Chemical, sensory and microbiological stability of freeze-dried Nile tilapia croquette mixtures

Abstract

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.

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.

Keywords:

• Nile tilapia mince
• flaxseed
• freeze-drying
• croquette
• storage

Palabras clave:

• Carne picada de tilapia del Nilo
• linaza
• liofilización
• croqueta
• almacenamiento

1. Introduction

The culture of Nile tilapia (Oreochromis niloticus) as an ornamental 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 approximately 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 (Kuhn & Soares, 2002). Tilapia production is expected to rise 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, leading 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 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, appearance, 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 ingredients and other high-value foods. However, with increasing concerns 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) demonstrated 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 formulation 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 establishments 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 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.

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

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 polyethylene 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 determined 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 equipment 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 thiobarbituric 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, 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 temperature (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 breadcrumbs, 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 croquette formulations were evaluated at 0, 120 and 240 days of storage.

2.5. Microbiological analysis

Microbiological counts of control and enriched freeze-dried croquette 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 methodology described by ICMSF (1982). Current Brazilian legislation (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 formulations (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	0.1666^Ga ± 0.0058	0.0554^Ib ± 0.0002
30	0.2705^Fa ± 0.0083	0.0921^Hb ± 0.0047
60	0.3176^Eb ± 0.0062	0.3269^Ga ± 0.0030
90	0.3171^Eb ± 0.0006	0.4035^Fa ± 0.0045
120	0.4376^Da ± 0.0027	0.4191^Eb ± 0.0008
150	0.4563^Ca ± 0.0002	0.4420^Db ± 0.0004
180	0.4681^Ca ± 0.0015	0.4642^Ca ± 0.0010
210	0.4812^Bb ± 0.0032	0.4904^Ba ± 0.0009
240	0.5392^Aa± 0.0029	0.5415^Aa± 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 formulations, 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.

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.

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 antioxidants (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 coumarins, in addition to a high content of lignans, such as secoisolariciresinol diglucoside components, which act as antioxidants.

Few published studies have evaluated the oxidation of freeze-dried fish products. It is known that many deterioration 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 consumption 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 oxidation 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 higher than at time 0. Odour decreased during storage, with an acceptable final average that corresponded to ‘like moderately’. The characteristics of the enriched mixture remained constant throughout the evaluation period, with the exception of the overall acceptance attribute.

Table 3. Mean hedonic values ​​(on a 9-point hedonic scale) assigned by the panellists to control and enriched freeze-dried mixtures of tilapia croquette during storage.

Tabla 3. Los valores promedio hedónicos asignados por los panelistas para la mezcla control y la mezcla enriquecida liofilizada de croquetas de tilapia durante el almacenamiento.

	Formulations
Time (days)	Control mixture	Enriched mixture
	Appearance
0	7.06^Aa ± 1.236	6.58^Aa ± 1.197
120	6.95^Aa ± 1.384	5.98^Ab ± 1.489
240	7.34^Aa ± 1.504	6.02^Ab ± 1.645
	Texture
0	7.04^Aa ± 1.212	7.04^Aa ± 1.442
120	7.34^Aa ± 1.328	6.85^Ab ± 1.412
240	7.47^Aa ± 1.369	6.63^Ab ± 1.680
	Flavour
0	6.86^Ba ± 1.294	6.94^Aa ± 1.449
120	7.79^Aa ± 1.380	6.77^Ab ± 1.309
240	8.02^Aa ± 0.908	6.42^Ab ± 1.453
	Odour
0	8.04^Aa ± 0.903	7.16^Ab ± 1.149
120	7.46^ABa ± 1.385	6.95^Aa ± 1.521
240	7.14^Ba ± 1.548	6.68^Aa ± 1.570
	Overall acceptance
0	7.26^Aa ± 1.175	7.20^Aa ± 1.370
120	7.69^Aa ± 1.104	6.85^ABb ± 1.195
240	7.68^Aa ± 0.819	6.39^Bb ± 1.390

Note: Values ​​with different letters (a–b) in the same row and (A–B) in the same column differ significantly (p < 0.05). The results are shown as mean ± SD (n = 50).

Nota: Los valores con diferentes letras (a-b) y en la misma fila (A-B) en la misma columna difieren significativamente (p < 0,05). Los resultados se muestan como promedio ± SD (n = 50).

At time 0, the two mixtures were considered equal in respect of all sensory attributes with the exception of odour. During the 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 the odour factor was considered equal between mixtures, suggesting that over time, volatiles had been lost. This flow of vapours between the freeze-dried mixtures and the external medium was also observed during the evaluation of aw, indicating the necessity of a more impermeable package to maintain the stability of the product.

The overall acceptance of the croquettes differed significantly with storage time. At time 0, the overall acceptance of the samples was equal (p < 0.05). Over time, the overall acceptance of the control mixture remained constant while the average for the enriched mixture decreased significantly. At the end of the evaluation, the overall acceptance of the control mixture was ~8 on a 9-point hedonic scale (‘like very much’) while the enriched mixture showed values ​​near 6 (‘like 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 recommended by Teixeira, Meinert and Barbetta (1987) for a product to be considered suitable for commercialization (70%). This suggested that 240 days was the time limit for sensory stability of enriched freeze-dried Nile tilapia croquette mixtures. When compared with other products made from Nile tilapia, such as nuggets (Kirschnik, 2007), sausage (Oliveira Filho, Viegas, Kamimura & Trindade, 2012), pate (Minozzo, Waszczynskyj & Boscolo, 2008) and fishburger (Marengoni et al., 2009), the acceptance index of both mixtures was similar or even superior.

3.3. Microbiological analysis

Both mixtures were analysed for total and faecal coliforms, Bacillus cereus, Salmonella sp., Staphylococcus coagulase, moulds and yeasts at 0, 120 and 240 days as required by international legislation (ICMSF, 1982).

At time 0, the control mixture showed the following counts: Salmonella sp., 25 g⁻¹; total and faecal coliforms, 10¹ CFU g⁻¹; and Bacillus cereus, yeasts, moulds and Staphylococcus coagulase, <10¹ CFU g⁻¹. At other time points, the microbial count for all microorganisms was less <10¹ CFU g⁻¹. For the enriched mixture, the microorganism counts were <10¹ CFU g⁻¹ at all time points and Salmonella was not detected at any time. These data indicated that the microbiological results were within the standards set by international law (ICMSF, 1982).

Preservation methods, such as freezing, drying and freeze-drying, are commonly used to control microbial contamination. After freeze-drying, for instance, the population of microorganisms may be either killed or sustain sublethal injury (Wu, Fung & Kang, 2001). Several authors have described the types of 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, Philippe & Mokhtar, 2009; Santivarangkna, Wenning, Foerst & Kulozik, 2007). Furthermore, some macromolecules are altered within these cells with consequent functional damage, causing potential metabolic injury (Jay, Loessner & Golden, 2005). Freezing damages the lipopolysaccharide molecules of the outer membrane of Gram-negative bacteria due to the destabilization of ionic bonds (Ray, 1986).

4. Conclusions

Control and enriched freeze-dried croquette mixtures can be stored for 240 days at room temperature without undesirable changes in chemical, sensory or microbiological quality. Lipid oxidation and microbiological analyses for both products suggested that the shelf life could be extended for an even a longer period. Water activity values ​​for both mixtures increased with storage time, indicating that a less porous type of packaging should be used for these products. All sensory attributes evaluated changed little for either formulation. Throughout storage, the overall acceptance of the mixtures was equal (time 0), before diverging, with that of the enriched mixture dropping to levels ​​close to the minimum acceptable for a marketable product, indicating the end of that product’s shelf life.

Disclosure statement

No potential conflict of interest was reported by the authors.