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Article

Effects of Deoxygenated Packaging and Super-Chilled Storage on Yellowtail (Seriola quinqueradiata) Quality Deterioration

by
Yajing Ji
1,
Yu Kondo
1,
Run Wang
2,
Akane Matsumoto
2,
Ayumi Furuta
2,
Genya Okada
2 and
Shota Tanimoto
2,*
1
Graduate School of Comprehensive Scientific Research, Prefectural University of Hiroshima, 1-1-71 Ujinahigashi, Minami-ku, Hiroshima 734-8558, Japan
2
Faculty of Regional Development, Prefectural University of Hiroshima, 1-1-71 Ujinahigashi, Minami-ku, Hiroshima 734-8558, Japan
*
Author to whom correspondence should be addressed.
Appl. Sci. 2025, 15(17), 9686; https://doi.org/10.3390/app15179686
Submission received: 11 July 2025 / Revised: 22 August 2025 / Accepted: 27 August 2025 / Published: 3 September 2025
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Abstract

This study investigated how super-chilled (SC) storage at −3 °C combined with deoxygenated packaging (DO) affects quality degradation in yellowtail (Seriola quinqueradiata), dorsal ordinary muscle, and dark muscle. Sensory evaluation showed that DO significantly suppressed spoilage odor intensity in both muscle types, with enhanced effects under SC conditions. Spoilage in air-stored samples was primarily driven by Pseudomonas growth, whereas DO (especially SC) maintained microbial diversity by inhibiting bacterial proliferation and delaying spoilage. Volatile compound profiles differed markedly between the DO and air-stored samples. Despite these changes, DO-induced volatile compound alterations in the dorsal ordinary and dark muscles had minimal effects on perceived odor. Although DO prevented the accumulation of thiobarbituric acid reactive substances in both muscles, it did not suppress trimethylamine formation. These results demonstrate that SC-DO synergistically extends the shelf life of yellowtail by mitigating microbial spoilage and lipid oxidation, particularly during odor deterioration.

1. Introduction

Regulation of seafood quality deterioration during storage remains a critical challenge in the food industry [1,2,3]. Fish are highly perishable owing to post-harvest microbial activity and biochemical changes, which accelerate quality changes and reduce marketability. Although conventional refrigeration and ice-storage methods are widely employed, their efficacy in preventing quality degradation is often limited, necessitating the development of advanced preservation strategies [4,5,6,7]. Therefore, integrating innovative preservation techniques is crucial for extending shelf life while maintaining seafood quality.
Among the emerging preservation technologies, deoxygenated packaging (DO) and super-chilled (SC) storage have demonstrated significant potential for mitigating seafood spoilage. SC storage, which maintains products slightly below their freezing point without complete solidification, further suppresses enzymatic activity and microbial metabolism compared with conventional refrigeration [8,9]. SC and ultra-cold storage have been successfully applied to various aquatic products, including Atlantic salmon, grouper, and grass carp, where they effectively suppress microbial growth, delay lipid oxidation, and extend shelf life [8,9,10,11]. Additionally, compared with freezing, there are benefits concerning energy consumption, specifically in terms of management costs. In contrast, DO, implemented using oxygen scavengers or modified atmosphere packaging (MAP), inhibits oxidative reactions and slows microbial proliferation, thereby delaying quality deterioration [12,13,14,15,16]. Recent studies have shown that oxygen reduction within packaging systems substantially decreases lipid oxidation and discoloration, thereby preserving the overall quality of stored seafood [13,15].
Odor changes are a major factor that degrades the quality of fish meat [17]. This odor may cause people to refuse to eat fish dishes. The proliferation of spoilage-causing bacteria plays a key role in the development of foul odors in fish and shellfish [18]. However, advances in next-generation sequencing (NGS) technology have made it possible to accurately assess microbial communities that are difficult to cultivate or present in small proportions [19]. Pseudomonas, Psychrobacter, Photobacterium, and Shewanella were identified using NGS as the bacteria involved in fish spoilage [19]. Additionally, changes in volatile compounds (VCs) resulting from lipid oxidation occur in fish meat and affect its sensory properties [20].
Yellowtail is one of the most important fish species farmed in Japan [21]. Given the economic significance of this species, it is imperative to elucidate the effectiveness of these preservation techniques to optimize post-harvest handling and storage. Kitabayashi et al. [12] have reported that the progression of lipid oxidation, browning, and odor development in raw yellowtail meat could be suppressed by refrigeration in nitrogen gas packaging for a brief time (seven days). In contrast, we have previously reported that in yellowtail meat subjected to SC storage, the retention period of the dorsal portion of ordinary muscle (OMC) and dark muscle (DMC) can be effectively extended by suppressing bacterial growth, VC changes, and the development of spoiled odors; however, the effect on the suppression of lipid oxidation and trimethylamine (TMA) accumulation is minimal [22].
The concurrent utilization of DO and SC storage has been shown to have synergistic effects in prolonging the seafood preservation period, as it simultaneously minimizes oxidative degradation and microbial spoilage [17,18]. The effects of these combinations on the microbial flora and VCs in fish meat are unknown. Given the economic significance of yellowtail, it is imperative to elucidate the effectiveness of these preservation techniques to optimize post-harvest handling and storage. Additionally, this species is representative of fish with a highly developed DMC. The OMC characteristics differ significantly from those of DMC [23]. We have previously reported that changes in VCs during the storage of yellowtail meat are significantly different between OMC and DMC [24,25,26]. Therefore, it is important to verify the inhibitory effects of DO combined with SC on changes in VC in yellowtail OMC and DMC.
The present study aimed to comprehensively evaluate the effectiveness of a combination of DO and SC storage on the quality preservation of yellowtail flesh (OMC and DMC) by assessing VC composition, bacterial flora, biochemical properties, and sensory attributes under different storage conditions. Based on these results, we sought to provide evidence-based insights for optimizing seafood preservation strategies and ensuring product safety.

2. Materials and Methods

2.1. Sample Preparation and Storage Conditions

Fresh yellowtail (nine farmed specimens) was procured from a retail outlet in Hiroshima City, Japan, and transported on ice to the laboratory within 9 h. The mean weight of the fish was 4.28 kg, with a standard deviation of 0.39 kg. Upon arrival, the samples were divided into three groups of three fish each, filleted, rinsed in saline, sliced into 1 cm-thick portions containing both OMC and DMC, and randomly assigned to four storage conditions: storage on ice under air (IC-AR), DO on ice (IC-DO), SC storage under air (SC-AR), and DO with SC storage (SC-DO). For ice-chilled storage, the samples were stored in Styrofoam boxes with ice replenished every 2–3 d. For SC storage (−3 °C), the samples were pre-cooled on ice before placement in a refrigerator (SC-DF25; Twinbird, Tsubame, Japan) for rapid cooling. For storage in air, the samples were wrapped in polyvinyl chloride film (0.01 mm thick, KitcheNista, Chikusei, Japan). In DO, the samples were sealed in low-oxygen-permeable retort pouches together with an oxygen absorber (AGELESS® ZP, Mitsubishi Gas Chemical Co., Tokyo, Japan). Samples were stored for a given period (14–133 days) according to the storage conditions. Based on a spoilage threshold of 7 log CFU/g, the maximum storage duration was determined to be 28 and 77 d for IC-AR and SC-AR, respectively. Because this threshold was not exceeded for IC-DO and SC-DO, the storage durations were extended to 56 and 133 d, respectively, while considering other quality indicators. Prior to the analysis, the samples were subdivided into OMC and DMC, homogenized, and stored at −80 °C, except for 5.0 g portions allocated for viable bacterial count (VBC) analysis.

2.2. VBC Analysis

The samples were homogenized in a peptone saline solution, serially diluted stepwise, and plated onto each medium. The colony-forming units (CFU/g) of the bacteria listed in Table 1 were measured as described previously [22]. Spoilage thresholds were defined as the bacterial counts of Mesophilic bacteria reaching 7 log CFU/g [19].

2.3. Biochemical Analysis

The total volatile basic nitrogen (TVB-N) was quantified using microdiffusion analysis [20]. One gram of OMC or 0.5 g of DMC was thoroughly mixed with 10 mL of 5% trichloroacetic acid. The mixture was centrifuged at 10,000× g for 20 min and analyzed according to established protocols [22]. TMA concentrations were determined using a column for volatile amines (0.32 mm id. × 30 m, RESTEK, Bellefonte, PA, USA) incorporated into a GC-MS apparatus (Shimadzu mass spectrometer GCMS-QP2010, Kyoto, Japan), based on Tanimoto et al. [26]. Thiobarbituric acid reactive substance (TBARS) levels, indicative of lipid oxidation, were measured following the method described by Kitabayashi et al. [12], with some modifications. Briefly, 1.0 g of OMC or 0.25 g of DMC was analyzed using spectrophotometry.

2.4. Browning Index

The degree of browning of the DMC samples was assessed using a colorimeter (CR- 400, Konica Minolta Optics Inc., Tokyo, Japan). The browning index was expressed as the b/a ratio, where b represents yellowness and a represents redness [12].

2.5. Sensory Test

A sensory test was conducted with 49 undergraduate and graduate students from the Prefectural University of Hiroshima (32 females, 17 males; mean age: 23.8 ± 4.4 years) following a standardized scoring method. This study was approved by the Research Ethics Committee of Prefectural Hiroshima University (approval no. 20HH003-1). The samples (5 g) were held on ice for 1 h, followed by an additional hour at room temperature before assessment to equilibrate to this temperature (approximately 20 °C). The panelists rated the spoilage odor on a 3-point scale (1, fresh; 2, slightly spoiled; 3, very spoiled) and the overall odor intensity on a 5-point scale (1, very weak; 2, weak; 3, normal; 4, strong; 5, very strong). These criteria were based on the results of the preliminary tests conducted by skilled evaluators and those of our previous studies [12,22,25]. For spoiled odor, OMC before storage had a score of 1, whereas OMC stored for 28 d in ice under air had a score of 3. Before storage, DMC received a score of 3, whereas DMC stored for 28 days on ice under air received a score of 5. The order of presentation of the samples was randomized in each session to eliminate order bias, and each panelist was blinded to the treatment groups. The panelists were trained using these criteria prior to the evaluation.

2.6. NGS for Microbial Analysis and Data Processing

NGS sample extraction and sequencing were performed as previously described [22]. Given that no distinction was observed between OMC and DMC in the VBC analysis, NGS was conducted exclusively on OMC. Each 5 g sample was homogenized with 45 mL of sterile saline (0.85% NaCl) and centrifuged (800× g for 5 min, followed by 12,000× g for 5 min at 4 °C), and the precipitate was redispersed in 1.5 mL of saline. DNA was extracted using a Quick-DNA Fecal/Soil Microbe MiniPrep Kit (Zymo Research Corporation, Irvine, CA, USA). Amplicon sequencing targeted the 16S rRNA V3-V4 region using primers 341F and 805R. PCR (25–45 cycles) was performed using TksGflex DNA Polymerase (Takara Bio Inc., Shiga, Japan). Gel electrophoresis (2% agarose) was used to confirm amplification. The second PCR and sequencing were outsourced to Bioengineering Lab Co., Ltd. (Kanagawa, Japan).
Metagenomic data were processed using QIIME 2(2019.7, https://view.qiime2.Org, accessed on 8 August 2025) with DADA2 for quality control, generating a feature table and phylogenetic tree construction. Various alpha diversity indices were computed, including observed operational taxonomic units (OTUs), Chao1, Good’s coverage, Shannon index, and Simpson’s index. Principal coordinate analysis of the weighted UniFrac distance by Bray–Curtis was performed and visualized using the QIIME platform (https://view.qiime2.org, accessed on 8 August 2025). Taxonomic classification at the genus level was performed using the Silva 132 database (99% OTUs). The relative abundance of microbial genera was depicted through hierarchical cluster analysis, employing Euclidean distance and the Ward method, as implemented in MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/, accessed on 8 August 2025). The relative abundances of the microbes were corrected using the second-lowest relative abundance and converted to common logarithms. For this analysis, 30 genera were selected, each with a relative abundance exceeding 1% in any given sample.

2.7. VC Analysis

The VCs were subjected to solid-phase microextraction using the method described by Mukojima et al. [27] with minor modifications. Briefly, 1.4 g of the sample underwent homogenization with 7.0 mL of a saturated saline solution, followed by centrifugation at 15,000× g for 10 min at 4 °C. Following the addition of 5.0 µL of 0.010% cyclohexanol solution, which served as an internal standard, the supernatant (5.0 mL) in a sealed vial was equilibrated at 40 °C for 60 min. Subsequently, a 65 µm polydimethylsiloxane/divinylbenzene SPME fiber (Merck KGaA, Darmstadt, Germany) was exposed to the headspace at 40 °C for 30 min before being injected into the Shimadzu GCMS-QP2010 system (Shimadzu, Kyoto, Japan). A capillary column (Intert Cap Pure-WAX, 0.25 mm i.d. × 60 m, 0.25 µm film thickness, GL Science, Tokyo, Japan) was employed for the analysis of VCs. The processes for identifying, estimating, and semi-quantifying the VCs were conducted as previously described [22]. Semi-quantitative VC data were analyzed using principal component analysis using SIMCA 16 (Sartorius Stedim Biotech, Auvergne, France).

2.8. Data Analysis

Data are expressed as averages (n = 3). Statistical comparisons were performed using a two-tailed Dunnett’s test (SPSS Statistics 23, IBM Japan, Ltd., Tokyo, Japan), with day 0 as the control (p < 0.05).

3. Results and Discussion

3.1. VBCs

The changes in VBCs in yellowtail muscles during storage under different conditions are shown in Table 1 and Table S1. Bacterial proliferation was greatly influenced by storage conditions. IC-AR and SC-AR resulted in the rapid growth of Mesophilic bacteria, Pseudomonas spp., marine bacteria, Aeromonas spp., and Enterobacteriaceae, reaching spoilage thresholds at 28 d and at 56 or 28 d, respectively, with certain exceptions. Conversely, DO, particularly under SC conditions, effectively suppressed bacterial growth and delayed spoilage beyond day 133. The suppression of these specific spoilage organisms by DO suggests that the removal of oxygen can inhibit the growth of aerobic bacteria such as Pseudomonas spp. as well as facultative anaerobes [28,29,30,31]. The combined use of oxygen removal and SC has been shown to have a greater inhibitory effect on the growth of specific spoilage organisms. In contrast, LAB, which are specific spoilage organisms found in deoxygenated MAP storage [31], did not grow under the present storage conditions, indicating that they were not involved in spoilage under the present conditions.

3.2. TVB-N

TVB-N levels increased significantly (p < 0.05) under all storage conditions (Table 2 and Table S2). Neither storage temperature nor oxygen availability significantly influenced this increase, which is consistent with previous findings on yellowtail meat [19]. Deng et al. [32] and Li et al. [33] have suggested that endogenous enzymatic activity rather than microbial metabolism predominantly drives TVB-N formation during extended storage, which explains the ineffectiveness of DO in suppressing its accumulation. Nonetheless, the TVB-N levels remained below the spoilage threshold (25–30 mg/100 g) under all conditions [26].

3.3. TMA

TMA levels increased significantly (p < 0.05) in OMC across all storage conditions, while in DMC, they plateaued after an initial rise (Table 2 and Table S2). Although low storage temperatures suppressed TMA production, DO unexpectedly promoted it. TMA is produced via the bacterial reduction of trimethylamine oxide (TMAO) by species such as Aeromonas spp., Pseudomonas phosphoreum, Shewanella putrefaciens, and psychrotolerant Enterobacteriaceae, generating the characteristic ammonia-like odor of spoiled marine fish [34,35,36,37]. Although DO suppressed overall bacterial growth, it may have facilitated the anaerobic reduction of TMAO, particularly in DMC, where TMAO reductase activity is naturally higher [36,37]. However, the underlying mechanisms remain unclear. Further investigations are required to substantiate this hypothesis.

3.4. TBARS

TBARS levels increased significantly (p < 0.05) in the AR samples at all temperatures, indicating active lipid oxidation (Table 2 and Table S2). In contrast, the DO samples exhibited minimal TBARS accumulation. SC further delayed TBARS increase in OMC compared with IC, although this effect was less pronounced in DMC. These results are consistent with those of Kitabayashi et al. [12], who have reported that nitrogen flushing inhibited lipid oxidation in yellowtail muscles during ice storage. Similarly, Ahn et al. [38] have demonstrated that oxygen exposure, rather than temperature, plays a dominant role in initiating lipid oxidation.

3.5. Browning Degree

The degree of browning of yellowtail DMC during storage is shown in Table 3. Color degradation was significantly more pronounced under AR, in which the autoxidation of oxymyoglobin to metmyoglobin was not mitigated (p < 0.05). In contrast, DO, particularly SC, effectively suppressed DMC browning. This suppression was due to the inhibition of metmyoglobin formation, as oxygen availability is a key factor in this discoloration. Previous studies have shown that nitrogen gas packaging inhibits the browning of yellowtail.
DMC is shown during seven days of ice storage [12]. It was shown that DO-IC inhibited the browning of DMC for a longer period than in the previous study and that DO-SC delayed browning progression.

3.6. Sensory Test

Sensory tests revealed that spoilage odor and odor intensity increased significantly (p < 0.05) across all storage conditions during the experimental period (Table 4 and Table S3). For OMC, spoilage odor was detected from day 28 in IC-AR and day 56 in both SC-AR and IC-DO, whereas for DMC, it was noticeable from day 14 in IC-AR and day 77 in SC-AR. The odor intensity showed a similar trend. However, no significant odor deterioration was observed in OMC stored under SC-DO or in DMC stored under DO. These results support previous findings [17] that DO, especially when combined with SC storage, effectively delays the development of spoilage-related odors and extends the shelf life of yellowtail muscle.

3.7. Microbial Community Analysis

Sequencing of the DNA extracted from stored yellowtail OMC yielded 875,555 reads. Variations in the observed OTUs and α-diversity indices throughout the storage period are detailed in Table 5 and Table S4. Good coverage remained at 100%, confirming adequate sequencing depth for the analysis of bacterial communities. ACE and Chao1 indices were used to assess species richness, whereas Shannon and Simpson indices were used to evaluate biodiversity. A significant reduction in α-diversity (p < 0.05) was noted in the AR-IC samples by day 28, suggesting a decrease in microbial complexity. In contrast, SC and DO mitigated this decline, preserving a higher microbial richness over prolonged storage.
At the genus level, as illustrated in Figure 1, Pseudomonas emerged as the predominant spoilage bacterium in the AR samples, reaching an abundance of over 90% by the spoilage threshold (day 28 in AR-IC and day 77 in AR-SC). In contrast, DO inhibited Pseudomonas proliferation, thereby preserving a more diverse microbial community. Additionally, other bacterial genera, including Sphingobium and Unclassified Enterobacterales, were also identified at higher abundances in DO samples at later storage stages, suggesting that spoilage progression was delayed without the dominance of any single genus.
The Weighted UniFrac principal coordinate analysis revealed distinct microbial clustering patterns contingent on the storage duration and packaging type (Figure 2). In AR samples, spoilage-associated bacteria such as Pseudomonas [22,26] became predominant after storage. Consequently, IC samples on day 28 and SC samples on days 56–77 were categorized within the same cluster. Conversely, the DO samples retained a more diverse microbial community throughout storage without the formation of any clusters.
Heatmaps generated from the hierarchical clustering analysis (Figure 3) revealed two distinct clusters at the genus level. Cluster I comprised spoiled samples, specifically the 28-day sample from IC-AR and the 56- and 77-day samples from SC-AR, all of which were predominantly characterized by Pseudomonas. Cluster II included AR samples with shorter storage durations and IC-DO samples (except for the 14-day sample). Cluster III included pre-spoiled SC-DO samples and a 14-day sample from the IC-DO group. These findings suggest that different storage conditions result in markedly different alterations in the microbial communities.
Specifically, spoilage in AR samples was attributed to the proliferation of spoilage-associated bacteria, such as Pseudomonas spp. In contrast, SC and DO effectively preserved microbial diversity and retarded spoilage by inhibiting bacterial growth. These findings align with the existing literature, which underscores the significance of the packaging atmosphere in modulating microbial communities during fish storage [39,40]. In the IC-AR samples, a marked reduction in α-diversity was observed on day 28, corroborating results that demonstrated rapid spoilage progression under aerobic conditions due to the proliferation of Pseudomonas.
Conversely, the preservation of microbial richness in SC and DO is consistent with research findings indicating that low-oxygen environments, such as MAP or vacuum packaging, can inhibit the proliferation of aerobic spoilage bacteria and maintain microbial diversity [41,42,43]. In the present study, unclassified Enterobacterales emerged as the predominant group in both IC-DO and SC-DO samples during the later stages of storage. Enterobacteriaceae and Carnobacterium were identified as the primary genera in grouper fillets stored under vacuum packaging at 4 °C [39]. The Enterobacteriaceaeles order comprises Gram-negative facultative anaerobic bacteria that are extensively distributed in soil, in water, and in association with living organisms [44]. Enterobacteriaceae is a member of this order. Consequently, these bacteria may play a role in the quality changes observed in the DO samples. The genus Sphingobium was relatively highly abundant in the DO samples during the later stages of storage in the present study. Members of this genus, characterized as mesophilic, strictly aerobic, and chemoorganotrophic, are predominantly isolated from contaminated soil and wastewater and are recognized for their roles in the bioremediation and biodegradation of pollutants [45]. Furthermore, this genus, which is closely related to Sphingomonas, was identified in pork steaks packaged under MAP [46]. However, the reasons for the dominance of this genus in DO samples remain unclear and warrant further investigation. Aeromonas emerged as a major spoilage organism in vacuum-packed crisp grass carp fillets during storage at 4 °C [47]. At a sensory rejection time of 4/8 °C, Brochothrix thermosphacta was found to dominate the microbiota of vacuum-packed tropical yellowfin tuna [40]. Consequently, the predominant bacterial genera in vacuum-packed fish meat after storage may vary according to the storage conditions, fish habitats, and other factors.
The effects of DO and SC storage on fish meat quality observed in the present study were partially consistent with previous findings in other fish species, such as grouper and grass carp [39,47,48]. Consistent with these studies, DO-SC treatment inhibited the growth of aerobic spoilage bacteria (e.g., Pseudomonas spp.), delayed the onset of off-odors, and reduced the rate of lipid oxidation. However, yellowtail meat exhibited more pronounced accumulation of TMA in the DO samples. Under DO conditions, the accumulation of TMA is likely driven by TMAO reductase activity rather than by the proliferation of H2S-producing bacteria (Shewanella spp.), which were not detected. DMC contains higher levels of TMAO and exhibits distinct enzymatic activity compared with OMC, which explains the more pronounced accumulation of TMA in DMC [49]. Additionally, DMC (dark muscle) is known to contain higher baseline levels of TMAO than OMC (ordinary muscle) and may exhibit distinct enzymatic activity, contributing to the more pronounced TMA accumulation observed in this muscle type [50]. In freshwater fish such as grass carp, no accumulation of TMAO was observed in the muscle tissue, suggesting differences in TMA formation during storage compared to the present study [51,52,53]. Therefore, given that the post-storage microbial profile varies depending on the fish habitat and that the characteristics related to quality changes during storage differ according to fish species and muscle parts, further research is necessary to generalize the findings of the present study.

3.8. VCs

A total of 162 VCs, including 29 unidentified, were detected in yellowtail meat under different storage conditions (Table S5a–d). The principal component analysis results for OM and DMC are shown in Figure 4 and Figure 5, respectively. For OMC, PC1 and PC2 accounted for 26.1% and 24.5% of total variance, respectively. The samples before storage (0 day) were separated from those after storage. As storage progressed, the score plots of the AR samples shifted toward the lower left quadrant, whereas those of the DO samples moved toward the upper left quadrant, suggesting distinct patterns of VC changes between the two packaging types. The score plots of the DO samples remained closer to those of the day 0 reference, indicating a strong suppression of VC changes in the OMC. For DMC, PC1 and PC2 accounted for 51.2% and 15.5% of total variance, respectively. The score plot of the AR sample was positioned to the right of the plot before storage, whereas that of the AR-SC sample on day 77 was situated further to the upper right. In contrast, the score plot of the DO samples was positioned to the left of that before storage, and the change in behavior during storage differed between the IC and SC samples. Thus, the patterns of VC changes during storage exhibited significant differences between the DMCs with DO and those with AR. Moreover, there was no significant increase in the spoiled odor and odor intensity of either muscle part after DO, or the significant increase required a longer duration than AR. These findings suggest that alterations in the VC profiles of OMC and DMC after DO did not substantially affect the odor.
From the loading plot results, during the AR, the predominant VCs in OMC were identified as 3-methyl butanoic acid, (E)-3-hexen-1-ol, and (E, Z)-3,5-octadien-2-one. Conversely, the DO samples for the OMC were characterized by the accumulation of 2-ethyl-1-hexanol, 1-nonanol, butanoic acid, and octanoic acid. However, the levels of many of these compounds also increased in the AR samples. In contrast, the levels of many aldehydes, such as (Z)-4-Heptenal, in OMC decreased significantly during both AR and DO. During AR, there was a significant increase in ethyl benzaldehyde, (E, Z)-3,5-octadien-2-one, and 1-penten-3-ol in the DMC, and numerous other compounds were linked to alterations in the VC profiles of the AR samples. Conversely, during DO, many of the compounds in the DMC either decreased significantly or showed no significant change. Additionally, 2-ethyl-1-hexanol and numerous unidentified compounds were linked to alterations in the VC profiles of DMC during DO. The concentration of 2-ethyl-1-hexanol in the DMC increased significantly during both DO and AR. In our previous study, VCs such as 2,3-butanedione, 2-methyl butanol, 3-methyl butanol, and 3-hydroxy-2-butanone were identified as potential indicators of spoilage in yellowtail muscles (DMC and OMC) [22].
The current study yielded comparable results for aforementioned VCs in both spoiled muscle parts after AR, with some exceptions. Conversely, after DO, these VCs either exhibited a delayed increase or were not detected. This trend was particularly evident for SC-DO. 3-Methyl butanoic acid was undetectable in both muscle parts before storage and increased after AR. Furthermore, given the significant correlation of the compound (0.727–0.913, p < 0.05) with the VBCs of Pseudomonas spp. in AR samples, it was identified as a potential novel indicator of spoilage. Additionally, this VC was either undetectable or only slightly detectable during DO storage. 3-Methylbutanoic acid as well as 3-methyl-1-butanol, associated with the metabolic activity of Pseudomonas, have been proposed as indicators of spoilage in meagre [54]. These alterations in VCs in the OMC and DMC during AR are consistent with the conclusions of our previous study [22]. The VCs (ex. (Z)-4-heptenal), which decreased during storage in OMC regardless of storage conditions, may be attributed to endogenous muscle enzymes rather than microbial activity or lipid oxidation.
Conversely, elevated levels of VCs (e.g., 1-penten-3-ol) in DMC after AR compared with those after DO were most likely due to lipid oxidation as well as the metabolism of bacteria such as Pseudomonas. In contrast, the increased or decreased levels of VCs (e.g., 1-penten-3-ol) in the DMC after DO compared with those after AR may have been caused by endogenous enzymes rather than lipid oxidation or bacterial processes. Collectively, these findings suggest that DO, especially when combined with SC, effectively alters the VC profile by suppressing spoilage-related compounds while minimizing the accumulation of novel odor indicators.

4. Conclusions

The present study investigated the effects of storage at 0 °C (Ice), −3 °C (SC), and DO on the quality deterioration of ordinary muscle and DMC in yellowtail (Seriola quinqueradiata), aiming to extend the preservation period of raw fish meat. Sensory evaluation results indicated that DO significantly suppressed the development of spoilage odors and odor intensity in both muscle types, particularly when combined with SC storage. Furthermore, DO, especially in conjunction with SC, maintained microbial diversity by inhibiting the proliferation of spoilage-associated bacteria such as Pseudomonas spp., thereby delaying microbial spoilage. The VC profiles exhibited markedly different patterns between DO and atmospheric storage in both muscle types; however, the underlying mechanisms require further investigation. DO storage also suppressed discoloration in DMC and reduced TBARS levels in both OMC and DMC, indicating the inhibition of metmyoglobin formation and lipid oxidation. However, DO had no significant effect on TVB-N or TMA accumulation. These findings suggest that combining SC storage with DO effectively extends the shelf life of fish meat by mitigating microbial growth and oxidative deterioration. Future studies should address strategies to inhibit nitrogenous spoilage indicators, such as TVB-N and TMA, which are not effectively controlled under current storage conditions.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/app15179686/s1, Due to space limitations, full data with standard errors are presented in Supplementary Tables S1–S5a–d. Table S1. Changes in visible bacterial counts (VBCs) of different yellowtail muscles during storage under various conditions (log CFU/g). Table S2. Changes in total volatile basic nitrogen (TVB-N), trimethylamine (TMA), and thiobarbituric acid reactive substances (TBARS) of yellowtail muscles during storage under different conditions. Table S3. Changes in sensory scores of yellowtail muscles during storage under different conditions. Table S4. OTUs and α diversity index of yellowtail ordinary muscle during storage under different conditions. Table S5a–S5d. Changes in volatile compounds (VCs) in raw yellowtail muscle (OM and DMC) stored under different conditions. Table S5a: IC-AR (ice storage under air). Table S5b: IC-DO (ice storage under deoxygenated packaging). Table S5c: SC-AR (super-chilled storage under air). Table S5d: SC-DO (super-chilled storage under deoxygenated packaging).

Author Contributions

Conceptualization, S.T., A.M., A.F., R.W. and Y.J.; methodology, S.T., A.M., A.F., R.W. and G.O.; software, Y.J., Y.K., G.O. and S.T.; validation, S.T. and A.F.; formal analysis, Y.J. and Y.K.; investigation, Y.J., Y.K., G.O., R.W. and S.T.; resources, S.T.; data curation, Y.J. and Y.K.; writing−original draft, Y.J., S.T. and Y.K.; writing−review and editing, Y.J., S.T., Y.K., A.M., A.F., R.W. and G.O.; visualization, Y.J. and Y.K.; supervision, S.T.; project administration, S.T.; funding acquisition, S.T. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by JSPS KAKENHI (grant number: JP20K02346).

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Research Ethics Committee of the Prefectural University of Hiroshima (protocol code 20HH003-1 and date (July 5, 2023) of approval).

Informed Consent Statement

Informed consent was obtained from all participants involved in the study.

Data Availability Statement

The original contributions of this study are included in the article and Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
CFUColony-forming unit
DODeoxygenated packaging
DMCDark muscle
IC-ARStorage on ice under air
IC-DODeoxygenated packaging on ice
MAPModified atmosphere packaging
NGSNext-generation sequencing
OMCDorsal portion of ordinary muscle
SCSuper-chilled
OTUsOperational taxonomic units
SC-ARSC storage under air
SC-DODeoxygenated packaging with SC storage
TBARSThiobarbituric acid reactive substances
TMATrimethylamine
TMAOTrimethylamine oxide
TVB-NTotal volatile basic nitrogen
VBCViable bacterial count
VCVolatile compound

References

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Applsci 15 09686 g001
Figure 1. Changes in the relative abundance of bacterial genera in yellowtail ordinary muscle during storage under different conditions; storage conditions: IA (ice storage under air), ID (ice storage under deoxygenated packaging), SA (super-chilled storage under air), and SD (super-chilled storage under deoxygenated packaging). Storage time: #d (days).
Figure 1. Changes in the relative abundance of bacterial genera in yellowtail ordinary muscle during storage under different conditions; storage conditions: IA (ice storage under air), ID (ice storage under deoxygenated packaging), SA (super-chilled storage under air), and SD (super-chilled storage under deoxygenated packaging). Storage time: #d (days).
Applsci 15 09686 g001
Applsci 15 09686 g002
Figure 2. Three-dimensional principal coordinate analysis based on weighted UniFrac distances of microbial communities at the genus level in yellowtail ordinary muscle during storage under different conditions. The storage conditions (IA, ice storage under air; ID, ice storage under deoxygenated packaging; and SA, super-chilled storage under air; under deoxygenated packaging) and storage time (#d) are indicated.
Figure 2. Three-dimensional principal coordinate analysis based on weighted UniFrac distances of microbial communities at the genus level in yellowtail ordinary muscle during storage under different conditions. The storage conditions (IA, ice storage under air; ID, ice storage under deoxygenated packaging; and SA, super-chilled storage under air; under deoxygenated packaging) and storage time (#d) are indicated.
Applsci 15 09686 g002
Applsci 15 09686 g003
Figure 3. Heatmap and hierarchical clustering of bacterial genera in yellowtail ordinary muscle during storage under different conditions. The class indicates the storage conditions (IA, ice storage under air; ID, ice storage under deoxygenated packaging; and SA, super−chilled storage under air; under deoxygenated packaging) and storage time (#d). Colors indicate the common logarithm of the relative abundance. Clusters A–E (rows) represent the hierarchical clustering of bacterial genera based on their similarity in relative abundance patterns across samples. These clusters group together genera that exhibit similar abundance trends during storage. Clusters I–V (columns) represent the clustering of samples stored under different conditions and time points, indicating similar microbial community structures among grouped samples.
Figure 3. Heatmap and hierarchical clustering of bacterial genera in yellowtail ordinary muscle during storage under different conditions. The class indicates the storage conditions (IA, ice storage under air; ID, ice storage under deoxygenated packaging; and SA, super−chilled storage under air; under deoxygenated packaging) and storage time (#d). Colors indicate the common logarithm of the relative abundance. Clusters A–E (rows) represent the hierarchical clustering of bacterial genera based on their similarity in relative abundance patterns across samples. These clusters group together genera that exhibit similar abundance trends during storage. Clusters I–V (columns) represent the clustering of samples stored under different conditions and time points, indicating similar microbial community structures among grouped samples.
Applsci 15 09686 g003
Applsci 15 09686 g004aApplsci 15 09686 g004b
Figure 4. Principal component analysis of volatile compounds in yellowtail ordinary muscle (OMC) during storage under different conditions. The data were pre-processed using an auto-scale. (A) The score scatter plot. The muscle type, storage conditions (IA, ice storage under air; ID, ice storage under deoxygenated packaging; and SA, super-chilled storage under air; under deoxygenated packaging), and storage time (#d) are indicated. (B) The loading plot. The numbers in the loading plot indicate the peak numbers (Supplementary Table S5a–d).
Figure 4. Principal component analysis of volatile compounds in yellowtail ordinary muscle (OMC) during storage under different conditions. The data were pre-processed using an auto-scale. (A) The score scatter plot. The muscle type, storage conditions (IA, ice storage under air; ID, ice storage under deoxygenated packaging; and SA, super-chilled storage under air; under deoxygenated packaging), and storage time (#d) are indicated. (B) The loading plot. The numbers in the loading plot indicate the peak numbers (Supplementary Table S5a–d).
Applsci 15 09686 g004aApplsci 15 09686 g004b
Applsci 15 09686 g005aApplsci 15 09686 g005b
Figure 5. Principal component analysis of organic volatile compounds in yellowtail dark muscle (DMC) during storage under different conditions. The data were pre-processed using an auto-scale. (A) The score scatter plot. The muscle type, storage conditions (IA, ice storage under air; ID, ice storage under deoxygenated packaging; and SA, super-chilled storage under air; under deoxygenated packaging), and storage time (#d) are indicated. (B) The loading plot. The numbers in the loading plot indicate the peak numbers (Supplementary Table S5a–d).
Figure 5. Principal component analysis of organic volatile compounds in yellowtail dark muscle (DMC) during storage under different conditions. The data were pre-processed using an auto-scale. (A) The score scatter plot. The muscle type, storage conditions (IA, ice storage under air; ID, ice storage under deoxygenated packaging; and SA, super-chilled storage under air; under deoxygenated packaging), and storage time (#d) are indicated. (B) The loading plot. The numbers in the loading plot indicate the peak numbers (Supplementary Table S5a–d).
Applsci 15 09686 g005aApplsci 15 09686 g005b
Table 1. Changes in visible bacterial counts of different yellowtail muscles during storage under various conditions (log CFU/g).
Table 1. Changes in visible bacterial counts of different yellowtail muscles during storage under various conditions (log CFU/g).
Packaging ConditionsMuscle TypeStorage Time at 0 °C (Days)Storage Time at −3 °C (Days)
0 142856028567798133
Mesophilic
bacteria		AtmosphereOMC2.2 4.0 *7.7 *–2.23.87.1 *8.2 *––
DMC3.03.9 *7.5 *–3.03.86.7 *7.7 *––
DeoxygenationOMC2.2 2.51.95.8 *2.23.42.6–2.52.7
DMC3.02.62.45.6 *3.03.82.8–2.93.3
B. thermosphactaAtmosphereOMC0.0 0.02.9 *–0.02.2 *3.1 *5.0 *––
DMC0.01.5 *2.8 *–0.02.9 *3.6 *3.8 *––
DeoxygenationOMC0.0 0.41.9 *0.00.01.7 *0.0–2.8 *2.3 *
DMC0.00.03.0 *0.10.02.6 *0.0–2.9 *2.8 *
Lactic acid bacteriaAtmosphereOMC0.0 0.01.5 *–0.00.41.8 *3.1 *––
DMC0.00.00.8–0.01.2 *1.7 *3.0 *––
DeoxygenationOMC0.0 0.00.00.00.00.00.1–0.10.6
DMC0.00.00.90.10.01.4 *0.3–0.30.9
EnterobacteriaceaeAtmosphereOMC0.0 2.3 *6.3 *–0.02.1 *6.1 *7.1 *––
DMC0.02.7 *6.6 *–0.02.4 *6.1 *6.9 *––
DeoxygenationOMC0.0 0.00.04.3 *0.01.40.6–0.01.1
DMC0.00.00.05.3 *0.02.50.0–0.01.5
Aeromonas spp.AtmosphereOMC0.0 4.3 *8.2 *–0.05.3 *7.6 *8.0 *––
DMC0.04.3 *8.1 *–0.05.0 *6.9 *7.9 *––
DeoxygenationOMC0.0 0.00.05.7 *0.04.5 *1.3–0.01.8
DMC0.00.00.55.9 *0.04.2 *0.0–0.01.4
Marine bacteriaAtmosphereOMC0.0 4.0 *7.7 *–0.04.4 *7.1 *7.7 *––
DMC0.03.9 *7.5 *–0.03.9 *6.7 *7.4 *––
DeoxygenationOMC0.0 0.00.53.4 *0.03.4 *0.6–2.7 *2.1
DMC0.00.01.33.6 *0.03.7 *0.7–3.1 *2.9 *
Pseudomonas spp.AtmosphereOMC0.0 3.3 *5.9 *–0.01.76.4 *6.8 *––
DMC0.03.85.2 *–0.03.8 *5.96.8––
DeoxygenationOMC0.0 0.70.81.30.03.3 *0.0–0.02.2 *
DMC0.00.03.13.10.00.00.0–1.93.0
H2S−producing bacteriaAtmosphereOMC0.0 0.00.0–0.00.00.00.0––
DMC0.00.00.0–0.00.00.00.0––
DeoxygenationOMC0.00.00.00.00.00.00.0–0.00.0
DMC0.0 0.00.00.00.00.00.0–0.00.0
The data are expressed as the average (n = 3). OMC, Ordinary muscle in dorsal part; DMC, Dark muscle. Asterisks denote a statistically significant difference from day 0 of storage for the same medium, flesh type, and storage conditions (p < 0.05). En dashes signify the absence of data for the storage days.
Table 2. Changes in biochemical index of yellowtail muscles during storage under different conditions.
Table 2. Changes in biochemical index of yellowtail muscles during storage under different conditions.
Packaging
Conditions		Muscle TypeStorage Time at 0 °C (Days)Storage Time at −3 °C (Days)
0 14 28560 28567798133
Total volatile basic nitrogen
(mg/100 g)		AtmosphereOMC6.58.310.7 *–6.58.06.913.4 *––
DMC2.99.4 *10.3 *–2.99.2 *10.7 *13.0 *––
DeoxygenationOMC6.510.7 *9.88.76.59.710.5 *–10.8 *12.5 *
DMC2.99.7 *13.3 *14.4 *2.910.4 *13.3 *–15.2 *15.3 *
Trimethylamine
(μg/g)		AtmosphereOMC0.4018.4 *36.7 *–0.4021.152.4 *51.4 *––
DMC18.7180.3 *146.7 *–18.7157.7 *133.0107.9––
DeoxygenationOMC0.4025.061.2 *109.5 *0.4035.474.8–170.2 *183.9 *
DMC18.7237.8 *277.8 *210.5 *18.7206.8 *188.4 *–181.2 *176.9 *
Thiobarbituric acid reactive
substances
(μmol/g)		AtmosphereOMC0.0040.031 *0.046 *–0.0040.026 *0.042 *0.054 *––
DMC0.0560.280 *0.321 *–0.0560.368 *0.420 *0.428 *––
DeoxygenationOMC0.0040.0050.0050.0060.0040.0030.005–0.0030.003
DMC0.056 0.149 *0.176 *0.173 *0.056 0.0450.132 *–0.238 *0.208 *
The data are expressed as the average (n = 3). OMC, Ordinary muscle in dorsal part; DMC, dark muscle. Asterisks denote a statistically significant difference from day 0 of storage for the same biochemical analysis, flesh type, and storage conditions (p < 0.05). En dashes signify the absence of data for the storage days.
Table 3. Changes in the b*/a* values of yellowtail dark muscle during storage under different conditions.
Table 3. Changes in the b*/a* values of yellowtail dark muscle during storage under different conditions.
Packaging
Conditions		Storage Time at 0 °C (Days)Storage Time at −3 °C (Days)
0142856028567798133
Atmosphere0.42 ± 0.11.20 ± 0.4 *1.20 ± 0.2 *–0.42 ± 0.11.30 ± 0.4 *1.60 ± 0.3 *1.40 ± 0.3 *––
Deoxygenation0.42 ± 0.10.70 ± 0.1 *0.70 ± 0.1 *0.70 ± 0.1 *0.42 ± 0.10.60 ± 0.1 *0.70 ± 0.1 *–0.70 ± 0.1 *0.70 ± 0.1 *
Values are expressed as mean ± standard deviation (n = 3). Asterisks denote a statistically significant difference from day 0 of storage under the same storage conditions (p < 0.05). En dashes signify the absence of data for the storage days. The b*/a* ratio indicates relative changes in yellowness to redness in dark muscle color during storage.
Table 4. The results of the sensory test of yellowtail muscles during storage under different conditions.
Table 4. The results of the sensory test of yellowtail muscles during storage under different conditions.
Packaging
Conditions		Muscle TypeStorage Time at 0°C (Days)Storage Time at −3°C (Days)
0142856028567798133
Spoiled
odor		AtmosphereOMC1.61.82.2 *−1.61.92.2 *2.5 *−−
DMC1.92.5 *2.6 *−1.92.42.42.7 *−−
DeoxygenationOMC1.61.71.72.1 *1.61.61.9−2.01.9
DMC1.92.21.81.91.92.02.3−2.12.1
Odor
intensity		AtmosphereOMC1.72.6 *2.9 *−1.72.32.7 *3.5 *−−
DMC2.84.0 *3.8 *−2.83.73.8 *4.4 *−−
DeoxygenationOMC1.72.02.02.1 *1.71.92.4−2.5 *2.4 *
DMC2.83.32.63.22.83.23.4−3.13.1
The data are expressed as the average of sensory scores (n = 3). OMC, Ordinary muscle in dorsal part; DMC, dark muscle. Asterisks denote a statistically significant difference from day 0 of storage for the same evaluation items, flesh type, and storage conditions (p < 0.05). En dashes signify the absence of data for the storage days.
Table 5. Observed operational taxonomic units (OTUs) and α diversity index of yellowtail ordinary muscle during storage under different conditions.
Table 5. Observed operational taxonomic units (OTUs) and α diversity index of yellowtail ordinary muscle during storage under different conditions.
Packaging
Conditions		Storage Time at 0 °C (Days)Storage Time at −3 °C (Days)
0142856028567798133
Observed OTUsAtmosphere83.053.016.3–83.031.721.014.3––
Deoxygenation83.058.312.727.083.063.057.3–43.036.7
ShannonAtmosphere5.014.052.09 *–5.013.901.75 *2.55 *––
Deoxygenation5.014.472.80 *3.805.014.494.53–4.253.21
Chao1Atmosphere83.054.718.3–83.033.023.315.7––
Deoxygenation83.061.312.727.083.068.359.0–44.038.0
ACEAtmosphere83.054.718.3–83.033.023.315.7––
Deoxygenation83.061.312.727.083.068.359.0–44.038.0
SimpsonAtmosphere0.940.880.59 *–0.940.910.49 *0.73––
Deoxygenation0.940.920.770.890.940.920.91–0.910.79
Good Coverage (%)Atmosphere100100100–100100100100––
Deoxygenation100100100100100100100–100100
The data are expressed as the average (n = 3). Asterisks denote a statistically significant difference from day 0 of storage for the same parameters and storage conditions (p < 0.05). En dashes signify the absence of data for the storage days.
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MDPI and ACS Style

Ji, Y.; Kondo, Y.; Wang, R.; Matsumoto, A.; Furuta, A.; Okada, G.; Tanimoto, S. Effects of Deoxygenated Packaging and Super-Chilled Storage on Yellowtail (Seriola quinqueradiata) Quality Deterioration. Appl. Sci. 2025, 15, 9686. https://doi.org/10.3390/app15179686

AMA Style

Ji Y, Kondo Y, Wang R, Matsumoto A, Furuta A, Okada G, Tanimoto S. Effects of Deoxygenated Packaging and Super-Chilled Storage on Yellowtail (Seriola quinqueradiata) Quality Deterioration. Applied Sciences. 2025; 15(17):9686. https://doi.org/10.3390/app15179686

Chicago/Turabian Style

Ji, Yajing, Yu Kondo, Run Wang, Akane Matsumoto, Ayumi Furuta, Genya Okada, and Shota Tanimoto. 2025. "Effects of Deoxygenated Packaging and Super-Chilled Storage on Yellowtail (Seriola quinqueradiata) Quality Deterioration" Applied Sciences 15, no. 17: 9686. https://doi.org/10.3390/app15179686

APA Style

Ji, Y., Kondo, Y., Wang, R., Matsumoto, A., Furuta, A., Okada, G., & Tanimoto, S. (2025). Effects of Deoxygenated Packaging and Super-Chilled Storage on Yellowtail (Seriola quinqueradiata) Quality Deterioration. Applied Sciences, 15(17), 9686. https://doi.org/10.3390/app15179686

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