Bacteriological Properties and Health Related Biochemical Components of Fermented Fish Sauce - An Overview.

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Bacteriological Properties and Health related Biochemical Components of

Fermented Fish Sauce: An overview
Article in Food Reviews International · June 2015

DOI: 10.1080/87559129.2015.1057844
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Bacteriological properties and health-related

biochemical components of fermented fish sauce:
An overview
Siddegowda Gopalapura Shivanne Gowda, Bhaskar Narayan & Shubha

Gopal
To cite this article: Siddegowda Gopalapura Shivanne Gowda, Bhaskar Narayan & Shubha
Gopal (2016) Bacteriological properties and health-related biochemical components
of fermented fish sauce: An overview, Food Reviews International, 32:2, 203-229, DOI:
10.1080/87559129.2015.1057844
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FOOD REVIEWS INTERNATIONAL
2016, VOL. 32, NO. 2, 203–229
http://dx.doi.org/10.1080/87559129.2015.1057844
Bacteriological properties and health-related biochemical

components of fermented fish sauce: An overview
Siddegowda Gopalapura Shivanne Gowdaa, Bhaskar Narayanb, and Shubha Gopalc
a
Postgraduate Department of Microbiology, Maharani’s Science College for Women, Mysore, India; bMeat and
Marine Sciences Department, CSIR–Central Food Technological Research Institute, Mysore, India; cDepartment
of Studies in Microbiology, University of Mysore, Mysore, India
Downloaded by [Central Food Technological Research Inst ] at 22:35 15 February 2016
ABSTRACT KEYWORDS
Fish sauce is an amber-colored salty liquid in Southeast Asian cuisine Desalination; fermentation;
used as an important condiment for improving the taste of foods. It is fish sauce; halotolerant;
produced by fermenting the fish with salt in the ratios of 1:1 or 3:1 proteolytic enzymes
(fish:salt, wet wt) in underground concrete tanks or earthenware for
6–12 months at ambient conditions. Proteins of the raw materials are
hydrolyzed into peptides and amino acids by microbial proteolytic
enzymes during fermentation. This paper reviews fish sauce diversity
and its bacteriological, biochemical, and biofunctional properties.
Additionally, efforts to accelerate fish sauce production by employing
halotolerant bacterial cultures and proteinases to reduce the fermen-
tation period and also to improve the acceptability of the product are
also reviewed. Further, the review provides an overview of bacterial
proteinases that have been employed to enhance the sensory and
microbiological quality of fish sauce. The review also outlines the
effect of fermented fish sauces on health, especially in reference to
several bioactive peptides and bacterial metabolites apart from dis-
cussing desalination technique to recover the metabolites from fish
sauce.
Introduction
The demand for fish material is on the rise globally, but there is no significant growth in
the annual world fish harvest.(1) This emphasizes the need to focus more on improved
utilization of fish. This situation also promotes better recovery and utilization of the by-
products as raw materials for foods and nutraceuticals and also for pharmaceutical and
biotechnological applications.(2) Fish and fishery products are excellent sources of high-
quality proteins in Asian countries. The total supply of amino acids provided by a meal
determines its nutritive value rather than the algebraic sum of the nutritive values of the
individual constituents. The fish and fish products not only provide protein, but in
addition provide polyunsaturated fatty acids, vitamins, and minerals that are important
in human nutrition. Fermentation is one of the oldest techniques in food preservation, as
it not only extends the shelf life but also enhances the flavor and nutritional quality of the
product.(3) The production and use of fermented fish products throughout the world is
closely related to the use of cereals, especially rice and vegetables.(4) Fermented fish are
CONTACT Shubha Gopal sguom@yahoo.com Department of Studies in Microbiology, University of Mysore,

Manasagangotri, Mysore 570 006, Karnataka, India.
© 2016 Taylor & Francis
204 S. G. S. GOWDA ET AL.
often produced according to family tradition and local geographic preferences, with large
differences existing in both production methods and proportion of raw material.(5)
Production does not include steps such as cooking or pasteurization, which kill pathogenic
bacteria. The microbiological safety of these products therefore mainly depends on rapid
adequate fermentation by lactic acid bacteria. Fermentation provides a combination of
reduced pH and production of organic acids.(6)
The fermented fish for human consumption has many benefits.(5,7) It is a low-cost,
convenient preservation method of fish muscle that does not require refrigeration. The use
of bacterial starters in food fermentation has become a means to increase processing rates
and product consistency. Starters were used to improve the sensory characteristics and
microbiological quality and also to shorten the fermentation time of fermented foods.(8)
More recently, starter cultures have become available as frozen concentrates, either dried
or lyophilized preparations produced on an industrial scale. According to Callewaert and
coworkers,(9) the use of starter cultures would be an appropriate approach for the control
and optimization of the fermentation process. These could be used in order to alleviate the
problems of variations in organoleptic quality and microbiological stability observed in
indigenous fermented foods.
This review will cover the diversity and manufacture of fish sauce, bacteriological
properties, and use of native halotolerant bacterial culture as starters to enhance the
process of fermentation. This article also overviews the production of biogenic amines
in fish sauce and their degradation as well as other healthy biochemical components of fish
sauce, enzyme-producing microorganisms, and their use as novel starter cultures to
accelerate the fish sauce fermentation.
Fish sauce: Diversity and production

Fermented fishes are considered to be a Southeast Asian product. These products are
highly salted and fermented until the fish flesh is transformed into simpler components.
Fish fermentation in the Southeast Asian subregion normally lasts for several months
(3–9 months), and the fish flesh may liquefy or turn into a paste.(10) The fermented fish
products, including fish sauce and fish paste, have recently been widely utilized in a
variety of processed products in Taiwan, and their imports to Taiwan continues to
increase year after year. Fish sauce is consumed by over 80–90% people in Southeast
Asia.(11) The annual production is estimated to be >400 million liters, with 20 out of
100 fish sauce producers contributing more than 80% of the global production.(12) Fish
sauce is a clear, brown liquid with a salty taste and mild fishy flavor that results from
the physical, chemical, and microbiological changes that occur at high salt concentra-
tion and low oxygen levels. Fish sauce is traditionally produced by using whole fish with
salt in a ratio of 1:1 to 3:1 and fermented anywhere between 6 and 12 months or even
longer. The fermented liquid is rich in fish soluble proteins, peptides, and amino acids
that are characterized by umami tastes.(13) They are produced during proteolytic
degradation by endogenous proteases in the muscles or digestive tracts of fish, and
various microorganisms exist in the fermentation broth.(14,15) Fish sauce is considered
as an important source of dietary proteins and amino acids, and has become a necessity
in the household in Southeast Asian countries.(16) Changes in the amino acid composi-
tion of fish sauces made from different species of fish at various salt concentrations and
FOOD REVIEWS INTERNATIONAL 205
Table 1. Amino acid composition (mg/mL) of fish sauces made from different species.
Amino acid Sauce A(17) Sauce B(18) Sauce C(19) Sauce D(20) Sauce E(21) Sauce F(22) Sauce G(23) Sauce H(24)
Essential
Arginine 2.81 2.01 2.97 2.11 0.84 2.80 3.68 0.25
Histidine 4.07 0.31 1.00 1.11 14.00 1.76 4.72 1.07
Isoleucine 3.59 3.18 1.30 0.29 4.74 8.26 2.69 5.94
Leucine 4.81 5.70 5.22 2.41 5.78 3.57 5.01 9.39
Lysine 5.87 5.69 3.12 0.63 23.30 11.17 6.71 4.52
Methionine 2.34 0.98 0.55 5.56 2.88 4.89 1.71 2.81
Phenyl alanine 2.39 2.20 1.34 2.14 3.59 2.14 1.64 2.97
Threonine 2.98 2.66 1.12 1.29 6.66 4.04 3.62 4.29
Tyrosine 0.98 0.83 1.05 4.76 2.27 7.62 0.09 2.04
Valine 4.30 2.70 3.21 1.42 4.74 8.02 3.02 12.63
Total 34.14 26.26 20.88 21.72 68.80 54.27 32.89 45.91
Nonessential
Alanine 4.26 2.01 3.11 0.92 4.52 9.60 5.01 9.51

Aspartic acid 1.28 4.55 5.16 12.48 7.31 5.87 6.88 2.53
Glutamic acid 5.82 4.67 8.11 9.65 11.35 12.22 11.00 15.56
Glycine 1.37 2.18 2.31 2.60 2.96 2.54 4.32 5.13
Proline ND ND 1.20 1.79 3.61 0.36 2.63 3.09
Serine 4.10 2.59 1.02 6.79 4.59 0.81 2.50 1.86
Cystein 0.07 ND 0.45 4.41 2.80 4.32 0.46 2.38
Total 16.90 16.00 21.36 38.64 37.14 35.72 32.80 40.06
Grand total 51.04 42.26 42.24 60.36 105.94 89.99 65.69 85.97
Note. Fish sauces: A = anchovies + 30% salt + 187 days; B = capelin + 10% salt + 250 days; C = clupeids + 20% salt + 90
days; D = Gambusia + 25% salt + 150 days; E = Indian anchovies + 15% salt + 90 days; F = sardine + 10% salt + 40 days;
G = sardine + 30% salt + 365 days; H = squid processing by-products + 20% koji + 8% salt + 0.2% enzyme + 30 days.
ND = not determined.
fermentation time is listed in the Table 1.(17–24) Depending on the degree of hydrolysis
or time of fermentation and the separation method, two types of sauce, namely, clear
and turbid, are produced. The clear type of fish sauces are jeot-kuk (Korea),(25) nampla
(Thailand),(25,26) bakasang (Indonesia),(24) yu-lu (China),(22) garum (Rome),(27) mahya-
veh (Iran),(28) nuoc-mam (Cambodia),(25) and ishiru or shottsuru (Japan),(25) whereas
budu (Malaysia),(29) paties (Philippines),(29) ketjap-ikan (Indonesia),(25) and ngan-pya-
ye (Cambodia)(25) are turbid types of sauce. These turbid-type fish sauces are obtained
from the exude liquid of cured fish, for example, paties from bagoong production in the
Philippines and jeot-kuk from jeot-kal production in Korea. The fish species used, fish
salt ratio, and fermentation time in the production of various fish sauces from around
the world are listed in Table 2.
The biochemical and microbiological changes at high salt concentration and reduced
oxygen levels produce the sauce from fish. There is high divergence in manufacturing
among fish sauce–producing countries throughout the world, although the fish and salt
are the major raw materials in general production. The leading fish sauce producer in the
world is Thailand. Nampla, a traditional Thai fish sauce, has become popular among
Western consumers, especially in the United States.(29) Chinese fish sauce yu-lu obtained
from fermentation of anchovies has been extensively studied by Jiang and coworkers(22)
regarding the mixed culture fermentation having the characteristics of many traditional
processes, and this favors the growth of halophiles. The enhancement of nutritive value of
the yu-lu with the changes in total soluble nitrogen, trichloroacetic acid (TCA)-soluble
peptides, formaldehyde nitrogen, total titratable acid, and free amino acid concentration
was observed. Yu-lu is widely consumed as a condiment for cooking in Guangdong and
Fujian provinces of the southern and eastern parts of China. The high-salt fermentation in
Table 2. Salt-fermented fish sauces from around the world.

Name Fish spp. used Fish:Salt Fermentation time Country References
Aek Jeot Astroscopus japonicas 3–4:1 12 months Korea (25)
Engraulis japonica
Anchovy Engraulis encrasicholus 2:1 6–7 weeks France (25)
Bakasang Sardinella spp. 5:1 5–6 weeks Indonesia (24)
Budu Stolephorus spp. 3–5:1 3–12 months Malaysia (29)
Colombo-cure Ristelliger spp. 6:1 12 months India and Pakistan (25)
Cybium spp.
Clupea spp.
Garum Tunnus thynnus 4:1 9 months Roman (27)
Scomber scombrus
Garos Scomber colias (liver) 9:1 8 days Greece (25)
Gau-ca Ophicephalus spp. 3:1–3:2 2–3 months Cambodia (25)
Ishiru Astroscopus japonicas 5:1 6 months Japan (29)

Clupea pilchardus
Ketjap-ikan Stolephorus spp. 6:1 6 months Indonesia (25)
Clupea spp.
Leiagnathus spp.
Osteochilus spp.
Mahyaveh Sardinella spp. 3:1 6 months China (28)
Nam-pla Stolephorus spp. 1–5:1 5–12 months Thailand (25, 26)
Ristrelliger spp.
Cirrhinus spp.
Nouc-mam Clarius spp 3:1–3:2 2–3 months Vietnam (25, 29)
Patis Stolephorus spp. 3–4:1 3–12 months Philippines (29)
Clupea spp.
Decapterus spp.
Leionathus spp.
Pissala Ahya pellucid 4:1 2–8 weeks France (29)
Gobius spp.
Engraulis spp.
Atherina spp.
Yeesui Sardinella spp. 4:1 3–12 months Hong Kong (29)
Engraulis pupapa
Yu-lu Stelophorus spp. 3:1 6 months China (22)
the production of traditional yu-lu(22) and use of exogenous enzymes to accelerate the
production of the Roman fish sauce “garum”(27) are schematically represented in Fig. 1.
Desalination of fish sauce

Fish sauce has a unique, pleasant flavor, but contains high levels of sodium chloride
(25.3 ± 0.0%, w/w), which is not desirable for health-conscious consumers. The
electrodialysis (ED) desalination of fish sauce was carried out by Chindapan and
coworkers.(30) The results indicated that an increase in the input voltage led to an
increase in the rates of salt removal, electrical conductivity, and total soluble solids. The
energy required increased, whereas the current efficiency and yield of desalinated fish
sauce decreased significantly with an increase in the input voltage and salt removal
level. At higher salt removal level, the total solids, viscosity, density, sodium, and
potassium ion concentrations decreased significantly. The color and total nitrogen of
the ED-treated fish sauce significantly were affected by input voltage and salt removal
level.(31) ED has been used to recover available substances (nutrients, flavor, and aroma
compounds) from many food by-products. For example, desalination of silage juice to
recover lactic acid and amino acid(32) and desalination of seafood cooking juice to
Fish and edible fish by product
Salt fermentation Enzyme fermentation
Anchovy
Frozen tuna liver and eviscerated mackerel (1:1)
Mixing with salt (30%–40%, Salt/Fish, w/w)

Defrost at room temperature
Fermentation for 12–18 months
Cut into small pieces of 3 g weight
Keeping at 40 ºC–50 ºC (for 1–2 weeks)
Salt (w/w) Ingredients Enzymes

Filtration
5 g/100 g Origan (1%) Fungal protease P31000
10 g/100 g Coriander (1%) Alcalase 2.4L
Fish residue
25 g/100 g Thyme (1%) Kojizyme MG

Saturated brine Saturated brine Citric acid (0.5%) Trypsin PTN3.0
BHA (0.001%) Neutrase 0.5L
Fermentation 1–4 months Boiling
Carmel or additives
Filtration Filtration
Fermentation with continuous stirring
Second-grade
Top-grade Dilute Xanthan gum solution
Fish sauce Concentrated fish sauce
Figure 1. Schematic representation of fish sauce production.(22,27)
recover flavor and aroma compounds.(32,33) Fish sauce prepared with sodium chloride
or natural salt was partially replaced by potassium chloride (KCl) at 90:10, 75:25, and
60:40 ratios. Volatile acids were collected by steam distillation under reduced pressure
and measured. Most of the volatile acids in all the samples with added KCl were higher
than in the controls where the samples mixed with only NaCl without added KCl,
suggesting the role of KCl in the formation of volatile acids in fish sauce manufacture.
There was no significant change in the pH values of the samples with added KCl and
controls. The pH of the controls for the natural salt: KCl and NaCl:KCl samples, were
5.3 and 5.35, respectively, and ranged from 5.32 to 5.37 for all the samples with added
KCl. There seems to be no significant difference in the concentrations of amino acids in
samples with the control. Glutamic acid showed a fairly high concentration, followed by
aspartic acid and lysine.
Sensory evaluation showed slight differences in odor between the samples with added
KCl and controls, but the differences did not affect the acceptability of the product.
However, acceptance of taste terminated at a 75:25 NaCl:KCl or natural salt:KCl ratio,
and up to this ratio could prove to be a possible replacement for an all-NaCl fish sauce.(17)
The characterization of volatile compounds and determination of characteristic fish sauce
aroma-impact compounds in fish sauce using solid-phase microextraction, gas chromato-
graphy–mass spectrometry (GC-MS), and solid-phase microextraction–osme (in Greek,
meaning “smell”)–gas chromatography olfactometry (SPME-osme-GCO) coupled with
Stevens’ power law, a psychophysical law that explains the relationship between perceived
intensity and the concentration of a sensory stimulus. The aromas of four fish sauce
samples determined by GCO and GCMS were verified using authentic standard com-
pounds. All the four contained the same aromas, but the odor intensity associated with
each compound or group of compounds was variable. Stevens’ power law exponents
determined for the samples were not consistent for the same compounds that were
found in all fish sauces. Stevens’ power law exponents ranged from 0.14 to 0.37, 0.24 to
0.34, 0.09 to 0.21, and 0.10 to 0.35 for dirty socks, fishy, buttery popcorn, and sweet
aromas, respectively.(34)
The laboratory-scale ED used for desalination has limitations, such as high energy
consumption and slower desalting process. Jundee and coworkers(35) developed a pilot-
scale ED and tested its desalination efficiency. The ED reduced the salt content of fish
sauce from an initial value of 25% (w/w) to the desired salt concentrations of 18, 16, and
14% (w/w) within 120, 210, and 300 minutes, the energy consumption was found to be
2.53 ± 0.05, 4.35 ± 0.05, and 6.15 ± 0.05 kWh, respectively. The study showed that the
pilot-scale ED produces low-sodium fish sauce with a residual salt concentration of less
than 18% (w/w) within 3 hours.
Bacteriological properties of fish sauce

Fish sauce has a very high concentration of salt (25–30%). Thus, microorganisms found
during fish sauce production are generally classified as halophilic. The important roles of
bacteria in fish sauce are protein degradation and flavor-aroma development.
Consequently, when fish sauce is produced under aseptic conditions, typical fish sauce
aroma is not developed.(29) Fish proteins are gradually hydrolyzed during fermentation by
both bacterial and fish proteinases.(36) Many bacteria are found in fish sauce fermentation,
including Bacillus, Micrococcus, Staphylococcus, Streptococcus, Pediococcus, and extremely
halophilic red Archaea, halophilic lactic acid bacteria, namely, Tetragenococcus.(37)
Tetragenococcus halophilus was reported to be isolated from soy sauce.(38) T. halophilus
from Thai fish sauce (nam-pla) was isolated by Thongsanit and coworkers.(39) Halophilic
lactic acid bacteria were the dominant microorganisms at the final stage of fish sauce
fermentation when color, aroma, and flavor are usually developed.(40) In Thailand,
Weissella formerly classified as the genera Leuconostoc and Lactobacillus have been iso-
lated from traditional fermented fish products, such as plaa-ra and plaa-som.(41,42)
However, a few strains of Weisella, such as W. cibaria 110, W. confusa Nh 02, and W.
paramesentroides Dfr 8, have been reported to produce antimicrobial substances in the
form of bacteriocins.(41–43) Tetragenococcus muriaticus is a moderately halophilic hista-
mine-forming lactic acid bacterium found in salted and fermented fish products.(44) A
gram-positive thermotolerant bacterium, Bacillus licheniformis RKK-04, was isolated from
a fermented Thai fish sauce broth having high proteolytic activity.(45) The strains belonged
to nine genera of microorganisms, namely, Bacillus, Lactobacillus, Pseudomonas,
Pediococcus, Staphlococcus, Klebsiella, Debaryomyces, Hansenula, and Aspergillus, have
been isolated from momoni, a Ghanaian fermented fish condiment.(46)
Selected strains of lactic acid bacteria isolated and screened from Thai traditional
fermented fish (pla-som) showed excellent antagonistic activity against Escherichia coli,
Staphylococcus aureus, and Salmonella sp.(47) Lactobacillus sakei has been reported in fish
products.(48) It has been associated with smoked salmon spoilage(49) and Trout spoilage.(50)
Changes in microbiological, biochemical, and physiochemical properties of Nham inocu-
lated with different inoculum levels of Lactobacillus curvatus was studied by Visessanguan
and coworkers.(8) Lactobacillus pobuzihii was isolated from tungtap, a traditionally fermen-
ted fish food and its bacteriocinogenic potentiality was analyzed.(51) L. pobuzihii has been
identified previously from pobuzihii-fermented seeds,(52) but its identification from tungtap
is reported for the first time. Pla-ra, a fermented fish product from Thailand, is rich in
various nutrients, particularly amino acids and peptides, and contains high concentration of
halophilic bacteria.(53,54) Nevertheless, no extremely halophilic bacteria, such as
Halobacterium sp., have been isolated from pla-ra and in particularly none of the genus
Halobacterium, family Halobacteriaceae.(55) A gram-negative, motile, rod-shaped, extremely
halophilic archaeon, designated strain HPC 1-2T, was isolated from pla-ra, a salt-fermented
fish product of Thailand.(56) Strain HPC 1-2T was able to grow at 20–60 °C, at 2.6–5.1 M
NaCl, and at pH 5.0–8.0. However, the hypotonic treatment with less than 1.7 M NaCl
caused lysis of cell. Based on the 16S rRNA gene sequence analysis, strain HPC 1-2T was
considered to represent a novel species of the genus Halobacterium named Halobacterium
piscisalsi, sp. nov.
Fresh water aquaculture fish are often fermented. Although the fermentation of raw fish
is a common process for improvement of shelf life and safety, little is known about the
survival of pathogenic bacteria in these products. Som-fak is an indigenous fermented fish
product composed of minced fish fillet, NaCl (2–5%), ground boiled rice (2–12%), and
minced garlic (4%).(57) Bernbom and coworkers(58) studied the survival and potential
growth of Salmonella enterica serovar weltevreden, S. enterica serovar enteritidis, Vibrio
cholera, and V. parahaemolyticus as influenced by the preservation parameters such as
NaCl, garlic, and lactic acid present in the som-fak. The inhibitory effects of sodium
chloride (0–4%), garlic (0–10%), and lactic acid (pH levels as in som-fak) were measured.
All bacteria were inhibited by 8–10% sodium chloride; Salmonella grew in all concentra-
tions of garlic, whereas Vibrio spp. was inhibited by 1.0–1.5% lactic acid. A distinct hurdle
effect was observed in the fermentation broth in combinations of sodium chloride, garlic,
and lactic acid. The combination of at least 6% garlic and 106 colony-forming units
(CFU)/g garlic fermenting Lactobacillus plantarum 509 prevented growth of the inocu-
lated Salmonella, whereas adding the Lactobacillus strain alone or in combination with
glucose was found to be insufficient to prevent growth.
Overall, the study of the importance of preventing contamination and the use of garlic
fermenting starter cultures in combination with garlic could improve safety of fermented
fish products. The histamine-forming bacteria Pantoea sp. and Enterobacter cloacae were
isolated from salted mackerel in Taiwan,(59) but they have rarely been isolated from
fermented fish products, such as fish sauce and fish paste.(60) The chemical, microbiolo-
gical, and sensory changes associated with sardine fermented fish sauce were examined by
Kilinc and coworkers.(61) The study showed that the fish sauces with spices had lower
bacterial counts than fish sauce without spices. The addition of glucose to the fish sauces
caused a significant increase in the bacterial counts. The fish sauces containing 100:100
(NaCl:glucose) g/kg had a higher total viable count, from 4.47 to 7.95 log CFU/g, than the
samples with 100:50 (NaCl:glucose) g/kg, with a count from 3.96 to 7.84 log CFU/g,
during the first 8 days of fermentation. Staphylococcus aureus and yeast and mold counts
were not detected during fermentation period. The use of spices in fermentation of
sardine enhanced color, aroma, and taste.
Bacterial starter cultures from fermented fish sauce

The use of bacterial starters in food fermentation has become a means to increase
processing and product consistency. Starters are used to improve the sensory character-
istics and microbiological quality and also to shorten the fermentation time of fermented
Table 3. Bacterial starters and their role in fish sauce fermentation.

Bacterial starter Role in fish sauce fermentation References
Bacillus amyloliquefaciens FS05 Reducing biogenic amines accumulation (68)
Halobacillus sp. SP1(1) Speed up the fermentation and additive effect on the existing (12)
natural microflora present in fish
Halobacillus thailandensis, sp. nov. Acceleration of fermentation (64)
Staphylococcus carnosus FS19 Reducing biogenic amines accumulation (68)
Staphylococcus sp. Sk1-1-5 Increase the α-amino content (15)
Staphylococcus xylosus Producing desirable notes of an odor (36)
Tetragenococcus halophilus Improve tissue solubilization and enrich free amino acids and (62, 66)
severely mitigate fishy odor without browning of the product
Virgibacillus sp. SK33 Decrease the histamine content (15)
Virgibacillus sp. SK37 Increase the proteolytic activity at high NaCl concentrations (65)
foods.(8) The acceleration of protein hydrolysis occurs during fish sauce fermentation with
the addition of proteinase-producing halophilic bacteria as a starter culture. The bacteria not
only increase the rate of protein solubilization, but also contribute to flavor development.
Bacterial starter cultures and their role in the fermentation of fish sauce are tabulated in the
Table 3. Staphylococcus xylosus isolated from fish sauce mash played a significant role in
producing desirable notes of an odor in fish sauce.(36) Starter cultures prepared from
Virgibacillus sp. SK33, Virgibacillus sp. SK37, and Staphylococcus sp. Sk1-1-5 were
inoculated during course of fermentation process of Thai fish sauce samples, and the
α-amino contents of all inoculated samples and the control (without the addition of starter
culture) were analyzed. The samples inoculated with Staphylococcus sp. Sk1-1-5 contained
the highest α-amino content of 733.37 ± 13.89 mM than that of the control, 682.67 ± 3.33
mM, after 4 months of fermentation. Virgibacillus sp. SK33 appeared to decrease the
histamine content by 50% when compared with the control. Staphylococcus sp. Sk1-1-5 is
a potential strain that can be applied to produce fish sauce with overall sensory character-
istics of traditional fish sauce in shorter time.(15) Tetragenococcus halophilus, a halophilic
lactic acid bacterium, at different salt concentrations such as 20% and 23% w/w affects the
autolysis, chemical composition, and organoleptic quality. Both autolytic release of protein
(including peptides and free amino acids) into the sauce fraction and the viability of T.
halophilus were highest at lowest salt concentration. To improve tissue solubilization and
flavor development, it may be necessary to add some hydrolytic enzymes and a mixture of
halophilic bacteria isolated from good-quality fish sauce.(62) Two strains of lactic acid
bacteria isolated from natural plaa-som fermentation were used as starter culture:
Lactobacillus plantarum IFRPD P15 and Lactobacillus reuteri IFRPD P17. These strains
have great potential for use as a mixed starter culture in plaa-som fermentation and may
possibly help to reduce fermentation time.(63) A gram-positive, bacillus, namely,
Halobacillus thailandensis, sp. nov., isolated from fish sauce has been successfully employed
to improve fermentation in industrial production of fish sauce in Thailand.(64)
Halobacterium sp. SP1(1) and its exogenous augmentation accelerated the fish sauce
fermentation process with an additive effect on the existing natural microflora present in
the fish during fermentation. The parameters used to monitor the progress of the
fermentation, such as proteinase activity, peptide release, and α-amino content, of
Halobacterium sp. SP1(1)–inoculated fish sauce samples were high at day 10 and day 20
compared with uninoculated controls. This revealed that the addition of Halobacterium
sp. SP1(1) accelerated the fish protein hydrolysis by 10 days even in the presence of
indigenous microflora during fermentation. Therefore, the strain can be used as an

important starter culture to accelerate the fish fermentation process.(12) Hydrolytic activity
of Virgibacillus sp. SK37, a moderately halophilic bacterium, accelerated proteolysis during
fish sauce fermentation and its cell-bound proteinases showed proteolytic activity at high
NaCl concentration (25% w/w) towards various protein substrates. α-Amino content of
fish sauce significantly increased when fermentation was supplemented with whole cells of
Virgibacillus sp. SK37 in conjunction with CaCl2 addition. At the 4th month of fermenta-
tion, the α-amino group content of fish sauce prepared by inoculating with Virgibacillus
sp. SK37 in the absence and presence of 0.2% (w/w) CaCl2 were 1395 ± 53 and 1520 ± 5
mM, respectively.(65) The halotolerant microorganisms, Zygosaccharomyces rouxii,
Candida versatilis, and Tetragenococcus halophilus, are employed as starter cultures in
chum salmon sauce. At the beginning of the fermentation, these microbes enriched free
amino acids and severely mitigated fishy odor without browning the product.(66)
Tetragenococcus halophilus MS33 and MRC5-5-2 isolated from fish sauce fermentation
showed high intracellular aminopeptidase activity. They produced important aldehydes,
namely, 2-methylpropanol and benzaldehyde, which contributed to meaty and almond-
like note. Therefore, T. halophiles may have potential as a starter culture for flavor
development in fish sauce fermentation.(67) Staphylococcus carnosus FS19 and Bacillus
amyloliquefaciens FS05 isolated from fish that possess amino oxidase activity were used as
starter cultures(68) in fish sauce fermentation process to reduce biogenic amine accumula-
tion. The overall biogenic amines concentration of the fish sauce inoculated with
Staphylococcus carnosus FS19 and Bacillus amyloliquefaciens FS05 after 120 days of
fermentation were 15.9% and 12.5% less, respectively, as compared with the control
(without starter culture). Histamine concentration was reduced by 27.7% and 15.4% by
Staphylococcus carnosus FS19 and Bacillus amyloliquefaciens FS05, respectively.
Health-related components of fish sauce

Good-quality fish sauce is high in high-quality proteins containing all the essential amino
acids that are required for growth and regeneration. The beneficial and healthful compo-
nents of fish sauce are showed in Fig. 2.
Biogenic amines and their degradation

Biogenic amines are basic nitrogenous compounds occurring in meat, fish, cheese, and
wine products, mainly due to amino acid decarboxylation activities of certain microbes.(69)
Foods such as sausages,(70) fish and fish products,(71) fermented vegetables,(72) wine,(73)
and cheese(74) contain high concentrations of biogenic amines. Fish proteins are broken
down rapidly by enzymes produced by bacterial growth and generates biogenic amines,
including histamine. Therefore, the quantification of amine in fish can be considered a
quality index and can be used as an important tool for sanitary surveillance.(75)
Commonly associated histamine poisoning fish species in the production of canned and
packaged fish are the scombroid fish (Spanish mackerel, tuna) and nonscombroid fish
(anchovy, sardine).(76) Eight biogenic amines (histamine, tryptamine, putresceine, 2-phe-
nylethylamine, cadaverine, tyramine, spermidine, and spermine) were determined in 13
species of fish and 49 fish products commonly consumed in southern China. In the fish
Fibrin clotting inhibitors
Corrinoid compounds Antioxidants
Fish sauce
Biogenic amines Bioactive peptides
Enzymes producing microbes

Aldehydes
Ammonia Amino oxidases Proteases Antimicrobial
H2O2 Antihypertensive
Figure 2. Flowchart illustrating the healthful biochemical components of fish sauce.(86, 88, 89–93, 106, 107,
128, 144, 148, 151, 163, 165)
samples, the total biogenic amines ranged from 5.03 to 156.17 mg/kg, with a mean value of
44.17 mg/kg in which the histamine levels were less than 21.85 mg/kg. The fermented fish
products and packaged fish products showed higher levels of biogenic amines. The total
level of biogenic amines of lightly cured horse mackerel, a traditional fish product in
southern China, showed 484.42 mg/kg where the levels of 2-phenylethylamine, cadaverine,
and tyramine were 57.61, 244.41, and 62.85 mg/kg, respectively.(77) Production of biogenic
amines was reported in dried and fermented fish(78) and vacuum packaged and cold
smoked fermented fish products.(79) The accumulation of some biogenic amines in some
fermented fish products are suppressed by starter lactic acid bacterial cultures.(80,81) Little
work has been done on the biogenic amine formation by mixtures of food borne pathogen
and lactic acid bacteria.(82) Red-flesh fishes, including mackerel and sardine, contain high
amount of histidine compared with one of the white-flesh fishes, such as codfish.(83) Raw
fish products were frequently found to contain high amount of histamine. The analysis of
549 commercial fish sauce samples for histamine content by solid-phase extraction and
high-performance liquid chromatography revealed that the range varied from 100 to 1000
mg/L, with the majority in the range of 200–600 mg/L.(84) It is known that
Tetragenococcus muriaticus, a moderate halophilic and lactic acid coccus, is a histamine-
forming bacterium in salted and fermented fish products.(85)
Fish sauce was reported to contain considerable amounts of biogenic amines in
addition to many nutritious compounds. Biogenic amine concentration in fish sauce
was predominated by histamine, putrescine, cadaverine, and tyramine. The highest ever
reported histamine, putrescine, cadaverine, and tyramine concentrations in fish sauce
were 1220, 1257, 1429, and 1178 ppm, respectively.(86) Tryptamine and phenyl ethyla-
mine are occasionally present at a low level, whereas spermine, spermidine, and agma-
tine are trace amines in fish sauce. Jiang and coworkers(87) analyzed commercially
produced yu-lu, a Chinese fermented fish sauce, for the biogenic amines by using pre-
column derivatization with dansyl chloride (Dns-Cl) and high-performance liquid

chromatography with fluorescence detection (HPLC-FLD). Putrescine, cadaverine, his-
tamine, and tyramine were the major biogenic amines (more than 100 mg kg−1), whereas
tryptamine, spermidine, and spermine were regarded as minor biogenic amines (less
than 25 mg kg−1). Figure 3 illustrates the production, types, and degradation of the
biogenic amines into aldehydes, ammonia, and hydrogen peroxide by amino oxidases
via oxidative deamination process.(88) Histamine oxidase is found in Staphylococcus
xylosus,(89) Staphylococcus carnosus,(90) Bacillus amyloliquefaciens,(91) and Lactobacillus
sakei.(92) Tyramine oxidase was found in Micrococcus variens(93) and Staphylococcus
xylosus,(89) putrescine oxidase in Micrococcus rubens,(88) and phenylethylamine oxidase
in Escherichia coli.(94) Common symptoms of biogenic amines intoxication in human are
Fish sauce
Amino acid decorboxylation by microbes
Biogenic amines
Predominant amines Less Predominant amines Trace amines

1. Histamine 1. Tryptamine 1. Spermine
2. Putrescine 2. Phenyl ethylamine 2. Spermidine
3. Cadaverine 3. Agmatine
4. Tyramine
Oxidative deamination by amino

oxidases from microbial sources
Sl.No. Amino oxidases Microorganisms Ref.
1. Histamine oxidase Staphylococcus xylosus (89)

Staphylococcus carnosus (90)
Bacillus amyloliquefaciens (91)
Lactobacillus sakei (92)
2. Tyramine oxidase Micrococcus variens (93)

Staphylococcus xylosus (88)
3. Putrescine oxidase Micrococcus rubens (88)
4. Phenylethylamine oxidase Escherichia coli (94)
Aldehydes, Ammonia, and H2O2
Figure 3. Production, types, and degradation of biogenic amines in fish sauce.

nausea, respiratory distress, hot flushes, sweating, heart palpitations, headaches, a bright
red rash, oral burning, hypertension, and hypotension.(95) To prevent or reduce the
accumulation of biogenic amines in fermented foods, microorganisms with amine
oxidase activity have been exploited. The effect of Staphylococcus xylosus 0538 to inhibit
biogenic amine formation in a salted and fermented anchovy was studied by Mah and
Hwang.(80) In that study, the researchers reported that 16% reduction in the biogenic
amine concentration in the fermented product inoculated with S. xylosus as compared
with the control. Staphylococcus carnosus FS19 and Bacillus amyloliquifaciens FS05
isolated from fish sauce that possess amino oxidase activity were used as novel starter
cultures to investigate the effect of starter cultures in reducing biogenic amines accu-
mulation during fish sauce fermentation. The study showed that S. carnosus FS19 and B.
amyloliquifaciens FS05 have the ability to degrade 29.1% and 59.9% histamine in a buffer
system, respectively.(68)
Histamine is well known as a mediator of anaphylactic reactions(96) and is a biogenic
amine that occurs in many foods, particularly histidine-rich fishes, such as tuna(76) and
mackerel,(59) as well as in fermented products, such as dry fermented sausage,(97) wine,(98)
miso,(99) and salted fermented fishes.(80) Impaired histamine degradation, based on reduced
diamine oxidase activity and the resulting excess of histamine, caused numerous symptoms
that mimic an allergic reaction.(100) High levels of histamines in foods can have important
vasoactive effects in humans.(101) The salting and fermenting time of ishiru is longer than 1
year at ambient temperature, and the fish sauce products, particularly those made from
sardine and mackerel, often contain large quantities of histamine, about 1000 mg/L or
greater.(102) There are several regulations that govern the concentration of histamine in
marine foods, such as 50 mg/L for tuna in the United States (Food and Drug Administration
[FDA], 2001) and 200 mg/L for enzyme-ripened products, including fish sauces, in Canada
(Canadian Food Inspection Agency [CFIA], 2009), although the histamine poisoning by the
salted and fermented fish products has not been reported. There are several reports
regarding bacterial histamine-reducing activity in traditional fermented fishes.(103)
However, there are no reports about clear histamine-reducing activity of food material
and compositions. Kuda and Miyawaki(104) studied the histamine-reducing activity of
fermented food products, including fish and radish-nukazuke, radish and turnip-kojizuke,
miso, natto, and yogurt, in fish sauce products. The nuka-paste portion of radish-nukazuke
showed the highest histamine-reducing activity. The addition of the nuka paste 20% (w/v) to
sardine fish sauce (iwashi-ishiru) reduced the histamine content to about 600–700 mg/L
from 1200 mg/L. The histamine-reducing activity of nuka paste was regarded as water
soluble, heat tolerant, and of high molecular weight. Of 156 extremely halophilic archaea
isolated from various salt-fermented fishery products, HDS3-1 from an anchovy fish sauce
sample exhibited the highest histamine degradation activity when cultured in halophilic
medium containing 5 mM histamine, followed by HDS1-1, HPC1-2, and HIS40-3. Based on
the 16S rRNA gene sequence similarity, HDS3-1 was classified as Natrinema gari and did
not exhibit decarboxylase activity towards all tested amino acids. The histamine-degrading
activity of HDS3-1 was located in the intracellular fraction and was tested on in vitro
cytotoxicity assay; the whole-cell extract of HDS3-1 treated with all cell lines tested resulted
in dose-dependent inhibitions the cell growth, with the IC50 values higher than 250 µg mL−1.
The histamine-degrading activity of HDS3-1 was most likely associated with salt-tolerant
and thermoneutrophilic histamine dehydrogenase.(105)
Enzymes in fish and seafood processing

Enzymes are used in many areas of fish and seafood processing, including quality assurance
and freshness testing, as well as various processing operations. They have been utilized for
centuries in the production of various cured and fermented fish products. They include
numerous sauces and paste produced in the Far East, such as paties, nam-pla, budu, and
bagoong. During the processing of fermented fish products, endogenous autolytic enzymes
(mostly proteases and lipases) degrade and solubilize the fish flesh and produce the desired
flavor compounds.(10) The fermentation process relies mainly on proteolytic activity of
endogenous proteinases in the digestive tract and muscle of anchovy as well as proteinases
secreted by halophilic/halotolerant bacteria that are naturally present in solar salt and fish.(25)
Degradation of proteins to amino acids and small peptides occurs slowly due to limited
proteolytic activity at high salt content.(106,107) Due to this, fermentation usually takes at least
12 months for complete hydrolysis and flavor/aroma development.
Proteinases, which catalyze the hydrolysis of proteins, constitute the most important
group of industrial enzymes used in the world and have several applications in the food
industry.(108) Proteinases applied in fish sauce manufacture shorten the fermentation period
and improve fish sauce nutrition and increase the profitability.(109) Proteinase, which can be
used as an accelerant for the fermentation of fish sauce, must have salt acid-tolerant
characteristics because fish sauce has high NaCl concentrations of 20–25%(38) and low pH
values of 4.3(110) to 5.8(111) due to accumulation of lactic acid. Fish endogenous proteinases
and microbial proteinases play an important role in protein hydrolysis during fish sauce
fermentation. The acceleration of fish sauce production would require the understanding of
the biochemical properties of endogenous proteinases. Siringan and coworkers(112) studied
the autolytic activity and biochemical properties of endogenous proteinases in anchovy.
Maximum autolytic activity of Indian anchovy was found at 60 °C, and the activity
decreased with increased NaCl concentration. The activity of crude proteinase extracts
decreased >50% when NaCl concentration was greater than 0.1 M. The results showed
that the autolytic activity of Indian anchovy at 10% NaCl was about 90%, but at 15% NaCl
the activity was 77% compared with the control (without NaCl). Endogenous proteinases
have been found to exhibit 52% of activity even at high concentration of NaCl (25% w/w).
Crude proteinase extracts exhibited the highest activity at 60 °C, using either casein or acid-
denatured hemoglobin as a substrate. Alkaline proteinases were predominant in fish muscle
and brine during the ripening of salted anchovy.(113) According to Choi and coworkers,(114)
the activities of trypsin, chymotrypsin, and cathepsin L purified from anchovy decreased
with increasing NaCl concentration. Exogenous proteolytic enzymes were used to accelerate
the elaboration process of fish sauce “garum,” and various fermentation technological
parameters such as salt percentage, temperature, enzyme dose, and continuous stirring
were assayed in order to optimize the process and obtain the best yield.(27)
Fish as a source of enzymes

The viscera and muscle of fish are the important sources of proteinases. Pepsin, trypsin,
chymotrypsin, carboxypeptidase, and aminopeptidase are the proteinases found in
viscera, whereas cathepsins, calpain, and neutral trypsin-like proteinases are found in muscle
tissue.(115) Siringan and coworkers(112) reported that trypsin-like proteinase exhibited the
highest activity in viscera and muscle of Indian anchovy (Stolephorus spp.). Trypsin-like,
chymotrypsin-like, and cathepsin-like proteinases were found in commercial fish sauce
samples throughout the fermentation period, suggesting that these proteinases were stable
at high salt concentrations (250–300 g kg−1 NaCl) and under acidic conditions (pH 5.2–5.8).
Internal organs are the important sources of fish proteases. The gastric mucosa secretes
pepsin, pancrease, and pyloric caeca, and the intestine secretes trypsin and chymotrypsin.(116)
Klomklao and coworkers(117) reported that tuna spleen is one of the organs possessing high
proteolytic activity, and trypsin-like serine proteinases were reported from spleen of skipjack
tuna. Fermentation period of fish sauce from minced capelin was shortened to 6 months with
addition of 5–10% enzyme-rich (trypsin and chymotrypsin) cod intestines,(1) and the addition
of spleen to salted sardine could accelerate the protein hydrolysis during fermentation due to
the high proteolytic activity in skipjack tuna spleen.(118) Trypsin from skipjack tuna
(Katsuwonus pelamis) degrades the sardine (Sardinella gibbosa) proteins by proteolytic
activity. The effect of NaCl on proteolytic activity was also studied by Klomklao and
coworkers.(119) The study revealed that the rate of liquefaction of fish sauce produced from
the sardine varied at the levels of spleen of skipjack tuna (0, 10, and 20%) with different salt
concentrations (15, 20, and 25%). The liquefaction rate of the lower-salt-treated samples was
generally faster than were those treated with higher salt content. The sardine with 20% spleen
and 15% salt exhibited the greatest protein hydrolysis, particularly at the early stage, but the
overall acceptance was poor due to the browning as well as the increase in the redness of the
liquid formed. Fish sauce samples containing 20% salt, without and with 10% spleen, had
similar acceptability to commercial fish sauce. So, the additive effect of spleen as well as
reduced concentration of salt can accelerate the liquefaction of sardine in the fish sauce
fermentation. Castillo-Yanez and coworkers(120) isolated trypsin from pyloric caeca of
Monterey sardine (Sardinops sagaxcaerulea) and found that the enzyme had an optimal
activity at pH 8.0 and at 50 °C. Kishimura and coworkers(121) purified and characterized the
trypsine from the viscera of true sardine and the pyloric caeca of arabesque greenling as having
optimal proteinase activity at pH 8.0 and 60 °C. Klomklao and coworkers(122) characterized
the autolytic profile of true sardine (S. melanostictus) and observed the involvement of heat-
activated proteinases, which are active at both acidic and alkaline pH values. Autolytic activity
decreased with increasing NaCl concentration (0–30%). This implied that pepsin is a pre-
dominant enzyme responsible for autolysis in true sardine during fish sauce preparation.
Enzymes from microorganisms isolated from fish

Some proteolytic enzymes produced by microorganisms isolated from fish sauce have been
reported. These were predominantly mesophilic and halophilic bacteria.(106,107) Real-time
polymerase chain reaction (PCR) quantification of protease-producing bacteria in tradi-
tional Chinese fish sauce was estimated to be (1.47 ± 0.73) × 103 colony-forming units (cfu)/
mL by Xiao and coworkers.(123) An alkaline protease produced by Bacillus megaterium
isolated from a fermented broth of Thai fish sauce was purified by hydrophobic interaction
and gel filtration techniques. The enzyme was purified 148-folds and its specific activity
increased from 0.09 to 13.33 U/mg protein.(126) Intracellular aminopeptidases have been
recognized as key enzymes responsible for flavor formation of lactic acid bacteria, such as
Lactobacillus sake, Lactobacillus plantarum, and Lactobacillus curvatus, in fermented sausage
and cheese. Aminopepetidases are parts of the proteolytic system of lactic acid bacteria.(127)
In addition to their role in lactic acid bacterial growth, these enzymes converted peptides
and/or oligopeptides to amino acids that further serve as a precursor for flavor
formation.(128) Similarly, aminopeptidases from Lactobacillus sake exhibited high activity
towards leucine and alanine substrates.(129) Use of halophilic/halotolerant starter culture
producing high amount of proteinase is the possible approach to reduce the time required
for the fermentation of fish sauce. Virgibacillus sp. SK37, a moderately halophilic bacterium
isolated from fish sauce, produced extracellular proteinases exhibiting proteolytic activity at
high salt concentration of 25%.(130) The extracellular enzyme showed activation and stability
at the salt content used in the fish sauce fermentation. The ability of cell-bound proteinase
from Virgibacillus sp. SK37 isolated from fish sauce fermentation increased with Ca2+
concentration. This was reported by Sinsuwan and coworkers.(131) Lactococcus sp., L.
helveticus, and Streptococcus sp. expressed Prt P, Prt H, and Prt S cell-bound proteinases,
respectively, which were subtilisin-like serine proteases.(132) Extracellular proteinase from
Virgibacillus sp. SK37 also displayed subtilisin-like characteristics, but showed different
biochemical properties from the cell-bound proteinases.(130) Activity of cell-bound protei-
nase from Lactococcus lactis subsp. Cremoris SK11 increased with NaCl concentrations up to
9.4 g/100 mL.(133) The activity of Virgibacillus sp. SK37 cell-bound proteinase decreased with
NaCl concentration when actomyocin was used as a substrate.(131) Gupta and coworkers(134)
reported that the activity of Bacillus proteinases decreased with increasing NaCl concentra-
tion. The enzyme activity of both crude and purified enzyme preparations was monitored in
the presence of 0–0.17 M salt concentration. The enzyme activity was slightly increased at
0.03 M NaCl. However, on increasing the salt concentration above 0.03 M, reduction was
observed. Relatively poor activity was observed in proteinases isolated from mesophilic
bacteria in extreme conditions. Exception to this was the serine proteinases from
Virgibacillus sp. SK33 isolated from Thai fermented fish sauce that showed increased activity
with NaCl up to 25%. The proteinase exhibited high stability in both the absence and
presence of NaCl up to 25%. Another distinct characteristic of the enzyme was high stability
at the optimal temperature, 55 °C,(135) in contrast to several microbial proteinases, which
were unstable in such a condition.(106) Udomsil and coworkers(67) isolated the proteinase-
producing halophilic lactic acid bacteria, Tetragenococcus halophilus ATCC 33315, from fish
sauce fermentation, which produced predominant volatile compounds, such as 1-propanol,
2-methylpropanol, and benzaldehyde, corresponding to major volatile compounds in fish
sauce. A salt-tolerant protease was purified from the culture broth of Bacillus megatarium
KLP-98, and its potential as a fermentation starter for the manufacture of fish sauce was
studied by Fu and coworkers.(136) The optimum temperature and pH for protease activity
were 60 °C and 5.5, respectively. Relative activity of protease was 73, 33, and 5% at 10, 20,
and 30% NaCl concentrations, respectively. Histamine-producing halophilic bacteria iso-
lated from fish sauce mashes possessed the complete sequences of the plasmids encoding the
pyruvoyl-dependent histidine decarboxylase gene hdcA.(137) Decomposition of lipids by
microbial and enzymatic activities produced alcohols and carboxylic acids. Esters are
derived from esterification of alcohols with carboxylic acid, which is usually found in
fermented sea foods.(138) The samples inoculated with T. halophiles contributed fruity
buttery note due to esterification.(134) The list of enzymes produced by microorganisms
isolated from different fish sauces is given in Table 4.
Table 4. Enzyme producing microorganisms isolated from fish sauce.

Microorganism Nature of enzyme References
Bacillus amyloliquefaciens FS05 Amino oxidase (68)
B. licheniformis RKK-04 Serine protease (45)
B. megaterium Alkaline protease (126)
B. megaterium KLP-98 Acid protease (135)
Bacillus sp. 11-4 Protease-I (143)
B. subtilis CN2 Alkaline protease (141)
B. subtilis JM-3 Acid protease (106, 140)
Filobacillus sp. RF2-5 Serine protease (124)
Halobacterium. piscisalsi, sp. nov. Alkaline phosphatase, esterase, lipase, acid phosphatase (142)
Halobacillus thailandensis, sp. nov. Extracellular serine metallo protease (64)
Halobacterium sp. F1, F2, and SP1(1) Extracellular protease (12)
Halobacillus sp. SR5-3 Serine protease (125)
Staphylococcus carnosus FS19 Amino oxidase (68)

Staphylococcus sp. SK1-1-5 Protease (15)
S. xylosus Tyramine oxidase (89)
Tetragenococcus halophilus Histidine decarboxylase (131)
T.halophiles strains MS33 and M11 Intracellular aminopeptidase (67)
T. muriaticus Histidine decarboxylase (139)
Virgibacillus sp. SK33 Protease (135)
Virgibacillus sp. SK37 Extracellular cell-bound protease (130)
Corrinoid compounds
A Japanese fish sauce, ishiru, made from squid by a traditional food manufacturing
process, contained the highest amounts of vitamin B12 amongst several fish sauces
evaluated.(144) The amounts of vitamin B12 (16.3 ± 5.8 µg/100 g) determined with
Lactobacillus microbiological method were about 5.4-fold greater than in various fish
sauces made in Japan (3.0 ± 2.0 µg/100 g).(145) Two corrinoid compounds were purified
and partially characterized from the fish sauce ishiru by the use of thin-layer chromato-
graphy (TLC) on silica gel as an important purification and analytical tool. The purified
corrinoid compounds were main corrinoid compound I (authentic B12) with the Rf value
of 0.61 and retention time 9.4 minutes, which were identical to the values of vitamin B12,
and minor corrinoid compound II (cyanocobamides) with Rf value of 0.55 and retention
time 14.5 minutes. Rf value and retention of minor corrinoid compound II were not
identical to any authentic corrinoids tested. This study emphasized that the fish sauce may
not be suitable for use as a good vitamin B12 source due to the occurrence of inactive
corrinoid compounds, as these compounds affect the absorption of B12 in mammalian
intestine.(144)
Bioactive peptides
Marine bioactive peptides have widely been produced by enzymatic hydrolysis of marine
organisms.(146,147) However, in fermented marine food sauces, enzymatic hydrolysis has
already been carried out using microorganisms; thus, bioactive peptides can be purified
without hydrolysis.(148) Depending on the amino acid sequence, bioactive peptides may be
involved in various biological functions, such as antihypertension, opioid agonist or
antagonist, immunomodulatory, antithrombotic, antioxidant, anticancer, and antimicro-
bial activities, in addition to nutrient utilization.(149–153) Some bioactive peptides have
demonstrated multifunctional activities based on their structure and other factors,
including hydrophobicity, charge, or microelement binding properties.(154) Some of these

bioactive peptides may have potential for human health promotion and disease risk
reduction.(155) Possible role of food-derived bioactive peptides in reducing the risk of
cardiovascular disease has been demonstrated.(156) Lee and coworkers(140) investigated the
anticarcinogenic activities of hydrophobic peptide fraction separated from anchovy sauce
and found an induction of apoptosis and increase of caspase-3 and -8 activities. The
induction of apoptosis by peptide fraction separated from anchovy sauce was studied in a
human lymphoma cell line (U937) using biochemical and flow cytometric methods. A
sub-G1 peak representing apoptosis was found in the cell cycle analysis of treated U937
with a hydrophobic peptide fraction, and the same was also confirmed by apoptotic DNA
fragmentation. Cathepsin L, which is the most active protease in anchovy muscle, is
important in the degradation of muscle proteins and may provide information for the
production of anchovy sauce.(157)
Kuroda and coworkers(158) quantified the γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly), a
potent kokumi peptide in fish sauces originating from various countries by the calcium-
sensing receptor assay. The analyses revealed that concentration of γ-Glu-Val-Gly was
ranging from 0.04 to 1.26 mg/dL. This indicates that γ-Glu-Val-Gly is widely distributed
among various commercial fish sauces. The γ-Glu-Val-Gly peptide activates the calcium-
sensing receptor and possesses the characteristics of kokumi substances, which modify the
five basic tastes, especially sweet, salty, and umami, when they are added to basic taste
solutions or food. A series of salt taste–modulating arginyl dipeptides were screened by the
enzyme digested fish protein protamine to enhance salt taste by sensomic approach.(159)
Sasaki and coworkers(160) purified a novel angiotensin-I–converting enzyme inhibitory
tripeptide, Leu-Ala-Arg, from fish sauce ishiru prepared from squid. The same researchers
observed that the tripeptide acted as an antihypertensive peptide, reducing systolic blood
pressure in spontaneously hypertensive rats.
Antioxidants and fibrin clotting inhibitors from fish sauce

During normal cellular activities, various processes inside the cell produce reactive oxygen
species (ROS). Some of the most common ROS are hydrogen peroxide, superoxide ions, and
hydroxide radical. These compounds when present in a high enough concentration, can
damage cellular proteins and lipids, or form DNA adducts that may promote carcinogenic
activity. Researchers have indicated that antioxidant nutraceuticals supplied from daily diets
quench the ROS, and they are required as cofactors for antioxidant enzymes in cellular
environments. Cellular antioxidants may be enzymatic (catalase, glutathione peroxidase,
superoxide dismutase) or nonenzymatic (glutathione, thiols, some vitamins, and metals, or
phytochemicals such as isoflavones, polyphenols, and flavonoids).(161) Antioxidants are very
important for human health, since the production of ROS is thought to be a significant
cause of aging and carcinogenesis.(162) Aoshima and Ooshima(163) studied the antioxidant
activity of Japanese liquid condiments, shoyu (soy sauce) and gyoshoyu (fish sauce), and
reported that both the condiments have high level of 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radical–scavenging activity. The DPPH radical–scavenging activity was greater for the shoyu
(32.3 ± 3.7%) than the gyoshoyu (87.7 ± 0.1%). Gyoshoyu is also useful for reducing level of
H2O2 in foods, when it is added as a liquid condiment. This anti–hydrogen peroxide activity
of gyoshoyu is due to the presence of a thermostable catalase.
Thongthai and Gildberg(164) analyzed the stimulation of the proliferation of human

white blood cells during in vitro cultivation with peptides from commercial Thai anchovy
fish sauce. Significant growth stimulation was obtained in the presence of 5 μg/mL of
small size fish sauce peptides, whereas no stimulation was detected at higher or lower
concentrations. A stable fibrin-clotting inhibitor was screened from an extract of salt-
fermented anchovy sauce after desalting by electrodialysis.(165) The fibrin-clotting inhibi-
tory activity increased with an increasing amount of extract. The active substance involved
in the fibrin-clotting inhibition was remarkably stable in a wide range of pH (2.0–12.0)
and even at boiling temperature. The fibrin-clotting inhibitory activity was fully retained
after treatment with hydrolytic enzymes, such as pepsin, trypsin endoprotease, and
exopeptidase (flavourzymes), and glycolytic enzyme mixture (viscozyme).
Conclusions
The hydrolysis of proteins of economically underutilized fish by mixing with NaCl to
produce soluble peptides and amino acids during the fermentation period for about 9–12
months varied greatly with species of fish, ratios of fish and salt, and fermentation period.
The taste and aroma of fish sauce are contributed by produced amino acids, peptides,
organic acids, and volatile compounds. Various novel halotolerant bacterial cultures have
been used to accelerate fermentation process, increase the α-amino content, enhance the
sensory characteristics, and improve the microbiological quality. Researchers have also
focused on effective inhibition of biogenic amines in fish sauces using novel bacterial starter
cultures, apart from effectively deciphering the formation of different biogenic amines and
their toxicity in fish sauces. The proteolytic enzymes from animal and microbial sources not
only shorten the fermentation period in fish sauce production, but also in turn will help
reducing the formation of biogenic amines. In addition, biological functions and desalina-
tion help to recover flavor and aroma compounds of fish sauce.
Future research studies are needed in order to investigate different bioactive peptides
from fish sauce and their biological effects in vivo. It is also very important to study
nonpathogenic bacteria that produce bacteriocins or biomedically important enzymes for
their use in fish sauce production. Another research area that needs to be addressed
includes isolation and characterization of healthful metabolites from fish sauces with
reference to their biofunctional and biomedical properties.
Acknowledgments
Dr. Shubha Gopal and Dr. Bhaskar Narayan are thankful to University of Mysuru,
Mysuru, and CSIR-CFTRI, Mysuru, respectively, for the permission to collaborate on
this work. The work forms part of the doctoral studies of G. S. Siddegowda.
Funding
G. S. Siddegowda thanks the Department of Collegiate Education for the partial funding to
carry out this work.
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Siddegowda GS Shubha Gopal

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Siddegowda Gopalapura Shivanne Gowda, Bhaskar Narayan & Shubha

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Bacteriological properties and health-related biochemical

CONTACT Shubha Gopal sguom@yahoo.com Department of Studies in Microbiology, University of Mysore,

Fish sauce: Diversity and production

Alanine 4.26 2.01 3.11 0.92 4.52 9.60 5.01 9.51

Table 2. Salt-fermented ﬁsh sauces from around the world.

Ishiru Astroscopus japonicas 5:1 6 months Japan (29)

Desalination of ﬁsh sauce

Fish and edible fish by product

Salt fermentation Enzyme fermentation

Mixing with salt (30%–40%, Salt/Fish, w/w)

Salt (w/w) Ingredients Enzymes

25 g/100 g Thyme (1%) Kojizyme MG

Figure 1. Schematic representation of ﬁsh sauce production.(22,27)

Bacteriological properties of ﬁsh sauce

Bacterial starter cultures from fermented ﬁsh sauce

Table 3. Bacterial starters and their role in ﬁsh sauce fermentation.

indigenous microﬂora during fermentation. Therefore, the strain can be used as an

Health-related components of ﬁsh sauce

Biogenic amines and their degradation

Fibrin clotting inhibitors

Corrinoid compounds Antioxidants

Biogenic amines Bioactive peptides

Enzymes producing microbes

Ammonia Amino oxidases Proteases Antimicrobial

column derivatization with dansyl chloride (Dns-Cl) and high-performance liquid

Predominant amines Less Predominant amines Trace amines

Oxidative deamination by amino

Sl.No. Amino oxidases Microorganisms Ref.

1. Histamine oxidase Staphylococcus xylosus (89)

2. Tyramine oxidase Micrococcus variens (93)

3. Putrescine oxidase Micrococcus rubens (88)

4. Phenylethylamine oxidase Escherichia coli (94)

Aldehydes, Ammonia, and H2O2

Figure 3. Production, types, and degradation of biogenic amines in ﬁsh sauce.

Enzymes in ﬁsh and seafood processing

Fish as a source of enzymes

Enzymes from microorganisms isolated from ﬁsh

Table 4. Enzyme producing microorganisms isolated from ﬁsh sauce.

Staphylococcus carnosus FS19 Amino oxidase (68)

including hydrophobicity, charge, or microelement binding properties.(154) Some of these

Antioxidants and ﬁbrin clotting inhibitors from ﬁsh sauce

Thongthai and Gildberg(164) analyzed the stimulation of the proliferation of human

M. Puriﬁcation and characterization of a halotolerant Bacillus licheniformis RKK-04 isolated

acteristics of endogenous proteinases in Indian anchovy (Stolephorus indicus). Food Chem.

in ﬁsh sauce. J. Liq. Chromatogr. Relat. Technol. 2003, 26, 2703–2707.

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