Compositions and methods for stimulating plant growth

Abstract

Disclosed are malt sprouts fraction extracts, their process of preparation and their use as a biostimulant agent.

Claims

1. A malt sprouts fraction extract for stimulating plant growth, wherein the malt sprouts fraction extract comprises at least 0.5%, and less than 5% of dry matter by total weight of the malt sprouts fraction extract and comprising at least: total nitrogen content from 25 to 60 g/kg of dry matter; free amino nitrogen content from 3 to 10 g/kg of dry matter; nucleic acid derivatives content from 2 to 5 g/kg of dry matter; total free auxin content between 500 and 8000 ng/g of dry matter; total gibberellin content between 0.1 and 3.2 ng/mg of dry matter; and total cytokinins content between 10 and 400 ng/g of dry matter; said malt sprouts extract having been obtained from an acidified aqueous extraction of the malt sprout fraction.

2. The malt sprouts fraction extract according to claim 1, wherein the malt sprouts fraction extract is derived from a plant selected from the group consisting of barley, wheat, rye, spelt, corn, millet sorghum, oat, triticale, rice and mixtures thereof, and obtained from an acidified aqueous extraction of said plant obtained by a sieving process and comprising a content of rootlets and acrospires higher than 50%, or higher than 60%, or higher than 65% or between 65% and 85% by total weight of fractions.

3. The malt sprouts fraction extract according to claim 1, wherein the malt sprouts fraction extract is derived from a plant selected from the group consisting of barley, wheat, rye, spelt, corn, millet sorghum, oat, triticale, rice and mixtures thereof, and obtained from an acidified aqueous extraction of said plant obtained by a sieving process and comprising a content of rootlets and acrospires higher than 50%, or higher than 60%, or higher than 65% or between 65% and 85% by total weight of fractions, obtained from an aqueous extraction of said plant comprising at least: dry matter by total weight of the plant from 92% to 98%, crude protein content from 25 to 37 g/100 g of dry matter, and free amino nitrogen content from 100 to 600 mg/100 g of said plant.

4. The malt sprouts fraction extract according to claim 1, wherein the malt sprouts fraction extract is derived from a plant selected from the group consisting of barley, wheat, rye, spelt, corn, millet sorghum, oat, triticale, rice and mixtures thereof, and obtained from an acidified aqueous extraction of said plant obtained by a sieving process and comprising a content of rootlets and acrospires higher than 50%, or higher than 60%, or higher than 65% or between 65% and 85% by total weight of fractions, obtained from an aqueous extraction of said plant comprising at least: dry matter by total weight of the malt sprouts from 92% to 98%, crude protein content from 25 to 37 g/100 g of dry matter, and free amino nitrogen content from 100 to 600 mg/100 g of malt sprouts, and containing: total fibers content from 43 to 52 g/100 g of dry matter, total carbohydrates content from 7 to 12 g/100 g of dry matter, total sugars content from 4 to 6 g/100 g of dry matter, fats content below 1 g/100 g, and ash content from 5 to 7 g/100 g.

5. A malt sprouts fraction extract, wherein the malt sprouts fraction extract comprises at least 0.5%, and less than 5% of dry matter by total weight of the malt sprouts fraction extract and comprising at least: total nitrogen content from 25 to 60 g/kg of dry matter, free amino nitrogen content from 3 to 10 g/kg of dry matter, nucleic acid derivatives content from 2 to 5 g/kg of dry matter, total free auxin content between 500 and 8000 ng/g of dry matter; total gibberellin content between 0.1 and 3.2 ng/mg of dry matter; and total cytokinins content between 10 and 400 ng/g of dry matter, obtained by the method of preparation, comprising the following steps: a. diluting isolated malt sprouts fractions in water, to obtain diluted malt sprouts fractions, b. diffusing the diluted malt sprouts fractions in aqueous phase, at a temperature from 20° C. to 60° C. in the presence of an acid, or sulfuric acid, to obtain two phases, an aqueous one and a solid one, c. optionally, separation of an aqueous phase from a solid phase to obtain a juice of malt sprouts, d. optionally, addition of preservatives.

6. The malt sprouts fraction extract wherein the malt sprouts fraction extract comprises at least 0.5%, and less than 5% of dry matter by total weight of the malt sprouts fraction extract and comprising at least: total nitrogen content from 25 to 60 g/kg of dry matter, free amino nitrogen content from 3 to 10 g/kg of dry matter, nucleic acid derivatives content from 2 to 5 g/kg of dry matter, total free auxin content between 500 and 8000 ng/g of dry matter; total gibberellin content between 0.1 and 3.2 ng/mg of dry matter; and total cytokinins content between 10 and 400 ng/g of dry matter, obtained by the method of preparation recited in claim 5, comprising the following steps: a. diluting isolated malt sprouts fractions in acidified water with a ratio malt sprouts fractions/water ranging from 1/15 to 1/4, or 1/10 with a final pH lower than 4, to obtain diluted malt sprouts fractions, b. diffusing the diluted malt sprouts fractions in acidified water between 1 and 5 h, or between 1 and 2 h, at a temperature from 20° C. to 60° C., c. Optionally, separation of an aqueous phase from a solid phase in order to get rid of the insoluble coarse compounds by using a centrifuge and/or a filter plate and/or a filter press and/or a press and/or a filter-candle, to obtain a juice of malt sprouts, d. Optionally, addition of preservatives.

7. A method for stimulating or promoting plant growth and/or for improving the natural plant defense, or for treating seeds or plants, comprising the application on plants of a malt sprouts fraction extract, said malt sprouts fraction extract having been obtained from an acidified aqueous extraction of malt sprout fraction or a juice of malt sprouts, said juice of malt sprouts comprising acidified water and malt sprouts fraction extracts, comprising rootlets and acrospires in a ratio malt sprout fractions/water ranging from 1/4 to 1/15, or from 1/8 to 1/10, said malt sprout extract comprising at least 0.5%, and less than 5% of dry matter by total weight of the malt sprouts fraction extract and comprising at least: total nitrogen content from 25 to 60 g/kg of dry matter, free amino nitrogen content from 3 to 10 g/kg of dry matter, nucleic acid derivatives content from 2 to 5 g/kg of dry matter, total free auxin content between 500 and 8000 ng/g of dry matter; total gibberellin content between 0.1 and 3.2 ng/mg of dry matter; and total cytokinins content between 10 and 400 ng/g of dry matter.

8. The method according to claim 7, wherein the malt sprouts fraction extract is obtained by a method of preparation comprising the following steps: a. isolating malt sprouts fractions, b. diluting the fractions in water, c. diffusing the fractions in aqueous phase, at a temperature from 20° C. to 60° C. in the presence of an acid, or sulfuric acid, d. separating an aqueous phase from a solid phase, and e. Optionally, addition of preservatives, or sorbate and benzoate.

9. The method according to claim 7, wherein the malt sprouts fraction extract is obtained by a method of preparation comprising the following steps: a. isolating malt sprouts fractions, b. diluting the fractions in acidified water with a ratio malt sprouts fractions/water ranging from 1/15 to 1/7, with a final pH lower than 4, c. diffusing the fractions in acidified water between 1 and 5 h, or between 1 and 2 h, or at a temperature from 20° C. to 60° C., d. separating an aqueous phase from a solid phase in order to get rid of the insoluble coarse compounds by using a centrifuge and/or a filter plate and/or a filter press and/or a press and/or a filter-candle, e. Optionally, addition of preservatives, or sorbate and benzoate.

10. The method according to claim 7, wherein the juice of malt sprouts is further diluted in acidified water with dilution ratios juice of malt sprouts/water ranging from 1/10 to 1/500.

11. The method according to claim 7, in combination with an additional plant growth stimulant and/or a plant defense stimulant.

12. The method according to claim 7, wherein the malt sprouts fraction extract or the juice of malt sprouts confers an improved nutrient absorption, confers an improved crop quality, improves resistance to biotic and/or abiotic stresses, improves nitrogen uptake by plants, enhances plant height, leaf area, biomass, plant vigor, or combination thereof.

13. The method according to claim 7, comprising the application on said plants by spraying, of a composition comprising the malt sprouts fraction extract, wherein the malt sprouts fraction extract comprises at least 0.5%, and less than 5% of dry matter by total weight of the malt sprouts fraction extract and comprising at least: total nitrogen content from 25 to 60 g/kg of dry matter, free amino nitrogen content from 3 to 10 g/kg of dry matter, nucleic acid derivatives content from 2 to 5 g/kg of dry matter, total free auxin content between 500 and 8000 ng/g of dry matter; total gibberellin content between 0.1 and 3.2 ng/mg of dry matter; and total cytokinins content between 10 and 400 ng/g of dry matter, in an amount in a range from 1 gram to 1000 grams per hectare of dry matter contained in the malt sprouts fraction extract.

14. The method according to claim 7, wherein the plant is chosen from the group consisted of commonly cultivated member of the kingdom Plantae, or in the group comprising the Plant SubKingdom of Spermatophytes, or the Phylum of Angiosperms, or in group consisted of all families constituting the Phyllum of Angiosperms, or in the group selected from the: Dicots: Arabidopsis, cucumber, rapeseed, sunflower, soya, bean, alfalfa, tomato vine and Monocots: Corn, wheat, barley, leek.

15. The method according to claim 7, wherein the application on plants is carried out pre-emergence, post-emergence or both.

Description

FIGURES

(1) FIG. 1: Graph representation of the effect of different products (juices of malt sprouts and positive control) on wheat plants growth by measuring fresh matter of wheat plants.

(2) FIG. 2: Graph representation of the effect of different products (juices of malt sprouts and positive control) on wheat plants growth by measuring dry matter of wheat plants.

(3) FIG. 3: Graph representation of the effect of different products (juices of malt sprouts and positive control) on wheat plants growth by measuring chlorophylls contents of wheat.

(4) FIG. 4; Schematic illustration of malt sprouts, with (1) representing an acrospire (=germ), (2) representing rootlets, (3) representing the husk (=hull), (4) representing a malted barley grain. A malt sprout is constituted of a mix of (1)+(2)+(3)+malt dust.

(5) The following examples illustrate the invention without limiting the scope thereof. Unless otherwise stated, the percentages are expressed by weight. The ratio are expressed in weight ratio.

EXAMPLES

Example 1: Preparation of the Juice of Malt Sprouts (Malt Sprouts—Extracted Juice)

(6) The mix of malt sprout fractions used to produce the juice is characterized below:

(7) TABLE-US-00003 TABLE 1 Physical characterization of malt fractions used for the examples 1 and 2. Malt sprout fractions (MF) - Physical characterization From crop 2014 Fractions composition (in %) Husk + whole grains 10 Acrospires 12 Rootlets 60 Dust 18

(8) TABLE-US-00004 TABLE 2 Physico-chemical analysis of malt fractions used for the examples 1 and 2 Physico-chemical analysis of Malt sprout Fractions (MF) Malt fractions blend Units Values Moisture % 5.4 Total nitrogen g/100 g 4.08 Total protein 25.5 Total fibers 53.13 From which insoluble fibers 50.77 From which soluble fibers 2.35 Starch 4

(9) During this first experimental step, various extraction processes were tested in order to determine the optimum extraction process.

(10) Different extraction procedures were tested with the malt fractions as described above:

(11) TABLE-US-00005 TABLE 3 Description of the different extraction procedures tested Further Stabilization of the Dilution Process used to produce the juice juice applied JMF dil. 10 >MF + tap water (ratio 1:10) Addition of 1/10 JMF dil. 100 >Diffusion during 1 h at 20° C. preservatives: 1/100 JMF dil. 200 >No pH adjustment - native pH = 6 potassium sorbate 1/200 Acidified JMF dil. 10 >MF + acidified water (ration 1:10) (3 g/liter of juice) 1/10 Acidified JMF dil. 100 >Diffusion during 1 h at 20° C. and or methyl 1/100 Acidified JMF dil. 200 benzoate (5 g/liter of 1/200 juice) JMF = Juice Malt Fraction (Juice of malt sprout) MF = Malt Fraction

(12) Witnesses: Negative control: without product (referenced in the figures as ‘untreated control’) Positive control: commercial natural biostimulant

(13) The process for extracting the juice of malt sprouts (also named juice of malt fraction JMF) according

(14) TABLE-US-00006 Further Stabilization of the Dilution Process used to produce the juice juice applied JMF dil.10 MF + tap water (ratio 1:10) Addition of 1/10  JMF dil.100 Diffusion during 1 h at 20° C. preservatives: 1/100 JMF dil.200 No pH adjustment - native pH = 6 potassium sorbate 1/200 Acidified JMF dil.10 MF + acidified water (ration 1:10) (3 g/liter of juice) 1/10  Acidified JMF dil.100 Diffusion during 1 h at 20° C. and or methyl 1/100 Acidified JMF dil.200 benzoate (5 g/liter of 1/200 juice)
to these conditions comprises the following steps: a) Isolation of malt sprouts fractions described in the above Tables 1 and 2; b) Dilution of theses fractions in aqueous solvent according to the different variable conditions, with a ratio malt fractions (MF)/water of 1/10, (detailed in Table 3). c) Diffusion in acidified water, or in tap water during 1 h, at temperature 20° C. d) Separation (filtration) of the aqueous phase from the solid phase by using a Buchner filtration (100 μm). e) Conservation by adding following preservatives potassium sorbate (3 g/liter) and methyl benzoate (5 g/liter) to keep the extract in time (6 months), f) Additional Dilution of the juice of malt sprouts from 1/10 to 1/200.

Example 2: Evaluation of the Malt Extracts on Plant Growth

(15) The different products obtained in example 1 were then tested on wheat seedlings to evaluate their performance as biostimulants.

(16) They are applied on wheat plants and evaluated according to the following conditions: Dose for each application: 250 ml Application method: root 1st application: 15 days after sowing 5 treatments (every 2 days for 8 days) Biological evaluation: the inventors observed general appearance of the wheat plant, fresh and dry biomass (FIGS. 1 and 2), and content of chlorophylls (FIG. 3).

(17) As shown in the FIGS. 1 to 3, it may be noted from the various parameters of biological evaluation that the most favorable conditions are found following application of the juice of malt sprouts obtained by extraction with acidified water and an additional dilution of the juice of malt sprouts at 1/100 or 1/200.

(18) This product appears to have a higher positive effect than the commercial natural reference.

(19) The process for extracting the malt fractions and preparation of the juice of malt sprout that has been identified as the most effective for this application is the solid/liquid extraction by acidified water comprising the following steps: a) Isolation of malt sprouts fractions enriched in anyone of rootlets, acrospires, hulk, dust and mixtures thereof, at the end of the malt drying step; b) Dilution of theses fractions in acidified water with a ratio malt fractions/water ranging from 1/15 to 1/7, preferably 1/10 with a final pH of the expected juice <2, the acid used being sulfuric acid, c) Diffusion in acidified water between 1 and 5 h, and more particularly between 1 and 2 h at temperature 20° C. d) Separation (filtration) of the aqueous phase from the solid phase in order to get rid of the insoluble coarse compounds by using a centrifuge and/or a filter plate and/or a filter press and/or a press and/or a filter-candle, e) Conservation by adding preservatives such as potassium sorbate or methyl benzoate to keep the extract in time (6 months), f) Preferably dilution of the juice malt sprouts, preferably in water, with dilution ratio juice of malt sprouts/water ranging from 1/10 to 1/500, and more particularly from 1/10 to 1/200, the ratios 1/100 and 1/200 being the preferred ones.

(20) These results show the advantage of using juices of malt sprouts-, preferably diluted, as a biostimulant agent or for preparing a biostimulant agent, for the plant nutrition, plant growth, natural plant defense and stimulation of plant seedlings.

Example 3: Malt Sprout Fraction Extract Used as Biostimulant to Activate Plant Seed Growth

(21) 1. Malt Sprout Fraction Characterization

(22) The malt sprout fraction used to prepare the acidified malt sprout fraction extract is characterized in Table 4 below:

(23) TABLE-US-00007 TABLE 4 Physico chemical characterization of malt sprout fraction used in examples 3 and 4. Physico-chemical analysis of Malt sprout Fractions (MSF) Batch no1611 Units Values Dry matter % 93.1 Total nitrogen % dm 5.7 Total protein (N*6, 25) 35.4

(24) 2. Malt Sprout Fraction Extract Preparation

(25) Process used to prepare the acidified malt sprout fraction extract as described below: a) Isolation of malt sprouts fraction described in the above Table 4; b) Dilution of the malt sprout fraction in acidified water with a ratio malt sprout fractions (MSF)/water of 1/10. The acidified water is composed of water and sulfuric acid and has a pH=2. c) Diffusion in acidified water during 1 h, at 20° C. d) Separation of the aqueous phase from the solid phase by using a Buchner filtration (100 μm), recovery of the aqueous phase constituting the malt sprout fraction extract. e) Conservation of the malt sprout fraction extract by adding following preservatives potassium sorbate (3 g/liter) and methyl benzoate (5 g/liter) to keep the extract over time (6 months), f) To be applied on plant seeds and used as biostimulant, the acidified malt sprout fraction extract is then further diluted in water at a 1/200 dilution.

(26) 3. Plant Seeds Treatment with Malt Sprout Fraction Extract (Biostimulant).

(27) The acidified malt sprout fraction extract (AMSFE) is tested as a biostimulant agent to treat plant seeds in order to demonstrate its plant growth stimulator effect at laboratory scale (versus two negative controls: water and a standard malt sprout extract).

(28) Plant seeds treated are: Arabidopsis, cucumber, rapeseed, sunflower, soya, bean, alfalfa, tomato Corn, wheat, barley, leek

(29) The seeds are treated through direct incorporation of biostimulant in an agar medium, or by directly steeping the seeds into juice of malt sprouts during 2 weeks.

(30) These experiments demonstrate that most of the growth of the different seeds are stimulated by the AMSFE (vs negative controls). The results show a higher length and weight of root system and the leaf area biomasses.

Example 4: Malt Sprout Fraction Extract Used as Biostimulant to Stimulate Wheat Growth Culture

(31) The acidified malt sprout fraction extract used in this example is the same than for example 3. Using the same malt sprout fraction as described in Table 4 and the same process of preparation.

(32) The acidified juice of malt sprouts is used as biostimulant to treat wheat culture parcels in open-field in the French Champagne area.

(33) The treatment plan used is:

(34) TABLE-US-00008 TABLE 5 Treatment plan applied on wheat plants Treatments done at the following wheat growing stage Cereal growing stages are defined in Cereals Witzenberger et al., 1989; Lancashire et al., 1991 Phenological growth stages and BBCH-identification keys of cereals Beginning of heading: tip of inflorescence 2 leaves First node at emerged from sheath, unfolded least 1 cm above first spikelet (stage tillering node just visible Products BBCH 12) (stage BBCH 31) (stage BBCH 51) Control water water water AMSFE 1 1 1 (liter of AMSFE 2 2 2 diluted at 3 3 3 1/200/hectare) 9 water water

(35) The acidified juice of malt sprouts according to the invention are diluted (1/200) and applied on wheat plants at three different stages of wheat growth, as defined in Table 5.

(36) The results show that the applications of the acidified juice of malt sprouts on wheat plants allow to stimulate wheat growth, especially by increasing plant vigor and increasing final biomass.

Example 5: Malt Sprout Fraction Extract Used as Biostimulant to Stimulate Corn Growth Culture

(37) The acidified malt sprout fraction extract used in this example is the same than for examples 3 and 4. Using the same malt sprout fraction as described in Table 4 and the same process of preparation.

(38) The acidified juice of malt sprouts is used as biostimulant to treat corn culture parcels in open-field in three different French regions. It is diluted (1/200) and applied on corn plants at three different stages of corn growth.

(39) TABLE-US-00009 TABLE 6 Treatment plan applied on corn plants Treatments done at the following corn growing stage Maize Weber and Bleiholder, 1990; Lancashire et al., 1991 Phenological growth stages and BBCH- identification keys of maize (Zea mays L.) 2 leaves First node Beginning of tassel unfolded detectable emergence: tassel (stage (stage detectable at top of stem Products BBCH 12) BBCH 31) (stage BBCH 51) Control water water water AMSFE 1 1 1 (liter of AMSFE 2 2 2 diluted at 3 3 3 1/200/hectare) 9 water water

(40) The results show that the applications of the acidified juice of malt sprouts on corn plants allow to stimulate corn vigor and growth, especially by increasing final yield (tons/hectare).