Microorganism biocapsule with fungal coating, method for obtaining same and associated uses

Abstract

The present invention relates to a microorganism biocapsule comprising: (a) a first filamentous fungus, and (b) a microorganism, wherein the biocapsule is coated with a second filamentous fungus layer. The invention further discloses a method for obtaining a biocapsule in accordance with the invention, wherein the method comprises the following steps: (a) providing a microorganism biocapsule comprising a first filamentous fungus, and a microorganism, (b) cultivating the microorganism biocapsule of step (a) in the presence of spores of a second filamentous fungus in a specific culture medium for the growth of the second filamentous fungus. Lastly, the invention discloses uses of the biocapsule for obtaining fermented beverages, clarifying liquids in industrial processes, performing bioremediation, producing alcoholic beverages, or providing high added value secondary metabolites as drugs, antibiotics or biofuels.

Description

[0001] FUNGALLY COATED MICROORGANISM BIOCAPSULES, PROCESS OF OBTAINING THEM AND ASSOCIATED USES

[0002] DESCRIPTION

[0003] FIELD OF INVENTION

[0004] The present invention falls within the field of biotechnology. More specifically, the object of the invention relates to a fungal-coated cell immobilization system, the process for obtaining it, and its associated uses.

[0005] BACKGROUND OF THE INVENTION

[0006] Cell immobilization systems aim to confine intact cells within a defined spatial region while preserving their biological activity. Cells contained in immobilization systems offer numerous advantages over freely suspended cells. For example, in fermentation processes, they facilitate the recovery and reuse of yeast, increase tolerance to alcohol and certain inhibitors, and simplify product recovery. Within immobilization systems, there are numerous types where cells are attached to a carrier substrate or a water-insoluble support. This support can be composed of materials such as polysaccharides (cellulose, dextran, agarose derivatives), proteins (gelatin, albumin), or synthetic polymers (resins, polystyrene, polyurethane).There are also entrapment methods where the cells are retained by a polymeric matrix (polyacrylamide, carrageenan), or encapsulated in semipermeable synthetic polymer microcapsules, in liposomes, membranes or reverse micelles.

[0007] Among cell immobilization systems, microorganism biocapsules (MBs) are a type of cell immobilization technology (CIT) that uses hollow granules made of filamentous fungi to support the cells (i.e., microorganisms) to be immobilized. Like other CITs, MBs simplify cell manipulation and the reuse of microorganisms in applications such as fermentations, while also increasing cell resistance to stress (physical and / or chemical) and improving metabolite production (e.g., ethanol), thus reducing costs in industrial operations. Unlike other CITs, MBs can be customized based on the following variables: filamentous fungal species, active or inactive filamentous fungus, and the specific microbial species to be immobilized.This versatility allows for diverse biotechnological uses such as bioremediation, the production of alcoholic beverages, or high value-added secondary metabolites such as drugs, antibiotics, or biofuels.

[0008] For the preparation of microorganism biocapsules (BM), a fungal spore-assisted encapsulation technique (FSA) can be used, in which the fungus and the cells to be immobilized are co-cultured in the same medium in a process in which the fungal mycelium is typically developed into small spherical granules or pellets and the cells are immobilized in a complex hyphal matrix.This assembly methodology, in the specific case of yeast encapsulation (ES 2204316 B1), has demonstrated certain limitations, such as reduced productivity, a high contamination rate in the biocapsule formation process, a limited cell retention capacity in the produced system, and the restriction to specific types of fungi and / or yeast strains that are compatible with each other and that have the ability to immobilize the yeast by binding to the fungus's phyta, which in turn restricts the applications of the encapsulation system to those uses involving yeasts, such as the manufacture of alcoholic beverages or bioethanol.

[0009] More recently, a new method for assembling BM has been developed (Lúquez-Caravaca et al. (US 63 / 411, 843; see also “Yeast cell vacuum infusion into fungal pellets as a novel cell encapsulation methodology”, Applied Microbiology and Biotechnology (2023) 107:5715-5726), which comprises the following steps:

[0010] (i) separately cultivate the filamentous fungal granules and the cells to be immobilized; (ii) mix the filamentous fungal granules and the cells to be immobilized,

[0011] (iii) infusing the cells inside the granules by means of a vacuum stage; and

[0012] (iv) cultivating the biocapsules thus obtained from filamentous fungal granules and cells (i.e., a microorganism), in a culture medium that specifically promotes the further growth of the latter.

[0013] This technique, compared to the previous co-encapsulation approach, offers significant advantages, such as higher biocapsule productivity (>35 times higher), a lower contamination rate, process scalability, a higher cell retention rate (2 times higher), and the ability to immobilize different cell types, not just yeasts, with various filamentous fungi. This last advantage broadens the spectrum of industrial applications, which can range from bioremediation to pharmaceuticals.

[0014] However, despite these promising improvements, certain limitations have been identified, including the persistent release of cells into the external environment. Therefore, there is a need in the current state of the art to provide more efficient microorganism biocapsules in terms of the degree of cell retention for use within a cell immobilization system.

[0015] BRIEF DESCRIPTION OF THE INVENTION

[0016] The present invention is aimed at overcoming the aforementioned limitation by means of a novel cell immobilization system using microbial biocapsules (MBs), where the MB comprises a hollow capsule (or one with a less dense interior) composed of a matrix of interwoven filamentous fungi, which houses a plurality of microbial cells, and where, furthermore, the MB is coated by a fungal layer that may comprise the same type of filamentous fungus or a different type of filamentous fungus. Thanks to this fungal layer, the persistent release of cells into the external environment is reduced.

[0017] Thus, in a first aspect, the invention relates to a biocapsule of microorganisms comprising: (a) a first filamentous fungus, and (b) a microorganism, wherein the biocapsule is coated with a layer of a second filamentous fungus, which may be the same as the first or different.

[0018] In a further aspect, the invention relates to a method for obtaining a biocapsule according to the present invention, wherein the method comprises the following steps: a) providing a biocapsule of microorganisms comprising a first filamentous fungus, and a microorganism, b) cultivating the biocapsule of microorganisms from step (a) in the presence of spores of a second filamentous fungus in a culture medium specific for the growth of the second filamentous fungus.

[0019] The invention also relates in another aspect to the use of a biocapsule of microorganisms according to the present invention for: (a) obtaining fermented beverages; (b) obtaining biofuels; (c) obtaining pigments; (d) obtaining organic acids; (e) obtaining pharmaceuticals; (f) obtaining antibiotics; (g) obtaining hormones; (h) clarifying liquids in industrial processes; (i) bioremediation.

[0020] DESCRIPTION OF THE FIGURES

[0021] • Figure 1. Examples of microorganism biocapsules: macroscopic images (a-b), and scanning electron microscope image (c).

[0022] • Figure 2. Diagram of (a) microorganism biocapsules (BM), and (b) microorganism biocapsules with fungal coating (BM + CF).

[0023] • Figure 3. Detail of image in conventional optical microscope (400 magnification) of the fungal covering of BM + CF, where no yeast cells are seen

[0024] • Figure 4. Sequence of breaking the BM + CF coating with a scalpel showing the detachment of retained yeast cells. Stereoscopic microscope image: bright field (a) and dark field (b).

[0025] • Figure 5. Macroscopic images of microorganism biocapsules (BM) and microorganism biocapsules with fungal coating (BM + CF) before (AF) and after (DF) an alcoholic fermentation.

[0026] • Figure 6. Cell immobilization rates (a) before (AF) and after (DF) an alcoholic fermentation using microorganism biocapsules formed with the filamentous fungus Aspergillus oryzae CECT 2095 and the untreated yeast Saccharomyces cerevisiae MYA-2451 (BM) and with the fungal coating made with the same filamentous fungus Aspergillus oryzae CECT 2095 (BM + CF); and fermentation efficiency (b). The lighter colors in (a) represent the yeasts released during the fermentation process, and the percentages represent the immobilized yeasts relative to the total. The values ​​represent the total cells counted in 100 mL of fermentation medium.

[0027] DETAILED DESCRIPTION OF THE INVENTION

[0028] As previously stated, the present invention proposes a new cell immobilization system by means of microorganism biocapsules (MB), where the MB comprises a hollow capsule (or solid ones with a less dense interior) composed of a matrix of intertwined filamentous fungi, which houses a plurality of microbial cells, and where, in addition, the MB is coated by a fungal layer that may comprise the same type of filamentous fungus or a different type of filamentous fungus.

[0029] Thus, in a first aspect, the invention relates to a biocapsule of microorganisms comprising: (a) a first filamentous fungus, and (b) a microorganism, wherein the biocapsule is coated with a layer of a second filamentous fungus, which may be the same as the first or different.

[0030] As used in the context of the present invention, the term "microorganism biocapsule" refers to a cell immobilization system shaped like a capsule, pellet, granule, or hollow sphere (or one with a less dense interior) of the filamentous fungus containing microorganism cells that may be loose within the mycelium or attached to the mycelium walls. Thus, the term "microorganism biocapsule" defines a hollow capsule or a solid capsule with a less dense interior, where the capsule is essentially spherical, and where the walls of the hollow capsule are formed by the mycelium of the filamentous fungi, forming a compact mycelium that acts as a framework or support to which the microorganism cells are attached.In the case of solid capsules (with a less dense interior) filled with cells, either from the filamentous fungus or the microorganism (e.g., yeast), the microorganism cells (e.g., yeast) are located between the fungal structures. The biocapsules of the invention are essentially spherical. However, those skilled in the art know that filamentous fungi can also grow in other shapes, for example, in biofilms or amorphous granules.

[0031] In the context of the present invention, the term “microorganism,” also known as a microbe, refers to unicellular organisms that, as such, can only be visualized under a microscope. However, those skilled in the art understand that microorganisms can also be observed without a microscope when present in large concentrations, although not individually. The term microorganism does not imply a specific taxonomic or phylogenetic affiliation, as it refers, in a non-exhaustive manner, to heterogeneous unicellular organisms that are not evolutionarily related to one another, such as bacteria, archaea (prokaryotes), microscopic algae, protozoa, and microscopic fungi (eukaryotes). In embodiments of the invention, the microorganism is selected from the group consisting of a yeast, a bacterium, an archaeon, a microalga, or any possible consortium thereof.As used herein, the term “yeast” refers to microscopic fungi ranging in size from 3 µm to 40 µm, generally unicellular (eukaryote cells), which, unlike filamentous fungi, do not form mycelia, or networks of fungi. Those skilled in the art know that yeasts are unicellular organisms whose cells do not form tissues, although they may remain attached after cell division. As used herein, the term “bacteria” refers to small prokaryotic microorganisms (between 0.5 and 5 µm) of diverse shapes (spherical, elongated, curved, helical, etc.) that differ from Archaea prokaryotes in their membrane lipid composition, among other differences. In the context of the present invention, the term “bacteria” encompasses the term “cyanobacteria” (i.e., cyanobacteria are grouped within the kingdom of bacteria).As used herein, the term “archaea” refers to a domain and / or kingdom of unicellular prokaryotic microorganisms that lack a nucleus (but possess a nucleolus) and internal membrane-bound organelles, but have a different evolutionary history from bacteria, as well as other relevant biochemical and genetic differences. As used herein, the term “microalga” refers to microscopic unicellular eukaryotic microorganisms ranging in size from 2–200 µm, which may occur individually, in chains, or in groups. As used herein, the term “consortium” may refer to any combination of the microorganisms described herein.In other words, the term “consortium” can refer to the combination of yeasts and bacteria, the combination of yeasts and archaea, the combination of yeasts and microalgae, the combination of bacteria and archaea, the combination of bacteria and microalgae, the combination of archaea and microalgae, the combination of yeasts, bacteria and archaea, the combination of yeasts, bacteria and microalgae, the combination of yeasts, archaea and microalgae, the combination of bacteria, archaea and microalgae, or the combination of yeasts, bacteria, archaea and microalgae.

[0032] In one embodiment of the invention, the microorganism is a yeast. In particular embodiments, the yeast belongs to a genus selected from the group consisting of Saccharomyces, Pichia, Wickerhamomyces, Candida, Trichosporon, Torulaspora, Metschnikowia, Lachancea, or combinations thereof. In particular embodiments, the yeast belongs to a species selected from the group consisting of Candida guillermondii, Candida utilis, Lachancea thermotolerans, Metschnikowia pulcherrima, Pichia fermentans, Pichia manshurica, Pichia membranifaciens, Pichia stipitis, Saccharomyces bayanus, Saccharomyces cerevisiae, Torulaspora delbrueckii, Trichosporon asahii, Wickerhamomyces anomalus, or combinations thereof. In one embodiment, the yeast is a flowering yeast.In a preferred embodiment of the invention, the yeast is Saccharomyces cerevisiae of the strain with internal notation G1, deposited in the “American Type Culture Collection” (ATCC) with identification number: MYA-2451. This yeast is commercially available: https: / / www.atcc.org / products / mya-2451.

[0033] In one embodiment of the invention, the microorganism is a bacteria. In particular embodiments, the bacteria belongs to a genus that is selected from the group consisting of Arthrospira, Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, Teragenococcus, Vagococcus, Weisella, Acetobacter, Gluconobacter, Basfia, Actinobacillus, Streptomyces, Carboxydothermus, Rhadobacter, Clostridium, Acinetobacter, Aminobacter, Variovorax, Arthrobacter, Massilia, Pseudomonas,

[0034] Cupriavidus and combinations thereof. In particular embodiments, the bacterium belongs to a species selected from the group consisting of Arthrospira maxima, Basfia succiniciproducens, Actinobacillus succinogenes, Streptomyces lividans, Carboxydothermus hydrogenoformans, Rhadobacter capsulatus, Clostridium acetobutylicum, Clostridium spp., Acinetobacter calcoaceticus (preferably strain JH-9), Aminobacter sp. (preferably strain MSH1), Variovorax sp. (preferably strain SRS16), Arthrobacter globiformis (preferably strain D47), Massilia sp. (preferably strain WF1), Arthrobacter sp. (preferably from strain ZXY-2), Pseudomonas stutzeri (preferably from strains GF2 or GF3), Cupriavidus sp. (preferably from strain H29) or combinations thereof.

[0035] In one embodiment of the invention, the microorganism is an archaeon. In particular embodiments, the archaeon belongs to a genus selected from the group consisting of Halobacterium, Thermococcus, Methanobrevibacter, Methanosphaera, and combinations thereof. In particular embodiments, the bacterium belongs to a species selected from the group consisting of Halobacterium salinarum, Thermococcus barosii, Thermococcus litoralis, Methanobrevibacter smithii, Methanosphaera stadtmanae, and combinations thereof.

[0036] In one embodiment of the invention, the microorganism is a microalga. In particular embodiments, the microalga belongs to a genus selected from the group consisting of Botryococcus, Chlamydomonas, Chlorella, Chroococcus, Dunaliella, Nannochloris, Nannochloropsis, Phaeodactylum, Pseudokirchneriella, Pyrocystis, Scenedesmus, Tetraselmis, Thraustochytrid, and combinations thereof. In particular modes of realization, the microalga belongs to a species that is selected from the group consisting of Botryococcus braunii, Chlamydomonas reinhardtii, Chlorella protothecoides, Chlorella pyrenoidosa, Chlorella sp., Chlorella vulgaris, Chroococcus sp., Dunaliella salina, Dunaliella tertiolecta, Nannochloris oculata, Nannochloropsis oceanica, Nannochloropsis oculata, Phaeodactylum tricornutum, Pseudokirchneriella subcapitata, Pyrocystis lunula, Scenedesmus quadricauda, ​​Tetraselmis chuii, Tetraselmis suecica, Thraustochytrid sp., and combinations thereof.

[0037] In the context of the present invention, the microbial biocapsules are capable of immobilizing millions of microbial cells. For example, in a particular embodiment of the invention where the microbial biocapsules are yeast biocapsules, it has been shown that each biocapsule, weighing between 0.01 and 0.1 mg dry weight, is capable of immobilizing from 0.85 to 8.5 million yeast cells.

[0038] As used herein, “filamentous fungus” (or “mold”) shall mean any eukaryotic organism, lacking chlorophyll, leaves, and roots, that reproduces by spores and is composed of a plurality of tubular branches called mycelia that combine to form mycelia. In one embodiment of the invention, the first and second filamentous fungi are of the same species. In another embodiment, the first and second filamentous fungi are of different species. The biocapsule of microorganisms may have applications in the food or pharmaceutical industries. Therefore, in some embodiments of the invention, it is important that the fungus be edible. Thus, the fungus may be a species generally regarded as safe (GRAS), particularly for its application in a food product.In embodiments of the invention, the first filamentous fungus and / or the second filamentous fungus belong to a genus of fungi capable of growing in pellets or spherical granules. In particular embodiments, the first filamentous fungus and / or the second filamentous fungus are fungi of a genus selected from the group consisting of Aspergillus, Rhizopus, Penicillium, Neurospora, Mucor, Mortierella, Fusarium, or combinations thereof. In embodiments of the invention, the first filamentous fungus and / or the second filamentous fungus are fungi of a species selected from the group consisting of Aspergillus sp., Aspergillus oryzae, Rhizopus sp., Penicillium sp., Neurospora intermedia, Mucor circinelloides, Mortierella isabellina, or Fusarium equiseti, or combinations thereof. In a particular embodiment of the invention, the first filamentous fungus and / or the second filamentous fungus are of the species Aspergillus oryzae.In a preferred embodiment of the invention, the first filamentous fungus and / or the second filamentous fungus are of the CECT 2095 strain, of the species Aspergillus oryzae, deposited in the “Spanish Type Culture Collection (CECT)” with identification number: CECT 2095. This strain is commercially available from the CECT collection: https: / / www.cect.org / vstrn.php?lan=es&cect=2095. Thus, in specific combinations of the microorganism biocapsule, the following combinations, among others, indicated in Table 1, can occur.

[0039] Table 1: Combinations of the microorganism biocapsule with fungal coating.

[0040] In the context of the invention, the microorganism biocapsule is coated with a layer of a second filamentous fungus, acting as a shell or covering. This fungal layer refers to the coating formed by the mycelium of the second filamentous fungus that envelops the underlying microorganism biocapsule, which is itself composed of the microorganism and the first filamentous fungus. In one particular embodiment of the invention, the encapsulation is complete. In another particular embodiment, the encapsulation is partial. In specific embodiments of the invention, the coating thickness is between 0.50 and 2.50 mm, between 1.00 and 2.00 mm, or between 1.20 and 1.50 mm. In other specific embodiments, the coating thickness is 1.00 mm, 1.10 mm, 1.20 mm, 1.30 mm, 1.40 mm, or 1.50 mm.

[0041] In microorganism biocapsules, the filamentous fungus can be provided in either an active or inactive state, depending on the context in which the invention will be used. Thus, it is advantageous for the filamentous fungus to be inactivated in applications such as winemaking and brewing, since in these cases it is beneficial to inhibit the fungus's own metabolism and avoid competition with the immobilized microorganism (for example, immobilized yeast). In other situations, it is preferable to keep the filamentous fungus active so that its own metabolism can be utilized, which may have a synergistic effect with the metabolism of the immobilized microorganism. For example, fungi, and not yeasts, are generally capable of hydrolyzing starch and lignocellulose from agricultural residues. In this regard, those skilled in the art know that some yeasts possess amylases and can therefore degrade starch.It is within the skill of the person skilled in the art to select the appropriate microorganisms depending on the specific effect of interest for particular applications. For these reasons, in embodiments of the present invention, the following situations may occur: (a) the first filamentous fungus is active and the second filamentous fungus is active; (b) the first filamentous fungus is active and the second filamentous fungus is inactive; (c) the first filamentous fungus is inactive and the second filamentous fungus is active; (d) the first filamentous fungus is inactive and the second filamentous fungus is inactive.

[0042] Methods for inactivating the first and / or second filamentous fungi are known in the prior art. Inactivation methods can be carried out by applying a physical treatment that includes pulsed high-voltage electric fields, high-to-moderate hydrostatic pressure, high shear pressure, and heat; more preferably, by autoclaving the cultured filamentous fungal granules at a temperature between 100 and 150°C (preferably 121°C), 1 atm overpressure, and for a period of time between 15 and 25 min (preferably 20 min). or by applying a chemical treatment, immersing the granules of cultivated filamentous fungi in 70% (v / v) ethanol and keeping the solution under agitation for a period of time between 1 and 5 h (preferably for 2 h) at a speed between 50 and 150 rpm (preferably 100 rpm).Finally, the solution can be washed with sterile deionized water.

[0043] In a further aspect, the invention relates to a method for obtaining a biocapsule according to the present invention, wherein the method comprises: a) providing a biocapsule of microorganisms comprising a first filamentous fungus, and a microorganism, b) cultivating the biocapsule of microorganisms of step (a) in the presence of spores of a second filamentous fungus in a culture medium specific for the growth of the second filamentous fungus.

[0044] In the context of the method of the invention, any culture medium that promotes the growth of the second filamentous fungus but does not promote the growth of the microorganism encapsulated in the biocapsule (i.e., yeast, bacteria, archaea, microalgae, or any possible consortium thereof) may be used. For example, gluconic acid is a consumable carbon source for the filamentous fungus (Penicillium or Aspergillus) but not consumable (or scarcely consumable) for yeasts. Specific examples include Sabouraud Dextrose Broth (SDB) when the immobilized microorganism is not a yeast, or a specific medium. In one embodiment of the invention, the specific culture medium has the following components:

[0045] • YNB without amino acids: 0.67% (m / v);

[0046] • Potassium dihydrogen phosphate (KH2PO4): 0.36% (m / v);

[0047] • Disodium hydrogen phosphate 2-hydrate (Na2HPO4'2H2O): 0.72% (m / v);

[0048] • Gluconic-D acid 50% (m / w): 1% (m / v);

[0049] • pH 7.

[0050] In step (a) of the method, an uncoated microorganism biocapsule has been previously obtained. That is, the fungal layer of a second filamentous fungus is applied onto an underlying microorganism biocapsule that has been prepared by any method known to the person skilled in the art. Thus, in particular embodiments of the present invention, the microorganism biocapsule has been previously obtained by an FSA ("Fungal Spore Assisted") technique where there is co-incubation between the first filamentous fungus and the microorganism as described in document ES2204316 B1 (in the FSA technique, yeast cells and fungal spores are co-inoculated so that the spores germinate and form the pellet as they trap the yeast cells);by an FPA ("Fungal Pellet Assisted") technique in which the microorganism cells and the fungal pellets are placed in the same medium at atmospheric pressure, i.e., it does not involve a vacuum stage (in the FPA technique, the already formed pellet is mixed with the yeasts in the same medium and the latter adhere to the fungal cells); or by a technique of infusing the microorganism into biocapsules of the first filamentous fungus by vacuum (as described in document US 63 / 411,843 A1, or in the document Lúquez-Caravaca et al., “Yeast cell vacuum infusion into fungal pellets as a novel cell encapsulation methodology”, Applied Microbiology and Biotechnology (2023) 107:5715-5726).

[0051] Thus, in one specific embodiment of the method of the present invention, when starting with biocapsules into which the microorganism is introduced by vacuum, it would consist of the following steps, in any technically feasible order: • Step (a): An aqueous suspension of spores of the filamentous fungal strain is agitated by vortexing (preferably at a speed of between 2000 and 2700 rpm) and sonication (preferably for a period of time between 2 and 10 minutes, more preferably for a period of time of 5 minutes). One of the advantages of this method is that, due to the application of vortexing and sonication to the aqueous suspension comprising the spores of at least one type of filamentous fungus, the spores are kept separate, allowing the subsequent formation of filamentous fungal granules. Otherwise, the spores would form amorphous agglomerations, which are advantageously avoided in the present method.

[0052] In a particular embodiment of the invention, the process may further comprise a preliminary step, prior to the cultivation of the filamentous fungus, of preparing the suspension comprising the spores of the filamentous fungus, preferably by pre-cultivating the filamentous fungus in a filamentous fungal sporulation medium for 7 days at a temperature between 25 and 35°C (preferably 28-30°C). This step may be carried out by any conventional method.

[0053] • Step (b): The suspension obtained in step (a) is inoculated into a suitable culture medium for filamentous fungi, preferably until a concentration of between 0.1 ■ 10 is reached 6 and 1 ■ 10 8 spores / ml.

[0054] • Step (c): The filamentous fungus culture from step (b) is incubated under high agitation (preferably at a speed of between 175 and 250 rpm) until a plurality of filamentous fungus spheres are obtained (where the filamentous fungus capsule is a matrix of interlocking strands that have a hollow, or low-density, interior).

[0055] Cultured filamentous fungal granules can be inactivated immediately after granule formation is complete. This inactivation process can be carried out, for example, by applying a physical and / or chemical treatment, as described above. Alternatively, it is also possible to inactivate the filamentous fungi once the biocapsule has already formed with the fungi and microorganisms (e.g., yeasts). In the specific case of yeast-filamentous fungal biocapsules, this can be achieved through alcoholic fermentation. The yeast itself inactivates the filamentous fungus at an advanced stage of alcoholic fermentation when oxygen is absent and there is a high concentration of ethanol. Furthermore, physical contact between the yeast and the filamentous fungus can induce fungal inactivation. Yet another alternative would be to add antibiotics to which the fungus is sensitive but the microorganism is not.In other words, it is possible in the context of the present invention to subject the system to a physical, chemical or physicochemical condition that inactivates the fungus but not the microorganism.

[0056] • Stage (d): A plurality of cells of a microorganism are grown separately, wherein the microorganism is preferably selected from the group consisting of a yeast, a bacterium, an archaea, a microalga or any possible consortium thereof, and wherein the culture medium is suitable for a specific type of microorganism.

[0057] In the case of yeast cells, step (d) of the method of the invention comprises cultivating said cells in a growth medium selected from the group consisting of YPD (Yeast Extract 20 Peptone Dextrose), YMB (Yeast Malt Broth), SBD (Sabouraud Dextrose Broth), or sterile grape must. In particular embodiments, step (d) comprises cultivating the yeast cells at a temperature of 20-35°C, 25-33°C, or 28-30°C. In particular embodiments, the temperature may be 20, 25, 26, 27, 28, 29, 30, 33, or 35°C. In particular embodiments, step (d) comprises cultivating the yeast cells for 12-72 hours, 24-72 hours, or 48-72 hours. In particular modes of implementation, the incubation time can be 12, 24, 48, 72 h.

[0058] In the case of bacterial cells, step (d) of the method of the invention comprises cultivating said cells in a growth medium consisting of NB broth (Nutrient Broth) or MRS (Medium for the Culture and Enumeration of Lactobacillus spp.). In particular embodiments, step (d) comprises cultivating the bacterial cells at a temperature of between 20 and 40°C, between 25 and 35°C, or between 25 and 30°C. In particular embodiments, the temperature may be 20, 25, 30, 35, or 40°C. In particular embodiments, step (d) comprises cultivating the bacterial cells for 12 to 24 hours. In particular embodiments, step (d) comprises cultivating the bacterial cells under agitation at 150 to 200 rpm.

[0059] In the case of archaeal cells, step (d) of the method of the invention comprises culturing said cells in an SBD (Sabouraud Dextrose Broth) culture medium. In particular embodiments, step (d) comprises culturing the archaeal cells at a temperature of 30-40°C, 35-39°C, or 36-38°C. In particular embodiments, the temperature may be 35, 36, 37, or 38°C. In a preferred embodiment, the temperature is 37°C. In particular embodiments, step (d) comprises culturing the archaeal cells for 1 to 10 days, 2 to 8 days, 3 to 6 days, or 4 to 5 days. In particular embodiments, the archaeal cells are cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In particular embodiments, step (d) comprises cultivating archaeal cells under shaking at 150 to 200 rpm.

[0060] In the case of microalgal cells, step (d) of the method of the invention comprises cultivating said cells in any commercial culture medium (e.g., MLA, TAP, etc.). The microalgal cells are cultivated under continuous light in a greenhouse (preferably between 30 and 60 PPFD, or “photosynthetic photon flux density”). In particular embodiments, step (d) comprises cultivating the microalgal cells at a temperature between 20 and 30°C, between 20 and 28°C, or between 23 and 25°C. In particular embodiments, the temperature may be 20, 23, 25, 28, or 30°C. In particular embodiments, step (d) comprises cultivating the microalgal cells for 2 to 3 weeks, for 15 to 20 days, or for 15 to 18 days. In particular embodiments, step (d) comprises culturing the microalgae cells for 14, 15, 18, 20, or 21 days.

[0061] • Step (e): An aqueous suspension is prepared with the filamentous fungal biocapsules obtained in step (c) and the microorganism cultivated in step (d), in a ratio between 1:1 and 1:3 by weight (w / w fungus: microorganism). In particular embodiments, the suspension with the biocapsules and cells is prepared in a ratio between 1:1 and 1:2, or between 1:2 and 1:3 (w / w).

[0062] • Step (f): The suspension obtained in step (e) is subjected to at least one vacuum cycle. Any vacuum system known to the person skilled in the art may be used in the context of the present invention. In one embodiment, the vacuum system is a Koch Ultravac system, operating at 0.01 atm of pressure. In a particular mode of use of this vacuum system, the filamentous fungus pellets and the microorganism cell suspension (e.g., yeast) are placed in a container (e.g., a Falcon tube) with the container lid slightly loosened and sealed with adhesive tape. The container is then placed in a vacuum bag (one container per bag). The vacuum program is set to: 99% vacuum, vacuum plus 5, seal 2, no gas. The bag is placed in the vacuum system, the system is closed, and when the ventilation reaches 0%, the lid is opened.The pellets containing microorganism cells are collected and rinsed with sterile deionized water over a strainer to remove any loose yeast cells. In another embodiment of the invention, the vacuum system is a Bonsenkitchen system (< 0.3 atm pressure), commonly used in kitchens. In one particular mode of use of this vacuum system, the vacuum adapter for jars must be sterilized with 75% (v / v) ethanol and dried before use. The sterilized adapter is placed in the opening of the container (e.g., a Falcon tube) containing the fungal pellets and the microorganism cell suspension (e.g., yeast), under aseptic conditions. The "suction switch" is pressed, and a vacuum is applied for approximately 1 second. The pellets containing microorganism cells are rinsed with sterile deionized water over a strainer to remove any loose yeast cells.

[0063] • Step (q): The product obtained in step (f) is incorporated into a growth medium suitable for the microorganism and incubated at a temperature optimized for each microorganism (e.g., between 20 and 40°C), so that the microorganism cells adhere to or are trapped by the filamentous fungus, thus forming microorganism biocapsules. In particular embodiments, the product obtained in step (f) is incubated at a temperature between 25-35°C or between 30-35°C. Optionally, step (g) may include an additional subsequent step in which the microbial biocapsules are subjected to a clarification process, removing the microorganisms located on the surface of said microbial biocapsules.In a particular embodiment of the invention, after the rinsing process, in order to inactivate the cells of microorganisms located on the periphery of the microbial biocapsules (i.e., on the surface of the biocapsules), said biocapsules can be treated by immersing said biocapsules in a 2.5 or 7.5% (v / v) formalin solution for three to six minutes; • Step (h): co-cultivating the microorganism biocapsules obtained in step (g) with a plurality of spores of a filamentous fungus in a culture medium that favors the growth of the filamentous fungus, but not of the microorganism.Examples of media that can be used in the context of the invention, already indicated above, are: Sabouraud Dextrose Broth (SDB) when the immobilized microorganism is not a yeast; or specific medium (YNB without amino acids: 0.67% (w / v); Potassium dihydrogen phosphate (KH2PO4): 0.36% (w / v); Disodium hydrogen phosphate 2-hydrate (Na2HPO4'2H2O): 0.72% (w / v); Gluconic-D acid 50% (w / w): 1% (w / v); at pH 7) when the immobilized microorganism is a yeast.

[0064] In a final aspect, the invention relates to the use of a biocapsule according to the present invention for: (a) obtaining fermented beverages; (b) obtaining biofuels; (c) obtaining pigments; (d) obtaining organic acids; (e) obtaining pharmaceuticals; (f) obtaining antibiotics; (g) obtaining hormones; (h) clarifying liquids in industrial processes; and (i) bioremediation. Thus, the filamentous fungus-coated biocapsule of the invention can be used as a fermentation agent in industrial fermentation processes or as a carrier for other biocatalysts in processes for obtaining and recovering high-value-added industrial products.The supported microorganisms include yeasts, bacteria, archaea and / or microalgae that are of interest and that can be used to produce different types of products of interest (alcoholic beverages, biofuels, pigments, organic acids, drugs, antibiotics, hormones, etc.) or services of interest (e.g., bioremediation decontamination).

[0065] The use of the coated biocapsules of the invention in the described processes can improve fermentation rates and reduce clarification costs, as the coating promotes anaerobic fermentation with the encapsulated microorganisms. The coated biocapsules of the invention also enable precision fermentations in which microorganisms can be easily inoculated and removed. Furthermore, in mixed applications, the biocapsules of the invention can be used to package microorganisms already employed in specific applications such as bioremediation (e.g., bacteria and microalgae), which, once supported, can be coated with the fungal layer and used for other biotechnological applications (e.g., biofuel production or highly nutritious foods).Specific applications of yeasts using the coated biocapsules of the present invention include the fermentation of alcoholic beverages with flor yeasts, where flor yeasts are strains of the species Saccharomyces cerevisiae used in the production of fine wines. These strains have demonstrated a high capacity for adhering to fungal structures. They can also be used in alcoholic fermentations and for lipid production. A specific example would be Saccharomyces cerevisiae G1 (ATCC: MYA-2451). Saccharomyces bayanus yeasts and non-Saccharomyces yeasts of oenological interest, such as Torulaspora delbrueckii, Metschnikowia pulcherrima, or Lachancea thermotolerans, can also be used for alcoholic beverage fermentations.For other uses, noteworthy examples include the yeast Pichia fermentans for the production of xylitol, Candida utilis for the production of microbial proteins, and pentose-terminating yeasts such as Pichia stipitis for the production of bioethanol from lignocellulosic waste.

[0066] Specific uses of bacteria in the coated biocapsules of the invention include cyanobacteria such as Arthrospira maxima (e.g., for biofuel production or production of highly proteinaceous fungus-cyanobacterium capsule food), lactic acid bacteria (e.g., for the production of lactic acid fermentation products or as probiotics), acetic acid bacteria (e.g., for vinegar production), succinogenic bacteria such as Basfia succiniciproducens or Actinobacillus succinogenes (e.g., for succinic acid production), bacteria that induce drug production by filamentous fungi such as Streptomyces lividans, or hydrogen-producing bacteria such as Carboxydothermus hydrogenoformans, Rhadobacter capsulatus, or Clostridium acetobutylicum, and methane-producing bacteria such as Clostridium spp.Furthermore, the filamentous fungal-coated microorganism biocapsules described in the present invention can be used to optimize the bioremediation use of bacteria already described in the context of fungal pellets that immobilize bacteria. The specific strains of these bacteria would be: Acinetobacter calcoaceticus JH-9; Aminobacter sp. MSH1; Arthrobacter globiformis D47; Arthrobacter sp. ZXY-2; Cupriavidus sp. H29; Massilia sp. WF1; Pseudomonas stutzeri GF2; Pseudomonas stutzeri GF3; and Variovorax sp. SRS16.

[0067] Additionally, there are interesting species of archaea that can be immobilized in the filamentous fungus-coated microorganism biocapsules of the present invention, such as Halobacterium salinarum for bioremediation, Thermococcus barosii and Thermococcus litoralis for the production of enzymes that remain stable at high temperatures, and Methanobrevibacter smithii and Methanosphaera stadtmanae for methane production.

[0068] All the terms and embodiments described above are applicable to any aspect and embodiment of the invention. According to the present invention, the singular term “the,” “a,” “one,” or “an” refers equally to its plural equivalent “the,” unless it is clear from the context that the term refers to a single species in the singular. The term “comprises” or “comprising,” as used herein, also describes “consisting of” or “consisting of” in accordance with generally accepted patent practice.

[0069] EXAMPLES

[0070] The following invention is described by means of the following examples, which should be interpreted as merely illustrative and not limiting to the scope of the invention.

[0071] Example 1: Production of filamentous fungus-coated biocapsules

[0072] For the preparation of the biocapsules of the invention (biocapsules coated with a layer of filamentous fungus), one can start from biocapsules of microorganisms previously obtained by any suitable technique, such as, for example: (a) co-incubation between a filamentous fungus and a microorganism, as described in ES 2204316 B1; or (b) infusion of the microorganism into the interior of filamentous fungus biocapsules by vacuum, as described in US 63 / 411 .843.

[0073] In one specific example, the procedure used involved immobilizing yeast cells inside pre-assembled filamentous fungal biocapsules using vacuum-assisted infusion. The following were used:

[0074] • the ATCC: MYA-2451 strain of Saccharomyces cerevisiae (strain with G1 notation in the University of Cordoba Collection, Cordoba, Spain), a flor-forming yeast used in the biological fermentation of Sherry wines, and

[0075] • The CECT 2095 strain of the filamentous fungus Aspergillus oryzae (strain with notation FST 76-2 in the UC Davis Phaff Culture Collection, Davis, CA, USA). To produce the biocapsules by the vacuum infusion method, the yeast cultures and filamentous fungus pellets were prepared separately prior to vacuum yeast infusion. The yeast strain was cultured in YPD (“Yeast extract Peptone Dextrose”) medium, containing: 10 g / L yeast extract; 20 g / L peptone; 20 g / L dextrose, as a preculture medium for overnight cultivation of yeast at 175 rpm, 28 °C. The filamentous fungus was cultured in a sporulation medium containing: 17 g / L cornmeal agar; 1 g / L yeast extract; 2 g / L glucose; 20 g / L agar; for 7 days at 28 °C. Spores were collected from the sporulation agar medium in a container with sterile deionized water, vortexed and sonicated for 5 min to avoid clumping and inaccuracy in inoculation.A controlled population of spores was inoculated to achieve a final population of 1 x 10. 6 spores / mL in a fungal pellet medium (FPM) composed of: 60 g / L glucose; 3 g / L yeast extract; 3 g / L NaNCh; 1 g / L K2HPO4; 0.5 g / L MgSCU; 0.5 g / L KCl; and 0.01 g / L FeSCU; and buffered to pH 5.5 with HCl. The filamentous fungal spores were cultured in FPM for 3 days at 175 rpm, 30°C; to form fungal pellets (Figure 1a). These filamentous fungal pellets acquire a hollow structure (Figure 1b), with an empty interior and a shell formed by long hyphal filaments, forming a mycelium. The fungal granules were inactivated in an autoclave (overpressure of 1 atm, 20 min, 121 °C).

[0076] Cultured yeast cells and inactivated filamentous fungal granules were collected, and a 1:1 (wet weight) mixture of yeast granules to filamentous fungal granules was prepared in a 50 mL Falcon tube with sterile deionized water. This suspension was infused under vacuum (< 0.3 atm pressure) for 1 min using a Bonsenkitchen system (Oakwood, GA, USA), thereby forcing the microbial cells into the tightly packed hyphal matrix of the filamentous fungal granules, as observed under scanning electron microscopy (Figure 1c). To confirm the infusion, the optical density at 580 nm (OD580) was measured in the cell suspension before and after the vacuum stage; a 20% reduction in OD580 was observed. Filamentous fungus granules infused with yeast cells were immersed in a YPD medium (“Yeast extract Peptone Dextrose”, described above) and cultured overnight at 175 rpm, 28 °C.Finally, the biocapsules obtained by this method (Figure 2a) were washed with sterile distilled water to remove as many cells as possible from the surface, thus preventing / minimizing potential leakage of free cells during subsequent use of the biocapsules, for example, during fermentation. Once the microorganism biocapsules were obtained, they were coated using the following procedure: the uncoated biocapsules were immersed in 50 mL of a medium with gluconic acid as the sole carbon source (detailed composition of the medium is YNB without amino acids (Difeo 291940): 0.67% (w / v); potassium dihydrogen phosphate or KH2PO4 (Panreac 131509.1210): 0.36% (w / v); disodium hydrogen phosphate 2-hydrate or Na2HPO4·2H2O (Panreac 122507).1210): 0.72% (w / v); gluconic acid-D 50% (w / w) (Sigma G - 1139): 1% (w / v); adjusted to pH 7) in 250 mL flasks, 1 million spores per mL were added and cultured for 3 days at 28°C, 175 rpm. The thickness of the filamentous fungal coating layer was 1.34 ± 0.58 mm. The fungal-coated biocapsules (Figure 2b) have an additional coating composed solely of filamentous fungus where no yeast cells are visible (Figure 3), which further minimizes the risk of possible leakage of free cells of the microorganism during subsequent use of the biocapsules, for example, during fermentation.

[0077] The effectiveness of retaining microorganism cells inside the fungal-coated microorganism biocapsules (BM + CF) of the present invention was verified by breaking the coating with a scalpel, in such a way that the release of the microorganism cells (e.g., a yeast) that were retained could be recorded (Figures 4a and 4b).

[0078] Example 2: Alcoholic fermentation with fungus-coated biocapsules

[0079] In one example of use, the coated biocapsules prepared in the previous section were used to carry out alcoholic fermentation. Cell immobilization rates were compared between uncoated (BM) and uncoated (BM+ CF) biocapsules formed with the filamentous fungus Aspergillus oryzae FST 76-2 and the yeast Saccharomyces cerevisiae MYA-2451, and biocapsules coated with a filamentous fungal layer, also called a fungal coating (BM+ CF), before (AF) and after (DF) alcoholic fermentation. Macroscopic images (Figure 5) show: a. a more yellowish color in the BM+ CF due to a greater accumulation of cells inside, b. an increase in volume between the BM and BM+ CF due to the filamentous fungal coating, and c. an increase in volume before (AF) and after (DF) fermentation, also due to the accumulation of yeast cells.It was observed that the initial immobilization percentage was 100% before fermentation in both systems, since all cells were immobilized and not free. From that value, it decreased to 39.31% in the case of untreated microorganism biocapsules (BM), or to 68.93% in the case of microorganism biocapsules incorporating a coating with a filamentous fungus (BM + CF) (Figure 6a).

[0080] Fermentation efficiency rates were also evaluated (Figure 6b), and it was found that the fermentation efficiency for the microorganism biocapsules without the fungal coating (BM) was lower than the fermentation efficiency for the biocapsules with the filamentous fungal layer (BM + CF), with efficiencies of approximately 70% versus over 80%, respectively. It is interesting to note that this efficiency also exceeds the value of conventional fermentation using free yeast cells (70.07 ± 6.37). Here, alcoholic fermentation efficiency is defined as the capacity of a fermentation process to convert a substrate (sugars) into a desired product (ethanol) and is calculated by dividing the amount of ethanol produced by the maximum theoretical amount of ethanol that can be produced (0.51 g of ethanol per 1 g of sugar) and multiplying by 100.

[0081] In this way, the persistent release of cells into the external environment could be evaluated. The object of the invention was to reduce cell escape from the system by coating the microorganism biocapsule with the extra fungal layer. The data indicate that this coating technique with a filamentous fungal layer (fungal layer, or CF) more than doubles cell retention and also shows greater alcoholic fermentation efficiency than untreated microorganism biocapsules, possibly due to the creation of an anaerobic environment within the system that favors anaerobic fermentation.

Claims

CLAIMS 1. A biocapsule of microorganisms comprising: a) a first filamentous fungus, and b) a microorganism, wherein the biocapsule is coated by a layer of a second filamentous fungus.

2. The biocapsule according to claim 1, wherein the first filamentous fungus and the second filamentous fungus are of the same species of filamentous fungus, or wherein the first filamentous fungus and the second filamentous fungus are of different species of filamentous fungus.

3. The biocapsule according to any one of claim 1 or 2, wherein: a) the first filamentous fungus is active and wherein the second filamentous fungus is active; b) the first filamentous fungus is active and wherein the second filamentous fungus is inactive; c) the first filamentous fungus is inactive and wherein the second filamentous fungus is active; d) the first filamentous fungus is inactive and wherein the second filamentous fungus is inactive; 4. The biocapsule according to any one of claims 1 to 3, wherein the first filamentous fungus and / or the second filamentous fungus is a fungus of a genus selected from the group consisting of Aspergillus, Rhizopus, Penicillium, Neurospora, Mucor, Mortierella, Fusarium or combinations thereof.

5. The biocapsule according to any one of claims 1 to 4, wherein the first filamentous fungus and / or the second filamentous fungus is a fungus of a species selected from the group consisting of Aspergillus sp., Aspergillus oryzae, Rhizopus sp., Penicillium sp., Neurospora intermedia, Mucor circinelloides, Mortierella isabelina, Fusarium equiseti or combinations thereof.

6. The biocapsule according to any one of claims 1 to 5, wherein the first filamentous fungus and / or the second filamentous fungus is Aspergillus oryzae, preferably Aspergillus oryzae of the CECT 2095 strain.

7. The biocapsule according to any one of claims 1 to 6, wherein the microorganism is selected from the group consisting of a yeast, a bacterium, an archaea, a microalga, or any possible consortium thereof.

8. The biocapsule according to any one of claims 1 to 7, wherein the microorganism is a yeast, preferably wherein the yeast belongs to a species selected from the group consisting of Candida guillermondii, Candida utilis, Lachancea thermotolerans, Metschnikowia pulcherrima, Pichia fermentans, Pichia manshurica, Pichia membranifaciens, Pichia stipitis, Saccharomyces bayanus, Saccharomyces cerevisiae, Torulaspora delbrueckii, Trichosporon asahii, Wickerhamomyces anomalus or combinations thereof, more preferably wherein the yeast is a Saccharomyces cerevisiae of strain ATCC: MYA-2451.

9. The biocapsule according to any one of claims 1 to 7, wherein the microorganism is a bacterium, preferably wherein the bacterium belongs to a species selected from the group consisting of Arthrospira maxima, Basfia succiniciproducens, Actinobacillus succinogenes, Streptomyces lividans, Carboxydothermus hydrogenoformans, Rhadobacter capsulatus, Clostridium acetobutylicum, Clostridium spp., Acinetobacter calcoaceticus (preferably strain JH-9), Aminobacter sp. (preferably strain MSH1), Variovorax sp. (preferably strain SRS16), Arthrobacter globiformis (preferably strain D47), Massilia sp. (preferably strain WF1), Arthrobacter sp. (preferably from strain ZXY-2), Pseudomonas stutzeri (preferably from strains GF2 or GF3), Cupriavidus sp. (preferably from strain H29) or combinations thereof.

10. The biocapsule according to any one of claims 1 to 7, wherein the microorganism is an archaea, preferably wherein the archaea belongs to a species selected from the group consisting of Halobacterium salinarum, Thermococcus barosii, Thermococcus litoralis, Methanobrevibacter smithii, Methanosphaera stadtmanae and combinations thereof.

11. The biocapsule according to any one of claims 1 to 7, wherein the microorganism is a microalga, preferably wherein the microalga belongs to a species selected from the group consisting of Botryococcus braunii, Chlamydomonas reinhardtii, Chlorella protothecoides, Chlorella pyrenoidosa, Chlorella sp., Chlorella vulgaris, Chroococcus sp., Du nal ¡ella salina, Du nal ¡ella tertiolecta, Nannochloris oculata, Nannochloropsis oceanica, Nannochloropsis oculata, Phaeodactylum tricornutum, Pseudokirchneriella subcapitata, Pyrocystis lunula, Scenedesmus quadricauda, ​​Tetraselmis chuii, Tetraselmis suecica, Thraustochytrid sp., and combinations thereof.

12. A method for obtaining a biocapsule according to any one of claims 1 to 11, wherein the method comprises the following steps: a) providing a biocapsule of microorganisms comprising a first filamentous fungus, and a microorganism, b) cultivating the biocapsule of microorganisms of step (a) in the presence of spores of a second filamentous fungus in a culture medium specific for the growth of the second filamentous fungus.

13. The method according to claim 12, wherein the microorganism biocapsule has been previously obtained by an FSA ("Fungal Spore Assisted") technique where there is co-incubation between the first filamentous fungus and the microorganism; by an FPA ("Fungal Pellet Assisted") technique in which the microorganism cells and the fungal pellets are placed in the same medium at atmospheric pressure; or by a technique of infusing the microorganism into the interior of biocapsules of the first filamentous fungus by vacuum.

14. Use of a microorganism biocapsule according to any one of claims 1 to 11 for: obtaining fermented beverages; obtaining biofuels; obtaining pigments; obtaining organic acids; obtaining pharmaceuticals; obtaining antibiotics; obtaining hormones; clarifying liquids in industrial processes; bioremediation.

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