Use of citric and lactic acids in ice to enhance quality of two fish
species during on-board chilled storage
Bibiana García-Soto1, Inmaculada Concepción Fernández-No2, Jorge
Barros-Velázquez2, and Santiago P. Aubourg3,*
1
2
Cooperativa de Armadores de Pesca del Puerto de Vigo (ARVI), Vigo (Spain)
Department of Analytical Chemistry, Nutrition and Food Science, School of
Veterinary Sciences, University of Santiago de Compostela, Lugo (Spain)
3
Department of Food Science and Technology, Marine Research Institute (CSIC), Vigo
(Spain)
* Corresponding author. Prof. Santiago P. Aubourg; C/ Eduardo Cabello, 6. 36208-Vigo
(Spain);
Phone:
+34
saubourg@iim.csic.es
986231930;
Fax:
+34
986292762;
e-mail:
ABSTRACT
This work focused on the on-board chilled storage of European hake (Merluccius
merluccius) and megrim (Lepidorhombus whiffiagonis). To enhance fish quality, an
aqueous solution including citric (1.25 g l-1) and lactic (0.50 g l-1) acids was prepared,
frozen, ground and employed as icing medium. Its effect on sensory, microbiological
and chemical changes was monitored after 9, 12 and 15 days of on-board storage.
Lower (p<0.05) bacterial growth was detected according to microbiological (aerobe,
anaerobe, psychrotrophe, proteolytic, and Enterobacteriaceae counts) and chemical
(trimethylamine content) assessments. An inhibitory effect (p<0.05) on autolysis
development (K value assessment) in hake was also detected. Finally, an enhancement
of sensory scores (eyes, external odour and gills) in both species was obtained. Results
described allow to conclude that on-board employment of such acid-mixture icing
system can provide a profitable strategy to obtain higher quality and safe products while
unloading.
Keywords: Merluccius merluccius; Lepidorhombus whiffiagonis; on-board chilling;
citric acid; lactic acid; shelf life.
Running Title: On-board storage of commercial fish
2
1. INTRODUCTION
Deterioration of marine species begins immediately upon capture or harvest, and
the degree to which it continues depends directly on storage conditions. Flake ice has
been the most employed method to cool and store fish products and partially inhibit
detrimental effects on the commercial value. However, significant deterioration of
sensory quality and nutritional value has been detected in chilled fish as a result of
microbial and biochemical degradation mechanisms (Whittle et al., 1990; Beaufort et
al., 2009). To retard fish damage as long as possible and accordingly extend shelf life, a
wide number of preservative strategies to be combined with flake ice chilling have been
tested satisfactorily such as previous chemical treatment (Manju et al., 2007),
employment of preservative packaging (Ruiz-Capillas et al., 2001) and presence of
preservative compounds (ozone) (Pastoriza et al., 2008) or plant extracts (thyme
hydrosol, rosemary extract) (Oral et al., 2008; Özyurt et al., 2012) in the icing medium.
Among previous chemical treatments used during chilling storage, natural low
molecular weight organic acids and their sodium salts represent a relevant choice
because of their easy availability, low commercial cost and wide range of permitted
concentrations for use. Thus, citric acid (CA) is widely known for its role as a chelator
and an acidulant in biological systems; its presence has resulted in a profitable effect on
fish fillet (Badii and Howell, 2002; Kilinc et al., 2009) and whole fish (Aubourg et al.,
2004) quality. Further, lactic acid (LA) has been reported to be effective in preserving
and extending shelf-life for fish fillets (Kim et al., 1995; Metin et al., 2001) and coated
fish (Gogus et al., 2006).
Unlike other muscle food, fish are usually harvested in remote locations. Among
them, the Grand Sole North Atlantic fishing bank has been exploited by a wide number
of European countries. Due to the fast post-mortem deterioration of fish species, most
3
problems are encountered because the time elapsed between catching in these locations,
and arrival at the ultimate destination can reach a 14-17-day period. Consequently, the
threat of having fish condemned, withdrawn from sale, or sold at low prices at harbour,
may limit the length of the voyage (Aubourg et al., 2006; Barros-Velázquez et al.,
2008). As a result, substantial efforts are needed for the optimisation of the refrigeration
systems employed on-board to meet the increasing consumer demand for high quality
and safe, fresh products.
The present work is focused on the on-board storage and commercialisation of
two abundant fish species (European hake, Merluccius merluccius; megrim,
Lepidorhombus whiffiagonis.) from the Grand Sole bank. Its basic objective was the
quality enhancement of fish captured during the first period of the trawler trip. Previous
research carried out at laboratory level showed that quality loss could be inhibited in
both species when applying ice prepared from an aqueous solution of CA and LA
(García-Soto et al., 2013a, 2013b). With the aim of attaining a quality enhancement, an
aqueous solution including both acids was prepared and employed on-board as an icing
medium. Its effect on sensory, chemical and microbiological changes was monitored at
different catching times during a trawler trip.
2. MATERIAL AND METHODS
2.1. Icing systems
An aqueous solution containing 1.25 g l-1 of CA and 0.50 g l-1 of LA was
prepared, packed in polythene bags and kept frozen at –20ºC until use. Traditional ice
was prepared starting only from tap water that was packed and kept frozen in the same
way as the ice including both acids. Before addition to individual fishes, the two ices
were ground to obtain common flakes. Organic acids encountered in the present
4
research are regarded as safe (GRAS) for use in foods according to European and
American administrations (Madrid et al., 1994; Giese, 1996).
Previous research was conducted onshore to assess a convenient concentration
of CA and LA to prepare the ice (García-Soto et al., 2013a, 2013b). Thus, solutions
combining the two acids in the 0.05-2.50 g l-1 concentration range were preliminary
tested. According the evaluation of sensory, microbiological and chemical indices
related to quality loss, the above-mentioned combination of both acids (1.25 g l-1 of CA
and 0.50 g l-1) of LA was chosen.
2.2. Fish material, processing and sampling
European hake (Merluccius merluccius; length 32-35 cm, weight 180-210 g),
and megrim (Lepidorhombus whiffiagonis; length 20-23 cm, weight 95-120 g) were
captured in the Grand Sole North Atlantic fishing bank throughout a single trip (MayJune 2012). All fish were gutted immediately after catching, but none were beheaded.
For each fish species, individuals were distributed on-board into acid (treated batch, T)
or traditional (control batch, C) icing treatments. Individuals were surrounded by ice at
a fish/ice ratio of 1/1 (w/w) and stored on-board in a refrigerated room at 0-1ºC. Each
fish species was captured at three different times of the trip. At each sampling time, and
for both C and T batches, individuals were separated into three groups (three individuals
per group) in order to be analysed separately (n=3).
Once the fishing boat arrived at Vigo harbour, fish specimens were transported
to the laboratory to be analysed. Consequently, sensory, microbiological and chemical
analyses were performed after 9, 12 and 15 days of on-board chilled storage from
catching time. Sensory analysis was conducted on the whole fish, while microbiological
and chemical analyses were done on the white muscle.
5
2.3. Sensory analysis
Sensory analysis was conducted by a sensory panel consisting of five
experienced judges (three male and two female with an age in the 30-55 yr range),
according to traditional guidelines concerning fresh and refrigerated fish adapted to the
species under study (Council Regulation, 1996). Before starting the present experiment,
the panel was trained on chilled hake and megrim. In this training, evaluation of chilled
specimens belonging to the two species and corresponding to different chilling times
(from starting material until the time the fish was no more acceptable) and quality
degrees were tested. These preliminary chilled experiments were carried out several
times, with a marked attention to the evolution of each of the different sensory
descriptors; special emphasis was given to descriptors that were found as limiting
factors of acceptability (namely, eyes, gills and external and muscle odours).
According to the mentioned Council Regulation (1996) procedure, four
categories were ranked: highest quality (E), good quality (A), fair quality (B), and
unacceptable quality (C). Sensory assessment of the fish included the following
descriptors: skin and mucus development, eyes, external odour, gills appearance and
odour, consistency, flesh odour (raw and cooked) and flesh taste (cooked). At each
sampling time, the fish were presented to panellists and were scored individually by
panellists; each descriptor of each sample was scored a single time by each member of
the panel. The panel members shared samples tested.
2.4. Microbiological analyses
Samples of 10 g of fish muscle were dissected aseptically from chilled fish
specimens, mixed with 90 ml of 1 ml l-1 peptone in water (Merck, Darmstadt,
Germany), and homogenized in sterilized stomacher bags (AES, Combourg, France) as
6
previously described (Ben-Gigirey et al., 1998). In all cases, serial dilutions from the
microbial extracts were prepared in 1 ml l-1 peptone in water.
The number of aerobic mesophiles was determined by surface inoculation on
plate count agar (PCA; Oxoid Ltd., London, UK) after incubation at 30ºC for 48 h. The
anaerobe counts were also determined in PCA at 30ºC, except that an anaerobic
atmosphere kit (Oxoid) was placed together with the plates inside the anaerobiosis jar.
Psychrotrophs were also investigated in PCA, but incubation was performed at 7-8ºC
for 7 d. Enterobacteriaceae were investigated by pour plating using Violet Red Bile
Agar (VRBA) (Merck, Darmstadt, Germany) after incubation at 37ºC for 24 h.
Microorganisms exhibiting a proteolytic phenotype were investigated in casein-agar
medium after incubation at 30ºC for 48 h, as previously described by Ben-Gigirey et al.
(2000).
In all cases, bacterial counts were transformed into log CFU g-1 muscle before
undergoing statistical analysis. All analyses were performed in triplicate.
2.5. Chemical analyses
Total volatile base-nitrogen (TVB-N) values were measured as previously
reported (Aubourg et al., 2006). Briefly, fish muscle (10 g) was extracted with 60 g l -1
perchloric acid in water (30 ml) and brought up to 50 ml. An aliquot of the acid extracts
was rendered alkaline to pH 13 with 200 g l-1 aqueous NaOH and then steam-distilled.
Finally, the TVB-N content was determined by titration of the distillate with 10 mM
HCl. Results were expressed as mg TVB-N kg-1 muscle.
Trimethylamine-nitrogen (TMA-N) values were determined by the picrate
method, as previously described by Tozawa et al. (1971). This involved the preparation
7
of a 50 g l-1 aqueous trichloroacetic acid extract of fish muscle (10 g/25 ml). Results
were expressed as mg TMA-N kg-1 muscle.
Nucleotides were obtained by extraction with 60 g l-1 aqueous perchloric acid
solution and analysed by HPLC according to Aubourg et al. (2005). Standard curves for
adenosine 5’-triphosphate (ATP) and each compound involved in its degradation
pathway, adenosine 5’-diphosphate (ADP), adenosine 5’-monophosphate (AMP),
inosine 5’-monophosphate (IMP), inosine (INO) and hypoxanthine (HX), were
constructed in the 0-1 mM range. Results obtained for each degradation compound were
calculated as mmol kg-1 muscle. The K value was determined according to the following
concentration ratio: K value (%) = 100 x (INO + HX) / (ATP + ADP + AMP + IMP +
INO + HX).
2.6. Statistical analysis
Data obtained from the different microbial and chemical analyses were subjected
to the ANOVA method to explore differences in two ways: icing condition effect and
chilling time effect. For these analyses, the PASW Statistics 18 software for Windows
(SPSS Inc., Chicago, IL, USA) was employed. The comparison of means was
performed using the least-squares difference (LSD) method. Differences between
batches were considered significant for a confidence interval at the 95% level (p<0.05)
in all cases.
3. RESULTS AND DISCUSSION
3.1. Microbiological analyses
The evolution of aerobic mesophiles in hake and megrim muscle throughout the
storage time under study is shown in Figure 1. Statistically significant (p<0.05) lower
8
mesophile counts were determined in hake and megrim stored in the treated batch (T),
compared with the control batch (C). Remarkably, the average differences after 15 d of
storage for hake and megrim between C and T batches were 0.63 log CFU g-1 in both
cases. With respect to the anaerobes, the T batch also showed lower counts than the C
batch both in hake and megrim (Figure 2). Statistically significant (p<0.05) differences
between T and C batches were determined in hake. Remarkably, differences up to 1.98
log CFU g-1 in the anaerobe counts between T and C hake batches were observed on day
9, while such differences decreased to 0.95 log CFU g-1 units on day 15. With respect to
megrim, the T batch exhibited a better control of anaerobes at all storage times,
although the differences with respect to the C batch were only statistically significant
(p<0.05) on day 9.
The development of psychrotrophs exhibited statistically significant (p<0.05)
differences between T and C batches in the case of hake on day 9 (Figure 3). Moreover,
the psychrotroph counts were lower in the hake T batch at all storage times compared
with the C batch. In the case of megrim, statistically significant (p<0.05) differences
were also observed on days 12 and 15 (Figure 3). With respect to proteolytic bacteria,
statistically significant (p<0.05) differences were observed between T and C batches
only in the case of hake after 15 d of storage (Table 1). In the case of megrim, no
statistically significant (p>0.05) differences between batches were observed at any
storage time (Table 1). However, the megrim T batch exhibited lower counts of
proteolytic bacteria than the C batch, with the highest differences between batches being
determined on day 12 (0.43 log CFU g-1 units). The role of proteolytic bacteria in the
spoilage of fish muscle has been reported (Rodríguez et al., 2003). Accordingly, the
inhibition of this microbial group in hake muscle by the acids included in the icing
system is a remarkable result in terms of fish quality.
9
Finally, the Enterobacteriaceae counts revealed statistically significant (p<0.05)
differences between hake T and C batches at all storage times (Table 1); the highest
difference between both batches (0.97 log CFU g-1 units) was observed on day 9. With
respect to megrim muscle, lower counts of Enterobacteriaceae were determined in the T
batch at all storage times (Table 1), although such differences were not statistically
significant (p>0.05).
The results of the microbiological analysis indicated that the T batch,
corresponding to the icing system that included 1.25 g l-1 of CA and 0.50 g l-1 of LA,
significantly (p<0.05) slowed down the growth of all microbial parameters analysed in
the case of hake, and this led to an improved microbial quality of this fish species even
after 15 d of storage. Indeed, the aerobic mesophiles, anaerobes, proteolytic bacteria and
Enterobacteriaceae did not reach 7, 5, 4 and 5 log CFU g-1 units in the hake T batch,
respectively, while the C batch was above such numbers. In the case of megrim, a
similar significant (p<0.05) beneficial effect in the presence of both acids in the icing
system was observed for aerobic mesophiles and psychrotrophs. Therefore, and
according to the results of the microbial analysis, the presence of organic acids in the
icing system evaluated in this study significantly slowed down the growth of certain
microbial groups in fish muscle, this result being especially relevant for hake and to a
lesser extent, for megrim.
This microbial activity inhibition agrees to previous research carried out
onshore, where both species were kept under the same icing conditions (García-Soto et
al., 2013a, 2013b); in such experiments, 0-13-day storage periods were analysed for
both species. Likewise, other authors reported the effectiveness on microbial activity
inhibition by including preservative compounds and natural compounds in the icing
system. These account for ozone during on-board chilled storage of megrim (Pastoriza
10
et al., 2008), rosemary extract during chilling storage of sardine (Sardinella aurita)
(Özyurt et al., 2012) and wild-thyme hydrosol in chilled Transcaucasian barb (Capoeta
capoeta capoeta) (Oral et al., 2008).
Previous studies performed with ice slurries prepared from marine water
indicated that when the microscopic ice crystals melt, the salt solution exerts a washing
effect of the fish surface, which reduces the microbial load of the fish surface, thus
reducing microbial diffusion through the skin towards the muscle (Campos et al., 2005;
Aubourg et al., 2006). Likewise, the melting of the ice flakes prepared with CA and LA
in the present study may exert a similar washing effect, which would also prevent the
formation of biofilms in the fish surface, thus limiting fish spoilage.
3.2. Chemical analyses
Volatile amine formation was observed by measuring the TVB-N and TMA-N
contents (Table 2). In all types of fish samples, increasing values for both parameters
could be observed by increasing the chilling time (p<0.05). In the storage period
analysed, this increase was found more relevant for trimethylamine (TMA) formation.
TVB-N values did not differ when acid was present in the ice (p>0.05). Our
results indicate that none of the batches analysed reached the legal limit of 300-350 mg
kg-1 that was set for this index (Directive 95/149/EEC) (Baixas-Nogueras et al., 2003).
Concerning the TMA assessment, lower mean values were obtained in fish
corresponding to the acid-icing treatment; differences were found significant at days 12
and 15 for hake and at day 12 for megrim. It is worth pointing out that hake stored
under traditional ice surpassed the legal limit established for this species (5 mg kg-1)
(Baixas-Nogueras et al., 2003), while megrim samples did not attain its legal limit (12
mg kg-1; Directive 91/493/EEC) (Aubourg et al., 2006).
11
Volatile amine compounds are produced partially by means of endogenous
enzyme activity but mostly as a result of microbial development (Whittle et al., 1990).
In the present case, agreement was found between microbiological (aerobes, anaerobes,
psychrotrophs, proteolytics and Enterobacteriaceae) and chemical (TVB-N and TMAN) parameters related to microbial activity development during the chilled storage in
both fish species.
Previous research shows the inhibitory effect on amine formation as a result of
including a preservative component in the icing system. Thus, the presence of wildthyme tyrosol in the icing medium led to a marked decrease in TVB-N content
formation during chilled storage of Transcaucasian barb (Oral et al., 2008). However,
the inclusion of a rosemary extract in the icing medium did not lead to a TVB-N content
decrease, but produced a lower formation of biogenic amines during the chilled storage
of sardine (Özyurt et al., 2012). Oregano and rosemary extracts were also included
successfully in the icing medium during Chilean jack mackerel (Trachurus murphyi)
chilled storage (Quitral et al., 2009); thus, a lower TVB-N content was detected in fish
stored under plant extract icing. A lower amine formation (TVB-N and TMA-N) was
also obtained in hake and megrim by applying the present icing conditions during a 013-day storage period (García-Soto et al., 2013a, 2013b).
Autolysis was measured by means of the K value (%) (Table 2). A progressive
increase (p<0.05) with time was observed in all sample types. A lower mean value was
obtained for acid-iced fish than for control fish in both species; differences were found
significant in the case of hake (p<0.05) at the end of the experiment.
During post-mortem fish storage, muscle nucleotides are known to degrade in a
series of stages as a result of endogenous biochemical changes, and the level of adenine
nucleotides and their related compounds have been used extensively as an index of the
12
freshness (K value) of fish muscle (Whittle et al., 1990). K values obtained for both
species can be considered as relatively low, especially in the case of hake. According to
the present results, hake muscle has already shown a low K value increase during
chilling storage (Losada et al., 2004).
3.3. Sensory analysis
Sensory evaluation was performed according to attributes mentioned in the
experimental section, and results are expressed in Table 3. For all types of fish samples,
a quality decrease was detected by the panel as a result of increasing the chilling time.
An extended shelf life time was obtained for hake stored under the acid-icing
system when compared to its traditional icing counterpart. Thus, control hake was found
unacceptable at day 15, while preserved hake was still valuable at that time. The
limiting factor was the flesh odour, both under raw and cooked conditions; better scores
were also obtained in treated hake when considering other attributes such as external
odour and gills appearance and odour. This sensory quality enhancement is in
agreement with the results previously mentioned for microbiological and chemical
quality indices. Previous research on chilled European hake (Ruiz-Capillas and Moral,
2001) observed longer shelf life times (20-25 days) than in the present work, which can
be explained by the relative smaller size of the hake specimens examined in the present
study in agreement with previous research (Rodríguez et al., 2003; Losada et al., 2004).
In the case of megrim, the effect of the acid presence in the ice on sensory
attributes was found lower than for hake. Both treated and control fish were found
acceptable at day 15; however, a better score was given to treated megrim at day 9 for
the external odour and gills appearance and odour and at day 15 for the eyes
appearance. This slight quality enhancement for the sensory appreciation is in
13
agreement with the microbiological and chemical results mentioned above.
Additionally, the sensory evaluation obtained for megrim is in agreement with a
previous on-board experiment (Aubourg et al., 2006); in it, megrim was acceptable at
day 16 under traditional icing and unacceptable at day 20. The differences found
between hake and megrim can be explained in terms of the longer shelf life that megrim
exhibits (Aubourg et al., 2006) with respect to hake (Rodríguez et al., 2003; Losada et
al., 2004). The fact that hake is a more perishable fish species than megrim may explain
that the improvement of storage conditions, in this case through the incorporation of LA
and CA in the icing medium, may exert a more significant effect in hake as compared to
megrim.
Previous research demonstrated an increased shelf life and a sensory quality
enhancement by means of including preservative compounds in the icing system. This is
the case with oregano and rosemary extracts during the chilled storage of Chilean jack
mackerel (Quitral et al., 2009), a wild-thyme hydrosol extract during the chilled storage
of Transcaucasian barb (Oral et al., 2008), a rosemary extract during the sardine chilling
storage (Özyurt et al., 2012), and ozone during the on-board chilled storage of megrim
(Pastoriza et al., 2008).
4. CONCLUSIONS
The presence of CA and LA in the icing medium led to a deteriorative activity
inhibition and a quality enhancement of hake and megrim during the on-board chilled
storage. A lower bacterial growth in both fish species has been detected according to
microbiological (aerobe, anaerobe, psychrotroph, proteolytic, and Enterobacteriaceae
counts) and chemical (namely, TMA-N) assessments that have led to an enhancement of
sensory appreciation. In this study, the acid icing showed to be more effective in hake
14
than in megrim, this result being explained in terms of the shorter shelf life of hake as
compared to megrim. Results described here allow us to conclude that on-board
employment of a CA-LA-icing system can provide a profitable strategy to obtain higher
quality and safer products so that increased commercial value while unloading and sale
can be attained.
ACKNOWLEDGEMENTS
The authors thank Mr. Alberto Fernández, Mr. Marcos Trigo and Mrs. Cristina
Nine for their excellent technical assistance and the Cachacho boat owners and staff for
their essential support to conduct the present study. This work was supported by the
Secretaría Xeral de I+D from the Xunta de Galicia (Galicia, Spain) through the
Research Project 10 TAL 018 E.
15
FIGURE LEGENDS
Figure 1
Aerobic mesophile count assessment* in chilled hake and megrim stored on-board
under different icing conditions**
* Mean values of three (n=3) replicates; standard deviations are indicated by bars. For
each species and chilling time, values accompanied by different letters (A, B)
denote significant differences (p<0.05) as a result of the icing condition. No
indication is provided when no significant differences are found (p>0.05).
** Icing conditions: Control batch (ice prepared only from water) and treated batch (ice
including the organic-acid mixture).
Figure 2
Anaerobe count assessment* in chilled hake and megrim stored on-board under
different icing conditions**
* Mean values of three (n=3) replicates; standard deviations are indicated by bars. For
each species and chilling time, values accompanied by different letters (A, B)
denote significant differences (p<0.05) as a result of the icing condition. No
indication is provided when no significant differences are found (p>0.05).
** Icing conditions as expressed in Figure 1.
16
Figure 3
Psychrotroph count assessment* in chilled hake and megrim stored on-board under
different icing conditions**
* Mean values of three (n=3) replicates; standard deviations are indicated by bars. For
each species and chilling time, values accompanied by different letters (A, B)
denote significant differences (p<0.05) as a result of the icing condition. No
indication is provided when no significant differences are found (p>0.05).
** Icing conditions as expressed in Figure 1.
17
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