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    • 专利摘要:
    Process for obtaining antimicrobial biopolymers comprising polyhydroxyalkanoates and metal nanoparticles. A process for obtaining an antimicrobial biopolymer comprising at least one polyhydroxyalkanoate (pha) and metallic nanoparticles with antimicrobial capacity, characterized in that the method comprises: a) inoculating a biomass comprising pha-producing microorganisms in a production medium suitable for production of phas; b) synthesizing metallic nanoparticles with antimicrobial capacity in situ by the reaction of a precursor compound and a reducing agent; and c) extracting the antimicrobial biopolymer comprising at least one polyhydroxyalkanoate and the metal nanoparticles. Antimicrobial biopolymer obtained by the procedure described above and uses of the biopolymer. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2647324A1
    申请号:ES201630829
    申请日:2016-06-20
    公开日:2017-12-20
    发明作者:Jinneth Lorena CASTRO MAYORGA；Jose Maria LAGARÓN CABELLO；María José FABRA ROVIRA；María Auxiliadora PRIETO JIMÉNEZ；Gloria SÁNCHEZ MORAGAS
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；
    IPC主号:
    专利说明:

    PROCEDURE FOR OBTAINING ANTIMICROBIAL BIOPOLYMERS UNDERSTANDING POLYHYDROXIALCANOATES AND METAL NANOPARTICLES SECTOR OF THE TECHNIQUE
    The present patent application is located in the polymer manufacturing sector. In particular, this application refers to a process for obtaining biopolymers comprising polyhydroxyalkanoates and metal nanoparticles, where these biopolymers are obtained by fermentation and have antimicrobial activity, in particular antibacterial, antifungal, antiviral or anti-fouling activity. Additionally, the present patent application refers to the antimicrobial biopolymer comprising polyhydroxyalkanoate and metal nanoparticles, as well as its use in the manufacture of plastic films or articles; particles such as capsules and / or fibers of micro, submicro or nanometric size; or coatings, all of them with antimicrobial properties. STATE OF THE TECHNIQUE
    Polyhydroxyalkanoates (PHAs) comprise a group of biopolyesters synthesized as energy reserve materials by a wide range of microorganisms. These biopolymers have physical-chemical and mechanical properties similar to those of conventional plastics, so they provide a good alternative to the use of materials derived from the petrochemical industry. In addition, PHAs are biodegradable, non-toxic, biocompatible and can be produced from renewable resources. These polyesters are used for applications in various areas, in the food and cosmetic packaging industry, in biomedicine as synthetic bone and surgical materials, as a coating for surfaces in contact with food, or as biodegradable carriers of medicines, hormones, insecticides and herbicides (Anderson, AJ, & Dawes, EA (1990). Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiological Reviews, 54 (4), 450-472 + iii).
    Of the PHA family, the most widely known is poly-3-hydroxybutyrate (PHB), this polymer has a melting point comparable to isotactic polypropylene, which allows it to be extruded and molded with conventional polymer processing equipment . Despite these advantages, the use of PHB is affected both by its high cost, and by its instability at high temperatures, high crystallinity and low tensile strength. In order to reduce the fragility of PHB, the introduction of hydroxyvaleric acid (HV) in poly-3'-hydroxybutyrate-co-3-hydroxivalerate (PHBV), mixing with other polymers and the addition of plasticizers, nucleating agents or nanorellenes (Ceccorulli, G., Pizzoli, M., & Scandola, M. (1992). Plasticization of bacterial poly (3-hydroxybutyrate). Macromolecules, 25 (12), 3304-3306; Martínez-Sanz, M., Villano , M., Oliveira, C., Albuquerque, MGE, Majone, M., Reis, M., Lopez-Rubio, A. & Lagaron,
    J.M. (2014). Characterization of polyhydroxyalkanoates synthesized from microbial mixed cultures and of their nanobiocomposites with bacterial cellulose nanowhiskers. New Biotechnology, 31 (4), 364-376).
    For its part, the use of polymeric materials with antimicrobial capacity is one of the technologies with the greatest impact in various sectors such as food preservation, biomedicine, water purification and pharmacology. One of the methodologies for incorporating antimicrobial agents in polymeric matrices, especially those that do not withstand high temperatures, is their fixation to the surface of the material through covalent bonds. This type of preparation requires the availability of functional groups, both on the surface of the polymer and on the structure of the antimicrobial agent to be immobilized, in addition to the use of additives that favor its dispersion and compatibility with the material, which makes the procedure to obtain the polymeric material with antimicrobial capacity is technically complex, the amount of antimicrobial to be used increases and the production costs.
    Among the most widely used inorganic antimicrobials, metals such as silver or copper, in the form of ions or nanoparticles, have emerged as one of the most researched technologies, since their incorporation into plastics and contact surfaces can help prevent appearance of pathogenic and / or altering microorganisms. The thermal stability of metals and their relative low cost make them ideal candidates for incorporation into a wide variety of materials. However, it is known that metals with antimicrobial capacity, especially silver and copper, undergo color changes caused by the presence of oxidizing-reducing environments and / or their agglomeration, and this fact limits their application possibilities.
    Recently, different systems based on the use of silver as an antimicrobial agent with application in the treatment of wounds have been developed, for example, US6592888 B1 (Composition for wound dressings safely using metallic compounds to produce anti-microbial properties; Jensen et al. , published 2003) uses adhesive formulations in the form of hydrocolloids and metallic silver for the preparation of antimicrobial bandages. Genetic Laboratories, Inc. currently markets a product called E-Z DERMTM, which consists of a biosynthetic bandage for the treatment of burns that has silver nitrate incorporated.
    The DuPont company has introduced the MicroFree ™, a product based on the incorporation of three types of silver or copper salts on inorganic supports and thermosetting agents that can be introduced into the polymer resin itself in the extrusion process. Currently, three types of MicroFree ™ are being offered, the Z-200 (silver on a zinc support), the T-558 (silver, copper oxide and zinc silicate on a titanium dioxide support), and B-558 (silver, copper oxide and zinc silicate on a barium sulfate support), which have been shown to have a biocidal effect on Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococus aureus.
    The Plasticopper company markets an additive based on copper nanoparticles, which can be placed on polymeric matrices or inert resins to provide them with antimicrobial action, against microorganisms such as the Resistant Vancomycin Enterococcus (VRE), the Staphylococcus Aureus Resistant Meticillin (MRSA) and Carbapenema Resistant Enterobacteria (CKD). This product is associated with patent applications 201401881, 201302101 and 201202350 of the National Institute of Industrial Property (INAPI) of Chile.
    On the other hand, patent application WO2015184561 (ALLUÉ) describes a gelcoat material made of resin and copper nanoparticles, which can be used to create surfaces with biocidal properties within vehicles of all types, but preferably in ambulances.
    Patent application US20150374610 develops a topical product for skin protection or decontamination applications based on micro and nanoparticles of metal oxides covalently associated with polymeric matrices.
    Patent application US20150336804 describes the development of a method for in situ synthesis of copper nanoparticles in paper by reduction with ascorbic acid and its application as an antimicrobial against Escherichia coli.
    Work has also been done on the incorporation of silver ions in inert supports such as synthetic zeolites, an example of this is the Apacider A® product in which silver ions are chemically and physically linked with calcium phosphate in a zeolite support. However, this agent presents problems in its incorporation as an extrusion additive since it requires a large number of surfactants to favor its dispersion in the plastic matrix and its migration towards the surface of the food. The Japan's Shinanen New Ceramics company together with Mitsubishi developed the Zeomic®, where silver ions are incorporated into a zeolite support, with a broad spectrum of action and with a relatively good behavior in the extrusion process. A similar product, known as BactiBlock®, based on clay functionalized with ionic silver, is marketed by the Nanobiomatters company as an antimicrobial additive for polymeric materials for application in the construction, electronics, health and food sector between others.
    In addition to the aforementioned patent documents, in scientific literature sources, the use of synthetic stabilizing agents such as polyvinyl alcohol, polyethylene glycol and polyvinyl pyrrolidone, Tween or Triton X-100, cetyltrimethylammonium bromide (CTAB), polysorbate (Tween) or has been reported sodium dodecyl sulfate (SDS) has proposed the synthesis of metal nanoparticles by microorganisms or plants with reducing capacity and the use of functionalized polymers (Moritz, M., & Geszke-Moritz, M. (2013). The newest achievements in synthesis , immobilization and practical applications of antibacterial nanoparticles. Chemical Engineering Journal, 228, 596-613).
    In the packaging sector for food preservation, there are several developments that have been made. In particular, patent application WO99049823 A1 (Light-activated antimicrobial polymeric materials; Wilson, JE et al., Published 1999) describes the method of obtaining different antimicrobial materials with application in the food packaging industry and the manufacture of covers Surgical and facial masks, based on dispersion methylene blue, which generates singlet oxygen as an antimicrobial agent. In this same area, US7981408 B2 (Method of preserving food using antimicrobial packaging; Schroeder, JD et al., Published 2011) describes the development of antimicrobial materials with quaternary ammonium and phosphonium salts covalently bonded to the polymeric material, these materials being antimicrobials useful in the production of films or packaging of food, cosmetics, equipment and medical devices.
    Patent application WO2013149356 A1 (Container that extends the shelf life of the food it contains, especially berries, by incorporating an antifungal agent on its surface. Particularly berries. Peparation procedure and uses; Junqueira, MP et. Al .; published 2013 ) refers to the incorporation of potassium sorbate as an antifungal agent in polyethylene terephthalate (PET) sheets for application in fresh fruit packaging. In this process, once the PET sheet is extruded, it is immersed in a solution containing the antimicrobial agent and silicone, the excess of the solution is then removed, and the sheet is rolled up and subsequently passed to the thermoforming and production process of the container.
    Patent application WO2001017356 A1 (Antimicrobial polymer; Collins A.N. et al., Published 2001) describes an antimicrobial polymer, polyhexamethyl biguanide, which carries a covalently linked chromophore marker. This polymer is capable of being used in the disinfection of industrial cooling waters, swimming pools, latex, surface coatings, geological drilling fluids and in personal care formulations, such as soaps and cosmetics. Said polymer is used in commercial products such as the Kendall ™ AMD foam antimicrobial dressing distributed by Covidien.
    Patent application WO2004056214 A2 (Method of preparation of bioactive packaging materials; Marek, M. et al., Published 2004) refers to the preparation of polymeric packaging materials with immobilized preservatives, where the application of a solution or dispersion of a Polymer with preservatives in the material is made by atomization, coating or immersion, followed by evaporation of the solvent at room temperature or higher. Antimicrobial compounds derived from phenol, bacteriocins, esters of p-hydroxybenzoic acid, monoglycerides of fatty acids, benzoic acid, sorbic acid, chitosan, lysozyme, nisin, among others, are used herein.
    Regarding the use of biopolymers as stabilizing agents of metal nanoparticles, there are only two preliminary studies, the one conducted by Phukon et al. (2011) (Phukon, P., Saikia, JP, & Konwar, BK (2011). Enhancing the stability of colloidal silver nanoparticles using polyhydroxyalkanoates (PHA) from Bacillus circulans (MTCC 8167) isolated from crude oil contaminated soil. Colloids Surf B Biointerfaces, 86 (2), 314-318) where the chemical synthesis of nanoparticles on PHA in suspension is described; and the one carried out by Castro-Mayorga et al. (2014) (Castro-Mayorga, JL, Martínez-Abad, A., Fabra, MJ, Olivera, C., Reis, M., & Lagarón, JM Stabilization of antimicrobial silver nanoparticles by a polyhydroxyalkanoate obtained from mixed bacterial culture. International Journal of Biological Macromolecules 71, 103-110) where PHBV is used as a stabilizing agent in the synthesis of antimicrobial silver nanoparticles.
    However, despite the many advances in the synthesis of metal nanoparticles, one of the main challenges for the production of metal-based antimicrobial materials is the production of stable nanoparticles, which do not present agglomeration due to the high processing temperatures of plastics, and also have a minimum size and a homogeneous distribution, since it has been shown that its biocidal effect depends largely on the size and state of agglomeration (Castro-Mayorga, JL, Martínez-Abad, A., Fabra , MJ, Olivera, C., Reis, M., & Lagarón, JM Stabilization of antimicrobial silver nanoparticles by a polyhydroxyalkanoate obtained from mixed bacterial culture. International Journal of Biological Macromolecules 71, 103-110).
    DESCRIPTION Brief Description of the Invention
    The invention presented here provides biopolymers, that is, polymeric materials of biotechnological origin, comprising at least one polyhydroxyalkanoate (PHA) and metal nanoparticles with antimicrobial capacity, where these nanoparticles have been incorporated into the biopolymer during the process of obtaining PHA by fermentation. These polymeric materials have ideal characteristics for use as a raw material in the production of plastics by conventional processing techniques, both for their thermal stability, as for their antimicrobial qualities and their optical properties. The antimicrobial biopolymers obtained from the process described herein overcome the problem of instability and agglomeration of the metal nanoparticles and, additionally, have an antimicrobial effect against pathogenic and / or altering microorganisms, in particular bacteria and / or viruses, even at lower doses. to those reported in the literature for antimicrobial PHA (0.1% -5% by weight) (Min, M., Shi, Y., Ma, H., Huang, H., Shi, J., Chen, X. , Liu, Y., & Wang, L. (2015). Polymer-nanoparticle composites composed of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) and coated silver nanoparticles. Journal of Macromolecular Science, Part B: Physics, 54 ( 4), 411-423; Yu, H., Sun, B., Zhang, D., Chen, G., Yang, X., & Yao, J. (2014). Reinforcement of biodegradable poly (3-hydroxybutyrate- co-3hydroxyvalerate) with cellulose nanocrystal / silver nanohybrids as bifunctional nanofillers. Journal of Materials Chemistry B, 2 (48), 8479-8489); or for other commercial polymers (0.3% -1% by weight) (Echegoyen, Y., & Nerín, C. (2013). Nanoparticle release from nanosilver antimicrobial food containers. Food and Chemical Toxicology, 62 (0), 16 -22.).
    The procedure referred to in the present application allows obtaining a biodegradable, renewable and biocompatible biopolymer with antimicrobial properties. Advantageously, this biopolymer can be obtained in a single production stage. The subsequent addition of the metal nanoparticles is not necessary, nor is the physical or chemical dispersion or stabilization of these nanoparticles necessary. In the process of the present invention, the synthesis of the metal nanoparticles takes place during the process of obtaining the PHA by fermentation and, consequently, this procedure allows to obtain an optimal dispersion and distribution of the nanoparticles in the polymer matrix.
    The antimicrobial biopolymer obtained by the procedure described in this patent application has excellent thermal stability and, therefore, is suitable for the manufacture of all types of thermoplastics without the optical properties being affected. Specifically, this biopolymer has a high transparency and the advantage of not presenting the black or brown color characteristic of polymer materials added with silver or copper salts or nanoparticles known to date. In addition, the incorporation of metal nanoparticles can favor the mechanical and barrier properties to oxygen or water vapor of the material manufactured from this biopolymer.
    When the antimicrobial biopolymer of the present invention is applied as a coating on commercial polymeric matrices, prior to preparation of a masterbatch of fibers and / or capsules of micro, submicro or nanometric size by means of the electrodynamic processing technique, the dispersion and distribution of the metal nanoparticles and therefore their antimicrobial effect. In this way, a coated material with excellent antimicrobial capacity can be obtained, even at concentrations lower than those required for the synthesis of materials by compression molding. Preferably, this coating does not modify the physical-chemical properties of the coated material and, additionally, presents a specific migration of the metal components below the limits established by European legislation (EFSA 2006, The EFSA Journal (2006) 395 to 401, 1-21 .; EFSA 2011, REGULATION (EU) No 10/2011 OF THE COMMISSION of January 14, 2011 on materials and plastic objects intended to come into contact with food).
    In the case of the synthesis of antimicrobial biopolymers containing silver nanoparticles, it is preferred to use the biomass reducing power for the biosynthesis of these nanoparticles. In this way, the use of external reducing agents, in particular synthetic reducing agents, which can become toxic can be avoided.
    In addition, the organic components present in the fermentation medium, including the same biopolymer, can be used as stabilizers of the metal nanoparticles, in particular copper and / or silver nanoparticles, which makes it possible to avoid the use of stabilizers of synthetic origin.
    The immobilization of metal nanoparticles with antimicrobial capacity, in particular of silver and / or copper, in the polymer matrix of polyhydroxyalkanoate helps control the release of active species from the surface to the medium with which they are in contact.
    Advantageously, the described process is easily scalable and attachable to the industrial polyhydroxyalkanoate (PHA) production infrastructure. In particular, this procedure does not require additional infrastructure in a processing plant, rather than solution pumping systems. Therefore, the process for obtaining an antimicrobial biopolymer comprising at least one PHA and metal nanoparticles of the present invention can be applied directly in the process of producing PHAs by fermentation on an industrial scale.
    Additionally, the antimicrobial biopolymers obtained by this process have potential applications in the area of renewable and biodegradable materials in sectors such as: active packaging, surface coating, sensors, cosmetics, pharmacy, protein and nucleic acid purification, and controlled release. of antimicrobial substances, in particular bactericidal or virucidal. In addition, in all those sectors that require the properties of the biopolymer presented here, as in the biomedicine sector for the development of products for the care of wounds, bandages and sutures; in the purification of water, as part of the process of elimination of pathogenic and / or altering organisms; in the manufacture of antimicrobial materials used as coatings; in paints and coatings for walls, floors, pipes, structural panels; as adhesives and sealants; in the manufacture of instruments and medical furniture; in building materials; textiles; footwear; technological devices for daily use, such as mobile phones, computers, appliances; and in the manufacture of plastic food containers or surgical material. In general, the biopolymers obtained by the process of the present invention can be used for all applications that require antimicrobial properties, as well as in applications that require an improvement in thermal resistance, biodegradability, compostability and / or biocompatibility of plastic matrices. in which the biopolymer is applied. Detailed description of the invention
    The present invention proposes a process that allows the development of biopolymers comprising at least one PHA and metal nanoparticles of effective and lasting antimicrobial action against pathogenic microorganisms. More specifically, the process of the invention allows to solve the problem of low stability and agglomeration of metal components during processing with polymeric matrices and, as a consequence, allows to avoid the loss of the biocidal capacity of the material, as well as the negative effects that these problems originate in the physical, especially optical, properties of the materials.
    In a first aspect, this document describes a method of obtaining an antimicrobial biopolymer comprising at least one polyhydroxyalkanoate and metal nanoparticles with antimicrobial capacity, characterized in that the process comprises:
    a) inoculating a biomass comprising polyhydroxyalkanoate producing microorganisms in a production medium suitable for the production of PHAs, preferably a production medium comprising a carbon source, a nitrogen source, macronutrients and micronutrients;
    b) synthesize metal nanoparticles with antimicrobial capacity in situ by reacting a precursor compound, preferably a metal salt, and a reducing agent; Y
    c) extracting the antimicrobial biopolymer comprising at least one polyhydroxyalkanoate and the metal nanoparticles.
    The polyhydroxyalkanoate (PHA) comprised in the antimicrobial biopolymer obtained by the process of this patent application may be formed by the monomers included in Table 1; it can be a functionalized PHA formed by the precursor monomers described in Table 1 and alkyl, cyclohexyl, halogenated, acetoxy, ester, alkoxy, epoxy, thio, cyano, nitro, benzoyl, methylphenoxy, phenoxy, phenyl radicals; or it may be a copolymer of the above polyhydroxyalkanoates.
    Table 1: Monomers that make up the PHA produced by microorganisms (Steinbüchel
    A. and Valentin H. (1995). Diversity of bacterial polyhydroxyalkanoic acids FEMS Microbiol. Lett. 128, 219-228)
    Due to its high productivity yields, thermal and mechanical properties, low
    For cost and high dispersion in water, it is preferred that the PHA comprised in the microbial biopolymer be selected from the group consisting of poly-3-hydroxybutyrate (PHB), poly-3-hydroxy-octanoctanoate (PHO), or a copolymer of 3-hydroxy butyric acid and 3-hydroxy valeric acid (PHVB) and a combination of the above.
    15 PHA can be obtained from pure or mixed wild microbial cultures, or pure or mixed microbial cultures modified by genetic engineering. For the culture medium, it is preferred to use agri-food waste, urban waste or wastewater as raw material, which provides the additional advantage of reducing production costs.
    In the present invention it should be understood that both the term "antimicrobial" and the expression "with antimicrobial capacity" refer to a substance or compound that has the ability to eliminate or reduce the proliferation of microorganisms. The microorganisms attacked by the antimicrobial can be bacteria, viruses, fungi, parasites or even encrustations caused by the formation of bacterial films or the accumulation of organic matter.
    More specifically, in the present invention it should be understood that the term "metal nanoparticles with antimicrobial capacity" refers to nanoparticles of any metal that has antimicrobial activity such as, for example, silver, copper, zinc or any combination thereof. Preferably, the metal nanoparticles are selected from the group consisting of silver nanoparticles, copper nanoparticles and a combination of the above.
    The metal nanoparticles generated in situ in the process of obtaining an antimicrobial biopolymer of the present invention can have a particle size between 1 and 500 nm, preferably between 1 and 100 nm, and more preferably between 1 and 20 nm, since in This interval favors its antimicrobial effect.
    The precursor compound of the metal nanoparticles with antimicrobial capacity used in the process of the present invention can be any metal compound in question, provided that it allows the synthesis of the metal nanoparticles in the polyhydroxyalkanoate production medium, optionally in the presence of compounds auxiliaries such as reducing and / or stabilizing agents. Preferably, the precursor compound is a metal salt or oxide comprising the metal with antimicrobial capacity in its ionic form. Even more preferably, the precursor compound is a silver metal salt, a copper metal salt, a copper oxide or a combination of the foregoing.
    In those embodiments of the invention in which the biopolymer comprises silver nanoparticles, the precursor compound may be a silver metal salt such as, for example, silver nitrate, silver bromide, silver chloride, silver iodide, acetate silver, silver phosphate, silver sulfadiazine, and silver organometallic compounds, such as perhalophenol derivatives, mesityl compounds or their derivatives, silver carboxylates such as silver stearate, silver octanoate or derivatives thereof, and any combination of the foregoing.
    In those embodiments of the invention in which the biopolymer comprises copper nanoparticles, the precursor compound may be a metal salt such as, for example, copper (I) sulfate, copper (II) sulfate, copper citrate, copper nitrate copper, copper acetate, copper phosphate, copper chloride or a combination of the above. Additionally, this copper nanoparticle precursor can be a copper oxide
    or a derivative thereof.
    As mentioned above, the synthesis of metal nanoparticles occurs in situ, that is, in the same production medium that is used for PHA biosynthesis by fermentation. The formation of these nanoparticles can be carried out using synthetic reducing agents such as, for example, ascorbic acid, sodium citrate, sodium borohydride or hydrazine hydrate. The use of these reducing agents is preferred when the synthesis of the metal nanoparticles requires a strong reducing power.
    Additionally, the synthesis of metal nanoparticles, in particular of silver and / or copper, can be carried out by biosynthesis with reducing microorganisms, with organic compounds derived from bacterial fermentation, or by using plant extracts, or other reducing agents of origin natural, such as marine polysaccharides, dextran, cellulose extracts or dextran. The use of reducing agents of natural origin is advantageous since they usually have a lower toxicity to reducing agents of synthetic origin.
    In preferred embodiments of the present invention, the synthesis of silver nanoparticles takes place in situ using as a reducing agent organic compounds derived from fermentation such as the polyhydroxyalkanoate itself, the microorganisms producing polyhydroxalcanoates inoculated in step a) of the process of the invention or a combination of both.
    In accordance with these preferred embodiments, the method of obtaining an antimicrobial biopolymer described in this patent application makes it possible to avoid the use of synthetic reducing agents that can become toxic, which represents a significant advantage. Additionally, the use of the reducing character of the PHA producing microorganisms and / or of the compounds derived from the bacterial fermentation is especially advantageous since it avoids the addition of additional reagents that could affect the stability of the system, in particular, of the producing microorganisms. PHA and, consequently, decrease the effectiveness (performance) of the procedure.
    In the procedure described in this patent application any PHA producing microorganism can be used by fermentation. For example, those described in the following documents: González García, Y., Meza Contsrerass, J. C., González Reynoso, O., & Córdova López, J. A. (2013). Synthesis and degradation of polyhydroxyalkanoates: Plastic microbial. International Journal of Environmental Pollution, 29 (1), 77-115; Khanna, S., & Srivastava, A. K. (2005). Recent advances in microbial polyhydroxyalkanoates. Process Biochemistry, 40 (2), 607-619.
    However, in particular in those embodiments in which the biopolymer to be synthesized comprises silver nanoparticles, it is preferred to use microorganisms that additionally have reducing capacity, because in this way the use of external inorganic reducing agents can be avoided. In particular, it is preferred that the PHA producing microorganisms are Ralstonia eutropha H16, Pseudomonas putida KT2440, Cupriavidus metallidurans (DSM 2839) or a combination of the above, since all these strains have the ability to reduce silver from its ionic form to metal nanoparticles
    Ralstonia eutropha H16 and Pseudomonas putida KT2440 are PHA producing microorganisms, while Cupriavidus metallidurans (DSM 2839) is a metalresistant microorganism that can be used in the production of poly-3-hydroxybutyrate (PHB), poly-3-hydroxyoctanoate (PHO) or a copolymer of 3-hydroxy butyric acid and 3-hydroxy valeric acid (PHBV).
    In preferred embodiments of the present invention, the biomass inoculated in step a) of the process described can be obtained by pre-culture of the polyhydroxyalkanoate producing microorganisms in a nutrient medium. Preferably, at the end of this pre-culture stage a concentration of microorganisms in the biomass is obtained between 1x108 to 1x109 CFU / mL or between 1-1.5 D.O.
    The process of the present invention comprises the production of polyhydroxyalkanoates by bacterial fermentation with pure or mixed cultures, of natural or synthetic origin, being able to use, alternatively, microorganisms producing polyhydroxyalkanoates with natural or metal-induced resistance.
    The process of the present invention can be carried out on a laboratory scale, pilot silver scale or on an industrial scale, in bioreactors operating under continuous regime, by batches or batches fed, under controlled temperature, pH and agitation conditions.
    Upon inoculation of the biomass, preferably with a concentration of PHA producing microorganisms between 1x108 to 1x109 CFU / mL or between 1-1.5 OD, in the production medium suitable for the production of PHAs, in particular a medium of Production comprising a source of carbon, a source of nitrogen, macronutrients and micronutrients, produces cellular stress that results in the accumulation of the biopolymer of polyhydroxyalkanoate and metal nanoparticles in the cell cytoplasm of microorganisms.
    In the present invention, any means of production commonly used in the production of PHAs by fermentation can be used, provided that the medium does not interact with the metal at a level that can be complexed and lose activity. The production medium can comprise any carbon source that allows the accumulation of PHA in the cell. In particular, natural or synthetic carbon sources can be used, as well as alternative carbon sources, for example, by-products of the agribusiness industry, urban waste, wastewater and, more preferably, agri-food waste such as whey, used vegetable oils or The waste of the fruit. It can also be used as a source of carbon, atmospheric carbon dioxide or monoxide or from the combustion of organic matter, methane (biogas), synthesis gas from industrial gases or from gasification and pyrolysis of waste.
    On the other hand, the composition and concentration of the sources of nitrogen, micro- and macronutrients included in the production medium may depend on the microorganism used. However, it is readily recognized to the person skilled in the art that in the process of the present invention the sources of nitrogen, micro- and macronutrients present in production media used for the production of PHA can be used.
    If limiting, the production medium used in the process of the present invention may comprise the following components: MgSO4-7H2O 0.39g / L, K2SO4 0.45g / L, H3PO4 12mL / L, FeSO4-7H2O 15mg / L, 0.01% NH4Cl, 1% sodium gluconate, and trace elements 24mL / L (CuSO4-5H2O 20mg / L, ZnSO4-6H2O 100mg / L, MnSO4-4H2O 100mg / L, CaCl2-2H2O 2.6g / L).
    The biopolymer can be extracted by lysis of the cell membrane, physically, chemically or biologically, preferably by an inert method of easy application at the industrial level such as the use of a high pressure homogenizer. The biopolymer obtained from the fermentation can be purified using organic solvents, the use of low environmental impact processes such as, for example, purification using ethanol and water being preferable. Advantageously, the organic matter produced during the fermentation process, including the biopolymer, can act as a stabilizing agent and support matrix for the metal nanoparticles.
    Additionally, the biopolymer obtained by the process described herein may be subjected to additional stages of centrifugation and drying, and / or lyophilization.
    Another aspect of the present invention relates to the antimicrobial biopolymer comprising at least one polyhydroxyalkanoate and metal nanoparticles, characterized in that it is obtained by the procedure described in this patent application. As described in detail above, in this process the formation of metal nanoparticles with antimicrobial capacity takes place in situ during the process of obtaining PHA by fermentation. Thus, the antimicrobial biopolymer of the present invention is characterized by comprising nanoparticles with a minimum size, more preferably between 1-20 nm, a low state of agglomeration and a homogeneous distribution in the polymeric matrix of PHA. In particular, the synthesis of metal nanoparticles in situ makes it possible to prevent them from aggregating between them and, as a result, the nanoparticles have a reduced size and a homogeneous distribution in the biopolymer that favors their antimicrobial activity.
    Additionally, the antimicrobial biopolymer described herein is characterized by greater thermal stability and improved optical properties, in particular high transparency. The process of the present invention avoids the characteristic black or brown coloration of other polymeric materials added with salts or metal nanoparticles such as silver or copper. Furthermore, the incorporation of metallic nanoparticles by means of the process of the present invention allows to favor the mechanical and barrier properties of oxygen or water vapor of the material. Consequently, the antimicrobial biopolymer obtained by this process can be used advantageously for the manufacture of all types of thermoplastics without affecting the optical properties of the material.
    In preferred embodiments of the present invention, the metal nanoparticles are silver nanoparticles, copper nanoparticles or a combination of the foregoing.
    The polyhydroxyalkanoate (PHA) comprised in the antimicrobial biopolymer described in this patent application may be formed by the monomers included in Table 1; it can be a functionalized PHA formed by the precursor monomers described in Table 1 and alkyl, cyclohexyl, halogenated, acetoxy, ester, alkoxy, epoxy, thio, cyano, nitro, benzoyl, methylphenoxy, phenoxy, phenyl radicals; or it may be a copolymer of the above polyhydroxyalkanoates. Preferably the antimicrobial biopolymer comprises at least PHA selected from the group consisting of poly-3-hydroxybutyrate (PHB), poly-3-hydroxyoctanoate (PHO) or a copolymer of 3-hydroxy butyric acid and 3-hydroxy valeric acid (PHVB) and a combination of the above. These PHAs are preferred for their high productivity yields, thermal and mechanical properties, low cost and high water dispersion.
    The antimicrobial biopolymer comprising PHA and metal nanoparticles described in this patent application can be used directly for the production of films, that is, polymeric films, or as a masterbatch in combination with other polymers, using conventional plastics processing techniques, such such as solvent deposition and evaporation, operations typically used during the formulation of thermosets and elastomers, by melt mixing processes such as extrusion, injection or blowing; and / or in-situ polymerization methods and, preferably, compression molding. On the other hand, the antimicrobial biopolymer can also be used for the manufacture of micro, submicro or nanoparticles, in particular fibers and / or capsules, by the technique of electrodynamic processing and subsequent coating of polymeric surfaces.
    The production of polymeric films (also called films herein) or antimicrobial plastic articles is described by incorporating metal nanoparticles, preferably of silver, copper or a combination of both, stabilized on biopolymeric polyhydroxyalkanoate (PHA) matrices, by biosynthesis or chemical synthesis of metal nanoparticles during the process of obtaining PHA by fermentation. More specifically, the production of antimicrobial plastic films or articles is described by any technique of processing plastics from the melt such as, for example, and without limitation, extrusion of film, blowing, injection, calendering, thermoforming, techniques of compression molding; as well as the manufacture of fibers and / or capsules of micro, submicro, or nanometric size by electrohydrodynamic processing techniques.
    The different materials obtained by the processes described in this application contain metal nanoparticles homogeneously distributed on the polymeric matrix of PHA and possess an antimicrobial activity, in particular of bactericidal and virucidal character, against pathogenic or altering agents. In addition, the products constituted by the antimicrobial biopolymer described herein, or those that contain them within their formulation, offer excellent thermal stability and may even present an improvement in their mechanical and barrier properties.
    Therefore, the present invention also relates to the use of the antimicrobial biopolymer described in this patent application as a masterbatch or nanoparticle concentrate in the biopolyester matrix, so that it can be used directly or more commonly as a mixing component to dilute using process techniques typically employed in the plastics industry, with the same or another polymer.
    The present invention also describes a process for obtaining a polymeric film.
    or plastic article comprising at least one polyhydroxyalkanoate and metal nanoparticles with antimicrobial capacity, where this process comprises: i) obtaining a biopolymer comprising at least one polyhydroxyalkanoate and metal nanoparticles as described in the present patent application; Y
    ii) obtain the polymeric film or plastic article from the biopolymer obtained in i) by a technique of processing plastics from the melt, such as, for example, and without limiting sense, film extrusion, blowing, injection, calendering, thermoforming , compression molding techniques.
    In particular, in stage ii) of the process of forming a film, the compression technique from the melt can be used, where the temperature and pressure used may vary according to the final application to which the plastic film or article is to be used, that is, depending on the crystallinity, thickness, thermal properties and barrier properties desired. Preferably, the compression molding step can take place at a temperature close to the melting point of the biopolymer, in particular between 130 and 200 ° C, and a pressure between 0.5 and 5 MPa.
    The present invention also relates to a film or plastic article comprising at least one polyhydroxyalkanoate and metal nanoparticles with antimicrobial capacity, preferably of silver, copper or a combination of both metals, where the film or plastic article is obtained by the process that is described in this patent application. The high thermal stability of the biopolymer allows the use of plastics forming techniques common in the sector, preferably compression molding, without negatively affecting the properties of the polymeric film or plastic article obtained. In particular, the process of the present invention allows, for example, to obtain antimicrobial films by compression molding at a temperature between
    130 and 200 ° C and a pressure between 0.5 and 5 MPa, without negatively affecting the antimicrobial activity of the biopolymer used for it, or its optical properties. Without limitation, this material can be processed to obtain monolayer or multilayer film using any plastics processing technique typically used in the industry. This material can also be used to form rigid or flexible containers.
    On the other hand, the present invention relates to a process for obtaining micro, submicro
    or nanoparticles, preferably fibers, capsules or a combination of the foregoing, comprising at least one polyhydroxyalkanoate and metal nanoparticles with antimicrobial capacity, characterized in that the process comprises:
    i) obtaining a biopolymer comprising at least one polyhydroxyalkanoate and metal nanoparticles as described in this patent application; and ii´) obtain micro, submicro or nanoparticles from the biopolymer obtained in i) by means of an electrodynamic processing technique.
    In particular embodiments of the present invention, the electro-stretching solution used in step ii ') may contain a concentration of between 3-10% by weight of total solids with respect to the total weight of the solution, the injector voltage may vary from 10 kV and 30 kV, the collector voltage can vary between 0 and 30kV and the distance between injector and collector can range between 5 cm and 40 cm. However, these parameters can be modified, for example, by the viscosity and surface tension of the electro-stretching solution, as well as by the temperature, relative humidity and humidity of the solvent used (for example: chloroform, dichloromethane, trifluoroethanol, acetone , their combinations, or any other in which the product is soluble and electrostable).
    Additionally, it may be necessary to adjust these parameters depending on the size and morphology characteristics of the particles to be obtained, in particular as it is intended to obtain fibers, capsules or a combination of the above.
    The present invention also relates to micro, submicro or nanoparticles comprising at least one polyhydroxyalkanoate and metal nanoparticles, preferably of silver, copper or a combination of both metals, obtained by the process described in this patent application. Preferably, these particles are fibers, capsules.
    or any combination of the above obtained by means of the electrodynamic processing technique. Electrodynamic processing allows the obtaining of solid microstructured or nanostructured material, which is dried without the use of high temperatures.
    This patent application also refers to the use of the antimicrobial biopolymer comprising PHA and metal nanoparticles, preferably of silver, copper or a combination of both metals described in this patent application, in the manufacture of packaging, surface coating, the manufacture of sensors, in cosmetics, biomedicine, purification of proteins and nucleic acids or in the controlled release of antimicrobial substances. Additionally, this application also refers to the use of the film-forming film, plastic article and the micro, submicro or nanoparticles described in this patent application for the applications mentioned above in relation to the biopolymer.
    The present patent application also refers to a coated material comprising a polymeric matrix coated with the antimicrobial biopolymer, the polymeric film or the micro, submicro or nanoparticles described herein. BRIEF DESCRIPTION OF THE FIGURES
    Figure 1 corresponds to an image obtained by Transmission Electron Microscopy (TEM) of Ralstonia eutropha bacterial cells containing granules of PHAs and silver nanoparticles in their cytoplasm
    Figure 2 shows photographs of PHA films containing silver and copper nanoparticles that are obtained by the procedure described in the present patent application: (a) PHAs film without metal nanoparticles (for comparative purposes), (b) film of PHA containing silver nanoparticles, (c) PHA film containing copper nanoparticles.
    Figure 3 contains the results of the evaluation of the antimicrobial activity of the films developed in the present invention against Salmonella enterica. The dotted line represents the detection limit (33 CFU / mL). The concentration of bacterial cells at the beginning of the evaluation corresponds to 5.48 ± 0.4 CFU / mL.
    Figure 4 corresponds to micrographs of Scanning Electron Microscopy of PHA microfibers and silver nanoparticles obtained by the electrodynamic processing technique.
    Figure 5 shows a photograph of a commercial PHBV film coated with a mat of submicron size fibers which has antivirucidal properties. Examples
    EXAMPLE 1: Incorporation of silver nanoparticles (example 1.1) or copper (example 1.2) during the fermentation process of polyhydroxyalkanoates
    In this example a polyhydroxyalkanoate producing microorganism, such as Ralstonia eutropha H16, Pseudomonas putida KT2440, or a PHA and metal-resistant producing microorganism, such as Cupriavidus metallidurans (DSM 2839), is used for the production of poly-3-hydroxybutyrate (PHB ), poly-3-hydroxyoctanoate (PHO) or a copolymer of 3-hydroxy butyric acid and 3-hydroxy valeric acid, and that for the biosynthesis of silver and copper nanoparticles.
    In a particular application (example 1.1), Ralstonia eutropha (DSM 428) was used as a microorganism producing PHB and silver nanoparticles. The strain was stored at -80 ° C in glycerol, reactivated by plate culture in Luria Agar and then transferred to nutritive broth (beef extract 3g / L, peptone 5g / L), incubated for 24 hours on a shaker orbital at 200 rpm and 30 ° C. At the end of this time, it was centrifuged at 4,000 rpm for 15 minutes and the biomass was recovered. Biomass is re-suspended in production medium (MgSO4-7H2O 0.39g / L, K2SO4 0.45g / L, H3PO4 12ml / L, FeSO4-7H2O 15mg / L, NH4Cl 0.01%, 1% sodium gluconate , trace elements 24mL / L (CuSO4-5H2O 20mg / L, ZnSO4-6H2O 100mg / L, MnSO4-4H2O 100mg / L, CaCl2-2H2O 2.6g / L)), and incubated at 30 ° C for an additional 24 hours . After this time, silver nanoparticles were synthesized in the fermentation medium. Silver nitrate at 3.5mM (333mL / L of culture medium) was added as a precursor to the nanoparticles and the biomass reducing power was used. The result is the biosynthesis of silver nanoparticles without the need for an additional reducing agent or a stabilizing agent. After 16 hours of incubation at 30 ° C, in continuous agitation at 100 rpm, the nanoparticles managed to cross the bacterial membrane and look at the biopolymer as confirmed in images of Transmission Electron Microscopy (TEM), in which it can be seen Ralstonia eutropha H16 cells containing silver nanoparticles and biopolymer within the cell cytoplasm (Figure 1).
    In another particular application (example 1.2), the procedure described in the previous paragraph was proceeded in a similar manner but a hydrazine hydrate (30 ml / L culture medium) was added as a reducing agent and a copper salt, in particular, sulfate of copper pentahydrate (CuSO4-5H2O, 200mL / L medium) to the fermentation medium in order to synthesize copper nanoparticles.
    The fermentation product (containing silver (example 1.1) or copper (example 1.2) nanoparticles) was passed through a high pressure homogenizer (2000 psi, 8 minutes in recirculation) to break the cell membrane and release the granules from biopolymer containing metal nanoparticles. The fermentation broth was centrifuged for 15 minutes at 9000 rpm and the supernatant was discarded. The biomass obtained was re-suspended in chloroform (10% dry weight / volume), for 16-20 hours at 50 ° C. Subsequently, water (50% volume / volume) was added, the phases were separated by centrifugation (2000 rpm, 2 min) and the organic phase was precipitated on 10 volumes of cold methanol. The precipitate was dried in a vacuum oven at 40 ° C for 24 hours. With the product obtained, polymeric films (films) of approximately 100 µm thick were made by compression molding at 180 ° C and 1.8 MPa for 5 minutes.
    The physical appearance of the films obtained, containing silver or copper nanoparticles, can be seen in the photographs of Figure 2, in which, for comparative purposes, the photograph of a commercial PHBV film and a coated film has also been included with a masterbatch manufactured with the same procedure, but without containing metal nanoparticles. From these photographs it is observed that the incorporation of the metal nanoparticles improves the optical properties of the material, while the films do not have the typical black (in the case of silver) or brown (in the case of copper) characteristic materials Polymers that are additive with silver or copper salts at high concentrations in order to achieve the desired antimicrobial (in particular, bactericidal or virucidal) effect.
    The antibacterial and virucidal activity of the polymeric films made was evaluated taking into account the JIS Z 2801: 2006 standard (ISO 22196: 2007) for evaluation of antimicrobial activity on plastic surfaces, with some modifications described below. 3 cm x 3 cm films were inoculated with a bacterial suspension of 5 x 105 CFU / mL or 1 x 105 virus of infectious dose 50 in cell culture (TCID50) and covered with inert 2.5 cm x 2 films , 5 cm of Low Density Polyethylene (LDPE). The assembly was left in incubation at 25 ° C and 90% relative humidity for 24 hours. After this time, a viable cell count was carried out on trypticasein soy agar plates (TSA) in the case of bacteria or the cytopathic effect of the virus on CRFK cells (CCL-94) was measured. The results of this evaluation are shown in Figure 3 and Table 3, where it is shown that both the silver contained in the films, which is equivalent to 0.084 ± 0.009% by weight, and copper, which corresponds to 0.201 ± 0.009% by weight, have a bactericidal effect against the analyzed bacteria and reduce the cellular damage caused during the enteric virus replication cycle. The exposed results, in addition to corroborating the stability and optimal dispersion of the metal nanoparticles in the biopolymeric matrices, facilitate their use in applications that require microbiological control, since the effective dose is decreased and the antibacterial and virucidal efficiency of the materials is guaranteed.
    EXAMPLE 2: Obtaining microfibers of polyhydroxyalkanoates with metal nanoparticles by electrodynamic processing and their use as an antimicrobial coating.
    The antimicrobial biopolymer obtained in the fermentation process described in the example
    1.1 was used for the preparation of a masterbarch consisting of sub-micrometer fibers manufactured using the electrodynamic processing technique (also known as electrospinning).
    In particular, a solution of the biopolymer was prepared containing the metal nanoparticles in an organic solvent, 2,2,2-trifluoroethanol, which contained a percentage of total solids of 3% by weight and microfibers were synthesized by the electrodynamic processing technique. Electrodynamic processing was carried out on a pilot scale FLUIDNATEK ™ device, operated under a steady flow regime and using a high performance injector equipped with 20 stainless steel needles 29mm in diameter. The process conditions in this example are: solution flow rate: 80mL / h, distance between the injector and the collector: 20cm, voltage in the collector 24kV, voltage in the injector 18kV, deposition time: 15min. The material obtained was used as a coating of polymeric surfaces. In particular, commercial PHBV films (3% HV, Tianan, Biopolymer, Ningbo, China) were coated with 80-100µm thick antimicrobial mats, 5-15% by weight with respect to the film to be coated. Figure 4 shows an image obtained by Scanning Electron Microcopy (SEM) of the biopolymer microfibers containing silver nanoparticles.
    In a variation of the electro-stretching process, a horizontal displacement motor was used, which moved the injector along the manifold, favoring the alignment of the microfibers and with it the dispersion and distribution of the metal nanoparticles and the vapor barrier of water and oxygen.
    The films developed in this example show bactericidal (Figure 3) and virucidal activity. (Table 3). For example, films containing silver nanoparticles reduce the number of viable Salmonella enterica cells by up to 5 logarithmic units and films containing copper nanoparticles reduce the cytopathic effect of Murine Norovirus by more than 2 log10 TCID50 / ml. In addition, the total amount of silver or copper that migrates from the material (over a period of 10 days at 40 ° C) to a liquid medium that simulates a food, such as 10% ethanol representing hydrophilic foods, is below the limits of current legislation. In the case of PHBV3 films coated with microfibers of the silver-based antimicrobial biopolymer, the specific migration is twice lower than that contemplated in European regulations (EFSA 2006, The EFSA Journal (2006) 395 to 401, 121 .; EFSA 2011, COMMISSION REGULATION (EU) No 10/2011 of January 14, 2011 on plastic materials and objects intended to come into contact with food). (0.05mg / kg of food, 83ng / cm2 for a hypothetical surface of 6dm2 / kg of food). Thus, the materials obtained by the procedure described above have bactericidal activity at lower concentrations than those required in the techniques used in Example 1.1, with the additional advantage of having excellent optical properties, as shown in Figure 5. In addition The incorporation of metallic nanoparticles by this route has little or no effect on the thermal, mechanical, barrier properties or on the crystallinity of the coated material.
     Temperature
    Kind of movie 37 ° C25 ° C
    Assessment obtained (log10 TCID50 / ml) Reduction Assessment obtained (log10 TCID50 / ml)Reduction
    Control 5.45 ± 0.00 A5.76 ± 0.08 A
    AgNP 3.44 ± 0.00 B2.015.32 ± 0.00 A0.13
    CUNP  <1.15 C> 3.593.19 ± 0.05 B> 2.57
    Table 3 contains the results of the evaluation of the virucidal activity of the polymeric films of PHAs containing silver or copper nanoparticles against murine Norovirus (MNV) at 25 ° C and 37 ° C.

    权利要求:
    Claims (7)
    [1]
    1.-A method of obtaining an antimicrobial biopolymer comprising theless a polyhydroxyalkanoate and metal nanoparticles with antimicrobial capacity,5 characterized in that the procedure comprises:
    a) inoculating a biomass comprising microorganisms producing polyhydroxyalkanoate in a production medium suitable for the production of polyhydroxyalkanoates;
    b) synthesize metal nanoparticles with antimicrobial capacity in situ by the reaction of a precursor compound and a reducing agent; and c) extracting the antimicrobial biopolymer comprising at least one polyhydroxyalkanoate and the metal nanoparticles.
    [2]
    2. The method of obtaining a biopolymer according to claim 1, wherein
    15 metal nanoparticles are selected from the group consisting of silver nanoparticles, copper nanoparticles and a combination of the above.
    [3]
    3. The method of obtaining a biopolymer according to claim 2, wherein the metal nanoparticles are silver and the reducing agent is selected from the group that
    20 consists of organic compounds derived from fermentation, microorganisms producing polyhydroxalcanoates inoculated in step a) and a combination of the above.
    [4]
    4.-The procedure for obtaining a biopolymer according to any one of the
    Claims 1 to 3, wherein the biomass comprises polyhydroxyalkanoate producing microorganisms selected from the group consisting of Ralstonia eutropha H16, Pseudomonas putida KT2440, Cupriavidus metallidurans (DSM 2839) and a combination of the foregoing.
    The method of obtaining a biopolymer according to any one of claims 1 to 4, wherein the polyhydroxyalkanoate is formed by monomers of 3-hydroxy-propionic acid, 3-hydroxy-butyric acid, 3-hydroxy-valeric acid, 3-hydroxyhexanoic acid, 3-hydroxy-heptanoic acid, 3-hydroxy-octanoic acid, 3-hydroxy-nonanoic acid, 3-hydroxy-decanoic acid, 3-hydroxy-undecanoic acid, 3-hydroxy-dodecanoic acid, acid
    3-hydroxy-tetradecanoic acid, 3-hydroxy-hexadecanoic acid, 4-hydroxy-butyric acid, 4

    hydroxy-valeric, 4-hydroxy-hexanoic acid, 4-hydroxy-heptanoic acid, 4-hydroxyoctanoic acid, 4-hydroxy-decanoic acid, 5-hydroxy-valeric acid, 5-hydroxy-hexanoic acid, 6-hydroxy-dodecanoic acid ; it is a functionalized polyhydroxyalkanoate formed by the monomers named above and alkyl, cyclohexyl, halogenated radicals,
    Acetoxy, ester, alkoxy, epoxy, thio, cyano, nitro, benzoyl, methylphenoxy, phenoxy, phenyl; or is it acopolymer of the above polyhydroxyalkanoates.
    [6]
    6. The method of obtaining a biopolymer according to any one of claims 1 to 5, wherein the biopolymer is extracted by physical, chemical lysis or
    10 biological cell membrane of polyhydroxyalkanoate producing microorganisms.
    [7]
    7.-Antimicrobial biopolymer comprising at least one polyhydroxyalkanoate and metal nanoparticles obtained by the process described in any one
    15 of claims 1 to 6.
    [8]
    8. Use of the antimicrobial biopolymer described in claim 7, as masterbatch or concentrate in mixtures with other polymers.
    9. Use of the antimicrobial biopolymer obtained by the method described in any of claims 1-6 for obtaining a film or plastic article comprising at least one polyhydroxyalkanoate and metal nanoparticles, by any technique of processing molten plastics .
    10. Use of the antimicrobial biopolymer obtained by the process described in any of claims 1-6 for obtaining micro, submicro or nanoparticles comprising at least one polyhydroxyalkanoate and metal nanoparticles, by means of an electrodynamic processing technique.
    11. Use of the biopolymer of claim 7 in the manufacture of packages, surface coating, sensor manufacturing, cosmetics, biomedicine, protein and nucleic acid purification or controlled release of antimicrobial substances.
    Fig. 1
    Fig. 2 Fig. 3
    Fig. 4 Fig. 5
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