Effects Of Plasma Functionalized Liquids On The Safety And Quality Of Fresh Fish Fillet

Fish and fishery products are highly perishable and presents viable medium for microbial propagation and spoilage,which contributes to possible foodborne illnesses and quality deterioration.Continuous evaluation and implementation of appropriate handling,preservation and storage techniques within the supply management chain is necessary for efficient and safe supply of products.Washing is an important step during fish processing for microbial load reduction,debris,exudates and dirt removal,and water can be functionalized through the use of chlorine-containing chemicals,organic acids,electrolysis,and ozone for ensuring the safety and quality of fish.However,the formation of halogenated disinfection by-products with health-related mutagenic concerns,undesirable quality effects,and regulatory issues have limited their applications.Cold plasma-based sanitizers obtained by exposing water,water mist,and solutions of organic acids and buffers to plasma discharge have emerged as eco-friendly washing alternatives to traditional chemical sanitisers.The plasma functionalized liquids（PFL）exhibit antimicrobial effects from a unique blend reactive chemistry,and their strong oxidative potential from reduced p H,etching and ionic bombardment of charged electrons and ultraviolet photons can be employed for manipulating and modifying biological materials.Owing to their promising potential,it becomes imperative to study the interactions between PFL and the properties of fish towards the successful development of a broad-spectrum sanitizer for seafood products decontamination.In the first part of the dissertation,PFL including plasma functionalized water（PFW）and plasma functionalized buffer（PFB）were generated from water and citrate-phosphate buffer（CBS）,respectively,using dielectric barrier discharge（DBD）plasma system.Their antimicrobial activities were investigated on grass carp under different conditions of applied voltage（0–70 V）,plasma exposure time（0–10 min）,and immersion time of the samples（3–10 min）.Increasing the voltage and exposure time led to an increase in the levels of reactive oxygen and nitrogen species（RONS）in PFW and PFB,while the presence of citric acid in the buffer accelerated possible reactions of active species with enhanced acidification,electrical conductivity（EC）and oxidation-reduction potential（ORP）when compared with PFW.Decontamination efficiency depended on voltage,exposure time and immersion time,which could be up to 1.21 and 1.52 log reductions for L.monocytogenes,and 1.44 and 1.75log reductions for S.Typhimurium for PFW and PFB,respectively.Reduced p H and increased total acidity of grass carp with no significant difference between PFW and PFB were also observed,while PFB greatly affected the colour change in fish fillets.The applications of PFL can be seen as adding nitrates（NO₃^-）and nitrites（NO₂^-）to products and appropriate amounts of NO₂^-is needed for antibotulism,while higher levels would favour potential formation of carcinogenic N-nitroso compounds.In the second part of the research,a novel approach of combining ultrasound treatment（US）and PFL（UPFL）at reduced levels of NO₃^-and NO₂^-for ensuring reduced risk of increased levels of residual NO₂^-in the product was explored.Potential synergy was demonstrated between PFL and US for reducing the populations of Escherichia coli and Shewanella putrefaciens on grass carp.The combined treatments of UPFW and UPFB presented additional reductions of 0.05–0.65log CFU/g,from reductions of 0.31-1.18 log CFU/g observed with applying PFW or PFB alone.The combined treatments were more effective for retarding the hardness reduction,but the colour change was significantly affected with increased whiteness of grass carp.The findings also suggested improved biomedical index and nutritional value of fatty acids and lipids,protein structural unfolding,increased lipid oxidation and protein degradation with values within the acceptable limits of freshness.Mathematical models can be employed for improving the process design of decontamination approaches and the objective assessment of food safety.Thus,the third part evaluated the suitability of linear（first-order）and non-linear（Weibull,biphasic,and log-logistic）models for predicting the inactivation dynamics of Escherichia coli and Listeria monocytogenes on grass carp during UPFB treatment.UPFB was more effective for inactivating bacteria when compared with individual applications of US or PFB with reductions of 3.92 and 3.70 log CFU/g for Escherichia coli and Listeria monocytogenes,respectively.Compared with the linear model,the three non-linear models presented comparable performances and were more suitable for describing the inactivation kinetics with superior adj-R²（0.962–0.999）,accuracies（0.970–1.006）and bias factors（0.995–1.031）,and by assessing the strengths of evidence,weights of evidence and evidence ratios for the models,the biphasic model was identified as the best fit model.In the fourth part,thermoultrasound-assisted PFB was evaluated for decontaminating grass carp using the Box–Behnken design（BBD）with processing variables including plasma generating voltage（P_V）,ultrasound treatment time（U_T）and temperature（T_P）.The predicted models were found to be significant（p<0.05）and displayed sufficient fitness with experimental data as indicated by non-significant（p>0.05）lack of fit and high coefficient of determination（R²≥0.97）values.The optimum decontamination conditions for the responses of S.putrefaciens and S.Typhimurium were P_V of 66 V,U_T of 14.90 min and T_P of60℃,achieving reductions of 4.40 and 3.97 log CFU/g,respectively,with a desirability of0.998.Temperature presented higher significance for inactivating bacteria and the production of volatile basic nitrogen and lipid peroxidation under the optimized conditions were within the limits of freshness for grass carp.The effects of PFB and the optimized thermoultrasound-assisted PFB decontamination were mild on the microstructure of grass carp with slight ruptures and loose myofibril structures,indicating the potential of thermoultrasound-assisted PFB for seafood products decontamination with reduced processing time.In the fifth part,the hurdle approach of UPFL treatments were evaluated on the quality of vacuum-packaged silver Pomfret stored at 4℃for 15 days.Conformational modifications in myofibrillar proteins,improvements in nutritional value of fatty acids and lipids,reduced p H of 5.70,increased K-value,TBARS,and TVB-N at values of 12.05%,0.576 mg MDA/kg,and 9.15 mg N/100 g,respectively,and 1.99 log reductions in spoilage microorganisms were evident immediately after treatments.UPFL presented better quality preservative effects when compared with individual applications of US or PFL,and vacuum packaging ensured optimal quality enhancement effects such as stability of myofibril fragmentation,inhibition of physicochemical quality degradation,and microbial growth control.The results also revealed the predominant cultivable spoilage microbiota of vacuum-packaged silver Pomfret treated with UPFL during refrigerated storage.Food processing techniques can modify protein structure for improving sensitivity to enzymolysis and influencing bioactive properties.In the sixth part,the objective of the study was to investigate the effects of the hurdle approach of UPFW on the functional and bioactive properties of small yellow croaker protein hydrolysates（SYPHs）produced from three different enzymes.Fluorescence and UV-Vis spectroscopy indicated that SYPHs tended to unfold with increasing intensity and shift in wavelengths to more flexible conformations under PFW and UPFW treatments.Particle size distribution and microstructure analysis revealed that treatments could disrupt aggregation of protein molecules to increase the roughness,specific surface area,and decrease the particle size of peptides during hydrolysis.The partially denatured structure of SYPHs induced by treatments increased the susceptibility of the fish proteins to exogenous enzymes,thereby accelerating the hydrolytic process to yield peptides with improved solubility,decreased emulsifying and foaming properties,and improved enzyme-specific antioxidant properties.The results revealed that the functionality of SYPHs was influenced by the treatment method and the enzyme type employed.In the last part,hybridising PFW and US pretreatment for the enzymatic hydrolysis （HPUEH）of small yellow croaker was evaluated by adopting Plackett-Burman design for parametric screening of six key variables,and Box-Behnken design for optimizing the three most significant variables including plasma generating voltage（P_V）,ultrasound treatment time（U_T）,and enzyme concentration（E_C）.The models developed for predicting the degree of hydrolysis（Do H）,protein recovery（PVY）,and soluble protein content（SPC）were sufficiently fitted to the experimental data（R²≥0.966）with non-significant lack of fit and used for determining the optimum conditions as P_V of 70 V,U_T of 15 min,and E_C of 1.787%,with predictive values of 27.74%,85.62%,and 3.28 mg/m L for Do H,PVY,and SPC,respectively.HPUEH presented hydrolysates with smaller peptide sizes and molecular weights,enhanced Do H,PVY,SPC,amino acids and antioxidant activity,but reduced emulsifying and foaming properties when compared with conventional enzymatic hydrolysis.In summary,the important aspects that ought to be considered during the evolution of broad-spectrum sanitizers for fish processing includes microbial safety,nutritional value,lipid composition,oxidative stability and sensorial properties.In this research,we demonstrated the potential of DBD atmospheric cold plasma for functionalizing and enriching water and citrate-phosphate buffer solution with abundant RONS,enhancing EC and ORP levels to profer antimicrobial properties.For the first time,the physicochemical properties of two PFL were comparatively evaluated,and the scientific basis for the application of cold plasma induced interactions in liquids for reducing microbial contamination on fish was provided.We also demonstrated for the first time,the positive interactions between ultrasound,thermoultrasound and PFL with regards to safety and quality maintenance for improving microbial reductions,extending shelf life,improving biomedical index and nutritional value of fatty acids and lipids,and reducing the formation of volatile compounds in fish.In addition,we established that these interactions could induce conformational changes and structural unfolding of fish protein molecules for the derivation of protein hydrolysates with improved biological activity.Furthermore,the reported models could serve as fundamental background for performance evaluation and optimization of the hygiene management systems during fish processing for guaranteeing safe supply,and scale-up to pilot decontamination studies may be useful for the final validation of the process to meet industrial requirements.