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 to other spoilage. The P. fluorescence cells can attach themselves 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. 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 food 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. 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. Recently, a novel emerging non-thermal processing by light called Photodynamic Inactivation (PDI) is gaining focus and is being applied for microbial growth control in the food industry.

Exposure to visible light in the presence of dyes was reported to inactivate the protozoan Paramecium caudatum in 1900 and the first practical application of Photodynamic Treatment (PDT) against tumour cells was proposed in 1905. The antimicrobial effects of PDT are due to the combination of visible or near-infrared light with a photosensitizer (PS) in the presence of molecular oxygen to generate reactive oxygen species (ROS), which ultimately leads to cell death. This is because ROS can react with a multiplicity of biomolecules and cell structures. Also, the use of non-toxic PS and harmless visible light makes PDT a promising alternative for food applications.

Principle mechanism of Microbial Inactivation
Fig 1: Principle mechanism of Microbial Inactivation

Principle Mechanism of Photodynamic Technology:

PDI is a photochemical reaction based on the combination of the simultaneous presence of light, photosensitizers (PSs) and oxygen. 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 electrons are transferred from photo-oxidized polyphenols to dissolved oxygen to produce H2O2, which is then photolyzed by blue Light to produce OH as the main reactive oxygen species (ROS). The ROS would interact with adjacent intracellular components, such as lipids, proteins and nucleic acids, leading to bacterial death.

The morphological changes of S. flexneri after photosensitization with curcumin were assessed by morphology using SEM. The curcumin-photosensitization bacteria were swollen and adhesive or shrivelled and distorted with a rough cell wall surface or ruptured membrane with leakage of intracellular material, which indicated that photosensitization treatment damaged bacteria. Protein oxidative damage by photodynamic treatment is also considered to be a cause of microbial death. Protein bands became shallower and reduced. Obvious changes in the protein spectrum indicated that PDT degraded most of the total protein in S. flexneri.

Mechanism of PDI Technology
Fig 2: Mechanism of PDI Technology

Significance of PDT:

Foodborne illnesses are usually infectious or toxic in nature and caused by bacteria, viruses, parasites or chemical substances entering the body through contaminated food or water. Food is presently sterilized mostly by thermal processing and non-thermal processing. However, thermal processing reduces the nutritional composition of food and affects the natural flavour and texture of food, thereby limiting its application to only some foods or parts of foods. Non-thermal processing includes ultrasonic vibration, high hydrostatic pressure and ultraviolet light. Although, it is possible to maintain the natural nutrition and colour of food, non-thermal processing cannot be widely applied, due to expensive equipment, strict application conditions and bacterial resistance to non-thermal processing. Therefore, Photodynamic Technology (PDT) that is currently used for medical purposes has attracted attention for its potential application for microbial food safety. The PDT sterilizes food without heat using appropriate excitation sources to activate photosensitized (PS) compounds that generate cytotoxic reactive oxygen species (ROS) to inactivate bacteria.

Components for Photodynamic Technology
Fig 3: Components for Photodynamic Technology

Light sources:

• Conventional Lamps (tungsten-halogen, metal–halide, xenon, etc.)
• Light-Emitting Diodes (LEDs)
• Lasers

Among all the light sources, LED had gained more attention, owing to several advantages such as low driving voltage, lightness, robustness, compactness, shock and vibration resistance, absence of toxic compounds (e.g., mercury), flexible assembly and narrow-band emission, coupled with no residue emission of undesirable spectral components. LED had become attractive to the food industry (mainly for food production, post-harvest storage and food safety) for its high benefit-cost ratio, inexpensive maintenance, durability and reduced heating and thermal effects. The current choice for LED means the delivery of a wide range of emission wavelengths from UVA (320–400 nm) to near-infrared (1100 nm) and the output irradiance can be as high as 150 mW/cm2.

Photosensitizer (PS):

Types of Photosensitizers used in the Food Industry

PSs generate ROS, including hydrogen peroxide (H2O2) and hydroxyl radicals (•OH) under the exposure of blue light (BL) in the presence of dissolved oxygen. Selection of an optimal PS is an important step in PDT optimization, as this can influence efficiency, selectivity and safety of the treatment. 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. 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 listed in Table 1 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 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. Electrons are transferred from photo-oxidized polyphenols to dissolved oxygen to produce H2O2, which is then photolyzed by BL to produce •OH radicals as the main contributor to the bactericidal activity of such PDI.

Factors affecting PDT:

• Characteristics of the PS (Dose);
• Characteristic of microorganism (Gram +ve, Gram -ve);
• Light source and exposure time (wavelength, Irradiance, Flux, Temperature);
• Composition of foods (pH, water activity);
• Geometry of foods (Surface).

A lower pH (4.5) was found to increase the efficiency of LED exposure against the Gram-positive bacterium L. monocytogenes. Conversely, inactivation of the Gram-negative bacteria E. coli and Salmonella after LED exposure was found to be higher in alkaline medium. There are three types of food surfaces, namely flat, spherical and complex. Significant inactivation of more than 3 log CFU/mL of E. coli was obtained on all flat surface products, which were represented by slices of cucumber, tomato and lettuce. Inactivation of 3.7 log CFU/mL and 5 log CFU/mL of E. coli was observed on the spherical surface of non-germinated mung beans and fenugreek seeds as long as the beans and seeds were rotated to allow light exposure throughout.

Application of PDT in food processing:

PDT is mostly used for Bacterial inactivation, biofilm destruction, yeast and molds inactivation in fruits, vegetables, milk, milk products, cereals and meat products. Some of the studies are discussed below for better understanding of PDT in food processing.

Bacterial Inactivation:

Curcumin-mediated as photosensitization exerts a bactericidal effect on S. flexneri. Incubating S. flexneri with curcumin for 1 hour followed by exposure to light for 20 minutes reduced the number of bacteria by 4.65 log CFU/mL. These findings were consistent with those of previous statement. The main biological targets of photosensitization are lipids, proteins and genes. On this basis, it is of great importance to explore the anti-bacterial mechanism in terms of cell leakage, protein damage and gene expression.

Biofilm Depletion:

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 not only prevent the formation of biofilms efficiently, but also scavenge the existing biofilms. Therefore, the obtained results also provide excellent insights into the design and fabrication of future alternative antimicrobial treatment and removing/reducing biofilm approaches that could be used in various situations associated with food sanitation.

Yeast and Molds Inactivation:

Many plant pathogenic fungi have been investigated for their susceptibility to aPDT. The susceptibility of the aflatoxin-producing fungi species Aspergillus flavus to aPDT using a light dose of 60 J/cm2 and curcumin at 50 μM was evaluated. In addition to microbial inactivation, levels of aflatoxin B1 were significantly reduced in treated (84.2 μg kg−1) maize kernels, as compared to the negative control group (305.9 μg kg−1). Therefore, PDT has multiple applications against fungi, including not only food decontamination, but also plant disease treatment and control, as well as oxidation or overall inactivation of mycotoxins in food matrices.

PDT Application in different food products

Advantages:

• Non-thermal sterilization;
• Potentiality of food safety applications;
• Attracts attention because it does not have dark toxicity;
• No drug-resistance development.

Disadvantages:

• Lower penetration depth – overcome by using PS;
• Colour changes – Overcome by process optimization using PS.

Compared to other anti-bacterial techniques, the major advantages of PDT are that existing or emerging PDT-resistant strains are unknown and it works against many common bacteria, viruses and fungi. Gram-negative bacteria have a negatively charged, dense outer membrane that can prevent photosensitizers from penetrating the cells. Therefore, PDT has limited capacity to inhibit such bacteria even with higher doses of photosensitizer, prolonged exposure to light and poor bactericidal effects. However, cell wall structures differ between gram-positive and gram-negative bacteria. This problem can be overcome by enhancing the interaction between photosensitizers and gram-negative bacteria using targeted molecules or modifying the photosensitizers. It can be also overcome by combination with cationic substances to promote the permeability of the membrane. The chitosan could increase the photodynamic efficacy of photosensitizers against gram-negative bacteria and the possible underlying mechanism could be the decomposition of cell membrane.

Food choices are significantly influenced by sensory attributes, an important example of which is colour. As with any light technology, colour is arguably the most sensitive parameter to photodynamic treatment. Both the orange juice and pineapples had experienced slight bleaching on account of the illumination. One way to minimize this problem is by process-optimization, wherein, colour change should be incorporated as one of the constraints while formulating the optimization problem. The colour of the food can also be compromised by the PS, as the PS absorbing in the visible range such as curcumin, chlorophyll and riboflavin can have a strong colour of their own. In such a scenario, the colour of the PS chosen should be compatible with that of the food to which it is applied.

Conclusion:

The PDT is a potential non-thermal technology used for preserving foods by inactivating the microorganism which cause harm to human health. The popularity of PDI as an antimicrobial method has increased significantly over the last 5 years. Researchers have gained a greater understanding of the scenario, where PDI can be applied and the microorganisms against which it can be successful. PDT is efficient at inactivating food spoilage microorganisms. Chlorophyllin and curcumin are the natural PS of choice. Fruits, vegetables and meat are the most studied food matrices using PDT. Therefore, this excellent technology provides insights into the design and fabrication of future alternative antimicrobial treatments and removing/reducing biofilm approaches that could be used in various situations associated with food sanitation.

References:

1. Temba, B. A., Fletcher, M. T., Fox, G. P., Harvey, J., Okoth, S. A., & Sultanbawa, Y. (2019). Curcumin-based photosensitization inactivates Aspergillus flavus and reduces aflatoxin B1 in maize kernels. Food microbiology, 82, 82-88.

2. Josewin, S. W., Kim, M. J., & Yuk, H. G. (2018). Inactivation of Listeria monocytogenes and Salmonella spp. on cantaloupe rinds by blue light emitting diodes (LEDs). Food microbiology, 76, 219-225.

3. de Oliveira, E. F., Tikekar, R., & Nitin, N. (2018). Combination of aerosolized curcumin and UV-A light for the inactivation of bacteria on fresh produce surfaces. Food Research International, 114, 133-139.

4. Correa, T. Q., Blanco, K. C., Garcia, E. B., Perez, S. M. L., Chianfrone, D. J., Morais, V. S., & Bagnato, V. S. (2020). Effects of ultraviolet light and curcumin-mediated photodynamic inactivation on microbiological food safety: A study in meat and fruit. Photodiagnosis and Photodynamic Therapy, 30, 101678.

5. Galstyan, A., & Dobrindt, U. (2019). Determining and unravelling origins of reduced photoinactivation efficacy of bacteria in milk. Journal of Photochemistry and Photobiology B: Biology, 197, 111554.

6. Tao, R., Zhang, F., Tang, Q. J., Xu, C. S., Ni, Z. J., & Meng, X. H. (2019). Effects of curcumin-based photodynamic treatment on the storage quality of fresh-cut apples. Food Chemistry, 274, 415-421.

About the Authors:
Sangeetha Karunanithi*, Gnana Moorthy Eswaran & Prem Prakash Srivastav
Agricultural and Food Engineering Department,
Indian Institute of Technology, Kharagpur, West Bengal – 721302.
*Corresponding Author Email ID: sangeetvp1996@gmail.com

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