Effect of Proteolysis on the Meat Quality of a Brand Fish, Red Sea Bream Pagrus major

Abstract

Building a brand fish is the key to success in business, as brand fish are generally sold at a higher price. We focused on one of the varieties of brand red sea bream, which is famous for its texture and aesthetic appeal. In this study, we evaluated the appearance and texture of red sea bream meat using organoleptic analysis to elucidate the relationship between degradation of muscle protein and meat quality. The appearance and texture were significantly superior in the brand red sea bream as compared to the non-brand variety. The brand red sea bream muscle showed greater resistance to apoptosis and autophagy. This suggested that the difference in the appearance and texture of the brand red sea bream was because of the suppression of muscle protein degradation. The results indicated that the brand fish was obviously tastier than the non-brand fish.

Introduction

Currently, regional agricultural and marine specialty products or traditional food products are certified as local brand products in many parts of Japan. Such products have been marketed in collaboration with the community to gain national recognition for these brands. Along with tourism, these products fulfill an important role in “Chiiki-okoshi” and “Machi-okoshi,” in other words, community revitalization. As of November 2015, 45 regional collective trademarks for fishery products were registered in Japan (Regional collective trademarks 2015, Japan patent office). Well-known products include Seki horse mackerel and Seki mackerel from Oita Prefecture, Oma tuna from Aomori Prefecture, Echizen crab from Niigata Prefecture, and Hiroshima oysters from Hiroshima Prefecture. In general, seafood branding refers to fishery products that have gained a favorable position in the market by establishing a brand, using strategies that differentiate them from similar available fishery products. Building a brand fish is the key to success within the fishery industry. Recognition of the distinction is absolutely necessary to build a brand. The superior aspects of a brand fish, including increased consumer benefits, distinguish brand fish from the other fish available in the market. Therefore, brand fish are generally sold at a higher price as compared to non-brand fish. Although terms such as “seafood branding” or “brand fish” are commonly used, they have not yet been clearly defined. Moreover, there are no scientific reports focusing on the differences in taste, texture, and composition of the meat and the reasons for a given brand fish.

According to Yamanaka (2009), a brand fish is defined as: 1) a high value-added fishery product; 2) a product with more emphasis on quality than freshness; 3) a fish that is instantly killed and drained of blood immediately after catching, and in red sea bream, tuna, and yellowtail, the spinal nerve is additionally destroyed; 4) a fish product that is not frozen or thawed; 5) a seafood product that is pre-rigor or alive when served; 6) a fish that is wild, because wild fish are often better suited as a brand fish than farmed fish and; 7) a product in which the freshness index (K value) is ≤20% or preferably ≤10%. With regards to the quality of brand fish, transparency, texture, and taste are the most important factors. In addition, freshness is important, particularly as the practice of consuming raw fish meat, known as “sashimi,” is gaining popularity worldwide. In general, palatability is mainly associated with taste, flavor, color, shape, and texture; however, when raw seafood is eaten, its texture is one of the most important factors. It is currently thought that a sticky texture for tuna and a crunchy texture for flounder are indices of palatability. Unfortunately, the crunchy texture of red sea bream and flounder is gradually lost during refrigeration, and the fish meat softens. Conversely, one of the varieties of brand red sea bream produced at Uwajima, Ehime Prefecture, Japan, is known for its transparency and the crunchy texture of its meat, and is thought to be more resistant to softening.

Texture is affected by ante- and post-mortem factors. The composition of the connective tissue and the muscle fiber density affect the texture (Hatae et al., 1986; Hurling et al., 1996; Johnston et al., 2000). Apoptosis, or a programmed cell death, plays an important role in homeostasis and normal development in multicellular organisms. Apoptosis has been widely reported to occur in post-mortem muscle of livestock (Cao et al., 2010, Kemp et al., 2006). Caspases plays an essential role in apoptosis, in which a programmed cell death is characterized by a series of dramatic perturbations to the cellular architecture (Kurokawa and Kornbluth, 2009). Some reports have indicated that active caspase-3 is related to the degradation of myofibrillar proteins in chicken (Chen et al., 2011). As apoptosis of myocytes is estimated to be involved in the degradation of muscle protein after fish death, we focused on caspase-dependent apoptosis in fish. In addition, elucidating the relationship between caspase-3 and the texture of red sea bream muscle was considered to be essential.

The ubiquitin-proteasome pathway is another intracellular proteolytic pathway, which is activated during autophagy in skeletal muscle (Sandri, 2010). Autophagy, which is a cellular self-degradation pathway, is important for maintaining cell homeostasis and removing aggregated or misfolded proteins, thereby clearing damaged organelles and removing cellular components (Mizushima, 2007; Rubinsztein, 2006). Therefore, in the present study, we compared the protein expression levels of autophagy related factors between a commercially available brand red sea bream and a non-brand red sea bream, focusing on autophagy in fish skeletal muscle to clarify the relationship with the degradation of muscle protein.

We also evaluated the appearance and texture of red sea bream meat using organoleptic analysis to determine the relationship between the degradation of muscle protein with red sea bream meat qualities, such as the appearance and texture of “sashimi.” Our results provided scientific evidence about the taste of brand fish.

Materials and Methods

Organoleptic analysis    We used commercially available brand live red sea bream and non-brand live red sea bream (approximately 1.5–2.0 kg) specimens originating from Uwajima, Ehime Prefecture, Japan. and obtained from the Tokyo Metropolitan Central Wholesale Market, Tsukiji Market, Tokyo, Japan (July, 2013). Both red sea bream were instantly killed and drained of blood immediately after capture. One side of the dorsal muscle was sliced and subjected to organoleptic analysis approximately 6 h after treatment (Day 0), whereas the opposite side of the dorsal muscle was de-skinned and refrigerated at 4°C as a fillet. Proximate composition analyses were performed on a portion of the muscle samples. On the following day, the sample fillet was used as the Day 1 storage sample, and was subjected to similar organoleptic analysis as Day 0. We conducted a triangle difference test using a candidate panel (Age: 22–58, Gender: male 24/female 21), and ten individuals who were able to distinguish between commercially-available brand red sea bream and non-brand red sea bream in the preliminary screening were selected for a sensory evaluation panel. These panelists were previously trained in sensory evaluation (Age: 22–30, Gender: male 5/female 5). The fish were evaluated in terms of four indices, including appearance, odor, texture, and umami, using a scale from 1 (dislike extremely) to 5 (like extremely). Moreover, a paired preference test was conducted for appearance, taste, and total evaluation.

Proximate composition analysis    Moisture content was determined by drying the sample in an oven at 103°C to a constant weight. Crude protein content was quantified by the Kjeldahl method (N × 6.25). Ash content was determined after heating the sample to 550°C in a muffle furnace. Crude lipids were extracted from the dried samples with a Soxhlet apparatus. Samples were homogenized in ice-cold distilled water. Crude homogenates were deproteinized by the addition of sulfosalicylic acid. The supernatant was collected and stored in liquid nitrogen until subjected to amino acids analysis. Free amino acid profiles were determined using an automatic amino acid analyzer (Amino Acid Analyzer L-8900; Hitachi, Tokyo, Japan). Total lipids were extracted according to the procedure of Bligh and Dyer (1959). Fatty acid composition was analyzed according to the American Oil Chemists' Society (AOCS) official method Ce1h-05. Fish muscle was extracted with 0.6 M perchloric acid and analyzed for K value. K values were determined with the super freshness meter KV-202 (Central Kagaku Corp., Tokyo, Japan).

Sampling for protein expression    The commercially available brand red sea bream and the non-brand red sea bream were obtained from Uwajima, Ehime Prefecture, Japan (June, 2011). Selected fish of red sea bream varieties (approximately 1.5–2.0 kg) were instantly killed, drained of blood, and the central nerves were destroyed. After 30 min, the dorsal muscles were instantly frozen using 99.0% acetone cooled by liquid nitrogen. The frozen muscles were homogenized using a Cool-mill (Tokken, Inc., Chiba, Japan). The muscles were lysed using RIPA buffer with a protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan), and then lysed in Laemmli sample buffer (Laemmli, 1970) for Western blotting analysis.

Effect of apoptosis and autophagy on brand fish    The proteins were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and 5 µg of protein was loaded in each lane of the separation gel. The separated proteins on the gel were electrotransferred to a polyvinylidene difluoride membrane using a semi-dry blotting system (Biocraft, Tokyo, Japan). The membrane was blocked using tris-buffered saline containing 5% skim milk and 0.1% Tween 20 for 1 h. Subsequently, the membrane was incubated overnight at 4°C in medium containing the following primary antibodies: anti-caspase 3 produced in rabbits (Sigma, USA); p-mTOR (Ser2448; Bioworld, USA); ribosomal protein S6 kinase-1 (p70pS6K; Phospho-Ser424; Signalway Antibody, USA); and ATG5 Autophagy Related 5 Homolog (LifeSpan Biosciences, USA), followed by secondary antibodies (Alexa Flour ^®680 goat anti-rabbit IgG(H+L); Invitrogen, USA). The polyclonal antibodies used in the present study successfully and dose-dependently recognized the accurate molecular weight of the target protein in SDS-PAGE and the subsequent western blotting analysis as described by Saera-Vila et al. (2016) and Khor et al. (2016). The immunoreactive bands were detected using Odyssey Infrared Imaging Systems (LI-COR Biosciences, USA). Band intensity was quantified using ImageJ software (National Institutes of Health, USA), and the immunoreactive band intensities were normalized to that of beta-actin [anti-actin antibody (Ab-5), Thermo Scientific, Waltham, USA].

Statistical analysis    A student's t-test and binomial test were utilized to assess significant differences at a 5% level. (*P < 0.05, throughout the paper). All data are expressed as the mean ± SD.

Results

Organoleptic analysis    The organoleptic attributes of the brand and commercially available red sea bream are presented in Figure 1a and 1b. The brand red sea bream fillets showed a significantly better appearance (3.53 ± 0.54) and texture (3.22 ± 0.79) compared to the non-brand red sea bream (2.87 ± 0.27, 2.56 ± 0.5, respectively) on Day 0 (Fig. 1a). No significant differences were found for odor and umami. On Day 1 (Fig. 1b), there were no significant differences between the brand and the non-brand red sea bream for any parameter.

Fig. 1.

Sensory attributes scores of non-brand red sea bream and brand red sea bream (**P < 0.01, *P < 0.05, n = 10). (a) day 0 (b) day 1. black column: non-brand red sea bream, white column: brand red sea bream

In the preference test, nine of the 10 panelists preferred the appearance of the brand red sea bream on Day 0 (Fig. 2a). In addition, on Day 1, nine panelists preferred the taste of the brand red sea bream (Fig. 2b). Thus, a significantly greater number of individuals selected the brand red sea bream in terms of the appearance and taste of the meat on Day 0 and taste on Day 1 (Binomial test *p < 0.05).

Fig. 2.

Number of panelists of paired preference test of non-brand red sea bream and brand red sea bream (*P < 0.05, n = 10). (a) day 0 (b) day 1. black column: non-brand red sea bream, white column: brand red sea bream, gray column: even

Proximate composition    Table 1 shows the moisture, protein, and ash contents of the brand and the non-brand red sea bream, and no differences were observed between the two varieties. There were no differences in free amino acid composition of the muscle for any of the examined amino acids (Table 2). Although there were no significant differences in muscle fatty acid composition between the two varieties, the n-3 HUFA content was higher in the brand red sea bream (26.3% ± 0.49%) as compared to the non-brand red sea bream (21.0% ± 0.14%) (Table 3). The obtained K values are given in Table 4. The K value of both varieties gradually increased up to approximately 15% over the period examined. However, there were no significant differences between the brand and the non-brand red sea bream.

Table 1. Muscle proximate compositions of non-brand (N1, N2) and brand red sea bream (B1, B2).

	Non-brand red sea bream				Brand red sea bream
	N1	N2	Mean ± SD	B1	B2	Mean ± SD
Moisture (%)	71.8	71.8	71.80 ± 0.00	73.4	71.3	72.35 ± 1.49
Protein (%)	21.8	22.1	21.95 ± 0.21	21.3	21.4	21.35 ± 0.01
Lipid (%)	5.5	5.4	5.45 ± 0.07	4.3	6.5	5.40 ± 1.56
Ash (%)	1.5	1.4	1.45 ± 0.07	1.4	1.5	1.45 ± 0.07

Table 2. Analyzed free amino acid composition of muscle of non-brand (N1, N2) and brand red sea bream (B1, B2). (%/100 g muscle)

	Non-brand red sea bream			Brand red sea bream
Amino acids	N1	N2	Mean ± SD	B1	B2	Mean ± SD
Alanine	5.00	6.79	5.90 ± 1.26	6.84	5.88	6.36 ± 0.68
Arginine	0.84	0.90	0.87 ± 0.04	0.78	0.56	0.67 ± 0.15
Asparagine	N.D.	0.09	---------	N.D.	N.D.	---------
Aspartic acid	0.12	0.17	0.15 ± 0.04	0.05	N.D.	---------
Glutamate	3.55	3.21	3.38 ± 0.24	3.30	3.30	3.30 ± 0.00
Glutamine	1.92	3.87	2.89 ± 1.38	4.26	2.78	3.52 ± 1.05
Glycine	3.52	4.25	3.88 ± 0.51	4.19	3.76	3.98 ± 0.30
Histidine	4.41	6.01	5.21 ± 1.13	1.18	1.02	1.10 ± 0.11
Isoleucine	0.66	0.58	0.62 ± 0.05	1.28	1.20	1.24 ± 0.06
Leucine	1.06	0.90	0.98 ± 0.11	1.95	1.82	1.88 ± 0.09
Lysine	5.71	8.05	6.88 ± 1.65	3.52	1.95	2.74 ± 1.11
Methionine	0.49	0.41	0.45 ± 0.06	0.85	0.80	0.82 ± 0.03
Phenylalanine	0.48	0.38	0.43 ± 0.07	0.97	0.79	0.88 ± 0.13
Serine	0.95	1.01	0.98 ± 0.04	1.35	1.22	1.29 ± 0.10
Taurine	68.30	60.31	64.30 ± 5.64	65.06	71.03	68.05 ± 4.22
Threonine	1.40	1.69	1.55 ± 0.20	1.47	1.25	1.36 ± 0.15
Tyrosine	0.68	0.59	0.64 ± 0.06	1.28	1.13	1.21 ± 0.11
Valine	0.90	0.79	0.84 ± 0.08	1.67	1.51	1.59 ± 012
Sum	100.00	100.00	---------	100.00	100.00	---------

N.D.; non detected, -------------; not calculated

Table 3. Selected fatty acid compositions of muscle filets of non-brand (N1, N2) and brand red sea bream (B1, B2).

Fatty acid	Non-brand red sea bream			Brand red sea bream
(%)	N1	N2	Mean ± SD	B1	B2	Mean ± SD
14:0	2.6	2.6	2.60 ± 0.00	3.4	3.9	3.65 ± 0.35
16:0	17.4	17	17.20 ± 0.28	18.9	18.5	18.7 ± 0.28
18:0	4.9	4.6	4.75 ± 0.21	5.4	5.6	5.50 ± 0.14
16:1	4.3	4.4	4.35 ± 0.07	5.3	5.6	5.45 ± 0.21
18:1	23.5	23.5	23.50 ± 0.00	20.2	20.3	20.25 ± 0.07
18:2n-6	11.2	11.4	11.30 ± 0.14	4.8	4.7	4.75 ± 0.07
18:3n-3	1.4	1.3	1.35 ± 0.07	0.8	0.9	0.85 ± 0.07
20:1	3.5	3.6	3.55 ± 0.07	2.4	2.4	2.40 ± 0.00
20:4n-6	0.8	0.8	0.80 ± 0.00	1.3	1.2	1.25 ± 0.07
20:5n-3	4.5	4.4	4.45 ± 0.07	6.8	7.7	7.25 ± 0.64
22:1	2.1	2.2	2.15 ± 0.07	1.3	1.2	1.25 ± 0.07
22:5n-3	2.8	2.9	2.85 ± 0.07	4.0	4.2	4.10 ± 0.14
22:6n-3	12.2	12.5	12.35 ± 0.21	15.1	13.2	14.15 ± 1.34
n-3 HUFA	20.9	21.1	21.0 ± 0.14	26.7	26.0	26.35 ± 0.49
n-6 HUFA	12	12.2	12.1 ± 0.14	6.1	5.9	6.00 ± 0.14

Table 4. The K values of non-brand (N1, N2) and brand red sea bream (B1, B2) stored in refrigerator for 0 to 72 h.

	Non-brand red sea bream			Brand red sea bream
Hours	N1	N2	Mean ± SD	B1	B2	Mean ± SD
0	0	0	0	0	0	0
24	5	3	4.0 ± 1.4	3	5	4.0 ± 1.4
48	10	8	9.0 ± 1.4	8	7	7.5 ± 0.7
72	14	13	13.5 ± 0.7	16	14	15.0 ± 1.4

Effect of apoptosis and autophagy on brand fish    The protein expression level ratio of caspase-3 to the active form in the brand red sea bream (0.912 ± 0.081) was significantly higher than that in the non-brand red sea bream (1.000 ± 0.116), (P < 0.05; Fig. 3).

Fig. 3.

The ratio of protein expression level of caspase-3 with active form in the red sea bream muscle. (n = 6, *P < 0.05)

Although there were no significant differences observed in the phosphorylated target of rapamycin (TOR) ratio between the brand (0.898 ± 0.425) and the non-brand (1.000 ± 0.528) red sea bream (Fig. 4), there were significant differences in the ratio of phosphorylated p70S6K protein expression between the brand (1.152 ± 0.136) and the non-brand (1.000 ± 0.237) red sea bream (P < 0.05; Fig. 5). Protein expression levels of ATG5–ATG12 conjugate showed a significantly greater suppression in the brand (0.819 ± 0.087) than in the non-brand (1.000 ± 0.246) red sea bream (P < 0.05; Fig. 6).

Fig. 4.

The ratio of protein expression level of phosphorylated TOR in the red sea bream muscle. (n = 6)

Fig. 5.

The ratio of protein expression level of phosphorylated p70S6K in the red sea bream muscle. (n = 6)

Fig. 6.

The ratio of protein expression level of ATG5–ATG12 in the red sea bream muscle. (n = 6, *P < 0.05)

Discussion

In the present study, the appearance and texture of the commercially available brand red sea bream meat were significantly superior to those of the non-brand variety. The brand red sea bream muscle showed greater suppression of apoptosis and autophagy than that of the non-brand red sea bream, suggesting that degradation of muscle protein in the brand red sea bream was suppressed. According to Taylor et al. (2002), degradation of cytoskeletal components causes the deterioration of myofibrils, and the disruption of muscle fiber interactions would result in the softening of fish meat. In the present study, we hypothesized that the difference in appearance and texture of the brand red sea bream was caused by the suppression of protein degradation in muscle tissue.

In this study, the brand red sea bream showed significantly improved appearance and texture on Day 0; however, there were no significant differences on Day 1 (Fig. 1). Previous studies showed the marked decrease in the firmness and texture of fish fillets during storage (Liu et al., 2013; Zang et al., 2017), which might be related to a reduction in the integrity of muscle cells (Lu et al., 2015). Moreover, it was reported that the extent and rate of energy metabolism, protein degradation, and the activity of endogenous proteases affected the texture of fish fillets (Duun and Rustad, 2008; Zang et al., 2017). In this study, it is thought that energy metabolism and proteases induced apoptosis, and autophagy affected the appearance and texture of brand red sea bream after storage as observed in previous studies. Further studies are needed to clarify the relationship between texture and protein degradation in brand fish.

Our findings indicated that the brand red sea bream appeared appetizing (Fig. 2a). Although the firmness decreased during refrigeration, ATP degradation contributed to an improvement in taste (Massa et al., 2005 and Kawai et al., 2002). Therefore, the taste of the brand red sea bream was enhanced after 1 day of refrigeration (Fig. 2b). No difference in the contents of glutamate, which contributes to the umami taste, was noted between the two varieties (Table 2). Sikorski (1990) stated that arginine, histidine, isoleucine, leucine, methionine, phenylalanine, and valine impart a bitter taste, whereas alanine, glycine, serine, and threonine contribute to a sweet taste. It was revealed that histidine, which contributes to a bitter taste, tended to be lower (no significant differences) in the brand red sea bream as compared to the non-brand red sea bream. Because of the lower histidine levels, the brand red sea bream may be tastier than the commercially available red sea bream. Khan (2013) revealed that dietary histidine levels affect histidine retention and body composition. Therefore, it was considered that different types of fish feed should be used for the brand red sea bream, for example, live bait. It was reported that n-3 HUFA, which include docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), modulates many diseases, such as cardiovascular diseases (Mozaffarian and Wu, 2011) and Alzheimer's disease (Laitinen et al., 2006). Our findings indicate that the brand red sea bream had higher n-3 HUFA levels as compared to the non-brand red sea bream, although not significantly so (Table 3). The n-3 HUFA are essential fatty acids, and marine fish are the main sources of n-3 HUFA in the diet (Pawlosky et al., 2001). Therefore, if the brand red sea bream were fed a more n-3 HUFA-rich diet, it could be considered a n-3 HUFA-rich fish, making it a high value-added product with a competitive advantage.

It is generally established that biochemical changes influence the texture of fish muscles. Yamashita and Konagaya (1991) determined that autolysis and proteolysis is additionally involved in the softening of fish meat. In general, higher autolytic activity of endogenous proteases in major muscles, which is caused by apoptosis and autophagy, induces fish muscle protein hydrolysis. These phenomena contribute to the softening of fish muscles during post-mortem storage. Post-mortem softening of muscle is related to connective tissue degradation, resulting from a decrease in the adhesion between the endomysium and myocytes during storage (Taylor et al., 2002). In addition, the soft texture of muscles is related to proteolytic degradation of cell membrane components and the extracellular matrix during storage (Bahuaud et al., 2010; Martinez et al., 2011). It is additionally claimed that muscle softening and myofiber detachment are correlated with the degradation of cytoskeletal proteins in the sea bream Sparus aurata (Caballero et al., 2009). According to Yoshida et al. (2009), the collagen in the muscle of red sea bream is broken down by serine protease and metalloproteinase, resulting in softening of the fish meat. It is assumed that myocyte apoptosis after fish death is involved in muscle proteolysis. Two major pathways of caspase activation have been elucidated in vertebrates undergoing apoptosis (Yamashita, 2003). Upstream signals of the apoptotic pathway induce a caspase family (Szegezdi et al., 2006). In the present study, a lower caspase-3 protein level was observed in the brand red sea bream muscle as compared to the non-brand red sea bream muscle (Fig. 3).

Degradation of structural proteins has been identified as the cause of the tenderness in pork and beef (Ahn et al., 2003). A previous study reported that caspase-3 was associated with proteolysis involved in meat conditioning (Kemp et al., 2006). An executioner caspase, which is involved in cell apoptosis, results from the proteolytic degradation of intracellular structural proteins (Fischer et al., 2003). According to Elmore (2007), caspase-3 is the most important executioner caspase, and is responsible for the proteolysis of proteins. In our results, we expected that caspase-3 would be associated with the decline in firmness and the acceleration of proteolysis in the non-brand red sea bream muscles during post-mortem conditioning.

Autophagy is known to be inhibited by TOR signaling (Noda and Ohsumi, 1998). Autophagy induction is associated with reduced phosphorylation of its downstream effectors, p70S6K at Thr389/Thr421/Ser424 (Klionsky and Emr, 2000). Moreover, activated p70S6K suppresses autophagy (Radimerski et al., 2002). In the present study, western blot analysis revealed that phosphorylated p70S6K at Ser424, which is located downstream of TOR, was significantly increased in the brand red sea bream muscle as compared to the non-brand red sea bream muscle (Fig. 5). Therefore, in the present study, autophagy was considered to be more suppressed in the brand red sea bream muscle than in the non-brand red sea bream.

Autophagy involves the sequestration of portions of cytoplasm within an enveloping double-membrane structure called the autophagosome. The formation of the autophagosome is induced by the inhibition of mTOR (Schmelzle and Hall, 2000). Autophagy related gene (atg) proteins are important for autophagic sequestration, and the ATG5–ATG12 conjugation step is key to autophagy (Mizushima, 2007). ATG5–ATG12 conjugate promotes autophagy by promoting the extension of the autophagosome membrane. Figure 2 b shows a significantly greater suppression of ATG5–ATG12 conjugate expression in the brand red sea bream than in the non-brand red sea bream. Therefore, the autophagosome expansion was suppressed to a greater extent in the brand red sea bream compared to the non-brand red sea bream, suggesting suppressed autophagy. The abnormal softening of the muscle of spawning salmon is caused by autophagy (Yamashita and Konagaya, 1991). The findings of the present study suggested that softening of the brand red sea bream muscle is inhibited through the suppression of muscle tissue proteolysis, resulting in the crunchy texture of the brand fish muscle. These results for protein expression were supported by the results of organoleptic analysis.

Gan et al. (2014) reported that dietary isoleucine deficiency led to the deterioration of flesh quality, for example muscle softening of grass carp, and high isoleucine concentration in the diet improved the fish meat quality because of antioxidant defense through the NF-E2-related factor 2 (Nrf2) signaling pathway. Ichimura et al. (2013) found that selective-autophagy pathways and Keap1-Nrf2 were connected.

Recently, Belghit et al. (2014) revealed that dietary methionine affects muscle protein turnover in rainbow trout. They found that methionine could modify the phosphorylation of p70S6K, which is mediated by the TOR signaling pathway. Moreover, a methionine-deficient diet induced the major proteolytic pathways, including autophagy. Further, the restriction of methionine or its metabolites affects the oxidative status, resulting in apoptosis and inflammation (Ozden et al., 2009; Devi and Anuradha, 2010).

As the isoleucine and methionine concentrations in fish muscle were reflected in dietary isoleucine and methionine levels (Espe et al., 2014; Gan et al., 2014), in the present study, isoleucine and methionine levels of the brand red sea bream muscle were higher than those of the commercially-available red sea bream (Table 2). Although we did not investigate the fish feed, it is possible that high isoleucine and methionine levels led to an improvement in fish quality and suppressed muscle softening.

We concluded that apoptosis and autophagy could be key factors affecting the skeletal muscle quality of red sea bream. The results indicated that the brand fish was obviously tastier than the non-brand fish. Because of its good taste and high quality, brand fish are usually more expensive than non-brand fish.

Although the key factor responsible for the good quality of fish muscle remains unknown, our study confirmed that the brand red sea bream muscle had a superior quality and taste as compared to the non-brand red sea bream. Future studies will reveal the mechanisms responsible for maintaining the good quality of brand fish in more detail.

Acknowledgments    This work was partially supported by JSPS KAKENHI Grant Number JP 25850142, Grant-in-Aid for Young Scientists (B; RN). We would like to thank Professor Hideki Ushio of the University of Tokyo for helpful discussions. We also thank Yoshihide Okazaki of Okazaki Fishery and Seiji Matsumoto of Scientific Feed Laboratory Co., Ltd. for supplying the red sea bream.

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