Introduction

Atmospheric cold plasma technology is an innovative technique that gains importance for the production of minimally processed, high-quality foods. Plasma technology requires lower processing times, less energy, and lower costs when compared to conventional technologies. It is a chemical-free technology, with a low impact on the internal product matrix. Therefore, it is especially convenient for the treatment of heat-sensitive foods (Coutinho et al., 2021). It has been reported that cold plasma can be successfully used for different purposes in food processing (Namjoo et al., 2022). Therefore, recent studies focused on the use of this technology in the field of food have gained importance. Múgica-Vidal et al. (2019) reported that atmospheric pressure plasma may produce antibacterial coatings on materials to combat gram-positive and gram-negative bacteria on food contact surfaces. Wang et al. (2016) studied the effect of in-pack discharge atmospheric cold plasma (DBD) on bacteria suspensions and reported higher effects on gram-negative spoilage bacteria than on gram-positive. Atmospheric pressure cold plasma is reported to have excellent potential to enhance shelf life and the quality of fresh produce (Dong & Yang, 2019). Panpipat and Chaijan (2020) used atmospheric pressure cold plasma to improve the biophysical properties of fish actomyosin and reported this technology as a possible approach to strengthen surimi gel. It has been reported that cold plasma may provide decontamination, preservation (Alves Filho et al., 2021),  microbial inactivation (Xu et al., 2017), and enhance bioactive compounds (Paixão et al., 2019) in juices.

The limitations of cold plasma are the variety of equipment used in different studies, the insufficiency of information about cold plasma–treated foods, the unexplored impacts on sensory properties of various food, and its early stage of technology development. Determination of the effects on different foods and a full understanding of the mechanisms of action are necessary for future commercial applications (Niemira, 2012). In recent studies, it has been stated that there is a need for extensive research using different plasma sources, working gases, and treatment times applied to various commodities (Laroque et al., 2022). Since factors such as the gas used to generate plasma, the application time, the power supply, and the structure of the food change the effect to be achieved, different parameters should be examined on various foods (Bourke et al., 2018). The effect of atmospheric pressure cold plasma is highly dependent on the type of gas in which the plasma is generated. Various carrier gases can be used to create cold plasma and the effects on foods may vary depending on the gas used (Kim et al., 2011). In former studies, helium has been used as a carrier gas to produce cold plasma, and different levels of antimicrobial effects have been reported depending on other parameters such as the applied medium or electrode design (Ulbin-Figlewicz et al., 2015; Hajhoseini et al., 2020, Bang et al., 2021). Air has also been used as a carrier gas, providing a low-cost alternative without the need for additional expenses to supply gas, and there is a high potential for industrial-scale applications (Rossow et al., 2018).

This study intended to determine the effect of different feed gases (air, helium) and exposure times on the sensory acceptability and quality of fish. A new device capable of producing air- or helium-plasma has been designed and manufactured. Air-plasma and He-plasma were applied onto whole sea bass with different treatment times (0.5, 1, 3, 5, 7, 10 min). It was aimed to determine (1) the immediate effect of plasma treatments on fish quality and (2) the effects after 5 days of cold storage, in order to monitor effects during marketing. Since it is one of the most perishable foods and has an important place in the world food trade, the effects of plasma treatments were studied on fish. Sea bass, farmed by Turkey, was chosen as the sample due to its importance in the European market.

Materials and Methods

Treatment with Cold Plasma

The atmospheric pressure cold plasma–generating device is designed to work with helium (He) or air (Air) as working gases. A power source with a frequency of 20 kHz and an output voltage of 30 kV was used to generate cold plasma. In order to generate cold plasma in a helium environment, the device is equipped with parallel-placed two copper electrodes (12 cm × 36 cm × 1 mm). The bottom base electrode is placed into a Plexiglas material to minimize charge accumulation. The gap between the two electrodes was 7 cm. When the system is filled with helium and energized, a visibly purple-colored plasma is formed (Fig. 1a). To generate cold plasma using air as the carrier gas, the same device is equipped with parallel-placed two copper electrodes. The bottom base electrode is the same as formerly described. The upper electrode consisted of a 1-mm-thick copper cable placed zig-zag on the Plexiglas material (Fig. 1b). Since no additional gas is used in this treatment, purple-colored atmospheric air-plasma is generated between the copper cable and upper electrode, when the system is energized. A fan is integrated into the device, and the particles produced in the plasma are circulated in the system. Therefore, the food placed in the system is exposed to the plasma products, even if it is not completely located in the plasma-generating area.

Fig. 1
figure 1

Treatment with cold plasma. a He-plasma is formed in purple color, b Air-plasma is formed in purple color between the zig-zag copper cable and the upper electrode

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Fresh sea bass (Dicentrarchus labrax) were obtained from an aquaculture plant in the Aegean Sea. They were iced after harvest and transferred to Istanbul overnight in a refrigerated truck. The average length and weight of the samples were measured as 29.63 ± 1.63 cm and 262.42 ± 32.70 g, respectively. Five randomly chosen individuals were immediately analyzed to determine their initial quality. Sea bass samples were treated with either atmospheric air-plasma or helium-plasma (He-plasma) for 0.5, 1, 3, 5, 7, and 10 min. Immediately after plasma treatments, samples were subjected to sensory, color, thiobarbituric acid reactive substance (TBARS), and microbiological analysis. Untreated samples were coded as control (C) and the same analyses were also performed for control samples. The samples were also cold-stored at + 2 ± 1 °C, and analyzed after 5 days, to determine possible effects during marketing. Whole, ungutted fish were used to imitate the sales conditions. For each treatment, the sample size was n = 3 and each sample was randomly selected individuals.

Sensory Analysis

The sensory characteristics of whole, ungutted sea bass samples were evaluated by 10 panelists, experienced in sensory assessment of fish quality. Samples were coded with randomly generated 3-digit numbers and placed in white plates, and the test was carried out in a well-ventilated environment, under daylight. To prevent the panelists to interact with each other, it was ensured that they evaluated samples at a physical distance.

To determine the sensory quality of fish, the color, smell, texture, and taste parameters were analyzed. For the evaluation of taste, fish meat was placed in a clean glass jar, cooked in a water bath, and served to the panelists. Thus, factors affecting sensory properties such as frying oil, spices, or salt during cooking are avoided. A 9-point scale was used in the sensory evaluation and the scoring was stated in the sensory test forms as 9: excellent, 8: very good, 7: good, 6: above average, 5: moderate, 4: below average, 3: bad, 2: very bad, and 1: extremely bad (Stone & Sidel, 2004). The effect of plasma treatments on the sensory properties of fish was determined by this sensory analysis, performed immediately after treatments. Sensory quality analysis was repeated after 5 days to determine the effect of plasma on sensory properties during cold storage.

A purchase intention test was also applied to the panelists. The panelists were asked, “Would you buy this fish?” It was aimed to determine whether the cold plasma application affects purchasing preference. The panelists were presented with the options “I definitely won’t buy (1 point), I probably won’t buy (2 points), I may or may not buy (3 points), I would probably buy (4 points), I would definitely buy (5 points)” (Dutcosky, 2011; Silva et al., 2021).

Since the firmness and juiciness of the fish are important factors affecting the consumer’s decision to purchase fish, sensory evaluations focused on the firmness and the juiciness of the fish were also questioned. Whole fish were presented to the panelists, and a sensory scale was given to test firmness. On this scale, 1 point indicates too hard/firm, 3 indicates exactly as it should be, and 5 indicates too soft (Solo, 2016). Similarly, the juiciness of the fish was evaluated before and after the plasma treatments. Here, the scale was as follows: 1 point for too dry, 3 for exactly as it should be, and 5 for too juicy/slimy (Solo, 2016).

Sensory evaluation forms containing the 9-point scale, purchase intention test, and firmness/juiciness test scores were prepared and given to the panelists. It was stated that they should also note if they felt a rancid taste in any of the samples.

Microbiological Analyses

Aseptically prepared fish samples (10 g) were placed in sterile stomacher bags with 90 mL of MRD (Maximum Recovery Diluent, Merck, Germany), and homogenized in a stomacher (Masticator, IUL Instruments, Spain) for 1 min; then, serial dilutions (1:9) were prepared. The total mesophilic and psychrophilic aerobic bacterial loads were determined on Plate Count Agar (Merck, Cat. No.: 1.05463.0500) after incubation at 37 °C for 24–48 h, and at 7 °C for 10 days, respectively (Baumgart, 1986). To determine net growth, the bacteria load of day 0 was subtracted from the load of day 5.

Thiobarbituric Acid Reactive Substances

Fish muscle (5 g) was homogenized with butylated hydroxytoluene (100 µL) and distilled water (50 mL) using Ultra Turrax, and then mixed with 4 N HCl (2.5 mL) in a glass balloon. Distilled water (97.5 mL) was added to the balloon and the mixture was heated and condensed. The condensed liquid (5 mL) and 2-TBAR (5 mL) were mixed in glass test tubes and heated to 70–80 °C in the water bath (30 min). After chilling, the optical density of this mixture was measured by using a spectrophotometer (PG Instruments, UV/VIS, T801, UK). The wavelength was 532 nm. Malondialdehyde concentration was calculated from the standard curve, prepared using solutions of the MDA precursor, and the TBARS values were expressed as mg MDA/kg of fish (Varlik et al., 2007). Hydrolysis of acetal was obtained by using 50 µL of TEP (tetraethoxypropane). The stock standard was prepared by containing 0.1 mM MDA (malondialdehyde).

Color Analyses

A Konica Minolta chromameter (Konica Minolta, CR 400/410, Japan) was used for color analysis. The L* (black at 0 and white at 100), a* (− values toward green, + toward red), and b*(− values toward blue, + toward yellow) values were measured before and after plasma treatments. The color change (ΔE) was determined by using the following formula (Sharma & Bala, 2003).

$$\Delta E=\sqrt{(\Delta {L}^{2+}\Delta {a}^{2+}\Delta {b}^{2})}$$
(1)

The color change was examined under two headings. First, the immediate changes in color were determined by comparing the colors of plasma-treated samples with the control, just after treatments. Then, all samples were cold-stored for 5 days and the color change of each group was examined. Thus, the effect of plasma treatment on the color change of cold-stored fish was determined. To accurately monitor the possible color change, the fish (3 individuals for each treatment) whose color was measured on the first day were put back into the cold storage and the colors of the same individuals were measured on the 5th day of storage. Here, the formula was used as follows:

$$\Delta L=L\ \mathrm{day}\ 5- L\ \mathrm{day}\ 0;\ \Delta \alpha=\alpha\ \mathrm{day}\ 5- \alpha\ \mathrm{day}\ 0;\ \Delta b=b\ \mathrm{day}\ 5- b\ \mathrm{day}\ 0.$$

Statistical Analysis

All analyses were performed in triplicate. Statistical analyses were carried out using SPSS 16.0 (SPSS version 16.00, Chicago, IL, USA) and the data were analyzed using analysis of variance (ANOVA). The level of significance was chosen as 0.05. Duncan’s multiple range test was used to compare the significant differences.

Results and Discussion

Sensory Characteristics

The initial sensory score of sea bass samples was 8.02 ± 0.31, according to the sensory analysis performed immediately after they arrived at the laboratory. The majority of plasma-treated samples scored above 7 (good), but the samples that were treated for 10 min received lower scores (p < 0.05) than the shorter treatment times in both applications (Table 1). The panelists reported that the 10-min processing time resulted in decreased brightness and drier appearance, which they would not prefer over shorter-treated samples. Similarly, Chen et al. (2019) reported undesirable effects of long treatment times on sensory properties. Chiper et al. (2011) packed fish in Ar/CO2 mixture, generated cold plasma in this package, and reported reduced sensory quality due to increased treatment times. Kim et al. (2013) treated bacon, a dried product, with plasma (5 and 10 min) and observed a significant decrease in sensory quality. Choi et al. (2017a) applied cold plasma to dried squid for 1, 2, and 3 min and stated that plasma treatment did not significantly change sensory properties. In another study, good sensory properties of semi-dried squid were reported after cold plasma application for up to 10 min (Choi et al., 2017b). No significant effect of cold plasma (0–3 min) has been reported on the sensory characteristics of dried fish (Choi et al., 2016). In general, it has been reported that cold plasma does not cause a significant change in the sensory properties of dried foods. However, it should not be ignored that the effects that may occur on the sensory quality of fresh fish may differ from those of dried foods.

Table 1 Sensory analysis of air-plasma- and He-plasma-treated sea bass, immediately after treatment and after 5 days of cold storage
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Mousavi et al. (2022) used gliding arc plasma, operated with argon (50–100%), a different system than atmospheric pressure cold plasma. They optimized discharge conditions and determined their effects on shrimp quality. However, although they examined the effects of treatment on sensory quality, they used agar shrimp culture instead of whole shrimp or peeled shrimp. The sensory acceptance of foods treated with any preservation technique is crucial for the applicability of the technique and the marketability of the product. Since fish are generally sold whole in the world food trade, it is important to determine the effects of plasma treatment on whole fish. In the present study, the effects of atmospheric pressure cold plasma were examined on whole fish and the results will be realistic for industrial applications.

To examine purchase intention, panelists were shown samples of sea bass treated with air- or helium-plasma and asked, “Would you buy this fish?” Samples treated with air- or helium-plasma for 0.5–7 min received positive responses. These treatments did not change the fresh appearance of the fish and did not adversely affect the consumer’s choice of purchase (Fig. 2). None of the panelists responded “I definitely won’t buy” or “I probably won’t buy” for the plasma-treated samples. On the other hand, the samples treated with cold plasma for 10 min were found to be less preferable for purchase than the others. After 10 min of plasma treatment, none of the panelists responded “I will definitely buy it,” and the number of responses “I may or may not take it” increased. Moreover, 1 and 2 panelists commented that “I probably wouldn’t buy it” for the air- and helium-plasma-applied samples, respectively (Fig. 2). The panelists commented about the samples treated with air- or helium-plasma for 10 min, “These samples are not too poor to buy but I wouldn’t prefer them compared to others.”

Fig. 2
figure 2

Purchase intention test of air-plasma- and He-plasma-treated sea bass

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Since the firmness and juiciness of fish are the affecting factors of purchase, the panelists were also asked about these properties. In this test, 3 indicates the expected firmness and juiciness (Fig. 3). It is seen that fish treated with air-plasma or He-plasma for 0.5–7 min maintained their acceptable characteristics, but hardness and dryness are higher in those treated for 10 min.

Fig. 3
figure 3

Firmness and juiciness of air-plasma- and He-plasma-treated sea bass

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A lack of understanding of the consumers’ perspective complicates the implementation of food preservation techniques. Understanding consumer perception is important in the early stages of new processing technologies to adapt them for industrial applications. Since the purchase intention and willingness to purchase are important for the implementation of new technologies, the sensory characteristics and acceptance of the product are important (Coutinho et al., 2021). The panelists’ ratings for sea bass, treated with air-plasma or He-plasma for up to 7 min, show consumer acceptability of cold plasma.

Microbiological Quality

The initial mesophilic aerobic bacteria count was 4.66 log CFU/g. As it is shown in Fig. 4, increased treatment times resulted in a lower number of mesophilic bacteria, and especially 7 and 10 min of air-plasma significantly reduced (p < 0.05) mesophilic aerobic bacteria counts. It was also observed that the air-plasma treatment resulted in significantly lower (p < 0.05) numbers of mesophilic aerobic bacteria compared to the control group after 5 days of cold storage. De Souza Silva et al. (2019) studied the antimicrobial effect of cold plasma on white shrimp. They reported that atmospheric cold plasma was successful in reducing the microbial load and stated that cold atmospheric plasma has a promising potential. The gas used to produce plasma has also been reported to be a factor affecting the antimicrobial properties of cold plasma (Olatunde et al., 2019a). Likewise, in our study, He-plasma was also found to be effective on mesophilic aerobic bacteria. The mesophilic bacteria load of samples treated with He-plasma for 0.5, 1, 3, 5, and 10 min was found statistically lower than that of the control group (Fig. 4).

Fig. 4
figure 4

Total mesophilic aerobic bacteria counts of air-plasma- and He-plasma-treated sea bass, immediately after treatment and after 5 days of cold storage. a,b,c,d,e,fDifferent letters on the same treatment and same day indicate significant differences (p < 0.05) between treatment times. *Shows the statistical difference (p < 0.05) between the same treatment times of air- and helium-plasma on the same day of storage

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As for the psychrophilic aerobic bacteria, a statistically significant decrease (p < 0.05) was observed especially after 10 min of air-plasma treatment (Fig. 5). In this study, although sea bass samples were transported directly to the laboratory after harvest, the initial load of psychrotrophic aerobic bacteria (6.14 CFU/g) was quite high. The microorganism load of food is associated with the efficiency of the cold plasma on microorganisms. Increased microorganism concentrations may limit the penetration of reactive plasma species (Liao et al., 2017). It can be concluded that the high initial bacterial load limits the success of the treatment. Likewise, Kulawik et al. (2018) studied the effect of cold plasma on cold-stored sushi with a high initial load and reported a limited effect on microbiological quality. On the other hand, when the increase in the number of bacteria was calculated after 5 days of storage, it was observed that the net growth of psychrophilic bacteria was better suppressed in the He-plasma-applied samples (Fig. 6). This shows that He-plasma treatment may help to slow psychrophilic aerobic bacteria growth during cold storage or marketing of sea bass.

Fig. 5
figure 5

Total psychrophilic aerobic bacteria counts of air-plasma- and He-plasma-treated sea bass, immediately after treatment and after 5 days of cold storage. a,b,c,dDifferent letters on the same treatment and same day indicate significant differences (p < 0.05) between treatment times. *Shows the statistical difference (p < 0.05) between the same treatment times of air- and helium-plasma on the same day of storage

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Fig. 6
figure 6

Net growth of a mesophilic aerobic bacteria and b psychrophilic aerobic bacteria in air-plasma- and He-plasma-treated sea bass after 5 days of cold storage

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The recent data on the use of cold plasma for seafood suggests that nonthermal plasma seems potent in reducing microbial load, but its decontamination potential is more pronounced in dried seafood than in fresh fish (Kulawik & Tiwari, 2019). It has been reported that voltage and treatment time are important factors in the effectiveness of plasma (Hatab, 2018). Albertos et al. (2017a) studied the effect of in-package atmospheric plasma (70 and 80 kV, 5 min) on the shelf life of herring, stored at 4 °C. According to their data, after treatment with 70 kV, the mesophilic and psychrophilic aerobic loads were approximately the same as the control, or even higher during cold storage. They reported higher reduction at higher voltage, but also noted that the key quality factors were better preserved in samples exposed to lower voltage. In a similar study (Albertos et al., 2017b), it was reported that in-pack atmospheric cold plasma did not provide a significant decrease in the total aerobic mesophilic count of mackerel, but decreased the numbers of psychrotrophic aerobic bacteria. It was also stated that these effects increased at high voltage (80 kV). However, they noted that high voltage or long treatment times may negatively affect the quality of fresh fish (Albertos et al., 2017b). In this study, 30 kV was used to generate air- and He-plasmas, and antibacterial effects were determined. Higher voltages may be studied for more effective results, but the effects of such treatments on quality parameters of fish such as sensory properties, color, and oxidation should also be examined.

TBARS Values

Lipid oxidation is one of the limitations of plasma treatment of foods (Coutinho et al., 2021), and TBARS value has been widely used as an objective index for lipid oxidation (Chen et al., 2019). Various limit values are given for the TBARS value in fish. Nunes et al. (1992) reported the acceptable limit of TBARS for fish as 5–8 mg MDA/kg. On the other hand, Schormüller (1968) defined TBARS values above 8 mg MDA/kg in fish as unacceptable, and Jeon et al. (2002) accepted the limit value as 10 mg MDA/kg. Choi et al. (2016) reported high levels of TBARS above 40 mg MDA/kg in dried fish treated with a plasma jet for 1–3 min. Increased processing times have been reported to result in higher oxidation in Asian sea bass, exposed to cold plasma generated in a gas mixture (argon/oxygen) for 5 min or longer (Olatunde et al., 2019a, b). Likewise, higher TBARS values (p < 0.05) were detected in samples exposed to He-plasma for longer than 5 min after 5 days of cold storage (Table 2). However, even the highest TBARS values detected were well below the acceptable limits and no rancidity was reported during the sensory tests by the panelists. Similarly, the TBARS values of in-package atmospheric plasma-treated herring were reported to be below 1 mg MDA/Kg, during cold storage (Albertos et al., 2017a). The TBARS values of in-pack atmospheric plasma-treated mackerels were also not significantly different from controls during storage at 4 °C (Albertos et al., 2017b). Kulawik et al. (2018) treated sushi with cold plasma for 5 min and studied its effect on lipid oxidation and microbial quality. They reported that the TBARS value of sushi samples did not reach 4 mg MDA/kg. Similarly, the TBARS values of shrimps treated with atmospheric cold plasma for different times (1–2.5 min) did not differ significantly between exposure durations (p > 0.05) and remained below 5.0–8.0 mg MDA/kg throughout the storage period (Elliot et al., 2021). The TBARS levels of semi-dried squid were reported to be below 1 mg MDA/kg, after various times (3–10 min) of plasma exposure with a corona discharge plasma jet (Choi et al., 2017b). Likewise, the TBARS value of smoked salmon was reported to be 2.84 mg MDA/kg, even after 15 min of atmospheric cold plasma treatment (Colejo et al., 2018). In this study, it was seen that air-plasma and He-plasma treatments did not cause a significant increase in TBARS values, which was consistent with the literature.

Table 2 TBARS values (mg MDA/kg) of air-plasma- and He-plasma-treated sea bass, immediately after treatment and after 5 days of cold storage
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Color

The color of food has an important effect on a consumer’s decision to buy any product. The properties of the fish (dried or fresh, whole or filleted), plasma treatment parameters (treatment time, working gas), and storage conditions may affect the color of the product (Pankaj et al., 2018). The treatment time is an important factor affecting the color of fish. This is probably related to the increased amount of generated ROS and RNS with increased time (Olatunde et al., 2019a).

Koddy et al. (2021) reported increased L* values (lightness), especially at longer plasma exposure times. In this study, L* values of plasma-treated samples were generally higher than of control samples (Table 3), similarly. In the present study, the color was measured just after plasma treatments, and ΔE was calculated, comparing with the non-treated control samples (Fig. 7). Thus, immediate changes in color as a result of plasma treatments were determined. Increased treatment times generally resulted in higher ΔE values, showing the color change. Choi et al. (2016) reported that ΔE values of dried fish treated with cold plasma increased with longer exposure time. They also noted that these color differences were not noticed visually. In this study, the panelists stated that they did not notice a change in the color of the samples in the sensory analysis, similarly.

Table 3 Color (L*, a*, and b*) of air-plasma- and He-plasma-treated sea bass, immediately after treatment and after 5 days of cold storage
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Fig. 7
figure 7

The immediate color change (ΔE) in sea bass fillets, due to air-plasma and He-plasma treatments. a,b,c,dDifferent letters on the same treatment indicate significant differences (p < 0.05) between treatment times. *Shows the statistical difference (p < 0.05) between the same treatment times of air- and helium-plasma

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Additionally, the effect of plasma treatment on the color change of cold-stored fish was determined by examining the color changes that occurred after 5 days of cold storage (Fig. 8). It was observed that the color change (ΔE) during cold storage generally increased with higher treatment times (Fig. 8) and the ΔEs of He-plasma-treated samples were higher than of air-plasma-treated ones (p < 0.05). Increased ΔE values with longer cold plasma times have also been reported for dried squid shreds (Choi, 2017a) and semi-dried squids (Choi, 2017b). Although the findings of our study are generally in harmony with this literature, the characteristics of the sample (fresh or dried) should also be considered when interpreting the color changes.

Fig. 8
figure 8

The total color change (ΔE) in air-plasma- and He-plasma-treated sea bass after 5 days of cold storage. a,b,c,dDifferent letters on the same treatment indicate significant differences (p < 0.05) between treatment times. *Shows the statistical difference (p < 0.05) between the same treatment times of air- and helium-plasma

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Conclusions

Although the initial microbial load was high, atmospheric pressure cold plasma reduced the bacterial load of fresh sea bass. Air-plasma, especially for 7 and 10 min, provided a higher reduction in mesophilic aerobic bacteria counts, but psychrophilic bacteria growth was better suppressed in He-plasma-treated samples after 5 days of storage. All samples, treated with air- or He-plasma, retained their fresh characteristics and maintained good sensory properties. Treatment of sea bass with cold plasma for up to 10 min did not increase TBARS values that would indicate rancidity. The longest treatment time, 10 min, resulted in the dryer and harder sensory characteristics and was less preferred. So, it can be said that 7 min of air-plasma provided the best results in terms of sensory appreciation and reducing bacteria. The findings obtained in this study showed that the potential of cold plasma to be used in fresh fish is promising. For the industrial use of cold plasma technology in fresh seafood, a wide range of studies is needed to examine different species, processing times, different gases, and voltage levels.