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    • 专利摘要:
    Inclusion complex comprising Streptomyces antimicrobial metabolites and chitosan amino-oligosaccharides, and biofertilizer comprising this complex and Streptomyces bacteria. An inclusion complex formed by antimicrobial compounds and plant growth promoters that can be obtained by fermentation of strains of bacteria of the genus Streptomyces and hydrolyzed chitosan oligo-saccharides. As well as a biofertilizer that comprises this inclusion complex and bacteria of the genus Streptomyces. Procedures for obtaining both products and their use for the treatment of pathogens that affect agriculture and, in general, agriculture and simultaneously stimulate plant growth. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2799848A1
    申请号:ES201930554
    申请日:2019-06-18
    公开日:2020-12-21
    发明作者:Gil Jesús Martin；Juan Celia Andres；Duran Laura Buzon；Lebena Eduardo Perez；Ramos Pablo Martin
    申请人:Universidad de Valladolid；
    IPC主号:
    专利说明:

    [0004] TECHNICAL SECTOR
    [0006] The present invention falls within the fields of Chemistry and Biology, and has direct application in the agricultural sector, in the phytosanitary field and that of fertilizers. More specifically, it provides products, in particular an inclusion complex and a biofertilizer comprising said complex, with antimicrobial characteristics that stimulate plant growth and, simultaneously, reduce and eliminate pathogens (fungi and bacteria) present in agriculture. and, in general, in Nature.
    [0008] More specifically, the biofertilizer with antimicrobial characteristics described here can be obtained by mixing solid, on the one hand, an inclusion complex (AOS-MS) formed between secondary metabolites produced during the fermentation of bacteria of the genus Streptomyces in a suitable culture medium for the selected strain and hydrolyzed chitosan amino-oligosaccharides and, on the other hand, the bacteria of the genus Streptomyces themselves. In the present invention, the Streptomyces bacterial strains are selected from those capable of emitting secondary metabolites with antimicrobial properties (antibacterial and / or antifungal). Thus, the present invention provides a product that can act as a biofertilizer by stimulating plant growth and, simultaneously, as a biocontrol agent for soil pathogens by Monitored Natural Attenuation (MNA).
    [0010] STATE OF THE ART
    [0012] Many microorganisms live within the roots of plants, as endophytes or in the rhizosphere, where they exert a great influence due to their physicochemical properties. A subset of these microorganisms, commonly referred to as plant growth-promoting rhizobacteria (RPCP), contribute greatly to plant health and productivity in a variety of direct and indirect ways. Direct mechanisms include enhancing nutrient availability, for example, through phosphate mobilization, nitrogen fixation, or iron acquisition, allowing plants to grow in soils. poor in nutrients. RPCPs can also affect plant growth through the production of plant hormones, such as auxins and cytokinins, or of enzymes and compounds that modify plant hormonal networks. In parallel, they can also contribute to growth by supporting plant health through direct biological control through the production of compounds harmful to neighboring phytopathological microorganisms. Indirectly, various RPCPs activate induced systemic resistance (RSI) which enhances immunity in a comprehensive way, making plants more resistant against a variety of diseases and pests. They can also protect plants against abiotic stresses, such as drought and salinity. Therefore, RPCPs can serve as an alternative to chemical fertilizers and pesticides.
    [0014] Several bacterial genera that act as RPCPs are known in the state of the art, for example, Pseudomonas, Bacillus and Azospirillum. Additionally, numerous strains of the genus Streptomyces have also been reported to promote plant growth. Within the phylum of Gram-positive Actinobacteria, Streptomyces is the largest genus (more than 600 species) and is known for its ability to produce various secondary metabolites, many of which have antibiotic activities that are used in medicine. In fact, these antibiotic-producing bacteria can exert biological control in the soil. In addition to antibiotics, they can also produce a large number of other bioactive metabolites, including auxin compounds such as indole butyric and indoleacetic acids, volatile organic (VOC) that stimulate plant growth directly and indirectly.
    [0016] In particular, indole butyric and indoleacetic acids and other auxins are used to initiate root formation in a procedure called micropropagation, in in vitro plant tissue culture . It is an asexual multiplication or propagation technique based on the organogenetic potential of plant cells, determined by culturing in vitro on appropriate substrates. Auxins can be used to cause the formation of masses of undifferentiated cells called calluses, and this is often used as a first step in the micropropagation process because by exposure to these auxin hormones, callus cells can be induced to form other tissues such as roots.
    [0018] Indole butyric and indoleacetic acids are frequently used to promote the rooting of cuttings, but since they are insoluble in water, they must be used dissolved in ethanol, which is a clear handicap for their general use.
    [0019] Actinobacteria have frequently been found as one of the five most dominant bacterial phyla in soils, occupying one tenth of the total microbiome. Within this, plants have a selective effect on the composition of microbial communities in the rhizosphere and in the endosphere. While soil geochemistry has been shown to be the main driver of microbial diversity, the composition of the microbial community of various plant and crop species differs greatly from the diversity found in soil samples without growing plants. Actinobacteria occupy an important part of the microbial communities in the roots of plants, although their abundance varies greatly between species. Furthermore, the root endosphere is significantly richer in Actinobacteria for several of these plant species than bulk soil and the rhizosphere compartment, indicating that these organisms have evolved adequate mechanisms to colonize and reside in the endosphere. A comparative analysis of microbial communities reveals that Actinobacteria in the above-ground endosphere are twice less abundant, and may not be present in plant leaves.
    [0021] Studies focusing on soil streptomycetes have shown that, as diversity among plant hosts (for grasses and legumes) increases, streptomycete communities become different, suggesting host-specific selectivity over the composition of the plant. streptomycete on the ground. It is confirmed that, among Actinobacteria, the bacterial sequences that belong to the genus Streptomyces are dominant and are usually found in the endosphere, but are absent in the control of incubated wood in the soil, which implies that there is an active recruitment by the roots. Therefore, the strains belonging to the Streptomycetaceae family can be selectively, but not exclusively, mobilized from the soil to the roots of the plants of many species, which shows that microbial trafficking in the roots of each crop or plant species it must be evaluated within its specific environment. By classical cultivation methods, thousands of strains belonging to the genus Streptomyces have been isolated from plant environments, soil or even compost.
    [0023] Host interactions with bacterial strains require the establishment of a specific chemical environment, a kind of niche, to be colonized by the bacteria. This niche includes the interface between the root and the soil, but bacterial strains can also colonize the interior of the root and establish endophytic populations that can even reach all the main organs of the plants through the vascular system. Although streptomycetes are present in the rhizosphere and / or endosphere of plants, it is known little about how and where root colonization occurs. The specific sporadic and filamentous life form of the strains within the genus Streptomyces is believed to provide a competitive advantage over other microorganisms in and around the roots. While the sporulated lifestyle helps streptomycetes survive harsh environments and harsh conditions, the ability to form multiple hyphae offers the benefit of easily colonizing plant roots and cells.
    [0025] On the other hand, when plants are infected by a fungus, they synthesize an enzyme called chitinase that is responsible for degrading chitin, the main component of the fungal cell wall. Not all plants produce chitinase, but it has been verified that the presence of glucosamine (chitosan monomer) in the composition of the soil causes an increase in absorbent trichomes, which leads to an increase in the vigor of the plant. The first reaction observed is a strengthening of the apices that take on an intense green color, with the edge of the leaves slightly curled. This is due to the fact that glucosamine induces in the plant a response similar to that which it would give rise to if it tried to defend itself from an attack by fungi, nematodes or insects, which results in increased vigor. In practice, glusocamine acts as a biotic elicitor, that is, an inducer of phytoalexin production by the plant, which contributes to maintaining their plant health.
    [0027] Among the compounds generated during the fermentation of strains of the genus Streptomyces, compounds technically called "secondary metabolites", include indole butyric and indoleacetic acids, different auxins and other molecular structures with an antibiotic character that are not soluble in water (for example, natamycin and others). This solubility deficit represents a technical problem in the application and use of these secondary metabolites by plants.
    [0029] The present invention solves this technical problem by forming an inclusion complex comprising secondary metabolites (MS) produced by the fermentation of Streptomyces bacteria , in particular antimicrobial compounds and plant auxins (preferably indole butyric and / or indoleacetic acids) capable of promoting growth. from plants, with hydrolyzed chitosan amino-oligosaccharides (OSA) that can act as elicitors and stimulators of phytoalexin production. Another additional advantage of this inclusion complex is that it increases the stability of secondary metabolites (in particular, antimicrobial compounds and plant growth promoters), preventing them from being easily degraded with sun exposure during foliar application.
    [0030] Additionally, the inclusion complex described here can act as a support for strains of the genus Streptomyces, increasing the stability of these strains before their application and, additionally, providing them with a specific chemical environment that allows colonization by the bacteria of the interface between the root and the soil.
    [0032] DESCRIPTION OF THE INVENTION
    [0034] A first aspect of the present invention refers to an inclusion complex comprising:
    [0035] - at least one antimicrobial compound obtainable by fermentation with at least one strain of the genus Streptomyces,
    [0036] - at least one plant growth promoting compound, and
    [0037] hydrolyzed chitosan amino-oligosaccharides, preferably amino-oligosaccharides with a molecular weight between 500 and 2000 Dalton, which can be obtained by hydrolysis of chitosan.
    [0039] In particular embodiments of the invention, the plant growth promoting compound is an auxin, preferably indole butyric acid, indoleacetic acid or a combination of the above. In particular, this growth-promoting compound can be obtained by fermentation with at least one strain of the genus Streptomyces, which can be the same or different from that used to obtain the antimicrobial compound.
    [0041] The mechanisms by which the antimicrobial and growth-promoting action takes place are interdependent, so that many of the antimicrobial compounds generated by the strains of the genus Streptomyces, in particular those specified in the next paragraph of this document, can also act as promoters of plant growth. Consequently, the inclusion complex of the present invention can comprise at least one antimicrobial compound and plant growth promoter obtainable by fermentation with at least one strain of the genus Streptomyces, and hydrolyzed chitosan amino-oligosaccharides, preferably with a molecular weight between 500 and 2000 Daltons.
    [0043] As mentioned above, the inclusion complex comprises at least one antimicrobial compound obtainable by fermentation of at least one Streptomyces strain . Due to its high production of metabolites with antimicrobial properties, as well as its ability to generate compounds with antimicrobial and promoter of the plant growth, bacteria of the genus Streptomyces are preferably selected from the group consisting of S. albidoflavus, S. albofaciens, S. althioticus, S. amphotericinicus, S. amritsarensis, S. anandii, S. atratus, S. atrovirens, S. bambusae, S. bellus, S. cacaoi, S. cellulosae, S. chattanoogensis, S. chrestomyceticus, S. colombiensis, S. crystallinus, S. cuspidosporus, S. fabae, S. filipinensis, S. flavofungini, S. fradiae, S. gamaensis, S. gilvifuscus, S. glomeratus, S. griseiniger, S. griseochromogenes, S. griseoviridis, S. griseus, S. halstedii, S. heilongjiangensis, S. hiroshimensis, S. hundungensis, S. hygroscopicus, S. kanamyceticus, S. kasugaensis, S. koyangensis, S. kurssanovii, S. laurentii, S. lavendofoliae, S. lavendulae, S. lushanensis, S. lydicus, S. macrosporus, S. matensis, S. narbonensis, S. netropsis, S. omiyaensis, S. phytohabitans, S. platensis, S. polymachus, S. prasinus, S. psammoticus, S. rochei, S. roseosporus, S. spectabilis, S. sulfonofacie ns, S. tunisiensis, S. variegatus, S. vitaminophilus, S. zaomyceticus and a combination of the above. Most preferably, the Streptomyces bacterial genus strain is selected from the group consisting of S. rochei, S. lavendofoliae, and a combination of the foregoing.
    [0045] These strains of the genus Streptomyces can generate antimicrobial compounds such as sesquiterpenes and / or polyphenols as secondary metabolites, as well as growth promoters such as indole butyric and / or indole acetic. Additionally, these Streptomyces strains can generate antimicrobial compounds such as, for example, oxytetracycline, spiramycin, albopeptin A, albopeptin B, alpomycin, neomycin B, neomycin C, valilactone, ansathiacin, awamycin, griseofulvin, phenamide, phenelhenelfamycin, acetylophanyclinic , Albanoursin, cycloheximide, actinomycin, ambiobactin, phoromazidin A, phoromazidin B, phoromazidin C, congocidin, amphotericin, pentaene G8, gilvocarcin V, gilvocarcin M, gilvocarcin E, cactinomycin, endophenacin B, endophenacin B, endophenacin B, endophenacin B, endophenacin B , epocarbazoline B, dextranase, telomestatin, actinomycin C, pentalenolactone, 2,5-dihydroxyphenylalanine, nafcillin, arenaemycin, atramycin A, leinamycin, mithramycin, chromocyclomycin, aurantin, panamycin 621, aurantimycin, or any combination of vanamycin 621, aurantimycin A, previous.
    [0047] In particular, the strains of the genus Streptomyces mentioned in this document can metabolize antifungal and / or antibacterial compounds that, additionally, are responsible for maintaining plant health and are plant growth promoters. Consequently, the inclusion complex and the biofertilizer described in this document are very versatile products for the treatment of pathogens present in the agriculture and / or nature, since it is possible to select a specific strain of Streptomyces depending on the infection to be treated.
    [0049] In general, it is always preferred that the antimicrobial compounds and growth promoters are water soluble, since thus the advantages they provide are significantly greater by requiring a lesser amount of water for the compound to solubilize and diffuse through the soil, from the area where the treatment is applied to the area close to the roots of the plants. However, many of the active metabolites generated by Streptomyces bacteria (in particular, many antimicrobial compounds and growth promoting compounds such as indole butyric acid or indoleacetic acid) have low water solubility. The inclusion complex described here represents a great advantage since it increases the solubility of these compounds.
    [0051] In a particularly advantageous and unexpected way, the inclusion complex of the present invention is maintained even for very low concentrations of the antimicrobial compounds and plant growth promoters included in said complex, and notably increases the solubility in water of the new preparations, even after consecutive dilutions, allowing the new biofertilizers to be applied to treat fungi and / or bacteria.
    [0053] Unlike chitosan which, being a macromolecule, is not soluble in water; The hydrolyzed chitosan aminooligosaccharides used in the present invention are soluble in water. These amino-oligosaccharides can be obtained by chemical or enzymatic hydrolysis treatment of chitosan, have a molecular weight of less than 2000 Daltons, preferably between 500 and 2000 Daltons, and are essential when forming water-soluble inclusion complexes described in this document.
    [0055] Among the hydrolyzed amino-oligosaccharides of chitosan (OSA) and secondary metabolites (MS), in particular, antimicrobial compounds such as sesquiterpenes or polyphenols and growth promoters such as indole-butyric or indoleacetic acid, the formation of inclusion complexes is evidenced. Due to the insertion reactions and the formation of AOS-MS inclusion complexes (see the proposed mechanism in Figure 1), the new complex that is formed is completely soluble in water and, additionally, new bands can be observed in the FTIR associated with the formation of the inclusion complex (see figure 2).
    [0056] Therefore, it can be established that among the hydrolyzed chitosan amino-oligosaccharides (OSA) and secondary metabolites (MS) obtainable by fermentation of strains of bacteria of the genus Streptomyces, in particular, antimicrobial compounds such as sesquiterpenes or polyphenols, and growth-promoting compounds such as indole butyric or indoleacetic acid, inclusion complexes are formed. The resulting product is a totally water-soluble poly-cation (AOS-MS) +, which allows to dramatically increase the solubility of antimicrobial compounds and plant growth promoters, generally insoluble in water.
    [0058] The present invention also refers to a process for obtaining the inclusion complex described in this document, wherein said process comprises:
    [0059] a) aerobic fermentation of one or more strains of bacteria of the genus Streptomyces in a culture medium to obtain secondary metabolites that comprise compounds with antimicrobial properties and compounds that promote plant growth such as indole butyric acid and / or indoleacetic acid;
    [0060] b) separating the bacteria from the rest of the fermentation mixture, obtaining a solid biomass that mainly comprises Streptomyces bacteria , and a liquid phase that comprises the secondary metabolites generated during fermentation; and
    [0061] c) forming an inclusion complex by adding hydrolyzed chitosan amino-oligosaccharides, preferably with a molecular weight between 500 and 2000 Dalton, to the liquid phase separated in step b) and stirring by ultrasound.
    [0063] In the procedure described here, fermentation is carried out aerobically, that is, in a closed system with control of the air that is introduced. In this way cumbersome anaerobic procedures are avoided, which must always be carried out in sealed environments. In this fermentation process, a culture medium comprising oligosaccharides can be used. For example, a conventional culture medium can be used such as that comprising malt extract, tryptone, soluble starch, glucose, yeast extract, and calcium carbonate in distilled water.
    [0065] In stage a) of aerobic fermentation, secondary metabolites generated by the strains of the genus Streptomyces present in the medium are obtained. In this patent application it is understood that the secondary metabolites (MS), which include the compounds with antimicrobial activity and growth promoters mentioned above in this document, are those compounds generated by fermentation of the Streptomyces strain that are present in the liquid phase obtained in step b) of the process of the invention and, more preferably, in the enriched liquid phase obtained after an additional step of separation of the oligosaccharides that may have remained from the culture medium used . In particular, when this oligosaccharide separation has taken place by precipitation of oligosaccharides with phosphate buffer, more specifically, with 0.02 M phosphate buffer at pH 7.
    [0067] In particular embodiments of the present invention, the aerobic fermentation takes place in the presence of a suitable culture medium (optimized) for the specific strain being used. In this way, the growth of Streptomyces bacteria is favored during the fermentation procedure and, when the inclusion complex is used as a support for the same strains in the biofertilizer described here, during the treatment of the soil where this product is applied. In particular, when using S. rochei bacteria , the optimized culture medium can comprise 2.5 g / l malt extract, 4 g / l tryptone, 5 g / l soybean meal, 3 g / l glucose, 25 g / l soluble starch, 3 g / l yeast extract and 2.5 g / l calcium carbonate in distilled water.
    [0069] The aerobic fermentation stage can take place under the usual conditions for this type of procedure. In particular, fermentation can take place at a temperature between 15 and 35 ° C, for a period between 7 and 15 days.
    [0071] After the fermentation period, the bacteria are separated from the rest of the fermentation mixture. This separation process can be carried out by any conventional solid-liquid separation process in the technical field of the invention. Preferably, the separation can be carried out by centrifugation, so that a solid biomass is obtained that mainly comprises the Streptomyces bacteria and, on the other hand, the supernatant (liquid) that contains the secondary metabolites (MS) in the fermentation, in particular , antimicrobial compounds and plant growth promoters.
    [0073] When required, the liquid supernatant (liquid phase) comprising the MS can be purified and enriched for secondary metabolites, including antimicrobial compounds and plant growth promoters. This enrichment step can take place by precipitation of excess oligosaccharides with 0.02 M phosphate buffer at pH 7 and stirring by means of ultrasound for at least 30 min, applying
    [0076] the ultrasounds in pulses of 5 min, spaced by periods without shaking also of 5 min.
    [0078] In order to reduce the moisture content of the solid biomass separated from the fermentation medium, it can be subjected to a drying process at a maximum temperature of 35 ° C or lyophilization, until obtaining a moisture content equal to or less than 5% by weight with respect to the total weight of the biomass. In this way, the stability of the bacteria is increased, since if they are left in the fermentation medium, they can become lysed due to lack of food. However, after the drying or lyophilization process they go into dormancy and are kept for a longer period of time.
    [0080] The procedure for obtaining the inclusion complex described here may comprise an additional stage, prior to the formation of the complex, where hydrolyzed chitosan amino-oligosaccharides (OSA) with molecular weight between 500 and 2000 Dalton are obtained by enzymatic hydrolysis or chemistry, chitosan (macromolecule with a molecular weight greater than 10 kDa). For example, this step can be carried out by hydrolysis with an acid such as citric or acetic acid at a temperature between 45-55 ° C, to form polycations of hydrolyzed amino-oligosaccharides of chitosan.
    [0082] The formation of the inclusion complex is carried out by shaking by ultrasound a mixture comprising secondary metabolites (MS) obtained from fermentation with Streptomyces bacteria (in particular antimicrobial compounds and plant growth promoting compounds such as indole butyric acid and / or acid indoleacetic acid), and chitosan hydrolyzed aminooligosaccharides. The formation of the inclusion complex is promoted by the application of ultrasound, preferably with a frequency between 20,000 and 60,000 Hz, for at least 30 min. Preferably, the ultrasound is applied in pulses of 5 min, spaced by periods without agitation also of 5 min. In relation to the above, the inventors have observed that the application of ultrasound under the conditions indicated above increases the speed of complex formation and gives rise to a much more intimate interaction between the different components (AOS and MS) than if another is used. type of shaking.
    [0083] Preferably, the ratio between secondary metabolites (MS) and amino-oligosaccharides (OSA) comprised in the inclusion complex described here can vary between 1:50 and 1: 6, expressed in weight of DM versus weight of OSA, where The MS value can be obtained from a sample of the liquid phase obtained after the separation of the biomass, after subjecting said sample to the enrichment treatment by separation of oligosaccharides described in this document and, subsequently, to a drying process to obtain the dry matter in the enriched liquid phase.
    [0085] The solution comprising the inclusion complex obtained after step c) of the procedure described herein can be used directly in the treatment of fungal or bacterial infections in agriculture or in nature. In particular, when the application is made by foliar or by contact.
    [0087] In other particular embodiments of the invention, the process for obtaining the inclusion complex comprises a step d), where drying takes place at low temperature (maximum 35 ° C) or lyophilization of the inclusion complex, giving rise to a solid product with a maximum humidity of 5% by weight with respect to the total weight. In this way the volume of the final product is reduced, which results in a reduction in costs associated with transport. In particular embodiments of the invention, the solid inclusion complex can be subjected to a physical post-treatment such as sieving, granulating or pelleting depending on whether it is desired to obtain an inclusion complex in the form of granules, pellets or, preferably, in powder form.
    [0089] The inclusion complex obtained by the process described in this document is also an object of the present invention.
    [0091] Additionally, the present patent application also refers to a biofertilizer with antimicrobial properties that comprises:
    [0092] - one or more strains of bacteria of the genus Streptomyces, in particular strains capable of generating secondary metabolites with antimicrobial properties; and
    [0093] - the inclusion complex described in this document.
    [0095] Thus, the present invention provides a new biofertilizer with antimicrobial properties (antifungal and / or antibacterial) that allows a sustainable growth of plants, reducing the risks associated with other phytosanitary products used to date and improving the management of agricultural resources. Among the fundamental objectives of the present invention it is worth highlighting the conservation and protection of the environment, as well as promoting the efficiency of natural resources, through the restoration of soil biodiversity and the promotion of ecosystems and the natural endosphere.
    [0096] The invention described in this document contributes to alleviating a problem in the protection of soil and crops in general. An improved biofertilizer is sought, aimed at the use of natural compounds that have antifungal and / or antibacterial activities, or act as rooters, growth promoters and elicitors for the formation of phytoalexins. In a particularly advantageous way, the formation of the inclusion complex between these components significantly improves their bioavailability and, consequently, provides a biofertilizer that can be a natural alternative to currently existing phytosanitary products, which affects the prevention of risks and the improvement management and sustainability of natural resources.
    [0098] In relation to the above, an additional advantage of the biofertilizer of the invention is that it is not necessary to incorporate surfactants or other chemical compounds to improve the solubility or bioavailability of the bioactive compounds.
    [0100] In addition to the aforementioned increase in the bioavailability of the different active components that are part of the biofertilizer, the inclusion complex fulfills other functions that are essential for the activity of the biofertilizer that is described in the present patent application. In particular, this inclusion complex provides a solid support where Streptomyces bacteria can remain stable until the use of the biofertilizer; It is beneficial for plant growth and helps it resist periods of stress. and contributes to the establishment of the so-called specific chemical environment, which allows colonization by strains of the genus Streptomyces of the interface between the root and the soil.
    [0102] The biofertilizer of the present invention is a natural product, harmless to human health and the environment, with fungicidal and / or antibacterial capacity. More specifically, this biofertilizer can be used for the treatment or prevention of the main diseases that affect crops and trees, as a solution to the complex problem of phytosanitary products and their harmful effects that affect both growing areas and numerous bodies of water.
    [0104] On the other hand, the presence of bacteria of the genus Streptomyces in the biofertilizer described here provides the following additional advantages:
    [0105] - These bacteria can be found naturally in the soil, so their addition does not represent the introduction of foreign bacteria;
    [0108] - They are harmless for people, animals and the environment in general (Risk group 1);
    [0109] - In particular the strains mentioned as preferred in this document (see below), are capable of producing antimicrobial metabolites and, additionally, plant growth promoting metabolites.
    [0111] The bacteria of the genus Streptomyces present in the biofertilizer described here may be the same as those used in the process for obtaining the inclusion complex comprised in said biofertilizer. The use of the same strains makes it possible to reinforce and prolong the antimicrobial activity against a specific type of pathogen, since after the application of the biofertilizer, the bacteria will generate the same metabolites included in the inclusion complex.
    [0113] Preferably, the Streptomyces bacteria present in the biofertilizer of the present invention are selected from the group consisting of S. albidoflavus, S. albofaciens, S. althioticus, S. amphotericinicus, S. amritsarensis, S. anandii, S. atratus, S. atrovirens, S. bambusae, S. bellus, S. cacaoi, S. cellulosae, S. chattanoogensis, S. chrestomyceticus, S. colombiensis, S. crystallinus, S. cuspidosporus, S. fabae, S. filipinensis, S. flavofungini, S. fradiae, S. gamaensis, S. gilvifuscus, S. glomeratus, S. griseiniger, S. griseochromogenes, S. griseoviridis, S. griseus, S. halstedii, S. heilongjiangensis, S. hiroshimensis, S. hundungensis, S. hygroscopicus, S. kanamyceticus, S. kasugaensis, S. koyangensis, S. kurssanovii, S.
    [0115]
    [0117] In preferred embodiments of the present invention, the biofertilizer described here has a humidity between 5% and 15%, a percentage expressed by weight with respect to the total weight of the biofertilizer.
    [0118] The present invention also refers to a process for obtaining the biofertilizer described in this document, which comprises mixing strains of Streptomyces bacteria with the inclusion complex, in particular when it has been obtained by the process described in this patent application. Preferably, the biofertilizer is obtained by mixing between 10% -50% of the solid biomass isolated in stage b) of the procedure for obtaining the inclusion complex (AOS-MS), and subjected to a drying process at a maximum temperature of 35 ° C or lyophilization until reducing the water content at max.
    [0119] 5% by weight, with between 90% -50% of the inclusion complex (AOS-MS) obtained after step d) of the procedure described above, where these percentages are expressed by weight with respect to the total solid weight of the biofertilizer.
    [0121] In those embodiments in which the inclusion complex is dissolved in a liquid, in particular the medium obtained after fermentation or, alternatively, an aqueous solution obtained after dissolving the solid inclusion complex (AOS-MS) in water, the mixture between the bacteria and the inclusion complex will be carried out in situ, that is, at the time of applying the biofertilizer for the treatment of fungal or bacterial infections in agriculture or, in general, in nature. Preferably this application is carried out in a root manner.
    [0123] However, in preferred embodiments of the present invention, the mixture between the Streptomyces bacteria and the inclusion complex is a solid mixture, that is, both components are in the solid state. This solid mixture can be carried out, under aerobic conditions, in a rotating container that allows an intimate mixture between the inclusion complex, in particular that obtained by the procedure described in this document, including a lyophilization step to obtain the AOS-MS complex in powder form; with Streptomyces bacterial strains , in particular those comprised in the solid biomass separated from the culture medium of the fermentation by centrifugation and subjected to a drying process at a maximum temperature of 35 ° C or lyophilization, to reduce the humidity to a maximum of 5 % by weight with respect to the total weight of the biomass.
    [0125] According to this embodiment, the inclusion complex provides a solid support where the Streptomyces bacteria can remain in a stable manner until the use of said product in agricultural soil or, in general, in nature. Once applied to the field, Streptomyces bacteria are rapidly released from the AOS-MS complex due to the solubility of the inclusion complex, while MS and AOS form a specific chemical environment for bacteria, making it easier for them to colonize the endosphere and can continue to generate compounds with antimicrobial activity, thus increasing the effectiveness of the treatment against pathogens such as fungi and / or bacteria.
    [0127] The procedure for obtaining the biofertilizer described in this patent application can be carried out in three separate containers: a first aerobic fermenter, with
    [0130] introduction of air from outside where aerobic fermentation takes place with the Streptomyces strains; a second container where the formation of the inclusion complex (AOS-MS) is carried out through the application of ultrasound; and a third rotating container where the solid mixture is made between the Streptomyces bacteria and the inclusion complex (both previously subjected to a freeze-drying or low-temperature drying process).
    [0132] The biofertilizer obtained by the process described in this document is also an object of the present invention.
    [0134] Finally, the present invention refers to the use of the inclusion complex or biofertilizer with antimicrobial properties, in particular when they are obtained by one of the procedures described here, for the treatment of pathogens present in agriculture and / or in the nature, as well as to improve the physical-chemical properties of the soil. In particular embodiments, the invention relates to the use of any of the products that are the object of this invention (inclusion complex or biofertilizer) for the treatment of fungi of the genus Aspergillus, Fusarium, Alternaria, Phitophtora, Penicillium; and / or bacteria of the genus Xylella fastidiosa, Pseudomonas.
    [0136] The products described in this patent application can be applied differently. Thus, in particular embodiments of the present invention, the inclusion complex can be applied foliarly to plants and / or trees. Preferably, this complex is applied dissolved in an aqueous solution, preferably water such as, for example, irrigation water. In particular, it is preferred that the range of working concentrations of the inclusion complex is comprised between 6 g / liter in the concentrated solution and 6 g / 100 liters of aqueous solution.
    [0138] On the other hand, the biofertilizer can be applied on the ground in solid form, for example in powder, granules or pellets. Later, due to the action of rain or irrigation water, the inclusion complex (AOS-MS) will dissolve, incorporating the different components of the biofertilizer into the soil.
    [0140] In other preferred embodiments, the solid biofertilizer can be dissolved prior to its application, either foliar or root, in a certain amount of water. In particular, it is preferred to dissolve an amount of biofertilizer such that the final solution to be applied comprises between 10 g / liter in the concentrated solution to 10 g / 100 liters of aqueous solution of the inclusion complex (AOS-MS).
    [0143] As mentioned above, the biofertilizer can be obtained in situ by mixing the Streptomyces strains with the inclusion complex in solution, either in the liquid phase (supernatant) obtained after separating the bacteria from the fermentation medium, or in an aqueous solution obtained. dissolving the solid inclusion complex in water.
    [0145] It is particularly beneficial if this treatment is applied by means of irrigation water, after sowing, since it will protect the germination of the seeds from the attack of fungi found in the soil.
    [0147] BRIEF DESCRIPTION OF THE FIGURES
    [0149] Figure 1: By way of illustration, the proposed (non-binding) mechanism for the formation of an inclusion complex (IV) formed by (I) hydrolyzed amino-oligosaccharides of chitosan (polycation) with (II) polyphenols and (III) is shown indoleacetic, where (II) and (III) can be obtained as secondary metabolites in fermentation with one or more of the Streptomyces strains specified in this document.
    [0151] Figure 2: FTIR of the AOS-MS inclusion complex obtained in Example 1.
    [0153] EXAMPLES OF REALIZATION
    [0155] EXAMPLE 1: Procedure for obtaining an inclusion complex (AOS-MS) and bacteria of the species S. rochei
    [0157] In one liter of water and with stirring, 20 g of high molecular weight chitosan (industrial) were added and, subsequently, 20 g of acetic acid were added until total solubilization was achieved, at a temperature of 50 ° C. Subsequently, they were chemically hydrolyzed using 30% H 2 O 2 (55 ml) until obtaining OSA with molecular weights between 500 and 2000 g / mol.
    [0159] On the other hand, fermentation was carried out for 7 days and subsequent centrifugation of a S. rochei strain in the culture medium. 1 liter of culture medium was started (see below) and distributed in 4 Erlenmeyer flasks of 250 ml each. The S. rochei strain was then inoculated with 1x105 CFU / mL. Once the fermentation period was over, the biomass with the strains was separated and approx. 200 ml of supernatant in which they were secondary metabolites MS. Subsequently, the liquid phase (supernatant) was enriched with the active products (that is, antimicrobial compounds and plant growth promoters) by precipitation of the oligosaccharides present in the supernatant with 0.02M phosphate buffer at pH 7. The amount of MS present in the enriched supernatant by weighing a small sample before and after lyophilization and approximately 1 gram was obtained.
    [0161] Culture medium for S. rochei:
    [0166] To make the inclusion complex, the supernatant obtained after the separation of the biomass was mixed with a quantity of aqueous solution containing 6 g of OSA for each gram of DM present in the supernatant. This mixture was sonicated for 5 minute periods, separated by 5 minute periods without agitation, for a total of 30 minutes.
    [0168] For the foliar application of the inclusion complex (AOS-MS), each liter of this complex formed in the ratio (6 to 1 in g / l), was dissolved in 50 liters of water and then nebulization was carried out on the plant .
    [0170] EXAMPLE 2: Procedure for obtaining a biofertilizer comprising the AOS-MS inclusion complex and bacteria of the species S. lavendofoliae
    [0172] In one liter of water and with stirring, 20 g of high molecular weight chitosan (industrial) were added and 20 g of acetic acid was added until total solubilization was achieved, at a temperature of 50 ° C; Subsequently, their chemical hydrolysis was carried out using 30% H 2 O 2 (55 ml) and until obtaining OSA with molecular weights between 500 to 2000 g / mol.
    [0175] As in the previous case, by means of the fermentation and subsequent centrifugation of a strain of S. lavendofoliae , a supernatant comprising the secondary metabolites MS was obtained on the one hand and, on the other hand, the centrifuged biomass was obtained. Subsequently, the supernatant was subjected to an oligosaccharide separation step by precipitation with a 0.02M phosphate buffer at pH 7, to enrich the supernatant with the active compounds. The amount of MS present in the enriched supernatant was determined by weighing a small sample of supernatant before and after lyophilization and approximately 1 gram was obtained.
    [0177] The inclusion complex was made by mixing 6 g of OSA for each gram of DM in one liter of water. The AOS-MS mixture was sonicated by ultrasound for 30 minutes in periods of 5 minutes of application and 5 minutes of rest.
    [0179] The above-described process was repeated twice, to serve as the starting product for Examples 2.1 and 2.2 detailed below.
    [0181] Example 2.1: The AOS-MS inclusion complex obtained as described above was lyophilized until obtaining a solid with a maximum humidity of 5% by weight relative to the total weight. Subsequently, the lyophilized AOS-MS complex was mixed with S. lavendofoliae bacterial strains , in a weight ratio (9: 1), that is: 9 g of AOS-MS per 1 gram of previously lyophilized biomass. The humidity of the gram of lyophilized biomass was maximum 5% by weight. The bio-fertilizer (solid) thus obtained was stored in refrigeration at 4 ° C until its application, which can be both foliar and root.
    [0183] Example 2.2: On the other hand, the AOS-MS complex in solution obtained as described above was mixed with strains of bacteria S. lavendofoliae in the liquid product just before being used in agriculture. To do this, all the centrifuged biomass obtained after fermentation (see above) and dried at a maximum temperature of 35 ° C was mixed with the AOS-MS inclusion complex solution. Each liter of the biofertilizer solution obtained was diluted in 50 liters of water and the root application was carried out on the ground.
    [0185] Example 3. Characterization of the inclusion complex and / or the fertilizer of the AOS-MS + complex by FTIR spectrometry and solubility tests.
    [0188] The characterization of the inclusion complex resulting from Example 1, as well as the biofertilizer obtained in Example 2.1, was carried out by FTIR spectrometry and solubility tests. The results obtained are very similar, so only the data obtained in relation to the inclusion complex of example 1 are included here.
    [0190] First, the FTIR spectrum of example 1 (AOS-MS) shows the formation of an inclusion complex AOS-MS, cationic complex, by the presence of a band of chitosan oligomers (AOS) at approx. 3227 cm-1 determined by u (OH) overlapped with us (N-H). Additionally, the following characteristic bands are observed:
    [0191] - 1457 cm-1 and 1441 cm-1 - indole butyric,
    [0192] - 1403 cm -1 - indole butyric and chitosan oligomers;
    [0193] - 1386 cm-1 - chitosan oligomers, attributed to 5 (-CH3) and
    [0194] - 1255 cm-1 - indistinctly, indole butyric or amide III (axial deformation vibrations of C-N bond and C = O bending vibrations).
    [0196] Secondly, the formation of the cationic inclusion complex AOS-MS + is evidenced, based on solubility tests of the following mixtures subjected to ultrasound stirring:
    [0197] 1) 6 g of OSA for each gram of lyophilized DM (obtained as described in example 1) in one liter of water is totally soluble,
    [0198] 2) The mixture formed by 9 g of AOS-MS and 1 gram of biomass previously lyophilized in 1 liter of water, is totally soluble in water.
    [0199] 3) The mixture that contains the active products of the MS, is not totally soluble in water, even in concentrations of mg / L. These compounds are only fully solubilized when the inclusion complex AOS-MS- is formed.
    2
    权利要求:
    Claims (15)
    [1]
    1. - An inclusion complex comprising:
    - at least one antimicrobial compound obtainable by fermentation with at least one strain of the genus Streptomyces,
    - at least one plant growth promoting compound, and
    - chitosan hydrolyzed amino-oligosaccharides.
    [2]
    2. - The inclusion complex according to claim 1, where the hydrolyzed amino-oligosaccharides of chitosan have a molecular weight between 500 and 2000 Dalton.
    [3]
    3. - The inclusion complex according to any one of claims 1 to 2, wherein the plant growth promoting compound is selected from the group consisting of indole butyric acid, indoleacetic acid and a combination of the foregoing.
    [4]
    4. - The inclusion complex according to any one of claims 1 to 3, wherein the strains of the genus Streptomyces are selected from the group consisting of S. albidoflavus, S. albofaciens, S. althioticus, S. amphotericinicus, S. amritsarensis, S. anandii, S. atratus, S. atrovirens, S. bambusae, S. bellus, S. cacaoi, S. cellulosae, S. chattanoogensis, S. chrestomyceticus, S. colombiensis, S. crystallinus, S. cuspidosporus, S. fabae, S. filipinensis, 5. flavofungini, S. fradiae, S. gamaensis, S. gilvifuscus, S. glomeratus, S. griseiniger, S. griseochromogenes, S. griseoviridis, S. griseus, S. halstedii, S. heilongjiangensis, S. hiroshimensis, S. hundungensis, S. hygroscopicus, S. kanamyceticus, S. kasugaensis, S. koyangensis, S. kurssanovii, S. laurentii, S. lavendofoliae, S. lavendulae, S. lushanensis, S. lydicus, S. macrosporus, S. matensis, S. narbonensis, S. netropsis, S. omiyaensis, S. phytohabitans, S. platensis, S. polymachus, S. prasinus, S. psammoticus, S. rochei, S. rose osporus, S. spectabilis, S. sulfonofaciens, S. tunisiensis, S. variegatus, S. vitaminophilus, S. zaomyceticus and a combination of the above.
    [5]
    5. A process for obtaining an inclusion complex as described in any one of claims 1 to 4, wherein the process comprises:
    a) aerobic fermentation of one or more strains of bacteria of the genus Streptomyces in a culture medium to obtain antimicrobial secondary metabolites and plant growth promoters;
    b) separating the bacteria from the rest of the fermentation mixture; and
    c) forming an inclusion complex by adding hydrolyzed chitosan amino-oligosaccharides to the liquid phase separated in step b) and stirring by ultrasound.
    [6]
    6. - The procedure for obtaining the inclusion complex according to claim 5, wherein step c) takes place for at least 30 min, applying ultrasound in pulses of 5 min, spaced by periods without stirring also of 5 minutes.
    [7]
    7. - The procedure for obtaining the inclusion complex according to any one of claims 5 or 6, where the ratio between secondary metabolites (MS) and hydrolyzed chitosan aminooligosaccharides (AOS) varies between 1:50 and 1: 6, expressed in weight of DM versus weight of OSA.
    [8]
    8. - The process for obtaining the inclusion complex according to any one of claims 5 or 6, comprising a step d) where the inclusion complex is lyophilized or dried at a maximum temperature of 35 ° C.
    [9]
    9. - A biofertilizer with antimicrobial properties that comprises:
    - one or more strains of bacteria of the genus Streptomyces capable of generating antimicrobial compounds; and
    - the inclusion complex as described in any one of claims 1 to 4.
    [10]
    10. - The biofertilizer according to claim 9, wherein the strain of Streptomyces bacteria is selected from the group consisting of S. albidoflavus, S. albofaciens, S. althioticus, S. amphotericinicus, S. amritsarensis, S. anandii, S. atratus , S. atrovirens, S. bambusae, S. bellus, S. cacaoi, S. cellulosae, S. chattanoogensis, S. chrestomyceticus, S. colombiensis, S. crystallinus, S. cuspidosporus, S. fabae, S. filipinensis, S . flavofungini, S. fradiae, S. gamaensis, S. gilvifuscus, S. glomeratus, S. griseiniger, S. griseochromogenes, S. griseoviridis, S. griseus, S. halstedii, S. heilongjiangensis, S. hiroshimensis, S. hundungensis , S. hygroscopicus, S. kanamyceticus, S. kasugaensis, S. koyangensis, S. kurssanovii, S.

    [11]
    11. - The biofertilizer according to any one of claims 9 to 10, where the humidity is between 5% and 15%, a percentage expressed by weight with respect to the total weight of the biofertilizer.
    [12]
    12. - A process for obtaining a biofertilizer as described in any one of claims 9 to 11, which comprises mixing bacteria of the genus Streptomyces with an inclusion complex as described in any one of claims 1 to 4.
    [13]
    13. - Use of an inclusion complex as described in any one of claims 1 to 4, or of a biofertilizer as described in any one of claims 9 to 11, for the treatment of pathogens present in the agriculture and / or nature.
    [14]
    14. - The use according to claim 13, for the treatment of:
    i) fungi selected from the group consisting of fungi of the genus Aspergillus, Fusarium, Alternaria, Phitophtora, Penicillium and a combination of the above;
    ii) bacteria selected from the group consisting of bacteria of the genus Xylella fastidiosa, Pseudomonas and a combination of the above; or
    iii) a combination of any of the fungi and bacteria mentioned above.
    [15]
    15. - Use according to any one of claims 13 to 14, to further promote the growth of plants.
    2
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    同族专利:
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    ES2799848B2|2021-05-18|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    KR100967143B1|2009-05-07|2010-07-05|김의수|Fertilizer for promoting plant growth and improving fertilization of soil and thereof method thereof|
    US20130255338A1|2012-03-27|2013-10-03|Agrinos, AS|Microbial Composition Comprising Liquid Fertilizer and Processes for Agricultural Use|
    CN108300681A|2018-04-20|2018-07-20|海南大学|One plant of Lou Che Shi streptomycete and its application|CN108587981A|2018-05-30|2018-09-28|西南大学|Multi-functional molten algae streptomycete Streptomyces amritsarensis and its application|
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