Supercooling with stepwise algorithm preserves the freshness of salmon and olive flounder during extended storage

doi:10.21203/rs.3.rs-1500361/v1

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Supercooling with stepwise algorithm preserves the freshness of salmon and olive flounder during extended storage

https://doi.org/10.21203/rs.3.rs-1500361/v1

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Supercooling storage reduces the temperature of a product by lowering its freezing point without phase transition and may extend its shelf life. However, it is difficult to maintain the supercooled state of food as it is thermodynamically metastable. A slow cooling rate and minimal fluctuation are essential for achieving stable supercooling storage. Therefore, a stepwise algorithm was adopted for supercooling storage in this study. Salmon and olive flounder were stored at 3°C (refrigeration), -18°C (freezing), and − 2°C (supercooling) for 12 days. Samples were maintained in a supercooled state and unfrozen during the storage period. Samples stored after the supercooling treatment were superior with respect to drip loss and water holding capacity (WHC) compared to frozen samples, regardless of the type of sample. WHC and total volatile basic nitrogen values of olive flounder was higher than those in salmon owing to the higher water and protein content in olive flounder than in salmon. Moreover, the supercooled samples inhibited the increase in trimethylamine and volatile basic nitrogen levels. Microbial growth was slow. Thus, a stepwise algorithm for stable supercooled storage was achieved, which effectively preserved fish quality better than freezing and refrigeration storage.

salmon

olive flounder

supercooling

stepwise algorithm

freshness

Atlantic salmon (Salmo salar) and olive flounder (Paralichthys olivaceus) are widely consumed fish in East Asia; however, they are highly perishable foods (Shumilina et al., 2018; Hamidoghli et al., 2020). Owing to their perishable nature, storing fish at low temperatures is a widely used method to maintain quality and safety, which reduces enzymatic proteolysis, microbial deterioration, and lipid oxidation during storage periods (Cropotova et al., 2019). However, the shelf life of fish is only 7–10 days in refrigerated conditions, and that of purchased fish is further decreased in household refrigeration conditions (Park et al., 2021a). Therefore, the period of maintaining the freshness of fish stored under home refrigeration will be shorter. Freezing is the most efficient method of preserving food and maintaining freshness for extended periods (Lee et al., 2021). Nevertheless, this preservation technology has the disadvantage of the food quality being degraded by ice crystals generated during the freezing process. (Choi et al., 2018). Therefore, researchers are interested in other low-temperature storage methods.

Superchilling and supercooling are subzero preservation techniques that are used for foods. The superchilling process lowers the freezing point of food by 1–2°C, which results in the freezing of approximately 30–50% of water in food (Park et al., 2021b). However, this storage method has disadvantages, such as quality loss owing to ice crystals (Duun & Rustad, 2007). Supercooling is a procedure for lowering the temperature of food below its freezing point without phase transition (Stonehouse & Evans, 2015). Thus, this preservation technique has the advantage of extending shelf life without the destruction of tissue and degradation generated by ice crystals during the freezing process (Yun et al., 2021; Lee, 2020). Despite the expected advantages of supercooling, this technology is thermodynamically unstable, and phase transitions can occur at any moment (Yun et al., 2021). Therefore, various technologies such as pressure shift, high pressure, electromagnetic waves, and electric fields have been applied to improve supercooling storage. (Dalvi-Isfahan et al., 2017; Kim, 2021). However, these technologies for stable supercooling storage have certain limitations. Using high pressure to maintain a stable supercooled state exposes the protein to pressure- and cold-induced denaturation (Hong and Choi, 2016; Choi et al., 2016). Additionally, the application of electric and magnetic fields under stable supercooled conditions is controversial (Dalvi-Isfahan et al., 2017).

Strict temperature control during supercooling storage of diverse types of food may preserve stable supercooling conditions (Kim, 2021). Particularly, stepwise cooling adopted for supercooling storage has been effective in preventing structural damage in fish (Fukuma et al., 2012). According to Fukuma et al. (2012), various kinds of fish stored in incubators set to stepwise cooling efficiently retain the supercooled state during storage. The temperature was decreased by 0.5°C every 24 h, and the samples maintained a supercooled state until reaching − 5°C, depending on the conditions. Park et al. (2021b) also reported that supercooled state of mackerel was successfully retained during storage. Furthermore, few investigations on supercooling storage of foods using a slow cooling rate have been reported (James et al., 2009; Kim and Hong, 2016). However, these studies have focused on maintaining the supercooled state as strictly as possible, and few theoretical experiments have been conducted for retaining a stable supercooled state.

Thus, the aim of this experiment was to analyze the physicochemical properties of fish stored under refrigeration (3°C), freezing (-18°C), and supercooling (-2°C) treatments and compare their freshness. A stepwise cooling algorithm was adopted to maintain stable supercooling.

Sample preparations

Fresh salmon (Salmo salar) and olive flounder (Paralichthys olivaceus) were purchased from a local fishery market. The salmon was cut into 7 × 10 × 5 cm pieces (width × length × height, 130 g). Afterwards, they were placed in a polypropylene tray and wrapped using a packaging machine. The olive flounder was deboned and reformed into 8 cm × 12 cm × 5 cm pieces (width × length × height, 150 g). The olive flounder samples were also packaged and separated in the same manner. A total of 39 packages were prepared and divided into 12 groups (3 types of preservation × 12 packages × 3 replications). The remaining three packages were used as controls without storage treatments. Three packaged samples were stored in one device, and this storage process was conducted in four devices simultaneously (two devices used for salmon and the others used for olive flounder).

Storage conditions

Packed salmon and olive flounder were assigned to three types of storage treatments and their physicochemical properties were analyzed at 3, 6, 9, and 12 days. Refrigeration treatment was conducted in a refrigerator (R-F875HBSW, LG Electronics, Seoul, Korea) set at 3 °C. Supercooling treatment was performed in a refrigerator (K417SS13, LG Electronics) regulated by a 54 h cycle stepwise cooling algorithm. The initial temperature was set to -1 °C and decreased twice by 0.5 °C every 18 h, which was repeated every 54 h. A T-type thermocouple was attached to the surface of the packed samples and connected to a data logger (Data Acquisition-MX 100, Yokogawa, Tokyo, Japan) to measure temperature changes during the storage period. Freezing treatment was implemented in a refrigerator (R-F875HBSW) set to -18 °C. To analyze the physicochemical properties of the fish, frozen samples were thawed in a refrigerator (R-F875HBSW) set at 3 °C until the core temperature of the sample reached 0 °C.

Proximate composition analysis

The proximate compositions of fresh salmon and olive flounder were determined using the AOAC method (1995). The water content of the fishes was determined by drying the sample in an air oven at 105 °C and the crude protein content was measured using the nitrogen content acquired by the Kjeldahl method. The crude fat content of the samples was determined using the solvent extraction method in a Soxhlet system.

Quality analysis

Drip loss

Fresh fish were weighed before storage. After preservation, the moisture of the treated sample was wiped down using tissues and weighed again. Drip loss is expressed as the percentage ratio of the removed weight to the initial weight of the sample.

$$Drip loss \left(\%\right)= \frac{{W}_{1}- {W}_{2}}{{W}_{1}} \times 100$$

where W₁ is the initial weight of sample (g) and W₂ is weight of the sample (g), respectively.

Water holding capacity (WHC)

WHC was performed using a modified version of the method from Hultmann and Rustad (2002). Approximately, one gram of treated sample was wrapped in gauze and placed in a conical tube. The conical tubes were then centrifuged at 1500 × g for 10 min at 4 °C. The WHC of the samples was calculated as the ratio of water remaining after centrifugation using the following formula:

$$Water holding capacity \left(\%\right)=\text{ }\frac{{W}_{2}}{{W}_{1}} \times 100$$

where W₁is the sample weight before centrifugation (g), and W₂ is the sample weight after centrifugation (g).

ThepH of the treated samples was measured using a pH meter (Orion 3 Star, Thermo Fisher Scientific, MA, USA). Approximately 2 g of treated sample was homogenized with 18 mL of distilled water using a mixer (SMT pH91 SMT, Tokyo, Japan) for 30 s.

Color

The color parameters were determined using a CR-400 Chroma Meter (Konica Minolta, Tokyo, Japan) calibrated with a white standard plate (CIE (Commission Internationale de l’Eclairag) L^*=96.79, CIE a^*=0.30, and CIE b^*=1.67). The flesh of salmon was ground using a mixer (SMT pH91) and placed in a petri dish (35 × 10 mm). The color values of samples of salmon and olive flounder were measured on the surface (each sample measured 10 times). The results were expressed as lightness (CIE L^*), redness (CIE a^*), and yellowness (CIE b^*) values. The total color difference (ΔE) was calculated as follows:

$$\varDelta E= \sqrt{{\left(\varDelta \text{C}\text{I}\text{E} {\text{L}}^{*}\right)}^{2}+ {\left(\varDelta \text{C}\text{I}\text{E} {\text{a}}^{\text{*}}\right)}^{2}+ {\left(\varDelta \text{C}\text{I}\text{E} {\text{b}}^{*}\right)}^{2}}$$

The appearance of samples depending on storage treatment and period was observed using a camera (EOS 100D, Canon, Tokyo, Japan).

Freshness analysis

Trimethylamine (TMA)

The TMA value was measured in accordance with a modified method described by Park et al. (2021a). Two grams of treated samples were mixed with 8 mL of 4% TCA solution. Subsequently, the mixture was homogenized using a blender (SMT pH91) for 60 s. The homogenized sample was passed through filter paper (Whatman No. 41, GE Healthcare Life Science, Buckinghamshire, UK) and incubated for 30 min at ambient temperature. One milliliter of the filtrate was pipetted into the outer side of a Conway dish. Then, 0.1 mL of indicator solution (0.1% bromocresol green in ethanol: 0.2% methyl red in ethanol) and 1 mL of 0.01% boric acid solution were poured into the inner ring of the Conway dish. Next, 1 mL of 10% formaldehyde and saturated K₂CO₃ solution were dropped into the outer ring. The unit of the Conway dish was then closed and mixed gently. After mixing, the mixture was incubated at 37 °C for 1 h. The value of TMA was defined as the addition of 0.02 N HCl titrated into the inner side of the Conway dish. The TMA content was calculated using the following formula:

$$TMA (mg/100 g)= \frac{14.00 \times \left(a - b\right)\times f\times 100 \times (s \times \frac{m}{100}+e)}{s}$$

where a is the titer for the sample (mL), b is the titer for the blank (mL), f is a factor of HCl, s is weight of the sample (g), m is percentage moisture of the sample, and e is volume of 4% TCA solution used in extraction.

Thiobarbituric acid reactive substances (TBARS)

TBARS was determined using the TBARS assay following the method described by Kim (2021). Five grams of the treated samples was diluted in 45 mL of distilled water and homogenized using a blender (SMT pH91) for 60 s. The mixture was then filtered through Whatman No. 1 filter paper (GE Healthcare Life Sciences). The filtrate (0.5 mL) was transferred to a 15 mL test tube and mixed with 4.5 mL of TBA solution (0.25 N HCl, 15% TCA, and 0.375% TBA reagent). The samples were placed in a water bath (BF-30SB, BioFree, Seoul, Korea) at 95 °C for 15 min and centrifuged at 3,000 × g at 4 °C for 10 min. After that, 200 μL of supernatant was placed into a microplate and measured using a spectrophotometer (Multiskan^TM GO UV/VIS, Thermo Fisher, Massachusetts, USA) at 535 nm absorbance. The TBARS values (expressed as milligram malondialdehyde/kg (mg MDA/kg)) were calculated using a standard curve and the following formula:.

$$TBARS (mg MDA/kg)= \frac{ \left(a - b\right)+0.0159}{10.239}\times 100$$

where a is the optical density of the sample and b is the optical density of the blank.

Total volatile basic nitrogen (TVBN)

The TVBN content of the treated fish was determined using Conway’s micro-diffusion method (Park et al., 2021a). Treated samples were cut into approximately 5 g pieces, and distilled water was added to a stomacher bag. The samples were then homogenized using a slap-type homogenizer (WS-400, Shanghai Zhisun Equipment Co. Ltd., Shanghai, China) for 180 s. The homogenized sample was then passed through a filter paper (Whatman No. 2, GE Healthcare Life Science). After filtration, 1 mL of the filtrate was poured on the outer side. Then, 1 mL of 0.01 N H₃BO₃ and 100 μL of Conway solution (0.066% methyl red in ethanol: 0.066% bromocresol green in ethanol) was dropped into the inner side of a Conway dish. Next, 1 mL of 50% K₂CO₃ was poured into the outer side. The lid of the Conway dish was closed and the dish was incubated at 37 °C for 2 h in a thermo-hygrostat (IL3-25A, JEIO Tech, Daejeon, Korea). The TVBN content was determined following the addition of 0.02 N H₂SO₄ to the inner side of the Conway dish. A blank test was performed following the same procedure, without the addition of homogenized samples. The TVBN value was calculated as follows:

$$TVBN (mg/100 g)= \frac{14.007 \times (a - b) \times f\times 100 \times c}{S}$$

where a is the titer of the sample (mL), b is the titer of the blank (mL), f is the H₂SO₄ concentration, S is the weight of the sample used, and c is the dilution.

Microbial analysis

Two grams of treated samples were separated from the sample surface and homogenized with 18 mL of sterilized 0.85% NaCl solution using a slap-type homogenizer (WS-400) for 1 min. The homogenate was serially diluted, and the diluted solution was pipetted onto Petrifilm aerobic count plates (3M Petrifilm^TM, 3M, St. Paul, MN, USA) and incubated for 48 h in an incubator (SI-600R, Jeio Tech, Seoul, Korea) at 37 °C. After incubation, the colonies of the samples were counted and presented as log colony forming unis per 1 g of sample (log CFU/g).

Statistical analysis

All treatments were performed with at least three replicates. Data are expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was performed and mean comparisons were performed using Duncan’s multiple range test (p < 0.05).

Proximate composition

The moisture content of salmon and olive flounder was 65.3% and 75.8%, crude proteins were 20.2% and 22.2%, and crude lipids of fish were 15.7% and 3%, respectively (Table 1). These results were similar to those reported by Erdem et al. (2020) and Yoon et al. (2015). Overall, the crude protein and water contents of olive flounder were higher than those of salmon, but the crude lipid content was lower than that of salmon.

Time-temperature profiles

The time-temperature profiles of salmon and olive flounder during storage for 12 days are shown in Figure 1. Refrigeration treatment was implemented at 3 °C, and all salmon samples reached 3 °C after 1.5 h of storage. Stepwise algorithm cooling was applied to accomplish slow cooling for the supercooled storage of salmon and olive flounder. The limit lower temperature was set at -2.0 °C since the nucleation temperature of fishes were mostly from -1.0 °C to -2.5 °C (Sivertsvik et al., 2002). The fish were slowly cooled and sustained at -2 °C for 18 h. Thereafter, temperatures were restored to prevent nucleation by repeating the stepwise algorithm. While repeating the algorithm, the temperature of the sample followed the target temperature in the range of ±0.1 °C. Temperatures of samples were maintained -2.1 °C to -1.9 °C without phase transition at 54–72 h, which was in the range of the initial freezing temperature of fish (Kim et al., 2021). During this slow-cooling storage, all the samples stably maintained supercooled conditions. The freezing treatment was conducted at -18 °C and reached -18 °C after 6.5 h of storage. The samples were preserved at -18 °C to -19 °C for 12 days. The physicochemical properties of the samples were then evaluated to compare the frozen and refrigerated samples at 3, 6, 9, and 12 days.

Quality analysis

Drip loss

Drip loss is the amount of liquid lost from food and is an important indicator of quality (Hong et al., 2013). The greater the amount of drip, the more water-soluble nutrients, texture, and flavor compounds that are lost (Xu et al., 2020). The drip loss of treated salmon and olive flounder is shown in Figure 2 (A). Drip losses of all samples were 1.74–3.77% after 3 days of storage and increased with storage treatments and periods in salmon (p< 0.05). Fish stored after the refrigeration treatment showed sharply increased values of drip loss, with increases of 10.56% and 9.21% for salmon and olive flounder, respectively, after 12 days of storage. According to Kaale et al. (2014), refrigerated samples showed a higher drip loss value than frozen samples, which is in agreement with this experiment. However, samples stored in the freezing and supercooling treatments maintained their quality during the storage period compared with those stored in the refrigerated treatment. In particular, salmon and olive flounder stored in the supercooled treatment had a lower drip loss value than those stored in the freezing treatments after 12 days of storage (p < 0.05). The drip loss of salmon was higher than that of olive flounder, which is inconsistent with the results of a study where it was observed that the lower the moisture content and the higher the fat content, the lower the drip generation (Kim et al., 2013). The drip loss value after the freezing treatment was higher than that for the supercooling treatment. We hypothesize that cell tissues may have been damaged by ice crystals generated during freezing storage. Considering these results, the supercooling treatment of salmon was advantageous for drip loss in fish.

WHC

The changes in the WHC of fish during the cooling treatments and storage periods are presented in Figure 2 (B). According to Kaale and Eikevik (2015), WHC is one of the most important factors affecting the quality of food products. Generally, WHC is influenced by several factors, such as destruction of muscle fiber structure, myosin denaturation, and increased extracellular space (Kaale et al., 2014). The WHC values of fresh fish were 88.78% and 89.75% in salmon and olive flounder, respectively, which showed different patterns during storage with different cooling treatments and periods. Samples stored in the refrigeration conditions showed a constant decrease in the WHC value for 9 days and a rapid decrease after 12 days. During the last two storage days, the WHC values of salmon and olive flounder stored under refrigerated conditions were 82.93% and 82.33%. In comparison, the WHC content of fish stored in other treatments also showed a decrease with an increase in the storage period. Nevertheless, there were no significant differences between supercooling and freezing treatments (p < 0.05). Jung et al. (2016) reported that the WHC value of fish tended to be inversely proportional to drip loss. This result is consistent with that of our study. This may be due to degradation of the protein structure induced by proteolytic activity of enzymes (Wu and Sun 2013). Overall, the WHC of olive flounder with high water content was lower than that of salmon, which is due to the high proportion of loosely bound water in fish with high water content (Duun and Rustad, 2007). Considering this result, supercooling was considered beneficial for maintaining muscle fiber structures as a freezing treatment for 12 days.

The changes in pH during the storage period of salmon and olive flounder are shown in Figure 2 (C). According to Mazrouh (2015), the pH value of fish flesh is a good indicator of quality. The pH values of the fresh fishes were 6.11 and 6.21, respectively. However, there were no significant differences in the pH values during the storage treatment at 3 days of storage (p < 0.05). After this storage period, the pH values of the refrigerated samples showed an increasing trend. After 12 days of storage, the pH values of samples stored in the refrigerated conditions were 6.49 and 6.61 in salmon and olive flounder, respectively, which were significantly lower than those of samples stored in other storage treatments (p < 0.05). However, the pH value of the supercooled samples increased slightly and was higher than that of frozen salmon. The pH of olive flounder, particularly after the refrigeration treatment, was higher than that of salmon. Hernández et al. (2009) reported that the pH increase during storage may be related to the degradation of amino compounds caused by microbial reactions. Therefore, it appears that the high protein content of olive flounder indicates a high pH value. The increase in the pH value of the sample stored at -2 °C was lower than that of the refrigerated fish. This result indicated that biochemical and microbial reactions were faster at 3 °C. In contrast, the frozen fish showed a constant pH throughout the storage period after 12 days (p < 0.05). This means that the metabolic activity of bacteria might be inhibited at a lower temperature, resulting in a constant pH in fish. Therefore, a supercooling treatment was helpful in maintaining the quality of the fish without decomposition by microbial activity compared to the refrigeration treatment.

Color and appearance

Color is an important indicator of food because it generally affects consumer acceptance of a product (Tee & Siow 2015). The color of fish with different storage treatments and periods are presented in Tables 2 and 3. The CIE L^* values for fresh fishes were 39.77 and 58.64% in salmon and olive flounder, respectively. After 12 days of storage, the CIE L^* value of salmon stored under refrigerated treatment decreased by 36.76. However, salmon stored in other treatments showed a higher value than those stored in the refrigerated treatment. Refrigerated salmon showed a decrease in the CIE a^* values to 9.85 after 12 days. In contrast, samples stored under supercooling and freezing treatments did not change color during the storage period. However, the CIE a^* content of olive flounder did not show any trend and was lower than that of salmon. This difference may be due to the carotenoid (mainly astaxanthin and canthaxanthin) pigments in salmon (Ouahioune et al., 2022). the CIE b^* of fresh salmon was 11.95 and decreased to 9.62 after 12 days in the refrigeration treatment. While fish were stored at relatively high temperatures, water dehydration occurred on the surface of the fish, resulting in a darker flesh color (Drummond & Sun 2010). Therefore, the CIE b^* of salmon stored under the refrigerated treatment was significantly different from that of the other treatments (p > 0.05). According to Cropotova et al. (2020), a gradual increase in the CIE b* value is due to the accumulation of lipids and water-soluble aldehydes in fish muscle. Furthermore, Tolstorebrob et al. (2014) reported that refrigeration of fish with high TBARS values had a greater effect on the muscle color of fish compared to that of other fish. The ΔE values of the fish stored in the refrigeration treatment were higher than those in the other treatments. In particular, the color change of the refrigerated samples was remarkable compared with that of the other treatments (Figure 3). However, the ΔE of fish stored under refrigeration showed an increasing trend, but that of the other treatments did not present a pattern during the storage period. Accordingly, it is assumed that the reaction of salmon stored in refrigeration treatment is faster than that in freezing and supercooling treatments. Consequently, supercooling storage is beneficial for preventing color change compared to refrigeration storage.

Freshness analysis

TMA

The TMA of fish subjected to various storage treatments and periods is shown in Figure 4 (A). Generally, the TMA value is correlated with sensory evaluation and the main odor property of deteriorated seafood (Jääskeläinen et al., 2019). The TMA value of fresh salmon was 0.21 mg/100 g and the TMA values of fish stored for three days were not significantly different during storage treatments (p > 0.05). However, samples stored at refrigeration temperatures tended to increase sharply after six days. The values of refrigerated salmon and olive flounder were 3.76 mg/100 g and 2.75 mg/100 g respectively, whereas those of frozen and supercooled fishes were significantly lower than those of fresh fishes (p < 0.05). The TMA values of supercooled samples showed sharp increases and exceeded over 3 mg/100 g. However, the TMA values of frozen samples were 1.02 mg/100 g and 1.09 mg/100 g for salmon and olive flounder, respectively, at 12 days of storage but there was no significant difference compared to that of freezing treatment for 3 days (p > 0.05). Trimethylamine oxide (TMAO) is a natural and nontoxic substance found in aquatic species, such as fish and shellfish (Tee and Siow, 2015). The TMAO contained in defunct fish is converted to TMA by bacteria or enzymes, and a fishy smell is generated, which determines the degree of spoilage of fish (Park et al., 2016). During fish spoilage, there is not only an increase in TMA level, but also in total volatile basic nitrogen (TVBN) (Shin, 2008). The TMAO value of the samples stored in the refrigerated treatment increased rapidly from 3 to 6 days, which is consistent with Shin (2008). A large increase in TMA probably occurs as the activity of enzymes or bacteria increases (Park et al., 2016). Consequently, supercooling storage is more effective in slowing the spoilage of fish than refrigeration storage.

TBARS

The TBARS values of fish subjected to various storage treatments and periods are shown in Figure 4 (B). The TBARS values of salmon were also influenced by the storage temperature. Generally, salmon and olive flounder contain unsaturated fatty acids, such as docosahexaenoic acid (DHA), which are vulnerable to oxidative substances and form hydroperoxides that deteriorate secondary products such as MDA (Luan et al., 2018; Fidalgo et al., 2021; Jeong et al., 2021). The TBARS values of fresh salmon and olive flounder were 0.19 mg MDA/kg and 0.16 mg MDA/ kg, respectively, which were not significantly different from those of the other treatments for 6 days (p < 0.05). However, values of samples stored at 3 °C showed 0.31 mg MDA/kg and 0.28 mg MDA/kg for salmon and olive flounder, respectively, after 9 days. However, the values of frozen and supercooled samples were under 0.23 mg MDA/kg and presented no significant difference after 12 days of storage (p > 0.05). The TBARS levels of fish stored in the refrigerated treatment were higher than those of the other groups after 9 days of storage. Hydroperoxide, which is the primary product generated by oxidation of fat and produced by microbial metabolism and lipolytic enzymes, is more easily developed during refrigerated treatment (Joo et al., 2016). In this study, the TBARS values of refrigerated salmon and olive flounder increased more sharply than those of samples stored under the supercooled treatment. However, the TBARS content of salmon was slightly higher than that of olive flounder, which may be due to the difference in fat content. Therefore, preserving fish during supercooling is more effective than refrigerated treatment in preventing lipid oxidation.

TVBN

The TVBN content, which measures the amount of volatile compounds, is used as one of the parameters of fish freshness (Soares et al., 2015). The changes in TVBN during the storage of salmon and olive flounder are presented in Figure 4 (C). The TVBN values of fish were influenced by storage temperature, and the value of almost all treated samples increased compared to that of the controls. The TVBN values of fresh salmon and olive flounder were 8.68 mg/100 g and 9.48 mg/100 g, respectively, which was similar to the changes in TMA and TBARS during the storage period. In particular, the increase in TVBN was significant during refrigeration storage (p > 0.05). On the 9th day, the TVBN content of refrigerated salmon and olive flounder increased significantly, but those of the other treated samples were significantly lower than those of samples preserved under refrigeration (p > 0.05). After 12 days of storage, TVBN values of the samples stored in the supercooled treatment were higher than those stored in the freezing treatment, but there was no significant difference (p < 0.05). According to Lee et al. (2013), the value of TVBN increases as proteins are decomposed into low-molecular-weight substances such as peptides, amino acids, and peptones. These nitrogen-containing substances are produced by the proteolytic activities of microorganisms (Lee, 2020). Therefore, preserving fish at low temperatures is important for decreasing the amount of TVBN by prohibiting the reactions of microorganisms related to TVBN compound production (Park et al., 2021b). In this experiment, olive flounder had more protein content than salmon, which caused more protein degradation and led to a higher TVBN value. Obemeata et al. (2011) also observed that many bacteria were found in samples stored under refrigeration. Therefore, supercooling treatment was more effective than freezing treatment for preventing protein degradation in salmon.

Microbial analysis

The activity of microorganisms is a major factor related to the expiration date of fish and is affected by temperature and pH (Lu et al., 2019). The total aerobic count (TAC) for fish with various storage treatments and periods is shown in Figure 4 (D). The TAC of fresh salmon and olive flounder was 1.54 log CFU/g and 2.52 mg/100 g, respectively. The TAC values of the samples increased depending on the storage time for all the treatments. In particular, the TAC of refrigerated conserved samples was the highest for all storage periods (p > 0.05). The TAC value of salmon and olive flounder stored in the refrigeration treatments steeply increased to 4.02 log CFU/g and 6.08 log CFU/g, respectively, after 9 days of storage. After 12 days, the TAC values of refrigerated salmon and olive flounder were 4.15 log CFU/g and 6.6 log CFU/g, which was the highest value among all treatments (p < 0.05). In this experiment, the overall trend of TAC values related to the storage temperature was similar to that of TVBN values. The amount of TAC in olive flounder appeared faster than that in salmon, which may be due to the difference in the protein content required for the growth of microorganisms (Lee, 2020). According to Ye et al. (2020), storage at low temperatures is necessary to control the number of microorganisms present. Refrigeration caused microbial growth in fishes owing to higher storage temperature and the value of TAC reached 5 ± 0.5 log CFU/g, which satisfied the criteria for decomposition (Soares et al., 2015). Freezing treatment was effective in preventing microbial spoilage of samples during the storage period. Supercooling treatment also inhibited the increase in the TAC value of fish. Therefore, supercooling is a beneficial storage treatment for fish without a phase transition during 12 days of storage.

This study evaluated a stepwise cooling algorithm to maintain salmon and olive flounder a supercooled state for 12 days without phase transition. The supercooled preserved salmon showed a relatively low drip loss and showed higher water holding capacity than the frozen samples. After three days, the amount of trimethylamine exceeded the limit value of 2 mg/100 g in the sample stored under refrigeration. However, the amounts of trimethylamine and TVBN in the supercooled sample after 9 days increased rapidly. The frozen fish samples showed excellent quality within a short storage period; however, their water-holding capacity was the lowest. Based on these results, supercooled storage of fish was confirmed to inhibit quality deterioration compared to refrigeration storage. However, the WHC, pH, TBARS, and TVBN values were different for each type of fish, which may be due to the difference in composition between the two samples. The TAC values of fish preserved under supercooled conditions were more acceptable after 12 days than those of fish preserved with refrigeration. Therefore, the supercooling storage enabled by the stepwise cooling algorithm maintained a stable supercooled state and extended the shelf life of fish as a novel form of low-temperature preservation.

Acknowledgements

This study was financially supported by a grant from the Refrigerator Research of Engineering Division, Home Appliance, and Air Solution Company (LG Electronics Inc., Korea).

Author Contributions: Dong Hyeon Park: conceptualization, methodology, validation, formal analysis, investigation, resources, data curation, writing (original draft), visualization; SangYoon Lee: methodology, validation, investigation, visualization; Yeong Mi Byeon: methodology, validation, investigation, visualization; Eun Jeong Kim: conceptualization, investigation, resources, visualization, supervision, project administration; Mi-Jung Choi: conceptualization, resources, data curation, writing (review and editing), supervision, project administration

Funding: This research received no external funding

Data Availability Statement: The data presented in this experiment are available on request form the corresponding author

Conflicts of Interests: Authors declare no conflict of interest

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Table 1. Proximate composition of salmon and olive flounder

Sample	Proximate composition (%)
	Moisture			Crude protein				Crude lipid
Salmon	75.79	±	0.18		20.17	±	1.49		15.71	±	0.80
Olive flounder	65.33	±	3.54		22.23	±	0.41		3.00	±	0.71

Table 2. Changes in the color of salmon depending on various storage temperature and periods

Color

Storage

temperature

(°C)

Storage period

(days)

CIE L^*

CIE a^*

CIE b^*

ΔE

Control

39.77

0.53

9.85

0.47

11.95

0.94

Refrigeration

(3)

38.75

1.01^aB

8.39

0.44^bB

10.79

0.70^bC

2.33

0.90^cB

37.61

0.38^abB

9.24

0.81^aC

12.08

0.42^aB

2.10

0.16^cA

37.55

1.40^abB

8.04

0.96^bB

10.29

1.01^bB

3.65

1.24^bA

36.76

2.80^bC

8.09

0.41^bC

9.62

0.41^cC

4.90

1.60^aA

Freezing

(-18)

39.06

0.43^bcAB

9.11

0.56^cA

11.33

0.53^cB

1.30

0.66^bcA

38.63

0.91^cA

10.34

0.17^aA

12.86

0.44^aA

1.78

0.63^aA

39.48

0.48^bA

9.66

0.41^bA

12.39

0.39^bA

0.95

0.15^cB

41.17

0.48^aA

9.79

0.32^bA

12.36

0.41^bA

1.53

0.37^abB

Supercooling

(-2)

39.65

1.31^aA

8.88

0.16^dA

11.91

0.50^bA

1.56

0.33^abB

38.22

1.48^bAB

9.76

0.22^aB

12.07

0.14^abB

2.06

1.30^aA

39.40

0.80^aA

9.30

0.15^cA

12.25

0.20^aA

1.08

0.11^bB

38.87

0.81^abB

9.47

0.31^bB

11.46

0.28^cB

1.18

0.51^bB

^a-cMeans with different letters within the same storage temperature are significantly different (p < 0.05).

^A-CMeans with different letters within the same storage period are significantly different (p < 0.05).

Table 3. Changes in the color of olive flounder depending on various storage temperature and periods

Color

Storage

temperature

(°C)

Storage period

(days)

CIE L^*

CIE a^*

CIE b^*

ΔE

Control

58.64

0.29

-1.28

0.01

1.42

0.04

Refrigeration

(3)

57.04

6.10^abA

-1.06

0.24^aAB

1.54

0.91^bAB

6.00

1.55^abA

58.13

1.38^aB

-1.04

0.15^aAB

2.02

0.30^abB

1.34

0.90^cA

50.44

1.79^bC

-0.85

0.07^aA

1.29

0.21^bB

8.32

1.76^aA

55.39

2.96^abB

-0.92

0.10^aA

2.82

0.36^aA

4.21

1.87^bcA

Freezing

(-18)

55.70

2.16^cA

-1.24

0.13^bB

0.71

0.18^cB

3.21

1.86^abA

59.34

1.07^bAB

-1.13

0.03^abB

2.68

0.29^bA

1.77

0.46^bcA

62.03

0.57^aA

-1.16

0.19^abA

2.22

0.12^cA

3.50

0.55^abB

63.90

1.29^aA

-0.94

0.22^aA

2.74

0.28^aA

5.46

1.20aA

Supercooling

(-2)

59.82

2.71^abA

-0.80

0.20^aA

2.06

0.44^aA

2.66

1.50^abA

61.08

1.13^abA

-0.82

0.18^aA

2.88

0.25^aA

2.94

1.14^abA

58.71

1.12^bB

-1.26

0.14^bB

2.43

0.19^aA

1.43

0.16^bcB

62.24

1.10^aA

-1.18

0.07^bA

2.75

0.50^aA

3.86

1.14^aA

^a-cMeans with different letters within the same storage temperature are significantly different (p < 0.05).

^A-CMeans with different letters within the same storage period are significantly different (p < 0.05).

No competing interests reported.

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Supercooling with stepwise algorithm preserves the freshness of salmon and olive flounder during extended storage

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