Abstract
The physicochemical properties of recovered protein from Japanese anchovy using salt water treatment and pH-shift processing were investigated and compared. The protein recovery yield was the highest with the acid-aided treatment, followed by the salt water and alkaline-aided treatments. No significant differences were found between minced fish and the recovered protein using salt water treatment in L* value, b* value and Ca-ATPase activity. Moreover, myofibrillar protein extracted from the recovered protein using salt water and acid-aided treatments exhibited lower surface hydrophobicity than that using the alkaline-aided treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated a greater concentration of myosin heavy chain and actin in the recovered protein by the salt water treatment, while those proteins were degraded in the acid and alkaline-aided treatments. Overall, the results demonstrated that the physicochemical properties of the recovered protein using salt water treatment were superior to those using pH-shift processing.
Introduction
Japanese anchovy (Engraulis japonicus) is a small pelagic fish with a wide distribution and spawning area around South East Asia and Japan (Funamoto et al., 2004). It is one of the most important fish harvested from cold and temperate waters in Japan and is used mostly for food (Kishimura et al., 2005). The total catch reported for this fish was 1, 304, 484 tons in 2016 (FAO, 2018). Its body length at maturity is approximately 80–90 mm (Takasuka et al., 2005). In Japan, most of this fish is boiled and subsequently dried to produce niboshi products (Haratake et al., 2007). Anchovies have high nutritive value, mainly attributed to essential amino acids, minerals (including calcium and iron) and highly unsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid (Kim and Heu, 2011). On the other hand, they contain high amounts of myoglobin in the muscle. This component exerts a negative effect on several qualities, such as color, shelf-life and lipid oxidation (Chaijan and Undeland, 2015). Moreover, anchovy degrades rapidly due to its high endogenous protease activity (Sato and Takagi, 2017) and meat separation is difficult due to its small size. Therefore, alternative processing methods are needed to drive increases in the utilization of Japanese anchovy.
In an earlier study, Hultin et al. (2005) showed that pH-shift processing can be used for protein recovery. This process solubilizes and precipitates proteins based on their isoelectric behavior. Additionally, protein was recovered from raw materials in which it is difficult to obtain the meat, such as krill, fish, chicken and by-products of beef processing (Matak et al., 2015). Furthermore, Chen et al. (2016) pointed out that the recovered protein is denatured during acid and alkaline treatments. Therefore, it is apparent that new methods for protein recovery from difficult-to-obtain meats should be explored. It is well known that myofibrillar protein is soluble in mild salt solutions, such as NaCl, KCl, or LiCl at neutral pH conditions (Tan et al., 2019). Amano et al. (2017) reported a new method, salt water treatment, for the recovery of protein from North Pacific krill (Euphausia pacifica). Tan et al. (2019) reported that the electrophoretic pattern of protein isolated using a salt solution extraction method was similar to commercial surimi products and that a cooked protein gel produced using the salt solution extraction method was more viscoelastic. Thus, this method might be an alternative method for protein recovery from raw materials such as difficult-to-obtain meats. Many studies have reported on the composition, nutritional and physicochemical properties of recovered protein from many fish species. However, there have been no reported studies on the extractability of proteins from whole small fish or the comparative study of physicochemical properties of proteins yielded by pH-shift processing and salt water extraction. Therefore, the objective of this study was to investigate the effects of a salt solution extraction method on the physicochemical properties of proteins recovered from whole minced Japanese anchovy in comparison to pH-shift processing.
Materials and Methods
Raw material Japanese anchovy (Engraulis japonicus) was caught on November 2017 offshore of Nagasaki Prefecture. The fish were anesthetized by immersing in an ice-slurry, then placed on ice and transported to our laboratory. After arriving at our laboratory, the fish were ground with 7.5% (w/w) sucrose using a meat chopper (M-22A, Nantsune, Osaka, Japan). The minced fish meat was packed into polyethylene bags and stored at −30 °C until use.
Determination of protein solubility at various concentrations of NaCl Ten grams of minced fish meat was mixed with 100 mL of 20 mM Tris-HCl buffer (pH 7.0) and then homogenized on ice at 10 000 rpm for 1 min. The homogenate was centrifuged at 1 500 × g, 4 °C for 5 min and then the supernatant was discarded. The pellet was washed three more times as above. The pellet (1 g) was mixed with 20 mL of NaCl − 20 mM Tris-HCl buffer (pH 7.0) at various NaCl concentrations (final concentration of NaCl between 0 and 10% at 1% intervals). The mixture was homogenized on ice at 10 000 rpm for 1 min and then shaken for 1 h using a seesaw shaker (Wave-SI, Taitec, Saitama, Japan). The mixture was centrifuged at 15 000 × g for 30 min. The protein concentration of the mixture before and after centrifugation was determined by Lowry's method (Lowry et al., 1951; Peterson, 1979). Solubility (%) was calculated using the following equation.
Where PA and PB are the protein concentrations of the mixture after and before centrifugation, respectively.
Protein recovery by salt water treatment Recovered protein by salt water treatment was prepared following the method of Amano et al. (2017) with slight modifications. Minced fish was homogenized with cold ion exchanged water (IEW) and 8% salt water at a ratio of 1:1:2 (w/v/v), respectively, (final concentration of NaCl = 4%) and continuously shaken for 10 min using a seesaw shaker. The mixture was then centrifuged at 15 000 × g, 2 °C for 10 min to precipitate insoluble matters, including scales, eyeballs, bones and connective tissue. Next, the supernatant was diluted 10 times with cold IEW and stirred for 30 min at 4 °C to precipitate the protein. The recovered protein was collected by centrifugation at 15 000 × g, 2 °C for 10 min.
Protein recovery by acid-aided treatment Protein was recovered from minced fish by acid-aided treatment according to the method of Rawdkuen et al. (2009). Minced fish was homogenized with cold IEW (1:9, w/v) for 1 min at 10,000 rpm on ice. The homogenate was adjusted to pH 3.0 with 2 N HCl, stirred at 4 °C for 10 min and then centrifuged at 15 000 × g, 2 °C for 10 min to remove insoluble material. The supernatant was collected and adjusted to pH 5.5 with 2 N NaOH, followed by stirring at 4 °C for 30 min to precipitate the protein. The centrifugation was performed as described previously. The precipitate was mixed with cold IEW water (1:3, w/v), adjusted to pH 7.0 and centrifuged again.
Protein recovery by alkaline-aided treatment The alkaline-aided process was performed according to the method of Rawdkuen et al. (2009) with slight modifications, as described previously, except that the sample pH was adjusted to 11.2 with 2 N NaOH. Soluble proteins were recovered by isoelectric precipitation at pH 5.5 and collected as described above.
Proximate analysis Moisture, ash and protein contents of minced fish and recovered protein were determined according to standard AOAC (AOAC, 1990) methods. The samples were oven-dried (DRA430, Advantec, Tokyo, Japan) at 105 °C until a constant weight was achieved. Ash content was determined by the dry ashing method at 550 °C for 24 h in a muffle furnace (FO300, Yamato Scientific, Tokyo, Japan). Crude protein content was determined by the Kjeldahl (AOAC, 1990) method. Percentage of crude protein was calculated by multiplying%N with a factor of 6.25. Crude lipid was determined according to the method of Folch et al. (1957).
Protein recovery yield The protein recovery yield was expressed as the weight of protein in recovered protein divided by the weight of protein in minced fish. The protein recovery yield was calculated using the following formula.
Where WRP and WM are the weight of protein (g) in recovered protein and raw minced fish, respectively.
Color properties A color reader (CR13, Konica Minolta, Osaka, Japan) was used to determine the color of minced fish and recovered protein. The values for the CIE (International Commission on Illumination) color system using L* a* b* tristimulus color values were determined. The color difference (ΔE) was calculated using the following equation.
Where L*RP, a*RP and b*RP are the color values of recovered protein and L*M, a*M and b*M are the color values of minced fish.
Total heme protein Total heme protein was determined according to the method of Chaijan and Undeland (2015). Minced fish or recovered protein (5 g) was homogenized with 15 mL of 0.1 M phosphate buffer (pH 7.0, 5% sodium dodecyl sulfate (SDS) (w/v)). The homogenate was then incubated at 85 °C for 1 h. After cooling, the mixture was centrifuged at 5 000 × g for 15 min at room temperature. The absorbance of the supernatant was measured using a UV-spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan) at 535 nm. Total heme protein concentration was calculated according to a standard curve prepared from bovine hemoglobin (H2625, Sigma, St. Louis, MO, USA)
Myofibrillar protein extraction Myofibrillar protein was prepared according to the method of Amano et al. (2017) with slight modifications. The sample (5 g) was homogenized in 40 mL of chilled buffer solution (0.16 M KCl, 40 mM Tris-HCl, pH 7.5) for 1 min at 10 000 rpm. The homogenate was centrifuged at 3 000 × g for 7 min at 4 °C. The pellet was washed three more times with the same buffer using the same homogenization and centrifugation condition as indicated above. The pellet was suspended in 20 mL of chilled buffer solution (0.16 M KCl, 40 mM Tris-HCl, pH 7.5).
Ca-ATPase activity Ca-ATPase activity was determined according to the method of Amano et al. (2017). One hundred microliters of 0.5 M Tris-maleate (pH 7.0), 100 µL of 0.1 M CaCl2, 440 µL of 2.0 M KCl and 1 060 µL of IEW were added to 200 µL of myofibrillar suspension (2.5 mg/mL). The reaction was started by adding 100 µL of 20 mM ATP to the mixture and then incubated for 8 min at 25 °C. The reaction was terminated by adding 1 mL chilled 15% (w/v) trichloroacetic acid. One milliliter of the supernatant was removed after centrifugation at 4 000 × g for 10 min and reacted with 1.0 mL of molybdic acid sulfate, 500 µL of Elon reagent and 2.5 mL of IEW. The mixture was allowed to react for 45 min. The absorbance of the liberated inorganic phosphate was measured using an UV-spectrophotometer at 640 nm.
Surface hydrophobicity Hydrophobicity was determined using a hydrophobic fluorescence probe, 1-anilino-8-naphthalene sulfonate (ANS), according to the procedure of Yongsawatdigul and Park (2003). To obtain a series of protein concentrations from 0 to 1 mg/mL at 0.2 intervals, the supernatant of myofibrillar protein was diluted with 20 mM potassium phosphate buffer (pH 7.0) containing 0.6 M KCl. Then, the samples (4 mL) with different protein concentrations were mixed with 20 µL of 8 mM ANS in 0.1 M potassium phosphate buffer (pH 7.0) and placed at room temperature for 10 min. Fluorescence intensities were measured using a spectrofluorometer (FP-8600, Jasco, Tokyo, Japan) at excitation and emission wavelengths of 374 and 485 nm, respectively, with 5 nm width for both the excitation and emission slits. The initial slope (S0) of relative fluorescence intensity (R) versus the percentage (w/v) protein concentration was calculated by linear regression analysis and represented as protein surface hydrophobicity. The relative fluorescence intensity (R) was calculated using the following formula.
Where F is the fluorescence of the protein–ANS conjugate and F0 is the reading of the ANS solution without protein.
Reactive and total sulfhydryl groups (SHs) Total SHs were determined according to the method of Yongsawatdigul and Park (2003). To 1 mL of myofibrillar suspension (4 mg/mL), 9 mL of buffer (pH 7.0) containing 50 mM potassium phosphate, 10 mM ethylenediaminetetraacetic acid, 0.6 M KCl, and 8 M urea were added and the mixture was centrifuged at 10 000 × g for 15 min. A 4.0-mL aliquot of the supernatant was added to 0.4 mL of Ellman's reagent (0.1% DTNB in 0.1 M sodium phosphate, pH 8), and the mixture was incubated at 40 °C for 25 min. The absorbance was measured using an UV-spectrophotometer at 412 nm to calculate the total SH groups using the extinction coefficient of 13,600 M−1 cm−1 (Ellman, 1959). Reactive SH groups were measured by incubating the reaction mixtures in the absence of urea at 4 °C for 1 h.
Protein pattern Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of raw minced fish and recovered protein was performed according to the method of Laemmli (1970) and Takahashi et al. (2016) using 5–20% gradient polyacrylamide gels (e-PAGEL, ATTO Corporation, Tokyo, Japan). A 0.5-g portion of sample was dissolved in 20 mL of 20 mM Tris-HCl (pH 8.8), 2% SDS, 8 M urea and 2% 2-mercaptoethanol. In each sample, 10 µg of protein was loaded onto the precast gel and subjected to electrophoresis at a constant current of 20 mA per gel. PageRuler Unstained Protein Ladder (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used as a standard for protein molecular weight determination. After electrophoresis, the gel was stained in methanol/acetic acid/water (30:10:60% v/v) containing 0.1% Coomassie Brilliant Blue R-250, and then destained in methanol/acetic acid/water (30:10:60% v/v).
Statistical analysis Statistical analysis on a completely randomized experimental design was performed using Excel XLSTAT 2018 software (Addinsoft Inc., Boston, MA, USA). One-way analyses of variance (ANOVA) were carried out and mean comparisons were made with Duncan's multiple range test. All reported data are shown as mean values ± standard deviation.
Results and Discussion
Solubilization of myofibrillar protein Figure 1 shows the effect of NaCl concentration on the protein solubility of Japanese anchovy. The protein solubility increased significantly, from 10.63 to 74.17%, with increasing NaCl concentration from 0 to 4%. However, there were no significant differences in protein solubility between the 4 and 5% NaCl concentrations (p > 0.05). Thus, the 4% NaCl concentration was optimal for the solubilization of myofibrillar protein in Japanese anchovy. This result is comparable to that for recovering the myofibrillar protein from catfish (Ictalurus punctatus) as previously reported by Tan et al. (2019). Myofibrillar protein is a major protein in fish and accounts for 40–60% of the total crude protein in fish species (Hui et al., 2007). Additionally, this protein is generally solubilized in high ionic-strength solution (Sun and Holley, 2011).
Chemical composition and protein recovery yield The proximate composition of minced fish without sucrose and recovered proteins is shown in Table 1. Following protein recovery, significant differences (p < 0.05) were found in moisture, protein, lipid and ash contents between minced fish and recovered protein samples. The highest protein content was obtained with the acid-aided treatment (88.74%), followed by the alkaline-aided (80.87%) and salt water (78.36%) treatments. The reason for the high recovery of the acid-aided process is likely attributable to the recovery of sarcoplasmic proteins. In general, sarcoplasmic proteins, including enzymes and heme protein, are water soluble and can be removed by water washing (Chang et al., 2016). The ash content of the recovered protein using salt water treatment and pH-shift processing was lower than for minced fish. This result indicates that impurities, such as bones and scales, in the minced fish were removed during the recovery process.
Table 1.
Proximate composition and protein recovery yield of minced fish and recovered protein prepared by different treatment.
|
Minced fish |
Salt water |
Acid-aided |
Alkaline-aided |
| Moisture (g/100 g sample) |
74.59c ± 0.07 |
93.77ab ± 1.37 |
93.19b ± 0.30 |
94.64a ± 0.81 |
| Protein (g/100 g dry basis) |
55.65c ± 6.83 |
78.36b ± 4.46 |
88.74a ± 2.70 |
80.87b ± 1.32 |
| Lipid (g/100 g dry basis) |
5.97b ± 0.53 |
11.37a ± 0.95 |
6.36b ± 1.24 |
6.16b ± 1.68 |
| Ash (g/100 g dry basis) |
9.89a ± 0.16 |
8.03b ± 1.58 |
1.84c ± 0.27 |
1.96c ± 0.35 |
| Protein recovery yield |
- |
42.93b ± 1.43 |
54.60a ± 2.62 |
41.94b ± 5.80 |
Means ± standard deviation (n = 4).
Means with different letters within the row are significantly different (p ≤ 0.05).
“Salt water”, “Acid-aided”, and “Alkaline-aided” mean protein recovered by salt water treatment, acid-aided treatment and alkaline-aided treatment, respectively.
The protein recovery yields for each process are shown in Table 1. The protein recovery yield was approximately 43, 55 and 42% for the salt water, acid-aided and alkali-aided treatments, respectively. The acid-aided treatment resulted in the highest protein recovery (p < 0.05). Several researches also reported that the protein recovery yield using the alkaline method was higher than that of the salt extraction method, since the pH adjustment enhances the repulsive force among protein molecules compared with adding salt (Stefansson and Hultin, 1994; Tan et al., 2019). The high yield of the acid-aided process was likely due to the recovery of sarcoplasmic proteins from the muscle. The acid-aided treatment leads to higher protein denaturation and more co-aggregation of proteins when readjusted to pH 5.5 for the precipitation of proteins (Kristinsson and Liang, 2006). Chang et al. (2016) studied the recovered protein from bighead carp (Aristichthys nobilis) using pH-shift processing and found that greater water-soluble sarcoplasmic proteins and myofibrillar protein were recovered compared to conventional surimi processing (Kristinsson and Liang, 2006).
Color property and total heme protein content The color (L*, a* and b* values) of minced fish and recovered protein from each process were determined (Table 2). The L* value (lightness) was increased from 37.50 (minced fish) to 41.12 for the acid-aided treatment, and decreased to 33.90 for the alkaline-aided treatment, while there were no significant differences in the L* value between meat using the salt water treatment and minced fish. The recovered protein with the acid-aided treatment had the highest L* value (p < 0.05) and less redness (p < 0.05) than the other recovery methods and minced fish, while the yellowness was increased significantly (p < 0.05) by both pH-shift processes. Color differences were increased in the rank order: salt water < acid-aided < alkaline-aided treatments. The recovery process significantly influenced the color of the recovered protein, especially for the alkaline-aided treatment. The changes of color during the protein recovery process might be associated with the formation of protein-protein complexes (Undeland et al., 2002). Tan et al. (2019) reported that there were no significant differences in the lightness of recovered proteins using salt water extraction and alkaline extraction from each part of catfish (head and frame). The changes in redness (a*) and yellowness (b*) values of recovered protein from minced fish may be due to the removal of heme proteins during the protein recovery process (Surasani et al., 2017) or changes in myoglobin and hemoglobin to the oxidized forms to exhibit a brown color (Abdollahi et al., 2016).
Table 2.
Color, color difference (ΔE), total heme protein of minced fish and recovered protein prepared by difference treatment.
|
Minced fish |
Salt water |
Acid-aided |
Alkaline-aided |
| L* |
37.50b ± 1.32 |
35.15bc ± 0.84 |
41.12a ± 0.57 |
33.90c ± 2.96 |
| a* |
−0.25a ± 0.13 |
−1.25b ± 0.10 |
−2.12c ± 0.3 |
−0.81b ± 0.47 |
| b* |
4.40c ± 0.49 |
4.64c ± 0.52 |
5.94b ± 0.32 |
7.95a ± 0.68 |
| ΔE |
- |
2.56 |
4.35 |
5.09 |
| Total heme protein (mg/g dry basis) |
21.46c ± 0.32 |
92.05a ± 12.04 |
29.16c ± 5.51 |
47.32b ± 3.92 |
Means ± standard deviation (n = 4).
Means with different letters within the row are significantly different (p ≤ 0.05).
“Salt water”, “Acid-aided”, and “Alkaline-aided” mean protein recovered by salt water treatment, acid-aided treatment and alkaline-aided treatment, respectively.
Total heme protein of minced fish and recovered protein by salt water, acid-aided and alkaline-aided treatments is shown in Table 2. Myoglobin and hemoglobin are the two major pigments in muscle (Rawdkuen et al., 2009). The retained heme protein content in the recovered protein was 92.05, 47.32 and 29.16 mg/g (on a dry basis) for the salt water, alkaline-aided and acid-aided treatments, respectively. The highest content of heme protein was found in protein recovered using salt water treatment. Heme protein can be removed from fish muscle by water-washing (Venugopal and Shahidi, 1996). In the present study, the heme protein was washed out only once in the salt water treatment, whereas two washings were carried out in pH-shift processing. The efficiency of heme protein removal depends on the fish species, muscle type, storage time and washing process (Rawdkuen et al., 2009). Therefore, the increased heme protein content in the recovered protein with salt water and alkaline-aided processing might be due to a concentration effect and co-precipitation with myofibrillar protein during the recovery process (Chaijan et al., 2010).
Ca-ATPase activity The Ca-ATPase activity of myofibrillar protein extracted from minced fish and the recovered protein samples using salt water, acid-aided and alkaline-aided treatments is shown in Fig. 2A. The highest Ca-ATPase activity was found in myofibrillar protein extracted from minced fish. The obvious decrease in Ca-ATPase activity of myofibrillar protein from recovered protein samples using pH-shift processing compared to that using salt water treatment might be due to myosin denaturation, induced by acid and alkaline solubilization processes (Chaijan et al., 2010). However, no differences in Ca-ATPase activity were observed between recovered proteins using both pH-shift processes (p > 0.05).
Surface hydrophobicity Hydrophobic interaction is one of the important interactions to stabilize the native protein structure (Hrynets et al., 2011). Changes in surface hydrophobicity can be used to indicate conformational changes in tertiary protein structure (Chaijan et al., 2010). The surface hydrophobicity of proteins correlates with the extent of protein unfolding during different processing methods; the higher the hydrophobicity, the greater the protein unfolding (Omana et al., 2010). The surface hydrophobicity of myofibrillar protein extracted from minced fish and the recovered protein using salt water, acid-aided and alkaline-aided processing is shown in Fig. 2B. Increases in the surface hydrophobicity of the alkaline-aided treatment might be due to unfolding of proteins during the recovery process. Copeland (1994) proposed that the embedded amino acid residues in the hydrophobic interior are exposed to the exterior environment when protein unfolds (Yongsawatdigul and Park, 2003). Notably, myofibrillar protein extracted from both recovered protein samples using salt water and acid-aided treatments showed a marked decrease in surface hydrophobicity. However, in the case of acid-aided treatment, the exposed hydrophobic residues might form hydrophobic interactions and decrease the surface hydrophobicity (Chaijan et al., 2006).
Reactive and total sulfhydryl groups Sulfhydryl groups are the most reactive functional group in proteins (Omana et al., 2010). The reactive and total sulfhydryl contents of myofibrillar protein extracted from minced fish and recovered protein samples using salt water, acid-aided and alkaline-aided treatments are shown in Fig. 2C. The lowest reactive and total sulfhydryl contents were found in the alkaline-aided process (p < 0.05). Reactive sulfhydryl was significantly decreased following the recovery process, likely due to the oxidation of sulfhydryl groups. However, the ratio of reactive sulfhydryl to total sulfhydryl of minced fish was similar to that of the recovered protein using salt water processing (29 and 28%, respectively), while the ratio for pH-shift processing was lower (11 and 15%, respectively). Results from this study indicate that salt water treatment has less effect on recovered proteins than pH-shift processes. Moreover, the sulfhydryl groups may remain unchanged even though hydrophobicity increases. This might be due to thiol and disulfide exchange reactions (Monahan et al., 1995).
Protein patterns The protein patterns of minced fish and recovered protein using salt water, acid-aided and alkaline-aided treatments are shown in Fig. 3. Myofibrillar protein, predominantly composed of thick filaments (mainly myosin) and thin filaments (mainly actin, troponin, and tropomyosin), are generally water-insoluble, but are soluble in concentrated saline solutions. They are responsible for functional properties such as gelation. Myosin consists of a heavy chain (220 kDa) and a light chain (18–25 kDa). Sarcoplasmic proteins (36–97 kDa) are considered to be water-soluble (Shi et al., 2017). In this study, myosin heavy chain and actin (approximately 220 and 45 kDa, respectively) were detected in the recovered protein using salt water treatment and in the minced fish. These were the major proteins detected in the recovered protein using salt water processing. The myosin heavy chain band was found only in the recovered protein using salt water treatment. Conversely, the protein band at a molecular weight of approximately 150 kDa was observed only in the recovered sample using pH-shift processing. The formation of low molecular weight bands might be due to the hydrolysis of myosin heavy chain by proteolytic enzymes (Sun et al., 2013) as well as protein hydrolysis under acid and alkaline conditions (Azadian et al., 2012). These results indicated that protein recovery using salt water is an excellent method for concentrated myofibrillar protein.
Conclusion
This study proved that salt water, acid-aided and alkaline-aided treatments can be efficiently employed in the recovery of muscle protein from small whole Japanese anchovy. The highest protein yield was achieved with acid-aided treatment. However, Ca-ATPase activity of the recovered protein using salt water treatment was significantly higher, whereas surface hydrophobicity was lower than those of recovered protein using pH-shift processing. In the SDS-PAGE analysis, both the myosin heavy chain and actin were observed only in the recovered protein using salt water treatment. This study confirmed that salt water treatment is applicable as an alternative method for the recovery of nutrients, especially protein, from under-utilized and low-value sources.
Acknowledgements The authors would like to acknowledge the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) and financial support from Japan Society for the Promotion of Science (KAKENHI) Grant Number 19K06233.
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