Salt-Reduced Fish Sauce Produced under Pressurized Carbon Dioxide Treatment Using Sardinops melanostictus, Trachurus japonicus, Konosirus punctatus, Odontamblyopus lacepedii, Their Collective Mixture, and Unused Fish Mixture

Fish sauce is produced at high salt concentrations (>20%) to inhibit the growth of harmful microorganisms. The salt-reduced fish sauce (10% salt) was prepared under pressurized CO2 (pCO2) conditions at 30 °C and 5 MPa for 3 months (FSCO2), from Sardinops melanostictus, Odontamblyopus lacepedii, Trachurus japonicus, Konosirus punctatus, and their collective mixture, as well as unused fish mixture obtained from the Ariake Sea in Japan. FSCO2 exhibited significantly better microbial quality and free amino acid content, lighter color, standardized odor (dashi-like odor), and umami richness qualities compared to fish sauces prepared using the conventional method (FScon) (20% salt), as previously demonstrated, after a fermentation period of 2 months. Bacterial flora analysis implied that the standardization of odor and umami richness may not be the result of specific microbial metabolism. Even when using previously unused fish, it was possible to produce FSCO2 equivalent to that produced by conventional sardines and other fish. These results indicate that the quality of fish sauce can be improved. The flavor of FSCO2 became similar regardless of the type of fish and fermentation period using pCO2 during fermentation, leading to the effective utilization of unutilized fish as a resource for high-quality salt-reduced fish sauce.


Introduction
Fish sauce is a liquid seasoning made by marinating fish in a high salt concentration and allowing it to digest.It is produced through protein hydrolysis by the action of endogenous proteases in the fish body and the metabolism of contaminating bacteria, which contribute to the formation of a unique flavor during the fermentation process [1].High salt concentration is essential to inhibit the growth of spoilage bacteria that produce unfavorable flavors and amines in fish sauce [2].However, consumer demand for salt reduction is increasing, and high salt content hinders the decomposition of fish meat.Salt reduction has been attempted through electrodialysis and the addition of acetic acid [3,4].However, these methods do not address the retarded decomposition rate of fish meat, despite meeting consumer demand.
We attempted to reduce salt concentration by applying pressurized CO 2 (pCO 2 ) during fermentation.Both acidic and anaerobic conditions can be created under pCO 2 treatment, which is expected to inhibit the growth of aerobic mesophilic bacteria and prevent the oxidation of fish sauce components.The use of pCO 2 eliminates the need for CO 2 removal after fermentation because the CO 2 dissolved in fish sauce is naturally released after depressurization.Noma et al. [5] demonstrated that reduced-salt (10% of the final salt concentration) sardine fish sauce could be prepared under pCO 2 treatment (30 • C and Sardinops melanostictus, Odontamblyopus lacepedii, Trachurus japonicus, and Konosirus punctatus used in this study were purchased from a local supermarket or fresh fish store on the day the fish sauce preparation was started (Thursday, 4 March 2021), and brought back to the laboratory while being kept on ice.In another set of experiments, fish species that are unused and/or rarely used because of their low commercial value, caught in the Ariake Sea (unused fish), were acquired from a fisherman on the day of fish sauce production (Wednesday, 13 July 2022).S. melanostictus was repurchased on the same day to prepare fish sauce for comparison with the unused fish sauce.The weights and lengths of the fish used in this study are listed in Tables 1 and 2, respectively.Fish sauce was produced according to the method described by Tagawa et al. [5].Fish sauce using the unused fish was prepared by mixing each fish species according to their composition ratio at the time of fishing in the Ariake Sea (Pennahia argentata:Cynoglossus abbreviatus:Pampus puntatissimus:O.lacepedii = 5:2:2:1 by weight).The fish mixture prepared in the experiment started on 4 March 2021, and 13 July 2022, and was referred to as "collective mixture" and "unused fish mixture", respectively.

Fish Sauce Characterization
The degree of fish meat decomposition in the fish sauce mashes was visually observed, and the odor impression was evaluated.

Viable Mesophilic Bacteria Count
The fish sauce moromi was coarsely filtered through a drainage net and centrifuged (1000× g, 15 min, 4 • C).A volume of 100 µL of fish sauce sample was serially diluted in 0.85% NaCl solution, plated on Tryptic Soy agar (TSA; Difco, Detroit, MI, USA), and incubated at 30 • C for 2 days.Colonies formed on the plate (10-1000 colonies/plate) were counted, and viable counts were expressed as colony-forming units (CFU/mL).

Biogenic Amine Content
The proteins and fats contained in the fish sauces were removed according to the method described by Noma et al. [5] after rough filtration and centrifugation.The biogenic amine contents of the resulting fish sauces were analyzed using a pre-column derivatization method with dansyl chloride.Briefly, amines in the fish sauce samples and standard amines derivatized with dansyl chloride (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were separated by isocratic elution using 75% acetonitrile (Sigma-Aldrich Co. LLC, St. Louis, MO, USA) as the mobile phase on an Intersil ODS-SP column (GL-science Inc, Tokyo, Japan) at a flow rate of 1.0 mL/min and a temperature of 40 • C, and detected using a UV detector at a wavelength of 254 nm.The concentration of each amine in the fish sauce samples was determined by comparing the areas of the peaks with retention times matching those of the standard sample.The analysis was performed with an HPLC system, an L-6200 intelligent pump, an L-5090 degasser, and an L-2400 UV detector (Hitachi High-Technologies Co., Tokyo, Japan).

Free Amino Acid Content
The free amino acid composition of fish sauces without proteins and fats was analyzed using the method described by Noma et al. [5].Briefly, free amino acids in fish sauces and standard amino acid mixture (FUJIFILM Wako Pure Chemical, Osaka, Japan) were derivatized using phenylisothiocyanate (PTC, FUJIFILM Wako, Osaka, Japan) and analyzed using the HPLC system with Wakopak ® Wakosil-PTC (4.0 mm × 250 mm, FUJIFILM Wako) under a gradient condition from 0% (0 min) to 70% (15 min) of solvent B at a flow rate of 1.0 mL/min at 40 • C, and detection was performed at 254 nm.Eluents A and B were specifically designed for analysis using a column (FUJIFILM Wako).The concentration of free amino acids was determined by comparing the peak areas in fish sauces with a standard amino acid mixture.

Organic Acid Analysis
After defatting and deproteinizing the fish sauce, 25 µL of the fish sauce samples were analyzed with the Nexera XR organic acid analysis system (Shimadzu, Kyoto, Japan) according to the manufacturer's protocol.The concentration of each organic acid in the fish sauce samples was calculated by comparing the peak areas with those of a standard organic acid mixture.

Sensory Evaluation
The odor of the fish sauce was evaluated using a two-point test.Ten non-trained panelists (four males and six females, ages [21][22][23][24] participated in the test.The test was conducted to confirm whether an untrained panel representing typical consumers could distinguish between FS con and FS CO 2 .Two milliliters of the fish sauce sample was placed in a test tube and covered with aluminum foil so that the type of sample could not be determined visually from its appearance.Each test tube containing a fish sauce sample was randomly assigned a three-digit number, and the samples were allowed to stand at room temperature for 30 min before sensory testing.The panels were asked to select the one fish sauce with stronger putrefactive odor, fishy smell, rancid odor, shore scent, soup stock-like scent, and preferable odor when comparing between FS CO 2 and FS con prepared from each fish species.A binomial distribution with p = 1/2 was used for the significant difference test.

Analysis of Volatile Compounds
Volatile compounds in the fish sauce were analyzed by solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) according to Tagawa et al. (2022) [6].In brief, fish sauce samples in glass sample bottles were absorbed at 37 • C for 40 min on SPME fiber (divinylbenzene dispersion/dimethylsiloxane, SUPELCO, MilliporeSigma, Darmstadt, Germany) and desorbed at 210 • C for 10 min within the GC-MS (GC-MS-QP2010, Shimadzu, Kyoto, Japan) for analysis.DB-WAX (30 m × 0.25 mm i.d.× 0.25 µm film thickness) (Agilent Technologies, Santa Clara, CA, USA) was used for the separation of the volatiles.After GC-MS, a similarity score was calculated for the detected peaks (peak area > 100,000) according to the following formula, where n FS and m FS represent the number of peaks in each fish sauce and the number of fish sauces containing the compound (m FS takes the value 1-5), respectively:

Taste Evaluation Using a Taste Sensor
The taste of each fish sauce was evaluated using a TS-5000Z system (Intelligent Sensor Technology, Inc., Kanagawa, Japan).

Bacterial Flora Analysis by Next-Generation Sequencing
Total DNA was extracted from the fish sauce using an extraction kit (DNA Suisui-E without skim milk, RIZO Inc., Ibaraki, Japan) according to the manufacturer's protocol.16S rRNA (V4) amplicon metagenomic sequencing and determination of operational taxonomic units as relative abundances were performed by Novogen Co., Ltd.(Beijing, China).

Statistical Analysis
Results are expressed as averages of the triplicate measurements ± standard deviation.Principal component analysis (PCA) of free amino acid content and taste was performed using JMP Pro 17.0.0(JMP Statistical Discovery LLC, Cary, NC, USA).Significant differences in viable count, amine content, and organic acid content were determined using Student's t-test and/or Tukey-Kramer's method at p < 0.05, using Bell Curve for Excel 3.21 (Social Survey Research Information Co., Ltd., Tokyo, Japan).

Results and Discussion
In this study, fish sauces were prepared from various fish under pCO 2 conditions for three months.For FS CO 2 , mesophilic bacteria were not detected.The brighter color and more similar and less distinctive flavor, regardless of the raw material, were characteristics of the FS CO 2 .These improved qualities were equal to or greater than those of 2-month FS CO 2 [6], suggesting that extending the fermentation period was favorable for improving the quality of FS CO 2 .Interestingly, the quality of FS CO 2 prepared from the unused fish mixture was comparable to that of sardines, the most popular raw material for fish sauce.The appearance and odor impressions of FS CO 2 and FS con mashes are summarized in Table 3.The fish sauce mash was not subjected to the conventional residue separation procedures used in the preparation of fish sauce, such as filtration, and thus exhibited pronounced turbidity.This observation suggests that the fish may still be undergoing decomposition.Fish meat decomposition was better in FS CO 2 mash than in FS con mash.FS CO 2 mash commonly had a soup stock-like odor, although FS CO 2 prepared from O. lacepedi has a characteristic odor.However, the characteristic fishy and pungent odor impressions of each raw material were observed in FS con mashes.The odor tended to be more average in both the FS con and the FS con mashes when the collective mixture was used as the raw material.As shown in Figure 1, the color of FS CO 2 was lighter than FS con regardless of fish species.Color formation during fish sauce fermentation can be attributed to the Maillard reaction.The Maillard reaction is initiated by the formation of a Schiff base by the reaction between amino and carbonyl compounds and produces melanoidin at the end of the reaction series, resulting in browning.FS CO 2 was prepared under CO 2 -generated anaerobic conditions that suppressed aldehyde formation via lipid oxidation.Horikawa [7] reported that in the middle stage of the Maillard reaction, the Amadori rearrangement proceeds relatively close to neutral.pCO 2 caused the acidification of the fish sauce, which retarded the progress of the reactions after the middle stage.[8] reported that a salinity of more than 20% inhibit of bacteria to an extent that did not adversely affect the quality of fish s

Microbial Quality
Lopetchant et al. [8] reported that a salinity of more than 20% inhibited the growth of bacteria to an extent that did not adversely affect the quality of fish sauce.Figure 2 shows the viable mesophilic bacterial counts in each fish sauce.The counts in FS con prepared from O. lacepedii, T. japonicus, or their collective mixtures exceeded 10 6 CFU/mL.In an experiment comparing S. melanostictus and unused fish mixture, mesophilic bacteria were detected in both FS con .Therefore, 20% salinity was insufficient to inhibit mesophilic bacterial growth.Wang et al. [9] reported that the microbial flora in fish sauce changed dramatically during each fermentation period.In addition, bacterial flora and fish vary greatly with the season [10,11].The growth inhibition effect of 20% salinity was not consistent with that observed in our previous report, with a shorter fermentation period (2 months) and the season at the start of fermentation [6].
FS CO 2 did not contain any detectable mesophilic bacteria, despite 10% salinity.The difference in viable counts between FS con and FS CO 2 was significant for O. lacepedii, T. japonicus, and their mixture.Mesophilic bacteria in FS CO 2 may be inactivated under acidic pH, anaerobicity, and high pressure caused by pCO 2 [12].Higher CO 2 pressure increases the permeability and fluidity of bacterial membranes, causing a decrease in phosphoglycerides relative to phosphatidylethanolamine, which may be essential for microbial growth, and its reduction may lead to microbial inactivation.Changes in the charge balance on the membrane surface owing to a decrease in the pH of the membrane may also cause inactivation.Furthermore, pCO 2 causes destruction of cell surface or cellular tissue [13][14][15] and inactivation of microbial enzymes [16].Anaerobic conditions were generated during the FS CO 2 fermentation process as CO 2 was dissolved in the salt solution with fish and filled the pressure-resistant vessel.As many mesophilic bacteria grow aerobically, anaerobic conditions under pCO 2 conditions may inhibit their growth.Mesophilic bacteria were not detected regardless of the fish species in FS CO 2 .This result is in agreement with Tagawa et al. [6], indicating that factors such as the length of the fermentation period and fishing season have no effect on the suppression of mesophilic bacteria in FS CO 2 , and the pCO 2 condition is suitable for producing fish sauce with more stable quality.

FS
did not contain any detectable mesophilic bacteria, despite 10% salinity.The difference in viable counts between FS and FS was significant for O. lacepedii, T. japonicus, and their mixture.Mesophilic bacteria in FS may be inactivated under acidic pH, anaerobicity, and high pressure caused by pCO2 [12].Higher CO2 pressure increases the permeability and fluidity of bacterial membranes, causing a decrease in phosphoglycerides relative to phosphatidylethanolamine, which may be essential for microbial growth, and its reduction may lead to microbial inactivation.Changes in the charge balance on the membrane surface owing to a decrease in the pH of the membrane may also cause inactivation.Furthermore, pCO2 causes destruction of cell surface or cellular tissue [13][14][15] and inactivation of microbial enzymes [16].Anaerobic conditions were generated during the FS fermentation process as CO2 was dissolved in the salt solution with fish and filled the pressure-resistant vessel.As many mesophilic bacteria grow aerobically, anaerobic conditions under pCO2 conditions may inhibit their growth.Mesophilic bacteria were not detected regardless of the fish species in FS .This result is in agreement with Tagawa et al. [6], indicating that factors such as the length of the fermentation period and fishing season have no effect on the suppression of mesophilic bacteria in FS , and the pCO2 condition is suitable for producing fish sauce with more stable quality.
Biogenic amines are commonly present in fermented foods [17,18].According to the Codex standards [19], the concentration of histamine should not exceed 400 ppm, as it has the greatest effects on the human body, including nausea and headache among the amines [20,21].Histamine production in fish is primarily attributed to histidine decarboxylase enzymes of histamine-producing bacteria [22].High concentrations of tyramine cause headaches and other physiological effects at high concentrations [22][23][24].Putrescine, cadaverine, and spermidine do not have physiological effects by themselves, but they enhance the physiological effects of histamine and tyramine or inhibit enzymes that decompose histamine [25,26].Table 4 presents the biogenic amine content of each fish sauce sample.The average contents of putrescine and cadaverine in FS and FS were significantly (p < 0.05) higher than those of the other three amines.The mean putrescine and cadaverine levels were also higher in FS than in FS Histamine was detected at levels below the Codex specification (40 mg/mL) in both FS and FS .Uehara et al. [27] reported that histamine production was higher when fish with internal organs underwent fermentation.Noma et al. (2020) reported that histamine was not detected in FS and FS after 6 months of fermentation of sardines with the removal of internal organs under pCO2 [5].Biogenic amines are commonly present in fermented foods [17,18].According to the Codex standards [19], the concentration of histamine should not exceed 400 ppm, as it has the greatest effects on the human body, including nausea and headache among the amines [20,21].Histamine production in fish is primarily attributed to histidine decarboxylase enzymes of histamine-producing bacteria [22].High concentrations of tyramine cause headaches and other physiological effects at high concentrations [22][23][24].Putrescine, cadaverine, and spermidine do not have physiological effects by themselves, but they enhance the physiological effects of histamine and tyramine or inhibit enzymes that decompose histamine [25,26].Table 4 presents the biogenic amine content of each fish sauce sample.The average contents of putrescine and cadaverine in FS con and FS CO 2 were significantly (p < 0.05) higher than those of the other three amines.The mean putrescine and cadaverine levels were also higher in FS CO 2 than in FS con Histamine was detected at levels below the Codex specification (40 mg/mL) in both FS con and FS CO 2 .Uehara et al. [27] reported that histamine production was higher when fish with internal organs underwent fermentation.Noma et al. (2020) reported that histamine was not detected in FS con and FS CO 2 after 6 months of fermentation of sardines with the removal of internal organs under pCO 2 [5].The volatile compounds in each fish sauce were analyzed using GC-MS, and the obtained peaks with areas larger than 100,000 were compared.For FS con , the numbers of S. melanostictus, O. lacepedii, T. japonicus, K. punctatus, and their collective mixture were 63, 55, 44, 57, and 63, respectively.For FS CO 2 , the numbers of peaks were 42, 37, 43, 35, and 37 for S. melanostictus, O. lacepedii, T. japonicus, K. punctatus, and their collective mixture, respectively.The numbers of peaks commonly observed for each fish sauce are shown in Figure 3.For example, 16 peaks observed in FS con prepared from S. melanostictus were also detected in the three types of FS con .For FS con prepared from S, O, T, and M, the number of volatile compounds contained in the sample was greater than 17.The peaks common to all FS CO 2 were not specific to FS CO 2 , and were also common to all or some of the FS con .The ratio of the number of peaks specific to one fish species or common to two fish species was 47.5% in FS con , which was higher than that in FS CO 2 (35.0%).The ratio of the peaks specific to FS con was calculated as 66.3%.Therefore, the results suggest that FS con contains fish-specific odor components, while FS CO 2 enables the production of fish sauce with a reduced fish-specific odor.

Odor
The volatile compounds in each fish sauce were analyzed using GC-MS, and the obtained peaks with areas larger than 100,000 were compared.For FS , the numbers of S. melanostictus, O. lacepedii, T. japonicus, K. punctatus, and their collective mixture were 63, 55, 44, 57, and 63, respectively.For FS , the numbers of peaks were 42, 37, 43, 35, and 37 for S. melanostictus, O. lacepedii, T. japonicus, K. punctatus, and their collective mixture, respectively.The numbers of peaks commonly observed for each fish sauce are shown in Figure 3.For example, 16 peaks observed in FS prepared from S. melanostictus were also detected in the three types of FS .For FS prepared from S, O, T, and M, the number of volatile compounds contained in the sample was greater than 17.The peaks common to all FS were not specific to FS , and were also common to all or some of the FS .The ratio of the number of peaks specific to one fish species or common to two fish species was 47.5% in FS , which was higher than that in FS (35.0%).The ratio of the peaks specific to FS was calculated as 66.3%.Therefore, the results suggest that FS contains fish-specific odor components, while FS CO 2 enables the production of fish sauce with a reduced fish-specific odor.The peak similarity score, which indicates how many fish species the volatile compounds present in each FS and FS were common to on average, was calculated.Fish sauces with higher scores indicated higher similarity in volatile compounds among fish sauces.The score for FS was 3.27 and that for FS was 2.77.Thus, the fish sauce preparation methods with higher scores indicated a higher similarity of volatile compounds among the fish sauces, indicating that the volatile compound profiles of FS were more similar than those of FS .This result was consistent with the observation that the unique odor of the fish species was suppressed, and the common odor of fish sauce was a soup stock-like odor in FS (Table 3).A 16% increase in the percentage of peaks commonly present in FS was observed in the unused fish mixture, indicating that the types of volatile compounds tended to be similar.The odor characteristic of FS showed a decreased fish-specific odor and an increased soup stock-like odor.This result is consistent with those observed in the sensory analysis in this study.
Table 5 shows the odor attributes with significant differences (p < 0.05) and significant trends (p < 0.11) in FS by sensory evaluation.The quantification of the odor impressions is shown in Table 3.In FS , putrefactive and rancid odors were reduced, The peak similarity score, which indicates how many fish species the volatile compounds present in each FS CO 2 and FS con were common to on average, was calculated.Fish sauces with higher scores indicated higher similarity in volatile compounds among fish sauces.The score for FS CO 2 was 3.27 and that for FS con was 2.77.Thus, the fish sauce preparation methods with higher scores indicated a higher similarity of volatile compounds among the fish sauces, indicating that the volatile compound profiles of FS CO 2 were more similar than those of FS con .This result was consistent with the observation that the unique odor of the fish species was suppressed, and the common odor of fish sauce was a soup stock-like odor in FS CO 2 (Table 3).A 16% increase in the percentage of peaks commonly present in FS CO 2 was observed in the unused fish mixture, indicating that the types of volatile compounds tended to be similar.The odor characteristic of FS CO 2 showed a decreased fish-specific odor and an increased soup stock-like odor.This result is consistent with those observed in the sensory analysis in this study.
Table 5 shows the odor attributes with significant differences (p < 0.05) and significant trends (p < 0.11) in FS CO 2 by sensory evaluation.The quantification of the odor impressions is shown in Table 3.In FS CO 2 , putrefactive and rancid odors were reduced, preferred odors were enhanced regardless of the fish species, and the soup stock-like scent tended to be enhanced.In addition, when comparing the S. melanosticus and the unused fish mixtures, similar differences were observed.These trends were in agreement with those of some 2-month fish sauces [6], and the longer fermentation period of 3 months in the present study tended to improve the odor of FS CO 2 .This suggests that a fermentation period of 3 months or longer is preferable for the production of FS CO 2 in terms of odor.Furthermore, all panelists distinguished between FS CO 2 and FS con prepared from S. melanostictus and an unused fish mixture.However, more than half of the panelists could not distinguish between FS CO 2 prepared from a mixture of S. melanostictus and the unused fish mixture.The results of both GC-MS and sensory analyses demonstrated that the FS CO 2 exhibited a Foods 2024, 13, 2646 9 of 14 comparable odor profile, suggesting that pCO 2 can be employed to generate fish sauces with a similar odor profile, even when utilizing unspecified fish as the raw material.

Taste
The taste analyzers recognized three types of first tastes (salty, umami, bitterness and miscellaneous), and two types of aftertastes (bitter and umami).Figure 4 shows a radar plot comparing the tastes detected in FS CO 2 and FS con .The radar shape varied with FS con , and the bitterness of miscellaneous tastes commonly decreased in FS CO 2 .This tendency was more pronounced in 3-month fish sauce compared than in 2-month fish sauce [6]. Figure 4 shows the PCA of the tastes detected in FS CO 2 and FS con .The distribution of FS con is scattered, whereas that of FS CO 2 is similar.These results suggest that the taste of FS con reflects the characteristics of each raw fish, whereas in FS CO 2 , regardless of the raw fish, the tastes converged to the same family, characterized by enhanced umami richness and reduced bitterness.
Table 6 shows the amount of free amino acids in each fish sauce sample.The total amounts of free amino acids in FS CO 2 prepared from S. melanostictus, O. lacepedii, T. japonicus, K. punctatus, and their collective mixture, respectively, were 1.9, 2.2, 1.3, 1.7, and 1.4 times and significantly (p < 0.05) higher than those in FS con .Furthermore, a significant (p < 0.05) increase in total free amino acid content was observed in the FS CO 2 prepared from the unused fish mixture.Free amino acids are produced by endogenous proteases and the contaminating bacteria [1].Endogenous acid proteases can effectively hydrolyze fish proteins in a pCO 2 environment because the number of contaminating bacteria may be reduced under such conditions.
Therefore, the enrichment of total free amino acid content is a universal phenomenon in FS CO 2 regardless of fish type.Figure 5 shows the principal component analysis of the proportions of free amino acids in each fish sauce.The free amino acid composition appeared to be divided into two groups, depending on PC1 between FS CO 2 and FS con .Glutamic acid, a typical umami amino acid, does not always constitute this group.The PCA results indicate that the free amino acid composition did not provide sufficient evidence for enhanced umami taste and umami richness.Imai et al. [28] reported that the bitter taste was not caused mainly by free amino acids but by peptides such as carnosine and glutathione.Kuroda et al. [29] reported that a tripeptide, glutamylvalylglycine, detected in fermented foods such as fish sauce, has umami richness.
Jung et al. [30] and Ohshima et al. [1] reported that microbial metabolism during fermentation plays an important role in flavor formation in fermented fish-based foods.Wang et al. [31] reported that the peptides in fish sauce are formed by the hydrolysis of fish proteins by microbial proteases.Therefore, the bacterial flora of each fish sauce was analyzed using 16S rRNA amplicon analysis and presented as relative ratios of the bacterial flora (Figure 6).In all raw fish, the bacterial flora changed with the different methods of fish sauce preparation.However, the dominant species in both the FS CO 2 and FS con collective mixture were staphylococci.The diversity of microflora appeared to increase in CO 2 fish sauces.The bacterial flora in fish sauce could not be clearly characterized by FS CO 2 and Foods 2024, 13, 2646 10 of 14 FS con .It is difficult to infer which bacteria contribute to the common flavor observed in FS CO 2 .The bacterial flora in fish sauce changes during fermentation [8].The fermented foods contained dead cells, which were also detected using 16S rRNA amplicon analysis.In addition, 16S rRNA amplicon analysis could not quantify the absolute number of viable cells.Therefore, it is necessary to examine viable cells quantitatively and over time to identify the bacterial species that have a significant impact on flavor similarity.
The organic acid composition was similar for all types of FS CO 2 , and a high lactic acid content was commonly observed in FS CO 2 (Table 7).The types of organic acids used varied depending on the raw material used in FS con .The average lactic acid content of FS CO 2 tended to be higher (p = 0.07) than that of FS con .Lactic acid has a peculiar soft acidity and a low-acid smell.Acidity was not detected in FS CO 2 (Table 3 and Figure 4), suggesting that the organic acids had no significant effect on the flavor of FS CO 2 .Lactic acid in fish sauce is produced by lactic acid bacteria or rigorous mortis [32,33].However, positive evidence that lactic acid bacteria accumulated lactic acid at high concentrations was not obtained from the microflora analysis (Figure 6).In addition, production via the rigor mortis process provides a clear explanation for the difference in lactic acid content between FS CO 2 and FS con prepared from O. lacepedii and T. japonicus.Table 6 shows the amount of free amino acids in each fish sauce sample.The total amounts of free amino acids in FS prepared from S. melanostictus, O. lacepedii, T. japonicus, K. punctatus, and their collective mixture, respectively, were 1.9, 2.2, 1.3, 1.7, and 1.4 times and significantly (p < 0.05) higher than those in FS .Furthermore, a significant (p < 0.05) increase in total free amino acid content was observed in the FS prepared  Therefore, the enrichment of total free amino acid content is a universal p in FS regardless of fish type.Figure 5 shows the principal component an proportions of free amino acids in each fish sauce.The free amino acid appeared to be divided into two groups, depending on PC1 between FS Glutamic acid, a typical umami amino acid, does not always constitute thi PCA results indicate that the free amino acid composition did not provi evidence for enhanced umami taste and umami richness.Imai et al. [28] repo bitter taste was not caused mainly by free amino acids but by peptides such and glutathione.Kuroda et al. [29] reported that a tripeptide, glutamyl detected in fermented foods such as fish sauce, has umami richness.Jung et al. [30] and Ohshima et al. [1] reported that microbial metab fermentation plays an important role in flavor formation in fermented fish- Funding: This research was supported by the Japan Society for the Promotion of Science (grant number 20K02406) and the 2017 Preliminary Research Subsidy Project related to functional research of agriculture, forestry, and fishery products, foods, etc., by the Foundation of the Saga Prefecture Regional Industry Support Center.
Institutional Review Board Statement: Ethical review of sensory testing in this study was waived by Ethics Review Committee, Saga University Faculty of Medicine.
Informed Consent Statement: Ethical review of sensory testing in this study was waived by Ethics Review Committee, Saga University Faculty of Medicine.However, for ethical considerations, the information related to the experiment was fully explained to the panelists who might participate in the experiment before the start of the experiment, and only those who agreed to participate in the sensory testing were allowed to do so.

Figure 4 .
Figure 4. Comparison of tastes detected in FS and FS with radar charts and their PCA.Blue and red lines/dots in the graphs indicate FS and FS , respectively.Blue and red dots on PCA graphs show FS and FS , respectively.S, S. melanostictus; O, O. lacepedii; T, T. japonicus; K, K. punctatus; M, collective mixture; S*, S. melanostictus; U*, unused fish mixture.* The set of experiments comparing FS prepared from S. melanostictus (S*) and an unused fish mixture (U*).

Figure 4 .
Figure 4. Comparison of tastes detected in FS CO 2 and FS con with radar charts and their PCA.Blue and red lines/dots in the graphs indicate FS CO 2 and FS con , respectively.Blue and red dots on PCA graphs show FS CO 2 and FS con , respectively.S, S. melanostictus; O, O. lacepedii; T, T. japonicus; K, K. punctatus; M, collective mixture; S*, S. melanostictus; U*, unused fish mixture.* The set of experiments comparing FS CO 2 prepared from S. melanostictus (S*) and an unused fish mixture (U*).

Figure 5 .
Figure 5. PCA analysis of free amino acid relative composition of fish sauces.Blue a the graphs indicate FS and FS , respectively.S, S. melanostictus; O, O. lacepedii; K, K. punctatus; M, collective mixture.

Figure 5 .
Figure 5. PCA analysis of free amino acid relative composition of fish sauces.Blue and red dots in the graphs indicate FS CO 2 and FS con , respectively.S, S. melanostictus; O, O. lacepedii; T, T. japonicus; K, K. punctatus; M, collective mixture.

Table 1 .
Average weights and lengths of fishes used for fish sauce production.

Table 2 .
Weights and lengths of fishes (S. melanosticus and unused fish mixture) used for each fish sauce preparation.

Table 3 .
Appearances and odor impressions of FS CO 2 and FS con mashes.
CO Almost completely soup stock-like, shore-like, and a little rancid * The descriptions below the double lines are for the set of experiments comparing FS CO 2 prepared from S. melanostictus and an unused fish mixture.Foods 2024, 13, 2646 6 of 14 Foods 2024, 13, x FOR PEER REVIEW Figure 1.Appearances of fish sauce mashes.S, S. melanostictus; O, O. lacepedii; T, T punctatus; M, collective mixture; S*, S. melanostictus; U*, unused fish mixtur experiments comparing FS prepared from S. melanostictus and an unused fish m Table 3. Appearances and odor impressions of FS and FS mashes.Raw Materials FS Decomposition Degree of Fish Meat Odor S. melanostictus FS Almost completely thick FS Completely a little savory, soup stock-like, and stronger than O. lacepedii FS Incomplete fishy, soup stock-like, and characteristic smell o FS More than the FS a little savory, soup stock-like, and light T. japonicus FS Completely fishy, a little soup stock-like, and pungent FS Almost completely savory, soup stock-like, stronger than the FS K. punctatus FS Almost completely FS of S. melanostictus-like, a little savory, and smell of K. punctatus FS Almost completely a little savory, stronger than the FS , and ligh * Unused fish mixture FS sour and pungent FS Almost completely soup stock-like, shore-like, and a little rancid 3.1.2.Microbial Quality Lopetchant et al.

Table 5 .
Odor attributes for which a significant difference or significant tendency was observed in FS CO 2 .S. melanostictus; O, O. lacepedii; T, T. japonicus; K, K. punctatus; M, collective mixture; S*, S. melanostictus; U*, unused fish mixture.* The set of experiments comparing FS CO 2 prepared from S. melanostictus (S*) and an unused fish mixture (U*).