Agricultural composition of indoleacetic acid with improved light stability, method for producing the same, and use thereof

Abstract

The present invention relates to a method for the preparation of an agricultural composition comprising the photostabilization of indole acetic acid (IAA) of biological and / or synthetic origin in a mixture comprising said IAA, a nitrogen source and reducing sugars. The photostabilization of the IAA plant hormone for application to agricultural crop targets according to the invention is achieved by applying an elevated temperature to said mixture to convert said nitrogen source and reducing sugars into melanoidin type pigments via the Maillard reaction, reducing IAA photodegradation.

Description

Technical Field

[0001] The present invention relates to an agricultural composition for promoting plant growth, which contains bio-derived and / or synthetic-derived indole acetic acid (IAA) with improved photo-stability, as well as a method for producing the same and its use.

Background Art

[0002] The production of plant hormones by bacteria is one of the main factors governing plant growth promotion. Plant hormones (auxin, cytokinin, gibberellin, ethylene, and abscisic acid) are organic substances that play a role in regulating plant growth (Raven et al., 2001). The main naturally occurring auxin is indole acetic acid (IAA), which has great potential for use as a plant growth regulator. Some microorganisms such as bacteria and fungi in the soil and / or associated with plants synthesize the same growth hormones found in plants, including indole acetic acid (IAA). Therefore, the use of agricultural compositions containing plant hormones such as IAA is desirable for promoting crop growth.

[0003] In plants, microbial IAA is produced by bacteria of the genera Azospirillum spp., Alcaligenes faecalis, Klebsiella sp., Enterobacter sp., Xanthomonas sp., Pseudomonas spp., Bacillus spp., Herbaspirillum seropedicae, Rhizobium spp., and Bradyrhizobium spp., which has been associated with plant growth stimulation (Patten and Glick, 1996; Idris et al., 2007; Kochar et al., 2011; Shao et al., 2015).

[0004] Microorganisms are thought to be able to select a specific IAA biosynthesis pathway from among several available IAA biosynthesis pathways depending on the environment. Also, in many bacteria, it has been reported that there are multiple IAA synthesis pathways, and most of them are tryptophan-dependent pathways (Patten and Glick, 1996). The induction of IAA biosynthesis via the tryptophan-dependent pathway is well known and has been studied (Tullio et al., 2019; Iamada et al., 2017). The production of indoleacetic acid by fermentation of Azospirillum is described, for example, in CN104975052. Although IAA molecules are relatively stable against physical factors such as the temperature and pH of the medium (Hiratsuka et al., 1989), photodegradation of plant hormones is one of the major obstacles regarding the use of agricultural compositions containing IAA (Dunlap and Robacker, 1988; Stasinopoulos and Hangarter, 1990 and Hiratsuka et al., 1989). Photodegradation can reduce the synthesized IAA by up to 90% depending on the wavelength and intensity.

[0005] Therefore, in the art, there is a need for an agricultural composition containing an IAA plant hormone with improved photo-stability so that the compound can remain stable and active for a long time after application of the composition to plant crops on a commercial scale.

[0006] [Brief Description of the Drawings] [Figure 1] Shows the concentrations of synthetic IAA and biological IAA at various exposure times to UV light that have undergone a stabilization process under specific temperature and pressure conditions. It serves as evidence of the involvement of melanoidin in the photo-stability of the compound. [Figure 2] Shows the concentration of biological IAA under various stabilization times and exposure to UV light. [Summary of the Invention]

[0007] Surprisingly, the present invention teaches that in a composition containing indoleacetic acid (IAA), by combining IAA (indoleacetic acid) with a melanoidin type pigment, an IAA-containing composition for agricultural use with improved photo-stability can be achieved. The IAA used in the compositions and methods of the present invention can be of biological and / or synthetic origin.

DETAILED DESCRIPTION OF THE INVENTION

[0008] As shown herein, the stabilization by combining a melanoidin type pigment and IAA provides photo-protection to IAA from biological and / or synthetic sources.

[0009] In one embodiment, the present invention provides a composition comprising a combination of IAA and a melanoidin type pigment. The composition of the present invention may optionally contain additives or agriculturally acceptable carriers, such as additives for treating seeds and sowing furrows, cytoprotective agents for spray tanks, soil conditioners, grout aids, polymers for seed coating, polymers for coating granules of chemical fertilizers or organic fertilizers, liquid fertilizers, solid fertilizers, endospore activators, and other items for other purposes.

[0010] The composition of the present invention can be packaged by appropriate packaging methods known in the art. Preferably, bags and / or plastic bottles that do not require oxygen exchange for the final product can be used. Since the packaging of the product of the present invention has photo-stability, the need for light shielding is eliminated. Preferably, the volume of the packaging is about 1 to 20 L and can be stored in a refrigerated environment or at room temperature in the range of about 10 to 35°C.

[0011] The present invention further provides a method for producing an IAA agricultural composition with improved photo-stability by combining IAA with melanoidin. In a particular embodiment of the present invention, the combination of IAA and melanoidin-type dye is achieved by subjecting a mixture containing IAA, a nitrogen source, and a reducing sugar to temperature and pressure conditions that promote the conversion of the nitrogen source and reducing sugar to melanoidin-type dye via the Maillard reaction. In another embodiment, IAA and melanoidin-type dye are combined after the formation of the dye by the Maillard reaction.

[0012] The term "nitrogen source" includes, but is not limited to, amino acids, peptides, and proteins of biological or synthetic origin known in the art, as well as mixtures or biological extracts containing them.

[0013] The term "reducing sugar" includes, but is not limited to, any sugar having a free aldehyde group or free ketone group known in the art and capable of acting as a reducing agent, as well as mixtures or biological extracts containing them.

[0014] In a preferred embodiment of the present invention, the mixture containing the IAA, and the nitrogen source and reducing sugar is obtained by fermenting IAA-producing bacteria in a tryptophan-containing medium. The broth obtained from the fermentation contains IAA produced by the bacteria, and reducing sugar and nitrogen source from the fermented medium. After the fermentation, the broth is subjected to temperature and pressure conditions that promote the conversion of the nitrogen source and reducing sugar to melanoidin-type dye via the Maillard reaction, whereby the melanoidin-type dye provides photo-protection to the IAA in the composition.

[0015] The term "IAA-producing bacteria" includes, but is not limited to, bacteria of the genus Azospirillum spp., Alcaligenes faecalis, Klebsiella sp., Enterobacter sp., Xanthomonas sp., Herbaspirillum seropedicae, Rhizobium spp., and Bradyrhizobium spp. (Patten and Glick, 1996). Preferably, the IAA-producing bacteria is Azospirillum brasilense.

[0016] Accordingly, preferred specific embodiments of the present invention include an industrial manufacturing method of an agricultural composition, comprising the steps of fermenting a culture containing one or more IAA-producing bacterial strains and tryptophan for the biosynthesis of indoleacetic acid (IAA); and stabilizing under temperature and pressure conditions that promote the conversion of a nitrogen source and a reducing sugar into a melanoidin-type pigment via the Maillard reaction.

[0017] The present invention further provides a composition produced by the method of the present invention and its use.

[0018] Advantageously, in a preferred embodiment, the present invention provides an IAA agricultural composition with improved photo-stability that does not contain synthetic components and is obtained only by biological pathways.

[0019] The present invention further provides a preferred embodiment for producing a high-concentration IAA concentrate, including additional parameters of a method for industrially manufacturing an IAA agricultural composition, such as pressure, temperature, oxygenation (air volume and agitation), and medium parameters for the fermentation of IAA-producing bacteria.

[0020] As will be understood by those skilled in the art, various IAA-producing bacterial strains, preferably Azospirillum brasilense, can be used, and various culture parameters and fermentation parameters can be combined with the present invention.

[0021] In a preferred embodiment, the present invention provides the following steps: (a) fermenting a culture containing one or more IAA-producing bacterial strains and tryptophan for the biosynthesis of indole acetic acid (IAA); and, (b) stabilizing under temperature and pressure conditions that promote the conversion of the nitrogen source and reducing sugar to a melanoidin-type pigment via the Maillard reaction, to provide a method for producing an agricultural composition.

[0022] Surprisingly, the photo-stability of IAA in the compositions of the present invention is achieved by combination with melanoidin, which eliminates the inability to use bioproducts in agriculture due to photo-degradation. The above-mentioned pigment can filter the light hitting the final product to protect the IAA plant hormone, which may be biosynthesized via the tryptophan-derived pathway or of synthetic origin.

[0023] In a preferred embodiment of the present invention, the melanoidin pigment is obtained from a process known as the Maillard reaction. This reaction has three stages depending on the intensity, and in the most advanced stage, polymerization of an amino compound and a sugar fragment occurs, producing melanoidin, a brown pigment (Van Boekel, 1998). In an embodiment involving the use of a mixture consisting of a broth obtained by fermenting IAA-producing bacteria, this reaction occurs after the fermentation by converting the nitrogen source and reducing sugar inserted during the fermentation process into a pigment that provides the stability of IAA. Thereby, the high plant hormone levels in the final product are maintained very stably without impairing agricultural efficiency, whether under light irradiation conditions, within the packages in which the product is stored and sold, or in agricultural applications to seeds, planting furrows, irrigation, or leaves, or in an environment under strong irradiation of UV wavelengths.

[0024] In certain embodiments, the IAA-producing bacteria are of the genus Azospirillum sp., more preferably Azospirillum brasilense. In a preferred embodiment, one or more strains of Azospirillum brasilense are selected from the group consisting of Ab-V5, Ab-V6, Sp6, Sp7, Sp245, Cd, 8-I, Az39. In a more preferred embodiment, both the Ab-V5 strain and the Ab-V6 strain are used.

[0025] In a preferred embodiment, the stabilization of the plant hormone is carried out within the fermenter environment. Preferably, the stabilization process is carried out at a temperature of about 100°C to about 130°C for about 15 minutes to 120 minutes. Preferably, the stabilization of the product is carried out at a pressure of about 0.5 to 2.0 kgf / cm 2 of.

[0026] In a preferred embodiment of the present invention, the batch fermentation or fed-batch fermentation of the Azospirillum brasilense culture is carried out for about 18 to 96 hours.

[0027] In a preferred embodiment, the method of the present invention includes sequential scaling up of an Azospirillum brasilense culture for inoculation of a fermentation culture. Preferably, the sequential scaling up starts with a volume of 100 mL that functions as an inoculum for about 10 L. Next, these are inoculated into about 180 L and then finally transferred to a reactor of about 2,000 L.

[0028] In a preferred embodiment, the Azospirillum brasilense strain culture is grown by incubating in a 100 mL flask on an orbital shaker at about 80 rpm to about 150 rpm. The incubation time is preferably about 18 hours to about 96 hours. Preferably, the Azospirillum brasilense strain is then grown in a stainless steel round flask containing about 10 L of medium. The incubation time is preferably about 0.25 Nm 3 / h to about 1.0 Nm 3 / h (= 0.41 to 1.66 vvm) at an air flow rate for about 8 to about 96 hours.

[0029] In a preferred embodiment, the culture temperature for culturing and growing the Azospirillum brasilense strain according to the present invention is about 22°C to about 38°C.

[0030] In a preferred embodiment, in the upscaling process from the 180 L culture described for the present invention, the Azospirillum brasilense Ab-V5 strain and Ab-V6 strain are inoculated together. For this purpose, in a preferred embodiment, after growing those strains in two stainless steel round flasks of about 10 L, the flasks are inoculated into a tank containing about 180 L of medium and incubated for about 18 to about 96 hours. The air flow rate is preferably about 1.25 to about 3.0 Nm 3 / h (= 0.1 to 0.25 vvm).

[0031] In a preferred embodiment, the fermentation process is carried out at a temperature of about 22 °C to about 38 °C. The air flow rate is preferably about 1.0 Nm 3 / h to about 2.5 Nm 3 / h (= 0.0085 - 0.021 vvm). The pressure is preferably about 0.5 to about 1.2 kgf / cm 3 . Stirring is preferably about 40 hz to about 45 hz. The fermentation process is preferably carried out for about 18 hours to about 96 hours.

[0032] In a preferred embodiment, the medium used to scale up the cultivation and / or fermentation of Azospirillum brasilense (A. brasilense) for a 100 mL scale is the NFb medium (Dobereiner, 1995). For other production scales, namely 10 L, 180 L, and 2000 L, in a preferred embodiment, the media described in Table 1 are used.

[0033] In another embodiment, the present invention provides a photo-stabilized IAA agricultural composition that can be obtained by the method according to the present invention described above.

[0034] In another embodiment, the present invention further provides the use of the agricultural composition of the present invention for application to agricultural crops as a plant growth promoter or biochemical substance. Preferably, the use is for application by spraying for seed, planting furrow, irrigation, or foliar application.

[0035] In another embodiment, the present invention provides the use of the photo-stabilized IAA plant hormone of the present invention for manufacturing an agricultural composition for optimizing the growth of agricultural crops and / or increasing the yield of agricultural crops.

[0036] In another embodiment, the present invention provides plants, plant parts and / or seeds coated with the composition obtained according to the present invention.

Examples

[0037] Example 1: Scale-up of Cultivation The Azospirillum brasilense Ab-V5 strain and Ab-V6 strain are separately inoculated into flasks containing 100 mL of NFb medium (Dobereiner, 1995) and incubated on an orbital shaker at 80 - 150 rpm and 22 - 38°C for approximately 18 - 96 hours. The next scale-up step consists of inoculating into a stainless-steel round flask containing 10 L of medium (Table 1) and growing the strains separately therein, with an air flow rate of 0.25 - 1.0 Nm 3 / h (=0.41 - 1.66 vvm) and a temperature of approximately 22 - 37°C for approximately 18 - 96 hours. Thereafter, each culture is inoculated into a tank containing 180 L of medium and incubated at an air flow rate of 0.25 - 3.0 Nm 3 / h (=0.1 - 0.25 vvm) and a temperature in the range of 22 - 38°C for approximately 18 - 96 hours.

[0038]

Table 1

[0039]

Table 2

[0040] Example 2: Fermentation in a Bioreactor For growth in a 2,000 L fermenter, a medium containing the amino acid tryptophan shown in Table 1 is used. The growth parameters of Azospirillum brasilense in a 2,000 L fermenter are as follows: pressure 0.5 - 1.2 kgf / cm 3 , oscillation range 40 - 45 hz, temperature 22 - 38°C, air flow rate 1.0 - 2.5 Nm 3 / h (=0.0085 - 0.021 vvm), for 18 - 96 hours.

[0041] Example 3: Stabilization After 18 - 96 hours of fermentation, the medium containing the microorganisms is at 100 - 130°C, 0.5 - 2.0 kgf / cm 2A process for stabilizing metabolites is carried out by sterilizing at a pressure of for about 15 to 120 minutes.

[0042] Example 4: Photo stability To verify formulations that provide photo stability of the indole acetic acid plant hormone, two procedures were carried out. In the first assay, the following were compared: i) synthetic IAA with water (without protection by melanoidins); ii) synthetic IAA using the same medium as used in the biological IAA production process of the present invention, and iii) biological IAA; for the purpose of stabilizing indole compounds by the formation of melanoidins by the Maillard reaction, all samples were subjected to a sterilization process at 121 °C for about 1 hour. After the time required for stabilization had elapsed, the IAA concentration was evaluated using the Sakwolski reagent according to the method proposed by Glickmann and Dessaux (1994).

[0043] The results shown in Graph 1 indicate that synthetic IAA without the stabilization process of this claim is completely decomposed after 4 hours of exposure to UV light. Within the first hour after exposure, photodegradation already reaches 74.5%. However, when evaluating IAA biologically produced by Azospirillum brasilense, only 0.7%, 17.2%, and 29.1% degradation occurred after 1, 2, and 4 hours of UV light exposure, respectively. Compared to IAA without stabilization (diluted with water), the concentration decrease was less, so higher stability was confirmed. Similarly, when synthetic IAA was added to the formulation shown in the technical solution and then the stabilization process was carried out, the same behavior of light protection against UV light was obtained. This shows the surprising ability of melanoidin, a pigment produced by the Maillard reaction, to protect indole acetic acid, a plant hormone of biological and synthetic origin, from photodegradation. Leasure et al. (2013) reported that indole acetic acid is difficult to use in plants due to its high photodegradation rate, which demonstrates the importance of the practical solution of this claim to enable the agricultural use of IAA, a powerful plant growth promoter of natural origin that can contribute to sustainable agribusiness, on a commercial scale.

[0044] In the second procedure (Graph 2), to evaluate the role of melanoidin concentration in light protection, the process of stabilizing the formulation using biological IAA was varied in time and evaluated in the range of 15 minutes, 1 hour, and 1 hour 30 minutes.

[0045] Similar to the first assay, the stabilization of the IAA plant hormone was effective, and the stability of the compound against UV light irradiation was improved. Also, when the indole compound was exposed to different stabilization times, photoprotection occurred in a directly proportional ratio, that is, the longer the process time, the greater the stability obtained. In particular, when the treatment time of biological IAA stabilization was 1 hour and 30 minutes, the high stability of IAA synthesized by Azospirillum brasilense was also noted. The surprising effect of the improvement in IAA stability was confirmed by the fact that there were significant differences at all times of exposure to ultraviolet light tested in this assay by applying the Scott Knott mean test (p≤0.05). It is important to note that when the stabilization process is not carried out, photodegradation of biological IAA occurs at 54.85%, and the partial protection of biological IAA is due to the sterilization of the medium before microbial growth.

[0046] The results obtained demonstrate the effectiveness of melanoidin, a pigment produced during the stabilization process, shown by the technical solution for improving the stability of the irradiated IAA plant hormone. This solution brings several possibilities for the application of plant growth-promoting compounds and is positively influenced by an innovative and applicable solution that is environmentally friendly and brings improvements to important crops, contributing to the sustainability of agriculture, which is essential for the social-environmental balance.

Claims

1. An agricultural composition comprising a combination of indoleacetic acid (IAA) and a melanoidin-type pigment, having improved photo-stability compared to an IAA composition not containing melanoidin, wherein the composition further comprises a medium fermented with IAA-producing bacteria, and the melanoidin-type pigment is a product obtained by converting a nitrogen source and a reducing sugar contained in the fermentation medium into a melanoidin-type pigment by the Maillard reaction.

2. The composition according to claim 1, wherein the IAA-producing bacteria comprise one or more Azospirillum brasilense strains.

3. The composition according to claim 2, wherein the one or more Azospirillum brasilense strains are selected from the group consisting of Ab-V5 and Ab-V6.

4. The composition according to claim 1, further comprising an agriculturally acceptable additive or carrier.

5. The additive is selected from the group consisting of additives for treating seeds and sowing furrows, cytoprotective agents for spray tanks, soil conditioners, mixing aids, polymers for seed coating, polymers for coating granules of chemical fertilizers or organic fertilizers, liquid fertilizers used in foliar preparations, solid fertilizers, and endospore activators. The composition according to claim 4.

6. A method for producing an IAA agricultural composition with improved photo-stability, comprising formulating a combination of IAA and a melanoidin-type pigment in the composition, wherein the composition exhibits improved photo-stability compared to an IAA composition not containing melanoidin, and the combination of IAA and the melanoidin-type pigment is (a) fermenting a culture containing one or more IAA-producing bacterial strains, tryptophan, and a reducing sugar for the biosynthesis of indoleacetic acid (IAA); and (b) stabilizing the fermentation culture of step (a) under temperature and pressure conditions that promote the conversion of a nitrogen source and a reducing sugar into a melanoidin-type pigment via the Maillard reaction. A method created by

7. The method according to claim 6, wherein the IAA-producing bacteria comprise one or more Azospirillum brasilense strains.

8. The method according to claim 7, wherein the one or more Azospirillum brasilense strains are selected from the group consisting of Ab-V5 and Ab-V6.

9. The method according to claim 6, wherein the Maillard reaction occurs in the fermenter environment.

10. The method according to claim 6, wherein the fermentation is batch culture or fed-batch culture.

11. The stabilization step (b) is (i) for 15 minutes to 120 minutes; (ii) at a temperature of 100°C to 130°C; and / or (iii) 0.5 gf / cm ２ to 2.0 Kgf / cm ２ at a pressure of The method according to claim 6, which is carried out.

12. The fermentation step (a) is (i) at a pressure of 0.5 kgf / cm ２ to 1.2 kgf / cm ２ ; (ii) under stirring at 40 Hz to 45 Hz; (iii) at a temperature of 22°C to 38°C; (iv) at an air flow rate of 1.0 Nm ３ / h to 2.5 Nm ３ / h (= 0.0085 to 0.021 vvm); and / or (v) for 18 hours to 96 hours, The method according to claim 6, which is carried out.

13. Use of the agricultural composition according to claim 1, wherein the use is for application to crops.

14. The use according to claim 13, wherein the composition is applied to seeds, planting furrows, irrigation or foliage.

15. A plant, plant part and / or seed coated with the composition according to claim 1.

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