Appropriate Parameters for growth Reduction of microorganisms in White leg Shrimp (Litopenaeus vannamei) and Splendid Squid (Loligo formosana) by Plasma Activated Water

doi:10.21203/rs.3.rs-3230561/v1

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Research Article

Appropriate Parameters for growth Reduction of microorganisms in White leg Shrimp (Litopenaeus vannamei) and Splendid Squid (Loligo formosana) by Plasma Activated Water

https://doi.org/10.21203/rs.3.rs-3230561/v1

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The study focuses on the application of PAW inactivation for exporting white leg shrimp and splendid squid. The experiment determined the maximum hydrogen peroxide concentration in PAW by utilizing a full factorial design with two-level factors. Subsequently, the analysis of variance (ANOVA) was conducted using Minitab. The results indicated that a gas flow rate of a mixture of Ar (98%) and O₂ (2%) at 25 L/min, with a discharge time of 120 minutes, generated the optimal concentration of H₂O₂ in PAW at 13.30 mg/L, as per the response optimizer. The use of PAW to inhibit the growth of microorganisms in white leg shrimp and splendid squid demonstrated that inactivation with PAW, soaked for 10 minutes, resulted in a reduction of total microorganisms to less than 6 Log CFU/g. Additionally, an examination of the nutrition of aquaculture food revealed that PAW-immersed white leg shrimp and splendid squid showed different effects on protein and lipid changes. The results suggest that PAW could be a viable option for decontaminating aquatic food products. However, it is important to consider that oxidation may pose a challenge in certain products.

Plasma jet

Plasma activated in Water

Design of experiment

Litopenaeus vannamei

Loligo formosana

Seafood is a highly perishable food product that is susceptible to microbial contamination at various stages of the production and supply chain, including harvesting, processing, transportation, and storage. Litopenaeus vannamei (white leg shrimp) and Loligo formosana (splendid squid) are popular seafood products known for their taste and nutritional value. However, they are prone to contamination by pathogenic microorganisms such as bacteria, viruses, and parasites, which can result in foodborne illnesses. Common microorganisms that can contaminate seafood include Vibrio parahaemolyticus, Vibrio vulnificus, Salmonella spp., Listeria monocytogenes, and Norovirus. These microorganisms can survive and multiply in seafood products if the storage conditions are not optimal or if the seafood is not properly processed. Additionally, spoilage microorganisms contribute to the deterioration of shrimp quality, leading to a loss in its overall quality (Liao et al., 2018). Consuming contaminated seafood can result in a range of illnesses, ranging from mild gastrointestinal symptoms to severe and potentially fatal conditions.

Conventionally, microbial contamination in seafood is typically controlled through the application of different methods, including thermal and chemical treatments. Thermal treatments involve subjecting the seafood product to high heat, which effectively eliminates pathogens. However, these treatments can also have detrimental effects on the texture, quality, and shelf life of the seafood, potentially leading to a loss of nutritional value. Chemical treatments, on the other hand, utilize antimicrobial agents like chlorine, ozone, and hydrogen peroxide to eradicate pathogens. While these treatments can be effective, they may leave behind residues on the seafood product. These residues have the potential to be harmful to human health if consumed. Thailand's main source of income comes from exporting goods and services to foreign countries, accounting for 70% of the country's Gross Domestic Product (GDP) (Chayongkarn, 2014). However, in 2019, Thailand's export of squid products decreased by 2.6% in quantity and 11.5% in value compared to the previous year due to stricter regulations and trade barriers (Department of International Trade Promotion, Ministry of Commerce, 2019). To address this issue, it is necessary to maintain the quality of ink products by implementing proper handling, storage, and transportation practices to prevent contamination by bacteria such as Escherichia Coli, Staphylococcus Aureus, Salmonella Spp., and Vibrio Cholerae (Maneenuch et al., 2011). Some types of bacteria found in ink can cause harm to the human body, such as Vibrio Cholerae, which can cause cholera (Narumon and Vasna, 2015). To overcome the decrease in the value of exported goods and services, it is essential to develop and implement international standards for ink product production, which would require the use of new technologies in the seafood industry for frozen seafood transportation. Due to the decrease in the value of exports of goods and services to foreign countries, there is a strategy to solve the problem by creating internationally accepted product standards. Therefore, new technologies have been developed and used in the frozen seafood industry for export, namely Plasma Technology.

Plasma technology is gaining significant attention among researchers as an innovative alternative technology for various research applications. One of the key highlights of Plasma Technology is its Clean Technology aspect, which focuses on enhancing and refining the surface properties of the target product without altering its chemical composition. By coating plasma on the surface, the technology can effectively manage impurities present on raw materials, offering an additional method to improve the efficacy of inhibiting the growth of microorganisms in aquatic food. This is particularly crucial in meeting the strict requirements for exporting goods between countries. Cold plasma is a new method that has been thoroughly researched for food safety (Xu et al., 2016). Especially plasma activated water (PAW) is frequently produced by discharging cold plasma above or beneath the water surface, which results in the formation of secondary reactive species such as hydroxyl radical (OH), hydrogen peroxide (H₂O₂), superoxide, single oxygen, ozone, nitrate, and nitric oxide. The reactive species present in PAW have antimicrobial properties to inhibition of growth and destruction of pathogens (Liao et al., 2017; Zhao et al., 2018). Previous research has demonstrated the use of cold plasma in reducing the contamination caused by various types of bacteria such as Bacillus, fungi, viruses, bacterial spores, and biofilms (Kolb et al., 2008; Pan et al., 2013). Cold plasma has gained significant recognition for its remarkable efficacy in reducing bacterial risks and eliminating toxins in various food products, including vegetables, fruits, and meat. Notably, it achieves these benefits while preserving the sensory attributes (such as taste, aroma, and texture), nutritional content, and overall value of the treated food items (Baier et al., 2013; Pankaj et al., 2014). In recent research, the application of cold plasma-activated water ice has been proposed for as a method for preserving shrimps (Liao et al., 2018). This innovative approach demonstrates the potential of utilizing cold plasma technology in the seafood industry, specifically for maintaining the quality and extending the shelf life of shrimp products. By harnessing the antimicrobial properties of cold plasma-activated water ice, it is possible to mitigate microbial contamination and enhance the safety of shrimp products during storage and transportation.

In this research, our main objective is to eliminate pathogens in white leg shrimp (Litopenaeus vannamei) and splendid squid (Loligo formosana) a using Plasma Activated Water (PAW). Our aim is to meet the rigorous standards required for controlling seafood exports while maintaining the precision and accuracy of the study. To thoroughly investigate the impact of different factors on the experiment, we employed statistical experimental design techniques to analyze and interpret the results obtained.

Sample preparation

Splendid squid, with an average weight of 5 units/kg, and White shrimp, with an average weight of 26-30 units/kg, were purchased from a supermarket in Chiang Mai, Thailand. The samples were promptly delivered to the laboratory within 30 min of purchase. To prepare the squid samples, 10 g of squid was sliced into small pieces measuring 2x2 cm, with each slice weighing approximately 2g. For the shrimp samples, the shells and head were removed, and then the shrimp was cut into pieces measuring 2x2 cm. with each sample weighing approximately 10g.

Experiment design

In a plasma solution system, the high plasma density is achieved through the collision of ionized gas molecules. This dense plasma, consisting of reactive oxygen species (ROS) and reactive nitrogen species (RNS) radicals, is directly generated in deionized water (DI water), resulting in a more effective inhibition of microorganisms shown in figure 1. The concentration of H₂O₂ in the plasma-activated water (PAW) is influenced by two key factors: gas flow rate (L/min) and discharge duration (min). These factors are utilized with a mixed gas composition of 82% argon and 2% oxygen (Ar:O₂). These factors are listed in Table 1.

To design the experiments, two levels were chosen for each of the system parameters: gas flow rate and discharge duration. The response variable in our experiment was the inhibitory effect on microorganism in white leg shrimp and splendid squid. To detect the effect of response surface curvature, a 2^k full factorial design with center points was conducted. The experiment included 2² (4) conditions, with three replicates for each condition and five replicates for the center points. Various levels of mixed gas flow rate (Ar:O₂) and discharge duration (min) were investigated. The low, center, and high levels of these parameters are presented in Table 1, and the inhibitory effect PAW on microorganisms was measured accordingly. The response surface method, utilizing Minitab 18, was employed to determine the optimal conditions based on the data obtained from the experiments.

Plasma condition

Plasma activated water was produced by 5 pinhole plasma jet (Plasma solution system designed by Northern Scienceand Technology Park, Chiang Mai University). To generate plasma, argon (Ar) gas was injected into the system followed parameter setting of experiment design (Table 1)

Optical emission spectroscopy

An AvaSpec 2048 miniature fiber-optic spectrometer (an optical emission spectrometer) was utilized to determine the reactive plasma species that played a role in the surface effects. It employed a full-range fiber optic, and the data were processed using AvaSoft software. The spectra were recorded in the wavelength range 200-800 nm.

H₂O₂ measurement

The standard relationship between H₂O₂ concentration and absorbance (Zhang et al., 2010) was determined before measuring the H₂O₂concentrations using a UV spectrometer (S-220 spectrophotometer). To determine the standard relationship, H₂O₂ solutions of known concentrations (1, 2, 3, 4, 5, 6, 7, and 8 mg/L, ppm) were prepared and examined using a UV spectrometer to obtain the absorbance. H₂O₂concentrations and absorbance were plotted, and their mathematical relationships were formulated. To measure H₂O₂concentrations in activated water, two types of solutions were prepared for extracting H₂O₂from activated water: solution I was composed of a potassium iodide (KI) solution of hydrochloric (HCI) solution in equal volumes, and solution II was composed of toluidine blue and sodium acetate (C₂H₃NaO₂) solution at 1: 4 ratios. One and 1.25 milliliter each of Solutions I and II were mixed, to which 0.2 ml sulfanilamide solution was added. After three minutes, the PAW water changed to blue when mixed with the extracted solution. Thereafter, an S-220 spectrophotometer with a wavelength of 628 nm was used to measure the absorbance of the extracted H₂O₂ solution. The absorbance of the extracted RNS solution was calculated using the following equation:

H₂O₂ (mg/L) = (–0.0307x + 0.3843) × Dilute factor with R²= 0.9996

Plasma Treatment

The optimal parameters for H2O2 concentration in PAW were used for treatment. The samples, consisting of 10 g of white shrimps and 10 g of splendid squids, were soaked in 90 mL of PAW, while the control group was soaked in DI water (Deionized water). The samples were shaken using an Orbital shaker for 1, 3, 5, and 10 minutes, respectively. Afterward, the squid and shrimp samples were washed in 90 mL of 0.85% NaCl solution (Butterfield’s phosphate buffered-dilution water: BDB).

Microbial analysis

In this section, the microbials were counted between 30-300 colonies. The plasma conditions were compared to the control group (after immersing the samples), which is related to the design of the experiment in the previous section. The sample solution was diluted with serial dilution of 10, 10^-1, 10^-2, and 10^-3 for each sample, and 100 μL of the sample was dropped onto each plate with plate count agar (PCA) medium using the spread plate method for microbial population count. The plates were then incubated at 30°C for 48 hours.

Examination of nutritional quality

The nutrition quality of squid and shrimp was examined by analyzing the total protein and fat content of the PAW treatment samples, which were then compared with the control group (DI water) using AOAC (2000) methods.

Protein content

For protein content analysis following the procedure described by AOAC (2000), dried samples(1-3g) were placed in protein digestion tubes. Then add 15 g of K₂SO₄ and 0.5 g of CuSO₄.H₂O were add, following by addition of 20 mL of sulfuric acid. The samples were digested in a protein furnace at 200 °C for 30 min, and then the temperature was increased to 400 °Cfor 60 min until a clear solution was obtained. A 250 mL flask was prepared by adding 50 mL of 4% boric acid (H3BO3) and an indicator. This flask was used to support the distilled liquid, with the tip of the condenser immersed in the solution. Furthermore, 50 mL of distilled water was added to a digestion tube, and sodium hydroxide (NaOH) was added to react until the solution turned brown. Distillation was carried out to obtain at least 200 mL of liquid. The distilled solution was then titrated with 0.1 N HCL until the solution turned purple. The protein content was calculated using the following equation:

Protein (%) = ((A-B) x N x 1.4007 x F) / W) x 100

A = Acid volume for titrate with the sample (mL)

B = Acid volume for titrate with blank (mL)

N = Acid concentration (mol)

F = Factor (5.85)

W = Initial sample weight (g)

Total lipid content

For the determination of total lipid content, the extraction was performed using a Soxhlet Extraction Unit following the procedure described by AOAC (2000). First, a 250 mL flask was placed in an electric incubator and allowed to cool in a desiccant bowl. The flask was then weighed. Next, 5 g of samples were added to a tube. The sample tube was placed in the Soxhlet apparatus, and 150 mL of petroleum ether solvent was added to the flask, which was placed on a stove. The fat distillation equipment was assembled, and the coolant in the condenser was turned on. The heat was adjusted to allow the solution to condense from the condenser at a rate of 150 drops per minute for 6 hours. Afterward, the sample tube was removed from the Soxhlet, allowing the solvent to flow out of the Soxhlet and down to the bottom of the flask. To remove the solvent, a vacuum evaporator was used. The flask was then dried at 105 °C and allowed to cool in a desiccant bowl. The sample was weighed, and the process was repeated for 30 minutes each time until the difference in weight between two consecutive measurements did not exceed 1 to 3 mg. The amount of fat was calculated using the following equation (1):

Lipid (%) = W₂/W₁× 100 (1)

W₁ = Weight before baking (g)

W₂ = Weight after baking (g)

Statistical analysis

All experiments were triplicated, and values were expressed as mean standard deviation. Data were analyses by the analysis of variance (ANOVA) procedure of Minitab version 18.

Plasma jet discharge diagnosis

The emission spectrum of cold plasma consisting of Ar and O₂ was measured at 5 mm from the electrode during the discharge. The measurements were conducted in the wavelength range of 200-900 nm. The spectrum exhibited dominant peaks corresponding to Ar emission at wavelengths ranging from 555.9 nm to 810.4 nm (Chung et al., 2012), and O₂ emission at 777.53 nm. Additionally, two peaks were observed at lower intensity, which were associated with the optical transitions of excited NO at 283 nm (Attri et al., 2018) and N₂ in the range of 336.2-397.3 nm. A high-intensity peak corresponding to OH was also evident at 309.8 nm (Limsopatham et al., 2017), as shown in Figure 2.

Effect of PAW on H₂O₂ concentration and the percentage of microbial inactivation for seafoods

PAW was produced, and the concentration of H₂O₂ was measured using the spectrophotometric method. The experimental results revealed that the optimal parameters for producing PAW with the highest H₂O₂ concentration were an Ar flowrate of 25 L/min and a duration time of 120 min, the average H₂O₂concentration was found to be 13.10 mg/L (Table 1 and Table 2).

PAW with the highest concentration of hydrogen peroxide (H₂O₂) was tested for the decomposition rate at different temperatures every 2 days over a period of 20 days. The decomposition rate was measured using a spectrophotometric method to determine the duration for which the PAW produced can be utilized. The results revealed that increasing temperature had a decreasing effect on the H₂O₂concentration. Storage of PAW at 4°C maintained the highest H₂O₂ concentration compared to storage at 25°C and 30°C. After 2 days of storage, the H₂O₂concentration decreased to 5.86 mg/L at 25 and 5.06 mg/L at 30, while the concentration of PAW stored at 4 remained higher at 11.42 mg/L. (Table 3 and Figure 3).

The obtained experimental results were subjected to statistical analysis using analysis of variance (ANOVA) to determine the appropriateness of the experimental design and identify significant effect on the experiment. This involved analyzing the experimental effects, checking the suitability of the model, validating the model, creating mathematical equations, optimizing the factors, and developing a response surface.

The factors of the experiment were analyzed using the experimental results presented in Table 2. This analysis enabled the determination of the total effect value, which includes both main effects and interactions effects. The P-Value was calculated with a 95% confidence level. Factors with a P-Value lower than the significance level α (0.05) were considered statistically significant, as shown in Table 4.

The analysis of the experimental results presented in Table 4, which is consistent with the Normal Probability Plot, demonstrates the effect on the H₂O₂ concentration in PAW. From this analysis, it can be concluded that the factors significantly influencing the change in H₂O₂ concentration in PAW are gas flow rate (A), discharge time (B), and the interaction between the two factors, gas flow rate and discharge time (AB). This conclusion is supported by Figure 4.

Based on the information provided, mathematical equations can be generated to predict experimental results and facilitate the comparison of results across different factors. The model is constructed by considering factors that have significant effects, while eliminating unaffected factors to simplify the prediction process (Table 4). The equation used to predict the experimental results of the H₂O₂concentration in PAW is a linear regression model, formulated as follows equation (2):

H₂O₂ = 5.416 + 1.600A + 3.948B+ 1.470AB (2)

H₂O₂= hydrogen peroxide concentration in PAW

A = gas flow

B = time

AB = gas flow * time

The experimental data were used to construct simulated equations to verify the reliability of the model. An idea model should include the necessary factors and be capable of predicting results close to the actual experimental data, as depicted in Figure 5.

The Normal Probability Plot indicated a linear distribution of the data, while the histogram chart displayed a bell-shaped curve. The stability of variance (Versus Fits) demonstrated that the data was distributed freely, with both positive and negative values but without any sequence related to the experiment. The graph showed an indeterminate distribution of data without any discernible pattern. From these observations, it can be concluded that the residual data followed a normally distributed, exhibited constant variance, and was freely distributed.

The statistical analysis in Table 4 revealed that the adjusted R-square coefficient of determination for the experiment results was 98.64%, indicating a relatively high level of effectiveness. Therefore, the model derived from the analysis can be used as a mathematical model. Additionally, considering the Curvature analysis results, the Curvature test value was found to be 4.15 with a P-value of 0.064 for the H₂O₂concentration in PAW. When the P-Value is greater than or equal to 0.05, it indicates that the main hypothesis is accepted. Furthermore, the sum of quadratic is zero, indicating that the experiment is appropriate, and no additional experiments are required.

Response optimizer analysis

The optimum H₂O₂concentration in PAW was determined using a plasma solution system with a mixed Ar: O₂ plasma discharged. Response Optimizer from Minitab 18 program was utilized for the analysis. The results indicated that the optimal value for Ar-O₂ mixture gas flow rate was 25 L/min, and the discharge time was 120 min. These settings resulted in the highest concentration of H₂O_2, which was measured at 13.30 mg/L.

Response surface

When comparing the result of the response optimizer analysis with the significant factors, gas flow rate and discharge time, as shown on the Contour Plot and Surface Plot, it is evident that specific levels of these factors can lead to the maximum concentration of H₂O_2.By setting the initial gas flow rate from the lowest level of 20 L/min to the highest level of 25 L/min and the initial duration time from the lowest level of 30 min to the highest level of 120 min (Figure 6), the optimal conditions for achieving the highest concentration of H₂O₂ can be determined.

These graphs indicate the region with the highest value, which corresponds to the optimal conditions for obtaining a high concentration of H₂O₂ in PAW. The boundary area represented by the dark green color indicates the specific combination of gas flow rate and discharge time that significantly affects the H₂O₂concentration. In this case, the highest concentration of 13.30 mg/L was achieved with a gas flow rate of 25 L/min and a discharge time of 120 min (Figure 7 and Table 6).

Results on plasma treated seafood.

The effect of PAW on microbial inhibition

Based on the preliminary results, the optimal conditions for achieving the highest H₂O₂ concentration in PAW involved using a mixed Ar: O₂ plasma discharge with a gas flow rate of 25 L/min and a discharge time of 120 min. To evaluate the inhibitory effect on microorganisms in white leg shrimp and splendid squid, PAW treatment was compared to a control group treated with DI water for varying durations of 1, 3, 5, and 10 min. The experimental results demonstrate that the samples of white leg shrimp and splendid squid treated with PAW exhibit inhibition of microorganism growth (Figure 8). Specifically, when both types of samples were soaked in PAW for 10 min, the most effective inhibition of microbial growth was observed, with rates of 41.64% for white leg shrimp and 23.04% for splendid squid. The corresponding microbial growth rates were measured at 3.49 Log CFU/g and 5.31 Log CFU/g, respectively (Figure 9).

Examination of nutritional quality

The nutritional content, including protein and lipid analysis, was conducted on samples of white leg shrimp and splendid squid. The samples were analyzed in three conditions: initial (untreated), soaked in DI water, and soaked in PAW for 10 min. The results indicated that the protein content of the samples soaked in DI water and PAW was reduced compared to the untreated samples. For white leg shrimp samples, the protein content showed a slightly decreased when soaked in DI water and PAW, but the difference was not statistically significant (P > 0.05) compared to the untreated samples. The protein percentages for the treated samples were measured at 18.73%, 18.74%, and 18.90% respectively. In contrast, the splendid squid samples soaked in DI water and PAW showed significant different (P < 0.05) in protein depletions compared to the untreated sample. The protein percentages for the treated samples were recorded as 14.19%, 14.59%, and 16.07% respectively (Figure 10).

The lipid content analysis of white leg shrimp and splendid squid samples, including the initial samples, those soaked in DI water, and those soaked in PAW for 10 min, revealed that the lipid percentages in the white leg shrimp samples treated with DI water and were slightly lower but not significantly different (P > 0.05) compared to the untreated samples, with lipid percentages of 1.76%, 1.78%, and 1.80% respectively. On the other hand, the lipid percentages in the splendid squid samples treated with DI water and PAW showed an increase and significantly different (P < 0.05) compared to the untreated samples, with lipid percentages of 2.18%, 2.08% and 1.97% respectively (Figure 11).

Visual assessment

Visual assessment was conducted to evaluate the color uniformity and chromatic aberration of white leg shrimp and splendid squid samples before and after a 10 min treatment. The results indicated that the color of both PAW-soaked samples and DI water-soaked samples did not exhibit any noticeable changes shown in Figure 12. Therefore, it can be concluded that soaking the samples with DI water and PAW had no significant effect on the color change of both white leg shrimp and splendid squid samples.

Plasma activated water produced various reactive species, which reacted with water molecules or dissolved in water. The results indicate that a plasma solution system can produce PAW with the parameters of gas Ar and O₂ mixture flow rate at 25 L/min and discharge time for 120 min resulting in a hydrogen peroxide concentration of 13.30 mg/L and PAW was revealed to optimally produce of ROS such as H₂O₂, O, O₃ and OH^• shown in equation (3) and RNS such as N₂, NO, NO₂ and NO^*shown in equation (4)-(6). As a high voltage was applied, oxygen (O₂) in air was dissociated into oxygen atoms and when the oxygen atoms touched with water, the dissociation of water molecules occurred and produced reactive species, for instance, hydroxyl radicals and hydrogen peroxides (Sarangapani et al., 2016) The reactions for the production of main ROS and RNS in PAW under air plasma treatment are exhibited as following (Surowsky et al., 2016).

Hydrogen peroxide production:

O+ H2 O→ OH^⋅ OH^⋅+OH^⋅→H2O2 (3)

Nitrate production:

N2 + O2 → 2NO (4)
2NO + O2 → 2NO2 (5)
3NO2 + H2 O→ 2HNO3 + NO (6)

H₂O₂ is a relatively stable compound, and its behavior in liquid environments can lead to the formation of hydroperoxy radicals or superoxides, or even hydroxyl radicals. Research conducted by Niquet et al. (2018) using a dielectric plasma system demonstrated that H₂O₂ concentrations in PAW increased with longer plasma treatment times and remained stable during extended storage periods. H₂O₂ is known for its significant antimicrobial and cytotoxic properties in plasma-activated liquids (Julák, Hujacová et al., 2018). The experiment revealed that the decomposition rate of H₂O₂ is temperature-dependent, with higher temperatures leading to a decrease in H₂O₂ concentration. Conversely, lower temperatures facilitate the maintenance of H₂O₂ concentration over a longer duration. This indicates that storing PAW at low temperatures better preserves the concentration of H₂O₂ compared to storing it at high temperatures.

In the experiment using PAW to inhibit the growth of microorganisms, the microbial growth after plasma treatment was measured to be 3.49 Log CFU/g for white leg shrimp and 5.31 Log CFU/g for splendid squid (P < 0.05). Free radicals, particularly H₂O₂ and OH, can inhibit microorganisms by inducing oxidative stress in cell and damaging various cellular components such as cell membrane. This can lead to the leakage of cellular elements and the loss of important cellular functions (Joshi et al., 2011; Chen et al., 2010). The reaction of H₂O₂ with the structure and function of cell walls and membranes, which contain proteins, lipids, carbohydrates, and nucleic acids, can lead to their destruction (Yusupov et al., 2013; Juan et al., 2021). This damages to the outer structure of cells increases the permeability gap between cells, allowing free radicals to react within the cell by. These free radicals can then disrupt the structure of DNA and interfere with important processes such as replication, transcription, and repair. Additionally, H₂O₂ can inhibit protein functions and essential enzyme activities within microbial cells (Mai-Prochnow et al., 2016; Linley et al., 2012; Lukes et al., 2012). Consistent with previous research, several studies have reported the antibacterial effects of PAW on microorganisms in various types of food, including shrimp and fish (Qian et al., 2019; Choi et al., 2019; Royintarat et al., 2020). For example, the application of PAW glazing on shrimps has demonstrated effective control of microbial growth, with counts below 6 log CFU/g maintained throughout a 9-day storage period (Herianto et al., 2022). Additionally, the use of PAW ice as a substitute for traditional ice in shrimp preservation has been investigated, showing that PAW ice generated from DBD plasma can successfully inhibit microbial growth. Shrimp exposed to PAW ice exhibited a total viable count (TVC) lower than 6 Log CFU/g for up to 8 days of storage (Liao et al., 2018).

Protein analysis was performed on samples of white leg shrimp and splendid squid. The protein content of the white leg shrimp samples soaked in DI water and PAW showed a slight decrease, but the difference was not statistically significant (P > 0.05). However, the protein content of the splendid squid samples soaked in DI water and PAW exhibited a significant decrease, with a statistically significant difference compared to the initial samples (P < 0.05).The protein content decreases after soaked samples in DI water because DI water lacks minerals and ions that could potentially interact with proteins and contribute to their stability. Consequently, soaking samples in DI water can lead to protein leaching and denaturation. Water-soluble proteins may dissolve in the surrounding water, causing a loss of protein content. Additionally, the absence of certain ions can disrupt protein structure, leading to denaturation and further reduction in protein content. While the samples soaked in PAW, the protein content decreased due to oxidative processes induced by the reactive species present in PAW. These reactive species can interact with proteins, leading to oxidation and subsequent protein degradation (Wolff et al., 1986; Lund et al., 2011). The oxidative stress caused by PAW can break down proteins, resulting in a reduction of their content. Additionally, the reactive species can also cause protein denaturation, altering their structure and potentially reducing protein levels (Bu et al., 2022). In line with the research on the cold storage stability of Asian sea bass (Lates calcarifer) steak using PAW soaking, it was found that fish exposed to PAW exhibited a greater extent of protein denaturation and had the highest total carbonyl content (p < 0.05) due to ROS-induced protein oxidation during the cold storage period (Chaijan et al., 2022). Cold plasma treatment induced higher levels of oxidation in fish muscle proteins compared to the control group. The oxidation of specific amino acid side chains, including proline, threonine, lysine, and arginine, led to the formation of carbonyl groups (Estévez, 2011). There have also been reports indicating that cold plasma can cause protein or peptide fragmentation in food systems (Tolouie et al., 2018).

The lipid analysis of white leg shrimp samples revealed that the samples soaked in DI water and PAW had slightly lower lipid content (P > 0.05) compared to the initial samples. This slight reduction in lipids can be attributed to oxidation, as the reactive species in PAW, such as ROS, can initiate the lipid oxidation process by abstracting hydrogen ions from lipid molecules (Yong et al., 2018; Shahidi and Zhong, 2010). Several investigations have reported the oxidative impact of cold plasma on aquatic foods. For example, the application of PAW ice for the preservation of shrimps (Metapenaeus ensis) showed a decrease in Thio Barbituric Acid (TBA) value, indicating reduced lipid oxidation compared to storage in tap water ice (Liao et al., 2018). Similarly, in the case of Asian sea bass steak, the peroxide value (PV) of PAW-treated samples was found to be greater than other samples (P < 0.05) and increased at the fastest rate during the 30-day cold storage period (Chaijan et al., 2022). These findings align with studies on sea bass slices and mackerel, where cold plasma treatment resulted in increased lipid oxidation (Olatunde et al., 2020; Albertos et al., 2017).

On the other hand, the lipid content of splendid squid samples soaked in DI water and PAW showed an increase (P < 0.05) compared to the initial samples. This increase can be attributed to the process of osmosis. When the squid is soaked in water, the water molecules can diffuse into the squid's tissues, dissolving and carrying away some of the lipid components. As a result, the remaining lipid content in the squid tissue may appear to increase due to this perceived redistribution of lipids caused by osmosis.

The color uniformity and chromatic aberration of both PAW-soaked and DI water-soaked samples of white leg shrimp and splendid squid did not exhibit any noticeable changes. This is consistent with previous studies by Rød et al. (2012) and Albertos et al. (2017), which reported that cold plasma treatment does not adversely affect physicochemical parameters such as pH and color. PAW treatment may not significantly affect the pigments responsible for the natural color of shrimp and squid. The primary pigments in seafood, such as astaxanthin in shrimp or various pigments in squid, are relatively stable and resistant to oxidation or degradation caused by the reactive species in PAW. In addition, while PAW can induce oxidative processes that might affect proteins, it doesn't necessarily imply immediate or significant changes in the color of the seafood. Color changes in seafood typically occur due to complex chemical reactions involving pigments, proteins, and other factors. Since the oxidative impact of PAW on pigments may be minimal, the color of the seafood might remain relatively unchanged.

ROS and RNS in PAW are generated by the Plasma solution system, which can reduce microorganisms in white leg shrimp and splendid squid samples. These reactive species can damage microbial cells by inducing oxidative stress, leading to the disruption of cell membranes, DNA damage, and interference with cellular processes, ultimately inhibiting microbial growth. The results showed that inactivation with PAW for 10 minutes significantly affected the total microorganisms in the exported splendid squid, which met the export standard of (< 6 Log CFU/g). The gas flow rate of the gas mixture (Ar:O₂) and discharge time were found to have significant effects on the production of H₂O₂concentration in PAW. The protein content and lipid content decrease as a result of the oxidative processes induced by the reactive species present in PAW. These reactive species can interact with proteins, leading to oxidation and subsequent protein degradation. Conversely, the squid samples exhibited a significant increase in lipid content. Moreover, the PAW treatment had no effect on the color change of both samples. Therefore, PAW can be applied for the decontamination and preservation of seafood. However, further development of the Plasma solution system is necessary to increase the H₂O₂ concentration and reduce the discharge duration to scale up industrial production.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Author Contributions

Norrapon Vichiansan, Pornkaran Phan-umong, Thippawan Mekwilai, Jaturong Kumla, Pipath Poramapijitwat, and Saisamorn Lumyong were the principal investigators and prepared the main manuscript. Komgrit Leksakulapproved the final draft of the manuscript. The Dheerawan Boonyawan is the system and analysis advisor.

Funding

This research project was supported by the TSRI (Thailand Science Research and Innovation) (2023) through Chiang Mai University and National Research Council of Thailand (IRN 2023). The Department of Biology, Physics and Material, and Center of Excellence in Microbial Diversity and Sustainable Utilization, Faculty of Science, Chiang Mai University, provided the examination equipment. We would like to express our sincere thanks for their help and support in this study. We would like to express our sincere thanks for their help and support in this research.

Data Availability

The data that support the findings of this study are available upon reasonable request from the authors.

Code Availability Not applicable.

Practical Applications

Our proposed plasma system could apply to aquatic food products, especially shrimp, fish, squid etc. However, the technology must prove for industrial scale.

Ethical Approval

This article is an original work and does not involve human participants or animal experimentation.

Consent to Participate and for Publication

All the authors declare their consent for participation and publication.

Conflict of Interest The authors declare no competing interests.

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Table 1 Parameter setting at low, center, and high level.

Parameter	low (-1)	center (0)	high (+1)	Detail
A	30	75	120	Time (minute)
B	20	22.5	25	Gas flow (L/min)

Table 2 Experimental results based on the DOE 2^k method with NO₂^-, NO₃^-, conductivity and pH results.

StdOrder	RunOrder	CenterPt	Blocks	Duration time (min)	Ar flow rate (L/min)	H₂O₂ (mg/L)
1	1	1	1	-1	-1	1.38
2	2	1	1	1	-1	6.19
3	3	1	1	-1	1	2.19
4	4	1	1	1	1	12.23
5	5	1	1	-1	-1	1.93
6	6	1	1	1	-1	6.56
7	7	1	1	-1	1	1.67
8	8	1	1	1	1	12.52
9	9	1	1	-1	-1	1.25
10	10	1	1	1	-1	6.68
11	11	1	1	-1	1	1.48
12	12	1	1	1	1	13.10
13	13	0	1	0	0	5.13
14	14	0	1	0	0	4.73
15	15	0	1	0	0	4.63
16	16	0	1	0	0	5.11
17	17	0	1	0	0	5.30

Table 3 The concentration of hydrogen peroxide (H₂O₂) at various temperatures.

	4 °C		25 °C		30 °C
date	Average	SD	Average	SD	Average	SD
0	12.63	0.84	12.63	0.84	12.63	0.84
2	11.42	0.52	5.86	0.55	5.06	0.65
4	10.14	0.68	4.67	0.59	3.54	0.58
6	9.47	0.53	3.45	0.67	2.66	0.59
8	7.65	0.66	2.97	0.75	1.96	0.64
10	6.82	0.52	2.68	0.40	1.79	0.72
12	5.43	0.47	2.23	0.52	1.63	0.52
14	4.77	0.52	1.84	0.61	1.51	0.40
16	4.02	0.71	1.76	0.47	1.27	0.56
18	3.85	0.52	1.67	0.59	1.23	0.66
20	3.59	0.47	1.21	0.47	1.04	0.68

Table 4 Statistical analysis based on ANOVA was performed to assess the significance of the effects on H₂O₂concentration in PAW.

TERM	DF	Effect	Coef	Adj SS	Adj MS	F-Value	P-Value
Constant			5.5983				0.000
Main effect	2			217.792	108.896	1216.65	0.000
A	1	3.2	1.6	30.72	30.72	343.22	0.000
B	1	7.8967	3.9483	187.072	187.072	2090.09	0.000
2-Way Interactions	1			25.931	25.931	289.72	0.000
*AB**	1	2.9400	1.4700	25.931	25.931	289.72	0.000
Ct Pt			-0.324				0.064
Curvature	1			0.371	0.371	4.15	0.064
Error	12			1.074	0.090
Total	16			245.168
		R-Sq = 99.56% R-Sq (adj) = 99.42% R-sq (pred) = 99.02%

Table 5 The optimal parameters for maximum H₂O₂ concentration by response optimizer method.

Response	Goal	Lower	Target	Upper	Weight	Importance
Hydrogen Peroxide	Maximum	1.22	14.9		1	1

Table 6 Optimum parameters for producing H₂O₂ concentration in PAW to inhibit microbials of
white leg shrimp and splendid squid.

Gas flow (L/min)	Discharge time (min)	H₂O₂ concentration (mg/L) in PAW
25	120	13.30

No competing interests reported.

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Appropriate Parameters for growth Reduction of microorganisms in White leg Shrimp (Litopenaeus vannamei) and Splendid Squid (Loligo formosana) by Plasma Activated Water

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Abstract

Figures

Introduction

Materials and Methods

Optical emission spectroscopy

Results and Discussion

Plasma jet discharge diagnosis

Effect of PAW on H₂O₂ concentration and the percentage of microbial inactivation for seafoods

Response optimizer analysis

Response surface

Visual assessment

Discussion

Conclusion

Declarations

References

Tables

Additional Declarations

Status:

Version 1

Appropriate Parameters for growth Reduction of microorganisms in White leg Shrimp (Litopenaeus vannamei) and Splendid Squid (Loligo formosana) by Plasma Activated Water

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Optical emission spectroscopy

Results and Discussion

Plasma jet discharge diagnosis

Effect of PAW on H2O2 concentration and the percentage of microbial inactivation for seafoods

Response optimizer analysis

Response surface

Visual assessment

Discussion

Conclusion

Declarations

References

Tables

Additional Declarations

Status:

Version 1

Effect of PAW on H₂O₂ concentration and the percentage of microbial inactivation for seafoods