13 Fermented fish and fish products

e.G. BEDDOWS

13.1 Introduction

Fermented fish products contribute significantly to the protein intake of

a large number of the World's population. In some countries, such as
Thailand, Kampuchea, Malaysia, Cambodia, Philippines and Indonesia in
South East Asia, these products are a staple part of the diet. Amano (1962)
reported that in Cambodia, some 7.5% of the total dietary protein is
derived from fish sauce.
The preservation depends mainly on the use of salt. This may be used
with other methods, such as sun-drying that speed up the process but
introduce other changes. The principle of 'salt' fermentation is that the
initial step is dehydration brought about by the osmotic effect. This reduces
the moisture content to a level where spoilage may no longer occur. In
addition, salt exhibits its own anti-bacterial action.
In this work, the term fermentation is defined as 'the transformation
of organic substances into simpler compounds by either the action of
microorganisms or by enzymes'. In the fermentation of fish, enzymes
produce the textural change and assist in producing some of the flavour.
However, bacteria are involved in the development of aroma and
flavour.
The manufacturing processes involve fish and salt being packed in layers
and left for a long period; the cellular liquid that is extracted forms the
'pickle'. The proteolytic enzymes in the fish are liberated within the cells
and attack the internal membranes and muscle. The resulting solubilized
protein exudes and is attacked in turn, by the enzymes as they are liberated,
to produce afish sauce. However, if the pickle is drawn off at intervals or the
period of contact is kept short, then a fish paste may be produced. If the
period is much shorter or the amount of salt used is limited, then salted
fish may result. These will have undergone some softening and flavour
development.
In this chapter, attention is paid first to fish sauces, as this is where
the major changes involved with fish fermentation can be demonstrated.
However, the same processes apply to fish pastes and salted fish.
B. J. B. Wood (ed.), Microbiology of Fermented Foods
© Thomson Science 1998
 FERMENTED FISH AND FISH PRODUCTS 417

13.2 Fish sauces

Fermented fish sauces have been consumed since ancient times. Badham
(1854) reported that both 'Garum' and 'Alec' were highly prized by the
Romans. Even today, a sauce, 'Garos', is made in Greece from fish viscera,
particularly the liver of Scomber colias (Kelaiditis, 1949a). A sauce of
ancient Greece was 'Aimeteon', made from viscera and blood of tunny.
Badham (1854) reported that two other fish sauces, 'Botargue' and
'Ootarides', were still being produced in Italy and Greece in the 19th
century. A number of traditional fish sauces are listed in Table 13.l.
'Nuoc-mam' is produced in Vietnam and consumed by a large proportion
of the population, mostly added to rice to overcome the blandness of such
cereals. The sauce is a clear brown liquid with a distinctive meaty, sharp
aroma. The taste is predominantly salty, but many compounds make a
significant contribution.
The sauce is prepared using species of small fish or shrimp (Truong Tan
Quan, 1951). Fish, caught by seine-netting, are kneaded and pressed by
hand. They are layered with salt in earthenware jars almost buried in the
ground. After 6 to 18 months the jars are removed and the supernatant
liquor decanted off to give first-quality nuoc-mam or 'nuoc-nhut' (Rose,
1921; Bremond, 1919).
The residual mass is then extracted with boiling sea water to lixivate the
fish and the liquid is sold as second-quality product. The extraction proce-
dure may be repeated to give poorer qualities of nuoc-mam. Generally,
these lower quality sauces have poorer keeping properties. Often, these
products are improved by the addition of caramel, molasses, roasted maize
or roasted barley to the fish before the second and subsequent extractions.
The undissolved residue is used for animal feedstuffs.
With some fish species, such as Dorosoma nasas or other fatty fish, the oil
which comes to the top, is separated off to avoid rancidity due to the
oxidation of the fats. The oil is sold and is of good nutritive value since it is
high in polyunsaturated fats.
Nam-Pla made in Thailand is very similar (Van Veen, 1965). The process
is more commercialized than that of nuoc-mam. The fish: salt mix is stored
in loosely covered large concrete vats for 6-12 months (Dougan & Howard,
1975; Beddows et ai., 1979). The pickle is run off and exposed to the sun for
1-3 months (ripening) (Saisithi et ai., 1966) before blending with 'dark' or
'light' meiki, which is a concentrated by-product obtained from the bacte-
rial production of monosodium glutamate (Beddows et ai., 1979).
Budu, is similar and is produced in the north-eastern states of Malaysia.
The species of fish used is restricted almost totally to Stoiephorus spp. The
manufacture and the changes that occur during its production have been
reported by Beddows et ai. (1979,1980). Usually a mixture of three fish to
two of salt is used, which is a higher concentration of salt than others,
418 MICROBIOLOGY OF FERMENTED FOODS

Table 13.1 Fish sauces

Conditions and time of

Country and name Species fermentation

Japan
Shottsuru Astrocopus japanicus (sandfish) 5: 1 fish: salt + malted rice
Uwo-shoyu Clupea pilchardus (sardine) and koji (3:1) (6 months)
Ika-shoyu Omnastrephis sloani (squid)
Omnastrephis paeificus
Korea
Ieot-kal Various 4: 1 fish: salt (6 months)
Vietnam
Nuoc-mam Stolephorus spp., Ristrelliger spp. 3:1-3:2 fish:salt
Engraulis spp., Decapterus spp. (4--12 months)
Dorosoma spp., Clupea spp.
Nuoc-mam-gau-ca Clarius spp., Ophicephalus spp. Livers only, 10: 1 salt for
8 days then boiled
Thailand
Nam-pla Stolephorus spp., Ristrelliger spp. 5: 1 fish:salt (5-12 months)
Cirrhinus spp.
Malaysia
Budu Stolephorus spp. 5: 1-3: 1 salt + palm sugar +
tamarind (3-12 months)
Bakasang Stolephorus spp., Sardinella spp. 5: 2 fish: salt (3-12 months)
Burma
Ngapi Various 5: 1 fish: salt (3-6 weeks)
Philippines
Patis Stolephorus spp., Clupea spp. 3:1-4:1 fish: salt
Decapterus spp., Leionathus spp. (3-12 months)
Indonesia
Ketjap-ikan Stolephorus spp., Clupea spp. 5: 1 fish: salt (6 months)
Leionathus spp., Osteochilus spp.
Puntius spp., Ctenops spp.
IndialPakistan
Colombo-cure Ristrelliger spp., Clupea spp. Gutted fish + tamarind
Cybium spp. 6: 1 fish: salt (12 months)
Hong Kong
Sardinella spp., lelio spp. 4: 1 fish: salt (3-12 months)
Carangidae spp., Engraulis spp.
Teuthis spp.
Greece
Garos Scomber colias Liver only, fish 9: 1 salt (8 days)
France
Pissala Aphys pellucida, Gobius spp. 4: 1 salt (2-8 weeks)
Engraulis spp., Atherina spp.
Me/etta spp.
Anchovy Engraulis encrasicholus Beheaded/gutted
2: 1 fish: salt (6-7 months)

tamarind and caramelized palm sugar may be added to the pickle, to

sweeten and give the sauce a darker appearance.
Patis, produced in the Philippines is similar to nuoc-mam in the type of
fish used and its general manufacture. The analysis is comparable, although
 FERMENTED FISH AND FISH PRODUCTS 419

a greater variation in the distribution of nitrogen compounds can occur

(Subba Rao, 1967).
Shottsuru produced in Japan, may be prepared from sardine, anchovy,
molluscs or sandfish using salt in a similar manner to other sauces (Subba
Rao, 1967).
Tareeh and mehiaweh prepared and consumed in the Middle East are
similar to these far eastern products even though they are derived from
sardine which has a higher fat content. The details and analysis of the sauces
are given by Musaiger et al. (1990).

13.2.1 Biochemical changes; hydrolysis of protein

Many changes take place during sauce manufacture. An understanding of
the changes is essential if the production rate is to be faster or if less valued
species are to be used. The tendency is to assume that all of the sauces
undergo the same changes, which must be far from what actually happens as
the fish species do vary and the microorganisms will differ according to the
environment. However, the main part of the production of all fish sauces is
the conversion of insoluble fish protein into a soluble or 'suspended' form.
The agents are the enzymes present in the fish, although enzymes from
microorganisms may assist.
The main products are amino acids and polypeptides. These contribute
to the flavour quite significantly. However, side products are ammonia
and other low molecular mass nitrogenous compounds. These will be
covered later. Ijong & Ohta (1995) have given a full amino acid analysis of
bakasang which is produced in Indonesia and is typical for this type of
product.
Uyenco et al. (1953) showed that the total nitrogen increases steadily
during nuoc-mam and patis fermentation over 120 days and that the organic
nitrogen, which represents mainly the soluble protein, polypeptides, amino
acids and ammonia, reached a maximum of approximately 2.0% with a total
nitrogen of 2.38%.
The free amino acid and polypeptides content increased steadily until
it reached a maximum after 45 days and maintained that level. When
fermentation was complete, approximately 84 % of the total nitrogen was
organic and of this 63 % titrated in the presence of formol. As the free
amino acid content was 49%, then the polypeptide was 14% of the total
organic nitrogen and ammonia was 17%. The decomposition of the fish
flesh was complete after 4-5 months, although the ammonia concentration
continued to increase. Beddows et al. (1979) reported similar results with
Budu, except that the amino acids continued to increase over the full period
of fermentation and 78% of insoluble nitrogen was converted to soluble
nitrogen. A comparison of the nitrogen distribution in various sauces is
given in Table 13.2.
420 MICROBIOLOGY OF FERMENTED FOODS

Table 13.2 Comparison of the distribution of nitrogenous compounds in various fish sauces

Sauce Total-N Amino-N Volatile-N Formol-N Polypeptide-N

(Reference) (Organic; g%) (g%) (g%) (g%) (g%)

Budu (1) 1.77 1.17 0.12 1.85 0.48

Nam-pla (2) 1.96 ND 0.21 ND ND
Nuoc-mam (3) 1.90 1.05 0.40 1.45 0.45
" (4) 2.00 0.94 0.48 1.41 0.47
" (5) ND 0.2-1.0 0.2-0.7 ND ND
" (6) 3.20 ND 0.25 1.32 ND
" (10) 1.24 0.57 0.38 ND 0.38
Patis (4) 2.02 0.99 0.18 1.18 0.84
" (7) 1.55 ND 0.15 ND ND
" (8) 0.14-1.90 0.70-1.43 ND ND ND
Shottsuru (9) 0.30-1.56 ND 0.03-0.17 ND ND

ND = not determined.
(1) Beddows et at. (1979a); (2) Saisithi et ai. (1966); (3) Rose (1921); (4) Uyenco et at. (1953);
(5) Truong-van-Chom (1957); (6) Nguyen-Nhu-Nghi (1960); (7) Fujii et at. (1980); (8) Rosario
& Basaran (1984); (9) Fujii et at. (1992); (10) Nguyen-Thi-Lau & Richard (1959).

The enzymes derived from the fish are responsible for the proteolysis.
Mesnard & Rose (1920) used aseptic preparations and showed that the
hydrolysis of the fish proteins in nuoc-mam occurred under sterile condi-
tions. This was confirmed by Beddows et at. (1979b) who used broad-
spectrum antibiotics during fermentation; no microbial growth occurred
and the rate of formation of nitrogenous products was not altered com-
pared with that of a control fermentation. The maximum volume they
obtained was 760cm3/kg of fish after 140 days.
Further evidence for the fish enzymes being responsible for digesting the
protein comes from the work on methods to speed up proteolysis. In gen-
eral, they may involve lowering the pH to rely on acid proteases and to
prevent or alter the microftora. (Beddows et at., 1979b; Gildberg et at., 1984;
Bae et at., 1990). Beddows et at. (1979) suggested that in a normal fermen-
tation of budu, the changes in nitrogen distribution, can be considered to
occur in three stages.
• The first phase (0-25 days) was an increase in volume of the supernatant
liquid and soluble nitrogen due to osmosis.
• In the second phase (80-120 days), the cellular tissue begins to break
down and cellular protein comes into contact with the enzymes; the
autolysed protein products are released. Virtually all the fish tissue
disappears after 120-140 days.
• In the third phase (140-200 days) the enzymes are liberated and attack
the partially solubilised proteins. This causes the change in the distribu-
tion of nitrogen compounds.
Many enzymes have been implicated in the proteolysis. It is important to
identify the detailed mechanisms since a range of products could be formed.
 FERMENTED FISH AND FISH PRODUCTS 421

Different amino acid mixtures have various flavours and also will provide
different substrates for any microbial action that may take place. Studies
have not yet advanced very much in this respect.
Some progress has been made on identifying the enzymes involved.
Orejana & Liston (1982) concluded that the main enzyme involved ap-
peared to be 'trypsin'-like; other proteases such as cathepsin B are signifi-
cant and increase the amount of soluble protein. The trypsin activity in a
fermented shrimp sauce was higher in the early months. The major changes
occurred during the first 2 months. Gradually, trypsin activity decreased,
possibly due to inhibition by the end-products. Noda et al. (1982) identified
a number of proteinases from sardines. One acid and one alkaline
proteinase were stable in strong salt solutions and were liberated in the later
stages of sauce manufacture. Murakami & Neda (1981) isolated the
enzymes from the digestive organs of sardines.
The identification of enzymes from the fermenting sauces is of particular
value in that they must still be active. Yamashita et al. (1991) identified
those still present in shottsuru mash and Heu et al. (1991) isolated two
alkaline a-chymotrypsin-like proteinases that were still active in the
fermentation of anchovy.
Other enzymes have been implicated in the breakdown of polypeptides
to amino acids. The aminopeptidase activity of a fish sauce from sardines
has been shown to be quite high during the early period of fermentation
(Vo et al. 1983,1984). Again, such enzymes could playa significant role in
flavour development. However, some microorganisms survive in sauces and
may contribute to the proteolytic breakdown. A very hydrophilic strain of
Archaeobacterium has been isolated from nam-pla by Thongthai et al.
(1992) and is reported to produce a salt-stable extracellular proteinase. This
may not be the main agent of proteolysis, but it could contribute to the
development of particular peptides and amino acids involved in flavour and
should be included in such studies.

13.2.2 Aroma and flavour

Aroma and flavour are the most important factors in consumer acceptabil-
ity of traditionally produced sauces. Colour can be significant and can be
influenced by the addition of caramel.
(a) Aroma. The nature of the compounds that contribute to the aroma
have been investigated whereas the compounds involved in flavour are far
less well-characterized. Obviously, many aroma compounds contribute to
flavour perception. Dougan & Howard (1975) classified the aroma of
nam-pla into three major contributing notes:

1. The 'ammoniacal' note due mainly to ammonia and trimethylamine

(Rose, 1921; Truong-Van-Chom, 1957). Many investigators have shown
422 MICROBIOLOGY OF FERMENTED FOODS

the build-up of these compounds during fermentation in a range of

sauces. (Uyenco et al., 1953; Beddows et al., 1979).
2. A 'cheesy' aroma due to the low-molecular weight fatty acids (VFA)
which are present in a characteristic ratio with ethanoic and n-butanoic
acids predominating. VFA have been reported to be present in all sauces
(Abe & Tsuyaki, 1969; Beddows et al., 1980).
3. A 'meaty' aroma which as yet is not attributable to a single or group of
particular compounds: it appears to be due to a large number of volatiles.
When the fish sauce was extracted with ether, the meaty note was absent
until air was bubbled through, which would suggest that the volatiles
were formed in situ. However, when air was passed through a sauce
prepared asceptically and which did not have the 'meaty' aroma to start
with, no such note developed; presumably the required precursors were
not present (Dougan & Howard, 1975).
Saisithi et al. (1966) examined the rate of formation of VFA, volatile
bases and ketones. All increased during fermentation. They extracted the
sauce with an ether-alcohol mixture and separated the amino acids by ion
exchange. The amino group fraction did not contain any typical fish sauce
aroma. However, they eluted trimethylamine and a fraction which was
similar to that obtained from the Strecker degradation of phenylalanine,
which they claimed was fairly typical of the aroma of fish sauce. Attempts
to reproduce the aroma constituents by subjecting amino acids to the
Strecker degradation met with little success.
The development of the VFA in budu was investigated by Beddows et al.
(1980). They found that the levels of the individual acids were similar to
those for nam-pla except that most significantly, n-butanoic, propanoic and
iso-pentanoic acid concentrations did not increase from day 1. Only acetic
acid increased during budu fermentation, a situation they showed to be due
to bacterial action before the addition of salt.
Many other workers have identified other compounds involved in the
aroma (Table 13.3). Brooks & Reineccius (1976) extracted fermented fish
sauce with dichloromethane and analysed for neutral and basic volatile
compounds (see Table 13.3). Lactones predominated and represented over
75% of the basic and neutral volatiles.
Sanceda et al. (1986a) extended their earlier work on patis (Sanceda et al.,
1983,1984) and compared three different sauces. The steam distillates were
subjected to further separation and GC-MS analysis to identify many new
compounds, which form the basis of the data in Table 13.3. The contribu-
tion of these to the typical aroma has not yet been clarified. Another useful
appropriate approach was that of Tanaka & Shoji (1994) who analysed the
components of the head space of nuoc-mam; these are listed in Table 13.4.
Microorganisms are involved in aroma development, as the 'sauce' pro-
duced in the presence of antibiotics did not have the characteristic aroma of
Table 13.3 Volatiles of fish sauces

Nam-pla Nuoc-mam Shottsuru Budu Patis References

Acids
Ethanoic (acetic) + + + + + 1-6,8,10
Propanoic + + + + + 1,3-8,10
Isobutanoic + + + + + 1,2-6,8,10
n-Butanoic" + + + + + 1--8,10
Iso-pentanoic + + + + + 1,3--8,10
2 Methyl butanoic + 6,10
3 Methyl butanoic + 10
2-Ethyl-butanoic + 10
n-Pentanoic + + + + + 1,3-6,8,10
4-Methyl valeric + 8
n-hexanoic + + + + 1,4,6,8
Iso-hexanoic + + + + + 1,6,8
n-Heptanoic + 1,6,8
Iso-heptanoic (+) + 1,8
Phenylacetic + 5,6,8
3-Phenylpropionic + + (+) 6,8
Benzoic (+) (+) 1,6,8
Laevulinic + 6
Alcohols
Methanol + + 5
Ethanol + + + 1,5,6,8,10
Ethylene glycol + + + 8
Propan-1-o1 + + + 5,10
Propan-2-ol + + + 5,10
Propan-1,2-diol + 6
2-Methyl propan-1-o1 + + + 5,6
Glycerol + 6
Butan-1-ol" + + 5,6,8,10
Butenol + 10
Butan-2,3-diol + 6
Pentanol" + + + 5,6
2-Methyl butan-1-ol' + + 5,10
3-Methyl butan-1-ol' + + 6,8
3-Methyl-3-butan-2-o1 + 10
Hexanol' + (+) 8
Heptanol" (+) 1,8
2-Furan-methanol" + 6
Benzyl alcohol' (+) 1,8
2-Phenyl ethanol" (+) (+) 1,8
4-Methyl cyclo-hexanol + 6
Lactones
y-Butanolactone + 6
4-Hydroxypentanoic + + 6,9
acid y-lactone
y-Caprolactone (+) (+) 6,8
Aldehydesiketones
Formal + + 5
Ethanal + + 5,6
Propional + + 5
Propionone + + 5,10
Propan-dial + + 5
2-Butenal' + + 10
2-Methyl-butanal" + + 5,10
2-Methyl propanal + 10
Butan-2-one + + 5,10
n-Pentan-2-onea + 10
2-Ethyl-butan-2-one + 5
Iso-Butan-2-one + 10
Table 13.3 Continued

Nam-pla Nuoc-mam Shottsuru Budu Patis References

Benzaldehyde" + + 5,6,8
Cyclopentanone (+) (+) (+) 1,8
3-Hydroxy-butan-2-onea + 6
3-Hydroxy-3-methyl- (+) (+) (+) 8
butan-2-one
5-Methyl furan-2-one + 6
Piperidinone + 6
2-Pyrrolidinone + 5,6
N-containing compounds
Ammonia + + + + + 1,3,4,6,9
Trimethylamine + + + + + 1,3,4,6,9
Dimethylamine + + + + + 1,3,4,6,9
Methylamine + + + + 1,4,5,9
Ethylamine + 5
Propylamine + 5
2-Aminopropane + + 5
n-Butylamine + 5
1-Aminopentane + + 5
Diethylamine + 5
Dipropylamine + 5
2 Methyl pyrazine" + + 5,6,9
2,5 Dimethyl pyrazine" (+) + 1,5--8
2,6 Dimethyl pyrazine" (+) (+) 1,8
2,3 Dimethyl pyrazine" (+) (+) 8
2,3,5 Trimethyl Pyrazine" + 5
Indole + 6
3-Methyl indole + 6
2-Ethyl imidazole + + 6
Phenols
Phenol" (+) (+) 1,8,9
4-Ethyl phenol (+) 8
Esters
n-Butyl formate (+) (+) 8
Diethyl oxalate (+) (+) 8
Ethyl acetate" (+) (+) (+) 1,8
n-Propyl acetate + + 5
Ethyl propanoate (+) (+) (+) 8
Ethyl n-butanoate" (+) (+) (+) 8
Ethyl lactate (+) (+) (+) 8
Dibutyl pthalate (+) 8
Hydrocarbons
Decane" (+) (+) (+) 8
Dodecane (+) (+) (+) 8
cis-3-pentene (+) (+) (+) 8
S-containing
Methyl mercaptan + + 5,6
Dimethyl sulphide" + 5
3-Methyl thio-propan-1-ol + 6
2-( 4-Methyl-5- + 6
thiazoJidyl) ethanol

A number of compounds have been tentatively identified by Sanceda et al. (1986) but have not
been included.
"See analysis of fish paste (Cha & Cadwallader 1995), page 431.
References: 1: Sanceda et al. (1984); 2: Truong Van Chom (1957); 3: Dougan & Howard (1975);
4: Beddows et al. (1979); 5: Nonaka et al. (1975); 6: McIver et al. (1982); 7: Ide et al. (1982);
8: Sanceda et al. (1986); 9: Brooks & Reineccius (1976); 10: Tanaka & Shoji (1994).
After Sanceda et al. (1986a).
 FERMENTED FISH AND FISH PRODUCTS 425

Table 13.4 Headspace analysis of nuoc-mam

Component ppb Component ppb

Ethanol 46.0 Butenol 0.5

Acetone 39.0 2-Pentanone 1.2
2-Propanol 2.9 Propanoic acid 4.1
2 Methyl propanal 12.0 2-Methyl propanoic acid 8.3
I-Propanol 1.5 Butanoic acid 9.7
2-Butanone 4.7 3 Methyl butanoic acid 13.0
Ethanoic (acetic) acid 3.6 2 Methyl butanoic acid 8.1
3 Methyl-3 buten-1-o1 tr n-Pentanoic acid 6.0
2-Methyl butenal 8.5 2-Ethyl-butanoic acid 4.9
Butanone 9.7

ppb, parts per billion.

From Tanaka & Shoji (1994).

budu and the levels of the volatile fatty acids (VFA) were very low
(Beddows et al., 1979) and aseptically produced fish sauces did not give the
typical aroma (Uyenco et al., 1953). A number of investigations have been
made to identify the specific microorganisms responsible.
Saisithi et al. (1966) isolated species of Bacillus, Streptococcus,
Micrococcus, Staphylococcus and Coryneform bacteria from nam-pla and
which may have derived from the solar salt used since it contained an
average of 27300 bacteriaig: this confirmed an earlier report (Bain et al.,
1958). All the bacteria were halophilic with an optimum salt concentration
of about 20%. The pH optima was 6.5-7.5. The bacteria grew better in
enclosed containers and many of the species isolated had the ability to
produce VFA from nutrient media although the profile of the acids was not
determined. They suggested that bacterial action could produce the VFA
present in nam-pla after 9 months. The total viable count decreased to 2 x
103 cells/cm3 over this period.
Further work on nam-pla and other sauces suggested that other bacteria
may be involved in aroma production (Van Veen, 1953; Amano,
1962; Visco & Fratoni, 1963, Crisan & Sands, 1975). Sanchez &
Klitsaneephaiboon (1983) identified a succession of microorganisms that
they believed could be involved. Bacillus pumilus, B. coagulans and B.
subtilis predominated initially, but were then overtaken by B.lichenformis,
Micrococcus colpogenes and Staphylococcus epidermis in the middle stages.
In the final period of patis production, M. roseus, M. varians and S.
saprophyticus predominated. However, the levels may not be sufficient to
produce significant biochemical changes.
This in part confirms the work of Crisan & Sands (1975) who examined
the microflora of four fermented fish sauces. They isolated B. cereus and a
strain of B. lichenformis after 7 months of nam-pla fermentation but ulti-
mately, they isolated another strain of B. lichenformis. This suggests that
426 MICROBIOLOGY OF FERMENTED FOODS

the microfiora is not constant. Similar results were obtained with patis.
After 1 month, no organisms grew on 20% salt, but single strains of B.
pumila, M. copogenes, M. varians and Candida clausenii were isolated from
a 10% salt medium. No indication was given as to their concentration in the
sauce.
Itoh et al. (1985a,b) isolated a number of lactic acid bacteria from
nam-pla and shottsuru, including Pediococcus halophilus, Staphylococcus
saphrophyticus and Micrococcus varians. These were not investigated
for their ability to produce a typical aroma. Fujii et al. (1980, 1984a,b,c,d)
and, Nakayama et al. (1990) as part of their studies on shottsuru,
reinoculated sterilized fish and identified the causative spoilage
microorganisms.
This may be important, as indicated by the work of Beddows et al. (1980),
who looked at the origin and development of the volatile fatty acids in
Budu. They showed that VFA did not appear to arise from the autoxidation
of fatty acids but from the action of a bacterial species that increased
markedly in concentration prior to salting and continued to work in a
halophilic environment. Microbiological examination of the spoiled mate-
rial showed only one strain of bacteria to be present; this could not be
identified. The bacterium produced VFAs on unheated Stolephorus spp.
only, although it grew well on other fish and protein-rich media. It is not a
Clostridium spp., which is able to produce n-butanoic acid.
VFAs were produced rapidly when a radiolabelled amino acid mixture
was incubated with the fish and the bacterium for 24 hours at 30°C. Al-
though ethanoic and n-butanoic acids were the major products, most of the
radioactivity appeared in the isobutanoic, n-butanoic and iso-pentanoic
acids. Using labelled acetic acid, they showed that the n-butanoic acid was
not formed from acetic acid (Barker 1958), and also they eliminated the
route from a 'Strickland' type of reaction from glutamate. The mechanism
is not known and a new metabolic route may be involved.
Sanceda et al. (1986b) found that bacterial action was required to produce
the volatile fatty acids of Patis, and the same workers looked at the deriva-
tion of VFA (Sanceda et al., 1990b) and also established that the mechanism
needed to be anaerobic (Sanceda et al., 1992). Very few studies have been
carried out on the fats present, but Kim et al. (1994) examined the changes
that occurred when anchovy sauce was prepared at different temperatures.
This type of study could be of increasing importance with the greater
nutritional emphasis being placed on the polyunsaturated fatty acids
present. These workers found that the C22:6 (n-3) did not appear to dete-
riorate at the temperatures used.
Because of the complex nature of the aroma, no simple involvement of
microorganisms can be ascribed. Although the ammonical note does not
involve bacteria, as both ammonia and TMA are formed under aseptic
conditions, such action could increase the amount formed.
 FERMENTED FISH AND FISH PRODUCTS 427

(b) Flavour. Some work has been reported on the flavour of fish sauces.
The over-riding taste is salty with a contribution from the volatile com-
pounds, but other constituents must contribute. Amano (1962) and
Kelaiditis (1949b) suggested that the flavour is affected by the combination
of the amino acids. Since amino acids and many di- and polypeptides are
known to have particular flavours, these observations are not surprising.
With, Nam-pla, tyrosine has been shown to appear as crystals (Thongthians,
1990).
Proteolytic enzymes vary in their specificity in relation to their point of
attack and as such will have a profound impact on flavour, since this will
determine the relative concentrations of individual amino acids and
peptides. Thus, all the factors which can influence the relative activities of
these enzymes could affect the taste. Consequently, the enzymes present in
the material used, the pH of the pickle, the temperature and the salt
concentration, for example, could influence the taste.
Raksakulthai & Haard (1992a) attempted to relate the concentration of
peptides and amino acids. Other workers have studied various aspects of
sensory evaluation (Sanceda et al., 1990a, 1994; Minoza-Gatchalia et al.,
1994). Sanceda et at. (1995) evaluated the development, acceptability and
value of a histidine-enriched sauce.
No systematic study has been undertaken to identify the role of
nucleotides/nucleosides nor the role of nucleases in fish sauces. However,
the levels of particular compounds derived from nucleic acid have been
reported. For example, Fujii et al. (1992) found that commercial shottsuru
contains 4.5-9.7mg/100ml of inosinic acid as well as glutamic acid,
histamine, tyramine and other related compounds.
Biogenic amines are of some concern. Histamine was identified in Nam-
pIa by earlier workers and later by Hsu & Chen (1987). Saisithi et at. (1966)
found tyramine and tryptamine to be present in addition to histamine.
Beddows et at. (1985) identified putrescine, cadaverine and histidine in both
budu and nam-pla. Although the levels were relatively low and may not
contribute to the aroma at the pH of 5.2-5.6, they could have a significant
physiological effect.
Nutritionally, Chayavan et at. (1983a,b) investigated the reduction or
replacement of sodium and found that partial replacement with potassium
chloride was acceptable organoleptically. Nitrosamines have been investi-
gated, but do not appear to present a major problem (Lee, 1982; Soo et al.,
1994).

13.2.3 Acceleration and extension offish sauce production

Fish sauce production is time consuming and is costly in the plant that is tied
up. Consequently, many attempts have been made to speed up the process.
The obvious route is to alter the salt concentration and the temperature.
428 MICROBIOLOGY OF FERMENTED FOODS

However, a balance must be reached. If too Iowa salt concentration is used,

the degree of osmosis is reduced and undesirable microflora flourish.
Similarly, too high a temperature can denature essential proteolytic en-
zymes and be expensive, as large tanks need to be warmed for an extensive
period.
Ngo-Ba-Thanh (1953) used finely ground salt and stirred the fish after the
initial tissue degradation. Initial pressures were kept low to prevent the fish
tissue from hardening too much and reduce some of the loss in enzymic
activity. In contrast, Hamm & Clague (1950) reduced the fermentation time
in patis and in bagoong to 22-24 days by using elevated temperatures
(45°C) and reduced salt concentration to reduce the loss of the enzymic
activity. A similar temperature was used by Kataoka et al. (1987), but they
found that the inclusion of bromelain to be of value.
Similarly, Embisan (1977) designed a fermentation unit for rapid patis
production using ground fish. This used a reduced amount of salt and the
mixture was stirred at 37°C. Sauces could be produced in 4-13 days. Simi-
larly, Mabesa et al. (1985) developed a pilot plant system that reduced the
fermentation time of Patis from 12 to 2 months. No data have been pro-
vided on the hydrolysis or on the acceptability of the product with either
development.
Digestion of insoluble protein can be completed within a few days by
using an anti-bacterial agent instead of salt (Amano, 1962) and the yield of
solubilized nitrogen was increased by 50%; however, the patis did not have
the typical aroma or flavour.
Other agents have been used; for example, Lee et al. (1989) produced a
Korean fish sauce more rapidly using a mixture of fish and koji, while a
mixture of koji and proteolytic enzymes have also been investigated by
Chae et al. (1989).
Hale (1969) found the plant enzyme, ficin to be better than microbial
enzymes for solubilizing fish protein. Sen et al. (1962) investigated papain
but found that fermentation still took 340-350 days. A rapid breakdown
was obtained by Kim et al. (1986) using ficin, bromelain or papain with
sardines. Trypsin and chymotrypsin have also been used; the hydrolysed
product was added to nam-pla and was as acceptable to a trained panel
(Ravipim et al., 1993). Higashi et al. (1965) used a protease from
Streptomyces spp. and Nakano et al. (1986) used another protease partially
purified from a Pseudomonas spp. and to reduce the fermentation period
from 2-3 years to 3-6 months. In a different approach, a continuous column
reactor method using immobilized yeast cells was developed by Seong et al.
(1993) to produce a fish sauce with l.72g% total nitrogen after 40 days.
However, these latter attempts did not include salt as bacterial action could
take place; hence, a preservative may need to be present.
Beddows & Ardeshir (1979a) extended their earlier work with bromelain
(Beddows et al., 1976) by using the traditional mix of minced fish: salt and
evaluating the naturally occurring plant proteases, which are probably more
 FERMENTED FISH AND FISH PRODUCTS 429

readily available in countries where fish sauces are traditionally made.

Bromelain gave better results than either papain or ficin in that greater
amounts of protein are solubilized using minced fish in 18-21 days at 33°C.
The nitrogen distribution was similar to nuoc-mam or nam-pla, but very
little aroma was present. These mixtures could be added to traditional fish
sauce to increase volume without loss of protein.
Ooshira et al. (1981) examined the use of papain, bromelain and trypsin
to produce a fish sauce from sardine, mackerel and ami, with 25% salt
added directly, or in three stages over 24 hours. Similarly, Miyazawa et al.
(1979) treated Engraulis spp. with 2% salt or 55% koji and 0.5% pronase
for 150 days at 30°C and obtained a good conversion of insoluble protein.
The use of acid ensiling has certain advantages for rapid production since
if hydrochloric acid is used, neutralization with sodium hydroxide gives salt.
Beddows & Ardeshir (1979b) showed that proteolysis occurred much more
rapidly, without microbial infection, under such conditions Inclusion of salt
in the mixture improved filtration and handling. A similar method was
employed by Gildberg et al. (1984) who examined the product using expe-
rienced panellists. Unfortunately, most of the sauces were inferior even
when added to patis, though this may be related to the distribution of
nitrogenous compounds produced. To investigate, this would require closer
investigation of the precise action of cell of the proteaser at lower PM and
compariser to neutral conditions.
The fish sauce manufacturing process has been extended into being used
for less valuable or less used species. Often, the processes have examined
the use of added enzymes or other means of increasing the rate protein
hydrolysis. One series deals with using male Capelin. Sauces were produced
quite rapidly by adding squid hepatopancreatic tissue which is rich in
cathepsin B (Raksakulthai & Haard 1986, 1992b). This enzyme was com-
pared with others such as pronase, trypsin and, chymotrypsin and a sauce as
acceptable as patis was produced (Raksakulthiai et aI., 1986). Visceral
enzymes of sardine have been used to produce a fish sauce with similar
acceptability to commercial sauces (Yoshinaka et al., 1983). Gildberg & Shi
(1994) reported on a purification method for recovering the tryptic enzymes
from fish sauce to give a product that can be re-used. Other workers have
prepared sauces from filefish scrap (Lee et al., 1988; Lee, 1990), trash fish
(Velayutham et al., 1987), deep-sea fish (Lekshmy et al., 1989), whitebait
and carangid (Hiremath et al., 1985).

13.3 Fermented fish pastes

Fish pastes are far more widely produced and eaten than fish sauces, usually
being consumed as a condiment. The large variety of pastes obtainable are
very amenable to blending with carbohydrates such as rice, flours, bran,
roasted cereals and spices.
430 MICROBIOLOGY OF FERMENTED FOODS

Not all pastes are fermented, even in relation to degradation by the fish
enzymes. However, traditionally fermented pastes will normally have a
shorter period of fermentation than sauces. Large fish and also other fish
unsuitable for sauce production may be used.
Bagoong is an example that is produced in the Philippines from whole or
ground Stolephorus spp., Sardinella spp., Decapterus spp., fish roe, small
shrimp (Atya spp. or its roe), with 25% salt (Hamm & Clague, 1950). The
fish are cleaned and mixed with ground salt in a ratio of 3 : 1 and placed in
vats to ferment. As with patis, the tissue breakdown is due to proteolytic
enzymes of the fish (Uyenco et al., 1953). Excess liquid may be drained off
and this constitutes patis.
When the fish has attained a pasty consistency, it may be aged and mixed
with other materials. Its keeping qualities may be limited as some of the salt
may be lost through draining. The paste contains 35% solids, 13% protein
and 20-25% salt. Fujii et al. (1980) reported that the pH is 5.1 and the paste
contains 66.2% water, 1.55% total nitrogen, 0.151 % volatile-N, 0.015%
TMA, 0.013% HzS and 4.5 x 103 viable cell count per gram.
Sometimes bagoong is coloured by adding angkak - a rice product fer-
mented with Monascus purpurea (see Chapter 12). Bagoong can be blended
with other spices, flavouring and approved colouring materials. Fujii et al.
(1980) isolated 40 strains of which the major species were Bacillus spp.,
Micrococcus spp. and Moraxella spp. However, in another study, Fujii
(1980) reported that only two species of the bacterium, Halobacterium patis
will grow in 20% salt and not in 10% salt.
Tinabal and balbakwa are other pastes produced in the Philippines from
larger fish such as Stolephorus indicans. Greater bacterial action is allowed
to take place and a stronger flavour is produced (Van Veen, 1965).
The chemical composition of the paste Ngapi, produced from sun-dried
Puntius sophore has been reported by Sarojnalani & Vishwanath (1994).
The paste has lower moisture at 18% and contains 45% protein, 18.5% lipid
and 11 % ash. A nutritional evaluation was carried out. Choorit et al. (1991)
showed that a delay of 1 day before salting and a fish: salt ratio of 4: 1 was
beneficial for good-quality product. This suggests that bacterial action is
important in flavour development of such products, as shown by Beddows
et al. (1980) for budu.
Very little data are available on the precise changes brought about by
microorganisms in fish paste manufacture, but they probably assist more in
the breakdown of tissue than with sauces and are involved in the develop-
ment of aroma and flavour. For example, with Padec, produced in Laos,
sliced raw fish is piled onto hanging baskets, with some pressure applied for
a 3-day period before salting. Westenberg (1951) refers to the juice which
runs off as being malodorous, and this must be the result of bacterial action.
After mixing with salt, the juice is allowed to run off again. This reduces the
salt concentration of the mass, making it more aerobic. More salt and rice
 FERMENTED FISH AND FISH PRODUCTS 431

is added after a further 3 days and allowed to ferment. The resulting paste
contains only about 1% salt; this low salt level encourages padec to be used
as a staple in the diet in combination with rice.
The chemical and bacterial composition of Trassi have been investigated
by Surono and Rosono (1994), who found fairly high levels bacteria (lOs/g)
and identified a number of species. As might be expected, some interest has
been taken into the hygienic quality of a number of such pastes and fer-
mented products. Eisgruber et al. (1995) consider that a risk exists with
fermented fish because of the presence of Clostridium spp.
The paste Rentak, is a little unusual in that the preparation does not use
salt but requires the use of a plant, Alocasia macrorhiza which is ground
with sun-dried Esomus danricus and left for very long periods. Vishwanath
& Sarojnalini (1989) report that the plant on its own is dangerous due to the
high levels of oxalate, but that fermentation reduces this considerably.
In Japan, a wide range of fermented products are available. A product
somewhere between a sauce and a paste is sbiokara which is described as a
dark brown liquid, generally containing lumps of solid tissue, although it
depends on the fish used. Slices of fish are stirred well with the viscera and
salt (4-10 fish: 1 salt) in a barrel for several days, after which it is sealed and
allowed to ferment for up to a year. In many cases, carbohydrates such as
malted rice are added. For squid, the salt content must be more than 20%
(Subba Rao, 1967). An analysis has been carried out by Mizutani et al.
(1992); this shows that the composition is similar to Bagoong. Shiokara
from squid was reported to contain 74.2% water, 7.8% salt, 11.6% protein
and 8.68% ash and from skipjack entrails 64.2% water, 23.9% salt, 8.2%
protein and 26.2% ash (Amano, 1962); the ash includes the sodium
chloride.
The proteolytic enzymes of the fish are involved; the visceral enzymes
from the stomach, pancreas and intestine are more active than the muscle
enzymes and amino acids are produced (Fuji, 1961). Zenitani (1955) identi-
fied 18 strains of bacteria from different shiokara and found that a
Micrococcus spp. was 'responsible' for the ripening, whereas halophilic
Vibrio spp. and Achromobacter spp. were responsible for spoilage.
Ooshira & Koreeda (1960) and Teshima et al. (1967) investigated
the volatile compounds and identified formic, ethanoic, propanoic, iso-
butanoic and n-pentanoic acids, as well as ammonia, TMA, mono-and di
-methylamines. They also identified amino-butane and 2-methyl propyla-
mine which they suggested came from microbial action.
The volatile components in shrimp and fish pastes have been identified by
GC-MS of the steam distillates (Cha & Cadwallader, 1995), some 155
compounds being identified. Some are similar to those found in fish sauces
and are included in Table 13.3. The range of compounds present might
suggest that many could contribute to the aroma. If so, then it would be very
difficult to find the correct balance of components.
432 MICROBIOLOGY OF FERMENTED FOODS

Comparison with fish sauces is inevitable and these authors do not report
the presence of many of the volatiles associated with sauces. Such volatiles
include the VFA and the TMA, DMA, methylamine and ammonia. These
might be expected to be present as the analysis was on the materials
obtained from a steam distillation-solvent extraction. However, the
samples had been purchased in the local market and no indication was
given about the method of manufacture. If the pastes had been prepared as
for Padec, where the main sauce is allowed to drain away, this would affect
the water-soluble acids as well as other metabolites. The pastes are worthy
of closer attention since, apart from the complex interactions taking place,
some have little fermented flavour (Adnan & Owens, 1984). These could
present a means of increasing the protein content of the diet of poorer
populations.
Anchovy pastes are consumed in a number of countries. These have not
been investigated fully with respect to microbial activity. However, Cha
(1994) and Cha & Lee (1990) used koji to speed up the fermentation in low
salt conditions. They report enhanced breakdown and an increase in
proteolytic bacteria. Cha (1994) followed the changes in the volatile com-
pounds of this mixture in which some 79 compounds were identified.
Another method of producing a paste is to add miso. Shimomura &
Matsomuto, (1987) showed that the breakdown appeared to be due to the
proteolytic enzymes of the miso. However, they did not appear to have used
an untreated fish control and some of the activity noted could be due to the
fish enzymes.

13.4 Salted fish

Not all salted fish involve a fermentation stage. However, a number of

salted fish products are prepared in such a way that microbial and biochemi-
cal action can occur. The degree of proteolysis required is much less than
that for paste production.
Westenberg (1951) described pedah siam or pedah-kemburg. The higher
quality is a fatty partly-dried fish that has a characteristic brown colour.
Whole Rastrelliger spp., Scomber neglectus and Scomber kangurtal which
have 15-16% fat have the entrails removed. Salt is added (3:1, fish: salt)
and stored for 24 hours. Some osmotic dehydration occurs which removes
some of the water. The residual salt often contains fish oil in addition to
other low-molecular mass compounds. The fish is packed carefully in crates
in which the ripening takes place and is virtually anaerobic. The pickle
drains away, aiding dehydration.
A degree of breakdown of protein occurs in that the highest quality
merah basah does have a somewhat pasty consistency and a fresh red
colour. The volatiles are low in concentration. The initial fishy smell is lost
 FERMENTED FISH AND FISH PRODUCTS 433

on ripening. Whole fish are produced, that contain 21-22% protein, 15-
17% salt and 7-15% fat.
In the production of pedah, the aim is to retain as much fish as possible
with the correct texture. To aid in this, many enzymes are removed with
the viscera and also the pickle is taken off as it is formed, thus reducing the
salt concentration.
Other products, similar to pedah siam are prepared elsewhere. For exam-
ple, fish are 'pit-cured' or 'Kench-cured' in India, although less dehydration
occurs. However, large amounts of free amino acids are produced and may
be lost in the pickle (Krishnapillai et al., 1956; MacKie et aI., 1970).
In the south of India, certain fish are 'pickled' or 'colombo-cured'.
Usually fish and salt (3: 1) are mixed in a concrete tank and dried tamarind
fruit is added, which is believed to lower the pH to 6.0. Improvements in the
quality are obtained if 5% acetic acid is added (Vasavan & Varma, 1959).
Sushi prepared in Japan from gutted Crucian carp, sea bream and
sandfish are cured with 20-30% (w/w) salt. The fish is mixed with a ferment-
ing cereal such as rice. Lactic acid bacteria thrive and decrease the pH over
a period of 1-2 months. The fish is then desalted and the liquid drained off.
Boiled rice and koji are then mixed in and a secondary fermentation occurs
giving a particular flavour to the product, as the yeasts in the koji become
active. In this particular case, the lactic acid has aided preservation, but
proteolysis occurs.
Sometimes fish is used with vegetables. For example, in Korea, kimchi is
prepared from radish root, chinese cabbage and either fermented shrimp or
anchovy. The microflora have been identified by Crisan & Sands (1975),
although the role of the 54 different strains were not established.
Salted fish products are not restricted to tropical areas. In Scandinavia,
sustr!1lmming is made from whole herring (Schmidt-Nielson & Bohmer,
1937) and rakorret is made from trout (Schmidt-Nielson & Bohmer, 1935).
The production of anchovies in various countries usually involves some
fermentation. In France, beheaded Engraulis encrasicholus are layered with
salt, and weighed down to extract the pickle. The fish are matured at 15-
17°C for 6-7 months, after which the salt is washed off and the fish kept in
oil overnight before eating.
In Scandinavia, whole sprats are packed with salt, sugar, spices, and
sometimes with preservatives such as sodium nitrite or sodium benzoate.
After 48 hours, all the sugar and salt is dissolved. Maturation is at 12-15°C.
(Christianson, 1962). The fish are filleted and packed with a sauce contain-
ing salt, sugar and spice before they are pasteurized. If whole herring is
used, the product is known as tidbits.
The salting of herring can involve some fermentation where the practice
is to gut the fish, although some viscera is always retained. Luipjen (1959)
reported that the pyloric caeca, milt or roe are required for true flavour
development. The unwashed fish are mixed with salt and placed in layers in
434 MICROBIOLOGY OF FERMENTED FOODS

vats. In some cases the pickle is 'drawn off' after 1-2 weeks and more fish
and salt are added before the vats are topped up with pickle and sealed to
reduce fat oxidation. The rate of ripening depends on the quality and type
of fish, salt concentration and temperature (Voskresensky, 1965).
Bain et al. (1958) examined the microbiology of pickled herring and
found halophilic and halotolerant micrococci, yeast and spore-forming
bacteria, but the load was low. It is debatable as to whether the flavour is
due to fish enzymes or microbial activity (Omland, 1955). Voskorensky
(1965) suggested that early autolysis was important and that microbial
intervention was significant at later stages.

13.5 Conclusions

The manufacture of traditional fermented fish products depends a great

deal upon the action of the enzymes contained within the fish for the
essential changes in texture. They can also contribute to the flavour al-
though research in this area is very limited. Microorganisms playa signifi-
cant role in aroma and flavour development, but their precise action needs
further clarification. More investigation is required before attempts are
made to influence the rate of production.
As for future developments, it is of interest to learn from these traditional
processes in order to utilize fish or fish products more fully. However, the
traditional product does not have to be copied for consumer acceptance,
since the supply of such products is often adequate for those areas that
have long been using them. It is of greater importance to make full use
of all potential fish resources more fully. Fish 'waste' is ever present and
methods to utilize this are always in demand. Many of the present-day
proposals are expensive and often result in products with low consumer
acceptability.

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