Food and Nutrition Sciences, 2013, 4, 1229-1238
Published Online December 2013 (http://www.scirp.org/journal/fns)
http://dx.doi.org/10.4236/fns.2013.412157
Open Access FNS
Development of Model Fermented Fish Sausage from
Marine Species: A Pilot Physicochemical Study
Sarim Khem, Owen A. Young*, John D. Robertson, John D. Brooks
School of Applied Sciences, AUT University, Auckland, New Zealand.
Received September 4th, 2013; revised October 4th, 2013; accepted October 11th, 2013
Copyright © 2013 Sarim Khem et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accor-
dance of the Creative Commons Attribution License all Copyrights © 2013 are reserved for SCIRP and the owner of the intellectual
property Sarim Khem et al. All Copyright © 2013 are guarded by law and by SCIRP as a guardian.
ABSTRACT
Marine fish, hoki (Macruronus novaezealandiae), kahawai (Arripis trutta) and trevally (Pseudocaran x dentex) were
used to develop fermented fish models to emulate Asian examples. The formulations comprised ground fish, carbohy-
drate, garlic and salt, but no added culture. The carbohydrate was cooked rice or glucose. The mixtures were extruded
into open-ended 50 mL plastic syringes, sealed and incubated at 30˚C for 96 h. The syringe piston was progressively
advanced to yield test samples. The endogenous lactic acid bacteria were capable of fermenting glucose, but not cooked
rice. After glucose fermentation, the treatments contained around 8.7 log cfu·g−1 (from 3.3) with the pH range of 4.38 to
5.08 (from around 6.3), depending on the species. Hardness, springiness and cohesiveness of treatments all increased
with fermentation, except for hoki, which was subject to an endogenous proteolytic activity. Color development varied
with species: light reflectance (L*) of the trevally and kahawai treatments increased, while the a* (redness) and b*
(yellowness) values were static. Hoki exhibited the least color changes except for yellowness, which increased mark-
edly. Possible reasons for this are discussed. Proteolysis was greatest for trevally. Lipid oxidation was least for hoki,
notably the species with the lowest fat content. The trevally treatment generated the highest concentration of amines,
but values were lower than reported for fermented fish sausage in Asia, possibly because of raw material hygiene status.
The physiochemical outcomes indicated that trevally (as pieces unsuited for sale as fillets) would be best suited to this
food product class. Suitable species could be identified in other fisheries.
Keywords: Marine Fish; Lactic Fermentation; Texture; Sausage; Biogenic Amines
1. Introduction
Lactic acid fermentations are often used to preserve
foods, and at the same time to provide the consumer with
a wide variety of flavors, aromas and textures. Ferment-
able sugars are converted to lactic acid by a wide range
of lactic acid bacteria (LAB), often in the presence of
added salt [1]. These fermented foods are usually safe to
eat, which can be traced to antimicrobial mechanisms of
LAB, including bacteriocins, and the direct antimicrobial
effects of organic acids [2]. Preservation can be further
enhanced by partial drying to lower water activity and, in
the case of fermented meat sausage common in Asia, by
the addition of humectants like sucrose.
There is a huge range of food based on this principle
throughout the world. Among the product categories of
lactic fermentations are raw mammalian meats in the
West and raw fish in Southeast Asia. Fish meat is very
susceptible to spoilage by microorganisms, and fermen-
tation is a common way of preventing deterioration in the
hot climates of Southeast Asia where preservation by
refrigeration is often not available [3].
In its Thai expression, fermented ground fish (FGF)
comprises raw fish ground with cold steamed rice,
ground garlic and salt. The mixture is tightly packed in
banana leaves or plastic bags to exclude air and left to
ferment by endogenous LAB for two to five days at
around 30˚C [4,5]. The temperature is dictated by the
climate, because FGF is mainly a domestic activity. The
active LAB are those occurring naturally on the fish and
other ingredients, and on hands and equipment. Lactoba-
cillus sp. and Pediococcus sp. were identified as the
dominant LAB in commercial FGF [6]. FGF emerges as
*Corresponding author.

Development of Model Fermented Fish Sausage from Marine Species: A Pilot Physicochemical Study 1230
a sliceable gel that is eaten as is, rather than being semi-
dried as is common with fermented meat products. In
common with fermented mammalian meats, FGF is
slightly sour and salty and has a firm and springy texture.
Also in common with fermented meats, proteolysis and
fat oxidation occur during and after fermentation, and
contribute to flavor [7,8]. The glucose and maltose in
steamed rice and inulin in garlic act as the carbohydrate
substrates for fermentation [9]. In addition to the sub-
strate role, garlic is believed to act as an antimicrobial
agent particularly against Gram-negative bacteria. This is
believed to be due to garlic’s allicin content, and garlic
reportedly promotes the growth of lactic acid bacteria
[10-12].
In Southeast Asia the main fish species used for FGF
are freshwater. In Thailand for example, a range of fresh
water species such as Ophicephalus micropeltes, Notop-
terus spp., Probarbus jullieni, and Puntius gonionotus
are used to prepare FGF [13]. In a study to investigate
the changes during fermentation of FGF made from sev-
eral marine species, Riebroy et al. [4] reported that FGF
from a snapper, Priacanthus tayenus, showed compara-
ble acceptability to the commercial FGF from freshwater
species. Although there is a Scandinavian tradition of
fermenting whole fish flesh with salt, fermented fish
products are uncommon in Western cultures [14]. On the
face of it there is no technical reason why products simi-
lar to Southeast Asian FGF could not be produced and
sold in Western markets.
Remote Pacific countries like New Zealand have huge
exclusive economic zones for commercial fishing, but
are far from their food export markets. Thus, storage sta-
bility and its associated cost is a challenge for fish pro-
ducers. Most fish caught in these zones are marketed in
relatively unprocessed forms, typically chilled around
0˚C, frozen or canned. FGF would represent another
product option, and one that would require a less de-
manding refrigeration regime to be acceptable in distant
markets.
In New Zealand, most commercial fish are marine
species and three have been used here for FGF trials. The
species used were the low-fat pink-fleshed trevally
(Pseudocaranx dentex), the high-fat red-fleshed kahawai
(Arripis trutta), and the low-fat white-fleshed hoki (Ma-
cruronus novaezelandiae) (Table 1).
These species represent a wide range of fish skeletal
muscle types, but it is emphasized that the experiments
have no statistical base in terms of fish biology. In ex-
ploratory research of this type it is better to explore
widely (three species bought at retail as required) than to
focus on one species with a defined biological status. The
outcomes measured were texture, color, pH, microbial
counts, soluble protein, fat oxidation, and biogenic
amines; these outcomes do have a statistical base. The
Table 1. Approximate chemical composition of trevally,
kahawai and hoki.
Fish species Protein
(g·100 g−1)
Fat
(g·100 g−1)
Iron
(mg·100 g−1)
Trevally 120.9 2.8 0.9
Kahawai 21.2 8.6 2.1
Hoki 17.0 1.0 0.2
1Data are derived from [15-17].
work also included experiments on the role of added folic
acid that purportedly accelerates lactic fermentation [18].
2. Materials and Methods
2.1. Chemicals
Man-Rogosa-Sharpe (MRS) medium was obtained from
BD Difco (France). Malonaldehyde was obtained from
Fluka (Buchs, Switzerland). Dansyl chloride, histamine
dihydrochloride, tryptamine hydrochoride, tyramine as
free base, and 1,7-diaminoheptane were purchased from
Sigma (St. Louis, MO, USA). Folin-Ciocalteau reagent
was obtained from Scharlau Chemie (Barcelona, Spain).
Bovine serum albumin was obtained from Serva Fein-
biochemica, Germany. Food grade folic acid was donated
by Sanitarium, New Zealand. Other chemicals were ana-
lytical grades sourced from a range of suppliers.
2.2. Sausage Casings and Novel Equipment
Sausage casing prepared from collagen were donated by
Globus, Sydney, Australia. The novel casing equipment
were 50 mL (nominal) plastic syringes (300865, BD
Drogheda, Ireland), 25 mm in diameter. The needle end
was excised on a lathe (Figure 1). The interior surface of
the syringe barrel was then lightly lubricated with petro-
leum jelly, and the piston minimally reinserted to the 60
mL mark. The barrel was completely filled with the FGF,
unfermented at that point, then tightly sealed to exclude
air with Parafilm® overlaid with aluminium foil. A rub-
ber band secured the cover. The idea was that samples of
FGF could be extruded and excised over time, in each
case followed by resealing.
2.3. FGF Preparation
Unfrozen fillets of the three species were bought as re-
quired from retail outlets and held on ice. Processing
equipment was thoroughly cleaned to a domestic stan-
dard but with no attempt at sterilization. Using chilled
equipment at ambient temperature, the fillets were
ground through a 4-mm plate and blended with other
ingredients (Kenwood KM210, Kenwood, Havant, UK).
These were the carbohydrate source, ground peeled raw
garlic cloves (4-mm plate), and salt. Mass ratios were 1
(fish):0.15:0.05:0.03, respectively [13]. Folic acid was
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Development of Model Fermented Fish Sausage from Marine Species: A Pilot Physicochemical Study 1231
Figure 1. A syringe modified to extrude fermented ground
fish.
also added at 20 ppm in some experiments. The mixtures
were extruded into the collagen casings or more com-
monly into the 50-mL syringe barrels described above.
After sealing, the syringes were incubated horizontally at
30˚C for 96 hours. Every 24 hours, flat cylinders of sau-
sage, with a range of heights depending on the test, were
extruded and excised for analyses. Typically, three repli-
cate barrels were prepared for each species for each ex-
periment. The initial carbohydrate source was a generic
long-grain white rice cooked in the volume ratio of 1:1.5
water to a firm end point in a domestic rice cooker, and
then cooled. This carbohydrate source was later replaced
by glucose.
2.4. Physical Analysis of FGF
A TAXT Plus Texture Analyser (Stable Microsystems,
UK) was used to perform texture profile analysis at 0
hours and 96 hours, using extruded cylinders of FGF, 30-
mm high. The analyzer was fitted with a 50-mm-diame-
ter cylindrical aluminium probe whose flat surface cov-
ered the top of each sample’s flat surface, while the bot-
tom lay flat on a glass plate on the instrument base. The
tests were performed with two compression cycles at
room temperature under these conditions: crosshead
speed 5 mm·s−1; 50% strain; surface sensing force 0.971
N; threshold 0.294 N; time interval between first and
second stroke was 1 second. Analyses were defined and
calculated as described by Bourne [19]. Thus, hardness,
adhesiveness, springiness and cohesiveness were calcu-
lated from the force-time/distance curves generated for
each excised cylinder.
The color of the FGF was measured in the L*, a*, b*
space using a Hunter meter (Model 45/0 Hunterlab Col-
orFlex, Reston, Virginia, USA). The extruded cylinders
of FGF were sharply cut 10 mm high and placed cen-
trally on the base of a clear glass crystallizing dish that
sat beneath the meter’s black shroud. The measured val-
ues were corrected for the color of the empty crystallize-
ing dish. The color of samples was measured three times
from each of three replicate barrels.
2.5. Microbiological Analysis of FGF
The lactic acid bacteria (LAB) were determined using
MRS agar [20]. A 10-g sample was aseptically trans-
ferred to a sterile plastic pouch and stomached for 1 min-
ute in a Lab-blender (Seward Medical, London, UK), to
which 90 mL of 0.1% sterile peptone water had been
added. Aliquots (0.1 mL) of decimal dilutions in peptone
water were plated in triplicate on MRS agar and incu-
bated anaerobically at 30˚C for two days. LAB counts
are reported as log colony forming units per g (cfu·g−1).
2.6. Chemical Analysis of FGF
The pH of extruded samples were determined by the
method of Benjakul et al. [21]. A 4-g sample was gently
dispersed with 40 mL of deionized water using an Ul-
tra-Turrax homogenizer (IKA-Werke, Germany) and the
pH was measured by meter.
Soluble peptides were determined according to Green
and Babbitt [22]. The FGF sample (1 g) was dispersed
for 2 minutes with 9 mL of 15% (w/v) trichloroacetic
acid (TCA) using an Ultra-Turrax homogenizer at 9500
rpm. The homogenate was kept on ice for 1 hour then
centrifuged at 12,000 gravities for 5 minutes. Soluble
peptides were determined in the supernatant [23]. Values
are expressed as mg of bovine serum albumin equiva-
lents·kg−1 of FGF.
Fat oxidation was determined as thiobarbituric acid
reactive substances (TBARS) [24]. A 2.5-g sample was
dispersed at 9500 rpm for 2 minutes with 12.5 mL
TBARS solution (0.375% thiobarbituric acid, 15% TCA
and 0.25 M HCl). The mixture was heated for 10 minutes
in boiling water to develop color. The mixture was
cooled under running cold water, centrifuged and the
absorbance measured at 532 nm. TBARS values are ex-
pressed as mg of malonaldehyde equivalents·kg−1 of
FGF.
The determination of biogenic amines largely followed
Mah et al. [25]. A 2-g sample was added to 10 mL of 0.4
M perchloric acid and the mixture was dispersed at 9500
rpm then centrifuged at 3000 gravities for 15 minutes.
The precipitate was re-extracted with another 10 mL of
acid. The combined supernatants were made to 25 mL.
The extract was filtered through Whatman paper No.1
prior to derivatisation [26]. A 1 mL aliquot was mixed
with 0.2 mL of 2 M NaOH, 0.3 mL of saturated sodium
bicarbonate, 0.1 mL of 1,7-diaminoheptane as internal
standard (0.5 mg·mL−1), 2 mL dansyl chloride solution
(10 mg·mL−1 in acetone), and then incubated at 40˚C for
1 hour. Residual dansyl chloride was eliminated with 0.1
mL of 25% v/v ammonia solution. After 30 minutes at
room temperature, the extract was filtered (0.45 µm pore)
prior to injection on a Nova-Pak C18 column (3.9 × 300
mm) (Waters, Ireland) fitted to a Shimadzu LC-10AD
liquid chromatograph (Shimadzu, Japan). The biogenic
amines were resolved with an isocratic mobile phase
comprising 50:50 acetonitrile and 0.1 M ammonium ace-
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Development of Model Fermented Fish Sausage from Marine Species: A Pilot Physicochemical Study 1232
tate at 1 mL·min−1. Absorbance due to dansylated amines
was monitored at 254 nm.
2.7. Data Analysis
Experiments were conducted with triplicate barrels of
FGF. Where multiple values were recorded within barrel,
these were averaged to derive the value for each barrel
replicate, the basis of further statistical analysis. Data
were analyzed for variance by the one way routine in
Minitab 15 (Minitab Inc., State College, PA). Compari-
sons between individual means were done with the
Tukey test in that routine.
3. Results
3.1. Attempted Fermentations with Rice and
Glucose
Ground hoki was blended with cooked white rice, garlic
and salt, and the mixtures were extruded into the colla-
genous casing and held suspended at 30˚C in a dry incu-
bator. Under these conditions the desired lactic fermenta-
tion did not occur. At 0 hours the pH was 6.3 and was
unchanged at 48 hours with spoilt fish smell attributed to
formation of biogenic amines [27]. The failure to ferment
may have been due to a lowering of water activity in the
dry incubator environment, which contrasts with the hu-
mid conditions of traditional banana or plastic bag
methods employed in Cambodia. In a subsequent trial
with kahawai and trevally, the mixtures were extruded
into generic water-impermeable polypropylene vials and
sealed. At 72 hours the LAB counts were between 7 and
8 log colony forming units (cfu)·g−1 and the pH was
around 5.4. It was concluded that the endogenous lactic
bacilli present in these mixtures were limited in their
ability to hydrolyze gelatinized starch in cooked rice to
simpler fermentable carbohydrates. This was tested by
substituting 15% glucose for the 15% rice, while realis-
ing that 15% glucose would be far in excess of that re-
quired for successful fermentation. However, it was pro-
posed that the excess glucose would also serve as a hu-
mectant and provide a mildly sweet taste to balance the
acidity arising from fermentation.
Incubations in the syringe extrusion system with the
three species and 15% glucose showed that for all species,
LAB counts as log cfu·g−1 typically increased from be-
tween 0 and 3 at 0 hours to between 8 and 9 at 72 hours.
By 96 hours all pH values were below 5 and each prepa-
ration had a distinct lactic acid smell indicating success-
ful fermentation. Fifteen percent glucose was used in all
subsequent experiments.
3.2. LAB Changes during Fermentation
Figure 2 shows the typical pattern of LAB growth kinet-
Figure 2. Kinetics of LAB growth in FGF produced from
trevally, kahawai and hoki with and without folic acid. Bars
indicate standard deviation of three replicate fermentation
barrels.
ics over 96 hours, in the presence and absence of added
folic acid. The initial values were just above 3 log cfu·g−1
and increased sharply for all treatments, reaching a simi-
lar count of between 8 and 9 log cfu·g−1. Hoki FGF was
slower to ferment than the other two species, but the
reason for this is unknown and probably unimportant.
Although there were numerical differences between the
control and folic acid treatments, they were inconsistent
and irrelevant by the time fermentation was complete.
3.3. Color Changes during Fermentation
Although the only colors that matter commercially are at
retail sale and consumption, monitoring of reflected color
during incubation revealed some differences between the
species. Summed over all analysis times, hoki had the
highest L* values (light reflectance), followed by trevally
and kahawai (P < 0.001) (Figure 3). While the L* values
of hoki FGF were essentially static, those of trevally and
kahawai increased significantly with time (P < 0.001 for
each). If there were any significant differences in L*
values due to folic acid, they were commercially unim-
portant.
As expected, the a* values reflected the flesh color of
the original fish, red for kahawai, pink for trevally and
white for hoki. Thus summed over all times, the mean a*
of kahawai was 4.27 ± 0.60, of trevally 1.22 ± 0.51 and
of hoki −0.13 ± 0.28, the last being red/green neutral.
Changes with time of fermentation were minor, and folic
acid had no important effect (data not shown).
The kinetics of b*, yellowness/blueness, were different
between the species. At 0 hours all had b* values be-
tween 13 and 17 (Figure 4). Whereas the b* values for
kahawai and trevally were essentially static, those for
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Development of Model Fermented Fish Sausage from Marine Species: A Pilot Physicochemical Study 1233
Figure 3. Kinetics of reflectance (L*) in FGF during fer-
mentation, in the presence and absence of folic acid. Bars
indicate standard deviations of three replicate fermentation
barrels.
Figure 4. Kinetics of yellowness/blueness (b*) of FGF dur-
ing fermentation. Bars indicate standard deviations of three
replicate fermentation barrels.
hoki steadily increased from about 15 to 20. Again, folic
acid had no effect, and was not included in subsequent
experiments.
3.4. Proteolysis and Fat Oxidation in FGF during
Fermentation
Proteolysis was determined as TCA-soluble peptides,
expressed as bovine serum albumin equivalents (Figure
5). Generally, protein hydrolysis increased with increase-
ing fermentation time (P < 0.001 within species). FGF
from trevally showed the highest concentration of soluble
peptides and amino acids at all times, followed by kaha-
wai and hoki FGF in that order (P < 0.001 between spe-
cies).
Expressed as malonaldehyde equivalents, hoki had the
lowest TBARS (P < 0.001), with trevally and kahawai
Figure 5. Kinetics of soluble peptides and amino acids in
FGF during fermentation. Bars indicate standard devia-
tions of three replicate fermentation barrels.
showing equal and higher TBARS at 96 hours (Figure 6).
Compared with the other two species, the kinetics of
TBARS for hoki were essentially static, with minimal
variation between replicates.
3.5. Biogenic Amines
No biogenic amines were detected before fermentation.
At 96 hours, histamine was present at 209 mg·kg−1, 16
mg·kg−1 and 12 mg·kg−1 in trevally, kahawai and hoki,
respectively (Table 2). The only species to generate
tryptamine and tyramine was trevally.
3.6. Textural Properties of FGF
Table 3 shows the effect of 96 hours of fermentation on
the textural properties of FGF made from the 3 species.
For trevally and kahawai, the hardness, adhesiveness,
springiness and cohesiveness of the FGF increased nu-
merically during fermentation, at various levels of statis-
tical significance. Whereas the adhesiveness of hoki FGF
increased numerically over 96 hours, the three other pa-
rameters decreased. At 96 hours, the FGF from trevally
was clearly the hardest, with little difference between
kahawai and hoki. Kahawai was the most adhesive at 96
hours (−1421 mN s) compared with −676 (trevally) and
−872 (hoki) (P < 0.05 for both comparisons). At 96 hours,
trevally FGF was the springiest and most cohesive of the
three species (P < 0.05 for both comparisons). Saisithi et
al. [13] reported that the gel-forming ability of FGF de-
pends on the kind of fish used, and that was certainly the
case here.
4. Discussion
4.1. Fermentation with Rice and Use of Starter
Cultures
There may be several reasons for the slow fermentation
with rice. First, the initial microbial load of the FGF
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Development of Model Fermented Fish Sausage from Marine Species: A Pilot Physicochemical Study
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Table 2. Changes in biogenic amines of FGF produced from trevally, kahawai and hoki.
Fish species Fermentation (hours) Histamine (mg·kg−1) Tryptamine (mg·kg−1) Tyramine (mg·kg−1)
0 n.d. n.d. n.d.
Trevally
96 1209 ± 27 48 ± 18 169 ± 32
0 n.d. n.d. n.d.
Kahawai
96 16 ± 0 n.d. n.d.
0 n.d. n.d. n.d.
Hoki
96 12 ± 2 n.d. n.d.
1Data are means ± standard deviation from three replicate fermentation barrels. n.d., not detected.
Table 3. Changes in texture of FGF produced from trevally, kahawai and hoki at 0 and 96 hours of fermentation.
Fish species Fermentation (hours)Hardness (N) Adhesiveness (mN s)Springiness Cohesiveness
0 17.71 ± 0.28 −1666 ± 118 0.54 ± 0.13 0.34 ± 0.06
Trevally
96 28.01 ± 0.34 −676 ± 314 0.65 ± 0.03 0.46 ± 0.01
Statistical effect of time *** ** NS *
0 5.82 ± 0.18 −1921 ± 196 0.31 ± 0.01 0.24 ± 0.01
Kahawai
96 9.19 ± 0.15 −1421 ± 353 0.44 ± 0.04 0.26 ± 0.02
Statistical effect of time *** NS ** NS
0 11.13 ± 0.47 −1352 ± 872 0.91 ± 0.48 0.51 ± 0.08
Hoki
96 8.99 ± 0.72 −872 ± 235 0.50 ± 0.04 0.28 ± 0.02
Statistical effect of time * NS NS **
1Data are means ± standard deviation from three replicate fermentation barrels. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.
Figure 6. Kinetics of fat oxidation of FGF during fermenta-
tion, measured as malondialdehyde equivalents. Bars indi-
cate standard deviations of three replicate fermentation
barrels.
mixture prepared here was around 3 log cfu·g−1, com-
pared those of FGF in Southeast Asia where the initial
microbial load was between 4 and 6 log cfu·g−1 for
products made from marine species [4], and between 5
and 7 log cfu·g−1 for fresh water species [13]. These high
initial loads may be a consequence of the tropical envi-
ronment of Southeast Asia where the hygiene is difficult
to maintain. Thus the tropical examples of FGF had a
“head start”. Second, in the production of fermented fish
product in Southeast Asia repeated for many years, it is
likely that a stable LAB population capable of hydrolyze-
ing starch to glucose (amylolytic LABs) has developed in
the domestic environment. This contrasts with New Zea-
land’s chilled fish production chain, where hygiene stan-
dards are high (witnessed by the low initial load) and
where carbohydrate is rigorously excluded from the pro-
duction chain.
Amylolytic LAB that can hydrolyze gelatinized starch
and also ferment the glucose to lactic acid have been well
described [28,29]. Thus, starter cultures could be chosen
to ferment starch or starch/glucose combinations. More-
over, it is well known that starter cultures can be chosen
that improve the quality of fermented foods. In the case
of fish, Yin et al. [30], Riebroy et al. [5] and Hu et al.
[31] showed that inoculation could variously reduce fer-
mentation time, and suppress TBARS values, total vola-
tile base nitrogen, trimethylamine production, and the
growth of spoilage bacteria and pathogens compared

Development of Model Fermented Fish Sausage from Marine Species: A Pilot Physicochemical Study 1235
with wild-type controls. Flavor was also improved by the
use of cultures.
4.2. LAB Growth and the Effect of Folic Acid on
Fermentation
Even though the initital bacterial load was a low 3 log
cfu·g−1, there were sufficient endogenous LAB on the
fish/clean hands/clean equipment to achieve a 5 log in-
crease in LAB in 96 hours, resulting in successful fer-
mentations. Claims made in the patent literature [18] for
folic acid enhancing the growth of LAB could not be
reproduced here. However, the folic acid requirements of
different LABs may vary and if a starter culture were
used for FGF production, the outcome may have been
different.
4.3. Color
Hoki flesh is notably white, which explains the highest
reflectance among the three species. It is well known that
fish flesh becomes more reflective when denatured. This
is obvious from cooking and from acidification by lime
juice for example. The increase in L* values of trevally
and kahawai presumably resulted from myosin denature-
tion as acidification developed from fermentation. This
poses the question as to why there was no obvious
change with hoki? The answer may lie in the likely pro-
teolysis that hoki undergoes during storage [32]. It is
proposed that any tendency to create cavities in FGF
structure would create light traps that would counter in-
creased reflectance as acidification developed. Proteoly-
sis is further discussed later.
The reason for the increase in yellowness in hoki FGF
during fermentation is unclear (although it may not be
commercially important). As stated earlier, kahawai flesh
has the highest iron content of the three species. Under
deteriorative conditions such as occur during fermenta-
tion at 30˚C, the original myoglobin and oxymyoglobin
in the fish muscle tends to oxidize to the brown pigment
metmyoglobin [33] that would increase b* values, and
this is favoured by acidic conditions such as experienced
here [34]. Thus, kahawai might be expected to be the
most prone to metmyoglobin formation. However, the
oxidoreductive status of muscle is also important in met-
myoglobin formation [35]. The average status of these
species and the specific status of the fillets chosen are
unknown.
4.4. Texture and Proteolysis
Three studies have reported the textural properties of
FGF in South East Asia [4,5,36]. The hardness data re-
ported here at 96 hours were in the same range as those
reported by those authors, but the test conditions were
slightly different and importantly rice was absent from
the present FGF. It is expected that retrogradation of rice
starch would tend to increase hardness values, and that is
likely to be desirable. In a comparison of FGF from
tropical marine fish, Riebroy et al. [4,5] found that FGF
with the highest hardness were the most preferred by a
sensory panel. By this criterion, the FGF made from tre-
vally was the best.
The adhesiveness (stickiness) values reported by Rie-
broy et al. [36] and Riebroy et al. [4] were of the order of
0 to −100 mN s, less adhesive than values reported here
at 96 hours, between −676 and −872 mN s. While the
nature of the test surfaces (probe and base) are important
for adhesiveness, this large difference is probably attrib-
utable to the use of rice compared with glucose, of which
only rice can retrograde and become less adhesive. The
ratio values for springiness and cohesiveness at 96 hours
were lower than those reported for South East Asian FGF,
but again would be affected by rice inclusion.
Hoki FGF was the hardest at 0 hours, but at 96 hours
had deteriorated in marked contrast to the other two spe-
cies. This might be related to the integrity of the hoki
myosin. Fish muscle proteins are hydrolyzed by endoge-
nous proteolytic enzymes at a rate 10 times higher than
those of mammalian muscle but that also varies with
species [37]. In a study on hoki muscle, Bremner and
Hallett [32] concluded that the fibrils that connect the
muscle fibres are destroyed by endogenous collagenases
and/or other proteinases during chilled storage. The fer-
mentation temperature in the present study was 30˚C
which would increase the rate of these texturally unfa-
vorable reactions. In surimi production, minced fish flesh
is washed to remove the proteolytic enzymes that reduce
gel strength after cooking, the so-called modori effect
[38]. In a study on the gel strength of surimi made from
hoki, MacDonald et al. [39] and Guenneugues and Mor-
rissey [40] reported that the gel strength from washed
mince was greater than from unwashed mince. Mac-
Donald et al. [39] also reported that surimi made from
unwashed hoki mince underwent textural deterioration.
Thus the inferior textural properties of hoki FGF were
probably due to proteolysis, although nothing is known
of the proteolytic status of the other two species. There is
also another factor that may be important for hoki as
discussed next.
If extensive proteolysis is occurring during hoki fer-
mentation, this raises the question as to why hoki had the
lowest soluble peptides concentration of the three species
(Figure 5). In a study on the protein degradation by en-
dogenous fish enzymes, Benjakul et al. [21] showed that
myosin heavy chain was most susceptible to proteolysis
among all the proteins. Microbial peptidases further de-
grade the protein fragments to small peptides and free
amino acids [41]. Now, when myosin is cleaved by, for
example trypsin, it splits into two fragments of about 100
kDa and 360 kDa that retain the α-helical character of the
Open Access FNS

Development of Model Fermented Fish Sausage from Marine Species: A Pilot Physicochemical Study 1236
parent molecule. These fragments have the physico-
chemical behaviour of the parent myosin and would be
expected to precipitate in TCA [42]. Any damage to my-
osin is likely to contribute to loss of favourable texture
(Table 3), because the myosin heavy chain is central to
gel formation due to heat or acidification [43], and thus
textural properties. However, cleavage of myosin may be
independent of proteolysis yielding TCA-soluble pep-
tides, which was most evident in trevally at all incubation
times.
Peptides (and amino acids) produced by proteolysis
are flavor active [44]. On the face of it, trevally FGF
should be the most flavorful from a peptide perspective.
However, the amino acid/peptide profiles may differ be-
tween species, and until sensory trials are performed, this
proposal can only be conjecture.
4.5. Fat Oxidation
The low TBARS values for hoki presumably reflect the
fact that hoki was the least fatty of the three species (Ta-
ble 1). Kahawai is a fatty fish (8.6 g·100 g−1) with the
highest iron content (2.1 mg·100 g−1) in the form of
heme [16]. When lost from the heme, as happens in de-
grading muscle foods, the free iron as Fe2+ is a strong
pro-oxidant through the Fenton reaction [44]. Hoki by
contrast contains typically 0.2 mg of iron 100 g−1 [16],
the least of the three. Values for trevally lie between
these markedly different flesh types. Thus the TBARS
values (kahawai > trevally > hoki) reflect the iron and fat
contents of the three species. All values were below 12
mg·kg−1, whereas Riebroy et al. [4] found that fat oxida-
tion in FGF from six tropical marine species had TBARS
values from 10 to 40 mg·kg−1. Compared with the com-
mercial FGF produced from tropical fresh water species,
which have TBARS values from 5 to 14 mg·kg−1 [8],
FGF produced from tropical marine species appear to be
more prone to fat oxidation, although the oxidative status
of those raw fish entering the FGF production chain was
not described. In the present study TBARS values were
low enough to be commercially acceptable. It must be
noted that these FGF preparations would have been an-
aerobic for most of the 96 hours of fermentation, and
how they would behave when eventually exposed to air
is untested. Although FGF may never be dried, fat oxida-
tion is always possible on exposure to air. Fermented
mammalian meats are often exposed to air in the course
of post-ferment maturation, but there fat oxidation is
controlled by phenolic antioxidants in spices and by ni-
trite curing. The same techniques could be applied to
FGF tailored to different culinary markets but that is be-
yond the scope of this pilot study.
4.6. Biogenic Amines
TCA-soluble peptides—and by implication free amino
acids—increased in all three species with fermentation
time, and the concentration of these was in the order tre-
vally > kahawai > hoki. Free amino acids are the sub-
strates for amino acid decarboxylases that generate
amines [27], and it may be significant that the same se-
quence applies to the biogenic amines, although not in
direct proportion; availability of free amino acids is only
one of several factors governing biogenic amine forma-
tion [45]. The only species to generate tryptamine and
tyramine was trevally. It may be that the trevally treat-
ment yielded more free amino acids due to endogenous
and microbial hydrolytic activity, but this was not deter-
mined. Riebroy et al. [8] showed that seven commercial
FGF preparations in Southeast Asia had histamine, tryp-
tamine and tyramine concentrations up to 291, 71 and
225 mg·kg−1, respectively, although concentrations in
fish flesh prior to fermentation were not reported. The
present results were below these values. The allowable
upper limits for these amines vary with jurisdictions and
specify fish rather than fermented fish. However, the
values reported here would be close to acceptable for
retail fish in Australasia (<200 mg·kg−1) [46], and in
many other jurisdictions.
5. Conclusion
By a number of physiochemical criteria, particularly
hardness, cohesiveness and TCA-soluble, a FGF pre-
pared from trevally (Pseudocaranx dentex) appears to
have the best commercial prospects, subject to future
sensory assessment that was beyond the scope of this
study. Future work should address the use of starter cul-
tures, addition of cost-reducing hydrocolloids, and flavor
development to suit regional flavor preferences.
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