Effects of modified atmosphere and vacuum on the shelf life of tilapia (Oreochromis niloticus) fillets

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AUTORES
AUTHORS
PALAVRAS-CHAVE
KEY WORDS
Braz. J. Food Technol., v.8, n.1, p. __-__, jan./mar., 2005 7Recebido / Received: 10/12/2002. Aprovado / Approved: 13/01/2005.
Effects of Modified Atmosphere and
Vacuum on the Shelf Life of Tilapia
(
Oreochromis niloticus
) Fillets
Efeitos da Atmosfera Modificada e do
Vácuo sobre a Vida Útil de Filés de
Tilápia (
Oreochromis niloticus
)
SUMMARY
The objective of the present work was to develop a convenience product, minimally
processed tilapia, by determining its shelf-life from the monitoring of its chemical properties,
as well as performing sensory and microbiological evaluations. With this intent, aquacultured
Nile tilapia (
Oreochromis niloticus
) raised on farms located in the Jau region, São Paulo State,
Brazil, was depurated, gutted, filleted, packed in polystyrene trays and covered with EVOH
(copolymer of ethylene of vinyl alcohol film) (control). Some of the fish were chemically treated
by immersion in 1% acetic acid and packed under a 60%CO2/40%O2 modified atmosphere
(MAP) and vacuum packing and some of the fish were just packed under 60%CO2/40%O2
modified atmosphere (MAP) and vacuum packing. The packaged samples were analyzed at
the beginning of the experiment and after 7, 13 and 20 days of refrigerated storage at around
1±1°C. No significant differences amongst the treatments were found for non-protein nitrogen
- NPN, total volatile base nitrogen - TVB-N or pH. The use of MAP, whether associated with
acetic acid or not, promoted greater amounts of thiobarbituric acid reactive substances -
TBARS. Moreover, rancidity was detected in the sensory analysis of the MAP fillets. The fillets
treated with acetic acid + EVOH, MAP and MAP associated with acetic acid were discolored
and presented a softer texture, when compared to the non-treated ones.
Salmonella
, sulfite-
reducing
Clostridium
and
E. coli
were not detected during the storage period. MAP and vacuum
packing, associated with acetic acid inhibited the development of
S. aureus
, total coliforms and
psychrotrophic bacteria, lengthening the shelf life by 20 days. Vacuum packing associated with
acetic acid was the treatment that promoted the best chemical, microbiological and sensory
stability up to the end of the experiment.
Marcilene C. Heidmann SOCCOL
Mestre em Ciência e Tecnologia de Alimentos
Esalq/USP, Piracicaba-SP
Bolsista da FAPESP
mchsocco@esalq.usp.br
Marília OETTERER
Profa associada do Departamento de Agroindústria,
Alimentos e Nutrição, Esalq/USP.
moettere@esalq.usp.br
Cláudio Rosa GALLO
Prof. Dr. do Departamento de Agroindústria,
Alimentos e Nutrição, Esalq/USP.
Marta Helena Fillet SPOTO
Profa. Dra. do Departamento de Agroindústria,
Alimentos e Nutrição, Esalq/USP.
Denise Oliveira BIATO
Mestranda em Ciência e Tecnologia de Alimentos, Esalq/USP.
Endereço para correspondência:
Av. Dom Daniel Hostin, 462.
88508-200 – Lages, SC.
Telefone: (49) 91036165 / (49) 2255082
Órgão Financiador: Fundação de Amparo à
Pesquisa do Estado de São Paulo (Fapesp)
Fresh-water fish, tilapia, minimum processing,
modified atmosphere, vacuum packing,
acetic acid.
Pescado de água doce, tilápia, minimamente
processado, atmosfera modificada,
embalagem a vácuo, ácido acético
RESUMO
Este trabalho teve como objetivo a obtenção de um novo produto, do tipo alimento
de conveniência – tilápia minimamente processada –, estabelecendo a vida útil deste, por
monitoramento dos componentes físico-químicos e avaliação sensorial e microbiológica. Para
isso, foram utilizados peixes provenientes de áreas de cultivo do Estado de São Paulo, da
região de Jaú, da espécie
Oreochromis niloticus
, os quais foram submetidos à depuração,
eviscerados, filetados e embalados em bandejas de poliestireno, recobertos com filmes de
EVOH (controle); parte dos peixes foi submetida ao tratamento químico, por imersão em
ácido acético 1%, e embalada sob atmosfera modificada (EAM) (60% CO2+40% O2) e a vácuo.
As amostras embaladas foram mantidas sob refrigeração, na faixa de 1±1°C, quando foram
submetidas às análises, no início do experimento: 7, 13 e 20 dias. Os tratamentos aplicados
não apresentaram diferenças significativas para NNP, BNVT e pH. Os produtos embalados
em EAM, associados ou não ao ácido acético, apresentaram valores mais elevados de TBA,
sendo detectada a presença de ranço pelos provadores. Os filés tratados com ácido acético
apresentaram-se descoloridos e com textura mais macia em relação aos demais tratamentos.
Durante o período de armazenamento não se detectou a presença de
Salmonella
,
Clostridium
sulfitorredutores e
E. coli
. O embalamento em EAM e a vácuo associados ao ácido acético
inibiram o desenvolvimento de
S. aureus
, coliformes totais e psicrotróficos, proporcionando
uma vida útil de 20 dias. Porém o embalamento a vácuo associado ao ácido acético foi o
tratamento que manteve as características físico-químicas, microbiológicas e sensoriais mais
estáveis até o término do experimento.

Braz. J. Food Technol., v.8, n.1, p. 7-15, jan./mar., 2005 8
SOCCOL, M.C.H.
et al.
Effects of Modified Atmosphere and
Vacuum on the Shelf Life of Tilapia
(
Oreochromis niloticus
) Fillets
Braz. J. Food Technol., v.8, n.1, p. 7-15, jan./mar., 2005 9
SOCCOL, M.C.H.
et al.
Effects of Modified Atmosphere and
Vacuum on the Shelf Life of Tilapia
(
Oreochromis niloticus
) Fillets
1. INTRODUCTION
Fish production in Brazil has been increasing as a result
of the expansion in freshwater aquaculture activities. Due to the
country’s great freshwater potential, fish cannot be regarded
only as an excellent source of food, but also as a source of
exportation revenue (OETTERER,, 1991). Fish has a relatively
short shelf life (12 days) under refrigerated conditions, not
presenting hygienic quality hazards when properly packaged
(OETTERER, 1999).
Fish shelf life is determined by the intensity of enzymatic
reactions and by the number and species of microorganisms,
affecting the product’s perishability. Other determining
characteristics are the storage temperature, which must be
evaluated throughout the several production stages, the
temperature during capture, delay in refrigeration, variation
in the storage temperature and the retail temperature. Food
preservation is based on combined methods, which can be
used for the quality improvement of conventional products or
the development of new products. They assure stability and
safety, resulting in products presenting adequate sensory and
nutritional properties (LEISTNER, 1992).
The renewal of interest in extending fish shelf-life is due
to the increasing demand for fresh products which, in turn,
has led to a greater diversification of products packed under
modified atmosphere, in which the air composition is changed
or modified. Extending fish shelf life is greatly advantageous to
industry, as it reduces losses during product distribution and
display, which may result in marketing improvements for fresh
products and in a regular supply at reasonable prices (LIOUTAS,
1988).
The techniques for modified atmosphere packed
products involve the use of several gases, such as CO2, N2 and
O2, CO2, whether alone or associated with other gases, being
the most effective and common amongst them.
CO2 is the gas with the greatest bacteriostatic effect in
modified atmosphere packaging. Such an effect is influenced
by the CO2 concentration, initial bacterial population, storage
temperature and product type (REDDY
et al
., 1992).
In general O2 may inhibit the growth of exclusively
anaerobic bacteria, although anaerobic microorganisms
show different sensitivity levels to oxygen (FARBER, 1991).
Davis (1995) supports the use of O2 in modified atmosphere
packaging for fish, stating that there is evidence that the use of
O2 reduces exudation in fish during storage. Many researchers
are increasingly concerned about the increase in the growth
potential of
Clostridium
in MAPs (modified atmosphere
packaging) and vacuum packaging. This concern is justified
due to the pathogenic importance of such a microorganism.
Clostridium botulinum
produces a neurotoxin that causes facial
paralysis, and is classified into A, B, C, D, E, F and G types.
The A, B and F types are important to humans (HINTLIAN &
HOTCHKISS, 1986).
Some research has claimed that storing fish under
modified atmosphere delays bacterial growth and increases the
shelf life. A variable mixture of gases has been used to inhibit
microbial growth in MAP systems (LINDSAY, 1981). Different
species of fish, storage temperatures and MAPs have been
used. Generally, MAPs using high CO2 proportions enhance
the stability of fresh fish, extending its shelf life (BAKER
et al
.,
1986).
REDDY
et al
. (1994, 1995) evaluated the effect of
modified atmospheres (75%CO2/25%N2; 50%CO2/50%N2;
25%CO2/75%N2) on the shelf life of tilapia (
Tilapia
spp) fillets
packed in high barrier film at 4°C. The authors observed that
tilapia fillets packed in 75%CO2/25%N2 showed an increased
shelf life of more than 25 days, presenting acceptable sensory
characteristics.
SIVERTSVIK
et al.
(1999) studied the quality of
refrigerated (≤1°C) gutted salmon (
Salmo salar
) stored in plastic
bags containing 50% and 100% CO2 and 60%CO2/40%O2, as
well as in conventional packaging material (polystyrene), during
transport. The authors observed that the microbial growth was
greater in the conventionally packed salmon. The MAP salmon
presented better sensory quality than the conventionally packed
one after 13 days of storage.
Whole gutted hake (
Merluccius merluccius
) was ice
stored in boxes under controlled atmospheres (CA) presenting
different gas mixtures (60%CO2/15%O2/25%N2; 40%CO2/
40%O2/ 20%N2; 60%CO2/40%O2 and 40%CO2/60%O2) for 33
days at 0±1°C. Using physical-chemical and sensory analyses,
RUIZ-CAPILLAS & MORAL (2001) found that the 60%CO2/40%O2
CA promoted a better product as to sensory acceptance, being
more effective than the other mixtures.
The use of good hygienic-sanitary quality raw materials
represents an important factor for the successful use of modified
atmosphere packaging. In addition, the observance of good
hygiene practices during fishing, the selection of the right
packaging material, an adequate gas proportion and good
temperature control are also necessary (STAMMEN
et al
.,
1990).
Some studies on the possibility of combining MAP with
preservatives to preserve fresh fish have been conducted, in
order to develop fish products presenting better quality and
longer shelf life. Acetic acid and its salts are very efficient and
widely used as acidulating agents and preservatives for food.
The presence of 1-2% of non-dissociated acid in meat, fish or
vegetable products is generally sufficient for bacterial inhibition,
as long as good hygiene practices are observed (PARDI
et al
.,
1994).
According to MAREL
et al
. (1988), the superficial
application of organic acids is used for meat decontamination,
aiming mainly at reducing the deteriorating and pathogenic
microorganisms naturally found in food.
The purpose of the present work was to develop
a convenience product, minimally processed tilapia, by
determining its shelff life through monitoring its chemical
properties, as well as performing sensory and microbiological
evaluations.

Braz. J. Food Technol., v.8, n.1, p. 7-15, jan./mar., 2005 8
SOCCOL, M.C.H.
et al.
Effects of Modified Atmosphere and
Vacuum on the Shelf Life of Tilapia
(
Oreochromis niloticus
) Fillets
Braz. J. Food Technol., v.8, n.1, p. 7-15, jan./mar., 2005 9
SOCCOL, M.C.H.
et al.
Effects of Modified Atmosphere and
Vacuum on the Shelf Life of Tilapia
(
Oreochromis niloticus
) Fillets
2. MATERIAL AND METHODS
Aquacultured Nile tilapia (
Oreochromis niloticus
) raised
in farms located in the Jau region, São Paulo State, Brazil, was
used. The fish was harvested from net-ponds and depurated in
ponds under running water for an average period of 78 hours.
The fish were then placed, still alive, directly into polystyrene
boxes, in layers interleaved on the ice prepared from drinking
water, where they suffered thermal shock.
2.1 Fish preparation
The fish were gutted, skinned and filleted. Part of the
fillets underwent chemical treatment by immersion in 1% acetic
acid at the proportion of 1.2:1, that is, 1.2 kg of fish per 1 liter
of solution, for 2 minutes at room temperature; the remaining
fish was immersed in distilled water under the same conditions.
The fillets were then laid on racks for 2 minutes to drain the
solution.
2.2 Product packing
The fillets were arranged in polystyrene trays, about
500 g in each package, wrapped in 6.92 µm thick EVOH
(copolymer of ethylene and vinyl alcohol film) with an oxygen
transmission rate for O2 of 28.18 cm3/m2/day at 23 °C, 80% RH
and 1 atm. Soon after it was heat sealed under atmospheric
air (control). Another lot was packed under 60%CO2/40%O2
modified atmosphere at a 2:1 (gas/fish) proportion, that is,
1000 mL of gas mixture per 500 g of fish; a third lot was
vacuum packed in EVOH (copolymer of ethylene and vinyl
alcohol film) (635 mmHg) in a TEC MAQ automatic vacuum
sealer AP-500. The products were kept at 1±1°C and evaluated
at the beginning of the experiment and after 7, 13 and 20 days
of refrigerated storage.
2.3 Microbiological analyses
The microbiological analyses for each treatment were
carried out in triplicate. A rinsing technique was used for the
microbiological determinations in which 100 g of tilapia were
filleted to about 0.5 cm thick, soaked in 100 mL of buffered
peptone water (1:1) and agitated. One mL of rinsing solution
was used for each gram of fish. Dilutions from 10–1 to 10–4 were
achieved by diluting each sample with 1% peptone water. The
methodology used for the analyses was that recommended by
VANDERZANT & SPLITTSTOESSER (1992).
Sulfite-reducing
Clostridium
: petri dishes containing
Tryptose Sulfite Cycloserine (TSC) agar were incubated at 46 °C
for 24-48 hours in an anaerobic atmosphere using an Anaerobac
jug (Probac).
Total coliforms and
Escherichia coli
: the total coliform and
E. coli
counts were according to the “Simplate Test Procedures”,
which uses kits of disposable dishes containing substrate
suitable for each microorganism. The chart accompanying the
kit was used to determine the most probable number (MPN)
for total coliforms and
E. coli
per g of fish.
Staphylococcus aureus
: petri dishes containing Baird-
Parker Agar (BPA) were used with the inoculum being spread
on the agar surface using a Drigalsky loop. The dishes were
incubated at 35-37 °C for 24-48 hours. In order to confirm
the typical colonies, Gram, catalase and coagulase tests were
performed.
Psychrotrophic bacteria: petri dishes containing Standard
Agar were used for the PCA count. The dishes were incubated
for 10 days at 7 °C.
Salmonella
spp: the Oxoid
Salmonella
Rapid Test kit was
used for the presumptive detection of
Salmonella
. A serologic
test using the Oxoid Latex Test (polyvalent latex agglutination)
was carried out to confirm the presence of
Salmonella
(SILVA
et al
., 1997).
2.4 Chemical analyses
Non-protein nitrogen (NPN): Determined by the
precipitation of muscle proteins using trichloroacetic acid
(TCA), followed by evaluation of non-protein nitrogen in the
TCA extract using the Micro Kjeldhal method, according to
AOAC (1995).
Total volatile base nitrogen (TVB-N): Determined by
protein precipitation using trichloroacetic acid (TCA) and
evaluation of the total volatile base nitrogen in the TCA extract
using the Micro Kjeldhal method, according to MORGA (1975).
Thiobarbituric acid reactive substances (TBARS):
Determined by the precipitation of proteins associated with
lipids and phospholipids. The spectrophotometer reading was
taken at 535 nm, using a 7.8 conversion factor to transform
mg of malondialdehyde to kg of food, according to TARLADGIS
et al
. (1960).
pH: determined by means of a Digimed digital
potentiometer, using muscle homogenized in distilled water in
a proportion of 1:1
2.5 Sensory analysis
Fillets were sensory evaluated by a team of 30 untrained
tasters, divided into three blocks of 10 tasters each. To assess
the acceptance test (color, aroma, texture and appearance),
a scoring system ranging from “liked extremely” (score 9)
to “disliked extremely” (score 1) was used, according to
DUTCOSKY (1996). The experimental design adopted was one
of randomized incomplete blocks with 5 replications and 3
samples per block, with each taster corresponding to a block,
according to COCHRAN & COX (1964).
2.6 Statistical analysis
The experimental design adopted was the 6x4x3 factorial
randomized block design, and the variable treatments studied

Braz. J. Food Technol., v.8, n.1, p. 7-15, jan./mar., 2005 10
SOCCOL, M.C.H.
et al.
Effects of Modified Atmosphere and
Vacuum on the Shelf Life of Tilapia
(
Oreochromis niloticus
) Fillets
Braz. J. Food Technol., v.8, n.1, p. 7-15, jan./mar., 2005 11
SOCCOL, M.C.H.
et al.
Effects of Modified Atmosphere and
Vacuum on the Shelf Life of Tilapia
(
Oreochromis niloticus
) Fillets
were the storage periods and the interaction among them. The
data were submitted to an analysis of variance using the PROC
GLM of the SAS 8.0 (1999) software. The means were compared
with one another using the Tukey test at 5% probability.
3. RESULTS AND DISCUSSION
3.1 Chemical analyses
The data for NPN, TVB-N, TBARS and pH in tilapia fillets
during the storage period at 1±1 °C are shown in Table 1.
3.2 Non-protein nitrogen (NPN)
The values for NPN were not significantly affected
(p>0.05) by the treatments and the storage period. They
showed irregular behavior (increases and decreases) during
the storage period (Table 1). According to MUJICA (2000),
a probable explanation for such variations could be the
simultaneous use of the substances generated by the microbial
activity on sulfurated amino acids, especially those in their free
form, such as the components of the NPN pool, causing their
reduction, while generating and replacing several non-protein
constituents as a result of autolytic reactions and proteolytic
activity of microorganisms.
The lower initial values for NPN observed in the control
may be due to the consumption of nitrogenous compounds
by the existing bacterial flora. According to LOAIZA (1996), the
initial drop in NPN amounts is evidence of an initial consumption
of low molecular weight nitrogenous compounds by the existing
bacterial flora before an intense proteolysis starts. Only when
the existing nitrogenous compounds are insufficient to attend
their nutritional needs, will the microorganisms start to degrade
proteins.
MOORJANI
et al
. (1962), studying several fresh-water
fish species stored in ice, observed a decrease in NPN amounts
during 16 days of storage. The authors concluded that if the
proteins had been hydrolyzed by bacterial and enzymatic action,
the NPN amount would have increased significantly during the
storage period.
3.3 Total volatile base nitrogen (TVB-N)
The TVB-N values for all the treatments were below
the limit established by the Brazilian legislation (30 mg/100 g)
(BRASIL, 2002). High initial values followed by decreases
were observed for some treatments. However, they were not
significantly (p>0.05) affected by the treatments and storage
periods (Table 1). According to YEH
et al
. (1978), such initial
increases in the ammonium content may be initially offset by
lixiviation, especially if the exposed area is large (fish fillets or
slices). However, after some days, the increase becomes evident
and generally coincides with the increase in pH. The more
alkaline the medium becomes, the more desaminase activity
is favored.
The 30 mg/100 g relation has been shown to be
compatible with other evaluation parameters, leading some
countries, including Brazil, to adopt this relationship as the
maximum limit for commercialization (BRASIL, 2002). In turn,
KUAYE (1982) reported that the amounts of TVB-N varied as
a function of the methodology used, fish species and fish
alteration stage. The amounts vary little for fresh-water fish, in
which important changes are not observed, even after several
days under storage on ice.
BANKS
et al
. (1980) stated that the differences in TVB-N
amounts must have been caused by a smaller number of bacteria
and/or their lower ability to act on the oxidative desamination of
non-protein nitrogen compounds. A second explanation refers
to the anaerobic conditions found in the CO2 MAP, as they may
inhibit this reaction due to the lack of atmospheric oxygen. The
kind of bacteria present may depend on atmospheric and pH
alterations caused by the absorption of CO2. Such bacteria show
low ability to produce ammonia, when compared to ordinary
deteriorating bacteria. Therefore, the deterioration mechanism
of CO2 MAP fish is extremely different from the one observed
for ice stored fish, and the TVB-N production is not a good
deterioration index.
3.4 Thiobarbituric acid reactive substances (TBARS)
The TBARS amounts were significantly (p<0.05) affected
by the treatments and storage periods, with interactions being
observed between these factors (Table 1). The products packed
under MAP, whether associated with acetic acid or not, presented
higher TBARS amounts, differing from the other treatments
(p<0.05). Rancidity started being sensorially detected by tasters
on the 7th day of storage (1.36 mg MA/kg).
The TBARS values observed for the vacuum packing
treatment were lower at the end of the storage period, when
compared to the values obtained for the other treatments. This
was probably caused by the absence of O2, which retarded
the oxidative process of the polyunsaturated fatty acids in this
treatment.
Oxidative rancidity may be a problem in O2 modified
atmosphere packing and is caused by the oxidation of
polyunsaturated fatty acids. O2 reacts with the fatty acids to
produce hydroperoxide without degrading the odoriferous
components (CHURCH, 1998).
RUIZ-CAPILLAS & MORAL (2001) observed greater TBARS
amounts for atmospheres richer in CO2. This was probably due
to a synergistic action between CO2 and O2, which made the
autoxidation of polyunsaturated fatty acids easier. Yet, FEY &
REGENSTEIN (1982), studying hake (
Merluccius merluccius
) and
salmon (
Salmo
spp) stored under 60% CO2 at 1 °C, suggested
that the changes in TBARS amounts were not significant.

Braz. J. Food Technol., v.8, n.1, p. 7-15, jan./mar., 2005 10
SOCCOL, M.C.H.
et al.
Effects of Modified Atmosphere and
Vacuum on the Shelf Life of Tilapia
(
Oreochromis niloticus
) Fillets
Braz. J. Food Technol., v.8, n.1, p. 7-15, jan./mar., 2005 11
SOCCOL, M.C.H.
et al.
Effects of Modified Atmosphere and
Vacuum on the Shelf Life of Tilapia
(
Oreochromis niloticus
) Fillets
3.5 pH
The pH values were significantly affected (p<0.05)
by the treatments, but not (p>0.05) by the storage periods
(Table 1), remaining stable and varying very little during the
storage period. Only the control showed pH values above the
legal limit after 13 days of storage, which led to an increase in
psychrotrophic organisms to levels above 106 CFU/g after 20
days of storage, evidencing proteolytic and lipolytic activities.
The Official Regulations on Industrial Sanitation and
Inspection of Animal Products – RIISPOA (BRASIL, 2002)
established the maximum pH limit for the internal part of fresh
fish to be 6.5. Based on the results, this criterion seems to be
suitable for the qualitative evaluations of minimally processed
tilapia.
According to SIKORSKI
et al.
(1994), the pH stability
might have been caused by the buffering effect of the fish
muscle. This effect is attributed to the presence of soluble
proteins, peptides, amino acids, ammonia, trimethylamine and
low molecular weight substances in the fish muscle, which may
mask pH changes, slowly increasing pH values at the beginning
of the deterioration process and more quickly toward its end.
DEBEVERE & BOSKOU (1996) reported that CO2
diffusion in fish muscle showed a somewhat contrary effect
to that of the increase in pH due to the production of TVB-N,
resulting in pH stabilization.
The pH decrease observed for the vacuum plus acetic
acid (6.1-6.0) and the MAP plus acetic acid (6.2-6.1) treatments
on the 7th and 13th days of storage, respectively, was related to
the decrease in psychrotrophic bacteria count, probably due to
the antimicrobial action of the acetic acid and the CO2 in the
fish muscle.
3.6 Microbiological analyses
3.6.1 Total coliforms and
E. coli
Total coliform counts in the tilapia fillets during the
storage period were not significantly affected (p>0.05) by the
treatments and storage period (<2 - 3.3x101). The only treatment
promoting the growth of total coliforms was vacuum packing
(0.3 - 1.0x102), while the others presented a decrease in growth,
probably due to the combination of the low temperatures and
use of acetic acid, as well as a low contamination level in the
water from the net-ponds.
VIEIRA
et al
. (1986), studying ice stored lobsters
(
Pacifastacus
spp), also failed to observe a progressive increase
in the number of total and fecal coliforms, which presented an
irregular growth behavior. This may have happened because
fecal coliforms are mesophyllic and, therefore, are inhibited by
low temperatures (0 °C) (MUJICA, 1988).
REDDY
et al.
(1994) reported that CO2 amounts ≥ 50%
delayed coliform growth in tilapia (
Tilapia
spp) fillets stored
under MAP.
E. coli
was not detected in any of the samples during
the storage period. This was probably because the tilapias
TABLE 1. Chemical parameters and pH of tilapia fillets stored at 1±1 °C for 20 days.
Days Control Acetic acid + EVOH Vacuum Vacuum + acetic Acid MAP MAP + Acetic Acid
NPN (mg/100 g)
1 534.72Aa 642.76Aa 590.76Aa 581.59Aa 635.29Aa 614.02Aa
7 534.63Aa 560.35Aa 590.29Aa 522.48Aa 536.25Aa 493.64Aa
13 595.96Aa 522.17Aa 589.81Aa 533.01Aa 568.21Aa 524.86Aa
20 568.52Aa 538.64Aa 525.90Aa 517.26Aa 511.82 Aa 470.13 Aa
TVB-N (mg/100 g)
1 14.00Aa 13.07Aa 14.70Aa 12.13Aa 13.07Aa 12.13Aa
7 13.30Aa 11.20Aa 12.60Aa 13.07 Aa 11.90Aa 12.13Aa
13 14.00Aa 12.37Aa 15.17Aa 14.00 Aa 13.07Aa 13.07Aa
20 18.90Aa 17.97Aa 17.03Aa 17.03 Aa 14.93Aa 14.93Aa
TBARS (mg MA/kg)
1 0.04Aa 0.84Aa 0.00Aa 0.71Aa 0.57Aa 0.79Aa
7 0.78Aa 1.58Aa 0.17Aa 0.73Aa 1.36Aa 3.00Aab
13 0.79Aa 2.38Aa 0.57Aa 0.93Aa 2.22Aa 6.16Bbc
20 0.98Aa 2.56Aa 0.48Aa 1.29Aa 8.07Bb 9.23Bc
pH
1 6.5Aa 6.2Aa 6.4Aa 6.1Ba 6.4Aa 5.9Ba
7 6.4Aa 6.1Aa 6.4Aa 6.0Ba 6.4Aa 6.2Aa
13 6.6Aa 6.2Ba 6.5Aa 6.2Ba 6.5Aa 6.1Ba
20 6.6Aa 6.3Aa 6.5Aa 6.2Ba 6.5Aa 6.2Ba
*Means followed by the same lowercase letters (columns) or the same capital letters (lines) do not differ according to theTukey test at 5%.

Braz. J. Food Technol., v.8, n.1, p. 7-15, jan./mar., 2005 12
SOCCOL, M.C.H.
et al.
Effects of Modified Atmosphere and
Vacuum on the Shelf Life of Tilapia
(
Oreochromis niloticus
) Fillets
Braz. J. Food Technol., v.8, n.1, p. 7-15, jan./mar., 2005 13
SOCCOL, M.C.H.
et al.
Effects of Modified Atmosphere and
Vacuum on the Shelf Life of Tilapia
(
Oreochromis niloticus
) Fillets
underwent a depuration period, promoting fish gut cleaning
and avoiding contamination during fishing. Besides, the water
used in the fish ponds showed low contamination levels by
this kind of bacteria. Similar results were also obtained by LIMA
et al
. (1998).
3.6.2 Psychrotrophic bacteria
Figure 1 presents the psychrotrophic bacterial count (log
CFU/g) during the storage period, which was not significantly
(p>0.05) affected by the treatments or by the storage period.
The development of psychrotrophic bacteria increased
during the storage period, with values above 106 CFU/g being
verified for the control. Although the limits for psychrotrophic
bacteria are not within the legislative scope, such high counts
for this group of bacteria must contribute to the reduction in
product shelf-life. The increase in the psychrotrophic bacterial
count for the control is also related to the increase in pH,
despite the product being sensorially accepted by the tasters,
who considered the appearance to be the most important
attribute.

   















FIGURE 1. Psychrotrophic bacterial count (log CFU/g) in tilapia
fillets stored at 1±1 °C for 20 days.
Vacuum and MAP packing associated with acetic acid,
presented a decrease in the bacterial count on the 7th and
13th days of storage, respectively, the MAP packing being the
treatment presenting the lowest count at the end of the storage
period. Probably, this fact was a consequence of the high CO2
concentration associated with the use of acetic acid, as both
show antimicrobial action.
The values obtained for treatments using acetic acid +
EVOH and vacuum, as well as for the control, were similar to
those found by RANDELL
et al
. (1999), when studying salmon
(
Salmo salar
) fillets stored at 2°C under 60%CO2/40%N2 MAP
and vacuum packs.
SILLIKER & WOLFE (1980) observed that high CO2
concentrations inhibited the growth of psychrotrophic
microorganisms when the fish was stored under low
temperature conditions, evidencing that psychrotrophic bacteria
are sensitive to CO2. REDDY
et al
. (1992) and SILVA
et al
. (1993)
verified that the lag phase was retarded and growth of such
deteriorating bacteria reduced by CO2.
According to CAI
et al.
(1997) a change in environment
may result in the growth of facultative anaerobic and anaerobic
bacteria in overwrapped packages, since there may be free O2
between the fillets, and the gas barrier properties of the EVOH
film may have been compromised by its sensitivity to the high
moisture content of the product.
3.6.3 Staphylococcus aureus
None of the treatments showed counts above the
Brazilian legislation limits (Table 2). The
S. aureus
count was not
significantly (p>0.05) affected by the treatments or the storage
period. All the treatments presented a decrease in the
S. aureus
count, except for the vacuum treatment, which allowed bacterial
growth up to the 7th day of storage, probably due to sampling
problems. This result differs from those obtained by PASSY
et al
.
(1983), who observed an increase in
S. aureus
growth, from 101-
102 log CFU/g to 103-104 log CFU/g, in prawns (
Macrobrachium
rosenbergii
) stored under CO2, after 12 days of storage. The
Compendium of Norms and Standards for Food (BRASIL, 2001)
established the following microbiological standards for fresh
refrigerated fish: a maximum of 103
Staphylococcus
coagulase
(+)/g of fish.
3.6.4 Salmonella spp and sulfite-reducing
Clostridium
The Compendium of Norms and Standards for Food
(BRASIL, 2001) establishes the following microbiological
standards for fresh refrigerated fish: absence of
Salmonella
in
25g of fish. The presence of
Salmonella
and sulfite-reducing
Clostridium
was not detected in any of the samples analyzed
in the experiment.
TABLE 2.
Staphylococcus aureus
count in tilapia fillets stored at 1±1 °C for 20 days.
Days Staphylococcus aureus (CFU/g)
Control Acetic acid + EVOH Vacuum Vacuum + Acetic acid MAP MAP + Acetic acid
1 1.0x102Aa* 5.3x101Aa 5.3x101Aa 1.7x101Aa 4.3x101Aa 3.0x101Aa
7 9.0x101Aa 6.7x101Aa 3.1x102Aa 2.3x101Aa 3.7x101Aa 1.7x101Aa
13 < 10Aa 5.7x101Aa 8.7x101Aa < 10Aa < 10Aa < 10Aa
20 < 10Aa < 10Aa < 10Aa < 10Aa < 10Aa < 10Aa
*Means followed by the same lowercase letters (columns) or the same capital letters (lines) do not differ according to the Tukey test at 5%.

Braz. J. Food Technol., v.8, n.1, p. 7-15, jan./mar., 2005 12
SOCCOL, M.C.H.
et al.
Effects of Modified Atmosphere and
Vacuum on the Shelf Life of Tilapia
(
Oreochromis niloticus
) Fillets
Braz. J. Food Technol., v.8, n.1, p. 7-15, jan./mar., 2005 13
SOCCOL, M.C.H.
et al.
Effects of Modified Atmosphere and
Vacuum on the Shelf Life of Tilapia
(
Oreochromis niloticus
) Fillets
In the same way, PASSY
et al.
(1983), SILVA & WHITE
(1994) and RANDELL
et al.
(1999) did not detect the presence
of such bacteria in fresh water prawns (
Macrobrachium
rosenbergii
), catfish (
Ictalurus punctatus
) and salmon (
Salmo
salar
) during MAP storage.
According to LEITÃO (1977), fish from non-polluted
waters are free from
Salmonella
because this bacteria is not
naturally found in the fish flora and its presence in fish is mainly
due to handling or contact with poorly disinfected surfaces.
Besides, this bacteria hardly proliferates in food containing other
microorganisms.
3.7 Sensory Evaluation
Table 3 shows the average scores given by the tasters
for the attributes color, aroma, texture and appearance. All the
attributes, except texture, were significantly (p<0.05) affected
by the treatments during the storage period.
According to the sensory evaluation results, MAP
packed fillets that underwent pretreatment with acetic acid
were considered unsuitable for consumption from the 7th day
of storage onwards, receiving scores below 5 for the attributes
color, aroma and appearance. From the first day of evaluation,
the MAP packed fillets that underwent pretreatment with acetic
acid were evaluated as presenting a softer texture, whitish
color and exudation, when compared to fillets from the other
treatments. From the 7th day of sensory evaluation onwards,
some evaluators detected rancidity, related to an increase in
TBARS values in fillets from the acetic acid + EVOH, MAP and
acetic acid plus MAP treatments. However, for the acetic acid
plus vacuum packing treatment, rancidity was detected only on
the last day of evaluation, probably due to the low proportion
of O2 in the packaging, which retarded the oxidative process
of polyunsaturated fatty acids.
However, fillets from vacuum packing and vacuum
packing plus acetic acid were similar to the control fillets,
being considered suitable for consumption up to the 20th day of
storage. Although the control fillets presented a psychrotrophic
bacterial count above 106 CFU/g and pH above the limits
established by the Brazilian legislation (6.5), the limiting
factor causing rejection of the product by the evaluators was
the change in appearance, caused by variations in the water
retention capacity, as well as changes in color due to the acetic
acid and O2 effects, characterized by a whitish color, fading and
exudation, which are directly related to the increase in TBARS.
The same samples also presented off-odors and rancidity.
4. CONCLUSIONS
The technology generated by the present study could
be made available to the fish productive sector in their search
for new products using tilapia as the raw material.
From the results of the chemical and microbiological
analyses, it can be concluded that all the treatments presented
TABLE 3. Scores (means) given by the sensory evaluation team for the attributes color, aroma, texture and appearance of tilapia fillets
stored at 1±1 °C for 20 days.
Days
Sensorial Scores
Control Acetic Acid + EVOH Vacuum Vacuum + Acetic
Acid MAP MAP +Acetic Acid
Color
1 6.40a* 7.00a 7.40a 5.90a 6.90a 6.90a
7 6.70ab 5.50b 7.90a 6.40b 6.60ab 4.20c
13 7.50a 4.00b 6.90a 6.80a 6.60a 3.10b
20 6.40a 4.50ab 6.70a 5.80ab 4.10ab 2.90b
Aroma
1 7.10a 6.50a 6.90a 5.70a 6.60a 6.10a
7 6.00a 5.00a 6.40a 5.90a 6.20a 4.50a
13 6.40a 5.10ab 5.70a 6.10a 5.50ab 3.90b
20 5.90a 3.70bc 4.90abc 5.30ab 2.90c 3.30bc
Texture
1 7.30a 7.80a 7.90a 6.60a 7.40a 7.00a
7 6.70a 6.50a 7.10a 7.50a 6.90a 6.10a
13 6.90a 5.30a 6.80a 7.00a 6.70a 5.30a
20 6.90a 6.70a 6.30a 6.70a 5.00a 4.70a
Appearance
1 6.80a 7.30a 7.50a 6.20a 6.70a 6.70a
7 6.20a 5.30ab 7.20a 6.10ab 6.40a 4.50b
13 6.90a 5.30a 6.20a 6.30a 6.10a 3.90a
20 6.30a 4.50ab 5.30ab 6.20ab 4.00ab 3.10b
Means followed by the same lowercase letters (lines) do not differ according to the Tukey test at 5%.

Braz. J. Food Technol., v.8, n.1, p. 7-15, jan./mar., 2005 14
SOCCOL, M.C.H.
et al.
Effects of Modified Atmosphere and
Vacuum on the Shelf Life of Tilapia
(
Oreochromis niloticus
) Fillets
Braz. J. Food Technol., v.8, n.1, p. 7-15, jan./mar., 2005 15
SOCCOL, M.C.H.
et al.
Effects of Modified Atmosphere and
Vacuum on the Shelf Life of Tilapia
(
Oreochromis niloticus
) Fillets
satisfactory results during the storage period, except for the
control, which showed psychrotrophic bacterial counts above
the acceptable levels and pH values above the limit established
by the Brazilian legislation, on the 13th and 20th day of storage,
respectively.
The treatments with acetic acid + EVOH, MAP and
MAP associated with acetic acid resulted in discolored fillets
that presented a softer texture than those from the other
treatments. Products packed under MAP, whether associated
with acetic acid or not, presented greater TBARS amounts and
rancidity could be detected by the tasters.
The presence of sulfite-reducing
Salmonella
,
Clostridium
and
E. coli
was not detected during the storage period. MAP
and vacuum packing associated with acetic acid inhibited the
development of
S. aureus
, total coliforms and psychrotrophic
microorganisms, lengthening the shelf-life by 20 days. Vacuum
packing associated with acetic acid was the treatment that
maintained the stability of the chemical, microbiological and
sensory characteristics of the fillets throughout the experiment,
extending the shelf-life by 20 days.
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