Ultra-efficient antimicrobial photodynamic inactivation system based on blue light and octyl gallate for ablation of planktonic bacteria and biofilms of Pseudomonas fluorescens

Highlights:

• Octyl gallate (OG) works as an effective photosensitizer and antibacterial agent.

• Antimicrobial photodynamic inactivation (PDI) system based on blue light and OG.

• The PDI was efficient at eradicating planktonic P. fluorescens and biofilms.

• Bacterial inactivation by PDI might be due to multi-damage to cellular components.

• Nanofibers in combination with PDI have superiorities for salamander preservation.

Abstract

Pseudomonas fluorescens is a Gram-negative spoilage bacterium and dense biofilm producer, causing food spoilage and persistent contamination. Here, we report an ultra-efficient photodynamic inactivation (PDI) system based on blue light (BL) and octyl gallate (OG) to eradicate bacteria and biofilms of P. fluorescens. OG-mediated PDI could lead to a > 5-Log reduction of viable cell counts within 15 min for P. fluorescens. The activity is exerted through rapid penetration of OG towards the cells with the generation of a high-level toxic reactive oxygen species triggered by BL irradiation. Moreover, OG plus BL irradiation can efficiently not only prevent the formation of biofilms but also scavenge the existing biofilms. Additionally, it was shown that the combination of OG/poly(lactic acid) electrospun nanofibers and BL have great potential as antimicrobial packagings for maintaining the freshness of the salamander storge. These prove that OG-mediated PDI can provide a superior platform for eradicating bacteria and biofilm.

Introduction

Pseudomonas fluorescence is a food spoilage bacterium largely responsible for the deterioration of aquatic products and dairy products, which can grow well at low temperatures and facilitate the spoilage of aquatic products in cold-chain transportation as compared with other spoilage (Caldera et al., 2016). Worse still, P. fluorescence cells can attach to food contact surfaces and form biofilms readily, making it more difficult to be eliminated. Meanwhile, the biofilm protects organisms against desiccation, biocides, some antibiotics and metallic cations, ultraviolet radiation (Flemming & Wingender, 2010). To avoid these problems, antibiotics are often used to reduce their threat, but indiscriminate use of antibiotics often might facilitate the emergence of drug resistance. Therefore, it is urgent to develop efficient strategies to kill P. fluorescence and eradicate its biofilm.

As distinguished from traditional thermal-based technologies used for foods decontamination, some non-thermal procedures including ultrasound, cold plasma, high hydrostatic pressure, pulsed electric field, and pulsed light processing have been developed to inhibit the growth of microorganisms and preserve nutritional quality and sensory acceptability of food (Ortega-Rivas & Salmeron-Ochoa, 2014). Despite such significant potentials, some limitations, such as low compatibility for broader food applications, higher processing requirements and costs, as well as the emergence of microbial tolerance, limit their wide applications (Cebrian et al., 2016). Recently, a novel emerging non-thermal processing by light called photodynamic inactivation (PDI) is gaining focus and are being applied for microbial growth control in the food industry (Ferrario et al., 2015). PDI is an athermal photochemical reaction based on the combination of the simultaneous presence of light, and photosensitizers (PSs) and oxygen (Luksienė & Zukauskas, 2009). Bacteria containing PSs have the ability to absorb light at specific wavelengths. Once the light is absorbed, the PS gets excited to a higher energy state under the presence of oxygen. On their way back to the ground state, they collide with oxygen in the cytoplasm, transferring energy and subsequently producing high reactive oxygen species (ROS). The ROS would interact with adjacent intracellular components, such as lipids, proteins and nucleic acids, leading to bacterial death (Nakamura et al., 2012). Although UV light has been widely used to decontaminate foods and inactivate various foodborne pathogens and biofilm cells, it has restricted application in the food industry due to its low penetration ability for solids or opaque liquids and causing serious eye and skin damage to food operators, as well as food sensory degradation (Kim et al., 2016). In contrast, light-emitting diodes (LEDs) can also effectively inactivate pathogens and preserve food in postharvest stages and avoid the mentioned issues related to UV radiation (D'Souza, Yuk, Khoo, & Zhou, 2015). LED technology being low-hazard (no mercury), low energy consumption, safe and high durability, as well as broader and higher antibacterial effect on microorganisms, LED-based PDI have recently been explored more and more as a novel preservation technology in food processing (Kim et al., 2017, Luksienė and Zukauskas, 2009).

Lots of studies in PDI focus on the synthesis or discovery of more effective PSs. They can be divided into endogenous and exogenous PSs from the source. Porphyrins are the most well-known natural endogenous PSs which are found in many bacterial and fungal cells (Rapacka-Zdonczyk et al., 2019). When the magnitude of inactivation with endogenous PSs is lower than desired, it is essential to use an exogenous PS to enhance it. At present, many artificially synthesized exogenous PSs, such as chlorine, phthalocyanines and phenothiazinium dyes, etc, show good photoactivity. However, safety considerations, organoleptic changes and consumer perceptions associated with the use of an exogenous PS are also crucial to the application of PDI in food processing. Thus, natural exogenous PSs such as hypericin, Vitamin K3 (Sheng et al., 2020), and curcumin are good candidates for food application, given that they have no toxic or genotoxic effects. Recently, phenolic compounds, especially phenolic acids, have been extensively studied in the food industry due to their various bioactive properties, especially antimicrobial activities. Limited studies have directly utilized them including gallic acid (GA), caffeic acid (CA), chlorogenic acid as PSs to generate ROS including hydrogen peroxide (H₂O₂) and hydroxyl radicals (•OH) under the exposure of blue light (BL) in the presence of dissolved oxygen, effectively inactivating bacteria (Nakamura et al., 2015, Nakamura et al., 2017, Nakamura et al., 2012). Electrons are transferred from photo-oxidized polyphenols to dissolved oxygen to produce H₂O₂, which is then photolyzed by BL to produce OH radicals (Nakamura et al., 2013), as the main contributor to the bactericidal activity of such PDI. Besides, PDI technology based on photo-oxidation of CA has been developed to eliminate S. mutans biofilm (Nakamura et al., 2017). On the other hand, our research group has long been engaged in the study of the antibacterial activities and mechanism of a variety of phenolic acids and their ester derivatives (Shi et al., 2021, Shi et al., 2020, Shi et al., 2018). Some of them have been found to exhibit stronger antibacterial and anti-biofilm activities against foodborne pathogens, compared to the corresponding phenolic acids. Among them, octyl gallate (OG) showed the superior interaction/affinity with membranes and antibacterial activity against E. coli and S. aureus (Shi et al., 2020), as well as P. fluorescence (Zhang et al., 2021). Surprisingly, the potential of OG with low concentration as a novel PS for PDI has been found in this work, which endows OG-mediated PDI with intriguing bactericidal efficacy to achieve rapid eradication of pathogens and biofilms in a relatively short time due to both photodynamic and intrinsic antibacterial properties of OG itself. Besides, since OG has been permitted for use as an antioxidant additive in food (FDA, 2001a, FDA, 2001b), it is supposed to be safe for humans and can be considered as an alternative exogenous PS of PDI. Additionally, electrospinning is one of the promising encapsulation methods in which a variety of active substances are encapsulated in the nanofiber matrix (Wen et al., 2017). Electrospun nanofibers with a large surface area to mass ratio have been proposed for stabilizing or controlling the release of the active compounds such as antibacterial agents in food processing and packaging (Kayaci & Uyar, 2012), showing longer lasting antibacterial activity. Recently, we successfully utilized OG as a multi-functionalized food additive combined with the advantages of electrospinning nanofibers for the preservation of Taihu icefish in China (Shi et al., 2021). Therefore, inspired by the combination of these ideas, we chose OG as a photosensitizer combined with BL to inactivate P. fluorescence, and further developed an ideal antibacterial strategy based on the combination of PDI and electrospinning nanofibers to protect sea foods from spoilage bacteria, avoid their quality degradation and flavor loss and extend the shelf life during storage.

Herein, the aim of the present study was to examine the synergistic effect of OG and BL to inhibit the growth of P. fluorescence planktonic bacteria as well as eradicate its biofilm, and further elucidate the mode of action. Besides, we investigated electrospun nanofibers embedded with the photosensitizer OG as a PDI-based packaging material and evaluated its antibacterial activity in the storage and preservation of Chinese giant salamander. The dual role of OG in improving the production of ROS as a novel PS on one hand and the enhanced antimicrobial activity as an effective antibacterial on the other, are highlighted for the first time.