Activators of Plant Metabolic Changes

Abstract

The present invention relates to a composition comprising saccharides extracted from blue-green algae biomass, the said saccharides comprisingfrom 55% to 60% of rhamnose by weight, from 6.5% to 10% of uronic acid by weight, from 5% to 15% of sulfate groups by weight, andless than 10% of xylose, glucose and galactose or a mixture thereof calculated by weight upon the total of the composition being 100%.

Claims

1. A composition comprising saccharides extracted from blue-green algae biomass, the said saccharides comprising from 55% to 60% of rhamnose by weight, from 6.5% to 10% of uronic acid by weight, from 5% to 15% of sulfate groups by weight, and less than 10% of xylose, glucose and galactose or a mixture thereof calculated by weight upon the total of the composition being 100%.

2. The composition according to claim 1, comprising saccharides extracted from genus Spirulina.

3. The composition according to claim 1, which is water soluble.

4. An enzymatic hydrolysate, preferably a pectinase hydrolysate of the composition according to claim 1.

5. The composition according to claim 1, which further comprises an alcohol soluble fraction extracted from genus Spirulina, which comprises beta-carotenes and fatty acids.

6. The composition according to claim 5, wherein the fatty acids are selected from the group consisting of C16 fatty acids, C18 fatty acids or a mixture thereof.

7. The composition according to claim 5, wherein the alcohol soluble fraction is an ethanol or methanol soluble fraction extracted from genus spirulina.

8. The composition according to claim 5, wherein the alcohol soluble fraction is a 30% ethanol or 30% methanol soluble fraction extracted from genus Spirulina.

9. The composition according to claim 5, wherein the alcohol soluble fraction comprises more than 440 mg/l polyphenols.

10. The composition according to claim 1, which further comprises a poly-cationic saccharide.

11. The composition of claim 10, wherein the poly-cationic saccharide is selected from the group consisting of a chitosan oligosaccharide, a functionally-modified chitosan oligosaccharide, chitosan polysaccharide, a functionally-modified chitosan polysaccharide, salt thereof, or combination thereof.

12. The composition of claim 11, wherein the chitosan or the functionally-modified chitosan has a degree of polymerization higher than 5 and a degree of acetylation lower than 50%.

13. The composition according to claim 1, which further comprises one or more of a saccharide, growth regulator or growth factor, vitamin, hormone, mineral, ion, nutriment, food additive, flavouring, colour, a phytosanitary product, phytonutrient, or combination thereof.

14. The composition according to claim 1, which is selected from the group consisting of a pharmaceutical composition, a veterinary composition, a nutraceutical composition, a food composition or feed composition including a functional food or a functional feed, a cosmetic composition, a phytosanitary product, an elicitor, a fertilizer, or an adjuvant of these products.

15. A biomaterial, preferably a textile, comprising the composition according to claim 1.

16. (canceled)

17. (canceled)

18. A method of inducing protection of a plant against infection by one or more of Phytophthora infestans, Phytophthora cinnamomi, Phytophthora capsici, Botrytis cinerea, Erwinia carotovora, Alternaria alternata, Alternaria solani, Fusarium spp., Erysiphe cichoracearum, Rhizoctonia solani or a combination thereof, the method comprising administering the composition of claim 1 to the plant.

19. A method for increasing protein content into a plant, the method comprising administrating the composition of claim 1 to the plant.

20. The method of claim 18, wherein the step of administrating the composition to the plant comprises foliar spraying of the composition upon the plant leaves, by coating the plant seeds with the composition, by dipping the plant roods with the composition or through amendment with the composition of the plant soil.

21. The method according to claim 19, wherein the composition is present in a solvent at a concentration higher than 0.1 mg/ml.

22. A method of treating wounds in a human or animal body comprising the step of administrating the composition of claim 1 to the human or animal body.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0073] FIG. 1 A represents the GC-MS chromatograms of the different XAD fractions and shows that the Spirulina alcohol extract comprises C16 and C18 fatty acids.

[0074] FIGS. 1 B and C represent respectfully the MS spectra of the XAD fraction in ethanol and in acetone.

[0075] FIG. 1.2 represents FTIR spectra and monosaccharide composition of Spirulina poly-anionic saccharides extract according to the invention.

[0076] FIG. 1.3 represents the thermogravimetric analysis of the Spirulina poly-anionic saccharides extract composition according to the invention.

[0077] FIG. 1.4 represents the relative viscosity of the (poly-anionicsaccharide extract solutions in function of the concentration.

[0078] FIG. 1.5 represents the Macro-Prep DEAE-chromatogram of spirulina (poly-anionic) saccharide extract solutions.

[0079] FIG. 2 represents the protein content and PAL activity in Arabidopsis thaliana cell suspension, 24 hours after treatments with increasing concentrations of Spirulina saccharides extract.

[0080] FIG. 3 shows that foliar spraying of the composition according to the invention increases protein production in the treated plant.

[0081] FIG. 4 represents the protein content and PAL activity in leaves of 10 days old wheat plants from seeds coated with increasing concentrations of Spirulina poly-anionic saccharides extract according to the invention.

[0082] FIG. 5 represents the protein content and PAL activity in leaves of 10 days old wheat plants growing on a soil pre-treated with a solution of Spirulina poly-anionic saccharides extract or Spirulina MeOH soluble extract.

[0083] FIG. 6.1 represents the protein content and PAL activity in leaves of tomato, wheat and cucumber plants from seedling pretreated with increasing concentrations of the spirulina poly-anionic saccharides extract and increasing dilution of spirulina MeOH extract according to the invention.

[0084] FIG. 6.2 represents the PAL activity in leaves of 10 days old tomato plants from seedling pre-treated by roots dipping with Spirulina poly-anionic saccharides extract according to the invention, chitosan or a combination of both.

[0085] FIG. 7 represents the protein and PAL activity in tomato leaves treated with spirulina poly-anionic saccharide extract alone or in combination with oligoglucan and chitooligosaccharides.

[0086] FIG. 8 represents the protein content and PAL relative activity obtained with increased concentrations of the added Spirulina poly-anionic saccharides fraction of the invention.

[0087] FIG. 9 represents the experimental steps applied in the examples

[0088] FIG. 10 represents the protein content and PAL relative activity according to increased concentration of added Spirulina poly-anionic saccharides fraction of the invention.

[0089] FIG. 11 represents the metabolite GC-MS analysis of the different hydrolysate fragments of the Spirulina poly-anionic saccharides fraction of the invention.

[0090] FIGS. 12A and 12B represents anion-exchange chromatographic profile of the hydrolysate fragments of the invention.

[0091] FIG. 13 represents the PAL relative activity obtained after addition of the poly-anionic saccharide extract and the fragments hydrolysate according to the invention.

[0092] FIG. 14 represents the production step of the different Spirulina ETOH fractions according to the invention.

[0093] FIGS. 15A to 15C represent the bioactive molecules profile of the obtained ETOH fractions of the invention.

[0094] FIG. 16 represent the protein content and PAL relative activity after extraction with increased Ethanol percentages.

DETAILED DESCRIPTION OF THE INVENTION

[0095] Two main fractions present in the composition according to the invention are obtained from a blue-green algae product, being Spirulina genus biomass: An alcohol, preferably a methanol soluble fraction and a (poly) saccharides fraction. This biomass being a cells pellet obtained after filtration of a Spirulina culture associated or not, to a drying step, such as lyophilisation or an air drying step. The measure of a plant biomass, which is basically the density or amount of plant-life, is also known to be directly related to crop yield and can be used to measure this crop yield.

[0096] In the Spirulina methanol (MEOH) and ethanol (ETOH) soluble fraction, fatty acids, preferably mostly the C16, C18 fatty acids, beta-carotenes and low molecular weight molecules are identified and show antimicrobial activity.

[0097] The (poly-anionic) saccharides are natural products (different from a synthetic polymer or oligomer), advantageously extracted from Spirulina genus biomass. They are acidic (poly-anionic) saccharides, preferably containing rhamnose, uronic acid and one or more sulphated groups and preferably of low viscosity in solution. As shown in FIG. 1.2, in these saccharides, rhamnose saccharide is identified as the major sugar residue, preferably representing more than 50%, preferably more than 55% by weight, more specifically between 55% and 60% by weight of the total (being 100% by weight) of the saccharides residues. Therefore, both fractions are obtainable from natural renewable source with little or no modifications.

[0098] Both fractions extracted from Spirulina genus, methanol-soluble and (poly) saccharides fractions, induce plant metabolic changes, in particular protein content variations, Phenylalanine Amnonia Lyase (PAL) activity increasing, proline accumulation and changes in the metabolites, advantageously triggering the induction of important plant defence reactions. The measure of the PAL activity, like the measure of the glucanase activity or of the chitinase activity is used to measure an elicitor effect of compounds applied upon plants and their capacity to trigger plant defence mechanisms against pathogen infections and aggression. In this way, an increase of the protein content of tomato leaves was observed 24 hours after foliar spraying with a solution of this Spirulina polysaccharides extracts.

[0099] Diverse application forms, in particular seed coating, soil amended, and plant roots dipping of these bioactive molecules and compositions comprising them were assessed in distinct plants species. However, treatment of whole plants, seeds, leaves, flowers, fruits and roots could be obtained by spraying, drenching, soaking, dipping, injection and via fertigation systems and means of the composition of the invention in dry form or as dilution of a solution comprising this composition. In divers crops used as plant models, the dose-response curves for both, methanol soluble and (poly) saccharides were established.

[0100] In divers experiments, chitosan saccharide, a well-known plant elicitor, has been used as positive control for comparative purpose. The inventors have discovered that another known elicitor, such as the chitosan saccharide and the Spirulina (poly) saccharides extract according to the invention can advantageously be used in combination (possibly through a stabilization of the applied polysaccharides) preferably under a suitable formulation, to enhance, in synergy, the characteristics of the applied (poly) saccharides according to the invention and extracted from Spirulina or to reduce the known side effects (toxicity) of the chitosan saccharides. Preferably, both compounds are used in synergy for improving plant biological activity, especially to increase plant growth or protein content in a plant as well as in other fields, including medical, pharmaceutical, neutraceutic, cosmetic, food, feed, beverage, industrial, including in textile and paper production, and/or environmental applications.

[0101] Preferably, the chitosan saccharide is either an oligosaccharide or a polysaccharide having a degree of acetylation lower than 50% and a degree of polymerization higher than 5 (five).

Example 1: Extraction and Characterization of Bioactive Molecules from Spirulina Genus Biomass

[0102] 1.1Extraction in Alcohol Solution

Commercial dried Spirulina genus biomass as described in ISBN 2-85744-853-X. (Ripley D. Fox. 1996. Editions Edisud, La Calade, R.N.7, 13090 Aix-en-Provence, France) and by Robert Henrikson (Apr. 20, 2011) AlgaelndustryMagazine.com) was subjected to lipid extraction as reported by Chaiklahan et al (Lipid and fatty acids extraction from the cyanobacterium Spirulina. Science Asia 34, 299. (2008)). Extraction was carried out in five time (20 minutes/each one) with a biomass-methanol ratio of 1:10 (w/v) at 25 C. After centrifugation at 4800g for 10 minutes, the supernatant (Methanol soluble) was concentrate by rotatory evaporation. A similar extraction was also obtained with addition of different alcohol percentages, especially with different ethanol (ETOH) percentages (20% ETOH, 30% ETOH and 40% ETOH), as described in FIG. 15.

[0103] 1.2Extraction and Selective Precipitation of (Poly) Saccharides

Commercial dried Spirulina genus biomass 100 g, were added to 800 mL of water. The extraction process was performed in a single stage at 100 C. for 2 hours under continuous mixing at 120 rpm using a stirrer. After centrifugation at 4800g for 10 minutes, the supernatant containing (poly) saccharides according to the invention was obtained.

[0104] (Poly) saccharides insoluble in CetylTrimethylAmmonium Bromide (CTAB): the (poly) saccharides according to the invention were precipitated with 1% (final concentration) CTAB solution. The precipitate was collected by centrifugation (10,000g, 10 minutes) and washed stepwise with saturated sodium acetate in 95% ethanol, and absolute ethanol, respectively.

[0105] (Poly) saccharides insoluble in Ethanol: the (poly) saccharides were precipitated with 80% ethanol solution, which is the final concentration. The precipitate was collected by centrifugation (10,000g, 10 minutes) and washed with absolute ethanol.

[0106] 1.3Characterization Methanol Soluble Fraction

[0107] 1.3.1GC-MS Analysis

[0108] GC-MS chromatogram of the MeOH soluble fraction obtained from Spirulina. GLC-MS analysis were carried out as follow: Dried samples were silylated by adding 10 l of pyridine and 50 l of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) and heating for 30 minutes at 60 C. in sealed vials. The trimethylsilylated derivatives were separated by Gas Chromatography (GCMS-QP2010, Shimadzu) using a 0.25 mm30 m optima 5 MS capillary column (0.25 m film thickness) (Macherey Nagel, Germany) and identified by their electron impact (70 eV) mass data. He (0.75 ml min-1) was used as carrier gas. GLC conditions were as follows: initial column temperature 100 C., held for 6 minutes, ramped at 30 C. min-1 to 320 C. and held for 8 minutes; injector temperature 310 C., split ratio 20, 3.

[0109] 1.3.2XAD-Fractionation and Identification by GC-MS

A solution (2 mL) of methanol soluble from Spirulina biomass was applied to a XAD-column (25 mm200 mm, BIO-RAD). The column was then eluted by using step elution with water, ethanol different concentrations and acetone at flow rate 2 mL/min. FIG. 1.1 A represents the GC-MS chromatograms of the different XAD fractions and shows that the alcohol extract comprises C16 and C18 fatty acids.

[0110] 1.3.3MS Analysis

MALDI-MS mass spectra were recorded using a Waters QToF Premier mass spectrometer equipped with a nitrogen laser, operating at 337 nm with a maximum output of 500 J.m-2 delivered to the sample in 4 nano-second pulses at 20 Hz repeating rate. Time-of-flight mass analyses were performed in the reflectron mode at a resolution of about 10,000. The samples were analyzed using dihydroxybenzoic acid/Dimethylaniline ionic liquid (DHB/DMA), that matrix was prepared as 25 mg in 250 L acetonitrile:water (1:1) plus 5 L of DMA. The matrix solution (1 L) was applied to a stainless steel target and air dried. 1 L aliquots of those samples were applied onto the target area already bearing the matrix crystals, and air-dried before adding 1 L of Nal solution (2 mg/mL, acetonitrile:water). For the recording of the single-stage MS spectra, the quadrupole was set to pass all the ions of the distribution, and they were transmitted into the pusher region of the time-of-flight analyzer where they were mass-analyzed with 1 s integration time. Data were acquired in continuum mode until acceptable averaged data were obtained. FIG. 1.1B and FIG. 1.1C represent respectfully the MS spectra of the XAD fraction in ethanol and in acetone.

[0111] 1.4Characterization of (Poly) Saccharides

[0112] 1.4.1FT-IR Analysis

FT-IR analyses of spiruline polysaccharides was carried out by the potassium bromide (KBr) pellet method with a Perkin-Elmer Spectrum One FT-IR spectrometer (Norwalk, USA) in the range 400-4000 cm-1. FIG. 1.2 represent the FT-IR-Analysis spectra of the saccharides of the composition according to the invention.

[0113] 1.4.2TGA Analysis

Thermogravimetric analysis (Thermo-gravimetric analyser TGAQ500, from TA instruments) was used to examine the thermal stability against temperature of (poly) saccharides. Analyses were performed using around 20 mg sample in a nitrogen gas atmosphere. After a first step at 110 C. to evaporate the residual water, the (poly) saccharides were equilibrated at 40 C. Samples were finally heated from 40 C. to 600 C. at a heating rate of 3 C.min-1. These low heating rate was applied to reach a good resolution about the derivative weight percent. FIG. 1.3 represents the thermogravimetric analysis spectra of the saccharides composition according to the invention.

[0114] 1.4.3Viscosity

CTAB insoluble (poly) saccharide was dissolved in distilled water and solutions at different concentrations were prepared by diluting in water. In FIG. 1.4, the relative viscosity of the (poly) saccharide solutions in function of the final concentration was determined using a microviscometer Rheosense (http://www.rheosense.com/products/viscometers/microvisc/overview).

[0115] 1.4.4Composition Analysis

Monosaccharide composition was determined by hydrolysis of the (poly) saccharide samples (50 mg) with H2SO4 1 M at 100 C. for 3 hours. The hydrolyzed samples were converted to alditol acetates by successive NaBH4 reduction and acetylation with anhydride acetic in presence of 1-methylimidazole following the slightly modified method described by Blakeney et al, 1983 (A simple and rapid preparation of alditol acetates for monosaccharide analysis. Carbohyd. Res. 113, 291-299 (1983)).

[0116] The resulting alditol acetates were analyzed by gas chromatography-flame ionization detection (GC-FID) using a Shimadzu model GC-2010A gas chromatograph, using a DB-225 capillary column (30 m0.25 mm i.d.), with helium as carrier gas. The analysis was carried out from 40 to 220 C. at 20 C. min-1, maintaining the temperature constant to the end of analysis (30 min). The monosaccharides (FIG. 1.2) were identified by their typical retention times.

[0117] The total sugars content of the spirulina polysaccharides is 44.1% as determined by the phenol-sulfuric and 6.5% in uronic acid content as determined according to the method of m-hydroxydiphenyl using glucuronic acid as the standard (Filisetti-Cozzi and Carpita, (Measurement of uronic acids without interference from neutral sugars. Analytical Biochemistry, 197, 157-162(1991)). The sulfate content is 11.7% as determined by the method of Jaques et al. 1968 (Jaques, L., Ballieux, R., Dietrich, C. & Kavanagh, L., 1968. A microelectrophoresis method for heparin. Canadian Journal of Physiology and Pharmacology, Issue 46, pp. 351-360).

[0118] 1.4.5Anion Exchange Chromatography

5 mg of (poly) saccharide of the invention was dissolved in 1 mL sodium acetate 0.02M at pH 5.5 and then applied to a Macro-Prep DEAE-column (10 mm50 mm, BIO-RAD). The column was first eluted by sodium acetate 0.02 M at pH 5.5 and then by a step gradient of sodium acetate 0.02 M containing NaCl at pH 5.5 at flow rate 2 mL/min. 2 mL fractions as represented in FIG. 1.5 were collected and assayed by the phenol-sulfuric method for neutral hexoses according to Dubois et al (Colorimetric Method for Determination of Sugars and Related Substances. Anal Chem 28, 350-356 (1956)) and uronic acids content estimated as described before.

Example 2: Induction of Plant Defense in Plant Cells Cultured In Vitro

[0119] The following Examples 2 to 7 present biological activity results obtained upon the induced plants and the Table 3 shows a summary of the biological trials.

Cell culture: Suspension-cultured cells of Arabidopsis thaliana strain L-MM1 ecotype Landsberg erecta were grown in Murashige and Skoog (Physiol. Plant, 15, 473-497 (1962)) medium (4.43 g/L) with sucrose (30 g/L) and 0.5 g/mL of N-AcetylAsparatate (NAA) and 0.05 g/mL of Kinetin, pH 5.7. Cultures were maintained under dark, at 24 C., on a rotary shaker at 100 rpm. Cells were diluted 10-fold in fresh medium every 7 days.

[0120] Bioassays: The (poly) saccharides were dissolved in distilled water, filtered through a 0.22 m membrane filter (Millipore) and aseptically added to 5 mL of 7 days-old suspension-cultured cells and incubated 24 hours at 24 C. under mild agitation. The reaction mixture was centrifuged for 5 minutes at 100 g and 4 C. to collect the cells (PAL activity measurement).

[0121] PAL activity: Cells were homogenized at 4 C. in 1 ml of 0.1 M borate buffer (at pH of 8.8) containing 2 mM mercaptoethanol. The homogenate was centrifuged at 4000 rpm for 10 minutes at 4 C. PhenylAlanine Ammonia lyase (PAL:EC 4.3.1.5) activity was determined in 0.125 ml supernatant in the presence of 1.37 ml 0.1 M borate buffer (at pH of 8.8) supplemented with 60 mM L-phenylalanine as described by Beaudoin-Eagan and Thorpe (Tyrosine and phenylalanine ammonia lyase activities during shoot initiation in tobacco callus cultures. Plant Physiology, 78(3), 438-441 (1985)). Protein concentration of the extracts was determined by the Bradford protein assay (Bio-Rad). FIG. 2 represent the protein content and PAL activity in Arabidopsis thaliana cell suspension, 24 hours after treatments with increasing concentrations of Spirulina saccharides.

Example 3: Foliar Spraying of the Composition of the Invention on Tomato Plants

[0122] Tomato plants of the variety Moneymaker were cultivated in soil under controlled conditions with a light/dark regime of 16 hours/8 hours respectively, at 24 C., during 18 days before being sprayed with solutions containing the Spirulina (poly) saccharides. As a control, water containing about 0.01% Tween 20 was sprayed on the leaves. After 24 hours, the true leaves from plants treated by spraying were collected and ground in liquid nitrogen. Powdered leaves were extracted in 50 mM sodium acetate buffer at pH 5.2 containing about 5 mM EDTA, about 14 mM of beta-mercapto-ethanol and about 1.0 M NaCl to the rate of about 1 g of powdered leaves per 2 ml of buffer. FIG. 3 shows that foliar spraying of the composition according to the invention increases protein production in the treated plant.

Example 4: Coating Wheat Seeds with (Poly) Saccharides of the Invention

[0123] Seeds of wheat plant of the variety Olivier were coated with a solution containing increase concentration of Spirulina (poly) saccharides, and water (as control). After drying, 10 seeds of wheat per pots (3 pots per treatment) were planted and wheat plants growth for 2 weeks on soil under controlled conditions (light/dark regime of 16 hours/8 hours respectively, at 24 C.). FIG. 4 present the protein content and PAL activity in leaves of 10 days old wheat plants from seeds coated with increasing concentrations of Spirulina saccharides according to the invention.

Example 5: Soil Amended with a Solution of (Poly) Saccharides of the Invention

[0124] A solution of Spirulina (poly) saccharides according to the invention as well as the MEOH soluble extract according to the invention were added at the soil (ratio of solution to soil (1:1.2, v/w) before being added to planter pots. Wheat plants (variety Olivier) were cultivated in pre-treated soil under controlled conditions with a light/dark regime of 16 hours/8 hours respectively, at 24 C., during 15 days; 10 plants per repetition, 3 repetitions per treatment. True leaves from plants were collected and ground in liquid nitrogen. Powdered leaves were extracted in 50 mM sodium acetate buffer at pH 5.2 containing about 5 mM EDTA, about 14 mM .beta.-mercapto-ethanol and about 1.0 M NaCl to the rate of about 1 g of powdered leaves per 2 ml of buffer. FIG. 5 present protein content and PAL activity in leaves of 10 days old wheat plants growing on a soil pre-treated with a solution of Spirulina saccharides or methanol (MeOH) soluble extract.

Example 6: Plant Roots Dipping

[0125] One week old seedlings of tomato (Lycopersicum esculentum), wheat and cucumber were carefully removed from the substrate and the roots immersed in a solution containing an increasing concentration of the Spirulina (poly) saccharide according to the invention, a solution of 5 mg/mL of chitosan saccharide or water being the Control for hours. Then, the seedlings were planted and cultivated during 10 days in soil at 25 C. in a 16 hours day light/8 hours dark regime. The true leaves from plants treated were collected and ground in liquid nitrogen. Powdered leaves were extracted in 50 mM sodium acetate buffer at pH 5.2 containing about 5 mM EDTA, about 14 mM beta mercapto-ethanol and about 1.0 M NaCl to the rate of about 1 g of powdered leaves per 2 ml of buffer. FIG. 6.1 represents protein content and PAL activity in leaves of tomato, wheat and cucumber plants from seedling pretreated with increasing concentrations of the saccharides and increasing dilution of methanol (MeOH) extract according to the invention. FIG. 6.2 represents PAL activity in leaves of 10 days old tomato plants from seedling pre-treated by roots dipping with Spirulina saccharides, chitosan or a combination of both.

Example 7: Induction of Proline Accumulation by Plant Roots Dipping

[0126] One week old seedlings of tomato plant (Lycopersicum esculentum) were carefully removed from the substrate and the roots immersed in a solution containing an increasing concentration of the Spirulina (poly) saccharide according to the invention, and water (Control) for hours. The seedlings were planted and cultivated during 7 days in soil at 25 C. in a 16 hours day light/8 hours dark regime. The true leaves from plants treated were collected and ground in liquid nitrogen. The amino acid Proline (Pro) content was determined in tomato leaves according to Bates et al. (Rapid determination of free proline for water-stress studies. Plant and Soil 39, 205-207 (1973)) based on Proline's reaction with ninhydrin. The proline concentration was determined from a standard curve using proline (sigma) and expressed as relative values respect to the content in control treatment as R/R control.

[0127] Table 7 shows the amount of proline in tomato leaves a week after tomato roots dipping in different solution of Spirulina (poly) saccharides. Values of the mean+/SE are reported (n=3). These results demonstrated the ability of Spirulina (poly) saccharides of the invention to induce advantageously the accumulation of proline in plants.

TABLE-US-00001 TABLE 7 Induction of proline accumulation proline in tomato leaves a week after tomato roots dipping in different solution of Spirulina (poly) saccharides Concentration of Proline content spirulina polysaccharides (R/R control) 1.25 mg/mL 1.13 0.02 5.00 mg/mL 1.22 0.05 10.00 mg/mL 1.44 0.05

Example 8. Effects of Spirulina Polysaccharides in Combination with Other Elicitor Molecules Foliar Spraying on Tomato Plants

[0128] Tomato plants of the variety moneymaker were cultivated in soil under controlled conditions with a light/dark regime of 16 h/8 h respectively, at 24 C., during 18 days before being sprayed with solutions containing the spirulina polysaccharides (5 mg/mL), chitooligosaccharides (COS) (0.1 mg/mL), oligoglucan (0.1 mg/mL) or combination spirulina polysaccharides (5 mg/mL) and chitooligosaccharides (COS) (0.1 mg/mL) and spirulina polysaccharides (5 mg/mL) and oligoglucan (0.1 mg/mL). As a control, water was sprayed on the leaves.

[0129] After 24 hours, the true leaves from plants treated by spraying were collected and ground in liquid nitrogen. Powdered leaves were extracted in 50 mM sodium acetate buffer pH 5.2 containing about 5 mM EDTA, about 14 mM beta-mercapto-ethanol and about 1.0 M NaCl to the rate of about 1 g of powdered leaves per 2 ml of buffer. Protein and PAL activity (FIG. 7) were determined as explained in previous examples.

Example 9: Spirulan Polysaccharides Induce Plant Defense in Arabidopsis thaliana Cell Suspension

[0130] Spirulina polysaccharides fraction according to the invention was prepared as explained in example 1.

Cell Culture:

[0131] Suspension-cultured cells of Arabidopsis thaliana strain L-MM1 ecotype Landsberg erecta were grown in Murashige T & Skoog F. (A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-97, 1962)4.43 g/L) with sucrose (30 g/L) and 0.5 g/mL of NAA and 0.05 g/mL of Kinetin, pH 5.7. Cultures were maintained under dark, at 24 C., on a rotary shaker at 100 rpm. Cells were diluted 10-fold in fresh medium every 7 days.

Bioassays:

[0132] The spirulina polysaccharides were dissolved in distilled water, filtered through a 0.22 m membrane filter (Millipore) and aseptically added to 5 mL of 7 days-old suspension-cultured cells and incubated 24 hours at 24 C. under mild agitation. The reaction mixture was centrifuged for 5 min at 100 g and 4 C. to collect the cells (PAL activity measurement).
PAL activity:
Cells were homogenized at 4 C. in 1 ml of 0.1 M borate buffer (pH 8.8) containing 2 mM mercaptoethanol. The homogenate was centrifuged at 4000 rpm for 10 minutes at 4 C. PAL (EC 4.3.1.5) activity was determined in 0.125 ml supernatant in the presence of 1.37 ml 0.1 M borate buffer (pH 8.8) supplemented with 60 mM L-phenylalanine as described by Beaudoin-Eagan L D, Thorpe T A. (Tyrosine and Phenylalanine Ammonia Lyase Activities during Shoot Initiation in Tobacco Callus Cultures. Plant Physiol. 1985 July; 78(3):438-41). Protein concentration of the extracts was determined by the Bradford protein assay (Bio-Rad).

Example 10: Induction of Plant Defense and Changes in Plant Metabolites Profiles after Foliar Spraying of Spirulina Polysaccharides

[0133] The inventors have conducted an experiment according to the method described here above and illustrated in the FIG. 9. The inventors have observed that changes in protein content and PAL activity are depending on spirulina polysaccharide concentration in the sprayed solution on tomato plants (see FIGS. 8 and 10)

This example 10 shows changes in different plant metabolites after foliar spraying of Spirulina polysaccharides. This means that Spirulina polysaccharide induce plant defense and also, changes in other plant metabolic pathways

Example 11: Enzymatic Treatment of Spirulan Polysaccharides Enhances its Biological Activity

[0134] Preparation of low molecular weight Spirulan fractions obtained by enzymatic hydrolysis depends on the enzyme screening and selection, on the hydrolysis conditions, in particular the substrate/enzyme used, pH, temperature, hydrolysis time and on the fractionation of the enzymatic hydrolysis obtained products needed. Preferred conditions are the following: An efficient hydrolysis is obtained by addition of one or more hydrolysase enzymes, preferably selected from the group consisting of xylanases, cellulases or pectinases, active at suitable pH values, preferably between (about) 2 and (about) 5 or between (about) 3 and (about) 4, preferably at a pH of 3.5 and preferably at room temperature, preferably between (about) 20 C. and (about) 40 C., preferably at about 37 C. and for an adequate period, preferably after at least 10 hours hydrolysis time, but less than 2 or 3 days, preferably less than 1 day, to recover oligosaccharides and not monomers or dimers. The aim of this experiment was to compare biological activity before and after molecular weight reduction.

[0135] Spirulan at 5 g/l concentration was dissolved by overnight shaking at room temperature in 0.05 M citrate buffer pH 3.5 and 100 ml of the obtained Spirulan solution was mixed in 50 ml of a pectinase rich enzyme (Lyvelin) containing a highly active fungal endopolygalacturonase enzyme and was incubated at 37 c. for 24 hours. Spirulina polysaccharides fraction was prepared as explained in example 1 and spirulan hydrolysate was obtained by an enzyme addition to form a hydrolysis mix which is purified by ultrafiltration (UF) step to remove the added enzyme and form the so called Spirulan hydrolysate of the invention.

Cell Culture:

[0136] Suspension-cultured cells of Arabidopsis thaliana strain L-MM1 ecotype Landsberg erecta were grown in Murashige and Skoog medium (4.43 g/L) with sucrose (30 g/L) and 0.5 g/mL of NAA and 0.05 g/mL of Kinetin, pH 5.7. Cultures were maintained under dark, at 24 C., on a rotary shaker at 100 rpm. Cells were diluted 10-fold in fresh medium every 7 days.

Bioassays:

[0137] The spirulina polysaccharides were dissolved in distilled water, filtered through a 0.22 m membrane filter (Millipore) and aseptically added to 5 mL of 7 days-old suspension-cultured cells and incubated 24 hours at 24 C. under mild agitation. The reaction mixture was centrifuged for 5 min at 100 g and 4 C. to collect the cells (PAL activity measurement).

Pal Activity:

[0138] Cells were homogenized at 4 C. in 1 ml of 0.1 M borate buffer (pH 8.8) containing 2 mM mercaptoethanol. The homogenate was centrifuged at 4000 rpm for 10 minutes at 4 C. PAL (EC 4.3.1.5) activity was determined in 0.125 ml supernatant in the presence of 1.37 ml 0.1 M borate buffer (pH 8.8) supplemented with 60 mM L-phenylalanine as described by Beaudoin-Eagan and Thorpe (1985). Protein concentration of the extracts was determined by the Bradford protein assay (Bio-Rad).

[0139] The anion-exchange chromatographic profile of the obtained low molecular weight spirulan (or obtained Spirulan hydrolysate) is presented in FIG. 13. According with this bioassay made upon Arabidopsis thaliana cell suspension and as shown in FIG. 13, reduction of spirulan MW (in the form of the spirulan hydrolysate) enhanced his biological activity in plants.

Example 12: Characterization of ETOH Extracts from Spirulina

[0140] Here, we provide additional information on the general composition of various ETOH extracts from spirulina biomass and on the composition of some specific compounds (fatty acids, sugars, organic acids) in these ETOH extracts obtained from spirulina biomass. These are GC-MS analysis after TMS derivatization The following table 8 presents proteins, polyphenols and polysaccharides content of each ETOH fraction.

TABLE-US-00002 TABLE 8 Component 20% ETOH 30% ETOH 40% ETOH Protein (mg/l) 850 30 760 70 780 30 Polyphenols (mg/l) 391 20 448 43 419 19 Polysaccharides (mg/l) 4200 200 3600 150 2800 150

[0141] One week old seedlings of tomato (Lycopersicum esculentum) were carefully removed from the substrate and the roots immersed in a solution containing ethanol extracts (20%, 30% and 40% ETOH extracts) from spirulina biomass or water (Control) for 0.5 hours. Then, the seedlings were planted and cultivated during 10 days in soil at 25 C. in a 16 hours daylight/8 hours dark regime. The true leaves from plants treated were collected and ground in liquid nitrogen. Powdered leaves were extracted in 50 mM sodium acetate buffer pH 5.2 containing about 5 mM EDTA, about 14 mM beta-mercapto-ethanol and about 1.0 M NaCl to the rate of about 1 g of powdered leaves per 2 ml of buffer. Applied alcohol extraction and analysis is presented in FIG. 15. The FIGS. 15A to 15C present the respective amounts of organic acids, fatty acids and saccharides present in each ETOH fraction.

Example 13: Comparison of Plant Defense Induction (PAL Activity) of Different Ethanol Extracts from Spirulina Biomass

[0142] The FIG. 16 presents the measured PAL relative response according to the different added Spirulina ETOH extracts of the invention obtained with increased alcohol %, respectively 20% ethanol (Spirulina ETOH 20%), 30% ethanol (Spirulina ETOH 30%) and 40% ethanol (Spirulina ETOH 40%).

According with these results, Spirulina ETOH 30% extract are showing slightly higher bioactivity than other Ethanol (ETOH) extracts. Similar results were obtained with Methanol (METOH) extraction.