Prototype of innovative minimally processed fresh oysters (Crassostrea gigas) treated with cold atmospheric pressure plasma

Introduction Oysters are abundantly harvested shellfish and are highly perishable due to their high water activity, neutral pH and chemical composition. Deterioration can be caused by enzymatic autolysis, oxidation and microbial growth of the natural spoilage microbiota. Refrigeration is used to limit the risk associated with potentially pathogenic microorganisms and to delay changes in freshness, undesirable odour, off-flavour and texture. However, maintaining low temperature throughout the supply chain can be difficult, making additional preservation measures desirable. Post-harvest treatments based on coatings, electrolyzed or ozonated water, rapid chilling, irradiation, hydrostatic highpressure processing, and vacuum or modified atmosphere packaging (MAP) have been investigated as additional measures to reduce the levels of pathogens and spoilage in oysters. Among the non-thermal intervention strategies, not extensively studied for fish products but considered promising for maintaining food quality and safety, cold atmospheric pressure plasma (CAP) has received increasing attention in recent years. Plasma, the “fourth state of matter,” is a neutral gas containing various species such as electrons, ions, reactive atoms, free radicals, neutral molecules, and photons in a metastable state with a roughly zero net electrical charge. If enough energy is added to a gas or gas mixture, it generates a plasma that produces a wide range of unique species, including reactive oxygen and nitrogen species (ROS and RNS) and UV radiation. These reactive species play a crucial role in microbial inactivation by damaging microbial DNA and causing oxidative damage to cell envelopes and membranes. The CAP configuration and processing conditions must be optimized to maintain nutritional and quality characteristics, depending on the food matrix composition and chemicalphysical properties. Plasma technology is a smart, green, non-thermal technology with relative advantages in extending the shelf-life of various foods, with limited side effects on quality parameters thanks to the low treatment temperature. The aim of the present research was to develop a prototype of fresh oysters treated post-harvest with CAP. The setup of protocols and identification of the best performing conditions were based on a preliminary screening by modulating different processing variables such as the feeding gas (argon or atmospheric air), the main reactive species generated during the treatments (ozone or NOx), and the processing time. The effects of the different conditions were checked by evaluating the reduction of the natural spoilage microbiota and retention of nutritional and quality features of the treated products. The selected conditions were then assessed through shelf-life tests by combining CAP treatments with two different MAP conditions. Materials and methods Pacific oysters (Crassostrea gigas) (shell length 11±1 cm) were harvested from France and transported to the laboratory under refrigeration within 24 hours. The live oysters, after cleaning and draining excessive drip solution, were shucked and the meat left on one side of the valve used for experiments. Two CAP prototype were used (Figure 1): - with argon gas or atmospheric air and treatment times of 20 and 45 minutes, - with ozone or NOx reactive species with treatment times of 20 and 45 minutes. Untreated (control) and treated oyster samples were analysed for quality parameters (pH, dry matter, oxidation, texture, colour and sensory proprieties) and natural microbiota (e.g., Pseudomonas spp., aerobic mesophilic and psychrotrophic bacteria). After plasma treatment, oysters were packed in high-density polyethylene (HDPE) boxes and high barrier film under two MAP conditions (20% CO2 + 80% N2 or 80% CO2 + 20% O2) and stored at 4 °C as an additional intervention to prolong the product shelf-life. Microbial load and pH were analysed at storage times of 0, 3, 7, 10, 15, 21, and 28 days, while the other quality controls were carried out up to 7 days of storage. Results During the application of cold plasma at atmospheric pressure to fresh oysters, different processing parameters were selected to control the generation of reactive species that can significantly affect the quality of the final product. The pH, dry matter and texture of the innovative oysters showed no significant changes in most cases immediately after the CAP treatments. However, after 45 minutes of treatment, the oysters showed a darker colour, regardless of the feed gas, which affected their visual quality. Despite this effect, the CAP treated oysters scored very well overall in terms of sensory freshness. Nutritional quality, assessed by lipid oxidation, showed that the 20-minute treatment with argon did not affect this parameter, while the 45-minute treatment with argon, ozone and NOx increased lipid oxidation, also if below the expected limit for seafood. From a microbiological perspective, CAP treatment with argon gas resulted in an immediate reduction (1-2 Log CFU/g) of Enterobacteriaceae, aerobic mesophilic and psychrotrophic bacteria. Similar results were obtained after treatments with ozone as reactive species, while those generating NOx mainly delayed and limited the extent of cell proliferation over storage of the microbiota that survived the treatments. Overall, the chosen CAP processing conditions in combination with MAPs helped to maintain the microbiological quality of the product within acceptable limits. Conclusions In summary, the results of the study showed promising effects of cold gas plasma treatment on some of the microbiological and physicochemical parameters investigated, with synergistic effects derived from the use of MAPs. However, given the inherent variability of the raw material and the multitude of parameters that need to be adjusted to optimize the quality and stability of the oysters during transport or storage, further studies are needed before considering actual industrial application of CAP. In addition, it is important to consider eventual potential toxicological concerns in future studies. Acknowledgements This research was conducted under the NewTechAqua project supported by the European Union’s Horizon 2020 Programme (grant agreement No 862658). The project aims to expand and diversify European aquaculture production by developing and validating technologically advanced, resilient, and sustainable applications for finfish, molluscs, and microalgae.