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Journal of Food Science and Technology logoLink to Journal of Food Science and Technology
. 2021 Mar 19;59(2):703–714. doi: 10.1007/s13197-021-05065-w

Effect of organic acids on the formation of biogenic amines in fermented anchovy sauce comprising raw anchovy materials with different levels of freshness

Kilbo Shim 1,✉, Jong Soo Mok 2, Yeongyeom Jeong 2, Kunbawui Park 2, Mi-Soon Jang 2
PMCID: PMC8814216  PMID: 35185186

Abstract

We investigated the effect of different levels of organic acids on the formation of biogenic amines in anchovy fish sauce. Fish sauce samples were prepared with fresh anchovies used immediately after being caught (F), and anchovies left at ambient temperature for 4 h (4 h), 18 h (18 h), or 24 h (24 h). Anchovies from each of the four groups were mixed with salt at a 4:1 ratio and then fermented at ambient temperature for varying periods of time. The F and 4 h anchovies contained higher levels of acetic acid, succinic acid, and lactic acid, and the levels increased during the fermentation process. The histamine content of the fish sauce samples prepared using F anchovies ranged from 44.0 to 9.2 mg/kg at 1 and 24 months of fermentation. The histamine content of fish sauce samples prepared using 4 h, 18 h, and 24 h anchovies was 111.5–37.0 mg/kg (4 h), 780.1–560.3 mg/kg (18 h), and 880.6–675.7 mg/kg (24 h). Our findings indicated that the histamine and other biogenic amine levels were closely associated with the degree of freshness of the raw anchovy material. These findings indicate that lower pH levels resulting from organic acids generated by the fresh raw material can effectively inhibit histamine formation.

Keywords: Freshness, Biogenic amine, Histamine, Organic acid, pH

Introduction

The anchovy (Engraulis japonicus) is the most abundant fish species in the Yellow Sea and southern coastal area of Korea, and an important target species of commercial fisheries in China, Japan, and Korea (Jung and Houde 2014). Pacific anchovies are caught throughout the Korean coastal waters, and most commercial catches are from shallow coastal areas with a water depth of less than 20-m, especially off Tongyoung, Busan, a city in the southern coastal area (Jung and Houde 2014).

In Korea, anchovies are prepared by sun-drying or salt-fermenting. The salt-fermented product is known as Aekjeot in Korea. Aekjeot, or fish sauce, is liquid extracted through a process in which the whole or parts of fish, crustaceans, mollusks, and echinoderm animals are used as raw material and fermented in 20% salt for periods ranging from 12 to 24 months (Lee et al. 2016; Mah et al. 2009). Aekjeot, which is rich in taste and aroma resulting from self-digestion of the muscles of raw fish by enzymes, is an ingredient in the traditional Korean traditional fermented food Kimchi (Lee et al. 2016). With the growing interest in natural seasoning, Aekjeot as a cooking ingredient is gaining acceptance around the world (Jung et al. 2013).

During the fermentation process, the free amino acid and volatile compound contents increase in the fish material and the degradation of fish protein to amino acids affects the taste of the resulting fish sauce. Saccharides are degraded into monosaccharides and oligosaccharides, and a variety of organic acids, alcohols, and esters are generated by microbial enzyme activities.

Myeolchi Aekjeot (anchovy fish sauce) is one of the most commonly consumed fish sauces in Korea. Fermentation of anchovy fish sauce is mainly associated with bacteria in the genera Micrococcus, Halobacterium, and Sarcina in the early stage of fermentation, with bacteria in the genus Pediococcus being dominant at the optimal fermentation time (Lee et al. 2015; Faisal et al. 2015).

Halanaerobium and Tetragenococcus are reportedly the main bacteria involved in fermentation of anchovy fish sauce with high levels of nitrogen and amino-nitrogen (Duan et al. 2016; Guan et al. 2011). Enterobacteriaceae transform amino acids like histidine in fish into hazardous amines like histamine, which can lead to food poisoning (Satomi 2016; Kimura et al. 2001). Histamine is a nitrogen compound generated by the decarboxylation of amino acids, amination of aldehyde and ketone, and transamination. Produced by bacteria or histidine decarboxylase, histamine is commonly found in protein-based fermented foods and is used as an indicator of the quality and safety of fermented foods (Kim et al. 2000).

Various methods have been applied to lower histamine formation, such as controlling decarboxylase activation in fish silage or controlling histamine-producing bacteria through gamma ray irradiation (Naila et al. 2010; Kim et al. 2003). Some researchers have utilized algae like Ecklonia cava and Eisenia bycyclis or garlic extract to remove histamine-producing bacteria (Mah et al. 2009), or lactobacteria as start strains to reduce histamine production (Mah and Hwang 2009; Yongsawatdigul et al. 2007).

In the present study, we prepared anchovy fish sauce samples using raw anchovy materials with different levels of freshness to investigate how physicochemical components, including organic acids generated during the fermentation process, reduce biogenic amines in the fish sauce. The results of this study are expected to be useful as basic information for developing techniques to reduce histamine generated during fish sauce fermentation.

Material and methods

Preparation of fish sauce samples using raw anchovies with different levels of freshness

Anchovies (Engraulis japonicus) used in this study were caught off the coastal region of Gijang-gun in Busan City, located in the south of Korea. The anchovies were maintained in a cooler at temperatures between 5 and 10 °C during transport to the laboratory. Upon arrival, the samples were randomly allocated into four portions, which were then left at ambient temperature for different time periods to induce varied degrees of decomposition.

The first portion was used immediately for fish sauce fermentation and designated fresh fish (F). The second, third, and fourth portions were left at ambient temperature for 4 (4 h), 18 (18 h), and 24 (24 h) h, respectively. The total volatile basic nitrogen (VBN) content in the anchovies was 12.3 mg/100 g (F), 35.1 mg/100 g (4 h), 74.4 mg/100 g (18 h), and 103.2 mg/100 g (24 h).

Each group of raw anchovy material was mixed with salt at a 4:1 (w/w) ratio and then fermented at ambient temperature (22 ± 3 °C) for use as experimental fish sauce samples.

Total nitrogen and amino acid nitrogen

Total nitrogen (TN) was measured using the micro Kjeldal method (AOAC 1995). Digestion was performed using a Kjeldtherm TT125 (Gerhardt, Germany). Distillation was achieved using a Vapodest 50S distillation unit (Gerhardt, Germany). Total nitrogen was expressed as g N/100 mL.

The Amino nitrogen (AN) content of the samples was measured in triplicate using the formol method (MFDS 2019). A 5 g sample was suspended in distilled water to obtain a final volume of 50 mL and then sonicated for 30 min. Thereafter, 10 mL of this sample solution was mixed with 20 mL of formalin solution, and then titrated to pH 8.3 with 0.1 N sodium hydroxide.

Volatile basic nitrogen

We used the microdiffusion method to analyze the total VBN content (Conway and Byrne 1936). Each sample was diluted 20 times with 20% trichloroacetic acid and then homogenized. The solution was then centrifuged (916 × g, 15 min, 4 °C) and the resulting supernatant was retained for use as the sample solution. We placed 1 mL of H3BO3 into the inner chamber of a Conway unit (Shibata Co. Ltd., Tokyo, Japan) and 1 mL of the sample solution and 1 mL of K2CO3 into the outer chamber. The lid of the unit was closed and the solution was incubated at 37 °C for 80 min. The resulting solution was titrated with 0.01 N H2SO4 before determining the VBN content.

pH

The pH was measured using a pH meter (Orion 3 star, Thermo Fisher Scientific Inc., Beverly, MA, USA).

Biogenic amines

The biogenic amine content in each sample was measured following food standard regulations and specifications for fish products and an analytical method described by The Pharmaceutical Society of Japan (2005). The samples (5 g) were transferred to 50-mL centrifuge tubes and homogenized with 25 mL of 0.1 N HCl for 3 min. The suspension was centrifuged at 4000 × g for 15 min at 4 °C (Supra 22K, Hanil Scientific Inc., Gimpo, Korea) and filtered through Whatman No. 2 filter paper (Whatman, Maidstone, England). The filtrates were placed in volumetric flasks, and 0.1 N HCl was added to bring to a final volume of 50 mL. The sample solution (1.0 mL) was placed in a test tube and 100 μL of internal standard (1,7-diaminoheptane, 100 μL/mL), 0.5 mL of saturated sodium carbonate solution, and 0.8 mL of 1% dansyl chloride acetone solution were added. The tube was capped and left for derivatization for 1 h at 45 °C. After derivatization, a 10% proline solution (0.5 mL) and ether (5 mL) were added to the tube, and the tube was shaken for 10 min. The resulting supernatant was reduced under a stream of nitrogen gas, reconstituted in acetonitrile (1 mL), and filtered through a 0.45-μm filter. The filtered solution was analyzed by ultra-high performance liquid chromatography (Acquity H-class system, Waters, Milford, MA, USA) with a Waters Acquity UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm). The analysis was carried out with a gradient elution of H2O/acetonitrile and a column temperature of 40 °C. The detection wavelength was 254 nm.

Organic acids

Analysis was performed in duplicate. The fish sauce samples (1 g) at room temperature were weighed and mixed with 100 mL of distilled water into a 1 mL centrifuge tube, then vortexed and centrifuged at 10,000 rpm for 15 min at 4 °C. The supernatant was filtered through a 0.45-μm pore Whatman nonsterile syringe filter into a high-performance liquid chromatography (HPLC) vial, and 10 μL of the sample was injected into a Thermo Series HPLC system (Surveyor Plus, San Jose, CA, USA).

An organic acid column (BIO-RAD, Aminex® HPX-87H Column, 300 mm × 7.8 mm) was used for isocratic separation of acids at 40 °C. The mobile phase was HPLC grade water acidified with 4 mM sulfuric acid, in which organic acid was determined at 215 nm.

Free amino acids

Each sample (2 g) was homogenized with 15 mL of cold 5% TCA solution at 11,270 × g for 3 min. The homogenized solution was then kept at ambient temperature for 30 min. The solution was centrifuged at 8000 × g for 15 min and the final volume was brought up to 50 mL with lithium citrate buffer (pH 2.2). The solution was filtered through 0.45-μm membrane filters and used for analyses of free amino acids in a Sykam S4300 amino acid analyzer (SYKAM, GmbH, Munich, Germany).

Microbiological analysis and isolation of histamine forming bacteria

Bacterial genomic analysis was performed using the MiSeq™ System (Illumina, San Diego, CA, USA). DNA was extracted using a PowerSoil® DNA Isolation Kit (Cat. No. 12888, MO BIO, Carlsbad, CA, USA) according to the manufacturer’s protocol (Li et al. 2012). Each sequenced sample was prepared according to the Illumina 16S Metagenomic Sequencing Library protocols. DNA quantity and quality were measured using the PicoGreen and Nanodrop kits, respectively (Thermo Scientific, Willington, DE, USA). The final products were normalized and pooled using the PicoGreen data, and the sizes of the libraries were verified using the LabChip GX HT DNA High Sensitivity Kit (PerkinElmer, Waltham, MA, USA). We then sequenced the DNA using the MiSeq™ System. Single long-reads were obtained by merging paired-end sequences created by sequencing both directions of the original library.

For precise Operational Taxonomic Unit (OTU) analysis, data containing sequence errors were removed. OTUs of the remaining reads were created using a cluster cutoff value of 97%. CD-HIT-OTU is a total package program that is based on the CD-HIT-EST program. QIIME (v1.8.0) was used for OTU analysis and obtaining taxonomy information The major sequence of each OTU was mapped by referring to the Ribosomal Database Project (RDP, Release 11, Update 4: May 26, 2015) database, and taxonomy information was obtained using UCLUST (v.1.2.22).

Statistical analysis

A general linear model was used to obtain least square means and standard error of the mean, and the Duncan test was used for pairwise comparisons of the times and treatments. A p value less than 0.05 was considered statistically significant.

Results and discussion

Effect of raw material freshness on total nitrogen, amino acid nitrogen, and volatile basic nitrogen content

Changes in the levels of TN, AN, and VBN in the four raw anchovy groups (F, 4 h, 18 h, and 24 h) during the fermentation process are shown in Fig. 1. After 1 month, the TN concentration was 1.34 (F), 1.33 (4 h), 1.51 (18 h), and 1.57 g/100 mL (24 h); at 12 months, it was 1.83 (F), 1.92 (4 h), 1.86 (18 h), and 1.82 g/100 mL (24 h); and at 24 months, it was 1.81 (F), 2.07 (4 h), 1.81 (18 h), and 1.83 (24 h) g/100 mL (Fig. 1A). The AN content showed a similar trend: at 12 months, it was 1081.91 (F), 1192.87 (4 h), 1189.70 (18 h), and 1131.53 (24 h) mg/100 mL; and at 24 months, it was 1289.01 (F), 1336.2 (4 h), 1439.17 (18 h), and 1432.2 (24 h) mg/100 mL (Fig. 1B). The TN and AN concentrations differed significantly among the anchovy groups in relation to both fermentation time and freshness of the raw materials (p < 0.05).

Fig. 1.

Fig. 1

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Changes in total nitrogen (TN), amino acid nitrogen (AN), and total volatile basic nitrogen (VBN) during fermentation of anchovy fish sauce prepared from different freshness of raw materials. a–g Identical letters on each bar that there is no significant difference in different fermentation period (p > 0.05)

The VBN content varied between experimental groups of fish sauce, with the content increasing from 62.9 mg/100 mL at 1 month to 113.1 mg/100 mL at 24 months of fermentation in fish sauce comprising the F anchovies, and from 226.1 mg/100 mL at 1 month to 267.9 mg/100 mL at 24 months of fermentation in fish sauce comprising the 24 h anchovies (Fig. 1C, p < 0.05).

Our findings indicated that the TN content was higher in fish sauce samples prepared with less fresh raw material, indicating better food quality. The fish sauce products distributed in Korea have TN and AN concentrations ranging from 0.84 to 1.79 g/100 mL and 637.42 to 1315.26 mg/100 mL, respectively (Lee et al. 2016). Yongsawatdigul et al (2004) found that the TN content of Indian anchovies (Stolephorus indicus) fermented at ambient temperature reached 2.1–2.3 g/100 mL at 25 weeks of fermentation, while in anchovies fermented at 40 °C, the TN content reached 2.0 g/100 mL at 7 weeks of fermentation. VBN is closely associated with odor-causing components and is utilized as an indicator of abnormal fermentation processes like decomposition (Mueda 2015).

The N content is considered an important factor for determining food quality and consequently the price of fish sauce products. In Thailand, the TN content is used as an indicator to determine the grade and price of fish sauce, with products containing over 2.0 g/100 mL TN classified as Grade I and those containing 1.5 to 2.0 g/100 mL TN classified as Grade II (TISI 1983). Korean authorities have established a regulatory limit of more than 1.0 g/100 mL for TN in fish sauce (MFDS 2019). The Korean Industrial Standards specifies fish sauce containing ≥ 1.6 g/100 mL TN and 1200 mg/100 g AN as high quality fish sauce, and that containing ≥ 1.2 g/100 mL TN and ≥ 900 mg/100 g AN as standard quality fish sauce (KSA 2016). In the Codex Standard for Fish Sauce (CODEX STAN 302-2011), the TN content should not be < 10 g/L (1.0 g/ 100 mL) and the AN content should not be < 40% that of TN (Codex Alimentarius Commission 2013). In the present study, the 18 h and 24 h raw material had a TN content of 2.0 g/100 mL at 18 and 24 months of fermentation, after which the level stabilized or slightly decreased. Therefore, considering only the TN and AN contents, raw material with a higher degree of decomposition results in higher-quality fish sauce.

Effect of raw material freshness on the biogenic amine content

Changes in the levels of biogenic amines, such as histamine, tryptamine, putrescine, cadaverine, tyramine, spermidine, and spermine, during fermentation of raw anchovy material groups with different levels of freshness (F, 4 h, 18 h, and 24 h) are shown in Fig. 2. The histamine content of raw anchovies was 44.04 mg/kg (F), 111.51 mg/kg (4 h), 780.11 mg/kg (18 h), and 880.61 mg/kg (24 h) (data not shown). Histamine levels at 1 month of fermentation were 10.17 mg/kg (F), 85.79 mg/kg (4 h), 882.95 mg/kg (18 h), and 990.07 mg/kg (24 h); and at 24 months, 9.52 mg/kg (F), 30.63 mg/kg (4 h), 422.03 mg/kg (18 h), and 558.13 mg/kg (24 h) (Fig. 2A). The histamine levels in most of fish sauce samples increased until 3 months of fermentation, after which the levels decreased. Histamine levels in fish sauce comprising the 18 h and 24 h anchovies were especially high: 1018.30 mg/kg (18 h) and 1148.93 mg/kg (24 h) at 3 months of fermentation, but then they declined over time.

Fig. 2.

Fig. 2

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Changes in biogenic amine during fermentation of anchovy fish sauce prepared from different freshness of raw materials. A histamine; B tryptamine; C 2-phenylethylamine; D putrecine; E cadaverine; F tyramine; G Spemidine; H spemine. a–g Identical letters on each bar that there is no significant difference in different fermentation period (p > 0.05)

Tryptamine levels were 3.82 mg/kg (F), 6.51 mg/kg (4 h), 95.45 mg/kg (18 h), 155.72 mg/kg (24 h) at 1 month of fermentation; and 10.65 mg/kg (F), 12.3 mg/kg (4 h), 74.51 mg/kg (18 h), and 124.3 mg/kg (24 h) at 24 months (Fig. 2B). The tryptamine levels in the fish sauce comprising 18 h and 24 h anchovies decreased from 3 to 15 months of fermentation (143.97 mg/kg and 275.60 mg/kg, respectively).

2-Phenylethylamine levels were 6.61 mg/kg (F), 6.93 mg/kg (4 h), 482.51 mg/kg (18 h), and 528.52 mg/kg (24 h) at 1 month of fermentation; and 2.86 mg/kg (F), 1.28 mg/kg (4 h), 158.25 mg/kg (18 h), and 136.11 mg/kg (24 h) at 24 months (Fig. 2C).

Putrescine was below the limits of detection in fish sauce samples comprising the F anchovies, 24.90 mg/kg (4 h), 687.12 mg/kg (18 h), and 1170.44 mg/kg (24 h) at 1 month of fermentation; and 11.12 mg/kg (F), 24.9 mg/kg (4 h), 332.37 mg/kg (18 h), and 625.69 mg/kg (24 h) at 24 months. The putrescine level increased rapidly until 1 month of fermentation and then declined (Fig. 2D).

Cadaverine levels were 90.75 mg/kg (F), 251.80 mg/kg (4 h), 1108.43 mg/kg (18 h), and 1314.20 mg/kg (24 h) at 1 month of fermentation; and 83.42 mg/kg (F), 279.74 mg/kg (4 h), 1336.05 mg/kg (18 h), and 1618.51 mg/kg (24 h) at 24 months, with no significant change in the content over time (Fig. 2E).

Tyramine levels were 5.99 mg/kg (F), 39.31 mg/kg (4 h), 433.15 mg/kg (18 h), and 557.93 mg/kg (24 h) at 1 month of fermentation; and 15.98 mg/kg (F), 60.76 mg/kg (4 h), 526.58 mg/kg (18 h), and 604.76 mg/kg (24 h) at 24 months (Fig. 2F).

The spermidine and spermine levels did not vary much during the fermentation process with measurements in all groups at 24 months of fermentation ranging from 5.32 to 17.47 mg/kg and 8.09 to 9.14 mg/kg, respectively (Fig. 2G, H).

Fish sauce samples prepared using less fresh raw material also contained higher levels of biogenic amines, including histamine, indicating poorer food quality. Fish sauce products distributed in Thailand are reported to contain the following: histamine (45–1220 mg/kg), tryptamine (no detection-177 mg/kg), 2-phenylethylamine (no detection-43 mg/kg), putrescine (2.0–243 mg/kg), cadaverine (no detection-243 mg/kg), tyramine (no detection-42 mg/kg), spermidine (no detection to 98 mg/kg), and spermine (no detection to 98 mg/kg) (Tsai et al. 2006). Most fish sauce products in Thailand contain histamine at levels ranging from 50 to 499 mg/kg (Tsai et al. 2006). One Korean study reported that anchovy fish sauce products contained the following: histamine (325.5–1127.6 mg/kg), putrescine (33.8–182.1 mg/kg), cadaverine (81.6–263.6 mg/kg), tyramine (60.1–296.8 mg/kg), spermidine (1.9–12.2 mg/kg), and spermine (4.7–27.1 mg/kg) (Park et al. 2001).

Fermented Indian anchovies (Stolephorus indicus) are reported to contain 32.8 mg/100 kg and 2007.0 mg/kg histamine after 8 h and 16 h of storage, respectively, at 35 °C, and fish sauce resulting from fermentation of the 8 h raw material and salt mixed at a 7:3 ratio showed increasing histamine levels during a 52-week fermentation period at ambient temperature and 13 weeks at 40 °C, while fish sauce from 16 h raw material at the same salt mixture ratio showed no significant change in histamine levels under the same fermentation conditions (Yongsawatdigul et al. 2004).

Our findings demonstrate that the freshness of raw anchovy material significantly affects the formation of biogenic amines in the early stage of fermentation; thus, in this study, levels were lowest in samples comprising the freshest raw anchovy material (F). In addition, after 3 months of fermentation, the biogenic amine levels tended to decrease regardless of the degree of raw material freshness. In particular, in the present study, the histamine level of the fish sauces comprising raw anchovy material supplied for fermentation within 4 h after catch was much lower than that in the other groups (18 h and 24 h), and lower than that of commercial fish sources in various countries such as Korea, Indian, and Thailand (Park et al. 2001; Yongsawatdigul et al. 2004; Tsai et al. 2006). The preventing biogenic amine formation in fish sauce can be achieved by using high-quality raw material.

Effect of raw material freshness on organic acid content

Variations in the levels of organic acid in the fish sauce samples during the 24 months of fermentation of raw anchovy material with different levels of freshness (F, 4 h, 18 h, and 24 h) are summarized in Fig. 3. Acetic acid was present at relatively higher levels in the F and 4 h groups, with the highest level in the 4 h group throughout whole examination period (Fig. 3A). In fish sauce samples prepared using the F group, the acetic acid levels did not differ significantly among fermentation periods, ranging from 97.11 to 99.45 mg/100 mL. In contrast, the acetic acid levels in the other groups (4 h, 18 h, and 24 h) were lowest at 1 month of fermentation, increased at 3 months, and remained rather steady thereafter.

Fig. 3.

Fig. 3

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Changes in organic acid during fermentation of anchovy fish sauce prepared from different freshness of raw materials. A Acetic acid; B succinic acid; C pyroglutamic acid; D lactic acid

The succinic acid content varied widely depending on the degree of raw material freshness (Fig. 3B). Fish sauce samples prepared using the F and 4 h anchovies contained higher levels of succinic acid than those prepared using the 18 h and 24 h anchovies. In samples prepared using F and 4 h anchovies, the succinic acid content was 22.44 mg/100 mL (F) and 31.64 mg/100 mL (4 h) at 1 month of fermentation; and 22.02 mg/100 g (F) and 35.68 mg/100 g (4 h) at 24 months, similar to the levels measured at 1 month. The succinic acid content in the 18 h and 24 h anchovies was 16.91 mg/100 mL (18 h) and 10.95 mg/100 mL (24 h) at 1 month of fermentation; and 17.19 mg/100 mL (18 h) and 7.45 mg/100 mL (24 h) at 24 months.

The pyroglutamic acid content range during the 24-month testing period was 13.57–97.92 mg/100 mL (F), 30.52–173.89 mg/100 mL (4 h), 9.36–60.53 mg/100 mL (18 h), and 14.87–82.06 mg/100 mL (24 h). The F and 4 h anchovies produced higher pyrolglutamic acid levels than the18h and 24 h anchovies.

The lactic acid content was 456.39 mg/100 mL (F), 516.5 mg/100 mL (4 h), 445.95 mg/100 mL (18 h), and 364.80 mg/100 mL (24 h) at 1 month of fermentation; increasing to 623.99 mg/100 mL (F), 680.21 mg/100 mL (4 h), 364.80 mg/100 mL (18 h), 544.69 mg/100 mL (24 h) at 24 months (Fig. 3D).

In fish sauces samples from Myanmar and China, acetic acid dominated among the organic acids detected, while in samples from other countries pyroglutamate and lactate acid dominated. In Korean fish sauce, levels of pyroglutamate ranged from 300 ± 125 mg/100 mL, lactate from 419 ± 214 mg/100 mL, acetate from 182 ± 104 mg/100 mL, and succinate from 41 ± 23 mg/100 mL (Park et al. 2001).

The organic acid content significantly varied among samples with different degrees of raw material freshness; samples from the F and 4 h groups contained higher levels of succinic acid, acetic acid, lactic acid, and pyroglutamate than those originating from the 18 h and 24 h groups. Further, fish sauce samples prepared from the 4 h group often contained the highest levels of organic acids, indicating very high levels of physicochemical food quality as well as high levels of TN and AN. This is attributable to the fact that organic acids such as acetate and lactate are generated through the Embden-Meyerhof-Parnas pathway in which bacteria like Chromohalobacter and Tetragenococcus degrade glucose into organic acids, and these bacteria are highly activated in raw anchovy material used within 4 h (Jung et al. 2013).

Effect of raw material freshness on histamine-generating bacteria

We investigated variations in bacterial counts and their relationship to biogenic amine formation in fish sauce samples comprising the F, 4 h, 18 h, and 24 h anchovies during fermentation (Fig. 4). Among the total of 1623 bacterial species detected in anchovy sauce, 16 species of histamine-generating bacteria were tested. We detected a total of 6 of these 16 species: Tetragenococcus muriatiscus, T. halophilus, Morgenalla morganii, Hafnia alvei, and Lactobacillus sp. in the fish sauce. A total of 31 strains related to the 6 species of bacteria were identified.

Fig. 4.

Fig. 4

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Changes in bacteria taxonomic composition at the species level during fermentation of anchovy fish sauce prepared from different freshness of raw materials

To assess changes in the bacterial counts during fermentation, we grouped the bacterial species into five families: Hafniaceae, Enterobacteriaceae, Lactobacillaceae, Morganellaceae, and Enterococcaceae. The raw F anchovies contained 76,982 copies of Lactobacillaceae (Fig. 4A). During fermentation, the copy number decreased dramatically for up to 9 months and then increased at 12 months to 15,952 copies, but then decreased again at 18 months, revealing no significant change in the count of Lactobacillaceae during fermentation. The Morganellaceae copy number ranged from no detection to 93 at 1 month of fermentation.

In the 4 h raw anchovy material, the Lactobacillaceae copy number was 22,122 (Fig. 4B). The copy number decreased dramatically at 1 month of fermentation to 445 and then increased to 6300 at 18 months. In the 4 h raw anchovies, the Morganellaceae copy number was 26, and the copy number increased during fermentation to 431 at 12 months and then decreased to 136 at 18 months.

In the 18 h raw anchovy material, the Lactobacillaceae copy number was 14,438 (Fig. 4C). During fermentation, the copy number decreased to 164 at 1 month and then increased to 333 at 18 months. Morganellaceae was not detected in the raw 18 h anchovies, but the copy number was 1216 at 1 month of fermentation, and then decreased to 155 at 3 months, after which the copy number ranged from 0 to 73.

In the 24 h raw anchovies, the Lactobacillaceae copy number was 9079. The copy number decreased to 333 at 18 months of fermentation. Morganellaceae was not detected in the raw 24 h anchovies, but the copy number was 464 at 1 month of fermentation, then decreased to 31 at 3 months, after which the detected levels ranged from 0 to 26 copies.

Effect of raw material freshness on free amino acid content

We also investigated variations in the free amino acid content in the F, 4 h, 18 h, and 24 h anchovies after different periods of fermentation (Fig. 5). The total free amino acid content increased with longer periods of fermentation regardless of the degree of freshness of the raw anchovies. The main amino acids included glutamic acid, alanine, leucine, histidine, ornithine, and lysine. Arginine was detected during fermentation of raw F anchovies, but not in the samples from the other groups.

Fig. 5.

Fig. 5

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Changes in free amino acid during fermentation of anchovy fish sauce prepared from different freshness of raw materials

The histidine content was 328.6 mg/100 g (F) and 321.48 mg/100 g (4 h) at 3 months of fermentation, and increased to 401.28 mg/100 g (F) and 394.16 mg/100 g (4 h) at 6 months, indicating a similar trend during fermentation. The histidine content of samples comprising 18 h and 24 h raw anchovies was 123.4 mg/100 g (18 h) and 96.15 mg/100 g (24 h) at 1 month of fermentation, and the content began to gradually increase at 3 months.

Effect of raw material freshness on the change in pH during fermentation

The pH of the raw anchovies was 6.32 (F), 6.6 (4 h), 6.79 (18 h), and 6.8 (24 h). During fermentation, the pH of the fish sauce decreased over time to 5.43 (F), 5.75 (4 h), 5.59 (18 h), and 5.86 (24 h) at 24 months (Fig. 6).

Fig. 6.

Fig. 6

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Changes in pH during fermentation of anchovy fish sauce prepared from different freshness of raw materials

The formation of organic acids decreases the pH in fish sauce. In the present study, the pH decreased during fermentation in all freshness conditions examined. Consistent with our finding, the pH of an anchovy (Stolephorus commersonii) sauce fermented for 270 days continuously decreased from 6.99 at the start to 6.05 at the end of fermentation (Mueda 2015).

The pH is used as an indicator of the quality of fish sauce and pH levels that continuously decrease during storage affect the micro flora of the sauce (Dagadkhair et al. 2016; Mueda 2015). The pH levels in fish sauce products in South East Asian countries range from 4.66 to 5.91 and the levels in anchovy fish sauce products in Korea range from 5.35 to 6.48 (Em et al. 2018; Lee et al. 2015; Nakano et al. 2017). The CODEX has established that the pH level should be within a range of 5.0–6.5. In generally, the optimal pH level for histidine decarboxylase approaches 6.5 and affects histamine formation (Lefèvre et al. 2016). The 18 h and 24 h groups had a high histamine content.

Fish sauce samples with high degrees of freshness had lower pH levels due to acetic acid and lactic acid generated in the early stage of fermentation, and low pH levels inhibit bacterial growth and activation of histidine decarboxylase, thus producing less histamine. Some studies have utilized starter strains to inhibit bacterial growth in the early stage of fermentation or additives that inhibit the formation of biogenic amines like histamine. Citric acid, succinic acid, d-sorbitol, and malic acid also inhibit the activation of decarboxylase, resulting in lower histamine levels in mackerel flesh stored at 10 °C and 25 °C. The addition of 1% citric acid drastically reduces the biogenic amine content in pickled cabbage samples fermented with 6%, 8%, and 10% salt (Yuecel and Ueren 2008). In that same report, the addition of 1% glucono-delta-lactone to meat samples reduced pH levels, inhibiting the formation of both histamine and putrescine (Maijala et al. 1993).

The histamine content significantly varied according to the degree of freshness of the raw material with the histamine levels declining after 3 months of fermentation. Histamine is degraded through two pathways: diamine oxidase and histamine-N-methyltransferase (HNMT). The diamine oxidase pathway results in imidazole-4-acetic acid and 1-ribosylimidazole-4-acetic acid, while the HNMT pathway generates N-methylhistamine 1-methylmidazoe-4-acetic acid (Maintz and Novak 2007). The observed drastic increases in the acetic acid content in fish sauce samples produced using the raw 18 h and 24 h anchovies after 3 months of fermentation are attributed to histamine degradation through the HNMT pathway.

Anchovy fish sauce samples fermented for 10 weeks with the addition of 20% salt and 5% glycine have 63–73% less putrescine, cadaverine, histamine, tyramine, and spermidine than the control (Mah et al. 2009). The addition of sucrose, glucose, sorbitol, lactic acid, citric acid, and c is mostly effective for inhibiting biogenic amines. In particular, adding 5% or 10% citric acid greatly reduces cadaverine, histamine, tyramine, and spermidine (Mah and Hwang 2009). Identifying the mechanism of the formation and degradation of histamine through further studies of the structures of these substances and the enzymes that are activated is important. As histamine formation in fish sauce is closely related to growth of histamine-generating bacteria in the early stage of fermentation, it is critical to manage the degree of freshness of the raw material. Maintaining the pH below 6.0 is thought to be effective for reducing the histamine content.

Conclusions

The study found that the generation of biogenic amines like histamine is closely associated with the freshness of the raw materials, with histamine levels reaching a maximum at 3 months of fermentation, and then decreasing. Further, reduced levels of histamine were attributable to enhanced levels of acetic acid and free amino acid.

On the basis of these results, using raw material within 4 h after catch (or material containing less than 40 mg/100 g VBN) and decreasing the pH of the raw material to less than 6.0 using organic acids can effectively inhibit histamine formation to levels below those in the CODEX and EU guidelines.

Acknowledgements

This work was supported by a grant from the National Institute of Fisheries Science (R2021062).

Footnotes

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References

  1. AOAC . Official method of analysis. Washington: Association of Official Analytical Chemists; 1995. [Google Scholar]
  2. Codex Alimentarius Commission (2013) Standard for fish sauce (Amendment 2012, 2013) (CODEX STAN 302-2011), Rome
  3. Conway EJ, Byrne A. An absorption apparatus for the micro-determination of certain volatile substances I. The micro-determination of ammonia. J Biochem. 1936;27:419–429. [PMC free article] [PubMed] [Google Scholar]
  4. Dagadkhair AC, Pakhare KN, Gadhave RK. Effect of storage on physicochemical properties of spiced fish sauce. J Nutr Food Sci. 2016;6:1000520. doi: 10.4172/2155-9600.1000520. [DOI] [Google Scholar]
  5. Duan S, Hu X, Li M, Miao J, Du J, Wu R. Composition and metabolic activities of the bacterial community in shrimp sauce at the flavor-forming stage of fermentation as revealed by metatranscriptome and 16S RNA gene sequencings. J Agric Food Chem. 2016;64:2591–2603. doi: 10.1021/acs.jafc.5b05826. [DOI] [PubMed] [Google Scholar]
  6. Em IS, Seo JK, Kim HD, Park KS. The quality of commercial salted and fermented anchovy Engraulis japonicas sauces produced in Korea. Korean J Fish Aquat Sci. 2018;51:667–672. doi: 10.5657/KFAS.2018.0667. [DOI] [Google Scholar]
  7. Faisal M, Noor-E-Islami S, Islam MN, Kamal M, Khan MNA. Study on microbial and physical changes in fish sauce during fermentation. Res Agric Livest Fish. 2015;2:375–383. doi: 10.3329/ralf.v2i2.25024. [DOI] [Google Scholar]
  8. Guan L, Cho KH, Lee JH. Analysis of the cultivable bacterial community in jeotgal, a Korean salted and fermented seafood, and identification of its dominant bacteria. Food Microbiol. 2011;28:101–113. doi: 10.1016/j.fm.2010.09.001. [DOI] [PubMed] [Google Scholar]
  9. Jung SK, Houde ED. Comparison of anchovy biomass estimates measured by trawls, egg production methods and hydro-acoustics in the Chesapeake Bay and the Korea Strait. Ocean Sci J. 2014;49:115–126. doi: 10.1007/s12601-014-0012-y. [DOI] [Google Scholar]
  10. Jung JY, Lee HJ, Chun BH, Jeon CO. Effects of temperature on bacterial communities and metabolites during fermentation of myeolchi-aekjeot, a traditional Korean fermented anchovy sauce. PLoS ONE. 2013;11(3):e0151351. doi: 10.1371/journal.pone.0151351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kim SH, Ben-Gigirey BA, Barros-Vela Zquez J, Price RJ, An HJ. Histamine and biogenic amine production by Morganella morganii isolated from temperature-abused Albacore. J Food Prot. 2000;63:244–251. doi: 10.4315/0362-028x-63.2.244. [DOI] [PubMed] [Google Scholar]
  12. Kim JH, Ahn HJ, Kim DH, Jo C, Yook HS, Park HJ, Byun MW. Irradiation effects on biogenic amines in Korean fermented soybean paste during fermentation. J Food Sci. 2003;68:80–84. doi: 10.1111/j.1365-2621.2003.tb14118.x. [DOI] [Google Scholar]
  13. Kimura B, Konagaya Y, Fujii T. Histamine formation by Tetragenococcus muriaticus, a halophilic lactic acid bacterium isolated from fish sauce. Int J Food Microbiol. 2001;70:71–77. doi: 10.1016/S0168-1605(01)00514-1. [DOI] [PubMed] [Google Scholar]
  14. KSA (Korean Standards Association) (2016) Anchovy sauce, KS H 6022. https://www.standard.go.kr/KSCI/standardIntro/getStandardSearchView.do?menuId=919&topMenuId=502&upperMenuId=503&ksNo=KSH6022&tmprKsNo=KSH6022&reformNo=09/. Accessed 10 Mar 2020
  15. Lee SH, Jung JY, Jeon CO. Bacterial community dynamics and metabolite changes in myeolchi-aekjeot, a Korean traditional fermented fish sauce, during fermentation. Int J Food Microbiol. 2015;203:15–22. doi: 10.1016/j.ijfoodmicro.2015.02.031. [DOI] [PubMed] [Google Scholar]
  16. Lee HW, Choi YJ, Hwang IM, Hong SW, Lee MA. Relationship between chemical characteristics and bacterial community of a Korean salted-fermented anchovy sauce, myeolchi-aekjeot. LWT Food Sci Technol. 2016;73:251–258. doi: 10.1016/j.lwt.2016.06.007. [DOI] [Google Scholar]
  17. Lefèvre S, Astier C, Kanny G. Histamine in tolerance or false food allergy with histamine mechanism. Rev Fr Allergol. 2016;57:24–34. doi: 10.1016/j.reval.2016.10.004. [DOI] [Google Scholar]
  18. Li W, Fu L, Niu B, Wu S, Wooley J. Ultrafast clustering algorithms for metagenomic sequence analysis. Brief Bioinform. 2012;13:656–668. doi: 10.1093/bib/bbs035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mah JH, Hwang HJ. Effects of food additives on biogenic amine formation in myeolchi-jeot, a salted and fermented anchovy (Engraulis japonicus) Food Chem. 2009;114:168–173. doi: 10.1016/j.foodchem.2008.09.035. [DOI] [Google Scholar]
  20. Mah JH, Kim YJ, Hwang HJ. Inhibitory effects of garlic and other spices on biogenic amine production in myeolchi-jeot, Korean salted and fermented anchovy product. Food Control. 2009;20:449–454. doi: 10.1016/j.foodcont.2008.07.006. [DOI] [Google Scholar]
  21. Maijala R, Eerola SH, Aho MA, Hirn JA. The effect of GDL-induced pH decrease on the formation of biogenic amines in meat. J Food Prot. 1993;56:125–129. doi: 10.4315/0362-028X-56.2.125. [DOI] [PubMed] [Google Scholar]
  22. Maintz L, Novak N. Histamine and histamine intolerance. Am J Clin Nutr. 2007;85:1185–1196. doi: 10.1093/ajcn/85.5.1185. [DOI] [PubMed] [Google Scholar]
  23. MFDS (Ministry of Food and Drug Safety) (2019) Korean Food Standards Codex. http://www.foodsafetykorea.go.kr/foodcode/01_03.jsp?idx=46/. Accessed 10 Mar 2020
  24. Mueda RT. Physico-chemical and color characteristics of salt-fermented fish sauce from anchovy Stolephorus commersonii. Aquac Aquar Conserv Legis Bioflux. 2015;8:565–572. [Google Scholar]
  25. Naila A, Flint S, Fletcher G, Bremer P, Meerdink G. Control of biogenic amines in food-existing and emerging approaches. J Food Sci. 2010;75:139–150. doi: 10.1111/j.1750-3841.2010.01774.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nakano M, Sagane Y, Koizumi R, Nakazawa Y, Yamazaki M, Watanabe T, Takano K, Sato H. Data on the chemical properties of commercial fish sauce products. Data Brief. 2017;15:658–664. doi: 10.1016/j.dib.2017.10.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Park JN, Fukumoto Y, Fujita E, Tanaka T, Washio T, Otsuka S, Shimizu T, Watanabe K, Abe H. Chemical composition of fish sauces produced in Southeast and East Asian countries. J Food Compos Anal. 2001;14:113–125. doi: 10.1006/jfca.2000.0963. [DOI] [Google Scholar]
  28. Satomi M. Effect of histamine-producing bacteria on fermented fishery products. Food Sci Technol Res. 2016;22(1):1–21. doi: 10.3136/fstr.22.1. [DOI] [Google Scholar]
  29. Thai Industrial Standards Institute (1983) Ministry industry, Bangkok, Thailand. UDC 641.88: 639.2
  30. The Pharmaceutical Society of Japan . Methods of analysis in health science. Tokyo: Kanehara & Co., Ltd.; 2005. pp. 180–182. [Google Scholar]
  31. Tsai YH, Lin CY, Chien LT, Lee TM, Wei CI, Hwang DF. Histamine contents of fermented fish products in Taiwan and isolation of histamine forming bacteria. Food Chem. 2006;98:64–70. doi: 10.1016/j.foodchem.2005.04.036. [DOI] [Google Scholar]
  32. Yongsawatdigul J, Choi YJ, Udomporn S. Biogenic amines formation in fish sauce prepared from fresh and temperature abused Indian anchovy (Stolephorus indicus) J Food Sci. 2004;69:312–319. doi: 10.1111/j.1365-2621.2004.tb06333.x. [DOI] [Google Scholar]
  33. Yongsawatdigul J, Rodtong S, Raksakulthai N. Acceleration of Thai fish sauce fermentation using proteinases and bacterial starter cultures. J Food Sci. 2007;72:382–390. doi: 10.1111/j.1750-3841.2007.00532.x. [DOI] [PubMed] [Google Scholar]
  34. Yuecel U, Ueren A. Biogenic amines in Turkish type pickled cabbage: effects of salt and citric acid concentration. Acta Aliment. 2008;37:115–122. doi: 10.1556/AAlim.2007.0022. [DOI] [Google Scholar]

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