Indoleacetic acid agricultural composition with enhanced photostability, production method and use thereof

Abstract

The present invention relates to a method for producing an agricultural composition, which comprises photostabilizing indoleacetic acid (IAA) of biological and / or synthetic origin in a mixture of a nitrogen source and a reducing sugar. The photostabilization of the plant hormone IAA applied to crops according to the present invention is achieved by applying a high temperature to the mixture so that the nitrogen source and reducing sugar are converted into melanoidin pigments through the Maillard reaction to reduce IAA photodegradation.

Description

[0001] Technical field of the invention

[0002] The present invention relates to an agricultural composition for promoting plant growth, comprising indoleacetic acid (IAA) of biological and / or synthetic origin with enhanced photostability, as well as a method for producing and using the same. Background of the Invention

[0004] Plant hormone produced by bacteria is one of the main factors promoting plant growth. Plant hormone (plant growth hormone, cytokinin, gibberellin, ethylene and abscisic acid) is an organic substance (Raven et al., 2001) that plays a role in regulating plant growth. The main naturally occurring plant growth hormone is indoleacetic acid (IAA), which has a huge application potential as a plant growth regulator. The growth hormone synthesized by several microorganisms (for example, in the soil and / or with the bacteria and fungi associated with plants) is identical to the growth hormone found in plants, including indoleacetic acid (IAA). Therefore, it is expected to use an agricultural composition containing plant hormones such as IAA to promote the growth of crops.

[0005] In plants, microbial IAAs produced by bacteria from the genera Azospirillum spp., Alcaligenes faecalis, Klebsiella sp., Enterobacter sp., Xanthomonas sp., Pseudomonas sp., Bacillus spp., Herbaspirillum seropedicae, Rhizobium spp., and Bradyrhizobium spp. have been associated with plant growth stimulation (Patten and Glick, 1996; Idris et al., 2007; Kochar et al., 2011; Shao et al., 2015).

[0006] It is believed that microorganisms are able to select a specific IAA biosynthetic pathway from several available pathways depending on the environment. It has also been reported that there is more than one pathway for synthesizing IAA in many bacteria, most of which are tryptophan-dependent pathways (Patten and Glick, 1996). Induction of IAA biosynthesis by tryptophan-dependent pathways is well known and has been studied (Tullio et al., 2019; Iamada et al., 2017). For example, the production of indoleacetic acid by fermentation of Azospirillum is described in CN 104 975 052. Although the IAA molecule is relatively highly stable to physical factors such as temperature and pH of the culture medium (Hiratsuka et al., 1989), photodegradation of plant hormones is one of the main obstacles to the use of agricultural compositions containing IAA (Dunlap and Robacker, 1988; Stasinotoulos and Hangarter, 1990 and Hiratsuka et al., 1989). Depending on the wavelength and intensity, photolysis can reduce the synthesized IAA by up to 90%.

[0007] Therefore, there remains a need in the art for agricultural compositions containing IAA plant hormones with enhanced photostability such that the compounds remain stable and active for a longer period of time after the compositions are applied to plant crops on a commercial scale.

[0008] BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 shows the concentrations of synthetic IAA and biological IAA at different times of exposure to UV light under specific temperature and pressure conditions for the stabilization method, confirming that melanoidins are involved in the photostability of the compounds.

[0010] Figure 2 shows the concentration of bio-IAA at different stabilization times and upon exposure to UV light.

[0011] Description of the invention

[0012] Surprisingly, the present invention teaches that by combining indoleacetic acid (IAA) with a melanoidin pigment in a composition, it is possible to obtain a composition comprising IAA with enhanced photostability for agricultural applications. The IAA used in the compositions and methods of the present invention may be of biological and / or synthetic origin.

[0013] As shown in this document, IAA is stabilized by combining melanoidin pigments with IAA to provide photoprotection to IAA from biological and / or synthetic sources.

[0014] In one embodiment, the present invention provides a composition comprising a combination of IAA and a melanoidin pigment. The composition of the present invention may optionally comprise an additive or agriculturally acceptable carrier, such as an additive for treating seeds and sowing furrows, a cell protectant for spray tanks, a soil conditioner, a grout adjuvant, a polymer for seed coating, a polymer for coating granules of chemical or organic fertilizers, a solid fertilizer, a liquid fertilizer used in a foliar product formulation, an endospore activator, among other purposes.

[0015] The composition according to the present invention may be packaged in suitable packaging known in the art. Preferably, bags and / or plastic bottles may be used in the absence of a finished product oxygen exchange. The packaging of the product of the present invention does not require light protection as it is light stable. Preferably, the packaging volume may comprise about 1 L to 20 L and may be stored in a refrigerated environment or at room temperature in the range of about 10° C. to 35° C.

[0016] The present invention also provides a method for preparing an IAA agricultural composition with enhanced light stability by combining IAA with melanoidins. In a specific embodiment of the present invention, the combination of IAA with melanoidin pigment is achieved by subjecting a mixture comprising IAA and a nitrogen source and a reducing sugar to temperature and pressure conditions that promote the conversion of the nitrogen source and the reducing sugar into a melanoidin pigment via the Maillard reaction. In an alternative embodiment, the IAA and melanoidin pigment are combined after the pigment is formed via the Maillard reaction.

[0017] 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 the same.

[0018] The term "reducing sugar" includes, but is not limited to, any sugar known in the art that is capable of acting as a reducing agent due to having a free aldehyde group or a free ketone group, and mixtures or biological extracts containing the same.

[0019] In a preferred embodiment of the present invention, the mixture comprising said IAA and said nitrogen source and reducing sugar is obtained by fermenting IAA-producing bacteria in a medium containing tryptophan. The broth obtained from said fermentation contains IAA produced by the bacteria and nitrogen source and reducing sugar from the fermentation medium. After said fermentation, the broth is subjected to temperature and pressure conditions that promote the conversion of nitrogen source and reducing sugar into melanoidin pigment by Maillard reaction, so that the melanoidin pigment imparts photoprotection to the composition IAA.

[0020] The term "IAA-producing bacteria" includes, but is not limited to, species of Azospirillum, Alcaligenes faecalis, Klebsiella, Enterobacter, Xanthomonas, Grass Spirillum, Rhizobium, and Bradyrhizobium (Patten and Glick, 1996). Preferably, the IAA-producing bacteria is Azospirillum brasilense.

[0021] Thus, a preferred embodiment of the present invention includes an industrial production method for an agricultural composition comprising the steps of: fermenting a culture comprising one or more IAA-producing bacterial strains and tryptophan to biosynthesize 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 pigment via the Maillard reaction.

[0022] The present invention also provides compositions produced by the method of the present invention and uses thereof.

[0023] Advantageously, in preferred embodiments, the present invention provides IAA agricultural compositions with enhanced photostability that are obtained solely through biological pathways and contain no synthetic ingredients.

[0024] The present invention also provides preferred embodiments that include additional parameters for the process of industrially producing IAA agricultural compositions, such as pressure, temperature, oxygenation (air volume and agitation), and culture medium parameters for fermenting IAA-producing bacteria to produce high IAA concentrations.

[0025] As will be appreciated by those skilled in the art, different IAA-producing bacterial strains, preferably Azospirillum brasiliensis, may be used, and different culture and fermentation parameters may be combined with the present invention.

[0026] In a preferred embodiment, the present invention provides a method for producing an agricultural composition, comprising the steps of:

[0027] (a) fermenting a culture comprising one or more IAA-producing bacterial strains and tryptophan to biosynthesize indoleacetic acid (IAA); and

[0028] (b) Stabilization under temperature and pressure conditions that promote the conversion of nitrogen sources and reducing sugars into melanoidin pigments via the Maillard reaction.

[0029] Surprisingly, the photostability of IAA in the composition according to the invention is achieved by the combination with melanoidins, without which the use of the bioproduct in agriculture is impossible, since it is degraded by light. The pigments described above are able to filter the light falling on the finished product, protecting the IAA phytohormone, whether it is biosynthesized by the tryptophan-induced pathway or of synthetic origin.

[0030] In a preferred embodiment of the present invention, the melanoidin pigment is derived from a method known as the Maillard reaction. The reaction is divided into three stages according to intensity, wherein the polymerization of amino compounds with sugar fragments into brown pigments, melanoidins (Van Boekel, 1998) occurs in the highest stage. In embodiments involving the use of a mixture consisting of broth obtained by fermenting IAA-producing bacteria, the reaction occurs after the fermentation by converting the nitrogen source and reducing sugar added in the fermentation method into a pigment that ensures the stability of IAA. Even under illumination conditions, in packaging where the product is stored and sold, or in agricultural applications for seeds, planting furrows, irrigation or leaves, in an environment with high incidence of UV wavelengths, it keeps the high plant hormone levels in the finished product highly stable without compromising its agronomic efficiency.

[0031] In a specific embodiment, the IAA-producing bacteria is a species of the genus Azospirillum, more preferably Azospirillum brasiliensis. In a preferred embodiment, one or more strains of Azospirillum brasiliensis are selected from the group consisting of Ab-V5, Ab-V6, Sp6, Sp7, Sp245, Cd, 8-I, Az39. In an even more preferred embodiment, both strains Ab-V5 and Ab-V6 are used.

[0032] In a preferred embodiment, the stabilization of the plant hormone is carried out in a 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 product is stabilized at about 0.5 Kgf / cm 2 Up to 2.0Kgf / cm 2 pressure.

[0033] In a preferred embodiment according to the present invention, the batch fermentation or fed-batch fermentation of the Azospirillum brasiliensis culture is carried out for about 18 hours to 96 hours.

[0034] In a preferred embodiment, the method of the present invention comprises the continuous expansion (amplification) of the Azospirillum brasiliensis culture to inoculate the fermentation culture. Preferably, the continuous expansion starts with a volume of 100 mL, which is used as an inoculum of about 10 L. These inocula are inoculated in turn in about 180 L, which are finally transferred to a reactor of about 2,000 L.

[0035] In a preferred embodiment, the Azospirillum brasiliensis strain culture is amplified in a flask of about 100 mL by incubation 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 brasiliensis strain is then grown in a stainless steel round flask containing about 10 L of culture medium. The incubation time is preferably about 8 hours to about 96 hours, with an air flow rate of about 0.25 Nm / s. 3 / h to about 1.0Nm3 / h (=0.41vvm to 1.66vvm).

[0036] In a preferred embodiment, the culture temperature for the proliferation of the Azospirillum brasiliensis strain according to the present invention is about 22°C to about 38°C.

[0037] In a preferred embodiment, in the 180 L culture scale-up method of the present invention, the Azospirillum brasiliensis Ab-V5 and Ab-V6 strains are inoculated together. To this end, in a preferred mode, after the strains are grown in two stainless steel round flasks of about 10 L, the flasks are inoculated into a tank containing about 180 L of culture medium and incubated for about 18 hours to about 96 hours. The air flow rate is preferably about 1.25 Nm 3 / h to about 3.0Nm 3 / h (=0.1vvm to 0.25vvm).

[0038] In a preferred embodiment, the fermentation step 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 2.5Nm 3 / h (=0.0085vvm to 0.021vvm). The pressure is preferably about 0.5kgf / cm 3 To about 1.2kgf / cm 3 The stirring is preferably about 40 Hz to about 45 Hz. The fermentation step is preferably carried out for about 18 h to about 96 h.

[0039] In a preferred embodiment, the medium used to expand the culture of Azospirillum brasiliensis and / or the fermentation scale of 100 mL is NFb medium ( 1995). For other production scales, i.e., 10 L, 180 L, and 2,000 L, in a preferred mode, the culture media described in Table 1 are used.

[0040] In an alternative embodiment, the present invention provides an IAA agricultural composition having enhanced photostability and obtainable by a method according to the present invention as described above.

[0041] In an alternative embodiment, the present invention also provides the use of the agricultural composition according to the present invention as a plant growth promoter or biochemical agent in crops. Preferably, the use is applied to seeds, planting furrows, irrigation or foliar application by spraying.

[0042] In an alternative embodiment, the present invention provides the use of a photostable IAA plant hormone according to the present invention for preparing an agricultural composition for optimizing the growth and / or increasing the yield of a crop.

[0043] In an alternative embodiment, the present invention provides plants, plant parts and / or seeds coated with a composition obtained according to the present invention. Example

[0044] Example 1 - Scaling up the culture

[0045] The Azospirillum brasiliensis strains Ab-V5 and Ab-V6 were inoculated into flasks containing 100 mL of NFb medium ( 1995) and incubated on an orbital shaker at 80 to 150 rpm at 22 to 38°C for about 18 to 96 hours. The next scale-up step consisted of: 3 / h to 1.0Nm 3 A stainless steel round flask containing 10 L of medium was inoculated at an air flow rate of / h (=0.41 vvm to 1.66 vvm) and a temperature of about 22°C to 37°C, in which the strains were cultured individually and incubated for about 18 hours to 96 hours. Thereafter, each culture was inoculated into a tank containing 180 L of medium and incubated at 0.25 Nm 3 / h to 3.0Nm 3 The mixture was incubated at an air flow rate of 0.1 to 0.25 vvm and a temperature range of 22° C. to 38° C. for about 18 to 96 hours.

[0046] Table 1. Culture medium for the cultivation of Azospirillum brasiliensis strains Ab-V5 and Ab-V6

[0047]

[0048]

[0049] Table 2. Micronutrient stock solutions and quantities required for 1 L of final micronutrient solution

[0050]

[0051]

[0052] Example 2 - Fermentation in a bioreactor

[0053] For growth in a 2,000 L fermenter, a medium containing the amino acid tryptophan was used as shown in Table 1. The growth parameters for Azospirillum brasiliensis in a 2,000 L fermenter were: 0.5 kgf / cm 3 Up to 1.2kgf / cm 3 pressure, 40 Hz to 45 Hz oscillation, 22°C to 38°C and 1.0 Nm 3 / h to 2.5Nm 3 / h air flow rate (=0.0085vvm to 0.021vvm) for 18h to 96h.

[0054] Example 3 - Stabilization

[0055] After 18h to 96h of fermentation, the 2 Up to 2.0kgf / cm 2 The method for stabilizing metabolites is performed by sterilizing a culture medium containing the microorganisms at a pressure of 100°C for about 15 minutes to 120 minutes.

[0056] Example 4 - Photostability

[0057] To verify the formulation that guarantees the photostability of the indoleacetic acid phytohormone, two procedures were performed. In the first assay, the following were compared: i) synthetic IAA with water (without the protection of melanoidins); ii) synthetic IAA with the same medium used in the bio-IAA production method of the present invention, and iii) bio-IAA; all samples were subjected to a sterilization method at 121°C for about 1 hour, as claimed that the indole compounds are stabilized due to the production of melanoidins through the Maillard reaction. After the required period of stabilization, the IAA concentration was evaluated using the Salkwolski reagent according to the method proposed by Glickmann and Dessaux (1994).

[0058] The results shown in Figure 1 prove that, in the absence of the stabilization method claimed, synthetic IAA is completely degraded after being exposed to UV light for 4 hours. Within the first hour of exposure, photodegradation has reached 74.5%. However, when evaluating the IAA produced by the Brazilian Azospirillum organism, greater stability was found, because compared with the unstabilized IAA (diluted in water), the concentration of this substance was reduced less, especially after being exposed to UV light for 1 hour, 2 hours and 4 hours, the degradation was only 0.7%, 17.2% and 29.1%, respectively. Similarly, when synthetic IAA is added to the preparation shown in the technical solution, and then the stabilization step is carried out, the same UV light protection performance can be obtained. It proves the amazing ability of the pigment (melanoid) produced by the Maillard reaction to protect the plant hormone indoleacetic acid from biological and synthetic sources from photodegradation. Leasure et al. (2013) reported the difficulty in using indoleacetic acid in plants due to its high rate of photodegradation, which demonstrates the importance of the claimed practical solution for achieving commercial-scale agricultural applications of IAA, a potent plant growth promoter of natural origin that can promote sustainable agribusiness.

[0059] In a second procedure (Figure 2), different times of the method used to stabilize the formulation with biological IAA were evaluated, ranging from 15 minutes, 1 hour, and 1 hour and 30 minutes, to assess the role of melanoidin concentration in photoprotection.

[0060] As in the first assay, IAA phytohormone stabilization was effective and increased the stability of the compounds towards UV light irradiation. It should also be noted that exposure of the indole compounds to different stabilization times resulted in proportional photoprotection, that is, the longer the method time, the greater the stability obtained, especially when the bio-IAA stabilization method time was 1 hour and 30 minutes, providing a high stability of IAA synthesized by Azospirillum brasiliensis. The surprising effect of the increased stability of IAA was confirmed by applying the Scott-Knott mean test (p≤0.05), with significant differences at all times of exposure to UV light in this assay. It is important to point out that, although 54.85% of the photodegradation of the bio-IAA occurred when no stabilization method was performed, part of the protection of the bio-IAA was due to the sterilization of the culture medium before the microbial cultivation.

[0061] The results obtained demonstrate the effectiveness of the pigments (melanoids) produced in the stabilization method proposed in the technical solution in increasing the stability of the IAA phytohormone subjected to light radiation. The solution is positively influenced by innovative and applicable solutions that provide improvements in important crops while being environmentally friendly, which provides multiple possibilities for the application of plant growth promoting compounds and contributes to agricultural sustainability, which is essential for the social-environmental balance.

Claims

1. Use of a melanoidin pigment for improving the photostability of an agricultural indoleacetic acid (IAA) composition, wherein the agricultural IAA composition comprises a combination of IAA and a melanoidin pigment and has increased photostability relative to an IAA composition without melanoidin.

2. The use according to claim 1, characterized in that The IAA is synthetic.

3. The use according to claim 1, characterized in that The IAA is of biological origin.

4. The use according to claim 3, characterized in that The IAA is obtained by fermentation of IAA-producing bacteria.

5. The use according to claim 1, characterized in that The agricultural IAA composition also includes an agriculturally acceptable additive or carrier.

6. The use according to claim 5, characterized in that The agriculturally acceptable additive is selected from the group consisting of additives for treating seeds and in-furrow seeding, cell protectants for spray tanks, soil conditioners, mixing adjuvants, polymers for coating seeds, polymers for coating granules of chemical or organic fertilizers, solid fertilizers, liquid fertilizers for foliar formulations, and endospore activators.

7. A method for increasing the photostability of an agricultural indoleacetic acid (IAA) composition, characterized in that The method includes formulating an agricultural IAA composition comprising a combination of IAA and a melanoidin pigment, wherein the agricultural IAA composition exhibits increased photostability compared to an IAA composition without melanoidin.

8. The method according to claim 7, characterized in that The method comprises subjecting a mixture comprising an IAA plant hormone and a nitrogen source and a reducing sugar to temperature and pressure conditions that promote conversion of the nitrogen source and reducing sugar into a melanoidin pigment via the Maillard reaction.

9. The method according to claim 7, characterized in that The IAA is synthetic.

10. The method according to claim 7, characterized in that The IAA is obtained by fermentation of IAA-producing bacteria.

11. The method according to claim 10, characterized in that The method comprises the following steps: (a) fermenting a culture comprising one or more IAA-producing bacterial strains and tryptophan to biosynthesize IAA; and (b) Stabilization under temperature and pressure conditions that promote the conversion of nitrogen sources and reducing sugars into melanoidin pigments via the Maillard reaction.

12. The method according to claim 11, characterized in that The IAA-producing bacteria include one or more strains of Azospirillum brasilense.

13. The method according to claim 12, characterized in that The one or more A. brasilense strains are selected from the group consisting of Ab-V5 and Ab-V6.

14. The method according to claim 11, characterized in that The Maillard reaction occurs in a fermenter environment.

15. The method according to claim 11, characterized in that The culture and fermentation are batch culture or fed-batch culture.

16. The method according to claim 11, characterized in that The stabilization step is carried out for 15 to 120 minutes.

17. The method according to claim 11, characterized in that The stabilization step is performed at a temperature of 100°C to 130°C and / or 0.5 Kgf / cm 2 Up to 2.0Kgf / cm 2 pressure.

18. The method according to claim 11, characterized in that The method also includes continuously expanding the Azospirillum brasiliensis culture to inoculate the fermentation culture.

19. The method according to claim 18, characterized in that Among them, Azospirillum brasiliensis Ab-V5 and Ab-V6 strains were inoculated separately.

20. The method according to claim 18, characterized in that Serial expansions were performed in volumes ranging from 100 mL to 10 L, and from 180 L to 2,000 L.

21. The method according to claim 20, characterized in that The Azospirillum brasiliensis strain was grown by incubation on an orbital shaker at 80 rpm to 150 rpm.

22. The method according to claim 21, characterized in that The Azospirillum brasiliensis strain was grown by incubation for 18 to 96 hours.

23. The method according to claim 22, characterized in that The Azospirillum brasiliensis strain was grown in a stainless steel round flask containing 10 L of culture medium.

24. The method according to claim 20, characterized in that The Azospirillum brasiliensis strain was incubated for 18 to 96 hours.

25. The method according to claim 24, characterized in that The Brazilian Azospirillum strain was 3 / h to 1.0Nm 3 The cells were incubated at air flow rates ranging from 0.41 vvm to 1.66 vvm / h.

26. The method according to claim 25, characterized in that After the Azospirillum brasiliensis strain was cultured separately in two 10 L stainless steel round flasks, the two round flasks were inoculated into a tank containing 180 L of culture medium.

27. The method according to claim 24, characterized in that The Brazilian Azospirillum strain was 1.25Nm 3 / h to 3.0Nm 3 The cells were incubated at air flow rates ranging from 0.1 vvm to 0.25 vvm / h for 18 to 96 hours.

28. The method according to any one of claims 21 to 27, characterized in that The incubation temperature is 22°C to 38°C.

29. The method according to claim 11, characterized in that The fermentation step is carried out at 0.5 Kgf / cm 3 Up to 1.2Kgf / cm 2 pressure.

30. The method according to claim 11, characterized in that The fermentation step is carried out under the following conditions: i) with stirring at 40 Hz to 45 Hz; ii) at a temperature of 22° C. to 38° C.; iii) at 1.0 Nm 3 / h to 2.5Nm 3 / h = air flow rate of 0.0085 vvm to 0.021 vvm; and / or (iv) for 18h to 96h.

31. Use of a melanoidin pigment for improving the photostability of an agricultural IAA composition, wherein the agricultural IAA composition is obtained by the method of any one of claims 7 to 30, and wherein the agricultural IAA composition has increased IAA photostability relative to an IAA composition without melanoidin.

32. The use of claim 31, wherein the agricultural IAA composition further comprises one or more agriculturally acceptable additives and / or carriers.

33. The use according to claim 31, characterized in that The agricultural IAA composition is for use in crop applications.

34. The use according to claim 33, characterized in that The agricultural IAA compositions are for seed, in-furrow, irrigation or foliar application.

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