The meat color fades and the taste deteriorates as the chum salmon matures (hereafter referred to as “buna salmon”), which lowers its commercial value. To expand the use of buna salmon, fish sauce was produced and its general and functional food components as well as antibacterial properties were investigated. The total nitrogen and amino nitrogen contents of the produced fish sauce were 2.10g/100g and 0.66g/100g, respectively, both of which were higher than those of soy sauce. The volatile basic nitrogen content of the fish sauce was low at 16.4 mg/100 g. This confirmed the good quality of the fish sauce. The free amino acids in the fish sauce were particularly high in isoleucine, serine, arginine, lysine, leucine, and glutamic acid. The antioxidant activity and the γ-aminobutyric acid content of the fish sauce were 127 μmol Trolox/100g and 1.8mg/100g, respectively, making it a highly functional fish sauce. Fish soy sauce demonstrated antibacterial properties against Escherichia coli, and fish soy sauce is presumed to contain factors that inhibit E. coli growth, demonstrating sufficient antibacterial properties. Using buna salmon as an ingredient in fish soy sauce, a highly functional fish soy sauce with antibacterial properties can be produced, and buna salmon has been confirmed to be used effectively.

Results of this study indicate that fish sauce can be effectively produced from aged chum salmon, and that using this fish sauce in processed foods and cooking can not only impart a salty and umami taste but also produce functional foods that are less likely to spoil.

Keywords: mature salmon “buna salmon,” effective use of underutilized resources, fish sauce, antibacterial effect against E. coli

VBN, volatile base nitrogen

Fish sauce is a seasoning made by marinating seafood in salt and aging it. Its demand as a secret ingredient in noodle soups and sauces is increasing.¹ Various methods are currently considered for fish sauce production, such as adding microbial protease preparations,^2-8 soy sauce koji,^9-11 amino acids,¹⁰ and organic acids,¹² to shorten the aging period and increase the amount of amino acids that are the umami components. In this study, we investigated the production of fish sauce and its antibacterial effect to assess the successful application of chum salmon that matured (buna salmon).

Chum salmon (Oncorhynchus keta) develop nuptial colors on their epidermis during the spawning season, which turn into buna salmon. There are five grading standards for the degree of buna salmon: silver hair, A-, B-, C-, and D-grade (Figure 1).^13-17 Silver-haired salmons have a silvery-white body surface. Silver-haired, A-, B-, C-, and D- grade buna salmons demonstrate none, some, slightly stronger, stronger, and very strong nuptial coloring, respectively. C-grade buna salmon swim upstream in rivers, and D-grade swim upstream. When fasting, buna salmon lose body components, which decrease sarcoplasmic and myofibrillar protein levels,¹⁸ the flesh softens and fades in color, and the flavor decreases, thereby reducing its commercial value. Hence, buna salmon is difficult to use as fresh fish, and it is used in processed foods such as kamaboko^17-19 as well as smoked products.^19-20 However, the majority of them are currently used as feed and fertilizer.^19-21

Figure 1  The mature chum salmon Oncorhynchus keta, S, A, B, C and D grade.

To expand the use of buna salmon, the author focused on fish sauce, which has recently become increasingly popular as a seasoning. In this study, we produced fish sauce by adding intestine homogenate to fish meat. Furthermore, we analyzed the general components and food functional components, investigated the antibacterial effects, and reported the results here.

Raw materials

The raw material for the fish sauce was A-grade buna salmon from chum salmon (Oncorhynchus keta) with a body length of approximately 80 cm and a weight of approximately 4.5 kg, caught along the coast of the Sea of Okhotsk.

Manufacturing method for fish sauce

The innards, head, fins, tail, and vertebrae were removed from the salmon, and the resulting fish meat was washed with saline and then chopped into approximately 3 cm × 3 cm pieces. Salt was added at 25% (w/w) of the amount of fish meat, and the homogenized innards were added to this, mixed, and then aged at 30°C for 120 days.

Method for component analysis Analysis of general components

Moisture²² was measured at 135°C using an infrared moisture meter (Mettler, LP16). Protein and total soluble nitrogen were measured employing the Kjeldahl digestion method,²³ fat with the Soxhlet extraction method,²⁴ and ash using the direct ashing method.²⁵ A pH meter (Horiba) was used to measure pH, and formol nitrogen, acidity, and salinity were measured based on the soy sauce analysis method.²⁶

Measurement of volatile base nitrogen

Volatile base nitrogen (VBN) was measured using the Conway micro diffusion absorption method²⁷ following the notes on the hygiene test method.

Measurement of free amino acid content

Samples were filtered using centrifugal filter micro tubes (Nihon Millipore Industrial Co., Ltd., molecular weight cutoff 4,000), and NBD labeling treatment was performed. Analysis was conducted with high-performance liquid chromatography using an ODS-80TS, 3.2 mm ID×150 mm (Tosoh Corporation) column and an F-1050 spectrophotometer (Hitachi, Ltd.) detector. The mobile phase used was a gradient solution of 0.1 M sodium citrate buffer (pH 6.2) and 50% acetonitrile solution-water (50:50), and detection was conducted at a flow rate of 1.0 ml/min, a column temperature of 43°C, an excitation wavelength of 470 nm, and a fluorescence wavelength of 530 nm.

Measurement of 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability

The antioxidant effect was investigated by measuring the DPPH radical scavenging ability.²⁸ The sample solution was the supernatant after centrifugation at 3000 rpm for 5 min. A mixture of 12 ml of 400 μM of DPPH, 12 ml of 200 mM of 2-morpholinoethanesulfonic acid buffer (pH 6.0), and 12 ml of 20% ethanol was prepared, and 0.3 ml of sample solution diluted with 80% ethanol was added to 0.9 ml of the mixture and reacted for 20 min. The reaction solution was measured at 520 nm, and the antioxidant activity was calculated from the absorbance decrease rate. Torolox was used to prepare the calibration curve, and the antioxidant effect was assessed employing the Torolox conversion method.

Measurement of γ-aminobutyric acid

The γ-aminobutyric acid (GABA) sample was prepared so that the protein content of fish sauce was 0.5 mg/100 ml, and 0.2 μl was passed through an aqueous filter. The column utilized was ShimPack AMINO-Na (6 mm ID×100 mmL), the mobile phase was 20 mM of citrate-sodium buffer (pH 5.9), the column temperature was 40℃, the flow rate was 0.4 mL/min, and the sample injection volume was 10 μl. The detector used was a spectrofluorometric RF-10AXL, and detection was performed using sodium hypochlorite and o-phthalaldehyde reagents.

coli antibacterial test

Desoxycholate medium (lactose of 10 g, peptone of 10 g, sodium chloride of 5 g, sodium deoxycholate of 1g, dipotassium hydrogen phosphate of 2g, ferric ammonium citrate of 2g, neutral red of 0.03g, agar of 15g, distilled water of 1L) was prepared at pH of 7.2 and poured into a petri dish at 45℃ to create a plate medium, which was then covered with E. coli (isolated from soil) on the surface. Sterilized filter paper disks were soaked in fish sauce diluted 1,000 times and incubated at 37℃ for 24h. As a control, a 1,000-fold dilution of 25% saline solution, which was the same salt concentration as the fish sauce, was used. If the filter paper disk demonstrated an antibacterial effect against E. coli, the growth of E. coli would be inhibited around the disk, forming an inhibition circle. This time, a qualitative test was performed to assess the presence or absence of an inhibition circle.

General composition of the raw material

The composition values of the flesh of the buna were measured (Table 1). The moisture content was 70.2%, protein was 22.7%, lipid was 5.9%, and ash was 1.2%. According to the standard tables of food composition in Japan 2020,²⁹ the composition values of the salmon were 72.3% moisture, 22.3% protein, 4.1% lipid, and 1.2% ash, which were not significantly different from the raw material. The moisture content increases and the lipid content decreases as the degree of bunafication increases,¹⁵ but the values were assumed to be similar since the raw material is a high-ranked buna.

Analysis of fish sauce composition

General analysis

Table 2 presents the general analysis of the fish sauce. The pH was 5.5, which was similar to that of commercially available fish sauce.³⁰ The total nitrogen amount was 2.10 g/100 g, and the amino nitrogen amount was 0.66 g/100 g, which was 1.2 times higher than that of soy sauce.³¹

Volatile base nitrogen amount

The amount of VBN was assessed using Conway’s microdiffusion absorption method (Table 2). VBN, such as trimethylamine, dimethylamine, and ammonia, is generated when protein decomposition products are decomposed by digestive enzymes. This is the component that gives fish sauce its unique aroma, but it causes a putrid odor if it is generated in excess. Further, it causes precipitation when it combines with minerals. The amount of VBN in the fish sauce was 16.4 mg/100 g, and the amount of VBN, such as ammonia, trimethylamine, and dimethylamine, was small. This confirmed that it was a high-quality fish sauce.

Free amino acids

Table 3 shows the analysis of free amino acids after 120 days of aging. The free amino acids in fish sauce were confirmed to be particularly high in isoleucine, serine, arginine, lysine, leucine, and glutamic acid. Further, it contained a large amount of branched-chain amino acids, which are an energy source for muscles.

Measurement of 1,1-Diphenyl-2-picrylhydrazyl radical scavenging ability

Recently, the antioxidant activity of foods has been attracting a lot of attention as a food functionality. Antioxidant activity eliminates active oxygen generated in the body and prevents aging and disease. Measurement of the antioxidant activity of fish sauce (Table 4) was 127 μmol Trolox/100 g, which is three times higher than that of general soy sauce.³² These results confirmed that components with antioxidant properties were produced during the aging process, making it a functional fish sauce.

Measurement of γ-aminobutyric acid

GABA has been attracting attention in recent years as it increases cerebral blood flow and function and is effective in improving the aftereffects of stroke. GABA is a neurotransmitter that has a tranquilizing effect and a blood pressure-lowering effect. The amount of GABA in the buna salmon fish sauce was assessed (Table 5) and was 1.8 mg per 100 g of fish sauce. GABA is generally found in salmon internal organs, but it is also produced by the action of digestive enzymes and was present in large amounts in fish sauce. This result was sufficient to attract attention as a functional fish sauce.

Antibacterial activity against E. coli

The examination of the antibacterial activity of fish sauce against E. coli (Figure 2) formed an inhibition circle, which was not seen in the control saline solution, and antibacterial properties were confirmed. Hence, a growth inhibitor for E. coli was inferred in the fish sauce. This result was obtained by diluting the fish sauce 1000 times, and the fish sauce was confirmed to have sufficient antibacterial properties.

Figure 2    The examination of the antibacterial activity of fish sauce against E. coli using paper disk.

This study aimed to produce high-quality fish sauce to confirm the effective use of buna salmon and to investigate its functional ingredients and antibacterial properties. Soluble total nitrogen, amino nitrogen, and amino acid levels in the produced fish sauce indicated that it contained sufficient umami components. Furthermore, the assessment of the functional components in the fish sauce during aging revealed that the antioxidant activity was 127 μmol Trolox/100 g, which is three times higher than that of regular soy sauce, and the GABA level was 1.8 mg/100 g, indicating that it was highly functional. Furthermore, the produced fish sauce demonstrated a low volatile basic nitrogen level and had antibacterial properties against E. coli, making it suitable for use in processed foods. The results of this study indicate that fish sauce can be effectively produced from aged chum salmon, and that using this fish sauce in processed foods and cooking can not only impart a salty and umami taste but also produce functional foods that are less likely to spoil.

None.

The authors declare that there are no conflicts of interest.

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