CyTA - Journal of Food

ISSN: 1947-6337 (Print) 1947-6345 (Online) Journal homepage: http://www.tandfonline.com/loi/tcyt20

Chemical, sensory and microbiological stability of

freeze-dried Nile tilapia croquette mixtures

Renata Hernandez Barros Fuchs, Ricardo Pereira Ribeiro, Makoto

Matsushita, Ailey Aparecida Coelho Tanamati, Cristiane Canan, Evandro
Bona, Leila Larisa Medeiros Marques & Adriana Aparecida Droval

To cite this article: Renata Hernandez Barros Fuchs, Ricardo Pereira Ribeiro, Makoto Matsushita,
Ailey Aparecida Coelho Tanamati, Cristiane Canan, Evandro Bona, Leila Larisa Medeiros
Marques & Adriana Aparecida Droval (2015) Chemical, sensory and microbiological stability
of freeze-dried Nile tilapia croquette mixtures, CyTA - Journal of Food, 13:4, 556-562, DOI:
10.1080/19476337.2015.1014431

To link to this article: http://dx.doi.org/10.1080/19476337.2015.1014431

© 2015 The Author(s). Published by Taylor & Published online: 06 Mar 2015.
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CyTA – Journal of Food, 2015
Vol. 13, No. 4, 556–562, http://dx.doi.org/10.1080/19476337.2015.1014431

Chemical, sensory and microbiological stability of freeze-dried Nile tilapia croquette mixtures
La estabilidad química, sensorial y microbiológica de la mezcla de croquetas de tilapia del Nilo
liofilizadas
Renata Hernandez Barros Fuchsa*, Ricardo Pereira Ribeirob, Makoto Matsushitac, Ailey Aparecida Coelho Tanamatia,
Cristiane Canand, Evandro Bonad, Leila Larisa Medeiros Marquesa and Adriana Aparecida Drovala
a
Food Technology Department, Federal University of Technology – Paraná (UTFPR), Via Rosalina Maria dos Santos, 1233 – CEP,
Campo Mourão 87301-899, PR, Brazil; bDepartment of Animal Science, Postgraduate Program in Food Science, State University of
Maringá, Av. Colombo, 5.790 – CEP, Maringá 87020-900, PR, Brazil; cDepartment of Chemistry, Postgraduate Program in Food
Science, State University of Maringá, Av. Colombo, 5.790 – CEP, Maringá 87020-900, PR, Brazil; dPost-Graduation Program of Food
Technology (PPGTA), Food Technology Department, Federal University of Technology, Paraná (UTFPR) Via Rosalina Maria dos
Santos, 1233 – CEP, Campo Mourão 87301-899, PR, Brazil
(Received 18 September 2014; final version received 29 January 2015)

The stability of tilapia croquette mixtures was evaluated for 240 days, through analyses of water activity (aw), lipid oxidation (TBARS) and
sensory and microbiological assays. The control mixture was composed of tilapia mince, wheat and rye flours and seasonings while the
enriched mixture also contained flaxseed flour. Aw increased significantly over time, from 0.1666 to 0.5392 for the control mixture and
from 0.0554 to 0.5415 for the enriched mixture. TBARS values increased from 0.08 to 0.38 mg kg−1 for the control mixture and from 0.11
to 0.40 mg kg−1 malondialdehyde (MDA) for the enriched mixture. Appearance, texture and overall acceptance of the control mixture
remained constant and the flavour improved during storage, while sensory attributes of the enriched mixture did not change. Overall
acceptance was nearly 8 for the control mixture and 6 for the enriched mixture using a 9-point hedonic scale, where 9 = ‘like a lot’ and
1 = ‘dislike intensely’. Microbiological parameters did not exceed the legal limits allowed for consumption. Both mixtures remained
suitable for consumption after 240 days.
Keywords: Nile tilapia mince; flaxseed; freeze-drying; croquette; storage

Se evaluó la estabilidad de la mezcla de croquetas de tilapia durante 240 días, mediante análisis de la actividad del agua (aw), la oxidación
lipídica (TBARS), además de ensayos sensoriales y microbiológicos. La mezcla control estaba compuesta por carne picada de tilapia,
harinas de trigo y centeno, además de sazonadores mientras que la mezcla enriquecida también contenía harina de linaza. Aw aumentó
significativamente con el tiempo, de 0,1666 a 0,5392 para la mezcla control y de 0,0554 a 0,5415 para la mezcla enriquecida. Los valores
TBARS aumentaron de 0,08 a 0,38 mg kg−1 MDA para la mezcla control y de 0,11 a 0,40 mg kg−1 MDA para la mezcla enriquecida. La
apariencia, la textura y el total de aceptación de la mezcla control se mantuvo constante y el sabor mejoró durante el almacenamiento,
mientras que los atributos sensoriales de la mezcla enriquecida no mostraron cambios. El total de aceptación fue cerca de 8 para la mezcla
control y 6 para la mezcla enriquecida. Los parámetros microbiológicos no superaron los límites legales permitidos para el consumo. Las
dos mezclas se mantuvieron aptas para el consumo después de 240 días.
Palabras clave: Carne picada de tilapia del Nilo; linaza; liofilización; croqueta; almacenamiento

1. Introduction (Kuhn & Soares, 2002). Tilapia production is expected to rise

The culture of Nile tilapia (Oreochromis niloticus) as an orna-
mental fish began in Egypt over 4000 years ago. Development of
sex-reversal techniques, nutrition and crop system research, as
well as market development and processing advances, led to the
rapid expansion of the sector from the mid-1980s (Rakocy,
2005). In Brazil, the production of tilapia in 2010 was approxi-
mately 160,000 tons (Brazil, 2012).
A by-product of the filleting process, tilapia mince, is a
product with high protein content (nearly 15 g 100 g−1) and a
mild flavour, and is an ideal ingredient for the development of
food products (Gehring, Gigliotti, Moritz, Tou & Jaczynski,
2011). Its use as a raw material for new products results in a
greater consumption or inclusion of fish in the diet and the
emergence of a consumer sector for this raw material that was
once considered a by-product of the fish-processing industry
in the near future, increasing the amount of mince produced to as
much as 33.2 g 100 g−1 of whole tilapia (Ninan, Bindu &
Joseph, 2010). Therefore, it is of great importance to devise
alternatives that strengthen the supply chain of tilapia, due to
the increased demand for this fish (World Bank, 2014). Value
addition could help to bring different forms of fish products to
the marketplace while reducing postharvest losses. Postharvest
losses in developing-country markets are as high as 30–40% of
the harvest (World Bank, 2014).
Most pre-packaged foods found on the market are frozen
(Adegoke & Olapade, 2012). However, even frozen fish and fish
products can undergo undesirable changes during storage, lead-
ing to reduced stability and loss in quality (Taub & Singh, 1998).
Undesirable changes that induce protein denaturation and lipid
oxidation continue to occur even at low temperatures, resulting

*Corresponding author. Email: renata@utfpr.edu.br

© 2015 The Author(s). Published by Taylor & Francis.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

in changes in texture and flavour (Kurade & Baranowski, 1987;
Tokur, Ozkütük, Atici, Ozyurt, & Ozyurt, 2006; Yerlikaya,
Gokoglu, & Uran, 2005).
Freeze-drying is a process of drying and preserving food that
maintains the structure of the material while removing moisture
at low temperatures, increasing product stability and minimizing
degradation during storage (Boss, Filho & Toledo, 2004;
Crapo, Oliveira, Nguyen, Bechtel & Fong, 2010; Krokida &
Philippopoulos, 2006). Freeze-drying is based on sublimation,
where water changes directly from a solid state to a vapour phase
without transitioning through the liquid phase. The combination
of low moisture and temperature, in addition to the presence of a
vacuum, minimizes product deterioration and microbial activity
during the drying process (Crapo et al., 2010). Compared with
classical dehydration techniques, the main advantages of the
vacuum freeze-drying process including the preservation of
most of the initial raw material properties, such as shape, appear-
ance, taste, colour, flavour, texture, biological activity and the
high rehydration capacity of the freeze-dried product (Hammami
& René, 1997). The freeze-drying process is currently not widely
used in the food industry due to its high operational costs.
Although improvements have been researched aimed at reducing
costs, vacuum freeze-drying is, to date, the only technology used
on an industrial scale to dry coffee, spices, meats, food ingredi-
ents and other high-value foods. However, with increasing con-
cerns about food quality, this process could be considered as a
valuable alternative in the preservation of other foods (Ratti,
2001). Wang, Zhang, Mujumdar and Mothibe (2013) demon-
strated that freeze-drying restructured fish meat has potential use
in the development of new dried fish products using optimal
drying conditions.
The aim of this study was to determine the shelf life of two
freeze-dried mixtures of Nile tilapia croquettes, stored at room
temperature, by evaluating the changes in their chemical, sensory
and microbiological characteristics.
2. Materials and methods
2.1. Ingredients
Nile tilapia mince was the main raw material used in the for-
mulation of croquette mixtures that were ultimately freeze-dried.
The pre-packaged, frozen mince was obtained from the
Cooperativa Agroindustrial Consolata (Copacol) Cafelândia
(Paraná – Brazil). Wheat (brand COAMO), rye and flaxseed
flour (brand Vitao), dried seasonings including onion, garlic,
parsley and chives (brand Kitano), and other ingredients used
in the formulations were purchased from commercial establish-
ments in the city of Campo Mourão (Paraná – Brazil).
2.2. Development and freeze-drying of mixtures of Nile
tilapia croquette
The basic formulation of tilapia croquettes was previously
defined using a simplex centroid experimental design. A multi-
objective optimization using the genetic algorithm (GA) with
desirability functions was performed to select the formulation
with the lowest cost and highest overall acceptance and fibre
content (Fuchs, Ribeiro, Bona & Matsushita, 2013). Although
most fish are a rich source of omega-3 (n-3) and omega-6 (n-6)
polyunsaturated fatty acids (PUFA), some studies have shown
that farmed fish, including Nile tilapia, have low amounts of the
main PUFA (n-3: α-linolenic acid, eicosapentaenoic acid and
CyTA – Journal of Food 557
Table 1. Composition of Nile tilapia croquette freeze-dried formulations
subjected to stability evaluation.
Tabla 1. Composición de las formulaciones de croquetas de tilapia del
Nilo liofilizadas sujetas a una evaluación de estabilidad.
Ingredient (g 100 g−1) Control formulation Enriched formulation
Nile tilapia mince 54.71 47.56
Water 27.35 23.78
Wheat flour 6.84 5.95
Rye flour 6.84 5.95
Flaxseed flour 0 12.50
Soybean oil 1.09 1.09
Dried seasonings 3.17 3.17
docosahexaenoic acid) compared with wild fish (Vieira,
Hilsdorf & Moreira, 2012). Therefore flaxseed flour was added
to the basic formulation of tilapia croquette to improve lipid
quality. Fuchs, Ribeiro, Matsushita, et al. (2013) found that the
upper limit to the amount of flaxseed flour that can be added to
Nile tilapia croquette, without reducing its acceptance, is
12.5 g 100 g−1. The formulations of both controlled and enriched
Nile tilapia croquette mixtures are shown in Table 1.
The mince was thawed at 4°C for 24 h, braised with soybean
oil (140°C for 3 min), combined with dried seasonings and
cooked (150°C for 3 min). Water was added and the mixture
brought to boil for 2 min. Then, the various flours were added
and the dough baked at 85°C for 2 min as described in Fuchs,
Ribeiro, Bona, et al. (2013). Portions of croquette dough (170 g)
were formed into 1 cm discs of diameter 17 cm, which were
frozen in an ultra-freezer (Brazil, Indrel CPH 35D) at −40°C for
24 h and then freeze-dried (Brazil, LIOTOP-L101, Liobras) at
−55°C and 290 Pa for 24 h.
After freeze-drying, the dried mass was ground in a food
processor (Philips Walita, 750 W) to yield a type of flour before
being vacuum packed in heat-shrinkable, low-density polyethy-
lene packaging (20 × 35 cm, 180 μm; Alviplast). The packaged
samples were stored in dark plastic containers protected from
light at room temperature (24°C ± 3°C) for up to 240 days.
2.3. Chemical parameters
2.3.1. Water activity
The water activity (aw) of the freeze-dried mixtures was deter-
mined over 240 days at 30-day intervals using an AquaLab
Series 3 TE (Decagon Devices Inc., Pullman, Washington,
DC). The sample temperature was 24°C (±3°C) and the equip-
ment was calibrated with a solution of lithium chloride
(aw = 0.250 ± 0.03).
2.3.2. Lipid oxidation determination
Lipid oxidation was analysed by determining the level of thio-
barbituric acid reactive substances (TBARS) according to the
method described by Vyncke (1970), with modifications. A
sample of the croquette mixture (10 g) was homogenized in
50 mL of an aqueous solution (7.5 g 100 g−1) of trichloroacetic
acid (Sigma-Aldrich) in a magnetic stirrer (1000 rpm) at 20°C
for 20 min. The suspension was centrifuged (4800 × g) at 5°C
for 20 min and 5 mL of the supernatant was added to 5 mL of
TBA reagent (Sigma-Aldrich; 0.02 M 2-thiobarbituric acid in
distilled water). The mixture was incubated in a water bath at 96°
C for 40 min then cooled in ice water for 5 min. Following this,
558 R.H.B. Fuchs et al.
the absorbance was measured at 538 nm using a FEMTO
spectrophotometer.
TBARS values were calculated from a standard curve of
MDA in 1,1,3,3-tetramethoxypropane (TMP) and expressed as
mg MDA kg−1 sample. The evaluation of lipid oxidation was
performed monthly over a period of 240 days.
2.4. Sensory evaluation
Appearance, texture, flavour, odour and overall acceptance of the
control and enriched freeze-dried mixtures were evaluated using
a 9-point hedonic scale, where 9 = ‘like a lot’ and 1 = ‘dislike
intensely’ (Meilgaard, Civille & Carr, 1999). Fifty untrained
adult panellists participated in the test and received samples
coded with three random digits. The panellists were students
and employees of the Federal University of Technology-Paraná
(UTFPR) who received instructions about the project and the
type of product being evaluated. The acceptance test for both
samples was carried out in one sensory analysis session. The
Ethics Committee in Research of the Integrado School (Campo
Mourão, Paraná, Brazil) approved this study under protocol
number 62/11 on 4 October 2011.
The mixtures were rehydrated in water at room tempera-
ture (22°C ± 1°C) using the following water/mixture ratios:
control croquette (1:1) and enriched croquette (1:1.2). After
the addition of water, the dough was mixed, shaped by hand
(portions of 15 g), breaded in wheat flour, egg and bread-
crumbs, deep-fried in soybean oil in an electric frying pan
(190°C for 2 min) and submitted for sensory analysis in a
randomized order. The control and enriched freeze-dried cro-
quette formulations were evaluated at 0, 120 and 240 days of
storage.
2.5. Microbiological analysis
Microbiological counts of control and enriched freeze-dried cro-
quette mixtures were performed. The microbiological analysis of
total and thermotolerant coliforms, Staphylococcus aureus,
Bacillus cereus, Salmonella sp., yeasts and moulds was carried
out on days 0, 120 and 240 of storage according to the metho-
dology described by ICMSF (1982). Current Brazilian legisla-
tion (Brazil, 2001) suggests analytical procedures for these types
of microorganism.
2.6. Statistical analyses
Measurements of water activity and TBARS (in triplicate) as
well as the results of the sensory analysis were evaluated by
ANOVA and Tukey tests (p < 0.05) using Statistica software 7.1
(Statsoft, Tulsa, USA, 2006).
3. Results and discussion
3.1. Chemical parameters
3.1.1. Water activity
The water activity values for the freeze-dried tilapia croquette
mixtures are shown in Table 2. Aw values found for the for-
mulations (on day 0) were within the ranges reported in the
literature for dried foods, which by their nature have a lower
aw, generally less than 0.30 (Vitali & Quast, 1996).
Table 2. Values of water activity (aw) of control and enriched freeze-
dried mixtures of tilapia croquette during storage.
Tabla 2. Los valores de actividad del agua (aw) de la mezcla control y
la mezcla enriquecida liofilizada de croquetas de tilapia durante el
almacenamiento.
Aw
Time (days) Control mixture Enriched Mixture
0 0.1666Ga ± 0.0058 0.0554Ib ± 0.0002
30 0.2705Fa ± 0.0083 0.0921Hb ± 0.0047
60 0.3176Eb ± 0.0062 0.3269Ga ± 0.0030
90 0.3171Eb ± 0.0006 0.4035Fa ± 0.0045
120 0.4376Da ± 0.0027 0.4191Eb ± 0.0008
150 0.4563Ca ± 0.0002 0.4420Db ± 0.0004
180 0.4681Ca ± 0.0015 0.4642Ca ± 0.0010
210 0.4812Bb ± 0.0032 0.4904Ba ± 0.0009
240 0.5392Aa ± 0.0029 0.5415Aa ± 0.0011
Note: Values with different letters (a–b) in the same row and (I–A) in the same
column differ significantly (p < 0.05). The results are represented as mean ± SD
(n = 3).
Nota: Los valores con diferentes letras (a-b) en la misma fila y (I-A) en la misma
columna difieren significativamente (p < 0,05). Los resultados se representan como
promedio ± desviación estándar (n = 3).
Table 2 shows that water activity increased significantly
(p < 0.05) during storage for both formulations. Aw values for
the control mixture increased from 0.1666 to 0.5392, and for the
enriched mixture from 0.0554 to 0.5415, over the 240 days of
storage. At 90 days of storage, the water activity of the two
formulations showed different behaviour because sometimes the
enriched mixture showed higher values of aw, and sometimes the
control mixture. The mean aw values for the control (r = 0.94)
and enriched (r = 0.81) mixtures showed a significant positive
correlation (p < 0.05) with time.
At the end of the study (240 days), the aw of the control
and enriched mixtures increased to 0.5392 ± 0.0029 and
0.5415 ± 0.0011, respectively, suggesting that the packaging
had not provided an adequate barrier to water vapour. Two
strategies can be used to control increase in aw: utilizing a
package with higher levels of impermeability or adding anti-
wetting agent(s) to the freeze-dried croquette mixtures.
However, by using freeze-drying, the assumption was that in
addition to preservation of the sensory characteristics of the
product, the end result would be a product free of chemical
additives, such as anti-wetting agents, stabilizers or
flavourings.
Unmodified foods have secured a market niche, justifying
the value added by the freeze-drying process. Therefore, the
packaging should ensure low aw values for a longer time without
the use of chemical additives. The use of packaging that includes
an aluminized middle layer may be an alternative that would
resist the passage of gases and moisture, protecting the mixture
from light and preventing oxidative processes (Souza &
Menezes, 2006).
Despite increased aw during the storage period
(240 days), the aw values for the two mixtures were lower
than the maximum recommended values (0.6) for dry foods,
which ensured the inhibition of microbial growth. It is known
that microorganisms require a minimum aw for growth.
Generally, bacteria are more sensitive and are growth-inhibited
at an aw of 0.90–0.91, while moulds and yeasts are more
tolerant of lower aw values, growing at an aw of 0.70–0.80
and 0.87–0.94, respectively. Water activity of 0.60 or less

prevents the growth of all types of microorganism (Abbas
Saleh, Mohamed & Lasekan, 2009).
3.1.2. Assessment of lipid oxidation
The level of TBARS, an index widely used to assess lipid
oxidation, was selected to evaluate the content of MDA in the
product. MDA is formed from hydroperoxides, which are the
initial products of the reaction between polyunsaturated fatty
acids and oxygen (Fernández, Pérez-Álvarez & Fernández-
López, 1997).
Lipid oxidation in the freeze-dried Nile tilapia mixtures
during storage is shown in Figure 1. The amount of TBARS in
the control mixture increased from 0.08 mg MDA kg−1 (time 0)
to 0.38 MDA kg−1 (240 days), and in the enriched mixture, from
0.11 (time 0) to 0.40 MDA kg−1 (240 days). In both formula-
tions, the amount of TBARS increased significantly (p < 0.05)
during the first 30 days of storage. In the control formulation,
this value remained stable between 30 and 150 days of storage,
rising from 0.16 to 0.19 mg MDA kg−1, while for the enriched
formulation, the value increased from 0.16 to 0.24 mg MDA
kg−1. TBARS values for the control mixture (r = 0.92) and the
enriched sample (r = 0.95) had a significant positive correlation
(p < 0.05) over time.
Although the enriched formulation had higher TBARS
values than the control (initially and at all time points), the
values were similar (p < 0.05) at 240 days. Because flaxseed is
a functional food rich in lipids, essential fatty acids and antiox-
idants (Tarpila, Wennberg & Tarpila, 2005), the addition of this
ingredient would have made the product more susceptible to
oxidation due to the presence of a higher concentration of
unsaturated fatty acids. However, this was not observed and
may be attributed to the presence of antioxidants in flaxseeds.
According Tarpila et al. (2005), flaxseed is naturally rich in
phytochemicals, such as flavonoids, phenolic acids and coumar-
ins, in addition to a high content of lignans, such as secoisolar-
iciresinol diglucoside components, which act as antioxidants.
Few published studies have evaluated the oxidation of
freeze-dried fish products. It is known that many deterioration
0.45
0.40
0.35
0.30
mg MDA kg–1
0.25
0.20
0.15
0.10
0.05
0.00
0 30 60
Control Formulation
Figure 1. TBARS values for control and enriched freeze-dried mixtures of tilapia croquettes enriched during storage.
Figura 1. Los valores TBARS en la mezcla control y la mezcla enriquecida liofilizada de croquetas de tilapia enriquecidas durante el almacenamiento.
CyTA – Journal of Food 559
reactions, including lipid oxidation, occur even at low aw values
(Labuza, McNally, Gallagher, Hawkes & Hurtado, 1972;
Labuza, Tannenbaum & Karel, 1970). Although low aw values
may have promoted lipid oxidation in this study, both freeze-
dried mixtures evaluated showed TBARS values lower than
those reported in the literature for products based on frozen
tilapia mince. Evaluating nuggets made from Nile tilapia
mince, Kirschnik (2007) found TBARS values ranging between
1.15 and 1.91 mg MDA kg−1 after storage at −18°C for
180 days, depending on the type of process used to obtain the
mince. Bonacina and Queiroz (2007) evaluated the lifetime of
batter made from corvina (Micropogonias furnieri) stored for
135 days at −18°C and reported TBARS values ranging from
0.33 to 1.59 mg MDA kg−1, indicating a significant increase in
the MDA value of 0.596 mg kg−1 recorded at 90 days of storage.
By comparison, the values reported in this work confirmed the
efficacy of freeze-drying in extending the shelf life of products
made from fish.
No legislation currently exists in regard to the levels of
oxidation in fish products. According to Al-Kahtani et al.
(1996), these products can be considered acceptable for con-
sumption at values below 3 mg MDA kg−1, while Ke,
Cervantes and Robles-Martinez (1984) indicated that MDA
values greater than 1.51 mg kg−1 are classified as unacceptable,
with those below 0.576 mg kg−1 indicating low rates of oxida-
tion or rancidity. The values reported in this study after 240 days
of storage for the two freeze-dried mixtures were lower than
those in the literature, and the rate of product rancidity was
very low even without the use of antioxidants.
3.2. Sensory evaluation
Table 3 shows the hedonic values given to the control and
enriched freeze-dried mixtures in acceptance tests performed
during the storage period. It was noted that appearance, texture
and overall acceptance of the control mixture remained constant
over the 240-day storage period. Throughout the evaluation
period, there was significant improvement (p < 0.05) in flavour,
as scores for this attribute at the second and third tastings were
90 120 150 180 210 240
Time (days)
Enriched Formulation
560 R.H.B. Fuchs et al.

Table 3. Mean hedonic values (on a 9-point hedonic scale) assigned by product to be considered suitable for commercialization (70%).
the panellists to control and enriched freeze-dried mixtures of tilapia This suggested that 240 days was the time limit for sensory stability
croquette during storage.
of enriched freeze-dried Nile tilapia croquette mixtures. When
Tabla 3. Los valores promedio hedónicos asignados por los panelistas compared with other products made from Nile tilapia, such as
para la mezcla control y la mezcla enriquecida liofilizada de croquetas de
tilapia durante el almacenamiento. nuggets (Kirschnik, 2007), sausage (Oliveira Filho, Viegas,
Kamimura & Trindade, 2012), pate (Minozzo, Waszczynskyj &
Formulations Boscolo, 2008) and fishburger (Marengoni et al., 2009), the accep-
tance index of both mixtures was similar or even superior.
Time (days) Control mixture Enriched mixture

Appearance
0 7.06Aa ± 1.236 6.58Aa ± 1.197 3.3. Microbiological analysis
120 6.95Aa ± 1.384 5.98Ab ± 1.489 Both mixtures were analysed for total and faecal coliforms,
240 7.34Aa ± 1.504 6.02Ab ± 1.645
Texture Bacillus cereus, Salmonella sp., Staphylococcus coagulase,
0 7.04Aa ± 1.212 7.04Aa ± 1.442 moulds and yeasts at 0, 120 and 240 days as required by inter-
120 7.34Aa ± 1.328 6.85Ab ± 1.412 national legislation (ICMSF, 1982).
240 7.47Aa ± 1.369 6.63Ab ± 1.680 At time 0, the control mixture showed the following
Flavour
0 6.86Ba ± 1.294 6.94Aa ± 1.449
counts: Salmonella sp., 25 g−1; total and faecal coliforms,
120 7.79Aa ± 1.380 6.77Ab ± 1.309 101 CFU g−1; and Bacillus cereus, yeasts, moulds and
240 8.02Aa ± 0.908 6.42Ab ± 1.453 Staphylococcus coagulase, <101 CFU g−1. At other time
Odour points, the microbial count for all microorganisms was less
0 8.04Aa ± 0.903 7.16Ab ± 1.149 <101 CFU g−1. For the enriched mixture, the microorganism
ABa
120 7.46 ± 1.385 6.95Aa ± 1.521
240 7.14Ba ± 1.548 6.68Aa ± 1.570 counts were <101 CFU g−1 at all time points and Salmonella
Overall acceptance was not detected at any time. These data indicated that the
0 7.26Aa ± 1.175 7.20Aa ± 1.370 microbiological results were within the standards set by inter-
Aa
120 7.69 ± 1.104 6.85ABb ± 1.195 national law (ICMSF, 1982).
240 7.68Aa ± 0.819 6.39Bb ± 1.390
Preservation methods, such as freezing, drying and freeze-
Note: Values with different letters (a–b) in the same row and (A–B) in the same drying, are commonly used to control microbial contamination.
column differ significantly (p < 0.05). The results are shown as mean ± SD After freeze-drying, for instance, the population of microorgan-
(n = 50).
isms may be either killed or sustain sublethal injury (Wu, Fung
Nota: Los valores con diferentes letras (a-b) y en la misma fila (A-B) en la misma & Kang, 2001). Several authors have described the types of
columna difieren significativamente (p < 0,05). Los resultados se muestan como
promedio ± SD (n = 50). injury sustained by microorganisms from these treatments
where the primary injury was related to a change in permeability
for barrier structures (surface and cytoplasmic membranes)
(Brennan, Wanismail & Ray, 1986; Manel, Sana, Abdeslam,
higher than at time 0. Odour decreased during storage, with an
Philippe & Mokhtar, 2009; Santivarangkna, Wenning, Foerst &
acceptable final average that corresponded to ‘like moderately’.
Kulozik, 2007). Furthermore, some macromolecules are altered
The characteristics of the enriched mixture remained constant
within these cells with consequent functional damage, causing
throughout the evaluation period, with the exception of the over-
potential metabolic injury (Jay, Loessner & Golden, 2005).
all acceptance attribute.
Freezing damages the lipopolysaccharide molecules of the
At time 0, the two mixtures were considered equal in respect
outer membrane of Gram-negative bacteria due to the destabili-
of all sensory attributes with the exception of odour. During the
zation of ionic bonds (Ray, 1986).
second evaluation (120 days of storage), panellists began to
detect significant differences (p < 0.05) between mixtures in all
sensory attributes with the exception of odour. After 120 days 4. Conclusions
the odour factor was considered equal between mixtures, sug-
Control and enriched freeze-dried croquette mixtures can be
gesting that over time, volatiles had been lost. This flow of
stored for 240 days at room temperature without undesirable
vapours between the freeze-dried mixtures and the external med-
changes in chemical, sensory or microbiological quality. Lipid
ium was also observed during the evaluation of aw, indicating
oxidation and microbiological analyses for both products sug-
the necessity of a more impermeable package to maintain the
gested that the shelf life could be extended for an even a
stability of the product.
longer period. Water activity values for both mixtures
The overall acceptance of the croquettes differed signifi-
increased with storage time, indicating that a less porous
cantly with storage time. At time 0, the overall acceptance of
type of packaging should be used for these products. All
the samples was equal (p < 0.05). Over time, the overall
sensory attributes evaluated changed little for either formula-
acceptance of the control mixture remained constant while the
tion. Throughout storage, the overall acceptance of the mix-
average for the enriched mixture decreased significantly. At the
tures was equal (time 0), before diverging, with that of the
end of the evaluation, the overall acceptance of the control
enriched mixture dropping to levels close to the minimum
mixture was ~8 on a 9-point hedonic scale (‘like very much’)
acceptable for a marketable product, indicating the end of
while the enriched mixture showed values near 6 (‘like
that product’s shelf life.
slightly’).
The acceptance index of the control mixture increased from
80.1% (day 0) to 85.3% (day 240), while the index for the enriched
mixture decreased from 80 to 71%, approaching the minimum Disclosure statement
recommended by Teixeira, Meinert and Barbetta (1987) for a No potential conflict of interest was reported by the authors.

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