Biochemical characterization of abnormally softened sardine “soggy meat” during the spring spawning migration

doi:10.21203/rs.3.rs-2214683/v1

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Biochemical characterization of abnormally softened sardine “soggy meat” during the spring spawning migration

https://doi.org/10.21203/rs.3.rs-2214683/v1

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“Soggy meat”, which is abnormally softened crushed sardine meat, is often detected in canned sardines seasoned with tomato chili sauce. As soggy meat represents lysed and decomposed tissue, we examined protease activity in the sardine meat during the spawning season. Abnormally soft sardine meat is often found in canned sardine meat from fish caught during the spring, and landed at Choshi Fishing Port in Japan, located on the Pacific Ocean. We measured cathepsin L activity in the muscle and protein, lipid, and moisture, as well as the trace elemental concentrations. High cathepsin L activity was detected in February and March during the Pacific sardine spawning season. In addition, 4 of 20 fish had extremely high protease activity of more than 30 unit/g

. Cathepsin L activity in the muscle was negatively correlated with selenium, protein, and lipid concentrations, indicating that sardines in the spring were starving during their spawning migration. Therefore, starved physiological conditions during the spawning season may be responsible for soggy meat.

Abnormally soft sardine meat or “soggy meat” is often found in canned sardines from fish caught during the spring and landed at Choshi Port in Japan, located on the Pacific Ocean. Frozen Pacific sardines are exported to Thailand to produce canned products seasoned with chili sauce. Thus, we characterized the cause of the abnormal sardine meat, to reduce the incidence of soggy meat.

Similar abnormally soft meat has been reported in chum salmon, and high proteolytic activity was detected in the muscle of fish caught during their spawning migration (Konagaya 1982; Yamashita & Konagaya 1990a,b). Such salmon meat reveals high cathepsin L activity because of the induction of autophagy induced by starvation and sexual maturation (Yamashita & Konagaya 1990a,b). Proteolysis proceeds extensively in muscle when autolytic activity is very high (Yamashita & Konagaya 1990a,b). The muscle softening is accelerated when the muscle undergoes a freeze-thaw cycle (Konagaya 1982; Yamashita & Konagaya 1990a,b). Cathepsin L is the most probable enzyme responsible for muscle autolysis as it causes extensive muscle softening that is often observed in mature chum salmon catch (Konagaya 1982; Yamashita & Konagaya 1990a,b). Immunohistochemical localization of cathepsin B and L in the white muscle of chum salmon during the spawning migration suggested the probable participation of phagocytes rich in cathepsins in the extensive muscle softening of mature salmon (Konagaya 1982; Yamashita & Konagaya 1990a,b). A similar phenomenon of muscle softening through activation of proteolytic activity in muscle has been reported in sweetfish (Yamashita et al. 1990), Japanese flounder (Toyohara et al. 1993), and mackerels (Aoki et al. 1997; Konagaya 1982; Yamashita & Konagaya, 1990; 1991; 2010; Yamashita & Yamashita 2010) during their spawning migrations. To understand the cause and biochemical mechanisms of this softening phenomenon of sardine meat during the spawning season, we determined cathepsin L activity and muscle chemical composition, such as protein, lipid, moisture, and selenium concentrations.

Selenium deficiency is one of the most common nutritional deficiencies in beef cattle (Combs & Combs 1986). It causes diarrhea, reduces weight gain, and can cause vaccines to be less effective. A selenium deficiency in calves can cause white muscle disease (WMD), also known as nutritional muscular dystrophy (WMD can also be caused by a vitamin E deficiency). In addition to cattle, WMD can occur in lambs, kids, poultry, and pigs in areas where soils are deficient in selenium and, therefore, the forage plants growing on those soils are deficient (Combs & Combs 1986). For example, volcanic soils are often deficient in selenium (Combs & Combs 1986). Even if a farm is not selenium deficient, it can be affected by WMD if the feed is imported from an area with soils that are deficient in selenium.

After catching fish, extensive apoptosis and autophagy occur in the white muscle, and hemolysis occurs in fish that contain blood selenium levels < 1 µg/g tissue (Yamashita & Yamashita, 2010). The antioxidant effects of selenoneine may be essential in enabling fish to adapt and survive in a low-oxygen marine environment. In rats, selenium deficiency induces hemolysis (Combs & Combs 1986). WMD in selenium-deficient animals exhibits similar features (Combs & Combs 1986).

Therefore, we focused on the enhanced cathepsin L activity induced by autophagy, related to the stress response and starvation during the Japanese sardine spawning season. We obtained frozen sardines caught from the Pacific Ocean off Choshi and measured cathepsin L activity in the muscle as well as protein, lipid, moisture, and selenium concentrations.

Materials

Sodium selenite was purchased from Fuji Film Wako Pure Chemical Corporation (Osaka, Japan).

Fish

The frozen samples of Sardinops melanostictus were obtained from the Chiba Prefectural Research Center (Table 1).

Table 1

Pacific sardines used in the experiment.
Fishing date	Fishing location	Body length (cm)	Body weight (g)	Individual number
Feb. 1, 2021	Kashima	19.6 ± 1.6	52.0 ± 14.9	20
Feb. 9, 2021	Izuoushima Southeast	21.4 ± 1.3	70.1 ± 12.4	10
Feb. 19, 2021	Izuoushima Southeast	18.4 ± 1.0	44.9 ± 6.0	10
March 26, 2021	Hitachi	21.2 ± 1.8	58.8 ± 13.7	20
May 7, 2021	Inubo Southeast	21.5 ± 1.2	75.1 ± 17.6	20
June 3, 2021	Kujukuri	21.6 ± 1.0	94.2 ± 15.8	20
June 7, 2021	Kujukuri	22.8 ± 0.8	103.1 ± 6.6	10
June 22, 2021	Kashima	20.2 ± 1.0	75.9 ± 14.7	10
July 16, 2021	Kashima	22.0 ± 1.0	107.8 ± 15.1	10
July 21, 2021	Kujukuri	21.8 ± 1.0	126.5 ± 8.9	10

Selenium Concentration Measurement

Total selenium concentration was determined by a fluorometric assay with 2,3-diamino-naphthalene, after digestion in 1.5 mL of nitric acid and perchloric acid (1:2 in volume) at 200–220℃ (Yamashita & Yamashita 2010).

Total lipid extraction

Total lipid was extracted according to the method of Bligh and Dyer (1959).

Protease assay

Cathepsin L activity was measured with Z-Phe-Arg-MCA (2 µM) as the substrate according to Yamashita & Konagaya (1990a,b). One unit of cathepsin L activity corresponds to the production of 1 pmol of methylcoumarylamine per min at 37 ˚C. Protein concentration was determined by the method of Lowry et al. (1951).

Moisture measurement

Moisture content was measured with a moisture analyzer (Adn MX-50) according to the manufacturer’s instructions.

Cathepsin L activity was measured as a biochemical indicator of autolysis induced under stress conditions in muscle tissues obtained from February to July 2021 (Fig. 1). Cathepsin L activity increased from 7.8 to 306 unit/g in March, and then decreased to 88 unit/g in May, and 5.8 pmol/min in June, which was later enhanced to 44.6–47.1 unit/g. In the spawning season, only 4 of 20 fish had extremely high protease activity of more than 30 unit/g. Thus, cathepsin L activity in the muscle was at high levels from February to May, particularly from February to March. Individual differences were detected among samples.

We also measured various chemical muscle components to characterize the fish meat quality and the nutritional and physiological condition of the sardines (Fig. 1).

Muscle lipid content ranged from 0.97% in May off Inubou to 3.0% in July off Kujuukuri. Low lipid levels (< 1% of muscle tissue) were detected from March to May (Fig. 1).

Selenium concentration ranged from 268 ng/g in February to 92 ng/g in May. The selenium concentration was lower (< 100 ng/g) during March and May than in other seasons (Fig. 1).

Muscle protein concentrations varied from 20.7 mg/g in February to 149 mg/g in July. The lowest levels of proteins were 20.7 mg/g in February and 23.8 mg/g in March, and then the protein concentration increased gradually to 149 mg/g in July (Fig. 1).

Moisture content ranged from 76.1 to 77.2% in February, 76.5% in March, 80.6% in May, 69.8–80.8% in June, and 65.0–70.5% in July. Moisture content was negatively correlated with lipid content (Fig. 1).

The condition factor depends on the marine environmental conditions. Figure 1 shows a condition factor of 6.83 on February 1, 7.09 on February 9, 7.21 on February 19, 6.13 on March 26, 7.50 on May 7, 8.78–9.17 in June, and 10.1–12.4 in July. The condition factor was highest in February and March. Figure 1 shows that the condition factor was positively correlated with muscle protein and selenium concentrations (Fig. 2). In contrast, moisture content was negatively correlated with the condition factor (Fig. 2).

“Soggy meat”, or abnormally soft crushed sardine meat, is often observed in canned sardines seasoned with tomato chili sauce. As the soggy meat is lysed and decomposed, we considered that the cause may be proteolytic degradation during the processing of the canned product before heating. As fish meat is enhanced during the spawning season, such as that of chum salmon, sweetfish, and mackerel (Yamashita 2010; Yamashita & Konagaya,1990a,b; Yamashita et al. 1991), we examined protease activity in sardine meat. Cathepsin L has optimal activity at a pH of 5.6 in chum salmon and mackerels, and its activity increases during the spawning season. The activity is induced in response to starvation and other stressors (Yamashita 2010; Yamashita & Konagaya 1990a,b; Yamashita et al. 1991).

High sardine muscle cathepsin L activity was detected in February and March. The highest activity was found on March 26. The Pacific sardine spawning season occurs in February and March. In addition, among 20 fish captured on March 26, four had more than 30 unit/g cathepsin L activity.

The meat of those fish also had high levels of other components, such as < 73.5 ng/g of selenium and < 73.7 mg/g of protein. From these findings and previous results reported in several fish species, starvation and nutritional depletion of sardines during the spring spawning migration may be related to the extensive proteolytic activity of cathepsin L and muscle softening, as in other fish species. Stress-induced autophagy accompanied chaperone-mediated autophagy and lysosomal cathepsin L activities during the thawing of the frozen fish and high-pressure sterilization during the canning process.

Therefore, the cause of the soggy meat from Pacific sardines caught during the spring spawning season was due to abnormally high muscle cathepsin L activity. When we measured cathepsin L activity in the muscle of sardine caught at Choshi, 4 of 20 fish had high cathepsin L activity (> 3 units/g). These fish also had low selenium concentrations of < 100 ng/g and protein concentrations of < 100 mg/g. Avoiding the use of fish containing high cathepsin activity would prevent abnormal soggy meat canned products. Thus, sardines may be not preferable for processing during their spring spawning season because of the high protease activity and low quality.

Conflicts of interest/Competing interests

The authors have no relevant financial or non-financial interests to disclose. The authors have no conflicts of interest to declare that are relevant to the content of this article.

Funding declaration

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Data availability statement

All data generated or analysed during this study are included in this published article.

Author contributions statement

Mayu Ito, Ryoga Noda, Sigetaka Honda, Yukari Matsuoka, Ruki Igura, Himawari Ueda, Kana Kinoshita, Michiaki Yamashita, and Masayuki Inoue measured biochemical components. M. Yamashita wrote the main manuscript text and prepared figures. All authors reviewed the manuscript.

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No competing interests reported.

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Biochemical characterization of abnormally softened sardine “soggy meat” during the spring spawning migration

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