A comparative study on properties of fish meat hydrolysates produced by an enzymatic process at high pressure

Abstract

Fish meat hydrolysates (FMHs) were produced from nine fishes at a high pressure of 300 MPa using Flavourzyme 500MG and a protease mixture including Flavourzyme 500MG, Alcalase 2.4L, Protamex, and Marugoto E. The electropherograms of the FMHs showed major far-migrating peptide bands in the vicinity of 5 kDa. The total soluble solids (TSS) and soluble nitrogen content in the FMHs were species-specific and were mostly higher in the case of four-enzyme hydrolysis. Most of the HPLC peptide peaks of the rockfish meat hydrolysates appeared within 10 min of elution, and total free amino acids in the hydrolysate increased abruptly as a result of four-enzyme hydrolysis. The FMHs, which were high in TSS and soluble nitrogen, may be applicable for use in food as seasoning, and could be produced efficiently via the enzymatic process used in this study.

Introduction

Fishes, which have a protein content of around 20%, are regarded as important protein sources for human consumption. They are currently consumed fresh and are used for processing, canning, freezing, smoking, or dehydration (Kristinsson and Rasco, 2000; Soriguer et al., 1997). With the progress of processing technology and increased understanding of fish proteins and peptides, a new industry has evolved exploiting the enzymatic hydrolysis of fish proteins. This industry aims to obtain new food ingredients and occupy relevant markets. Nowadays, these ingredients are extensively used in sports nutrition, food additives, and as seasoning materials, while simultaneously evidencing hygienic safety, and meeting sensory and functional requirements in prepared foods (Kristinsson and Rasco, 2000; Nutraingredients-usa.com, 2013; Tahergorabi et al., 2012).

Much evidence has been accumulated regarding the bioactive and antioxidant properties of fish protein powders with hydrolysates, including their positive effects on irritable bowel syndrome and Crohn’s disease, alleviation of hypertension, addition of lean muscle mass to humans, minimization of anti-inflammatory drug side effects, and inhibition of the spread of prostate and possibly other cancers (Guha et al., 2013; Kristinsson and Rasco, 2000; Ruvini et al., 2013; Ryan et al., 2011; Slonim et al., 2009). Additional benefits are expected to attend to the dietary needs of various subsets of the human population suffering from lactose intolerance, milk allergy, and gluten intolerance (Hacini-Rachinel et al., 2014; Heine et al., 2017; Kristinsson and Rasco, 2000; Wang et al., 2008).

It has been reported that some pressure-tolerant enzymes including trypsin and most industrial proteases can be used at high pressure with increased product yield. Moreover, the enzyme reaction at a high pressure of below approximately 300–400 MPa could exclude microbial contamination, which is detrimental to the quality of final products (Borda et al., 2004; Mozhaev et al., 1996; Raouche et al., 2011; Rupley et al., 1983). Based on this, some studies employing the application of high pressure during enzymatic hydrolysis have been reported. This has been done using various proteases at a high pressure of below 550 MPa, with the purpose of obtaining hydrolysates with improved bioactive and/or immunological properties, functional properties, and added values from proteins of peanut, egg, and soybean (Chicón et al., 2008; Dong et al., 2011; Singh and Ramaswamy, 2014; Yuan et al., 2012).

From our previous works, an enzymatic process at a high pressure of 300 MPa using a protease mixture composed of Flavourzyme 500MG, Alcalase 2.4L, Protamex, and Marugoto E showed conspicuously enhanced protein hydrolysis compared with the ambient-pressure counterpart, as measured by total soluble solids (TSS) and soluble nitrogen (Kim, 2017; Kim et al., 2016). As a subsequent work, the objective of this study was to produce fish meat hydrolysates (FMHs) high in TSS and soluble nitrogen from various fishes, based on the enzyme process described above. We also aimed to compare their properties according to hydrolysis. As a result, meaningful differences in these properties were found and are herein reported.

Materials and methods

Reagents

Four industrial proteases comprising an exo-type enzyme, Flavourzyme 500MG (aminopeptidase, from Aspergillus oryza, having the declared activity of 500 LAPU/g) and three endo-type enzymes, Alcalase 2.4L (subtilisin, from Bacillus licheniformis, having the declared activity of 2.4 AU/g), Protamex (from B. licheniformis and B. amyloliquefaciens, having the declared activity of 1.5 AU/g), and Marugoto E (from B. subtilis, having the declared activity of 100 PU/mg) were used for fish meat hydrolysis. From these, Flavourzyme 500MG, Alcalase 2.4L, and Protamex were products of Novozymes A/S (Bagsvaerd, Denmark), while Marugoto E was a product of Supercritical Technology Research Corporation (Hiroshima, Japan). They were selected because of their relative pressure tolerance at 300 MPa (Kim et al., 2013). Mini-PROTEAN^® TGX SDS-PAGE gels (12%, 10 wells, 30 μL), 10 × Tris/glycine/SDS buffer, and Bio-Safe™ Coomassie G-250 Stain (phosphoric acid, less than 5%) of Bio-Rad (Hercules, CA, USA) were used for SDS-PAGE. All other chemicals were guaranteed reagents from various suppliers, and double distilled water was used throughout this study.

Fishes hydrolyzed

In this study, nine fish varieties Paralichthys olivaceus (flatfish), Sebastes schlegeli (rockfish), Cyprinus carpio (carp), Pleuronichthys cornutus (finespotted flounder), Mugil cephalus (gray mullet), Scomberomorus niphonius (Spanish mackerel), Cololabis saira (Pacific saury), Tilapia sparrmanii (tilapia), and Sepioteuthis sepioidea (cuttlefish) were used for enzymatic hydrolysis. They were purchased from a local market in Seoul, Korea.

Production of FMHs

Fish meat was recovered after removing the bones and skin, and the meat was divided into 100 g portions, and stored in a deep freezer at -78 °C until use. Frozen samples were minced by comminuting with a mixer after thawing. Eighty grams of the resultant fish meat paste was hydrolyzed as follows, using the proteases described above at the high pressure of 300 MPa, according to a previously reported method, with slight modifications (Kim et al., 2016). Fifteen milliliters of distilled water was added to a vinyl pouch containing the meat paste. The temperature of the pouch contents was adjusted to 50 °C inside the vessel (150 mL capacity) of the high-pressure equipment (DP-SHPL-015L-400, Dima Puretech, Incheon, Korea) which was equilibrated with distilled water as the pressure-transmitting fluid at 50 °C. Following this, 0.383 g of Flavourzyme 500MG (for one-enzyme hydrolysis) or a protease mixture composed of 0.383 g, 53.36 μL (density; 1.2 g/mL), 0.096 g, and 0.125 g of Flavourzyme 500MG, Alcalase 2.4L, Protamex, and Marugoto E (for four-enzyme hydrolysis) were added, respectively. The amounts were determined mainly based on the recommended usages for these enzymes. The pouch was then submerged in the vessel, and the vessel cover was closed. To minimize adiabatic heating, the pressure was increased carefully until the pre-set pressure of 300 MPa was reached. The vessel was maintained at this pressure for 1 h for enzymatic hydrolysis. After the reaction, the reaction mixture was heat-treated for 20 min at 90 °C, followed by centrifugation at 16,300 × g for 30 min at 10 °C. The centrifugation was performed once more after suspending the residue in 50 mL distilled water. The combined supernatant was designated the corresponding FMH.

SDS-PAGE

SDS-PAGE was conducted using 12% Mini-PROTEAN^® TGX slab gel operated with a Power Pac™ Basic Power Supply (Bio-Rad). For use as a running buffer, 25 mM Tris-192 mM glycine (pH 8.3, containing 0.1% SDS) was used. Sample preparation and electrophoresis were conducted as described previously (Kim et al., 2016). For the stained gels, SDS-PAGE electropherograms were obtained with a Gel Doc™ EZ Imager (Bio-Rad) operated by Image Lab (version 4.1, Bio-Rad).

Determination of indices of enzymatic hydrolysis

Individual 10 mL samples of the FMHs were added to pre-weighed aluminum dishes overlaid with sea sand. The samples were then measured until reaching constant mass using a dry oven at 105 °C. TSS was calculated as follows:

$$\text{TSS}(\%)=(\text{total mass of dried matter in FMH}/\text{total mass of dried matter in fish meat paste hydrolyzed})\times 100$$

The Kjeldahl method was used to determine total water-soluble nitrogen (TWSN), trichloroacetic acid (TCA)-soluble nitrogen (TCASN), and total nitrogen (TN). The samples for TCASN measurement were prepared according to the method previously described (Kim et al., 2016). In this instance, degree of hydrolysis (DH) was calculated as follows (Ovissipour et al., 2009):

$$\text{DH}\left(\%\right)=\left(\text{total mass of TCASN in FMH}/\text{total mass of TN in fish meat paste hydrolyzed}\right)\times 100$$

HPLC

HPLC was conducted based on the method of Kim et al. (2016) using 1260 Infinity System of Agilent Technologies (Santa Clara, CA, USA) with two pumps, a variable wavelength UV detector with the wavelength set at 220 nm, and a 100 μL injection loop. An Ascentis^® Express Peptide ES-C₁₈ column (100 × 4.6 mm, Supelco, Bellefonte, PA, USA) with packed particle size of 2.7 μm was used for analysis, and a guard column (Supelco) packed with the same C₁₈ packing material was positioned in front of the column to avoid possible contamination. Data acquisition was performed with Agilent ChemStation (Rev. B.04.03.). For sample preparation, 1 mL of the standard mixture, rockfish drip, and rockfish meat hydrolysates produced by one- and four-enzyme hydrolysis were individually filtered through an Acrodisc^® LC 13 mm Syringe Filter with a 0.45 μm polyvinylidene difluoride membrane (Pall Life Sciences, Ann Arbor, MI, USA). The filtrates were loaded onto the column with an autosampler. Eluent A and B, with compositions of 10:90 (v/v) acetonitrile–water containing 1% trifluoroacetic acid (TFA) and 70:30 (v/v) acetonitrile–water containing 1% TFA, respectively, were used for gradient elution. After injecting a sample, a solvent gradient was applied from 0% eluent B to 50% eluent B in 15 min. Over the next 15 min, 50% eluent B was maintained, followed by returning to 0% eluent B in 5 min. This eluent composition was maintained for 10 min and the HPLC system was ready for another measurement. During analysis, the injection volume, flow rate, and eluent temperature were maintained at 5 μL, 1.5 mL/min, and 30 °C, respectively.

Determination of free amino acids

Free amino acids in the rockfish meat hydrolysates were determined based on the method of Kim et al. (2016) with Agilent 7890B GC-FID for the samples prepared by chloroformate derivatization using KG0-7167 Easy-fast Amino Acid Sample Testing Kit (Phenomenex, Torrance, CA, USA). A ZB-AAA (10 m × 0.25 mm) capillary GC column from the same company was used for analysis. Column injection was done in split mode (10:1) at 250 °C using a 2 μL sample. The carrier gas was helium, and the flow rate was 1.5 mL/min. The oven temperature was increased from 110 to 320 °C at 35 °C/min, and the FID temperature was set to 320 °C. Amino acid standards (Phenomenex) at 200 μM were used together with norvaline at 200 μM, eluting at 2.80 min as an internal standard.

Data analysis

When necessary, data were presented as mean ± standard deviation (SD). Analysis of variance (ANOVA) and Student’s t test were applied to the data of TSS and soluble nitrogen to determine significant differences at p < 0.05 using SPSS 20 (SPSS Inc., Chicago, IL, USA).

Results and discussion

Electrophoretic properties of FMHs

In this study, FMHs were produced at a high pressure of 300 MPa by one-enzyme hydrolysis using the exo-type protease Flavourzyme 500MG, or by four-enzyme hydrolysis. The latter enzyme mixture was composed of Flavourzyme 500MG and the endo-type proteases Alcalase 2.4L, Protamex, and Marugoto E. In this case, one-enzyme hydrolysis was conducted as a reference against which to evaluate the performance of four-enzyme hydrolysis.

As shown by the electropherograms of the drips prepared from nine fishes in Fig. 1A, very diffusive band patterns appeared in all fishes in the molecular mass range of 2–250 kDa. This indicated the presence of a great variety of proteins and peptides in the fish meat tested, differing in molecular size (Reed and Park, 2008). The band patterns of the drips were also partially different according to the fishes. After one- and four-enzyme hydrolysis in Fig. 1B and C, respectively, the molecular masses of the FMHs were mostly present at less than 50 kDa. Furthermore, the hydrolysates produced by four-enzyme hydrolysis revealed denser peptide bands in the vicinity of 5 kDa compared with those produced by one-enzyme hydrolysis. This could be evidence of more efficient hydrolysis progression in the case of four-enzyme hydrolysis, as previously reported when conducting hydrolysis of wheat gluten (Kim, 2017). In most hydrolysates, the band patterns of peptides were similar irrespective of the number of enzymes used for hydrolysis. However, carp meat hydrolysates showed a prominent band at 25 kDa after carrying out four-enzyme hydrolysis.

Fig. 1.

Electropherograms of drips (A) and fish meat hydrolysates produced by one- (B) and four-enzyme hydrolysis (C) according to fishes at 300 MPa. Lane description. 1 molecular mass markers; 2 flatfish; 3 rockfish; 4 carp; 5 finespotted flounder; 6 gray mullet; 7 Spanish mackerel; 8 Pacific saury; 9 tilapia; 10 cuttlefish. The drips were extracts separated from fish meat after centrifugation at 9000 × g. For one-enzyme hydrolysis, Flavourzyme 500MG was used. Meanwhile, a protease mixture including Flavourzyme 500MG, Alcalase 2.4L, Protamex, and Marugoto E was used for four-enzyme hydrolysis

Variation in TSS of FMHs according to fishes and protease usage

Since soluble fractions increase with the progression of enzymatic hydrolysis of proteinaceous substrates, TSS has been used as an important index for evaluating the degree of hydrolysis of resultant hydrolysates (Beuchat et al., 1975; Payne et al., 1978). When TSS in the FMHs was measured, variations in TSS content were found according to the fishes and protease usage (Table 1). However, the degree of variation was more conspicuous when considering the type of fish. In the case of one-enzyme hydrolysis, the maximum and minimum TSS contents of 28.41 and 9.37% were found for the flatfish and Pacific saury meat hydrolysate, respectively. For four-enzyme hydrolysis, the same trend was found with TSS content of 32.23 and 10.77% for the flatfish and Pacific saury meat hydrolysate, respectively. In addition, the rockfish, finespotted flounder, and cuttlefish meat hydrolysates were high in TSS irrespective of protease usage. When TSS in the FMHs was compared according to protease usage, four-enzyme hydrolysis was considerably superior to one-enzyme hydrolysis when considering the increasing TSS content of the FMHs, except for in the case of Pacific saury meat hydrolysate. This strongly indicated that four-enzyme hydrolysis at 300 MPa was the better choice for producing FMHs with good hydrolytic properties.

Table 1.

Changes in total soluble solids of fish meat hydrolysates according to fishes and protease usage at 300 MPa

Fish	TSS (%)
	One-enzyme hydrolysis1	Four-enzyme hydrolysis2
Flatfish	28.41 ± 0.32aB	32.23 ± 0.31aA
Rockfish	20.70 ± 1.36cB	24.95 ± 0.89cA
Carp	17.43 ± 0.43efB	22.21 ± 0.49dA
Finespotted flounder	25.34 ± 0.85bB	28.40 ± 0.52bA
Gray mullet	18.72 ± 0.85deB	21.73 ± 0.81dA
Spanish mackerel	12.57 ± 0.29gB	16.85 ± 0.29fA
Pacific saury	9.37 ± 1.11hA	10.77 ± 0.46gA
Tilapia	16.96 ± 0.70fB	18.40 ± 0.46eA
Cuttlefish	18.94 ± 0.61dB	24.67 ± 0.67cA

Values of TSS are mean ± SD (n = 3). Means within the same column with different lowercase letters are significantly different at p < 0.05 by ANOVA. Means within the same row with different capital letters are significantly different at p < 0.05 by Student’s t test

¹Flavourzyme 500MG was used

²A protease mixture including Flavourzyme 500MG, Alcalase 2.4L, Protamex, and Marugoto E was used

Variation in soluble nitrogen of FMHs according to fishes and protease usage

The TWSN and TCASN contents according to the fishes and protease usage were determined, followed by calculating the DH of the FMHs. These variables are regarded as important indices of protein hydrolysis such as TSS, and have been correlated well with TSS in protein hydrolysis (Kim, 2017). As shown in Table 2, the TWSN content was in the range of 0.69–1.31 and 0.78–1.51% in the cases of one- and four-enzyme hydrolysis, respectively. This indicated the presence of considerable variations in TWSN content in the produced FMHs. The fishes with higher levels of TWSN in the corresponding hydrolysates were flatfish and rockfish. Meanwhile, the TWSN content in the FMHs produced by four-enzyme hydrolysis was mostly higher than those produced by one-enzyme hydrolysis. All were statistically significant except for the hydrolysates of carp, gray mullet, and Pacific saury meat. As an estimate of peptide nitrogen, TCASN was determined (Adler-Nissen, 1979). In the cases of one- and four-enzyme hydrolysis, the TCASN content was in the range of 0.28–0.68 and 0.35–0.71%, respectively. Again, the flatfish and rockfish meat hydrolysates were conspicuously higher in TCASN in both protease usages. The TCASN content in the FMHs produced by four-enzyme hydrolysis was also higher than those produced by one-enzyme hydrolysis. All differences were statistically significant save for the gray mullet and cuttlefish meat hydrolysate. When TCASN/TWSN ratios for the produced FMHs were calculated, these values were in the range of 36.36–61.80 and 41.67–61.54% for one- and four-enzyme hydrolysis, respectively. As no meaningful difference in the TCASN/TWSN ratio was found, it could be concluded that enzyme hydrolysis increased TWSN and TCASN in the FMHs simultaneously upon usage of both proteases. In general, the DH is defined as the ratio of the number of peptide bonds hydrolyzed to the number of total peptide bonds (Adler-Nissen, 1979). As shown in Table 2, the hydrolysates of flatfish, rockfish, finespotted flounder, and cuttlefish meat showed relatively higher DH values for both protease usages. In addition, the DH values in the FMHs produced by four-enzyme hydrolysis were consistently higher than those produced by one-enzyme hydrolysis. This seemed to indicate that enzymatic hydrolysis with the protease mixture used in this study is an efficient method for producing FMHs from various fishes. Considering that a DH value of 30% or more is generally required for producing protein hydrolysates for use as flavoring components (Ohmori et al., 1991), the hydrolysates of flatfish, rockfish, and finespotted flounder meat produced by four-enzyme hydrolysis seemed to satisfy this criterion.

Table 2.

Changes in soluble nitrogen and degree of hydrolysis of fish meat hydrolysates according to fishes and protease usage at 300 MPa. TWSN and TCASN mean total water-soluble nitrogen and trichloroacetic acid-soluble nitrogen, respectively

Fish	One-enzyme hydrolysis1			Four-enzyme hydrolysis2
	TWSN (%)	TCASN (%)	DH (%)3	TWSN (%)	TCASN (%)	DH (%)
Flatfish	1.31 ± 0.02aB	0.68 ± 0.01aB	32.54	1.51 ± 0.01aA	0.71 ± 0.01aA	34.92
Rockfish	1.12 ± 0.01bB	0.53 ± 0.00bB	23.98	1.35 ± 0.00bA	0.62 ± 0.02bA	28.70
Carp	0.83 ± 0.02dA	0.41 ± 0.01 dB	21.47	0.87 ± 0.03eA	0.45 ± 0.01eA	27.18
Finespotted flounder	0.87 ± 0.02cB	0.47 ± 0.02cB	26.03	0.94 ± 0.02dA	0.55 ± 0.01cA	32.41
Gray mullet	0.83 ± 0.02dA	0.43 ± 0.01dA	20.35	0.79 ± 0.05fA	0.45 ± 0.02eA	23.23
Spanish mackerel	0.78 ± 0.02eB	0.38 ± 0.01eB	16.70	0.92 ± 0.02dA	0.47 ± 0.01dA	20.81
Pacific saury	0.77 ± 0.01eA	0.28 ± 0.02fB	12.50	0.84 ± 0.05eA	0.35 ± 0.01fA	15.05
Tilapia	0.69 ± 0.02fB	0.42 ± 0.00 dB	19.62	0.78 ± 0.02fA	0.48 ± 0.00dA	22.72
Cuttlefish	0.89 ± 0.01cB	0.55 ± 0.01bA	24.75	1.14 ± 0.02cA	0.57 ± 0.01cA	27.37

Values of TWSN and TCASN are mean ± SD (n = 3). Means within the same column with different lowercase letters are significantly different at p < 0.05 by ANOVA. Means within the same row with different capital letters are significantly different at p < 0.05 by Student’s t test

¹Flavourzyme 500MG was used

²A protease mixture including Flavourzyme 500MG, Alcalase 2.4L, Protamex, and Marugoto E was used

³TCASN/TN × 100

HPLC profiles of rockfish meat hydrolysates

Based on the aforementioned findings, it was presumed that the flatfish and rockfish meat hydrolysates had good hydrolytic properties, making them acceptable for use in food. As illustrative examples, the HPLC profiles and free amino acid compositions of the rockfish meat hydrolysates were further studied. First, the peptide profile of the hydrolysate produced by four-enzyme hydrolysis was compared with those of the drip and hydrolysate by one-enzyme hydrolysis by reversed-phase HPLC. This has been widely used for peptide analysis due to good resolution in peptide separation (D’Hondt et al., 2013; Mant et al., 2010). As shown in Fig. 2A, all standard peptides with molecular masses of 238–1031 Da eluted at a retention time (RT) of 1.051–8.089 min. In the elution profile of the rockfish drip in Fig. 2B, a major broad peak with some spikes appeared after 20 min of elution. In addition, four minor sharp peaks with RT 15–19 min were also found. For one-enzyme hydrolysis in Fig. 2C, the peaks found in the drip were reduced considerably. On the contrary, new major and minor peptide peaks appeared at RTs similar to those of the standard peptides of this study, together with an appreciable baseline rise. This was possibly due to the presence of the produced peptides. As depicted in Fig. 2D, the trends found for one-enzyme hydrolysis were further strengthened in the case of four-enzyme hydrolysis. That is, most of the major and minor peptide peaks appeared within 10 min of elution together with a steeper baseline rise, and the peaks found in the drip nearly disappeared. It was therefore found that the peptides liberated after the enzyme process of this study had shorter RTs than the peaks in the elution profile of the drip, and mostly eluted at RTs similar to those of the standard peptides. This appeared to support the results of SDS-PAGE.

Fig. 2.

Comparison of rockfish meat hydrolysate peptide profiles by HPLC at 300 MPa according to protease usage. (A–D) Indicate the elution profiles of the peptide standards, drip, and the hydrolysates produced by one- and four-enzyme hydrolysis, respectively. In (A), five peptide peaks corresponding to the retention times of 1.051 (1), 2.994 (2), 6.051 (3), 7.325 (4), and 8.089 min (5) indicate Gly-Val (FW 238), Val-Tyr-Val (FW 379), MT enkephalin (FW 573), angiotensin II (FW 1031), and Leu enkephalin (FW 569), respectively. For one-enzyme hydrolysis, Flavourzyme 500MG was used. A protease mixture including Flavourzyme 500MG, Alcalase 2.4L, Protamex, and Marugoto E was used for four-enzyme hydrolysis

Free amino acid composition of rockfish meat hydrolysates

In general, considerable amounts of free amino acids are liberated during the enzymatic hydrolysis of proteinaceous materials (Kim, 2017). The free amino acid content of the rockfish meat hydrolysate produced by four-enzyme hydrolysis was thus compared with that of the drip and hydrolysate by one-enzyme hydrolysis. As shown in Table 3, the total free amino acid content in the drip and rockfish meat hydrolysates produced by one- and four-enzyme hydrolysis were 790, 48,921, and 310,587 ppm, respectively. Compared with the hydrolysate produced by one-enzyme hydrolysis, a significant 6.3-fold increase in total free amino acids was found in that produced by four-enzyme hydrolysis. This might imply that most of the nitrogenous compounds in rockfish meat were degraded to peptides with low molecular mass and free amino acids (Adler-Nissen, 1979; Kim, 2017). This coincided well with the results described above. In decreasing order, alanine, lysine, histidine, glycine, aspartic acid, tyrosine, glutamine, leucine, and glutamic acid were present in low amounts in the drip of rockfish meat. In contrast, alanine, leucine, isoleucine, phenylalanine, glutamine, valine, lysine, asparagine, and glutamic acid were present as the major free amino acids in rockfish meat hydrolysate produced by four-enzyme hydrolysis. The pattern of free amino acids found in the hydrolysate produced by one-enzyme hydrolysis was very similar to that found in the hydrolysate by four-enzyme hydrolysis. However, the concentrations of the corresponding free amino acids in the former were far lower than those in the latter. Overall, alanine, glycine, aspartic acid, asparagine, glutamine, and glutamic acid with a sweet or savory taste increased considerably via enzymatic hydrolysis using this study’s protease mixture at a high pressure of 300 MPa. However, neutral (valine, leucine, isoleucine, and phenylalanine) and basic (lysine) amino acids increased simultaneously (Ohmori et al., 1991; Xu et al., 2013). It was presumed that the increase in free amino acids with a sweet or savory taste in the rockfish meat hydrolysates after enzymatic hydrolysis may be relevant to the good taste properties of these hydrolysates (data not shown). The increase in free amino acids with a sweet or savory taste also coincided with a previous study, reporting that rockfish meat itself contains plentiful taste compounds as constituents of protein, such as glutamic acid (Kim et al., 2000). For some other FMHs, including the flatfish meat hydrolysates, similar trends to those described above were found (data not shown).

Table 3.

Free amino acid contents in rockfish meat hydrolysates produced at 300 MPa

Amino acid	Content (ppm)
	Drip	One-enzyme hydrolysis1	Four-enzyme hydrolysis2
Alanine	157	6263	37,981
Glycine	55	1302	8492
α-Aminobutyric acid	5	28	ND
Valine	20	2175	21,726
β-Aminobutyric acid	15	ND	ND
Norvaline	23	23	117
Leucine	26	6682	37,352
Isoleucine	16	4225	25,578
Threonine	ND3	ND	1081
Serine	ND	6	ND
Proline	17	930	4420
Asparagine	ND	3451	20,690
Aspartic acid	48	1464	9072
Methionine	16	2380	14,447
4-Hydroxyproline	88	411	ND
Glutamic acid	23	3046	20,093
Phenylalanine	ND	4053	25,091
α-Aminoadipic acid	ND	106	1077
α-Aminopimelic acid	ND	151	1164
Glutamine	29	4546	24,608
Ornithine	ND	317	3447
Glycine-proline	ND	256	1953
Lysine	121	2850	21,237
Histidine	79	2066	14,497
Tyrosine	48	1141	8265
Proline-hydroxyproline	ND	163	2244
Tryptophan	4	665	4527
Cystine	ND	221	1428
Total free amino acids	790	48,921	310,587

¹Flavourzyme 500MG was used

²A protease mixture including Flavourzyme 500MG, Alcalase 2.4L, Protamex, and Marugoto E was used

³Not detected

It was concluded that the FMHs of nine fishes, produced by enzymatic hydrolysis at 300 MPa using the protease mixture of Flavourzyme 500MG, Alcalase 2.4L, Protamex, and Marugoto E, had characteristic hydrolytic properties. Of the resultant FMHs, flatfish and rockfish meat hydrolysate may be applicable for use in food as seasoning materials.

Compliance with ethical standards

Conflict of interest

The author declares no conflict of interest.