Smart Indicators for Alterations in the Metabolic Activity of Microorganisms

Since the metabolic products of microorganisms can alter the pH of the environment in which they live and multiply, their population can be monitored by the fabrication of high-performant pH colorimetric indicators. These materials are able to indicate the microbes’ presence by giving qualitative or semi-quantitative information through colorimetric changes of the pH-responsive molecules that they contain. Preferably, the molecules have to be of natural origin, and biocompatible in order to avoid toxicity as in the case of synthetic dyes. These natural-based indicators come in various structures, with films being the most popular due to their cheap and easy fabrication. However, they have the disadvantage of the low surface area, something that can impede the real-time monitoring. Moreover, most of the studies are focused on testing the color change of the materials with liquids. These two factors may lead to false estimations about the condition of the studied environment. Although the evaluation of their pH-induced color changes in the presence of vapors is scarcely reported, the volatility of the amines calls for an immediate tracing of such vapors in the packaging headspace at concentrations relevant to the high populations of microorganisms. Hence, in order to respond to these challenges, the main goal of my Ph.D. studies has been the fabrication of advanced polymeric natural-based smart colorimetric indicators able to trace in real time pH changes deriving from alterations in the metabolic activity of microorganisms. In the first part of my research activity, my colleagues and I tried to fill the gap of the current state-of-the-art by fabricating highly porous composites, thus trying to increase their surface area and investigate whether such increase can lead to the real-time detection of specific amines in the gas phase. To achieve this, we combined the biocompatible polymer Polycaprolactone with curcumin, a functional molecule of natural origin, able to change color in environments of different acidity, and tuned the structure of the composites in order to increase the pH-sensitivity in the presence of amine gases. This tuning was achieved by combining the non-solvent induced phase separation with electrospinning. The chemical, mechanical and thermal properties of the material were thoroughly investigated, while the antioxidant activity induced by curcumin was evaluated. Subsequently, the investigation of the porous structure of the material was performed with porosimetry and BET techniques and, most importantly, with the comparison of the porous fibers with their non-porous equivalents. The two materials were exposed to the vapors of the volatile Dimethylamine. The evolution of the color during and after exposure was followed by imaging and videos and studied with CIELAB color space analysis. In comparison with the non-porous fibers, the porous system showed significantly higher sensitivity and faster responsivity to the presence of Dimethylamine vapors, with a distinct color change even at very low vapor concentrations within the first seconds of exposure. After the comparative studies, we focused our experiments only on the porous composite, by testing its responsivity to the volatile amine Trimethylamine, and to the non-volatile amines Cadaverine, Putrescine, Spermidine, and Histamine. Results showed that the induced color changes can be easily perceived visually, as the differences between the initial and the final color of the indicator after its interaction with the modified environment, were well above the limit for visual perception, even by inexperienced users. Experiments with lower concentrations of the amines showed that the material is able to trace down to 2 ppm of volatile amines, and 11 ppm of the biogenic amines. Furthermore, the color changes upon exposure to volatile amines are spontaneously reversible, while in the case of biogenic amines a subsequent exposure to acid was needed. This color reversibility enables the use of the same indicator several times, making it, thus, a sustainable colorimetric indicator system. The second part of my research was focused on the extensive literature study of pH colorimetric polymeric indicators containing anthocyanins and on the exploration of new potential anthocyanin sources for such applications. We collected the scientific works of the last decade, listing their morphological and compositional characteristics and evaluating their efficacy in their various application fields. In accordance to our laboratory activity, we concluded that the structural properties play an important role to the indicators performance. Finally, yet importantly, the current challenges and the future perspectives of the use of anthocyanins in colorimetric indicator platforms were collected, in order to stimulate the exploration of new anthocyanin sources and the in-depth investigation of all the possibilities that they can offer. Bringing the future perspectives in the laboratory, we focused on studying the anthocyanins extracted from red leaves of Parthenocissus Tricuspidata. We experimented various extraction methods and conducted UPLC studies for the anthocyanins’ characterization. We also explored the incorporation of the leaves’ powder or their anthocyanin extract to Polyvinylpyrrolidone and gelatin based hydrogel films, in order to develop a pH-responsive and humidity absorbing material that can be used either in food packaging or wound healing. Results showed that the material was able to absorb high amounts of water, while its flexibility can be tuned by the leaves-PVP-gelatin ratio. Last but not least, the films made directly from the leaves were able to change their color in presence of alkaline vapors, while the ones made by the extract were able to exhibit color alterations both in acidic and alkaline environments. All in all, my Ph.D. studies aim to pave the way for the development of high-end colorimetric indicators and the expansion of their use to new application fields. The present thesis is articulated as follows: In Chapter 1, quality control and its importance in the fields of food preservation and prevention of infections will be discussed, and pH indicators will be introduced as a way the monitoring of the quality of such systems by tracing microbial metabolites. In Chapter 2, the main natural molecules used for pH sensing and the state of the art of the polymer based pH colorimetric indicators, along with the limitations of the currently existing materials in terms of structure, available surface area, and the techniques of their pH responsivity evaluation. In Chapter 3, the development of highly porous and non-porous polycaprolactone pristine or loaded with curcumin colorimetric indicators will be presented. The fabrication method of the fibers, their morphology, porosity, surface area, as well as their chemical, thermal, wetting, mechanical, and antioxidant properties will be discussed. In Chapter 4, the colorimetric response of the porous and non-porous fiber mats will be compared upon exposure to the volatile amine Dimethylamine, and, after the establishment of the porous fiber mats as a highly responsive system, the exposure of the porous mats to various amines, including Trimethylamine, Cadaverine, Putrescine, Spermidine and Histamine will be explored. The aspects that will be thoroughly investigated will be the response kinetics, the detection limit, and the color reversibility, aiming to prove that the porous Polycaprolactone-curcumin mats can be successfully used for the rapid detection of very low concentrations of alkaline vapors. In Chapter 5, the fabrication of pH colorimetric indicators based on polymeric hydrogels mixed with anthocyanins coming from alternative sources will be presented. Such anthocyanin sources will include red leaves from decedent trees and Malva Sylvestris flowers, which can potentially replace the current anthocyanin edible sources. In Chapter 6, a summary of the main results achieved through this research activity will be given, along with some guidelines for follow-up research and future perspectives.