Durability Assessment of a Plasma-Polymerized Coating with Anti-Biofilm Activity against L. monocytogenes Subjected to Repeated Sanitization

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).[EN] Biofilm formation on food-contact surfaces is a matter of major concern causing food safety and spoilage issues to this sector. The aim of this study was to assess the durability of the anti-biofilm capacity of a plasma-polymerized coating composed of a base coating of (3-aminopropyl) triethoxysilane (APTES) and a functional coating of acrylic acid (AcAc). Coated and uncoated AISI 316 stainless steel (SS) plates were subjected to five sanitization cycles with sodium hypochlorite (0.05%) and peracetic acid (0.5%). The effectiveness of the coating for the inhibition of multi-strain Listeria monocytogenes biofilm formation was confirmed using a three-strain cocktail, which was grown on the SS plates at 12◦ C for 6 days. Compared to the uncoated SS, relative biofilm productions of 14.6% on the non-sanitized coating, 27.9% on the coating after sanitization with sodium hypochlorite, and 82.3% on the coating after sanitization with peracetic acid were obtained. Morphological and physicochemical characterization of the coatings suggested that the greater anti-biofilm effectiveness after sanitization with sodium hypochlorite was due to the high pH of this solution, which caused a deprotonation of the carboxylic acid groups of the functional coating. This fact conferred it a strong hydrophilicity and negatively charged its surface, which was favorable for preventing bacterial attachment and biofilm formation.SIThis publication is based upon work from COST Action CA19110—PlAgri, supported by COST (European Cooperation in Science and Technology-www.cost.eu). XPS tests were conducted by the Advanced Microscopy Laboratory (LMA) of The Institute of Nanoscience of Aragón (INA), University of Zaragoza. The authors are thankful to the LMA-INA for the access to their equipment and their expertise. The AFM images were taken by the Central Research Support Service (SCAI) of the University of Málaga (UMA). The author P. Fernández-Gómez is grateful to Junta de Castilla y León and the European Social Fund (ESF) for awarding her a predoctoral grant (BOCYL-D-15122017-4). The author M. Oliveira is in receipt of a Juan de la Cierva contract IJC2018-035523-I awarded by the Spanish Ministry of Science, Innovation, and Universities MCIN/AEI/10.13039/501100011033. The author E. Sainz-García, as researcher of the University of La Rioja, is thankful to the postdoctoral training program funded by the Plan Propio of the University of La Rioja. The authors I. Muro-Fraguas and A. Sainz-García are thankful to the program of pre-doctoral contracts for the training of research staff that is funded by the University of La Rioja.This study is part of the Research, Development and Innovation projects AGL2017-82779- C2-R and PID2020-113658RB-C2, funded by MCIN/AEI/10.13039/501100011033 and by ERDF “A way to make Europe”

A Review on Non-thermal Atmospheric Plasma for Food Preservation: Mode of Action, Determinants of Effectiveness, and Applications

Non-thermal Atmospheric Plasma (NTAP) is a cutting-edge technology which has gained much attention during the last decade in the food-processing sector as a promising technology for food preservation and maintenance of food safety, with minimal impact on the quality attributes of foods, thanks to its effectiveness in microbial inactivation, including of pathogens, spoilage fungi and bacterial spores, simple design, ease of use, cost-effective operation, short treatment times, lack of toxic effects, and significant reduction of water consumption. This review article provides a general overview of the principles of operation and applications of NTAP in the agri-food sector. In particular, the numerous studies carried out in the last decade aimed at deciphering the influence of different environmental factors and processing parameters on the microbial inactivation attained are discussed. In addition, this review also considers some important studies aimed at elucidating the complex mechanism of microbial inactivation by NTAP. Finally, other potential applications of NTAP in the agri-food sector, apart from food decontamination, are briefly described, and some limitations for the immediate industrial implementation of NTAP are discussed (e.g., impact on the nutritional and sensory quality of treated foods; knowledge on the plasma components and reactive species responsible for the antimicrobial activity; possible toxicity of some of the chemical species generated; scale-up by designing fit-for-purpose equipment)

Organoleptic characterization of wines in contact with oak wood fragments immersed in plasma activated water (PAW)

Oak barrels are a valuable material for wine ageing, although their difficult cleaning and disinfection favours microbiological contamination causing wine quality depreciation. Atmospheric pressure cold plasma is a suitable technique to reduce microbiota, but there is little research on its impact on the nutritional and sensory characteristics of foods. The aim of this study was to analyse the organoleptic characteristics of red wines in contact with plasma-activated waters (PAW) treated wood, of different origins and toasting levels. These red wines were compared with others that had been in contact with two types of control wood; some immersed in distilled water and others subjected to sulphur dioxide combustion. The results showed that oak wood treated with PAW did not cause defects at the olfactory and gustatory level of the red wines, which presented good harmony, sufficient body and balanced fruity and spicy notes, sometimes even superior to those described for red wines in contact with control wood (sulphited or submerged in distilled water). Therefore, the treatment of the wood with PAW did not have a negative impact on the sensory quality of the wines, regardless of the origin and toasting of the wood

Recubrimientos anti-biofilm aplicados mediante plasma-polimerización destinados a la industria de manipulación de alimentos y al sector médico

La formación de biofilm en las industrias de manipulación de alimentos y en aplicaciones médicas es un motivo de preocupación debido a la capacidad de las bacterias para adherirse y reproducirse en diferentes entornos, materiales y superficies. Los microorganismos patógenos son muy difíciles de erradicar, puesto que pueden adherirse fácilmente a una superficie, desarrollar biofilm, sobrevivir a temperaturas extremas de refrigeración o condiciones de desecación y resistir a los desinfectantes. Todos estos problemas relacionados con la formación de biofilms provocan contaminación cruzada de productos, desechos y deterioro de alimentos, riesgos para la salud de los consumidores, daños en los equipos industriales y grandes pérdidas económicas para los productores. El inconveniente de los métodos convencionales de desinfección es que, además de emplear altas concentraciones de productos químicos tóxicos, son frecuentemente ineficientes ya que no logran la erradicación completa de los biofilms, aumentando así los problemas asociados con la posible generación de resistencia bacteriana. Por lo tanto, los investigadores y las empresas están realizando grandes esfuerzos para tratar de eliminar los biofilms de los entornos de producción. En esta tesis se ha empleado la tecnología de plasma a presión atmosférica con Descarga de Barrera Dieléctrica (Dielectric Barrier Discharge, DBD) para generar recubrimientos antibiofilm. En las dos primeras publicaciones científicas, el principal objetivo era la reducción del biofilm generado. Para ello, se aplicaron recubrimientos plasmapolimerizados a base de ácido acrílico (Acrylic acid, AcAc) y tetraetilo ortosilicato (Tetraethyl orthosilicate, TEOS) sobre muestras de ácido poli láctico (Polylactic acid, PLA) impresas en 3D. El rápido desarrollo de la tecnología de impresión 3D, y específicamente de los filamentos de PLA, para aplicaciones médicas (implantes, prótesis y dispositivos de protección) y en contacto con alimentos (utensilios de cocina con un diseño ergonómico para personas con problemas de movilidad), hace muy interesante el uso del PLA impreso en 3D como sustrato en esta tesis. Sin embargo, el empleo del PLA y de la tecnología de impresión 3D se están viendo limitados debido a la facilidad de proliferación bacteriana y la imposibilidad de someter las piezas impresas a un proceso de esterilización; provocados por la característica rugosidad superficial de las piezas impresas en 3D y la baja temperatura de fusión del PLA, respectivamente. La influencia del líquido precursor y el número de pasadas se caracterizaron mediante ensayos de microscopía de fuerza atómica (Atomic Force Microscopy, AFM), microscopía electrónica de barrido (Scanning Electron Microscopy, SEM), espectroscopia de fotoelectrones de rayos X (Xray Photoelectron Spectroscopy, XPS) y ensayos microbiológicos de cuantificación de biofilm. Se identificaron los mecanismos de adhesión y proliferación bacteriana. La formación de biofilm se debe a un efecto combinado de las propiedades químicas y morfológicas de la superficie del sustrato. En general, conforme mayor es la rugosidad y la hidrofilicidad de la superficie, mayor será la capacidad antibiofilm de los recubrimientos generados. Se puede concluir que los recubrimientos basados en AcAc resultaron más efectivos que los de TEOS. Sin embargo, no se puede establecer un número de pasadas específico como el mejor, puesto que la capacidad antibiofilm de los recubrimientos se ve influenciada por la forma y el tamaño de las bacterias que conforman el biofilm. Los recubrimientos de AcAc plasmapolimerizados más exitosos, redujeron la formación de biofilm de Pseudomonas aeruginosa, Listeria monocytogenes, Escherichia coli y Staphylococcus aureus en más de un 50%, con respecto a las muestras de PLA sin tratar con plasma. En los entornos industriales, coexisten múltiples cepas bacterianas y las superficies recubiertas requieren una higienización periódica para evitar la acumulación de bacterias a largo plazo y la contaminación cruzada de los productos alimenticios. Por ello, con el fin de dar un paso más hacia el escalado industrial, los objetivos fijados en la tercera publicación se basaron en analizar la magnitud y durabilidad de las propiedades antibiofilm de un recubrimiento plasmapolimerizado expuesto a un cóctel de múltiples cepas de Listeria monocytogenes (una de las bacterias más difíciles de controlar y eliminar).La durabilidad del recubrimiento bicapa, a base de aminopropiltrietoxisilano (Aminopropyltriethoxysilane, APTES) y AcAc, se evaluó sometiendo las muestras recubiertas a 5 ciclos de limpieza con desinfectantes de uso común, como el hipoclorito de sodio y el ácido peracético. Los recubrimientos se aplicaron sobre muestras de acero inoxidable (material predominante en las líneas de producción alimentaria). Las muestras recubiertas lograron reducir la producción de biofilm en más del 85% y se mantuvieron en niveles del 72% después de aplicar los ciclos de limpieza con hipoclorito de sodio. La efectividad antibiofilm después de la higienización con hipoclorito de sodio se debió al alto pH de dicha solución, que provocó una desprotonación de los grupos ácido carboxílico del recubrimiento funcional. Ello dotó a la superficie de una fuerte hidrofilia con carga negativa, favorable para prevenir la adhesión bacteriana y la formación de biofilm. Sin embargo, la capacidad antibiofilm perdió efectividad cuando se empleó ácido peracético como solución desinfectante. Se considera que se ha cumplido con los objetivos propuestos. Los recubrimientos aplicados en esta tesis no solo reducen el biofilm generado por diferentes bacterias presentes en instalaciones de manipulación de alimentos y en el ámbito clínico, sino que además son duraderos frente a procesos de limpieza con desinfectantes alcalinos. De modo que la tecnología de plasma a presión atmosférica se puede convertir en una alternativa innovadora a las soluciones actuales para aplicaciones de desinfección en la industria médica y alimentaria.Bacterial biofilms formation in food processing industries and medical applications is a matter of concern due to its ability to adhere and reproduce in different environments, materials and surfaces. Pathogenic microorganisms can easily attach to a surface, develop biofilms, survive at refrigeration temperatures or desiccation conditions and resist to disinfectants; which makes them very hard to eradicate. All these problems related to biofilms formation cause cross contamination of products, waste and food spoilage, damages of industrial equipment, economic losses for producers and health risks for consumers. Conventional methods for disinfection, in addition to employ high concentrations of toxics chemicals, are frequently inefficient because they do not achieve a complete eradication of biofilms, increasing the problems with the possible generation of bacterial resistance. Therefore, researches and companies are making great efforts to remove biofilms from production environments. Atmospheric pressure plasma technology with a Dielectric Barrier Discharge (DBD) was used in this thesis to generate antibiofilm coatings. In the first group of two scientific publications, the objective was to reduce bacterial biofilms formation. Plasmapolymerized coatings based on acrylic acid (AcAc) and tetraethyl orthosilicate (TEOS) were applied on 3D printed polylactic acid (PLA) samples. The high speed development of 3D printing technology for food contact (ergonomic cutely for disabled people) and medical applications (implants, prosthesis and protection devices), make interesting the use of 3D printed PLA as a substrate. However, the use of PLA and 3D printing technology are being limited due to the ease of bacterial proliferation due to its surface roughness and the impossibility of subjecting to sterilization process because of the low fusion temperature of PLA. The influence of the precursor liquid and number of passes were characterized by Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM), XRay Photoelectron Spectroscopy (XPS) measurements and biofilm quantification. The mechanism of adhesion and bacterial proliferation were identified. The biofilm formation is caused by a combined effect of the chemical and morphological surface properties. Generally, the more hydrophilic and roughness surfaces, the better the antibiofilm capacity of the coatings. It was concluded that AcAc coatings are more effective than TEOS coatings. However, no specific number of passes could be defined as the best, since it depends on the shape and size of the bacteria. AcAc plasmapolymerized coatings reduced biofilm formation more than 50% against Pseudomonas aeruginosa, Listeria monocytogenes, Escherichia coli and Staphylococcus aureus, regarding the untreated 3D printed PLA samples. In real industrial environments, multiple bacterial strains coexist, and the coated surfaces require periodical sanitization to prevent longterm bacterial accumulation and crosscontamination of food products. For that, in the third publication, with the purpose of taking one more step towards the industrial scaling, the aims were to analyze the antibiofilm character of a plasmapolymerized coating based on (3Aminopropyl) triethoxysilane (APTES) and AcAc exposed to a multistrain cocktail of Listeria monocytogenes (one of the most difficult bacteria to control and eliminate); as well as its durability after repeated 5 cycles of sanitization with commonly used disinfectants, such as sodium hypochlorite and peracetic acid, was also assessed. The coatings were applied on stainless steel (SS) samples (material most commonly used in food industry). Coated samples achieved to reduce the biofilm production more than 85% and remained at levels of 72% after sanitization cycles with sodium hypochlorite. The antibiofilm effectiveness after sanitization with sodium hypochlorite was due to the high pH of this solution, which caused a deprotonation of the carboxylic acid groups of the functional coating. This fact conferred it a strong hydrophilicity and negatively charged its surface, which was favorable for preventing bacterial attachment and biofilm formation. However, this antibiofilm capacity lost effectivity when peracetic acid was employed as disinfection solution. It is considered that the proposed objectives have been successfully fulfilled. The coatings applied in this thesis not only reduce the biofilm generated by different bacteria present in food and clinical field, but they are also durable against disinfecting processes with alkaline sanitizers. Therefore, this demonstrates the promising applications of atmospheric pressure plasma technology in food processing and medical industries and it could be an innovative alternative to the current industrial solutions