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The synergistic effect of dielectric barrier discharge plasma and phycocyanin on shelf life of Oncorhynchus mykiss rainbow fillets – Scientific Reports

Purity, concentration and absorption spectrum of PC extracted from spirulina microalgae

The concentration and purity of PC was measured both before and after dialysis. Initially, the concentrations were found to be 0.238 mg/ml and 0.063 mg/ml, respectively. After dialysis, there was a slight increase in concentrations to 0.251 mg/ml and 0.065 mg/ml, indicating a minor degree of purification. The specificity of PC was determined by constructing a graph (Supplementary Fig. 2). The peak of this graph was found to be 621.9, indicating a high degree of specificity.

Microbiological changes in Oncorhynchus mykiss rainbow fillets treated with DBD and PC during 18 days at 4°C

TVC and TPC

Notably, DBD treatment of P80:5 extended the shelf life of fish up to 9 days, while the combined effect of DBD treatment and PC in P80:5+PC the shelf life up to 12 days, indicating potential for the combination as a preservation method for fish products.

A significant increase in the TVC was observed across all samples during an 18-day storage period at 4 °C (p < 0.05) (Table 1). Notably, the C sample exhibited the highest increase in TVC, rising from 4.05 to 11.62 log CFU/g , while the P80:5+PC sample showed the lowest increase, rising from 3.52 log CFU/g to 9.66 log CFU/g (p < 0.05). Furthermore, on the third, sixth, ninth, and twelfth day of storage, the TVC exceeded the permissible limit of 6.00 log CFU/g for the C sample (6.39 log CFU/g ), PC-P (6.78 log CFU/g ), P80:5−PC (7.05 log CFU/g ), and P80:5+PC (6.69 log CFU/g ), respectively. Notably, DBD treatment of P80:5−PC extended the shelf life of fish up to 9 days, while the combined effect of DBD treatment and PC in P80:5+PC increased the shelf life up to 12 days.

Table 1 TVC changes with DBD treatment at 70 and 80 kV voltages and durations of 2 and 5 min, with and without PC treatment, on Oncorhynchus mykiss rainbow fillets during an 18-day storage period at 4°C.

A significant increase in the TPC was observed across all samples during an 18-day storage period at 4 °C (p < 0.05) (Supplementary Table 1). The C sample exhibited the highest increase in TPC, from 3.38 to 9.69 log CFU/g , while the P80:5+PC sample showed the lowest increase, from 2.53 to 7.55 log CFU/g (p < 0.05). DBD treatment effectively reduced TPC in all samples by increasing the voltage and the duration. However, the combined effect of DBD treatment with PC yielded more optimal results.

The rate of increase in TVC and TPC during the 18-day storage period in P80:5−PC and P80:5+PC samples was slower compared to the control sample. Moreover, the combined effect of DBD treatment with PC, especially in the case of P80:5+PC, inhibited the rate of increase in TVC and TPC significantly (p < 0.05).

Staph

There is a significant increase in Staph levels among all samples during the 18-day storage period at 4 °C (p < 0.05) (Supplementary Table 2). The C sample exhibited the highest increase in Staph rising from 0 to 9.33 log CFU/g. Conversely, P80:5+PC demonstrated the lowest increase in Staph levels, reaching only 7.09 log CFU/g. Moreover, on the 18th day, there is no significant difference observed between the C sample, P80:2−PC, P70:5−PC, and P80:2−PC (p > 0.05). Similarly, no significant difference was found on the 18th day between P80:5+PC, P70:5+PC, and P80:5+PC samples (p > 0.05). Interestingly, on the 18th day, the PC-P sample exhibited a significantly lower value of CPS (8.00 CFU/g ) than the C sample (p < 0).

Salmonella and E. coli

Throughout the 18-day storage period at 4 °C, neither the control nor the treated fish fillets showed any presence of Salmonella or E. coli bacteria. This finding underscores the effectiveness of the applied treatment in preserving the safety and quality of the fillets under refrigeration.

Lb

On the first day, the C sample contains 0.22 log CFU/g of LB. However, with DBD treatment (70 kV, 2 min), this value significantly decreased to 0.09 log CFU/g on the same day (p < 0.05) (Supplementary Table 3). As the voltage and treatment time increased, the Lb count in fish fillets decreased significantly, ultimately reaching zero (p < 0.05). Additionally, the presence of PC in DBD-treated fillets also resulted in a complete eradication of Lb.

The C sample exhibited the highest increase in Lb over 18 days, escalating from 0.22 to 9.39 log CFU/g (p < 0.05). However, PC alone effectively slowed down the growth of Lb. The lowest Lb count was observed on the 18th day, with a significant difference compared to the C sample observed in the P80:5+PC sample (7.88 log CFU/g).

Notably, on the 6th day, the Lb count in the control sample exceeded the spoilage limit of Lb (6 log CFU/g), reaching 6.36 log CFU/g. importantly, all treated samples surpassed this limit by the 12th day, except for the PC-P80:5 sample, which exceeded it on the 15th day, with a value of 6.84 log CFU/g (p < 0.05).

EB

The initial number of EB in all samples is zero on the first day (Supplementary Table 4). The highest and lowest levels of EB are observed on the 18th day of storage for the C sample (9.83 log CFU/g) and P80:5+PC sample (6.85 log CFU/g), respectively. The C sample exhibits the greatest increase in EB during the 18-day storage period, with levels rising from 0.00 to 9.83 log CFU/g (p < 0.05). DBD treatment, both with and without PC, leads to a decrease in the number of EB in all samples over time, with higher voltage and longer treatment time resulting in more significant reductions. Notably, DBD treatment with and without PC yields more favorable results on all days in all samples. Throughout the storage period, the P80:5+PC sample consistently demonstrates the lowest number of EB compared to the C sample, with a significant difference observed on the 18th day (6.85 log CFU/g).

Physicochemical changes in Oncorhynchus mykiss rainbow fillets treated with DBD and PC during 18 days at 4 °C

PH and acidity

The PH values increase significantly in all samples during the 18-days storage period (Supplementary Table 5). The C sample exhibited the highest increase, from 6.33 on the first day to 7.73 on the 18th day (p < 0.05). On the first day, the pH value of the PC-P sample significantly increased compared to the C sample, from 6.33 to 6.44 (p < 0.05). However, except on the first day, the pH value of the PC-P sample is consistently lower than of both the C sample and samples treated only with DBD (p < 0.05). The inhibitory effect of PC on PH is more potent than DBD treatment alone. Throughout the 18-day storage period, the C sample exhibited the highest pH value, while the lowest pH value is observed in the P80:5+PC sample (p < 0.05).

The pH values decrease significantly in all samples treated with DBD, with or without PC, over the 18-day storage period. However, the pH changes are slower in samples treated with both DBD and PC compared to those treated with DBD alone and the C sample. The combined effect of DBD treatment with PC in inhibiting pH is stranger than the independent effect of each treatment. Furthermore, increasing the treatment time at a constant voltage of 80 kV causes a significant decrease in the pH value in all samples (p < 0.05). However, this reduction rate is not significant on the third day for P70:2−PC and P70:5−PC samples and on the first and sixth days for samples P70:2+PC, P70:5+PC, P80:2+PC, and P80:5+PC at a constant voltage of 70 kV (p > 0.05). Increasing the voltage at a fixed time of 5 min causes a significant decrease in the pH value in all samples (p < 0.05). However, this reduction rate is not significant at a constant time of 2 min on the 9th and 12th days for samples P70:2−PC, P70:5−PC, P80:2−PC, and P80:5−PC, and on the first and sixth days for samples P70:2+PC, P70:5+PC, P80:2+PC, and P80:5+PC (p > 0.05).

The acidity of all samples increased significantly over the 18-day storage period (p < 0.05) (Supplementary Table 6). The C sample exhibited the highest rate of acidity increase, reaching from 0.13 to 0.24, whereas the P70:5+PC and P80:5+PC samples showed the lowest rate of increase, reaching from 0.13 to 0.19 (p < 0.05). PC-P sample consistently exhibits lower acidity levels than the C sample throughout the entire 18-day period (p < 0.05), indicating the effective ability of PC to reduce acidity levels. Furthermore, the C sample consistently exhibits the highest levels of acidity throughout the entire storage period, with only the P70:2−PC sample showing no statistically significant difference on the 15th day (p > 0.05). In contrast, the P80:5+PC sample consistently exhibited the lowest levels of acidity, with no statistically significant difference from the P70:5+PC, P70:5−PC, and P80:5−PC samples except on the third day (p > 0.05). Samples treated solely with DBD showed lower acidity levels than the C sample, with no statistically significant difference from the PC-P sample on the first, third, 12th, 15th, and 18th days (p > 0.05). Additionally, combined treatment with DBD and PC significantly slowed down the rate of acidity increase compared to the control sample over the 18-day period (p < 0.05).

PV and TBARS

The PV of all samples increases significantly during the 18-day storage period (Supplementary Table 7). On the 18th day, the C sample exhibits the highest PV (0.55), while the lowest PV is observed in the P80:5+PC sample (0.38) (p < 0.05). Except for the first day, the PV of the PC-P sample is significantly lower than that of the C sample and the P70:2−PC sample (p < 0.05). The rate of PV increase during the 18-day storage period is slower in samples treated with DBD than in the C sample, and it is even slower in samples treated with combined DBD and PC than in samples treated with DBD alone and the C sample.

On the first day, no statistically significant difference is observed between the C sample and the samples treated only with DBD and the PC-P sample (p > 0.05). However, the PV of the P70:2+PC, P70:5+PC, P80:5+PC, and P80:2+PC samples was significantly lower than that of the C sample (p < 0.05). On the 18th day, there is no statistically significant difference in PV between the PC-P and P80:2−PC samples (p > 0.05). Except for the first day, increasing the treatment time (from 2 to 5 min) at a constant voltage of 80 kV causes a significant decrease in PV (p < 0.05). On the third day, this reduction rate in the constant voltage of 50 kV for the P70:2−PC and P70:5−PC samples do not create a significant statistical difference (p > 0.05). Additionally, increasing the voltage causes a significant decrease in PV on all days except the first day (p < 0.05). On the first day, there is no statistically significant difference between the C sample and the treated samples (p > 0.05).

TBARS levels increase in all samples during the 18-days of storage period, and this increase is significant (Table 2). However, the rate of increase is much faster in the C sample (0.21–0.97) compared to the treated samples, and this difference is statistically significant (p < 0.05). The initial values of TBARS in fillets are consistent with those reported for high-quality fish products (1–2 mg MDA/Kg). Notably, on the first day, DBD treatment does not result in any significant difference (p > 0.05). on the subsequent days, DBD treatment, with and without PC, resulted in a significant decrease in TBARS values compared to the C sample (p < 0.05). This decreases is more pronounced in the samples treated with a combination of DBD and PC. By the end of the 18-days of storage period, the TBARS values decreased from 0.97 mg MDA/Kg (C sample) to 0.69 mg MDA/Kg (P80:5−PC) and then to 0.60 mg MDA/Kg (P80:5+PC) (p < 0.05). Moreover, DBD treatment itself significantly reduces TBARS values during storage. Nonetheless, the combined treatment of DBD and PC is more effective in reducing TBARS values compared to each treatment independently.

Table 2 TBARS changes with DBD treatment at 70 and 80 kV voltages and durations of 2 and 5 min, with and without PC treatment, on Oncorhynchus mykiss rainbow fillets during an 18-day storage period at 4°C.

Importantly, on the 18th day, the lowest TBARS value with significant differences compared to the C sample is observed in the P80:5+PC sample (p < 0.05). It is noteworthy that a TBARS value higher than 2.0 mg MDA/Kg in fish indicates spoilage. However, after 18 days of storage, this value is less than 1 in all samples, and very close to 1 in the C sample. Furthermore, increasing the treatment time at a constant voltage of 80 kV results in a significant decrease in TBARS values on all days except the first day (p < 0.05). However, the reduction rate at the constant voltage of 70 kV on the third day for P70:2+PC and P70:5+PC samples do not create a significant statistical difference (p > 0.05). Additionally, increasing the voltage always results in a significant decrease in TBARS values on all days except the first day (p < 0.05). Notably, on the first day, there is no statistically significant difference between the C sample and the treated samples (p > 0.05).

TMA and TVN

The results show a significant increase in TVN across all samples during the 18-day storage period (p < 0.05) (Table 3). The C sample demonstrates a faster increase compared to treated samples, nearing the permissible limit by the ninth day, while the PC-P and P80:5−PC samples exceed the limit on the twelfth and fifteenth days, respectively. However, the P80:5+PC sample remains below the limit by the 18th day. Initially, all samples exhibit TVN values below 10 mgN/100 g, indicating good quality with no significant difference observed (p > 0.05). By the third day, all samples surpass the 10 mgN/100 g limit, except for the P80:5+PC sample, which maintains freshness until the sixth day. DBD treatment, with or without PC, reduces TVN values compared to the C sample on the 9th, 12th, 15th, and 18th days (p < 0.05). The PC-P sample consistently shows lower TVN values than the C sample, while combined treatments exhibit even lower TVN values, suggesting effective mitigation of TVN in treated samples, particularly with DBD and PC combinations.

Table 3 TVN changes with DBD treatment at 70 and 80 kV voltages and durations of 2 and 5 min, with and without PC treatment, on Oncorhynchus mykiss rainbow fillets during an 18-day storage period at 4 °C.

The results show a significant increase in TMA values across all samples during the 18-day storage period, with the C sample exhibiting a notably faster increase than treated samples (Supplementary Table 8). Initially, no significant difference in TMA values is observed between treated samples and C (p > 0.05). By the 9th day, the C sample reaches 0.25 mg N/100g TMA, while the P80:5+PC sample remains below this value. By the 18th day, the C sample records the highest TMA value (53.76 mg N/100 g), whereas the lowest is observed in the P80:5+PC sample (33.69 mg N/100 g). Increasing treatment time at 80 kV voltage leads to a significant decrease in TMA values, more pronounced with longer treatment durations. However, this reduction is not significant for samples treated at 70 kV voltage on the ninth and twelfth days. Additionally, increasing voltage consistently decreases TMA values. Furthermore, PC, with or without DBD, significantly reduces TMA values compared to the C sample on all days except the first, indicating potential for TMA control in fish product storage with DBD and PC treatments.

FFA in Oncorhynchus mykiss rainbow fillets treated with DBD and PC during 18 days at 4 °C

The FFA composition of the C, PC-P, P80:5−PC, and P80:5+PC samples is analyzed on the first day. 24 types of FFA have been identified, divided into three groups: saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) (Table 4).

Table 4 FFA values for four samples (C, PC, P80:5 − PC and P80:5+PC) in Oncorhynchus mykiss rainbow fillets at 4 °C.

SFA

The values of all SFAs decrease significantly compared to the C sample when PC is added to the samples, except for C16:0 and C17:0. Conversely, these values increase significantly in all samples compared to the C sample with DBD treatment, except for C15:0 and C18:0. However, the combined effect of PC and DBD treatment reduces the SFA values of all samples significantly compared to samples treated only with DBD. It is noteworthy that across all four samples, C16:0 and C18:0 exhibit the highest values, while C15:0 has the lowest value. In terms of total SFAs, P80:5−PC, PC-P, P80:5+PC, and C rank from highest to lowest, respectively.

MUFA

The values of all MUFAs decrease significantly compared to the C sample when PC is added to the samples. Conversely, these values increase significantly in all samples compared to the C sample with DBD treatment, except for the C18:1 n9c sample. However, the combined effect of PC and DBD treatment reduces the MUFA values of all samples significantly compared to samples treated only with DBD, except for the C18:1 n9c sample. Notably, there is no statistically significant difference between the samples of C and P80:5+PC for C17:1. Across all four samples, C18:1 n9c has the highest value, while C17:1 has the lowest value. In terms of total MUFAs, P80:5+PC, C, PC-P, and P80:5−PC rank from highest to lowest, respectively.

PUFA

The addition of PC, DBD treatment, and their combined effect had varied impacts on the omega-3 and omega-6 groups. Generally, DBD treatment led to a decrease in total omega-6 values and an increase in total omega-3 values compared to the C sample. However, the combined effect of PC and DBD treatment increased the total amounts of both omega-6 and omega-3 compared to the C sample. Across all four samples, C18:3 n3 and C18:2 n6c exhibited the highest amounts, while C17:1 had the lowest amount. It’s crucial to note that if the value of n3/n6 is less than 0.2, it can be harmful to humans. This value increased to 1.17 in the sample treated with DBD compared to the C sample. Conversely, the combined treatment of DBD and PC increased this value to 0.22, suggesting a more balanced omega-3 to omega-6 ratio.

Changes of color in Oncorhynchus mykiss rainbow fillets treated with DBD and PC during 18 days at 4 °C

a*

There is a notable increase in the a* values, representing the redness/greenness, of all samples treated with PC, with and without DBD treatment, over the 18-day storage period (p < 0.05) (Supplementary Table 9). The C sample exhibits the greatest increase in a* values during storage, rising from 4.80 to 12.55 (p < 0.05). Despite all treated samples having significantly higher a* values than the C sample on the first day, by the end of the 18th day, the a* values of all treated samples are significantly lower than that of the C sample (p < 0.05). Additionally, increasing the voltage and time in DBD-treated samples results in an increase in the a* values (p < 0.05). On the 18th day, the sample with the lowest a* value is P80:5−PC, which is not significantly different from samples P70:2−PC, P70:5−PC, P80:2−PC, P80:2+PC, and P80:5+PC. Conversely, the C sample has the highest a* value on the 18th day.

b*

The values of b*, representing the yellow/blue color component, were measured regularly over the 18-day storage period (Supplementary Table 10). Results indicate a significant increase in b* value across all samples during storage, with the lowest value observed for the P70:2−PC sample (16/11) and the highest for the P80:5−PC sample (22/36) on day 18. The C sample exhibited the highest rate of increase in b* value (5.64 to 18.87), followed by the PC-P sample (22.90 to 34.18). Additionally, b* values were significantly higher for DBD-treated samples, both with and without PC, compared to the C sample. Moreover, the b* value increased with increasing voltage and time for all samples throughout the storage period. These findings collectively suggest that DBD treatment and PC contribute to enhancing the color stability of yellowtail fillets during storage.

L*

The results indicate a significant decrease in L* values for all samples until the 18th day of storage (p < 0.05) (Supplementary Table 11). Initially, DBD treatment alone led to a notable reduction in L* values compared to the C sample until the sixth day. However, by the end of the 18th day, samples treated with P80:2−PC, P70:2−PC, and P70:5−PC showed higher L* values than the C sample. Throughout the 18-day storage period, the rate of reduction in L* values were faster for C samples, P80:5−PC, and P80:5+PC compared to other samples. Nevertheless, by the 18th day, there was no statistically significant difference observed among these three samples (p > 0.05). Notably, the reduction rate of L* values in P70:2+PC and P70:5+PC samples was slower than in other samples over the 18 days, and by the 18th day, no significant statistical difference was observed between these two samples (p > 0.05). On the first day, pretreatment with PC, with or without DBD treatment, resulted in a decrease in L* value with a significant difference compared to the C sample. However, by the end of the 18th day, the L* value of samples pretreated with PC, with or without DBD treatment, was significantly higher than that of the C sample (72.57). Furthermore, the study investigated the effects of increasing voltage and treatment time on L* values. On the third, ninth, and twelfth days, no significant statistical difference was observed between P70:2+PC and P80:2+PC samples when increasing the voltage from 70 to 80 kV at a constant time of 2 min. However, in all cases, increasing the voltage from 70 to 80 kV at a constant time of 5 min resulted in a significant decrease in L* values. On the 6th, 9th, and 18th days, increasing the treatment time from 2 to 5 min at a constant voltage of 70 kV showed no significant statistical difference in L* values for P70:2+PC and P70:5+PC samples. However, at a constant voltage of 80 kV, increasing the time from 2 to 5 min led to a significant decrease in L* values for these two samples.

ΔE

Throughout the 18-day storage period, all samples display an increase in ΔE values, signifying browning (Supplementary Table 12). The C sample registers the highest ΔE value on the 18th day compared to the first day, while the P80:5+PC sample exhibits the lowest increase. Additionally, samples treated solely with DBD show higher ΔE values than those treated with both DBD and PC. Notably, the incorporation of PC reduces the ΔE value in the P80:5+PC sample from 12.29 to 8.14.

OES

The intensity of species and spectral lines of nitrogen molecules, oxygen atoms, and OH molecule in DBD plasma at 80 kV (Supplementary Fig. 3), with a focus on the wavelength range of 300–400 nm showing prominent peaks corresponding to excited nitrogen species. Additionally, a current voltage diagram is provided for voltages of 70 and 80 kV (Supplementary Figs. 4 and 5).

Changes of antioxidant activity in Oncorhynchus mykiss rainbow fillets treated with DBD and PC during 18 days at 4 °C

DPPH

The DPPH scavenging capacity of samples treated with DBD alone does not show a significant difference from the C sample until the third day (p > 0.05) (Table 5). However, starting from the sixth day, there is a significant decrease (p < 0.05) in the DPPH scavenging capacity of certain samples compared to the C, including P70:5−PC, P80:2−PC, and P80:5−PC. On the first day, the PC-P sample exhibits a significant decrease (p < 0.05) in DPPH scavenging capacity compared to the C. Throughout the 18-day period, samples treated with both DBD and PC show significantly lower DPPH scavenging capacity compared to the C and samples treated with DBD alone. However, there is no significant difference (p > 0.05) in the DPPH scavenging capacity between samples treated with PC with and without DBD treatment over the 18-day period. On the 18th day, the C sample exhibits the highest DPPH value (36.84), while samples treated with PC, with and without DBD treatment, show the lowest DPPH values (5.48). Results suggest that the combination of DBD and PC treatments may have a more pronounced effect on antioxidant activity than either treatment alone.

Table 5 DPPH changes with DBD treatment at 70 and 80 kV voltages and durations of 2 and 5 min, with and without PC treatment, on Oncorhynchus mykiss rainbow fillets during an 18-day storage period at 4 °C.

FRAP

FRAP of the PC-P sample significantly surpasses both the C sample and samples treated with DBD alone (p < 0.05) throughout the 18-day storage period (Supplementary Table 13). On the 18th day, the P80:5+PC sample exhibits the highest FRAP value (20.57), with no significant difference from the P80:2+PC sample (p > 0.05), while the C sample records the lowest FRAP value (9.26). Furthermore, the C sample experiences the highest increase in FRAP during the 18-day period (16.87–9.26), whereas the PC-P80:5 sample shows the lowest increase (32.74–20.57) (p < 0.05). These findings suggest a notable enhancement in the antioxidant activity of treated samples with the addition of P80:5−PC and PC-P, particularly evidenced by the substantial improvement in the P80:5+PC sample. Moreover, the study underscores the potential utility of these compounds as natural preservatives for extending the shelf-life of food products.

ABTS

The results indicate that ABTS values are not significantly different in samples treated solely with DBD compared to the C sample on the first day (p > 0.05) (Supplementary Table 14). However, from the third day onwards, ABTS values in the P80:2−PC and P80:5−PC samples decrease significantly compared to the C sample (p < 0.05). Furthermore, except for the first day, ABTS values in samples treated with DBD alone increase from 70 to 80 kV, resulting in a significant decrease compared to the C sample (p < 0.05). Conversely, samples treated with PC, with and without DBD, exhibit an improvement in ABTS radical scavenging capacity. Specifically, on the first day, ABTS values decreased significantly from 4.47 in the C sample to 1.54 in the PC sample (p < 0.05). Moreover, over the 18-day period, there is no statistically significant difference in ABTS values between samples treated with PC with and without DBD treatment (p > 0.05). On the 18th day, the C sample has the highest ABTS value of 12.58, while the P80:5+PC sample has the lowest value of 2.52. However, there is no significant statistical difference between the P80:5+PC sample and the P80:2+PC and P70:5+PC samples. Findings suggest that combining PC with DBD treatment could effectively enhance the ABTS radical scavenging capacity in food samples.

Changes of sensory properties (color, odor, texture, and overall acceptance) in Oncorhynchus mykiss rainbow fillets treated with DBD and PC during 18 days at 4 °C

Results indicate no significant difference in color, odor, texture, and overall acceptability between the C and treated samples (p > 0.05) (Fig. 3). However, by the 18th day, the color of P80:5−PC and P80:2+PC samples exhibited superior conditions compared to the C sample. Additionally, odor conditions of P80:5−PC, P70:5+PC, and P80:2+PC samples surpassed those of the C sample. Tissue quality of P80:5−PC, PC-P, P70:5+PC, and P80:2+PC samples also showed improvement over the C sample on the 18th day. Moreover, both P80:5−PC and P80:2+PC samples demonstrated better overall acceptance than the C sample.

Figure 3
figure 3

The sensory analysis of Oncorhynchus mykiss rainbow fillets over 18 days: (a) Odor, (b) Color, (c) Texture, and (d) Overall acceptance.