This study evaluated the potential use of an antimicrobial packaging system in combination with electron beam irradiation to enhance quality of fresh produce. Irradiated romaine lettuce up to 3.2 kGy showed negligible (p > 0.05) changes in color, but texture and sensory attributes were less acceptable with increased dose. We established the antimicrobial effectiveness of various active compounds incorporated into the low-density polyethylene (LDPE)/polyamide films to increase radiation sensitivity of surrogate bacteria (Listeria innocua and Escherichia coli). All films showed inhibition zones in an agar diffusion test. In the liquid culture test, the active compounds reduced the specific growth rate and decreased final cell concentration of strains. Films incorporated with active compounds increased the radiation sensitivity of the tested strains, demonstrating their potential to reduce the dose required to control microbial contamination using electron beam technology. The active compounds maintained their antimicrobial activity by exposure to ionizing radiation up to 3 kGy. Antimicrobial activity of LDPE/polyamide films incorporated with transcinnamaldehyde was tested with fresh-cut romaine lettuce. Total aerobic plate counts (APC) and yeast and mold counts (YMC) were determined as a function of dose (0, 0.5, and 1.0 kGy) for 14 days of storage at 4°C. Irradiation exposure significantly lowered APCs of lettuce samples by 1-log CFU/g compared to the non-irradiated controls; however, it only slightly reduced YMCs. The effectiveness of using irradiation with antimicrobial films was enhanced with increased radiation dose and transcinnamaldehyde concentration. Electron beam irradiation up to 20 kGy did not affect the tensile strength and toughness of the polymeric films. The film’s flexibility and barrier properties were significantly improved by exposure to 20 kGy. The addition of an active compound did not affect the tensile strength and barrier properties of the films, but decreased the percent elongation-at-break and toughness, making them slightly more brittle. Ionizing radiation affected the release kinetics of the antimicrobial agent from the packaging material into a model food system. Irradiated films exhibited slower release rates than non-irradiated film by 69%. In addition, release rate was lower at 4ºC by 62.6% than at 21-35ºC. The pH of the simulant solution affected release rate with pH 4 yielding higher rates than pH 7 and 10.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / An alternative for the recovery of industrial fish waste is the production of films, which can be applied in food packaging. However, a major challenge is to obtain films with antimicrobial properties aiding in increased food shelf life as well as films with good mechanical properties and barriers. Therefore, in order to improve these limitations, silver nanoparticles were used due to its potential antimicrobial in food, and tannic acid (TA) as crosslinker in order to improve the mechanical and barrier properties. This study focuses in the extraction of gelatin from the Nile tilapia skin (Oreochromisniloticus) in order to produce gelatin films with embedded nanosilver (AgNPs) generated by green process and evaluate the addition of tannic acid (TA) in synthesis of AgNPs and mechanical properties of the films and barrier. A pre-treatment was conducted to obtain gelatin followed by a collagen denaturing. The characterization was done by gel strength, isoelectric point, electrophoretic analysis, fourier transform infrared (FTIR) and thermogravimetric analysis (TGA). The incorporation was performed by in situ method, the films were obtained with and without the presence of TA and AgNO3 concentrations were 0, 45, 90 and 180 mmol.L-1.The films were characterized by UV-Vis, FTIR, TGA, Mechanical properties (tensile strength and elongation at break), water vapor permeability (WVP), scanning electron microscopy (SEM) and antimicrobial analysis. It was possible to use gelatin as a reducing agent and stabilizer in the reduction of silver nanoparticles. Films produced with the addition of AT showed more suitable properties for the usage as packaging material for food, showing better WVP, water solubility and were more resistant to applied loads. In addition, they presented better antimicrobial activity. Therefore, it was possible to obtain the green AgNPs method through the incorporationof thegelatin matrix and to obtain films with excellent properties after the TA addition. / Uma alternativa de valorizaÃÃo de resÃduo de pescado produzidos por indÃstrias à a produÃÃo de filmes, que pode ser aplicados em embalagens para alimentos. PorÃm, um dos grandes desafios à obter filmes com propriedades antimicrobianas, auxiliando no aumento da vida de prateleira dos alimentos, bem como obter filmes com boas propriedades mecÃnicas e de barreiras. Para isso, a fim de melhorar essas limitaÃÃes, nanopartÃculas de prata foram utilizadas devido a sua potencial aÃÃo antimicrobiana em alimentos, e Ãcido tÃnico (AT) como agente reticulante a fim de melhorar as propriedades mecÃnicas e de barreiras. Nesse estudo objetivou-se fazer a extraÃÃo da gelatina a partir de pele de tilÃpia do Nilo (Oreochromis niloticus), visando a produÃÃo de filmes de gelatina incorporados com nanopartÃculas de prata (NPAg) geradas por processo verde e avaliar a adiÃÃo de Ãcido tÃnico (AT) na sÃntese das NPAg e nas propriedades mecÃnicas e de barreira dos filmes. Para a obtenÃÃo da gelatina foram feitos prÃ-tratamentos e em seguida a desnaturaÃÃo do colÃgeno. A caracterizaÃÃo foi feita por ForÃa de Gel, Ponto IsoelÃtrico, DistribuiÃÃo de Massa Molar, Espectroscopia no Infravermelho pro Transformada de Fourrier (FTIR) e AnÃlise TermogravimÃtrica (TGA). A incorporaÃÃo foi feita pelo mÃtodo in situ, Os filmes foram obtidos sem e com a presenÃa de AT e as concentraÃÃes de AgNO3 foram 0, 45, 90 e 180 mmol.L-1. Os filmes foram caracterizados por: UV-Vis, FTIR, TGA, Ensaios MecÃnicos, Permeabilidade ao Vapor de Ãgua (PVA), Microscopia EletrÃnica de Varredura (MEV) e AnÃlise Antimicrobiana. Foi possÃvel utilizar a gelatina como agente redutor e estabilizante na reduÃÃo de nanopartÃculas de prata. Os filmes produzidos com a adiÃÃo de AT apresentaram propriedades mais adequadas para uso como embalagem para alimentos, apresentando melhores PVA, solubilidade em Ãgua e mostraram-se mais resistentes a esforÃos aplicados. AlÃm de apresentarem melhor atividade antimicrobiana. Logo, foi possÃvel, obter pelo mÃtodo verde a incorporaÃÃo de NPAg na matriz de gelatina, alÃm, de se obter filmes com excelentes propriedades apÃs a adiÃÃo de AT.

Tesis por compendio / This PhD dissertation thesis has been focus on the development and characterization of antimicrobial packaging films based on the incorporation in the polymer matrix or on the attachment to the film surface of naturally occurring antimicrobial compounds with the purpose of inhibiting the proliferation of microorganisms and extend the microbiological shelf life of packaged food products. The studied active films are based on the use of ethylene vinyl copolymers (EVOH) containing 29% (EVOH29) or 44% (EVOH44) molar percentage of ethylene as polymeric vehicle for the incorporation of several antimicrobial compounds -oregano essential oil (OEO), citral, ethyl lauroyl arginate (LAE), epsilon-polylysine (EPL), green tea extract (GTE) and lysozyme. These antimicrobial agents have been incorporated in the film-forming solution or immobilized to the film surface by covalent bonding. Prior to the preparation of the active films, the antimicrobial activity of the selected compounds against selected microorganism was demonstrated, confirming that they could be good candidates to be used as preservatives for active food packaging applications, and an alternative to synthetic additives. The effect of the incorporation of the antimicrobial agents on relevant functional properties of the developed EVOH films was studied. In general, the polymer properties as materials for food packaging were not relevantly affected. In order to evaluate the potential of EVOH matrices as sustain release systems of active compounds, the release kinetics of the active compounds from the film to different media was evaluated; for that the agent release rate and extend into food simulants was monitored, and it was concluded that the agent concentration, release temperature, type of EVOH, interaction of EVOH with the food simulant, and the solubility of the active compound in the release media were the main controlling factors. EVOH matrices have also shown good properties to be used for the attachment of active molecules. In this regard, lysozyme was successfully immobilized on the film surface of EVOH. Several experiments were conducted to determine the antimicrobial properties of the resulting films in vitro against different microorganisms responsible for foodborne illness and in vivo with real foods –minimally-process salad, infant milk, surimi sticks and chicken stock- to enhance their preservation. All the materials presented a strong in vitro antimicrobial activity. Although the results obtained through in vivo tests showed activity reductions caused by food matrix effects, all materials presented significant microbial inhibition and, therefore, great potential to be used in the design of active food packaging. They can be applied as an inner coating of the packaging structure, releasing the active agent or acting by direct contact, producing a great protection against contamination with a prolongation of the microbiological food shelf life. / Muriel Galet, V. (2015). Novel antimicrobial films based on ethylene-vinyl alcohol copolymers for food packaging application [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48522 / Premios Extraordinarios de tesis doctorales / Compendio

Dans le but de développer des matériaux actifs antimicrobiens biodégradables à libération contrôlée, des mélanges de deux polymères biodégradables incompatibles ont été mis en oeuvre par les voies classiques de transformation des matériaux plastiques (extrusion). L'originalité de ces mélanges réside dans la recherche d'une stratégie de contrôle de la libération d'espèces actives incorporées dans l'une des phases du système et générée par la tortuosité/disponibilité de chemins de diffusion sélectifs obtenue en jouant sur les proportions relatives des 2 polymères en présence. Les systèmes binaires étudiés sont : [amidon de maïs plastifié, PLS et poly(butylène succinate-co-adipate) ou PBSA] et un système référence [(poly(oxyethylène) PEO et PBSA]. Dans ces mélanges, les polymères polaires (PLS ou PEO) jouent le rôle de matrice hôte pour solubiliser les migrants actifs ou modèles, souvent polaires et le polymère semipolaire,le PBSA, compense la faiblesse des propriétés mécaniques et barrières à l'eau de la phase amylacée et /ou hydrophile tout en présentant une température de fusion basse (85°C) compatible avec une stratégie à plus long terme d'incorporation d'actifs peu thermostables. Les systèmes obtenus sont caractérisés afin de corréler les différentes morphologies obtenues en jouant sur la composition des mélanges (phase dispersées dans une matrice continue, ou continuité partielle ou totale, systèmes pseudo-multicouches) aux phénomènes de transport de diverses molécules. Les caractérisations effectuées ont alors pour objectif d'élucider les morphologies obtenues par i) extraction sélective par voie solvant (ou hydrolyse) enrichie par des observations microscopiques ii) par utilisation de sondes gazeuses via les propriétés de transport de vapeur d'eau (qui privilégiera les chemins offerts par la matrice polaire) ou de l'oxygène (qui, lui, privilégiera les chemins de diffusions offerts par le PBSA) iii) par la réalisation des isothermes et cinétiques de sorption d'eau et enfin iv) par l'étude de la libération dans l'eau de migrants actifs ou modèles préalablement incorporés dans la phase polaire (fluorescéinate de sodium, acides organiques, glycine, di-glycine). Il a ainsi pu être montré pour les mélanges PLS/PBSA que les phénomènes de diffusion sont contrôlés par la tortuosité générée par la présence de PBSA dans le cas du relargage de la fluorescéine et par l'hydratation limitée de la phase polaire en présence de fortes teneurs en PBSA dans le mélange pour les phénomènes de sorption d'eau. Ainsi, la diffusion de la fluorescéine, par exemple, est nettement plus influencée que celle de l'eau par la tortuosité (elle-même gouvernée par la composition du mélange), très probablement en raison de sa plus grande masse moléculaire. Pour les mélanges PEO/PBSA, des comportements distincts ont été mis en évidence selon la composition des mélanges. Pour les mélanges à faibles teneurs ou teneurs intermédiaires en PBSA, un transport rapide se produit principalement via la dissolution du PEO. La morphologie ne semble pas influencer les cinétiques de libération. Pour des teneurs élevées en PBSA, la cinétique de libération est cette fois dépendante de la morphologie présente un temps de latence caractéristique d'une cinétique de perméation (libération proportionnelle au temps). En conclusion, les matériaux élaborés par les voies migrants polaires tels que des conservateurs pour élaborer par exemple des emballages actifs antimicrobiens / In order to develop biodegradable and active materials s, blends of two incompatible biodegradable polymers have been implemented by conventiona plastic material processing (extrusion). The originality of these blends lies in the search for a strategy to control the release of active species included in one of the phases of the system. Therefore, the tortuosity / availability of selective diffusion paths obtained by varying the relative proportions of the two polymers involved will be exploited. Studied binary systems consist of: [plasticized corn starch, PLS and poly (butylene succinate-co-adipate) or PBSA] and a reference system [(poly (oxyethylene) PEO and PBSA]. In these blends, polar polymers (PLS or PEO) play the role of host matrix to solubilize the active migrants or model molecules which are often polar, and the semi-polar polymer (PBSA) compensates for the weakness of the mechanical properties of the starchy and / or hydrophilic phase while having a low melting temperature (85 ° C) consistent with a longer-term strategy of incorporation of thermostable active molecules such as lysozyme or nisin. The resulting systems were characterized to correlate the different morphologies obtained by varying the composition of the blends (dispersed phase in a continuous matrix, partial or total continuity, or pseudo-layer systems) to the transport phenomena of various molecules. The performed characterizations aim to elucidate the morphologies by i) selective solvent extraction method (or hydrolysis) enriched by microscopical observations ii) using gaseous probes via the determination of water vapor transport properties (water will favor the paths provided by the polar matrix) or oxygen transport properties (O2 will favor the diffusion paths provided by the PBSA matrix) iii) by determining water sorption isotherms and kinetics and finally iv) by the study of the release in water of active or model migrants previously incorporated in the polar phase (sodium fluoresceinate, organic acids, glycine, diglycine). It has been observed that the diffusion phenomena in PLS / PBSA blends are controlled by the tortuosity generated by the presence of PBS and by the limited hydration of the polar phase at high PBSA contents in the blends for water sorption phenomena. Thus, the diffusion of fluorescein, for example, is much more influenced by the tortuosity (itself governed by the composition of the blends) than that of water, which is likely due to its higher molecular weight. For PEO / PBSA blends, distinct behaviors were observed according to the blends composition. For the blends with low or intermediate content of PBSA, rapid transport occurs mainly via the dissolution of the PEO. The morphology did not seem to influence the release kinetics. For high contents of PBSA, the release kinetics were dependent on the morphology and a time lag which is characteristic of permeation kinetics (release proportional to time) appeared. In conclusion, the multiphasic materials prepared by plastic processing look promising for the controlled release of polar migrants such as food preservatives to develop antimicrobial active packaging

No description available.

Tesis por compendio / [ES] La presente tesis doctoral plantea distintas estrategias para el desarrollo y aplicación de envases activos portadores de agentes de biocontrol, con el fin de inhibir la proliferación de microorganismos como bacterias y hongos patógenos y alterantes y así, conseguir aumentar la seguridad y prolongar la vida útil de los alimentos. El desarrollo de envases activos antibacterianos se llevó a cabo incorporando bacterias ácido lácticas productoras de bacteriocinas, Lactococcus lactis y Lactobacillus sakei en diferentes matrices formadoras de películas. Previa a la preparación de las películas activas, se estudió la actividad antimicrobiana de los agentes naturales seleccionados frente a Listeria monocytogenes. Los resultados sugirieron que, a partir de una determinada concentración inicial de bacterias viables, estos microorganismos son buenos candidatos para ser utilizados como aditivos naturales en materiales de envasado de alimentos, siendo una alternativa al uso de aditivos de origen sintético. En este trabajo se observó que la viabilidad inicial de las bacterias ácido lácticas (LAB) es esencial para obtener una buena capacidad antibacteriana por lo que la optimización de la composición de las películas o recubrimientos fue crucial para su aplicación con éxito. El polímero escogido como base para hacer las películas y recubrimientos mediante la técnica de casting, fue el alcohol polivinílico, (PVOH) mezclado con diferentes proteínas, gelatina y caseinato sódico y sus versiones hidrolizadas añadidas en diferentes proporciones. En este estudio L. lactis demostró tener una mayor resistencia a la deshidratación durante la preparación y almacenamiento de las películas que L. sakei. Las películas de PVOH mezcladas con gelatina hidrolizada y caseína hidrolizada dotaron de protección a las bacterias ácido lácticas durante el secado y demostraron tener una mayor efectividad antilisteria. Por último, se varió la proporción de caseína hidrolizada obteniendo una mayor viabilidad de las bacterias en las películas prolongada en el tiempo a mayor proporción de HCas. Para desarrollar un envase activo antilisteria, se adaptaron las matrices para ser aplicadas como recubrimiento sobre ácido poliláctico (PLA) con el objetivo de desarrollar bolsas activas antilisteria para crema de setas y como separadores de lonchas de jamón cocido. Los recubrimientos demostraron tener una efectividad similar a la obtenida con las películas in vitro. Sin embargo, su eficacia disminuyó ligeramente al ser aplicadas en la crema de champiñones y el jamón cocido debido a la complejidad de las matrices alimentarias utilizadas, pero siendo una buena herramienta para, junto a otras estrategias de conservación, asegurar la salubridad de los alimentos. Se llevaron a cabo ensayos sensoriales con consumidores y se obtuvo una buena aceptación de ambos productos. Sin embargo, los consumidores percibieron una alteración del sabor debido a la generación de ácido láctico en la crema de champiñones, acidez que puede ser controlada mediante correctores de acidez sin modificar la efectividad antimicrobiana. Para desarrollar envases que aumentaran la vida útil, se trabajó para ampliar el espectro de acción de L. lactis frente a las bacterias Gram-negativas, normalmente responsables de la descomposición de los alimentos. Combinar el agente de biocontrol productor de nisina, L. lactis, con una baja concentración de ácido fítico, sustancia quelante y desestabilizadora de la membrana bacteriana, amplió el espectro antimicrobiano de L. lactis frente a las bacterias Gram-negativas obteniendo una nueva herramienta para garantizar la seguridad alimentaria y prolongar la vida útil de los alimentos. / [CA] La present tesi doctoral planteja diferents estratègies per al desenvolupament i aplicació d'envasos actius portadors d'agents de biocontrol, amb la finalitat d'inhibir la proliferació de microorganismes com a bacteris i fongs patògens i alteradors i així, aconseguir augmentar la seguretat i prolongar la vida útil dels aliments. El desenvolupament d'envasos actius antibacterians, es va dur a terme incorporant bacteris àcid làctics productores de bacteriocines, Lactococcus lactis i Lactobacillus sakei en diferents matrius formadores de pel·lícules. Prèvia a la preparació de les pel·lícules actives, es va estudiar l'activitat antimicrobiana dels agents naturals seleccionats enfront de Listeria monocytogenes. Els resultats van suggerir que, a partir d'una determinada concentració inicial de bacteris viables, aquests microorganismes són bons candidats per a ser utilitzats com a additius naturals en materials d'envasament d'aliments, sent una alternativa a l'ús d'additius d'origen sintètic. En aquest treball es va observar que la viabilitat inicial dels bacteris àcid làctics (LAB) és essencial per a obtindre una bona capacitat antibacteriana pel que l'optimització de la composició de les pel·lícules o recobriments va ser crucial per a la seua aplicació amb èxit. El polímer triat com a base per a fer les pel·lícules i recobriments mitjançant la tècnica de càsting, va ser l'alcohol polivinílic, (PVOH) mesclat amb diferents proteïnes, gelatina i caseinat sòdic i les seues versions hidrolitzades afegides en diferents proporcions. En aquest estudi L. lactis va demostrar tindre una major resistència a la deshidratació durant la preparació i emmagatzematge de les pel·lícules que L. sakei. Les pel·lícules de PVOH mesclades amb gelatina hidrolitzada i caseina hidrolitzada van dotar de protecció als bacteris àcid làctics durant l'assecat i van demostrar tindre una major efectivitat antilistèria. Finalment, es va variar la proporció de caseïna hidrolitzada obtenint una millor resposta de les pel·lícules no sols des d'un punt de vista antimicrobià, sinó també de les propietats fisicoquímiques de les pel·lícules, a major proporció de caseïna hidrolitzada.Per a desenvolupar un envàs actiu antilistèria, es van adaptar les matrius per a ser aplicades com a recobriment sobre àcid polilàctic (PLA) amb l'objectiu de desenvolupar bosses actives antilistèria per a crema de bolets i com a separadors de rodanxes de pernil cuit. Els recobriments van demostrar tindre una efectivitat similar a l'obtinguda amb les pel·lícules in vitro. No obstant això, la seua eficàcia va disminuir lleugerament en ser aplicades en la crema de xampinyons i el pernil cuit a causa de la complexitat de les matrius alimentàries utilitzades, però sent una bona eina per a, al costat d'altres estratègies de conservació, assegurar la salubritat dels aliments. Es van dur a terme assajos sensorials amb consumidors i es va obtindre una bona acceptació de tots dos productes, però una alteració del sabor degut a la generació d'àcid làctic en la crema de xampinyons corregible mitjançant correctors d'acidesa sense modificació de l'efectivitat antimicrobiana. Per a desenvolupar envasos que augmentaren la vida útil, es va treballar per a ampliar l'espectre d'acció de L. lactis enfront dels bacteris Gram-negatives, normalment responsables de la descomposició dels aliments. Combinar l'agent de biocontrol productor de nisina, L. lactis, amb una baixa concentració d'àcid fític, substància quelant i desestabilitzadora de la membrana bacteriana, va ampliar l'espectre antimicrobià de L. lactis enfront dels bacteris Gram-negatives obtenint una nova eina per a garantir la seguretat alimentària i prolongar la vida útil dels aliments. / [EN] This doctoral thesis proposes different strategies for the development and application of active packaging containing biocontrol agents, to inhibit the proliferation of microorganisms such as pathogenic and spoilage bacteria and fungi, thus increasing the safety and increasing the shelf life of foodstuffs. The development of antibacterial active packaging was carried out by incorporating bacteriocin-producing lactic acid bacteria, Lactococcus lactis and Lactobacillus sakei in different film-forming matrices. Prior to the preparation of the active films, the antimicrobial activity of the selected natural agents against Listeria monocytogenes was studied. The results suggested that, from a certain initial concentration of viable bacteria, these microorganisms are good candidates to be used as natural additives in food packaging materials, being an alternative to the use of additives of synthetic origin. In this work it was observed that the initial viability of lactic acid bacteria (LAB) is essential to obtain a good antibacterial capacity, so the optimization of the composition of the films or coatings was crucial for their successful application. The polymer chosen as a base to make the films and coatings by casting technique was polyvinyl alcohol (PVOH) mixed with different proteins, gelatin and sodium caseinate and their hydrolysed versions added in different proportions. In this study L. lactis showed higher resistance to dehydration during film preparation and storage than L. sakei. PVOH films mixed with hydrolysed gelatin and hydrolysed casein provided protection to lactic acid bacteria during drying and were shown to have a greater antilisteria effectiveness. Finally, the proportion of hydrolysed casein was varied, obtaining a better response of the films not only from an antimicrobial point of view, but also from the physicochemical properties of the films, the higher the proportion of HCas. To develop an antilisteria active packaging, the matrices were adapted to be applied as a coating on polylactic acid (PLA) with the objective of developing antilisteria active bags for cream of mushroom soup and as separator liner for cooked ham slices. The coatings showed similar effectiveness to that obtained with in vitro films. However, their effectiveness decreased slightly when applied to mushroom soup and cooked ham due to the complexity of the food matrices used, but being a good tool to, together with other preservation strategies, ensure food wholesomeness. Sensory tests were carried out with consumers and a good acceptance of both products was obtained. However, an alteration of taste due to the generation of lactic acid was detected in the mushroom soup that could be corrected by means of acidity correctors without modifying the antimicrobial effectiveness. Finally, life cycle analysis of conventional and active packaging for pastry cream was studied. In any case, despite the limitations regarding the waste product estimation, shelf-life extension through packaging innovation could significantly reduce the environmental impacts of the entire food packaging system. In the present thesis, the potential use of indigenous yeasts with antifungal capacity for the control of Penicillium expansum and the control of the mycotoxin they generate, called patulin, was also explored. In the present investigation, three new yeast strains isolated from the surface of apples were selected for their efficacy against P. expansum. The yeasts were identified as Metschnikowia pulcherrima being 3 different strains. The ability of these yeasts for patulin biodegradation was also demonstrated. Then, different studies on the viability of the yeast and its antifungal effectiveness on different films made with biopolymers naturally present in apples (pectin, cellulose ethers, and apple pomace) were carried out. / The authors acknowledge the financial support of the Spanish Ministry of Economy and Competitiveness (AGL2015-64595-R and RTI2018-093452-B-I00). / Settier Ramírez, L. (2021). Envases activos portadores de microorganismos para la bioconservación de alimentos [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/175807 / Compendio