The impact of zinc oxide particle morphology as an antimicrobial and when incorporated in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) films for food packaging and food contact surfaces applications

In this work, zinc oxide (ZnO) micron and nano sized-particles with different morphologies were synthesized by aqueous precipitation and evaluated as antimicrobial agents against foodborne pathogens. The most effective bactericide system was selected to prepare active poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) films by three different methods (i) direct melt-mixing, (ii) melt-mixing of preincorporated ZnO into PHBV18 (18 mol% valerate content) fiber mats made by electrospinning, and, (iii) as a coating of the annealed electrospun PHBV18/ZnO fiber mats over compression molded PHBV. Results showed that ZnO successfully improved the thermal stability of the PHBV18, being the preincorporation method the most efficient in mitigating the negative impact that the PHBV18 had on the thermal stability, barrier and optical properties of the PHBV films. Similar behavior was found for the coating structure although this film showed effective and prolonged antibacterial activity against Listeria monocytogenes. This study highlights the suitability of the PHBV/ZnO nanostructures for active food packaging and food contact surface applications.This work was financially supported by the Spanish Ministry of Economy and Competitiveness (MAT2012-38947-C02-01 and AGL2015-63855-C2-1-R). J.L. Castro-Mayorga is supported by the Administrative Department of Science, Technology and Innovation (Colciencias) of Colombian Government. M. J. Fabra is recipient of a Ramon y Cajal contract from the Spanish Ministry of Economy and Competitiveness.Peer reviewe

Antiviral properties of silver nanoparticles against norovirus surrogates and their efficacy in coated polyhydroxyalkanoates systems

Silver nanoparticles (AgNP) have strong broad-spectrum antimicrobial activity and gained increased attention for the development of AgNP based products, including medical and food applications. Initially, the efficacy of AgNP and silver nitrate (AgNO3) was evaluated for inactivating norovirus surrogates, the feline calicivirus (FCV) and the murine norovirus (MNV). These norovirus surrogates were exposed to AgNO3 and AgNP solutions for 24 h at 25 °C and then analyzed by cell-culture assays. Both AgNP and silver ions significantly decreased FCV and MNV infectivity in a dose-dependent manner between concentrations of 2.1 and 21 mg/L. Furthermore, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) films were prepared by depositing a coating of thermally post-processed electrospun PHBV18/AgNP fiber mats over compression moulded PHBV3 films. After 24 h exposure at 37 °C and 100% RH, no infectious FCV were recovered when in contact with the AgNP films while MNV titers decreased by 0.86 log. The morphology of the PHBV18 and PHBV18/AgNP fibers studied by SEM showed smooth and continuous fibers in both cases and the EDAX analysis confirmed the homogeneously distribution of AgNP into the coating and onto the PHBV3/PHBV18 layer. This study showed, for the first time, the suitability of the PHBV18/AgNP electrospun coating for antiviral surfaces.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) (RYC-2012-09950, RYC-2014-158, AGL2015-63855-C2-1-R and INIA grant RTA2014-00024-C04-03). GS and MJF were supported by the “Ramón y Cajal” Young Investigator from the MINECO (RYC-2012-09950 and RYC-2014-15842). JLC-M was supported by the Administrative Department of Science, Technology and Innovation (Colciencias) of Colombian Government.Peer reviewe

Efficacy of Cinnamaldehyde Against Enteric Viruses and Its Activity After Incorporation Into Biodegradable Multilayer Systems of Interest in Food Packaging

Cinnamaldehyde (CNMA), an organic compound that gives cinnamon its flavor and odor, was investigated for its virucidal activity on norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV), and hepatitis A virus (HAV). Initially, different concentrations of CNMA (0.1, 0.5 and 1 %) were individually mixed with each virus at titers of ca. 6–7 log10 TCID50/ml and incubated 2 h at 4 and 37 °C. CNMA was effective in reducing the titers of norovirus surrogates in a dose-dependent manner after 2 h at 37 °C, while HAV titers were reduced by 1 log10 after treatment with 1 % of CNMA. When incubation time was extended, HAV titers were reduced by 3.4 and 2.7 log10 after overnight incubation at 37 °C with 1 and 0.5 % of CNMA, respectively. Moreover, this paper analyzed, for the first time, the antiviral activity of adding an active electrospun interlayer based on zein and CNMA to a polyhydroxybutyrate packaging material (PHB) in a multilayer form. Biodegradable multilayer systems prepared with 2.60 mg/cm2 (~9.7 %) of CNMA completely inactivated FCV according to ISO 22196:2011, while MNV titers were reduced by 2.75 log10. When the developed multilayer films were evaluated after one month of preparation or at 25 °C, the antiviral activity was reduced as compared to freshly prepared multilayer films evaluated at 37 °C. The results show the excellent potential of this system for food contact applications as well as for active packaging technologies in order to maintain or extend food quality and safety.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) (RYC-2012-09950, AGL2015-63855-C2-1 and INIA Grant RTA2014-00024-C04-03). GS was supported by the “Ramón y Cajal” Young Investigator and MJF was recipient of a Juan de la Cierva contract from the MINECO. JLC-M was supported by the Departamento Administrativo de Ciencia, Tecnología e Innovación (Colciencias) of Colombian Government and WR by the “Student Mobility for Placement e SMP” Grant of the EU Life Learning Program.Peer reviewe

Use of electrospinning to develop antimicrobial biodegradable multilayer systems: encapsulation of cinnamaldehyde and their physicochemical characterization

In this work, three active bio-based multilayer structures, using a polyhydroxybutyrate-co-valerate film with a valerate content of 8 % (PHBV8) as support, were developed. To this end, a zein interlayer with or without cinnamaldehyde (CNMA) was directly electrospun onto one side of the PHBV8 film and the following systems were developed: (1) without an outer layer; (2) using a PHBV8 film as outer layer; and (3) using an alginate-based film as outer layer. These multilayer structures were characterized in terms of water vapour and oxygen permeabilities, transparency, intermolecular arrangement and thermal properties. The antimicrobial activity of the active bio-based multilayer systems and the release of CNMA in a food simulant were also evaluated. Results showed that the presence of different outer layers reduced the transport properties and transparency of the multilayer films. The active bio-based multilayer systems showed antibacterial activity against Listeria monocytogenes being the multilayer structure prepared with CNMA and PHBV outer layers (PHBV + zein/CNMA + PHBV) the one that showed the greater antibacterial activity. The release of CNMA depended on the multilayer structures, where both Fick's and Case II transport-polymer relaxation explained the release of CNMA from the multilayer systems.Acknowledgments: Miguel A. Cerqueira (SFRH/BPD/72753/2010) andAnaI.Bourbon(SFRH/BD/73178/2010)arerecipientofafellowship from the Fundação para a Ciência e Tecnologia (FCT, POPH-QREN and FSE Portugal). J.L. Castro-Mayorga is supported by the Administrative Department of Science, Technology and Innovation (Colciencias) of Colombian Government. M. J. Fabra is a recipient of a Ramon y Cajal contract (RyC-2014-158) from the Spanish Ministry of Economy and Competitiveness. This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and of the Project RECI/BBB-EBI/ 0179/2012 (FCOMP-01-0124-FEDER-027462). The support of EU Cost Action MP1206 is gratefully acknowledged

Biotechnological routes for the development of antimicrobial nano-metal based polyhydroxyalkanoates for active food packaging applications

Tesis por compendioThe development of novel bio-based materials with antimicrobial properties for active packaging applications is a topic of significant interest. The current PhD thesis deals with the development of biotechnologically derived polyhydroxyalkanoates (PHAs) based on nanometals for antimicrobial active food packaging applications. Initially, silver nanoparticles (AgNPs) were produced by chemical reduction and stabilized in situ within unpurified poly(hydroxybutyrate-co-hydroxyvalerate), PHBV18 (18 mol% valerate) suspensions previously obtained from mixed microbial cultures. The stabilized AgNPs were subsequently used to develop PHAs-AgNPs nanocomposites following two different strategies: 1) a direct melt-blending process where the AgNPs were added to the PHBV3 (3% mol valerate) from a highly dispersed and distributed enriched masterbatch form and, 2) as an annealed electrospun coating of PHBV3/PHBV18/AgNPs over compression molded PHBV3. The implementation of both strategies resulted in active nanocomposites with strong antimicrobial activity against food-borne pathogens, being the electrospinning coating technique the most efficient one in reducing the bacterial and virus population, even at very low AgNPs loading (from 0.002 to 0.04% wt.). As an alternative route, an integrated bioprocess for the biological synthesis of AgNPs and polyhydroxybutyrate (PHB) from the fermentation process with Cupriavidus necator was also carried out. Interestingly, this work demonstrated for the first time, the inherent capacity of C. necator to reduce silver nitrate and produce AgNPs without the need for adding a reducing agent. The process was successfully optimized and scaled-up to a fully automated 10 liters bioreactor. Finally, because of the limitations of the use of AgNPs in food applications, antimicrobial PHAs films based on zinc oxide (ZnO) and copper oxide (CuO) nanoparticles were prepared according to the previously developed strategies but in this case, a melt-mixing process of preincorporated ZnO into unpurified PHBV18 fiber mats made by electrospinning was also carried out to stabilize the metal nanoparticles. The effect of ZnO nanoparticles morphology and the method of ZnO/CuO incorporation on the morphological, optical, thermal, mechanical and barrier properties of the resulting active films as well as their influence on the antimicrobial (bactericide and virucidal) performance were studied. Thus, this PhD thesis represents a significant step forward in the understanding of the antimicrobial efficacy of highly dispersed and distributed nanometals and highlights the suitability of the developed PHAs/nanometals materials for antimicrobial applications and in particular for antimicrobial active food packaging applications.El desarrollo de nuevos biomateriales con propiedades antimicrobianas para aplicaciones de envasado activo resulta un tema de gran interés en la actualidad. La presente tesis doctoral estudia el desarrollo por vía biotecnológica de polihidroxialcanoatos (PHAs) conteniendo nanometales para aplicaciones de envasado activo antimicrobiano de alimentos. En primer lugar, se produjeron nanopartículas de plata (AgNPs) por reducción química y se estabilizaron in situ en una suspensión de poli (hidroxibutirato-co-hidroxivalerato) no purificado, PHBV18 (18% en moles de valerato), obtenido previamente a partir de cultivos mixtos microbianos. Posteriormente, las AgNPs estabilizadas se utilizaron para desarrollar nanocompuestos de PHAs-AgNPs siguiendo dos estrategias diferentes: 1) un proceso de mezclado-fundido en donde las AgNPs se añadieron al PHBV3 (3% mol de valerato) a partir de un masterbatch de nanopartículas altamente dispersas y distribuidas y, 2) como una estructura bicapa formada por un recubrimiento a base de PHBV/PHBV18/AgNPs depositado sobre un film de PHBV3 obtenido por moldeo por compresión. La aplicación de ambas estrategias dio lugar a nanocompuestos activos con una fuerte actividad antimicrobiana frente a patógenos transmitidos por los alimentos, siendo la estructura bicapa la más eficaz en la reducción de la población bacteriana y viral, incluso a una carga muy baja de AgNPs (de 0.002 a 0.04% en peso). Como ruta alternativa, también se llevó a cabo un proceso integrado de fermentación con Cupriavidus necator para la síntesis biológica de AgNPs y polihidroxibutirato (PHB). En este trabajo se demostró, por primera vez, la capacidad inherente de C. necator para reducir nitrato de plata y producir AgNPs sin la necesidad de añadir un agente reductor. El proceso fue optimizado y escalado satisfactoriamente a un biorreactor automatizado de 10 litros. Finalmente, debido a las limitaciones del uso de AgNPs en aplicaciones alimentarias, se prepararon films antimicrobianos de PHAs basados en nanopartículas de óxido de zinc (ZnO) y óxido de cobre (CuO) de acuerdo con las estrategias previamente desarrolladas. Adicionalmente, ambas estrategias se compararon con una tercera basada en la preincorporación de ZnO en fibras de PHBV18 no purificado y su posterior mezclado-fundido con polímero virgen. Se estudió el efecto de la morfología de las nanopartículas de ZnO y del método de incorporación de ZnO/CuO sobre las propiedades morfológicas, ópticas, térmicas, mecánicas y de barrera de los films activos resultantes, así como su influencia en el comportamiento antimicrobiano (bactericida y virucida). Por lo tanto, esta tesis doctoral representa un avance significativo en la comprensión de la eficacia antimicrobiana de nanometales altamente dispersos y distribuidos y destaca la idoneidad de los materiales desarrollados a base de PHAs y nanometales para aplicaciones antimicrobianas y, en particular, para aplicaciones de envasado de alimentos activos antimicrobianos.El desenvolupament de nous materials d'origen biològic amb propietats antimicrobianes per a aplicacions d'envasament actiu és un tema d'interès significatiu. La tesi doctoral actual s'ocupa del desenvolupament de polihidroxialcanoats (PHA) reforçats amb nanometals per via biotecnològicament per a aplicacions d'envasat actiu antimicrobià d'aliments. Inicialment, les nanopartícules de plata (AgNPs) van ser produïdes per reducció química i estabilitzades in situ dins en suspensions de poli (hidroxibutirato-co-hidroxivalerato) sense purificar, PHBV18 (18 mol% de valerat), prèviament obtinguts a partir de cultius mixtes microbians. Las AgNPs estabilitzades es van usar posteriorment per a desenvolupar nanocompostos de PHA's- AgNPs seguint dues estratègies diferents: 1) Procés directe de barreja en fusió que no utilitza dissolvents orgànics o estabilitzants addicionals i on es van afegir les AgNPs al PHBV3 (3% mol valerato) a partir d¿un masterbath on estaven perfectament disperses i distribuïdes 2) com una estructura bicapa formada per un recobriment de PHBV3 / PHBV18/AgNPS que es deposita sobre un film de PHBV3 obtingut per modelat per compressió. L'aplicació d'ambdues estratègies va donar lloc a nanocompostos actius amb una forta activitat antibacteriana enfront de patògens transmesos pels aliments, sent l'estructura de doble capa la més eficaç en la reducció de la població bacteriana i viral, fins i tot a una càrrega molt baixa de AgNPs (de 0.002-0.04% en pes). Com ruta alternativa, també es va dur a terme un procés integrat de fermentació amb Cupriavidus necator per a la síntesi biològica de AgNPs i polihidroxibutirato (PHB). En aquest treball es demostra, per primera vegada, la capacitat inherent de C. necator per reduir la sal de plata i produir AgNPs sense la necessitat d'afegir un agent reductor. El procés va ser optimitzat i escalat satisfactòriament a un bioreactor de 10 litres. Finalment, a causa de les limitacions de l'ús de nanopartícules de plata en aplicacions alimentàries, es van preparar films antimicrobians de PHA que incorporessin nanopartícules d'òxid de zinc (ZnO) i òxid de coure (CuO) d'acord amb les estratègies prèviament desenvolupades. Les dues estratègies es van comparar amb una tercera basada en la preincorporació de ZnO en fibres de PHBV18 no purificat i aquestes fibres es van barrejar posteriorment amb polímer verge. Es va estudiar l'efecte de la morfologia de les nanopartícules de ZnO i el mètode de la incorporació de ZnO/CuO sobre les propietats morfològiques, òptiques, tèrmiques, mecàniques i de barrera dels films actius resultants, així com la seva influència en el comportament antimicrobià (bactericida i virucida). Per tant, aquesta tesi doctoral representa un pas endavant significatiu en la comprensió de l'eficàcia antimicrobiana de nanometales altament dispersos i distribuïts i posa en relleu la idoneïtat dels materials desenvolupats basats en PHAs i nanometals per a aplicacions antimicrobianes i, en particular, per a aplicacions d'envasat d'aliments actius antimicrobians.Castro Mayorga, JL. (2017). Biotechnological routes for the development of antimicrobial nano-metal based polyhydroxyalkanoates for active food packaging applications [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/85678TESISCompendi

On the Use of the Electrospinning Coating Technique to Produce Antimicrobial Polyhydroxyalkanoate Materials Containing In Situ-Stabilized Silver Nanoparticles

Electro-hydrodynamic processing, comprising electrospraying and electrospinning techniques, has emerged as a versatile technology to produce nanostructured fiber-based and particle-based materials. In this work, an antimicrobial active multilayer system comprising a commercial polyhydroxyalkanoate substrate (PHA) and an electrospun PHA coating containing in situ-stabilized silver nanoparticles (AgNPs) was successfully developed and characterized in terms of morphology, thermal, mechanical, and barrier properties. The obtained materials reduced the bacterial population of Salmonella enterica below the detection limits at very low silver loading of 0.002 ± 0.0005 wt %. As a result, this study provides an innovative route to generate fully renewable and biodegradable materials that could prevent microbial outbreaks in food packages and food contact surfaces.This work was financially supported by the Spanish Ministry of Economy and Competitiveness (AGL2015-63855-C2-1-R). Jinneth Lorena Castro-Mayorga is supported by the Administrative Department of Science, Technology and Innovation (Colciencias) of the Colombian Government. Maria Jose Fabra is recipient of a Ramon y Cajal contract from the Spanish Ministry of Economy and Competitiveness. Maria Reis from Universidade Nova de Lisboa, Lisbon, Portugal is acknowledged for supplying the PHBV18 material. We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

Microbially Synthesized Polymer-Metal Nanoparticles Composites as Promising Wound Dressings to Overcome Methicillin-Resistance <i>Staphylococcus aureus</i> Infections

Antimicrobial resistance has been declared one of the top 10 global public health threats. Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of recurring skin and soft tissue infections in patients with chronic skin conditions such as diabetic foot infections, making the treatment of the ulcers challenging. Wound dressings combined with metal nanoparticles have been suggested to prevent and treat MRSA-infected wounds. However, these particles are commonly synthesized by chemical approaches. In this study, we developed bio-based silver (Bio-AgNPs) and copper oxide nanoparticles (CuONPs) polymer composites using a microbially produced polyester from the Polyhydroxyalkanoates (PHAs) family. Poly(3-hydroxyoctanoate)-co-(3-hydroxyhexanoate) (PHO) was synthesized by Pseudomonas putida and functionalized in-situ with Bio-AgNPs or ex-situ with CuONPs. PHO-CuONPs films did not inhibit MRSA growth, while a reduction of 6.0 log CFU/mL was achieved with PHO-Bio-AgNPs synthesized from silver nitrate (AgNO3) solution at 3.5 mM. Exposure of human fibroblast cells (HFF-1) to the bioactive films did not induce notable cytotoxicity and genotoxicity, as seen by a viability higher than 79% and no significant changes in basal DNA damage. However, exposure to PHO-Bio-AgNPs induced oxidative DNA damage in HFF-1 cells. No hemolytic potential was observed, while platelet aggregation was promoted and desired for wound healing. Here we demonstrate the biosynthesis of polymer-nanoparticle composites and their potential as bioactive films for MRSA treatment

Biosynthesis of silver nanoparticles and polyhydroxybutyrate nanocomposites of interest in antimicrobial applications

This study deals with the optimization and scaling up of the production of poly(3-hydroxybutyrate), PHB, nanocomposites containing biosynthesized silver nanoparticles (AgNPs) to generate materials with antimicrobial performance. First, a comparative study of the chemical and biological synthesis of AgNPs during the fermentation process of Cupriavidus necator at shake flask-scale was carried out. These experiments demonstrated the inherent capacity of C. necator to reduce the silver salt and produce AgNPs without the need for adding a reducing agent and, that the method of synthesis (with or without reducing agent) affects the dispersion of the AgNPs and their antimicrobial performance. Finally, the process was scaled-up to a 10 Liters bioreactor and the relevant physical properties of the PHB-AgNPs nanocomposites pressed into films were determined. From the characterization work, the AgNPs were found to be well dispersed and distributed into the polymer matrix, having a maximum frequency of particles with average diameter of 76–95 nm. Moreover, the presence of AgNPs did not cause any effect on the thermal properties of the biopolymer, although a slight reduction in crystallinity was seen. The developed materials presented a strong antimicrobial activity against the food-borne pathogens Salmonella enterica and Listeria monocytogenes, which makes them potentially suitable for active coatings and packaging applications. Complete biodisintegration of the samples occurred during composting conditions within the first 40 days. Interestingly, the presence of the AgNPs did not impair the profile of biodegradation of the microbial polymer.The authors would like to thank to the Active and intelligent fibre-based packaging – innovation and market introduction (ActInPak) COST Action FP1405 for the funding of the Short Therm Scientific Mission, to Octavio Cedenilla from the Instituto de Ciencias Agrarias-CSIC for the ICP-OES analysis and to Dr. Luis Cabedo, from Universitat Jaume I for his support with WAXD analyses and biodisintegration tests. Research leading to these results has received funding from the Comunidad de Madrid (P2013/MIT2807) and the Spanish Ministry of Economy and Competitiveness, (BIO2013-44878-R and AGL2015-63855-C2-1-R). Jinneth Lorena Castro-Mayorga is supported by the Administrative Department of Science, Technology and Innovation (Colciencias) of the Colombian Government.Peer reviewe

Antimicrobial nanocomposites and electrospun coatings based on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and copper oxide nanoparticles for active packaging and coating applications

Active biodegradable poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) melt mixed nanocomposites and bilayer structures containing copper oxide (CuO) nanoparticles were developed and characterized. The bilayer structures consisted of a bottom layer of compression molded PHBV3 (3% mol valerate) coated with an active electrospun fibers mat made with CuO nanoparticles and PHBV18 (18% valerate) derived from microbial mixed cultures and cheese whey. The results showed that the water vapor permeability increased with the CuO addition while the oxygen barrier properties were slightly enhanced by the addition of 0.05 wt % CuO nanoparticles to nanocomposite films but a negligible effect was registered for the bilayer structures. However, the mechanical properties were modified by the addition of CuO nanoparticles. Interestingly, by incorporating highly dispersed and distributed CuO nanoparticles in a coating by electrospinning, a lower metal oxide loading was required to exhibit significant bactericidal and virucidal performance against the food-borne pathogens Salmonella enterica, Listeria monocytogenes, and murine norovirus. The biodisintegration tests of the samples under composting conditions showed that even the 0.05% CuO-coated structures biodegraded within 35 days.This work was financially supported by the Spanish Ministry of Economy and Competitiveness (project AGL2015–63855-C2–1-R). J.L.C.-M. is supported by the Administrative Department of Science, Technology and Innovation (Colciencias) of Colombian Government. M.J.F. is recipient of a Ramon y Cajal contract from the Spanish Ministry of Economy and Competitiveness.Peer reviewe

Procedimiento para la obtención de biopolímeros antimicrobianos que comprenden polihidroxialcanoatos y nanopartículas metálicas

Procedimiento para la obtención de biopolímeros antimicrobianos que comprenden polihidroxialcanoatos y nanopartículas metálicas. Un procedimiento de obtención de un biopolímero antimicrobiano que comprende al menos un polihidroxialcanoato (PHA) y nanopartículas metálicas con capacidad antimicrobiana, caracterizado porque el procedimiento comprende: a) inocular una biomasa que comprende microorganismos productores de PHA en un medio de producción adecuado para la producción de PHAs; b) sintetizar nanopartículas metálicas con capacidad antimicrobiana in situ mediante la reacción de un compuesto precursor y un agente reductor; y c) extraer el biopolímero antimicrobiano que comprende al menos un polihidroxialcanoato y las nanopartículas metálicas. Biopolímero antimicrobiano obtenido por el procedimiento descrito anteriormente y usos del biopolímero.Peer reviewedCastro Mayorga, Jinneth Lorena; Consejo Superior de Investigaciones Científicas (España)A1 Solicitud de patente con informe sobre el estado de la técnic