.0 5.1 0 Y16 Spirulina Ethanol 90%, 1 extraction pass no 54.6 0.5 16.0 41.2 Y18 Spirulina Ethanol 90%, 2 extraction passes no 53.4 0.25 15.9 42.4 Y17 Spirulina Ethanol 90%, 1 extraction pass yes 84.2 −2.3 14.2 13.7 Y19 Spirulina Ethanol 90%, 2 extraction passes yes 81.3 1.9 18.8 19.1

[0360] Table 11 shows how L* increases and the AE-value decreases when a purification method is applied to the spirulina extract, which means the resulting emulsion gets closer to the whitish oat oil reference. There is still a visible difference between the oat oil emulsion and the emulsions after purifying the extracts with 10% charcoal but the colour perception by eye changes from green to yellow and the L value increases distinctly (from about 54 to about 83), meaning that the samples are more white.

Example 44. Production of an Ethanolic:Water (90:10) Crude Extract from Dried Parsley Leaves (Petroselinum crispum) by S/L Extraction, Decolorization Thereof with Filter Plate Coated with Activated Charcoal, and Use of 1% Thereof in a 10% Oil-In-Water Emulsion with a Continuous Phase at pH 7

[0361] Two hundred grams of dried parsley leaves (Petroselinum crispum) were mixed with 2 L of an ethanol:water (90:10) mixture for two hours at reflux and mechanically stirred (175 rpm). The extraction and decolorization were performed as described above in section 8 of the material and methods. One percent of the resulting extract (Y45) was then used to stabilize an oil-in-water emulsion at pH 7 made with medium chain triglycerides as the oily phase. The D.sub.v98 of this emulsion was 15.8 μm just after emulsification, indicating that the crude extract had the ability to form an emulsion. A quantification of the polar lipid content has been achieved on the extract as described in section 9 and gave a value of 5.6%.

[0362] Colorimetric properties of the decolorized parsley extract was then measured before drying with a device from Spectramagic NX in transmittance mode.

[0363] Table 12 shows that the L value increases distinctly from 11 to 93, which means the sample gets less dark. The b value also changes from 19 to 53 indicating that the sample is more yellow. The constant value of a might seem surprising however the large changes of L and b can also account for a perceived decrease of green in the sample.

TABLE-US-00012 TABLE 12 Colorimetric properties of a crude vs. a decolorized extract of parsley L* a* b* Parsley-EtOH90-Crude extract 11.2 −6.9 19.1 Parsley-EtOH90-Carbon sheet-R55S 92.6 −6.9 52.9

Example 45. The Use of 1% of Crude Extracts from Spinach as Emulsifiers in a 10% Water-In-Oil Emulsion with an Aqueous Phase at pH 7

[0364] In FIG. 22 the D.sub.v98 of emulsions prepared a commercially available soy lecithin (Topcithin, Cargill) is shown in comparison with three purified plant extracts from Spinach. Surprisingly spinach extract (extracted with 90% ethanol) is not only performing well as oil-in-water emulsifier but also for water-in-oil emulsions. The initial droplet size of spinach extract purified with charcoal sheet Filtrox is distinctly smaller than for Soy Lecithin. For all purified extracts the droplet size after one day is comparable or smaller than for soy lecithin. An emulsifier so versatile to be able to stabilize water-in-oil as well as oil-in-water emulsions is highly advantageous because it can be flexible used in many different applications of different emulsion type, dispersed phase content or oils of different polarity.

Example 46. Preparation of Coffee Creamer with 1% Purified Spinach Extract

[0365] A series of creamer emulsion according to the present invention were prepared by performing the steps of: [0366] 1. Preparing an oil phase by mixing a known amount (Table 13) of coconut oil (Kristal, AAK) and in example 37a, a known amount of Spinach extract containing polar lipids in a vessel equipped with a magnetic stirrer hotplate operating at 300 rpm and 50° C. (IKA, RET Laboratory); [0367] 2. Preparing a water phase by dissolving a known amount of pea protein (Pisane c9, Cosucra) in a known amount of water at 40° C. for 30 min while stirring with an overhead stirrer at 300 rpm. [0368] 3. Add a known amount of oat Syrup (Natu-Oat 35, Meurens) and a known amount of oat flour (Sweoat P19, Swedish Oat Fiber) to the water phase, and hydrate for another 30 min at room temperature while stirring with an overhead stirrer (Heidolph, HeiTorque) at 300 rpm [0369] 4. Combine a known amount of gellan gum (Kelcogel CG-HA, CP Kelco) with a known amount of guar gum (Keystone® 7555 Guar Gum, Main Street Ingredients) and a known amount of crystalline sugar (Magyar Cukor) and add this mix to the water phase while stirring with an overhead stirrer at 300 rpm at room temperature. [0370] 5. Heat up the water phase to 60° C., add oil phase while using Rotor-Stator mixer (Kinematica, PT-DA 3030-6060) operating at 8,000 rpm for 90 s, to obtain the pre-emulsion [0371] 6. Emulsifying the pre-emulsion by passing one time through a two-stage high-pressure homogenizer (GEA, Lab Homogenizer Panda Plus 2000), operating at a first stage valve pressure of 350 bar and a second stage valve pressure of 50 bar, in order to obtain the final emulsion. [0372] 7. Pasteurizing the final emulsion through a water bath operating at 95° C. for 12 min.

[0373] In all examples, step 5. resulted in oil-in-water emulsions.

TABLE-US-00013 TABLE 13 Composition of creamer emulsion Ex. 37a Ex. 37b Coconut oil [wt.-%] 7.5 7.5 Purified Spinach extract EtOH90 [wt.-%] 1 0 Pea protein [wt.-%] 0.3 0.3 Water [wt.-%] 60.74 61.74 Oat syrup [wt.-%] 9 9 Oat flour [wt.-%] 1.33 1.33 Crystalline Sucrose [wt.-%] 20 20 Gellan Gum [wt.-%] 0.03 0.03 Gum Guar [wt.-%] 0.1 0.1

TABLE-US-00014 TABLE 14 Droplet sizes of coffee creamers. The volume weighted mean diameter D[4, 3] and Dv90 of the droplet size distribution were then calculated using the software implemented in the measurement instrument. The Dv90, is defined as the diameter where 90% of the population (Volume) lies below this value. Droplet size D[4, 3] [μm] Droplet size d(90) [μm] Example 37a, fresh 4.62 7.71 Example 37b, fresh 9.76 18.22

[0374] In Table 14, it can be seen that the addition of Purified Spinach Extract led to a decrease in droplet size in the coffee creamer. This is advantageous for a longer shelf life as well as a better whitening effect in the coffee.