Original papers
Application of Pressurized Carbon Dioxide during Salt-Reduced Sardine Fish Sauce Production
2020 Volume 26 Issue 2 Pages 195-204
Details
2020 Volume 26 Issue 2 Pages 195-204
During sardine fish sauce production, salt concentration needs to be maintained at a specific level to inhibit the growth of undesirable microorganisms. Here, we employed pressurized CO2 to control microbial growth in fish sauce. Fish sauces prepared at 30 °C for 6 months under 1–5 MPa pressurized CO2 with 10% NaCl (FSCO2) were compared to those produced under atmospheric pressure with 10–20% NaCl (FScon). FScon with 20% NaCl had a slight putrefied odor, whereas FSCO2 exhibited a favorable odor despite its low NaCl concentration. In FSCO2, bacterial count, biological amine content, and pungent organic acids decreased while free amino acid content increased compared to the control. Sensory evaluation indicated the FSCO2 had weaker rancidity, fish-like, putrefactive, and pungent odors but an enriched dashi-like odor. These results show that pressurized CO2 application can produce a salt-reduced, amino acid-enriched, and sensorially ameliorated sardine fish sauce.
Fish sauce is a liquid seasoning with a clear brown color that it is broadly consumed in Southeast Asia. In some areas of Southeast Asia, fish sauce is a major source of protein (Xu et al. 2008). Fish sauce is produced by fermenting fish under high salt concentrations for 6–12 months. During this period, the whole fish (especially the muscles) is hydrolyzed by endogenous proteases, leading to amino-acid enrichment. For example, the Japanese fish sauce, Ishiri, has approximately twice the concentration of amino acids compared to soy sauce (Michihata et al. 2000).
The addition of salt (NaCl) concentrations above 20% during the fish fermentation process is essential for inhibiting the proliferation of unpleasant microorganisms that decrease fish sauce quality by producing of amines and organic acids with pungent odors. However, a high salt concentration does not increase the commercial value of the fish sauce because consumers are sensitive to excess NaCl consumption. Sasaki (2014) reported that the NaCl concentration of fish sauce can be reduced from 25% to 13% by electrodialysis without significant loss of amino acids; however, this salt concentration is still considered high. In addition, unsaturated fatty acids, alcohols, and aldehydes contained in fish are gradually oxidized during fermentation, which can reduce the sensory quality of fish sauce. Karahadian and Lindsay (1989) reported that aldehydes such as 2-methylpropanol, 2-methylbutanol, and 3-methylbutanol are formed from lipid oxidation during fermentation. Shimoda et al. (1996) detected oxidation-derived odors such as 2-methylpropanol and 2-methylbutanol by the headspace gas analysis of fish sauce.
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Meyssami et al. (1992) indicated that the pressurized dissolution of CO2 into water at room temperature decreased the pH to approximately 3.5, 3.3, and 3.2 at 1, 3, and 5 MPa, respectively. Gildberg et al. (1984) reported that lowering the pH to 4 using acetic acid and hydrochloric acid contributed to the reduction of salt concentrations by 5–15% in anchovy (Stolephorus sp.) fish sauce production. Therefore, we hypothesized that reducing the salt concentration of fish sauce could be achieved by the inhibition of microbial proliferation using CO2. Application of pressurized CO2 has the advantage of not requiring acidified pH neutralization after fermentation because the solubilized CO2 can be outgassed naturally or removed with mild heat treatment after the fermentation process. In addition, the replacement of air with CO2 and the following introduction of CO2 within the pressure vessel creates an anaerobic condition when fish are immersed into the NaCl solution. Under this anaerobic condition, additional benefits such as reduced fish oxidation and growth inhibition of aerobic and facultative anaerobic microorganisms, are promising.
The aim of the present study was to produce sardine fish sauces under pressurized CO2 conditions and to compare the efficiency of this process with that of the conventionally used method. The results may aid in providing a novel method to produce salt reduced food product using CO2.
Chemicals Most reagents were obtained from FUJIFILM Wako Pure Chemical (Osaka, Japan). The reagents purchased from the other suppliers are individually specified.
Fish sauce production Sardines (Sardinops melanostictus) were purchased at a local market in Saga city (Japan). The surfaces of seven sardines were thoroughly washed with tap water, and the head, internal organs, and guts were removed. The cleaned fish were cut into round slices and divided into 40 g portions. For producing fish sauce under a pressurized CO2 condition, a prepared 40 g portion was put into a cylindrical glass container (ϕ 36 mm × 130 mm) and 40 mL autoclaved NaCl solution was added to the container to a final NaCl concentration of 10% (w/v) taking the water content of the fish body into consideration. The glass container was set into a pressure vessel, as illustrated in Fig. 1. The pressure vessel with a 500 mL inner volume used for pressurized CO2 treatment was obtained from Taiatsu Techno (Tokyo, Japan). CO2 gas was allowed to flow into the pressure vessel for a certain amount of time, under the opening pressure control valve, to remove any remaining air in the vessel. Next, the valve was closed and CO2 gas was introduced from a CO2 gas cylinder until the vessel pressures of 1, 3, and 5 MPa were attained, and then the vessel was placed in an incubator at 30 °C for 6 months. If CO2 pressure decreased 0.3 MPa from the desired pressure, additional CO2 gas was introduced to recover the original pressure. For the control conditions, fish sauce was produced using a conventional method under atmospheric pressure. A 40 g portion of sardines was immersed in 10, 15, and 20% NaCl solutions and put into high-density polyethylene (HDPE) bags (8 µm thick, Nippon-Giken Industrial. Co., Ltd., Tokyo, Japan). The bags were heat-sealed and incubated at 30 °C for 6 months. However, heat treatment, hiire, was not carried out on the produced fish sauces before their analyses, because we intended to evaluate the effect of pressurized CO2 on the quality of the sauce. The produced fish sauce mushes were roughly filtered through a plastic mesh filter for cooking (approximately 0.6 mm pore size) and centrifuged at 1 000 × g and 4 °C for 15 min. The resultant supernatant was defined as fish sauce. This fish sauce was recovered with a Pasteur pipet and the fish sauce yield was expressed as its volume (mL) as determined with a measuring cylinder. The fish sauce was then subjected to further preparation and/or analyses (Fig. 2). Practical NaCl concentrations of the prepared fish sauce were determined as the chloride ion concentration using Mohr's method (http://www.chemteach.ac.nz/investigations/documents/chloride_ mohr.pdf). The pH of the fish sauces was measured with a pH meter (F-52, Horiba Co. Ltd., Tokyo, Japan), and color (L*a*b*) was evaluated by the transmission method using a spectral colorimeter (CM-5, Konica Minolta Japan, inc., Tokyo, Japan). A plastic cuvette with an optical path length of 2 mm was used for color measurement.
Schematic diagram of the pressurized CO2 treatment apparatus
Bacterial counts For counting mesophilic bacteria, fish sauces were serially diluted using a sterile 0.85% NaCl solution, plated onto tryptic soy agar plates (BD Difco, Franklin Lakes, NJ, USA) and incubated at 30 °C for 4 d before the colonies were counted. Lactic acid bacteria were detected by mixing 100 µL diluted fish sauces and 20 mL MRS agar (BD Difco), which was then poured into plastic petri dishes. The dishes were incubated at 35 °C for 1 week under anaerobic conditions using an AnaeroPack and anaerobic jars. Escherichia coli and coliform bacteria were counted using 3 M™ Petrifilm™ E. coli/Coliform Count Plates (Thermo Fisher Scientific, Waltham, MA, USA) that were incubated at 30 °C for 24 h according to the manufacture's protocol. Fish sauce was heated at 70 °C for 20 min, then interfused with Clostridia medium before being incubated for 24 h at 35 °C in an anaerobic pouch (Sugiyama-gen, Tokyo, Japan).
Biological amines Lipids and proteins were removed from the fish sauces using the following procedures before biological amines were measured: 2 mL prepared fish sauce and an equal volume of n-hexane were poured into a test tube and vortexed for approximately 1 min. The mixture was left to stand until it separated into a bilayer, the aqueous phase was then transferred to another test tube and 2.3-times the volume of ethanol was added. The mixture was vortexed, then centrifuged at 3 000 × g for 5 min at 4 °C. The resultant supernatant was filtered through a 0.45 µm syringe filter (Advantec, Tokyo, Japan).
To 100 µL aliquot fish sauce, 67 µL deionized water and 33 µL 10 mg/mL 1, 8-diaminooctane (internal standard) was added. To this mixture, 200 µL acetonitrile was then added, and the mixture was vortexed for several seconds. A saturated sodium carbonate solution (200 µL) was added, and the mixture was vortexed similarly. Then, 200 µL 10% dansyl chloride–acetone (Tokyo Chemical Industry, Tokyo, Japan) was added, and the mixture was again vortexed for 1 min. The mixture was incubated at 50 °C for 45 min before 300 µL toluene was added and vortexed for 15 s. The mixture was allowed to separate into two phases, and the oil phase was transferred to another tube and subjected to a centrifugal concentrator. To the resultant dried substance, 800 µL 10% proline/1.5 M sodium carbonate solution was added, sonicated at 28 kHz for 1 min, and then vortexed for 1 min. After adding 300 µL acetonitrile, the mixture was vortexed and allowed to stand for 1 min. The resulting oil phase was passed through a 0.45 µm syringe filter (Advantec) and subjected to the following reversed phase (RP)–HPLC analysis. Dansyl derivatives of the amines were separated with an Inertsil ODS-SP column (GL Science Inc., Tokyo, Japan; 5 µm, 250 × 4.6 mm) under an isocratic condition with 75% (v/v) acetonitrile–water as an eluent at a flow rate of 1.0 mL/min at 40 °C. The derivatives were detected at 254 nm. The mixture of standard amines (putrescine, cadaverine, histamine, tyramine, and spermidine) was also derivatized by the same method. The concentration of amines in each fish sauce was determined by comparing the experimental peak areas with those of the standard amine solutions. The HPLC system for biological amine analysis included an L-6200 intelligent pump, L-5090 degasser, and L-2400 UV detector (Hitachi High-Technologies Co, Tokyo, Japan). An exploratory experiment revealed that the NaCl concentration of fish sauces after lipid and protein removal had no effect on either the efficiency of derivatization with dansyl chloride or RP–HPLC analysis.
Hydrolysate After lipid and protein removal, the fish sauces were diluted 10 times with 0.1% trifluoroacetic acid and subjected to RP–HPLC analysis. The hydrolysates were separated using an Inertsil ODS-SP column (5 µm, 250 × 4.6 mm; GL Science Inc.) with a programmed gradient: 0% (0–3 min), 40% (20 min), 100% (25 min) of solvent B at a flow rate of 1.0 mL/min at 40 °C using the HPLC system for analysis of biological amines. The hydrolysates were detected by a UV detector at 220 nm. Solvents A and B were water and acetonitrile in 0.1% trifluoroacetic acid, respectively.
Free amino acid content Fish sauces with lipids and proteins removed were used for this analysis. Two microliters cysteic acid, which was prepared by dissolving 50 mg cysteic acid in 1 mL of 0.1 N HCl, were added as an internal standard to both 100 µL fish sauce and the amino acid mixture standard solution type H (FUJIFILM Wako Pure Chemical, Osaka, Japan). Five microliters 100 mM phenylisothiocyanate (PITC; Nacalai Tesque, Kyoto, Japan)–acetonitrile (Sigma, St. Louis, Missouri) solution was added to both 3 µL fish sauce and 10 µL amino acid mixture standard solution, after which 5 µL 1 M triethylamine-acetonitrile was added to both mixtures and incubated at 40 °C for 20 min. These mixtures were dried with a centrifugal concentrator, then the resultant dried matter was dissolved in 1.0 mL mobile phase A used in the Wakopak® Wakosil-PTC system and passed through a 0.22 µm syringe filter (Merck Millipore, Darmstadt, Germany).
The HPLC analysis was performed with mobile phases A and B (PTC-Amino Acids Mobile Phase A and B; FUJIFILM Wako Pure Chemical). The separation was conducted using Wakopak® Wakosil-PTC (4.0 mm × 200 mm, FUJIFILM Wako Pure Chemical) with a programmed gradient from 0% (0 min) to 70% (15 min) of solvent B at a flow rate of 1.0 mL/min at 40 °C. Detection was performed at a wavelength of 254 nm. The HPLC system employed for this analysis was the same as that described for the biological amine analysis. The concentration of each amino acid was determined by calculating the ratio of the peak areas of amino acids in fish sauce against those in the amino acid standard solution, after first standardizing the peak areas of cysteic acid in chromatograms of each fish sauce. The effect of NaCl on PTC-derivatization was corrected by calculating the ratios of peak areas obtained after derivatizations in the presence and absence of NaCl in fish sauce after lipid and protein removal.
Organic acid content Organic acid content of fish sauce was analyzed with a Nexera XR HPLC organic acid analysis system (Shimazdu, Kyoto, Japan) following the manufacturer's instructions. Briefly, organic acids (formic acid, pyruvic acid, succinic acid, malic acid, lactic acid, acetic acid, citric acid, butyric acid, and isovaleric acid) were used as standard solutions. The standard solutions and the fish sauces after removal of lipids and proteins were subjected to Nexera XR analysis. The analysis was performed at 40 °C. NaCl did not affect the organic acid content of fish sauces in this analysis.
Sensory evaluation Sensory evaluation of the fish sauces was conducted by smell test, which was approved by an ethical review committee at Saga University. Eight untrained panelists (five women aged 20–23 years old, and three men aged 21 to 61 years old) from the Saga University laboratory conducted the following sensory evaluation tests. Before the tests, a few members from among the panelists smelled the fish sauces and summarized their impression into six odor attributes, namely dashi-like, ocean-like, rancidity, fish-like, putrefactive, and pungent. After that, all the panelists sniffed the fish sauces in bottles covered with aluminum foil and identified them with random 3-digit numbers. They evaluated the strength of their impression of the six odor attributes of the fish sauces and provided an overall impression using a visual analog scale (Regan et al., 2019). The meaning of these odor attributes and overall impressions were not defined, and panelists were not provided with examples of each odor prior to their analysis. The panelists marked the strength of these impressions on a 10 cm bar with no scale after reading the sentence, “Please sniff each sample and mark the strength of odor attribute on the bar”. The left end of the bar was set to “0” which represented “not perceived”, and the right end was set to “10” representing “strongly perceived”. The length from the left end to the marked positions was measured, and the score was expressed as one-tenth of the measured value; the average of these values from the panels was used as the strength of expression.
Statistical analysis Significant differences of each odor attribute among the fish sauces were determined with Tukey–Kramer's method at p < 0.05 using BellCurve for Excel (Social Survey Research Information Co., Ltd., Tokyo, Japan).
The experimental design of this study is shown in Fig. 2. For the preliminary experiment, fish sauce was produced using a conventional method in the presence of NaCl at predicted final concentrations of 10%, 15%, and 20%. The fish sauces produced at 10% and 15% NaCl had putrefactive odors, whereas that at 20% NaCl had a distinct smell of normal fish sauce. Therefore, fish sauce produced using the conventional method at 20% NaCl served as a control for quality evaluation of fish sauces produced under pressurized CO2 conditions (1, 3, and 5 MPa). The predicted NaCl concentration for fish sauce production under pressurized CO2 was set to 10%, which is half that of the NaCl concentration used in the conventional method. The fish sauces produced under pressurized CO2 seemed to exhibit a pleasant smell despite the reduced NaCl concentration. Therefore, the qualities of the fish sauces produced by the conventional method with 20% NaCl (FScon) and by pressurized CO2 conditions with 10% NaCl (FSCO2 1 MPs, FSCO2 3 MPa, and FSCO2 5 MPa) were compared.
Experimental design of this study
NaCl concentration, pH, yield, and color of fish sauce fermented under pressurized CO2 Fish sauces were initially prepared by immersing sardine bodies into a 17% NaCl solution or 34% NaCl suspension. The NaCl concentrations at the start of fermentation were expected to give final concentrations of 10 and 20% when the water content of the fish was taken into consideration. NaCl crystals that remained undissolved in the 34% NaCl suspension completely dissolved during the fermentation process. After fermentation, the final NaCl concentration of the fish sauces was measured (Table 1). NaCl concentrations of the fish sauces were, as expected, approximately 22% and 10% for FScon and FSCO2, respectively. The pH of the fish sauces was also determined; the pH of FSCO2 was almost neutral, while that of FScon was weakly acidic (Table 1). The effect of the CO2 gas that remained in FSCO2 on pH was excluded because pH was measured after centrifugation.The FSCO2 yield was larger than that of FScon The a* value of FScon was slightly larger than that of FSCO2, and the b* value of FScon was clearly larger than that of FSCO2. These results indicate that FScon had a stronger brown color than FSCO2.
| Fish sauce Pressure (MPa) |
FScon | FSCO2 | |||
|---|---|---|---|---|---|
| 0.1 | 1 | 3 | 5 | ||
| NaCl concentration (%) |
21.6 | 10.2 | 9.86 | 10.4 | |
| pH | 5.5 | 6.3 | 6.2 | 6.4 | |
| Yield (mL) | 23.5 | 34.5 | 35.5 | 36.5 | |
| Color | L* | 95 | 99 | 99 | 99 |
| a* | −2.6 | −1.0 | −1.4 | −1.2 | |
| b* | 29 | 6.7 | 7.3 | 5.5 |
Bacterial counts under pressurized CO2 Each fish sauce was subjected to analysis for the detection of mesophilic and lactic acid bacteria, E. coli, coliforms, and Clostridia Mesophilic bacteria were detected in FScon at 3.46×105 CFU/mL, whereas no colony formation was observed in media for lactic acid bacteria, E. coli, coliforms, and Clostridia in FScon. These results indicated that 20% NaCl can inhibit the growth of lactic acid bacteria, E. coli, coliforms, and Clostridia; however, this concentration was not enough to suppress the proliferation of mesophilic bacteria. It is possible that the mesophilic bacteria detected were already contaminants of the fish body before fermentation or had grown during the fermentation process. Alternatively, no bacterial colony formation was detected in FSCO2, suggesting that CO2 application during the fermentation process reduced the NaCl concentration required to prevent mesophilic bacterial growth to at least 10% NaCl. Moreover, the detectable mesophilic bacteria may be inactivated during fermentation under pressurized CO2.
Biological amine content under pressurized CO2 The concentrations of amines, putrescine, cadaverine, histamine, tyramine, and spermidine in the prepared fish sauces were measured, and FScon contained almost all of these amines in high concentrations (Table 2). FSCO2 1 MPa also contained these amines, although their concentrations were slightly lower than those in FScon. FSCO2 3 and 5 MPa only contained cadaverine, and its concentrations were significantly lower than those in FScon and FSCO2 1 MPa. These results indicate that pressurized CO2 contributed to a reduction in biological amine production during the fermentation of sardine fish sauces.
| Fish sauce Pressure (MPa) |
FScon | FSCO2 | ||
|---|---|---|---|---|
| 0.1 | 1 | 3 | 5 | |
| putrescine | 19.1 | 16.9 | ND | ND |
| cadaverine | 111 | 118.7 | 24.1 | 20.0 |
| histamine | 54.0 | 40.0 | ND | ND |
| tyramine | 66.3 | 17.9 | ND | ND |
| spermidine | 91.0 | 22.5 | < 2.9 | < 2.9 |
ND: not detected
Hydrolysates in CO2 pressurized fermentation and traditional fermentation methods After protein and lipid removal, fish sauces were subjected to RP–HPLC with a UV detector (220 nm) to analyze hydrolysates in the fish sauce, particularly peptides. Overall, the detected peaks were those of peptides and the total peak area was smaller in FScon than in FSCO2. The area of the peak eluted at 11.0 min was larger in FSCO2 than FScon. The peaks at 14.8, 19.6 and 20.3 min were observed in FScon only, and those at 13.3 and 15.8 min were almost lost in FScon (Fig. 3). These results suggest that CO2 application can partially change the hydrolytic behavior in sardine fish bodies during fermentation.
RP-HPLC analysis of protein hydrolysates from the fish sauces fermented with or without pressurized CO2 treatment. *elution times of hydrolysates with clearly different peak areas.
Free amino acid content in CO2 pressurized fermentation and traditional production methods The free amino acid content in each fish sauce is shown in Table 3. FSCO2 1, 3, and 5 MPa contained approximately 1.8, 2.8, and 2.8-fold total free amino acids in FScon, respectively. This result indicates that pressurized CO2 can increase free amino acid content.
| Fish sauce Pressure (MPa) |
FScon | FSCO2 | ||
|---|---|---|---|---|
| 0.1 | 1 | 3 | 5 | |
| Gly | 18 | 156 | 211 | 344 |
| Ala | 161 | 618 | 783 | 847 |
| Val | 175 | 310 | 623 | 577 |
| Leu | 334 | 559 | 855 | 889 |
| Ile | 78 | 292 | 432 | 350 |
| Ser | 64 | 186 | 546 | 486 |
| Thr | 86 | 458 | 731 | 651 |
| Cys | ND | ND | ND | ND |
| Met | 52 | 205 | 852 | 395 |
| Phe | 50 | 192 | 319 | 295 |
| Tyr | 64 | 167 | 377 | 383 |
| Pro | 67 | 229 | 249 | 264 |
| Asp | 100 | 142 | 216 | 42 |
| Glu | 160 | 427 | 712 | 285 |
| Lys | 631 | 478 | 702 | 790 |
| Arg | 875 | 714 | 1 351 | 1 555 |
| His | 660 | 1 293 | 1 107 | 1 743 |
| Total | 3 576 | 6 426 | 10 065 | 9 896 |
Trp, Gln, and Asn were not tested.
ND: not detected
Organic acid content of fish sauce produced under pressurized CO2 The organic acid content of fish sauces is presented in Table 4, which shows that major organic acids in both FScon and FSCO2 samples included lactic acid. FScon samples contained succinic acid, formic acid, and acetic acid at higher amounts than the other fish sauces tested (FSCO2 3 and 5 MPa). All fish sauces did not contain citric acids, pyruvic acids, and butyric acids.
| Fish sauce Pressure (MPa) |
FScon | FSCO2 | ||
|---|---|---|---|---|
| 0.1 | 1 | 3 | 5 | |
| Malic | 6.3 | 8.7 | 4.2 | 3.3 |
| Succinic | 20 | 12 | 2.1 | 1.7 |
| Lactic | 290 | 490 | 420 | 390 |
| Formic | 59 | 2.3 | 4.4 | 4.1 |
| Acetic | 24 | 25 | 5.3 | 4.6 |
| Isovaleric | 11 | ND | ND | ND |
ND: not detected
Sensory evaluation of fish sauces produced with and without CO2 pressurized fermentation Odor sensory evaluation was performed for each fish sauce (Fig. 4a and b). An ocean-like odor (desirable) was not significantly different among the fish sauces; however, a dashi-like odor (desirable) was significantly stronger in FSCO2 1 MPa and 3 MPa samples than in FScon. Undesirable odors, especially rancidity, putrefactive, and pungent odors, were significantly lower in FSCO2 samples than in FScon. The overall impression of the odor was significantly higher in FSCO2 than in FScon (Fig. 4b), indicating that the odor quality of FSCO2 samples was superior to that of FScon.
Sensory evaluation of fish sauce odor attributes. (A) Intensity of odor impressions are presented. (B) Representative overall odor impression scores. Different characters indicate significant differences at p < 0.05 determined by the Tukey–Kramer method. Data in (B) represent the average value from eight panelists and error bars indicate the standard deviation of the average.
CO2 has been used in food processing such as drink carbonation and the selective removal of caffeine from coffee. In addition, the ability of CO2 to suppress microorganisms in food has been studied; for example, CO2 treatment in combination with mild heating can inactivate bacterial vegetative cells (Noma et al. 2010). Although bacterial spores are not inactivated by pressurized CO2 alone (Noma et al. 2015), the germination and following outgrowth of bacterial spores are inhibited under the acidic conditions created by dissolving CO2 in water. These findings motivated us to apply pressurized CO2 to control bacterial growth during the fermentation process of fish sauce production, which led to the reduction of NaCl required for fish sauce production without compromising the inhibition of unpleasant bacterial growth (above 20%). In the present study, we applied pressurized CO2 (1, 3, and 5 MPa) during the fermentation process of sardine fish sauce production in the presence of NaCl at half the concentration required in the conventional method. The resultant salt-reduced sardine fish sauces were almost free of harmful components and possessed superior odor sensory qualities.
The brown color depth of FScon was stronger than that of FSCO2, which may have been caused by the Maillard reaction during fermentation (Dissaraphong et al., 2006). This reaction is accelerated by heating, lower water activity (approximately 0.6–0.7), and alkaline conditions. Heating did not contribute to the difference in color among the fish sauces because the temperature during fermentation was standardized at 30 °C. FScon has lower water activity than FSCO2, which was attributed to the difference in NaCl concentration. The final pH of FScon was 5.5 and that of FSCO2 was approximately 6.3. The pH of FScon mush may have been higher than that of FSCO2 mushes at the early stage of the fermentation because the pH of the pressurized pure water decreased to approximately 3.2 in a pressure-dependent manner (Meyssami et al. 1992). Therefore, the reduced water activity and higher pH in the traditional fermentation method may be the major factor for the accelerated Maillard reaction in the fermentation process of FScon, and explain the observed color differences among the fish sauce samples. Although fish sauce contains low levels of reducing sugars, other carbohydrate derivatives can induce the Maillard reaction (Kawashima and Yamanaka, 1996). Alternatively, the deepened brown color may have been the result of reactions between free amino acids and products derived by lipid oxidation and lipolysis (Jiang et al., 2007). This was supported by the results of the odor sensory evaluation (Fig. 4), which suggested that oxidation was more pronounced in FScon compared to FSCO2. Fish sauce properties are controlled by consumer demand (Lopetcharat et al. 2001), and fish sauce with a light brown color can be prepared by applying pressurized CO2 during fermentation.
An increase in NaCl concentration decreases the water activity of fish sauce mush, which decreases bacterial cell growth. In addition, osmotic pressure generated by the high concentration of NaCl can kill or retard microbial cell proliferation by inducing plasmolysis (Lopetcharat et al. 2001). Hence, we sought to detect the number of viable cells using usual media to perform quality control analysis of the fish sauces. Our experiment revealed that the conventional production method for fish sauce required 20% NaCl to inhibit putrefaction, similar to that of commercially available fish sauces (Lopetcharat et al. 2001). Bacterial counts in FScon showed that growth of various bacteria was inhibited; however, mesophilic bacteria were still viable at this NaCl concentration during the fermentation process. In contrast, no bacteria were detected in the FSCO2 samples using the plate-counting method, despite their 10% NaCl concentration. The lower pH of the fish sauce mushes under pressurized CO2 likely contributed to this effective inhibition of microbial proliferation. CO2 molecules can penetrate microbial cells and release H+ in the cytoplasm (Dixon and Kell 1989; Spilimbergo et al. 2002). Microbial cells try to discharge excess protons through a respiration chain by consuming ATP (Klangpetch et al. 2011). However, the cytoplasm is eventually acidified when ATP is depleted by this active consumption, causing injury and loss of microbial cell viability (Kim et al. 2008). Hurdle techniques comprised of 10% NaCl and decreased pH may help in inhibiting the growth of these bacteria in pressurized CO2 conditions. NaCl could sensitize microbial cells to lower pH levels because NaCl-mediated injury to the cell membrane may reduce H+-efflux. Conversely, injury suffered due to pressurized CO2 can sensitize the cells to NaCl, as described by Bi et al. (2018), who reported the injury of Escherichia coli O157:H7 cells as pressurized CO2 decreased cellular resistance to NaCl.
It is generally recognized that halophilic and halotolerant bacteria are hardly detected by the normal plate-counting method. To address the matter of whether any bacteria existed and contributed to the odor of FSCO2, bacterial flora analysis using next-generation sequencing should be carried out for the detection of the non-culturable bacteria.
Some amines are formed by bacteria during food spoilage (Ibe 2014). Of the amines, putrescine, cadaverine, and spermidine affect fish sauce odor, and the histamines contained within these amines can cause food putrefaction and allergy-like food poisoning (Ibe 2014). The Codex standard states that the histamine content in fish sauce should be below 40 mg/100 g (https://www.fao.org/input/download/standards/11796/CXS_302e.pdf). Histamine was detected at concentrations above 40 mg/100 mL in both FScon and FSCO2 1 MPa fish sauce preparations. Lopetcharat et al. (2001) summarized that halophilic and/or halotolerant bacteria, including Tetragenococcus muriaticus, Acinetobacter, Photobacterium, Vibrio, Staphylococcus, Pseudomonas, and Lactobacillus contribute to histamine formation. Of these microorganisms, Staphylococcus and Pseudomonas can form colonies on TSA (Lin et al. 2016; Sousa et al. 2013), which was used to detect mesophilic bacteria in the current study. FScon contained bacteria that formed colonies on TSA, whereas FSCO2 1 MPa did not, implying that bacteria other than Staphylococcus and Pseudomonas generated histamine in FSCO2 1 MPa mush. For the FSCO2 3 and 5 MPa conditions, no bacterial colonies were detected, and amine content was dramatically reduced compared to the control, although these fish sauces contained larger amounts of the free amino acids that are amine precursors (Table 3). Ibe (2014) reported that the generation of biological amines is due to bacterial decarboxylase action. The increase of CO2 during fermentation under CO2 treatments at 3 and 5 MPa may indirectly decrease the decarboxylase activity of potential unculturable bacteria present in the sauce mushes.
The hydrolysis behavior of fish bodies during fermentation was investigated to evaluate the effects of salt concentration and pressurized CO2 on fish body hydrolysis. Basic amino acids were found to be major components in both FScon and FSCO2 samples, with a descending order of free amino acids that was roughly comparable between the treatment and control groups. These results suggest that hydrolysis behavior in the pressurized CO2 method was similar to that in the conventional method. However, there were several peaks that were observed only in FScon or FSCO2 (e.g., 14.7 min and 17.5 min), and decreasing orders of free amino acids such as Ala, Val, and Asp appeared to differ between FScon and FSCO2. These differences are possibly owing to changes in the kind of protease that was involved in fish body hydrolyzation. Within the endogenous proteases in fishes, metalloproteases can be inhibited by CO32- generated through CO2 solubilization; CO32- has the potential to precipitate divalent cations required for the activation of metalloprotease. In addition, cathepsin D, an aspartic acid protease, has an optimum pH of 3.2 (Osatomi 2016). From our results, we hypothesize that cathepsin D is involved in the hydrolyzation of fish bodies during fermentation under pressurized CO2.
The total peak area of the hydrolysates was smaller in FScon than that in FSCO2. Moreover, the free amino acid amount was lower in FScon than in FSCO2 samples (Table 3). Utagawa (2012) reported that the endogenous protease activity of fish decreases with increased NaCl concentrations. This may result in the reduced hydrolyzation activity observed in the FScon mush compared to that in the FSCO2 mushes. In addition, the lower free amino acid content in FScon may have resulted from amino acid decomposition to amines by microbial action. This hypothesis is supported by the presence of mesophilic bacteria and the higher content of amines in the fish sauces that were produced using the traditional method (Table 2). Furthermore, pressurized CO2 could denature large molecular weight proteins such as actomyosin, which is the major protein in fish muscle and could enhance sensitivity to proteases and increase free amino acid content. Indeed, compared to FScon, FSCO2 samples contained larger amounts of free amino acids that have sweet (Gly, Ala, Ser, Thr, and Pro), bitter (Val, Leu, Ile, Met, Phe, and Tyr), umami, and sour (Asp and Glu) tastes. Although the reason for the decreased content of Asp in FSCO2 5 MPa is still unknown, the enhanced content of taste-related amino acids may improve the commercial value of fish sauce.
Organic acids affect the sensory characteristics of fish sauce because some have high volatility and can affect the odor as well as induce a decrease in the pH. Moreover, some organic acids themselves have characteristic flavors. The formic acid and acetic acid contents in FScon were approximately 14, and 5.2 times larger than FSCO2 5 MPa, respectively (Table 4). The high contents of these organic acids may affect the sensory evaluation scores as well as decrease the overall odor impression score of FScon (Fig. 4). Lactic acid was detected in the highest amount compared to other parameters in all fish sauces prepared (Table 4). It is unclear whether the high lactic acid content was generated during fermentation or was transferred directly from the fish body. Michihata and Sado (1998) investigated the time dependent change of lactic acid content in sardine fish sauce produced by the conventional method and suggested that high amounts of lactic acid detected at the start of fermentation did not change over time. They further described that lactic acid was not generated during fermentation by lactic acid bacteria, but accumulated in fish bodies before fermentation.
Rancidity is an undesirable defect in food quality and leads to significant sensory quality deterioration. The main feature of rancidification is due to deterioration of lipid constituents (Pokorny 2007). Auto-oxidation of fatty acids is an important change in lipids, generating hydroperoxide and an unpleasant smell; oxygen plays a dominant role in this change. Production of fish sauce by conventional methods performed in an 8 µm-thick HDPE pouch does not provide a strong gas barrier and allows O2 gas to permeate at a rate of 2325 g 25 µm/m2/24 h (http://polymerdatabase.com/polymer%20physics/Permeability.html). Therefore, fish sauce oxidation can occur close to the inner surface of the film. Production of fish sauce in these pouches is expected to mimic the conventional and industrial production method that uses big tanks, where oxidization occurs at the surface of the tank. During pressurized CO2 treatment, the headspace air of the pressure vessel is replaced with CO2 gas before fermentation begins, which could decrease O2 gas. In addition, O2 gas cannot permeate into the vessel during fermentation due to its pressurization. Thus, under CO2 conditions, the rancidity of FSCO2 was significantly lower than that of FScon (Fig. 4).
To explore the factors that dictate safety and sensory qualities in FSCO2, fish sauce was produced in the presence of 10% NaCl for 2 months under (i) pressurized nitrogen conditions, (ii) low pH (3.2) using McIlvaine buffer, and (iii) low pH (3.2) using McIlvaine buffer plus pressurization by nitrogen or (iv) CO2 pressurization. Conditions (i) to (iii) were selected for examining the specific effects generated during pressurized CO2 treatment (iv). However, conditions (i)–(iii) did not yield fish sauces with similar characteristics to that of the CO2 treatment (iv). For example, the odor impressions for fish sauces produced under conditions (i), (ii), and (iii) were salted squid like odor, fishy odor, and fishy odor, respectively. These impressions differed to those of fish sauces produced under condition (iv). In addition, the fish sauces produced in (i)–(iii) had different volatile compounds and rates of protein hydrolyzation than the pressurized CO2 treatment (data not shown). Therefore, these results indicate that the properties of FSCO2 did not result from lowering the pH or pressurization alone, but that these conditions work in combination to produce fish sauce qualities. CO2 generates CO32- that can acquire divalent cations, and the citric acid in McIlvaine buffer has a strong chelating action against divalent cations. Proper control of divalent cations under acidic and pressurized conditions is possibly one of the key factors that regulate enzyme activity and microflora that affected the quality of FSCO2. Further investigation should focus on improving our understanding of the key factors that determine the specific quality parameters of fish sauce produced under pressurized CO2 conditions.
Application of a pressurized CO2 condition to the fermentation process of sardine fish sauce resulted in the fermentation process proceeding without putrefaction and with low rancidity in spite of the low NaCl concentration (10%) used. Therefore, the application of pressurized CO2 is a novel method for producing salt- and oxidation-reduced fish sauces. For practical applications, the pasteurization and storage conditions must be optimized after preparation of the salt-reduced fish sauce, because fish sauce with a reduced NaCl concentration may be highly perishable.
Acknowledgments This work was supported by the Japan Society for the Promotion of Science (grant number 17K00818). In addition, we would like to thank Konica Minolta, Inc. for assisting with the fish sauce color analysis.
