Study of antimicrobial and antioxidant properties of new materials for development of active food packaging

This work is focused on the design, production and characterization of sustainable active food packaging materials with antimicrobial and antioxidant properties to ensure the safety and quality of foods and prolong their shelf-life. Firstly, a microfluidic device combining dielectrophoresis (DEP) and Raman spectroscopy was developed for a fast and dynamic characterization of different bacterial strains, including food-related pathogens (i.e. E. coli and S. aureus), directly in planktonic suspension. Predictive models to identify bacterial cross-induced resistance to antibiotic within few hours were built using this method, overcoming the overnight incubation required by classical microbiological assays. Furthermore, Raman imaging was employed to detect the spatial distribution of different biomolecules at single cell level. Then, the antibacterial properties of innovative silver and carbon-based nanosystems and their inclusion in prototype packaging materials were studied. Differently sized silver nanoparticles, from 6 to 50 nm, were compared for their antibacterial efficacy in suspension and immobilized on glass. For the first time, the surface minimal bactericidal concentration (SMBC) of silver needed to kill 99.9999% of bacteria, was determined by ISO 22196, thereby facilitating the comparison between measurements and minimizing the amount of silver on the materials surface (0.023-0.034 μg/cm2) as well as their cost of production and toxicity. Colloidal carbon nanoparticles (CNP), obtained by a green chemistry synthesis, were tested against Gram + and a Gram – bacteria, by classical microbiological assays and the DEP-Raman system, revealing a rapid interaction with the bacteria but not significant bactericidal effects. Thus, CNP were loaded with an antimicrobial peptide which increased their antibacterial activity, especially against S. aureus. Finally, new antioxidant packaging modified with grape and olive industrial waste products and Moringa oleifera leaves obtained by different extractive procedures were produced and characterized. The antioxidant efficacy of many fractions of the plants extracts were analyzed by multiple standard assays and the results were correlated with their content of polyphenols obtaining higher values for anti-solvent and maceration extract fractions. The latter, resulting from a more sustainable extraction procedure, were included in cellulose-based active packaging systems. The antioxidant properties of such films were measured by indirect and direct analytical methods demonstrating good free radical scavenging properties for all the three kind of active agents and a higher radical reduction capacity of moringa. Additionally, the ability of the packaging coated with moringa (5% w/w) of delaying fresh ground beef oxidation was tested. This film was chosen as the best alternative to obtain the highest oxidative protection of meat on the basis of the in vitro results and ensuring a direct food-contact mechanism of action. This packaging revealed to prevent meat from lipid oxidation by at least 60% after 16 days compared to simple cellulose. Additionally, in situ analysis of the meat performed by vibrational spectroscopies evidenced also a protective action against protein and lipid degradation. This work could be considered valuable in the field of food packaging because the use of sustainable and degradable materials to prolong the food shelf-life perfectly fits in the actual compelling need to reduce pollution and global waste production. This is in accordance with the 12th Sustainable Development Goal of the European Green Deal purpose to halve the global food waste production per capita by 2030, ensuring an efficient and sustainable use of natural resources. Hence, an innovative way to recover food industry waste is proposed and their antioxidant efficacy in active food packaging was demonstrated even on real food matrices with many different techniques strengthening the reliability of the results

Hyperspectral Chemical Imaging of Single Bacterial Cell Structure by Raman Spectroscopy and Machine Learning

6openopenBarzan, Giulia; Sacco, Alessio; Mandrile, Luisa; Giovannozzi, Andrea Mario; Portesi, Chiara; Rossi, Andrea MarioBarzan, Giulia; Sacco, Alessio; Mandrile, Luisa; Giovannozzi, Andrea Mario; Portesi, Chiara; Rossi, Andrea Mari

Development of innovative antioxidant food packaging systems based on natural extracts from food industry waste and Moringa oleifera leaves

Active packaging that prolongs food shelf life, maintaining its quality and safety, is an increasing industrial demand, especially if integrated in a circular economy model. In this study, the fabrication and characterization of sustainable cellulose-based active packaging using food-industry waste and natural extracts as antioxidant agents was assessed. Grape marc, olive pomace and moringa leaf extracts obtained by supercritical fluid, antisolvent and maceration extraction in different solvents were compared for their antioxidant power and phenolic content. Grape and moringa macerates in acetone and methanol, as the most efficient and cost-effective extracts, were incorporated in the packaging as coatings or in-between layers. Both systems showed significant free-radical protection in vitro (antioxidant power 50%) and more than 50% prevention of ground beef lipid peroxidation over 16 days by indirect TBARS and direct in situ Raman microspectroscopy measurements. Therefore, these systems are promising for industrial applications and more sustainable farm-to-fork food production systems

Raman-dielectrophoresis goes viral: towards a rapid and label-free platform for plant virus characterization

An innovative spectroscopic method that allows to chemically and structurally characterize viruses directly in suspension within few minutes was developed. A library of five different plant viruses was obtained combining dielectrophoresis (DEP), performed with a device specifically designed to capture and agglomerate virus particles, and Raman spectroscopy to provide a chemical fingerprint of virions. The tested viruses, purified from infected plants, were chosen for their economic impact on horticultural crops and for their different morphological and structural features. Using the Raman-DEP device, specific profiles for each virus were successfully obtained, relying on chemical differences occurring even with genetically similar viruses belonging to the same taxonomic species and morphologically indiscernible by transmission electron microscopy (TEM). Moreover, we investigated the potentiality of Raman-DEP to follow dynamic changes occurring upon heat treatment of tobacco mosaic virus (TMV) particles. Raman peak deviations linked to TMV coat protein conformation were observed upon treatment at temperatures equal or higher than 85 degrees C, substantiating the rod-to-spherical shape transitions observed by TEM and the concomitant drastic loss of infectivity following plant inoculation. Overall, the Raman-DEP method can be useful for the characterization of virus (nano)particles, setting the basis to create a database suitable for the study of viruses or virus derived-nanoparticles relevant for the agricultural, medical, or biotechnological fields

Molecular Aspects of the Interaction with Gram-Negative and Gram-Positive Bacteria of Hydrothermal Carbon Nanoparticles Associated with Bac8c2,5Leu Antimicrobial Peptide

Molecular Aspects of the Interaction with Gram-Negative and Gram- Positive Bacteria of Hydrothermal Carbon Nanoparticles Associated with Bac8c2,5Leu Antimicrobial Peptide Giulia Barzan,⊥ Ida Kokalari,⊥ Giacomo Gariglio, Elena Ghibaudi, Marc Devocelle, Marco P. Monopoli, Alessio Sacco, Angelo Greco, Andrea M. Giovannozzi, Andrea M. Rossi, and Ivana Fenoglio* Cite This: https://doi.org/10.1021/acsomega.2c00305 Read Online ACCESS Metrics & More Article Recommendations *sı Supporting Information ABSTRACT: Antimicrobial peptides (AMPs) are widely studied as therapeutic agents due to their broad-spectrum efficacy against infections. However, their clinical use is hampered by the low in vivo bioavailability and systemic toxicity. Such limitations might be overcome by using appropriate drug delivery systems. Here, the preparation of a drug delivery system (DDS) by physical conjugation of an arginine-rich peptide and hydrothermal carbon nanoparticles (CNPs) has been explored, and its antimicrobial efficacy against Eschericia coli (E. coli) and Staphylococcus aureus investigated in comparison with the unloaded carrier and the free peptide. The mechanism of interaction between CNPs and the bacteria was investigated by scanning electron microscopy and a combined dielectrophoresis−Raman spectroscopy method for real- time analysis. In view of a possible systemic administration, the effect of proteins on the stability of the DDS was investigated by using albumin as a model protein. The peptide was bounded electrostatically to the CNPs surface, establishing an equilibrium modulated by pH and albumin. The DDS exhibited antimicrobial activity toward the two bacterial strains, albeit lower as compared to the free peptide. The decrease in effectiveness toward E. coli was likely due to the rapid formation of a particle-induced extracellular matrix. The present results are relevant for the future development of hydrothermal CNPs as drug delivery agents of AMP

Hyperspectral Chemical Imaging of Single Bacterial Cell Structure by Raman Spectroscopy and Machine Learning

In this work, biomolecules, such as membrane proteins, lipids, and DNA, were identified and their spatial distribution was mapped within a single Escherichia coli cell by Raman hyperspectral imaging. Raman spectroscopy allows direct, nondestructive, rapid, and cost-effective analysis of biological samples, minimizing the sample preparation and without the need of chemical label or immunological staining. Firstly, a comparison between an air-dried and a freeze-dried cell was made, and the principal vibrational modes associated to the membrane and nucleic acids were identified by the bacterium’s Raman chemical fingerprint. Then, analyzing the Raman hyperspectral images by multivariate statistical analysis, the bacterium biological status was investigated at a subcellular level. Principal components analysis (PCA) was applied for dimensionality reduction of the spectral data, then spectral unmixing was performed by multivariate curve resolution–alternating least squares (MCR-ALS). Thanks to multivariate data analysis, the DNA segregation and the Z-ring formation of a replicating bacterial cell were detected at a sub-micrometer level, opening the way to real-time molecular analysis that could be easily applied on in vivo or ex vivo biological samples, avoiding long preparation and analysis process

RAMAN spectroscopy applications in grapevine: metabolic analysis of plants infected by two different viruses

Grapevine is one of the most cultivated fruit plant among economically relevant species in the world. It is vegetatively propagated and can be attacked by more than 80 viruses with possible detrimental effects on crop yield and wine quality. Preventive measures relying on extensive and robust diagnosis are fundamental to guarantee the use of virus-free grapevine plants and to manage its diseases. New phenotyping techniques for non-invasive identification of biochemical changes occurring during virus infection can be used for rapid diagnostic purposes. Here, we have investigated the potential of Raman spectroscopy (RS) to identify the presence of two different viruses, grapevine fan leaf virus (GFLV) and grapevine rupestris stem pitting-associated virus (GRSPaV) in Vitis vinifera cv. Chardonnay. We showed that RS can discriminate healthy plants from those infected by each of the two viruses, even in the absence of visible symptoms, with accuracy up to 100 and 80% for GFLV and GRSPaV, respectively. Chemometric analyses of the Raman spectra followed by chemical measurements showed that RS could probe a decrease in the carotenoid content in infected leaves, more profoundly altered by GFLV infection. Transcriptional analysis of genes involved in the carotenoid pathway confirmed that this biosynthetic process is altered during infection. These results indicate that RS is a cutting-edge alternative for a real-time dynamic monitoring of pathogens in grapevine plants and can be useful for studying the metabolic changes ensuing from plant stresses