Journal of the Science of Food and Agriculture J Sci Food Agric 85:235– 242 (2005)

DOI: 10.1002/jsfa.1960

Sensory, microbial and chemical effects

of a slurry ice system on horse mackerel
(Trachurus trachurus)
Óscar Rodrı́guez,1 Vanesa Losada,2 Santiago P Aubourg2 and
Jorge Barros-Vela´ zquez1∗
1Food Technology Laboratory, Department of Analytical Chemistry, Nutrition and Food Science, School of Veterinary Sciences, University
of Santiago de Compostela, E-27002 Lugo, Spain
2Department of Seafood Chemistry and Technology, Institute for Marine Research (IIM-CSIC), Eduardo Cabello 6, E-36208 Vigo, Spain

Abstract: Slurry ice, a biphasic system consisting of small spherical ice crystals surrounded by seawater,
was evaluated in parallel with flake ice for the storage of horse mackerel (Trachurus trachurus). Storage
in slurry ice led to a signiftcant enhancement of shelf life (5 days for flake ice to 15 days for slurry ice),
better control of pH and lower counts of total aerobes and proteolytic and lipolytic bacteria, these reaching
an average difference between batches of 2, 1.43 and 1.98 log units respectively after 8 days of storage.
Storage in slurry ice also involved a signiftcantly slower formation of total volatile basic nitrogen and
trimethylamine after 8 days of storage. Staphylococcus xylosus and Proteus penneri were identifted as
the leading proteolytic and lipolytic organisms in horse mackerel muscle. Storage of horse mackerel in
slurry ice enhances the shelf life of this medium-fat ftsh species through better maintenance of sensory
and microbiological quality.
 2004 Society of Chemical Industry

Keywords: horse mackerel; slurry ice; shelf life; sensory quality; chemical analysis; microbiological activity

INTRODUCTION owing to the spherical geometry of its microscopic

Marine species deteriorate rapidly after death owing
to the effect of a wide variety of biochemical and
microbial degradation mechanisms. As a consequence
of this, both the rate at which heat can be removed
from seafood during chilling and the temperature of
chilled storage exert a direct effect on the maintenance
of fish quality and its shelf life. Accordingly, the
application of newer and more efficient refrigeration
systems to the operations involved in fish handling
and storage may enhance the quality of commercial
fish products. Thus, while conventional chilling has
traditionally involved the use of flake ice or refrigerated
seawater, more recently, slurry ice— also known as
fluid ice, slush ice, liquid ice or flow ice— has
been introduced as a promising technique for the
preservation of fish products at subzero temperature.
Slurry ice can be defined as a biphasic system
consisting of small spherical ice crystals suspended
in iced water at a temperature slightly above the initial
freezing point of fish (0 to− 2 ◦C). There are two main
advantages of slurry ice: its faster chilling rate due
to its higher heat exchange capacity, and the limited
physical damage that it causes to fish food products
ice crystals. Other advantages of slurry ice derive
from its complete coverage of the fish surface, which
affords better protection with respect to oxidation and
dehydration events. Slurry ice can also be pumped,
thus guaranteeing more hygienic handling of fish
products, and may be combined with other agents
such as ozone, to achieve an antiseptic surface effect,
or melanosis inhibitors, to prevent browning reactions
in shellfish.1
Other authors reported better maintenance of
quality of albacore tuna when stored onboard in
slurry ice as compared with flake ice. 2 Harada3 also
highlighted the advantages of slurry ice as a pre-cooling
method for fish. The scientific literature has recently
included accounts of the use of slurry ice systems for
the storage of Australian prawns 4 and shrimp.1 Other
authors have also reported that slurry ice represents a
good slaughter method for the sacrifice and storage of
farmed seabream.5
Horse mackerel is a medium-fat species abundant in
the Northeast Atlantic6 and has a potential role in the
prevention of heart disease owing to its high content
of n-3 polyunsaturated fatty acids (PUFAs) not

∗ Correspondence to: Jorge Barros-Vela´ zquez, Departamento de Qu´ımica Anal´ıtica, Nutricio´ n y Bromatolog´ıa, Facultad de Veterinaria,
Universidad de Santiago de Compostela, E-27002 Lugo, Spain
E-mail: jbarros@lugo.usc.es
Contract/grant sponsor: Xunta de Galicia; contract/grant number: PGIDTI02RMA18E
(Received 23 January 2004; revised version received 20 May 2004; accepted 19 July 2004)
Published online 15 October 2004
 2004 Society of Chemical Industry. J Sci Food Agric 0022– 5142/2004/$30.00 235
Ó Rodr´ıguez et al
synthesised by humans. Horse mackerel has not been
extensively used as a raw material by the fish industry
in the past; however, it is currently receiving increasing
attention from fish technologists and is considered to
be an under-exploited fish species of high commercial
potential as an effective functional food.7– 10 The
minimal seasonal variation of horse mackerel lipids
has been reported by Bandarra et al,11 who also
underlined the nutritional interest of this fish species
as an important year-round source of lipids of dietary
importance. Previous research carried out on chilled
storage has reported biochemical analyses 10 (amine
formation and lipid damage) and physicochemical
parameters,12 although no microbiological parameters
have been considered.
Here an advanced slurry ice system was applied to
the storage of horse mackerel (Trachurus trachurus)
over 22 days and the results were compared with those
obtained with a control batch stored in parallel in
conventional flake ice. With a view to investigating the
shelf life of horse mackerel, the effect of its storage in
slurry ice on sensory and microbiological quality was
investigated over 22 days. In addition, the isolation
and identification of the major bacteria involved in the
proteolytic and lipolytic breakdown of horse mackerel
muscle was undertaken.
EXPERIMENTAL
Slurry ice and flake ice systems
In the present work a slurry ice prototype (FLO-
ICE, Kinarca SAU, Vigo, Spain) was used. The
composition of the slurry ice binary mixture was 40%
ice and 60% water, prepared from filtered seawater
(salinity 33 g kg−1). The temperature of the slurry ice
mixture was 1.5− ◦C. Flake ice was prepared with an
Icematic F100 Compact device (Castelmac SPA,
Castelfranco, Italy).
Fish material, processing and sampling
Specimens of horse mackerel (T trachurus) were caught
at a local fishing bank close to northwestern Spain
and kept on ice until they arrived at the laboratory.
The length of time between capture and the start
of the experimental work was 10 h. Once the chilled
fish specimens arrived at the laboratory, they were
immediately placed in either slurry ice or flake ice
at a fish/ice ratio of 1:1 (w/w). The fish specimens
were neither headed nor gutted. The length of the
specimens was in the 16– 21 cm range and their weight
was in the 230– 270 g range. Both batches were stored
in a refrigerated room at 2 ◦ C for 22 days. In the case
of the flake ice batch, this was stored up to day 19,
since the fish material was too spoiled by that day.
When required, the flake ice and slurry ice mixtures
were renewed.
For each chilling treatment, three different batches
were used and studied separately for the same period
of time. The temperature of the fish specimens was
measured throughout storage. Samples were taken
236
from each batch on days 0, 2, 5, 8, 12, 15, 19 and
22. Once the intact specimens had been subjected
to sensory analyses, the white muscle was aseptically
separated and used for microbiological and chemical
analyses. All analyses were performed in triplicate.
Sensory analyses
Sensory analyses were conducted in triplicate: three
specimens were recovered from each batch at each
sampling time and evaluated by a taste panel of
five experienced judges according to the guidelines
presented in Table 1.13 All decisions were taken by
consensus. A four-category scale was used for sensory
assessment: highest quality (E), good quality (A), fair
quality (B) and unacceptable (C). Sensory assessment
of the fish included the following parameters: skin,
external odour, gills, eyes, consistency and flesh odour.
Microbiological analyses
Samples of 25 g of fish muscle were aseptically skinned
and dissected from chilled horse mackerel specimens
by means of sterile surgical blades under sterile air-
flow conditions to avoid cross-contamination. Samples
were then mixed with 225 ml of peptone water (1g l−1)
and homogenised in a stomacher (Seward Medi-
cal, London, UK) as previously described. 14,15 In all
cases, serial dilutions from the microbial extracts were
prepared in peptone water. Total aerobes were inves-
tigated in plate count agar (PCA, Oxoid Ltd, London,
UK) as described elsewhere.14 Anaerobes were inves-
tigated in the same way, except that an anaerobic
atmosphere kit (Oxoid) was placed together with the
plates inside the anaerobiosis jar. Lactose-fermenting
Enterobacteriaceae (coliforms) were investigated in
violet red bile agar (VRBA medium, Merck, Darm-
stadt, Germany) after incubation at 30 1 ◦C± for
24 ± 2 h as recommended by the manufacturer.16
Micro-organisms exhibiting a proteolytic phenotype
were investigated in casein agar medium 17 as
previously described.18 Bacterial colonies exhibiting
a lipolytic phenotype were detected in tributyrine agar
medium as described elsewhere.18
Routine microbiological tests included the investiga-
tion of colony morphology, cell morphology, motility,
Gram stain and the production of cytochrome oxidase
and catalase as described elsewhere. 19 Major prote-
olytic and lipolytic bacterial strains were isolated from
the muscle of fish stored in slurry ice for 22 days
and then left at room temperature (20– 25 ◦C) for
3 days, and identified using miniaturised biochemical
tests, API 20 E and API 20 NE for Gram-negative
micro-organisms and API 50CH and API STAPH
for Gram-positive micro-organisms, all of them from
BioMèrieux (Marcy L’Etoile, France). The results of
the identification tests were interpreted using API-
LAB PLUS software (BioMèrieux). The enzymic
profiles of the proteolytic and lipolytic bacterial iso-
lates were further characterised using the API ZYM
system (BioMèrieux).
J Sci Food Agric 85:235– 242 (2005)
 Storage of horse mackerel in slurry ice

Table 1. Scale employed for evaluating freshness of horse mackerel

Attribute Highest quality (E) Good quality (A) Fair quality (B) Unacceptable (C)

Skin Very intense Milky mucus; Slightly greyish mucus; Widely opaque mucus;
pigmentation; insignificant pigmentation important
transparent mucus pigmentation losses without shine pigmentation losses
External odour Sharply seaweedy and Weakly seaweedy and Incipiently putrid and Putrid and rancid
shellfish smell shellfish smell rancid
Gills Brightly red; without Rose coloured; Slightly pale; incipient Grey – yellowish colour;
odour; lamina without odour; fishy odour; lamina intense ammonia
perfectly separated lamina adhered in adhered in groups odour; lamina totally
groups adhered
Eyes Convex; transparent Convex and slightly Flat; opalescent Concave and milky
cornea; bright black sunken; slightly cornea; opaque cornea; internal
pupil opalescent cornea; pupil organs blurred
black cloudy pupil
Consistency Presence or partial Firm and elastic; Presence of Important shape
disappearance of pressure signs mechanical signs; changes due to
rigor mortis disappear elasticity notably mechanical factors
symptoms immediately and reduced
completely
Flesh odour Sharply seaweedy and Weakly seaweedy and Incipiently putrid and Putrid and rancid
shellfish smell shellfish smell rancid

Chemical analyses among the specialists. According to the results of the

The measurement of pH in horse mackerel muscle
during the storage period was carried out using a
6 mm diameter insertion electrode (Crison, Barcelona,
Spain). Total volatile basic nitrogen (TVB-N) values
were measured according to Aubourg et al.20 In this
method, fish muscle (10 g) is extracted with perchloric
acid (60 g l−1) and made up to 50 ml, the TVB-N
content being determined— after steam distillation
of the acid extracts rendered alkaline to pH 13
with NaOH (200 g l−1) — by titration of the distillate
with 10 mM hydrochloric acid. Results are expressed
as mg TVB-N per 100 g muscle. Trimethylamine
nitrogen (TMA-N) values were obtained according
to the method of Tozawa et al,21 which involves the
preparation of a 50 g l−1 trichloracetic acid extract of
fish muscle (400 g l−1) and reaction with picric acid.
Data are expressed as mg TMA-N per 100 g muscle.
sensory analyses (Table 2), horse mackerel stored in
slurry ice maintained good quality (E and A categories)
up to day 8, while the counterpart batch stored in flake
ice only maintained such good quality up to day 2. As
storage time progressed, sensory quality decreased and
by day 8 (flake ice batch) and day 19 (slurry ice batch)
the specimens were no longer acceptable. The external
features that limited the acceptability of the flake ice
batch were external odour, flesh odour and the aspect
of the gills. The shelf life of horse mackerel stored in
flake ice determined in this study is in agreement with
the values reported in previous work carried out with
this fish species10,23 and with other small species such
as mackerel.24 Of special relevance is the considerable
increase in shelf life obtained as a consequence of
the use of slurry ice, the horse mackerel specimens
remaining acceptable until at least day 15.

Statistical analyses −1 Quantitative microbiological analyses

Bacterial counts were calculated as log CFU g before
being subjected to statistical analyses. Data from
the different chemical measurements were analysed
using one-way analysis of variance; comparison
of means was performed using a least significant
difference (LSD) method. 22 SPSS 11.5 software for
Windows (SPSS Inc, Chicago, IL, USA) was also
used to explore the statistical significance of the
results obtained, including multivariate contrasts and
multiple comparisons by the Scheffé and Tukey tests.
In all cases a confidence interval at the 95% level
(p < 0.05) was considered.
RESULTS AND DISCUSSION
Sensory analyses
As stated above, the scores assigned to each batch
at each sampling time were determined by consensus
Figures 1– 3 show typical results concerning microbial
growth in horse mackerel stored in either slurry ice or
flake ice. Statistically significant (p < 0.05) differences
were observed between the two batches for aerobes and
proteolytic and lipolytic bacteria. In the case of total
aerobes (Fig 1) the average difference in the counts
between batches was 2.0 log units on day 8, and this
difference even increased to 2.61 log units after 12 days
of storage (Fig 1). At both sampling times the flake ice
batch had exhibited unacceptable quality according
to the sensory evaluation, but the slurry ice batch still
maintained acceptable quality (Table 2). Total aerobic
counts reached levels close to 10 6 CFU g−1 in the flake
ice batch after 8 days of storage, although these figures
are below those considered by other authors to elicit
the spoilage of fish stored aerobically. 25 The storage of
horse mackerel in slurry ice significantly

J Sci Food Agric 85:235– 242 (2005) 237

Ó Rodr´ıguez et al

Table 2. Comparative sensory evaluation of horse mackerel batchesa

Slurry ice batch (days of storage) Flake ice batch (days of storage)

Attribute 2 5 8 12 15 19 22 2 5 8 12 15 19 22

Skin E E A A B C C E A B C C C C
External odour E A A B B C C E A C C C C C
Gills E E A B B C C E B C C C C C
Eyes E A A B B C C E A B B C C C
Consistency E E A A B B B E A B B B C C
Flesh odour E A A B B C C E A C C C C C
a Freshness categories are as expressed in Table 1; initial quality on day 0 merited a score of E (highest quality) for all parameters.

to flesh spoilage. Microbial proteolysis of muscle

has also been reported to cause sensory spoilage in
horse mackerel homogenates stored at 10 ◦C.30 In our
work the evolution of the counts of micro-organisms
potentially involved in the proteolytic breakdown of
horse mackerel was investigated in both batches. As
seen in Fig 2, statistically significant (p < 0.05) lower
counts of proteolytic bacteria were determined in
the muscle of horse mackerel kept in slurry ice as
compared with flake ice. The average difference in the
counts between batches was 1.43 log units on day 8,
and this difference increased to 2.48 log units after
12 days of storage (Fig 2). The numbers of proteolytic
bacteria in the muscle of horse mackerel stored in flake
ice reached levels above 106 CFU g−1 by day 12, while,
in the slurry ice batch, counts of only 10 4 CFU g−1 or
lower were reached, even on day 22, clearly indicating
a significant decrease in the growth of this bacterial
group in horse mackerel muscle stored in slurry ice.
Figure 1. Total aerobes in horse mackerel muscle during storage in
The medium-fat nature of horse mackerel makes this
either slurry ice (squares) or flake ice (diamonds). fish species especially sensitive to mechanisms involved
in lipid damage. Among these mechanisms the
reduced bacterial growth, possibly contributing to the production of extracellular lipases by certain micro-
enhanced shelf life according to the sensory evaluation. organisms may play a role in the lipolytic breakdown
These findings agree with the results of recent work
reporting significantly lower bacterial counts in shrimp
stored in slurry ice as compared with counterpart
batches stored in conventional flake ice. 1 A recent
publication has reported aerobic bacterial counts as
high as 107 CFU g−1 in the skin of horse mackerel
stored for 7 days at 4 ◦C.26 In this sense the surface
wash caused by the liquid phase of the slurry ice
together with the subzero temperature achieved with
this advanced storage system can be invoked as the two
main reasons for the limited bacterial growth found in
the muscle of horse mackerel in the slurry ice batch as
compared with the flake ice batch.
Microbial metabolites such as peptides or amino
acids derive from protein hydrolysis and may
also contribute significantly to undesirable sensory
changes in seafood products. It is well known
that such modifications in odour, texture and
appearance are directly related to spoilage in aquatic
food products.19,27,28 Asakawa et al29 characterised a
protease from a Bacillus sp strain isolated from horse
mackerel that was thought to play a major role in Figure 2. Proteolytic bacteria in horse mackerel muscle during
the post mortem decomposition of skin tissue, leading storage in either slurry ice (squares) or flake ice (diamonds).

238 J Sci Food Agric 85:235– 242 (2005)


Figure 3. Lipolytic bacteria in horse mackerel muscle during storage
in either slurry ice (squares) or flake ice (diamonds).
of fish species such as albacore tuna. 18 Accordingly,
the numbers of micro-organisms exhibiting lipolytic
activity were investigated in both batches. The results
are shown in Fig 3. Statistically significant (p < 0.05)
lower numbers of lipolytic bacteria were determined
in the slurry ice batch than in the flake ice batch. On
day 8 the average difference in the numbers between
batches was found to be 1.98, this difference increasing
to 4.40 log units after 12 days of storage (Fig 3).
The numbers of lipolytic bacteria in the muscle of
horse mackerel stored in flake ice reached levels above
106 CFU g−1 by day 12, while, in the slurry ice batch,
counts of only 104 CFU g−1 were reached after 15 days
of storage. In the light of these results the growth of
bacteria potentially involved in the lipolytic breakdown
of horse mackerel can be said to be slowed down as a
consequence of storage in slurry ice. It is believed that
these are the first results describing the effects of slurry
ice on microbial lipolytic activity in a medium-fat fish
species such as horse mackerel.
The average counts of anaerobes in the mus-
cle of horse mackerel stored in slurry ice were
2.5 log CFU g−1, no statistically significant differences
at the p < 0.05 level being observed between the two
batches. The numbers of anaerobes in each batch
during storage did not show significant differences
with respect to the initial counts at day 0 either
(2.16 log CFU g−1). These results confirm the very
good initial quality of the fish specimens and the
limited growth of anaerobes during chilled storage,
regardless of the ice system employed. With respect to
the development of coliforms, similar results were
obtained. Thus the average numbers of coliforms
during storage were very low (<1 log CFU g−1), no
statistically significant (p < 0.05) differences being
observed between the two batches, nor with respect
to the initial counts on day 0. Similar low numbers
J Sci Food Agric 85:235– 242 (2005)
Storage of horse mackerel in slurry ice
of anaerobes and coliforms have been reported by
Figueroa et al31 for jack mackerel.
It should also be noted that the counts of
total aerobes and proteolytic and lipolytic bacteria
correlated well with the differences observed in the
sensory evaluation, a result that also agrees with
a previous report on shrimp 1 kept in slurry ice.
According to the results presented here, the slowing-
down effect of storage in slurry ice on the growth
of proteolytic and lipolytic bacteria would imply
a more reduced presence of microbial proteases
and lipases in the muscle of horse mackerel, thus
limiting the negative effects of such resistant enzymes
on the protein and lipid compounds of this fish
species, since proteases and lipases may retain
activity for long periods even at low temperatures. 32
Nevertheless, although sensory analyses correlated
well with microbial analyses, when the fish batches
obtained a C score (unacceptable quality) according
to the sensory study, the microbial numbers had not
reached 106 CFU g−1, these numbers being below
those considered to elicit the spoilage of fish stored
aerobically.25 These results would not allow us to
establish a quantitative cut-off point for the microbial
spoilage of horse mackerel, thus suggesting that,
although microbial activity was slowed down in the
slurry ice batch, this activity might not be the limiting
factor of acceptability of this medium-fat fish species.
Chemical analyses
Statistically significant (p < 0.05) differences were
observed in the pH in the muscle of horse mackerel
stored in slurry ice and flake ice (Fig 4). All pH
determinations were carried out in excised fillets.
Thus, while only slight increases in pH (from the
initial 6.38 to a peak of 6.59) were observed in the
slurry ice batch, significant increases in pH (up to 7.65)
Figure 4. pH in horse mackerel muscle during storage in either slurry
ice (squares) or flake ice (diamonds).
239
Ó Rodr´ıguez et al
were found in the flake ice batch during storage. The
marked increase in pH in the flake ice batch suggests
a more intense growth of alkalinising bacteria in this
batch, leading to a higher accumulation of ammonia
compounds, with the subsequent negative effects on
sensory quality, especially external and flesh odour.
The changes in the pH in the muscle of horse
mackerel specimens corresponding to each batch
correlated well with the evolution of TVB-N and
TMA-N (Figs 5 and 6 respectively). Thus the
formation of both TVB-N and TMA-N in horse
mackerel muscle was slowed down in the slurry
ice batch, especially after 8 and 5 days of storage
respectively. The TVB-N content (Fig 5) of horse
mackerel stored in slurry ice was very low, reaching
levels of only 31 mg per 100 g after 22 days of storage.
By contrast, the TVB-N content underwent a dramatic
increase after 8 days of storage in the muscle of horse
mackerel stored in flake ice, reaching concentrations
as high as 84 mg per 100 g after 19 days of storage.
Statistical analysis confirmed that the storage of
horse mackerel in slurry ice involved a significantly
(p < 0.05) lower formation of TVB-N as compared
with flake ice. Other authors 26 have also reported
TVB-N contents higher than 50 mg per 100 g in horse
mackerel stored for 9 days at 4 ◦ C, this also being far
above those determined in this study in horse mackerel
stored in slurry ice.
Likewise, the TMA-N content in muscle also
increased very slowly in the period between 0 and
22 days of storage in the slurry ice batch. After day 5 a
sharp increase in the TMA-N content was determined
only in the muscle of horse mackerel stored in flake
ice. Finally, average TMA-N values as different as 10.5
and 22.5 mg per 100 g were determined in the slurry
ice batch and flake ice batch respectively after 19 days
of storage (Fig 6). Thus, as expected from the results
Figure 5. Total volatile basic nitrogen (TVB-N) content in horse
mackerel muscle during storage in either slurry ice (squares) or flake
ice (diamonds).
240
Figure 6. Trimethylamine nitrogen (TMA-N) content in horse
mackerel muscle during storage in either slurry ice (squares) or flake
ice (diamonds).
obtained in the present study, storage in slurry ice
significantly (p < 0.05) slowed down the formation
of TMA-N, especially after 5 days of storage, in
comparison with storage in flake ice. Since TMA-N
has been reported to be the best chemical parameter
to determine quality loss in horse mackerel, 10 the
benefits of slurry ice for extending the shelf life of
horse mackerel should be noted.
As in the case of microbial analysis, sensory analyses
correlated will with the chemical parameters analysed
in each batch. Thus, although the results obtained
indicated a significantly slower formation of TVB-
N and TMA-N in the slurry ice batch, the values
obtained for these parameters on the first day that each
batch exhibited unacceptable quality according to the
sensory analysis were not high enough to be considered
limiting factors of acceptability of horse mackerel.
Identification of bacteria involved in proteolytic
and lipolytic breakdown of horse mackerel
There is little previous information available concern-
ing the identification of spoilage micro-organisms from
horse mackerel. Da Silva et al33 studied the effects of
the inoculation of five spoilage species into horse mack-
erel stored under an ozone atmosphere, but several of
the microbial species considered were typical spoilage
bacteria, not specific micro-organisms isolated from
horse mackerel. Thus in the present work a qualitative
analysis of the predominant proteolytic and lipolytic
bacterial strains isolated from horse mackerel muscle
stored in slurry ice over 22 days and then left at room
temperature (20– 25 ◦ C) for 3 days was carried out.
From the 26 initial microbial isolates exhibiting
proteolytic or lipolytic activity in plate bioassays,
13 isolates showing different phenotypes in the
preliminary microbiological study and the relative
abundance of the total proteolytic or lipolytic colonies
in each fish specimen were selected for further study
J Sci Food Agric 85:235– 242 (2005)
 Storage of horse mackerel in slurry ice

Table 3. Identification of micro-organisms involved in proteolytic and Table 4. Phenotypic differences among Staphylococcus xylosus and
lipolytic breakdown of horse mackerel muscle Proteus penneri strains isolated from horse mackerel

Proteolytic (P)/lipolytic Bacterial species and strain Phenotypea

Strain (L) activitya Bacterial species
S xylosus
P1 P(++)/L(++) Staphylococcus xylosus Group 1: strains P1 and P13 Trypsine (+ ), protease
P7 P(++)/L(−) Staphylococcus xylosus (w), lipase (w)
P8 P(+)/L(++) Proteus penneri Group 2: strains L2, L9 and L12 Trypsine ( ), protease
−
P9 P(++)/L(+) Proteus penneri (+), lipase (w)
P13 P(++)/L(++) Staphylococcus xylosus Group 3: strain P7 Trypsine (− ), protease
L1 P(+++++)/L(+) Proteus penneri (w), lipase (− )
L2 P(+++++)/L(++) Staphylococcus xylosus P penneri
L5 P(+++)/L(+++) Proteus penneri Group 1: strains P8 and P9 Cysteine aryl amidase
L7 P(+++)/L(+++) Proteus penneri (+), protease (w),
L8 P(+++)/L(+++) Proteus penneri lipase (w)
L9 P(+++)/L(+++) Staphylococcus xylosus Group 2: strains L5, L7 and L8 Cysteine aryl amidase
L12 P(+++++)/L(++) Staphylococcus xylosus L13 (−), protease ( +/w),
P(++++)/L(++) Proteus vulgaris lipase (+ /w)
activity: −= negative; += very weak; ++ =
a Proteolytic/lipolytic Group 3: strain L1 Cysteine aryl amidase
weak; +++ = moderate; ++++ = strong; +++++ = very strong. (−), protease (+),
lipase (−/w)

(Table 3). Except for strain P7, which exhibited only of tests on enzyme production: (+) = positive; (−) =
a Interpretation

a proteolytic phenotype, all the remaining 12 isolates negative; (w) = weak.

exhibited both proteolytic and lipolytic activities. Six
isolates were identified as Staphylococcus xylosus, while
six others belonged to the species Proteus penneri —an
indole-negative variant of Proteus vulgaris — the other
isolate being identified as Proteus vulgaris (Table 3).
While Proteus spp have previously been isolated from a
number of aquatic food products, S xylosus strains have
deserved the attention of fish technologists because of
their ability to biosynthesise and secrete extracellular
histidine decarboxylase, leading to the formation of
histamine in seafood products such as semi-preserved
anchovies.34
The phenotypic differences among the proteolytic
and lipolytic isolates belonging to the same species
were also investigated by the API ZYM system
(Table 4). The results obtained for S xylosus iso-
lates indicated that the six strains could be clas-
sified into three groups (Table 4). Accordingly, it
was concluded that at least three different S xylo-
sus strains, namely P1, L2 and P7, belonged to
the microflora of horse mackerel, the other S xylo-
sus strains being multiple isolates of any of these
three strains. Interestingly, all the S xylosus strains
isolated from horse mackerel biosynthesised and
secreted alkaline phosphatase, acid phosphatase, ure-
ase, leucine aryl amidase, naphtholphosphohydrolase
and α-glycosidase.
With respect to the P penneri strains, at least
three different strains— P8, L5 and L1— were isolated
from horse mackerel, as determined by phenotypic
analysis (Table 4). Interestingly, all P penneri strains
isolated from horse mackerel biosynthesised and
secreted alkaline phosphatase, acid phosphatase,
leucine aryl amidase, naphtholphosphohydrolase and
α-glycosidase.
The results obtained in this work suggest that P
penneri and S xylosus are involved in the proteolytic
and lipolytic breakdown of horse mackerel muscle.
In addition to these two species, a moderately
lipolytic/strong proteolytic strain of P vulgaris was also
isolated. The fact that slurry ice significantly slowed
down the growth of proteolytic and lipolytic bacteria in
the muscle of horse mackerel, as described above,
underlines the benefits that such a storage system may
exert on the maintenance of the quality and shelf life
of this fish species.
CONCLUSIONS
The results of the sensory analyses described here
show that the storage of horse mackerel in slurry ice
allows better maintenance of quality and enhances
the shelf life of this fish species by more than
10 days: from 5 days (flake ice batch) to 15 days
(slurry ice batch). Slurry ice, probably owing to
its higher heat exchange and its full coverage of
the fish material, significantly retarded the growth
of total aerobes and proteolytic and lipolytic bacte-
ria, also affording better control of pH and slowing
down both TMA-N and TVB-N formation. The
good results obtained in the microbiological, sen-
sory and chemical analyses strongly support the
use of slurry ice to improve the chilled storage
and thus assist in the commercial exploitation of
horse mackerel.
ACKNOWLEDGEMENTS
The authors wish to thank Kinarca SAU for providing
the slurry ice equipment. This work was supported by
a project granted by the Secretar´ıa Xeral de +I D from
the Xunta de Galicia (Project PGIDTI02RMA18E).
The authors also thank Ms Marta Torres, Mr Marcos
Trigo and Mr José M Antonio for their technical
assistance.

J Sci Food Agric 85:235– 242 (2005) 241

Ó Rodr´ıguez et al
REFERENCES
1 Huidobro A, López-Caballero M and Mendes R, Onboard
processing of deepwater pink shrimp (Parapenaeus longirostris)
with liquid ice: effect on quality. Eur Food Res Technol
214:469– 475 (2002).
2 Price RJ, Melvin EF and Bell JW, Postmortem changes in
chilled round, bled and dressed albacore. J Food Sci
56:318– 321 (1991).
3 Harada K, How to handle albacore. Aust Fish 2:28– 30 (1991).
4 Chinivasagam HN, Bremner HA, Wood AF and Notting-
ham SM, Volatile components associated with bacterial spoilage of
tropical prawns. Int J Food Microbiol 42:45– 55
(1998).
5 Huidobro A, Mendes R and Nunes ML, Slaughtering of
gilthead seabream (Sparus aurata) in liquid ice: influence
on fish quality. Eur Food Res Technol 213:267– 272 (2001).
6 FAO Inform, Fishery statistics, in Food and Agriculture
Organization of the United Nations Yearbook 1996. FAO, Rome,
pp 187– 188 (1998).
7 Garcı́a I, Pérez-Villarreal B and Pozo R, Processing of under-
utilized fish species. Alim Equip Technol 15:145– 149 (1996).
8 Tabara Y, Ueki S, Ito M, Watanabe T, Kan T, Hiraoka Y,
Nakajima S and Tsuchiya T, Changes in EPA and DHA
levels during manufacture of dried fish and shellfish. J Jpn Soc
Food Sci Technol 45:93– 99 (1998).
9 Smith J, McGill A, Thomson A and Hardy R, Preliminary
investigation into the chilled and frozen storage characteristics
of scad (Trachurus trachurus) and its acceptability for human
consumption, in Advances in Fish Science and Technology, Ed
by Connell J. Fishing News Books, London, pp 303– 307
(1980).
10 Aubourg S, Damage detection in horse mackerel (Trachurus
trachurus) during chilled storage. J Am Oil Chem Soc
78:857– 862 (2001).
11 Bandarra NM, Batista I, Nunes ML and Empis JM, Seasonal
variation in the chemical composition of horse mackerel
(Trachurus trachurus). Eur Food Res Technol 212:535– 539
(2001).
12 Monteagudo Torres S, Montaña Miguelez J and Mı́guez
Bernárdez M, Comparison of sensory and physicochemical
methods for evaluating the quality and commercial life of lean
fish (Trisopterus luscus) and fatty fish (Trachurus trachurus)
marketed in Pontevedra. Alimentaria 334:73– 79 (2002).
13 DOCE, Baremo de clasificación de frescura, in Diario Oficial de
las Comunidades Europeas. European Commission, Brussels,
pp 5– 6 (1989).
14 Ben-Gigirey B, Vieites Baptista de Sousa JM, Villa TG and
Barros-Velázquez J, Changes in biogenic amines and microbi-
ological analysis in albacore (Thunnus alalunga) muscle during
frozen storage. J Food Protect 61:608– 615 (1998).
15 Ben-Gigirey B, Vieites Baptista de Sousa JM, Villa TG and
Barros-Velázquez J, Histamine and cadaverine production by
bacteria isolated from fresh and frozen albacore (Thunnus
alalunga). J Food Protect 62:933– 939 (1999).
16 Merck Microbiology Manual. Merck, Darmstadt, p 273 (2000).
17 Phaff HJ, Starmer WT, Lachance MA and Ganter PF, Candida
caseinolytica sp nov., a new species of yeast occurring in
necrotic tissues of Opuntia and Stenocereus species in the
Southwestern United States and Baja California, Mexico. Int
J Syst Bacteriol 44:641– 645 (1994).
242
18 Ben-Gigirey B, Vieites-Baptista de Sousa JM, Villa TG and
Barros-Velázquez J, Characterization of biogenic amine-
producing Stenotrophomonas maltophilia strains isolated from
white muscle of fresh and frozen albacore tuna. Int J Food
Microbiol 57:19– 31 (2000).
19 Rodrı́guez O, Barros-Velázquez J, Ojea A, Piñeiro C and
Aubourg S, Evaluation of sensory and microbiological
changes and identification of proteolytic bacteria during the
iced storage of farmed turbot (Psetta maxima). J Food Sci
68:2764– 2771 (2003).
20 Aubourg S, Sotelo C and Gallardo J, Quality assessment of
sardines during storage by measurement of fluorescent
compounds. J Food Sci 62:295– 299 (1997).
21 Tozawa H, Erokibara K and Amano K, Proposed modification
of Dyer’s method for trimethylamine determination in codfish,
in Fish Inspection and Quality Control, Ed by Kreuzer R.
Fishing News Books, London, pp 187– 190 (1971).
22 Statsoft, Statistica for Macintosh. Statsoft, Tulsa, OK (1994).
23 Simeonidou S, Govaris A and Vareltzis K, Quality assessment
of seven Mediterranean fish species during storage in ice. Food
Res Int 30:479– 484 (1997).
24 Bennour M, El Marrakchi A, Bouchriti N, Hamama A and El
Ouadaa M, Chemical and microbiological assessments of
mackerel (Scomber scombrus) stored in ice. J Food Protect
54:784, 789– 792 (1991).
25 Gram L and Huss H, Microbiological spoilage of fish and fish
products. Int J Food Microbiol 33:121– 137 (1996).
26 Kuda T, Matsumoto C and Yano T, Changes in acid and
alkaline phosphatase activities during the spoilage of raw
muscle from horse mackerel (Trachurus japonicus) and gurnard
(Lepidotriga microptera). Food Chem 76:443– 447 (1996).
27 Shewan JM, The bacteriology of fresh and spoiling fish and the
biochemical changes induced by bacterial action, in Handling,
Processing and Marketing of Tropical Fish. Tropical Product
Institute, London, pp 51– 68 (1977).
28 Makarios-Laham IK and Lee TC, Protein hydrolysis and quality
deterioration of refrigerated and frozen seafood due to
obligately psychrophilic bacteria. J Food Sci 58:310– 313
(1993).
29 Asakawa M, Sadakata Y, Araki T, Sumi T and Nakagawa H,
Purification and characterization of the alkaline serine
protease produced by Bacillus sp N4 strain from fish skin
mucus. Fish Sci 64:793– 797 (1998).
30 Kobatake M, Kreger van Rij NJW, Placido MTLC and van
Uden N, Isolation of proteolytic psychrotrophic yeasts from
fresh raw seafoods. Lett Appl Microbiol 14:37– 42 (1992).
31 Figueroa G, Galeno H, Troncoso M and Aguilera JM, Analysis
of the microbial flora of jack mackerel (Trachurus murphyi)
minced products. Sci Alim 10:907– 912 (1990).
32 Alford JA and Pierce DA, Lipolytic activity of microorganisms at
low and intermediate temperatures. III. Activity of microbial
lipases at temperatures below 0 ◦C. J Food Sci 26:518– 524
(1961).
33 Da Silva MV, Gibbs PA and Kirby RM, Sensorial and microbial
effects of gaseous ozone on fresh scad (Trachurus trachurus). J
Appl Microbiol 84:802– 810 (1998).
34 Rodrı́guez-Jerez JJ, Mora-Ventura MT, López-Sabater EI and
Hernández-Herrero M, Histidine, lysine, and ornithine
decarboxylase bacteria in Spanish salted semipreserved
anchovies. J Food Protect 57:784– 787, 791 (1994).
J Sci Food Agric 85:235– 242 (2005)