Effect of frozen storage conditions (temperature and length of storage) on microbiological and sensory quality of rustic crossbred beef at different states of ageing

Meat Science 83 (2009) 398–404

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Effect of frozen storage conditions (temperature and length of storage) on microbiological and sensory quality of rustic crossbred beef at different states of ageing C. Vieira a,*, M.T. Diaz b, B. Martínez a, M.D. García-Cachán a a b

Estación Tecnológica de la Carne, ITACyL, Consejería de Agricultura y Ganadería de Castilla y León, Apdo, 58-37770 Guijuelo–Salamanca, Spain Instituto Nacional de Investigación Agraria y Tecnología Alimentaria (INIA), Ctra, Coruña, km, 7.5 Madrid, Spain

a r t i c l e

i n f o

Article history: Received 16 July 2008 Received in revised form 5 March 2009 Accepted 5 June 2009

Keywords: Frozen storage Freezing temperature Ageing Beef quality

a b s t r a c t The effect of frozen storage conditions on meat from 36 Morucha  Charolais crossbred yearlings was studied. Slices of M. Longissimus thoracis were randomly assigned to groups arising from the combination of experimental factors. These factors were: ageing extent (3 and 10 days), length of frozen storage (0, 30, 75 and 90 days) and temperature ( 20 and 80 °C). Regarding microbiological counts, although values were acceptable in all cases, longer storage time and longer previous ageing extent provided higher phychrotrophic bacteria counts. As frozen storage period increased, colorimetric parameters L*, a* and C* decreased, but H* increased. Regarding Warner–Braztler shear force and tenderness values, an interaction (p < 0.05) between frozen storage and post-mortem ageing resulted from larger differences between frozen storage periods at shorter ageing periods than those at longer ageing periods. Frozen storage for 90 days resulted in a reduction in water holding capacity, without differences in juiciness. No effect of freezing temperature was observed in any of the parameters studied. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction The limit of storage or shelf life of meat has been prevented from being reached by microbiological and physicochemical spoilage under conditions such as refrigeration and freezing. In this sense, in recent years, storing beef under freezing conditions has been increased in order to address market problems, and significant beef stocks have been generated. These stocks are often stored frozen for various times before being thawed to be consumed. Several investigations have reported that frozen storage could affect microbiological quality and physicochemical characteristics such as oxidative stability and sensory properties (Damen & Steenbekkers, 2007; Farouk & Weliczko, 2003; Hinton et al., 1998). In order to provide consumers with a high quality product from frozen/thawed beef, it is necessary to ascertain the effects of factors involved in the freezing process in relation to the quality of meat after thawing. Several authors have pointed out that one of the factors that affect beef quality after thawing is the frozen storage time (Farouk, Weliczko, & Merts, 2003; Shanks, Wulf, & Maddock, 2002). According to several studies (Ngapo, Babare, Reynolds, & Mawson, 1999; Shanks et al., 2002; Wheeler, Miller, Savell, & Cross, 1990), the quality of frozen meat deteriorates progressively over storage. Notwithstanding this, frozen storage has * Corresponding author. Tel.: +34 923 580 688; fax: +34 923 580 353. E-mail address: [email protected] (C. Vieira). 0309-1740/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2009.06.013

been claimed to improve tenderness, especially in unaged beef (Shanks et al., 2002; Wheeler et al., 1990). Also, freezing rate and storage temperature have been found to be important in the behaviour of ice crystals that could be detrimental to meat quality (Hildrum, Solvang, Nilsen, Froyetein, & Berge, 1999; Mousavi, Miri, Cox, & Fryer, 2007). However, Farouk et al. (2003) and Bertram, Andersen, and Andersen (2007) did not find an effect of frozen storage temperature on most sensory properties in frozen beef. Although recent works (Farouk & Weliczko, 2003; Lagersted, Enfält, Jihansson, & Lundström, 2008; Ngapo et al., 2002; Zhang, Farouk, Young, Weliczko, & Podmore, 2005) have tried to clarify the effects of the freezing process on beef muscle, findings from several studies, as mentioned, show an apparent lack of agreement on some of the points. On the other hand, it is important to note that the effects of frozen protocol on meat quality after thawing are dependent on the characteristics of meat itself. So, the stability of beef in frozen storage depends on its chemical composition because of the differences in colour stability, sarcoplasmic protein solubility and fat oxidation (Farouk & Weliczko, 2003; Zhang et al., 2005). In this sense, meat from rustic breed crosses such as Morucha  Charolais, could show differential behaviour under frozen storage, and not many studies have evaluated the effect of freezing conditions on this type of meat. Meat from rustic breeds such as Morucha and its crosses is characterized by a high amount of fatness, high water holding capacity and high heme pigment concentration

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(Albertí et al., 2005; Vieira, García-Cachán, Recio, Dominguez, & Sañudo, 2006), which could have an important influence on the effect of freezing on its quality. As indicated above, the effect of freezing on meat sensorial properties remains unresolved; especially that from rustic breeds. Thus, the purpose of this study was to study individual and combined effects of different factors in relation to frozen storage, on the characteristics of meat from the Morucha  Charolais breed. 2. Material and methods 2.1. Experimental design and sampling The experiment was carried out at the Meat Technological Station of the Agronomic Technological Institute of Castilla & Leon (ITACyL) in Guijuelo, Salamanca (Spain). Thirty-six Morucha  Charolais crossbred bulls were slaughtered on the same day at an EU authorized slaughterhouse. Taking into account local market preferences, slaughtering was fixed within a range of age of 13–14 months. Cold dressing percentage was expressed as chilled carcass weight – after chilling at 4 °C for 24 h – percentage of slaughter body weight. Conformation and fatness scores were graded visually following the European Normative (Council Regulation (EC) 1183/2006 and 103/2006). Conformation was assessed according to a scale ranking EUROP using a 15-point scale, from 15 (the best conformation) to 1 (the worst conformation). Fatness score was measured on a 5-point scale from 5 (very high fat) to 1 (very low fat). Carcass grading was determined by trained slaughterhouse staff. pH was measured at 24 h post-mortem in the M. Longissimus thoracis at the 6th rib level using a ‘penetration’ pH-electrode. At 48 h after slaughtering, L. thoracis muscle between the 6th and 11th ribs was removed from the right side of each carcass. In order to characterize the samples used, before sampling, a steak of about 100 g was removed from each 6th to 11th rib piece used, and dry matter, ash, ether extract and crude protein were determined according to official procedures (AOAC., 1990). A fatty acid profile was obtained from intramuscular fat by gas chromatography (Perkin-Elmer Auto syst-X.L). The lipid extraction from L. thoracis muscle was carried out according to the Bligh and Dyer (1959) technique, with the methyl esters being obtained according to Morrison and Smith (1964). A summary of carcass characteristics and muscle chemical composition is given in Table 1. The experimental design comprised three factors: ageing extent (3 and 10 days), storage conditions (fresh meat and meat Table 1 Carcass characteristics and Longissimus thoracis muscle chemical composition (means and standard deviations). Parameters

Mean

Standard deviation

Carcass characteristics Slaughter weight (kg) Carcass weight (kg) Cold dressing percentage (%) a Conformation score b Fatness score pH 24 h

595.7 342.0 57.4 3.8 3.0 5.6

35.33 22.95 1.57 0.15 0.00 0.04

Longissimus chemical composition Moisture (%) Ether extract (%) Crude protein (%) c SFA (g/100 g fatty acids identified) c MUFA (g/100 g fatty acids identified) c PUFA (g/100 g fatty acids identified) a

74.60 3.60 21.83 44.72 42.50 12.77

1.29 1.24 0.35 2.34 1.57 2.15

Conformation score: 1 = the worst conformation, 15 = the best conformation. Fatness score: 1 = the lowest fatness grade, 5 = the highest fatness grade. c SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acid. b

399

stored for 30, 75 and 90 days under frozen conditions) and temperature during frozen storage ( 20 and 80 °C). Once the steak used to evaluate meat composition was separated, L. thoracis sections were divided into three pieces, and each one was vacuum packaged in a total of 108 meat pieces. The sample pool of 108 meat pieces of L. thoracis from 36 bulls was divided randomly into two, in order to perform the ageing process (half of the samples were aged for 3 days and the remaining samples for 10 days). After a corresponding ageing period, two control groups of non-frozen meat, six samples of each ageing period, were collected to study the characteristics of non-frozen meat. The remaining samples were assigned to four groups according to the factors established in the experimental design: ageing extent (3 and 10 days) and temperature during frozen storage ( 20 and 80 °C). The effect of storage extent (30, 75 and 90 days) under frozen conditions was studied in each of the four groups of samples mentioned. After the corresponding storage time, frozen samples were removed from their respective storage packages and thawed for 48 h in a 4 °C cooler before being analyzed. In regard to samples which had not been kept under frozen conditions, analyses were performed just after removing from vacuum ageing packs.

2.2. Microbiological sampling and analysis In order to investigate the microbiological quality, all meat samples were treated as follows: the surface of each piece was sampled by swabbing an area of 20 cm2, with a cellulose acetate sponge that had been moistened with 0.1% peptone sterile water (Scharlau, Spain). Each sponge used for swabbing meat was mixed for 1 min with an additional 10 ml of 0.1% (w/v) sterile peptone water. Serial, 10-fold dilutions of each homogenised fluid were prepared with 1 ml each of the undiluted, 10-fold, 100-fold, and 1000-fold diluted fluids in 9 ml volumes of 0.1% peptone water. Then, the dilutions were plated onto growth media in duplicate. Culturing and incubation conditions for different microorganism groups were as follows. To enumerate aerobic psychrotrophic bacteria, aliquots of 1 ml of each dilution were placed on a plate of Plate Count Agar, PCA (Scharlau, Spain), which was incubated at 7 °C for 10 days. To count enteric bacteria, 1 ml of each dilution was placed on a plate of Violet Red Bile Glucose Agar, VRBGA, medium (Scharlau, Spain) and plates were incubated microaerobically at 37 °C for 2 days. Aliquots of 0.1 ml of each dilution were spread on plates of Man Rogosa Sharpe-MRS- agar (Scharlau, Spain) and incubated microaerobically at 30 °C for 2 days to enumerate lactic acid bacteria. Counts were expressed as the log10 cfu/cm2. 2.3. Meat colour Muscle colour was estimated using the M. L. thoracis at the 6th rib, after the newly cut surface was exposed to an artificial fluorescent light for 90 min at 10 °C. Colorimetric parameters (L*, a* and b*) were measured on four spots on each sample using a Minolta (Scharlau, Spain) (Minolta camera, Tokyo Minolta CM2002 spectrophotometer) in the CIEL*a*b* space under D65, 10° and SCI conditions. Hue angle (H*) and Chroma (C*) for meat were calculated as described by Liu, Scheeller, Arp, Schaefer, and Williams (1996). 2.4. Lipid oxidation (TBARs) Lipid oxidation was determined by thiobarbituric acid reactive substances (TBARs) value, using the method of Maraschiello, Sarraga, and García Regueiro (1999). Duplicate samples were used and the results were expressed in mg malonaldehyde (MDA)/kg muscle.

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2.5. Water holding capacity (WHC) Water holding capacity was measured by three methods: (1) thawing losses after thawing the sample (120 g approx.) for 48 h at 4 °C, (2) cooking loss after cooking the sample (100 g approx.) in open polyethylene bags in water at 75 °C, until the samples reached an internal temperature of 70 °C, which was measured with a thermocouple in the centre of the sample (Honikel, 1997) and (3) press losses using the filter paper press method (5 g, 5 min under 2.250 kg) (Hamm, 1960). In all methods, water losses were determined by difference in weight and were expressed as a percentage of initial weight. Obviously, in non-frozen samples, only cooking and press losses were evaluated. 2.6. Instrumental parameters of texture In relation to texture methods used, two procedures were performed: stress at 20% and 80% was carried out in raw meat (compression test) and shear force (Warner–Bratzler test) in cooked meat (Honikel, 1997). To develop the compression test, a minimum of 8 strips were obtained from each steak, each with a 1 cm  1 cm cross-section and the fibre parallel to a long dimension of at least 2 cm, so that the fibre axis was perpendicular to the direction of the compression plunger. A texture analyzer TA-XT2 was used with a cylindrical, flat-end plunger (diameter 2.5 cm) that was driven vertically 20% and 80% of the compression total. The compression procedure permitted transverse expansion of the samples. The speed of the assay was 50 mm/ min. The value taken from the force deformation curve was the force required to achieve the corresponding compression level. The samples used to determine cooking losses were also used in the shear force test. The TA-XT2 was used with a Warner– Bratzler blade and the dimensions and cut characteristics of the sample strips were the same as those used in the compression test. The value taken from the force deformation curve was the maximum force. 2.7. Sensory analysis To perform sensory analyses, steaks were wrapped in an aluminium foil and cooked in a convection oven (preheated at 220 °C for 10 min) to an internal temperature of 70 °C. Each steak, after structures containing visible fat and sinew were avoided, was cut into 2 cm2 samples and kept hot until the time of assessment. A trained eight-member sensory panel assessed the following sensorial attributes: beef odour intensity, tenderness, juiciness, beef flavour intensity and general palatability, using a ten-point descriptive scale (1 = the lowest attribute intensity, 10 = the highest intensity). In addition, panellists were asked for specific, detected off-flavours. 2.8. Statistical analysis The factors studied were ageing extent (3 and 10 days), storage (fresh meat and meat stored for 30, 75 and 90 days under frozen conditions), and temperature during freezing and frozen storage ( 20 or 80 °C). Obviously, to study the effect of temperature during freezing and frozen storage, non-frozen samples were excluded from the data. Data were subjected to variance analysis using GLM procedure to examine the effect of frozen storage length, postmortem ageing and frozen storage temperature. Although the effect of frozen storage temperature was initially included in the model, we removed it from the definitive statistical model because a previous statistical analysis including the three factors and the corresponding interactions showed no significant effect of this factor. Thus, a model including frozen storage length

and post-mortem ageing and the corresponding interactions was used. The statistical package used was SPSS 14.0 (SPSS Inc, 2006). 3. Results 3.1. Microbial counts Table 2 shows the results of microbiological analysis for the four storage times and two ageing periods studied. There was no significant interaction between ageing and storage time for any of the microorganisms (p > 0.05). Enteric bacteria were not detected in any treatment. In relation to the ageing process, a significant effect was observed on psychrotrophic bacteria counts (p < 0.01), with the highest value corresponding to samples which had been aged for a longer period before freezing. Psychrotrophic bacteria counts also increased (p < 0.05) gradually throughout the frozen storage period from a log10 value of approximately 1.7 in fresh meat (0 day), to 3.1 in meat stored under frozen conditions for 90 days. No significant (p > 0.05) effect of ageing period or frozen storage length was found for aerobic lactic acid bacteria. 3.2. Colour and TBARs No significant interaction between post-mortem ageing and frozen storage period was recorded (p > 0.05) for colorimetric parameters and TBARs values. Thus, the main effects are presented in Table 3. Longer ageing period resulted in lower L* (lightness), a* (indicative of red colour) and C* (colour intensity) values (p < 0.05), but no ageing effect was observed in TBARs, H* (hue) and b* (indicative of yellow colour) values (p > 0.05). Regarding frozen storage length, a decreasing trend was observed as regards L*, a*, b*, and C* values, together with an increase in H* values. The differences were more significant as the frozen storage period increased, thus, a*, b*, C* and H* just varied significantly at 90 days of storage. Lipid oxidation could have taken place since higher amounts of MDA (mg/kg) were found in meat stored under frozen conditions for 90 days than in meat stored under non-frozen conditions (p < 0.05). 3.3. Water holding capacity No interaction was observed between frozen storage and previous ageing process (p > 0.05), thus, mean values for both effects are

Table 2 Microbial counts (log10 cfu/cm2) according to the length of frozen storage and previous ageing period. Enteric bacteria

Psychrotrophic bacteria

Lactic acid bacteria

Ageing 3 days 10 days p Level

nd nd –

2.30 3.20

1.99 1.61 ns

Frozen storage length 0 days (not frozen) 30 days 75 days 90 days p Level

nd nd nd nd –

RSD

–

**

1.73a 2.04b 2.97b 3.11c *

23.30

1.54 1.65 1.613 1.71 ns 14.86

nd: not detected. RSD: residual standard deviation. : ** = 0.01; * = 0.05; ns: differences not significant. a,b,c Values with different superscripts in a column indicate significant differences between storage freezing periods.

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presented in Table 4. Samples aged before frozen storage for 10 days showed higher values (p < 0.05) for press and freezing losses, whereas cooking losses did not differ between ageing times (p > 0.05). Percentages of press and cooking losses were significantly higher (p < 0.01) for frozen samples stored for a longer period. Freezing losses were not affected by storage time. However, numerical trends for this attribute showed a slight increase throughout frozen storage. 3.4. Texture As shown in Table 5, in the compression test, ageing significantly affected (p < 0.05) 20% compression values, with lower values corresponding to samples aged for 10 days. The compression test revealed a significant effect (p < 0.01) of frozen storage length on compression at 20%, since lower values were found at 90 days of storage. However, no significant differences were found in compression at 80%. As expected, Warner–Braztler values were affected by post-mortem ageing (p < 0.01), decreasing as ageing period increased. Notwithstanding, an interaction existed between post-mortem ageing and frozen storage period (p < 0.05), indicating that the effect on Warner–Braztler values of the length of

Table 3 Colour parameters and TBARs values, according to the length of frozen storage and previous ageing period. Lightness Redness Yellowness Hue angle Chroma TBARs L* a* b* H* C* (mgMDA/kg) Ageing 3 days 10 days p Level

31.13 30.31

19.61 17.24

*

*

Frozen storage length 0 days 35.10a (not frozen) 30 days 31.90b 75 days 32.11b 90 days 29.06c ** p Level RSD

3.29

16.89 16.98 ns

39.2 40.72 ns

24.34 20.76

19.46a

15.75a

38.91a

23.35a

0.89a

19.65a 18.79ab 17.26b

15.64a 15.81a 14.47b

39.10a 38.29a 43.07b

22.63a 23.94a 19.75b

1.44ab 1.60ab 1.93b

*

***

*

*

***

1.73

1.71

0.14

4.56

1.96

*

1.72 1.80 ns

RSD: residual standard deviation. p Level: *** = p < 0.001; ** = p < 0.01; * = p < 0.05; ns: differences not significant (p > 0.05). a,b,c Values with different superscripts in a column indicate significant differences between storage freezing periods.

Table 4 Water holding capacity as a function of the length of frozen storage and previous ageing period. Freezing losses (%)

Press losses (%)

Cooking losses (%)

3.23 4.06

18.07 20.25

*

**

21.72 21.85 ns

Frozen storage length 0 days (not frozen) 30 days 75 days 90 days p Level

– 3.45 3.47 3.73 ns

17.81a 18.01a 17.87a 21.68b

18.05a 20.04b 19.81b 23.50b

**

**

RSD

1.59

2.95

4.07

Ageing 3 days 10 days p Level

RSD: residual standard deviation. p Level: ** = p < 0.01; * = p < 0.05; ns: differences not significant. a,b Values with different superscripts in a column indicate significant differences between storage freezing periods.

Table 5 Analysis of instrumental texture, as a function of the length of frozen storage and previous ageing period. Compression 20% (N)

Compression 80% (N)

Warner–Braztler (kg)

3 days ageing 0 days (not frozen) 30 days of frozen storage 75 days of frozen storage 90 days of frozen storage

1.20a 1.18a 1.35a 1.05b

108.70 97.51 84.80 90.10

7.40a 7.56a 6.70b 5.27c

10 days ageing 0 days (not frozen) 30 days of frozen storage 75 days of frozen storage 90 days of frozen storage

1.11a 0.87a 1.17a 0.55b

125.94 93.10 87.10 88.20

5.10a 4.94a 4.73a 4.80a

RSD

0.17

18.2

1.17

*

ns ns ns

**

ANOVA p Level (ageing) p Level (frozen time) p Level (ageing * frozen time)

**

ns

* *

RSD: residual standard deviation. p Level: ** = p < 0.01; * = p < 0.05; ns: differences not significant. a,b,c For each ageing period, values with different superscripts in a column indicate significant differences between storage freezing periods.

frozen storage was dependent on post-mortem ageing. In that regard, no effect of frozen storage was observed on Warner– Braztler values of beef aged for 10 days prior to freezing (p > 0.05). However, frozen storage affected Warner–Braztler values (p < 0.05) of beef previously aged for 3 days, since decreasing values were detected at 75 and 90 days of frozen storage. 3.5. Sensory analysis Table 6 shows the scores of sensory parameters given for frozen storage times and for ageing periods studied. The length of postmortem ageing only affected tenderness and general acceptability, samples aged for longer post-mortem periods had higher scores (p < 0.05). The length of frozen storage significantly affected odour intensity (p < 0.05) with values increased at 90 days of storage, but no significant differences were found in the case of flavour intensity (p > 0.05). In spite of the differences observed in water holding capacity, no effect of freezing protocol or ageing length was detected for juiciness (p > 0.05). Regarding tenderness, as in the case of Warner–Braztler values, an interaction between post-mortem ageing extent and frozen storage length was observed (p < 0.01). Thus, no significant effect of frozen storage resulted in samples previously aged for 10 days, whilst in samples aged for 3 days, increasing values (p < 0.01) for tenderness were observed from 75 days of storage. As has been indicated above, in that case, a frozen storage time in a vacuum package could be recommended. 4. Discussion 4.1. Effect of frozen storage length and post-mortem ageing on microbial count The results of microbiological quality after thawing revealed that frozen storage until 90 days in beef previously aged for 3 or 10 days did not cause spoilage of meat. Our values are far lower than those considered as the maximum acceptable for spoilage. Although the type of microorganisms present defines the pattern of spoilage, in general, spoilage occurs when the microbial population reaches 7 8 log10 cfu/cm2. In the case of frozen beef primal

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Table 6 Effect of frozen storage length period and previous ageing extent on sensorial parameters, according to a five-point scale (1 = minimum intensity, 5 = maximum intensity).

3 days ageing 0 days (not frozen) 30 days of frozen storage 75 days of frozen storage 90 days of frozen storage 10 days ageing 0 days (not frozen) 30 days of frozen storage 75 days of frozen storage 90 days of frozen storage RSD ANOVA p Level (ageing) p Level (frozen time) p Level (ageing * frozen time)

Odour

Tenderness

Juiciness

Flavour

Acceptability

2.69a 2.60a

2.75a 2.90ab

2.63 2.38

2.77 2.44

2.84 2.55

2.65a

3.15b

2.49

2.53

2.74

2.78b

3.35b

2.50

2.58

2.77

2.62a 2.61a

3.57a 3.69a

2.69 2.60

2.71 2.62

3.07 2.90

2.68a

3.75a

2.55

2.86

3.10

2.81b

3.65a

2.63

2.69

3.05

0.67

0.88

0.79

0.76

0.66

ns ns ns

ns ns ns

*

ns

**

*

***

ns

**

ns ns

RSD: residual standard deviation. p Level: *** = p < 0.001; ** = p < 0.01; * = p < 0.05; ns: differences not significant. a,b For each ageing period, values with different superscripts in a column indicate significant differences between storage freezing periods.

joints, Hinton et al. (1998), reported log10 5.0 cfu/cm2 of aerobic plate count as a satisfactory standard of carcass quality. It is possible that the counts for the enteric bacteria are an underestimate since the process of freezing reduces their number in primal joints (Hinton et al., 1998) and no resuscitation step was included in the isolation procedure. The numbers of these bacteria reflect the general microbiological status of the mean in relation to carcass handling, time of chilled storage and temperature control, all of which appeared generally satisfactory in this study. Our results showed an increase in psychrotrophic bacteria counts at longer frozen storage periods. Although muscle structure damage has not been evaluated, longer frozen period is generally associated with greater damage in muscle fibres that could have permitted faster microbiological growing during the thawing process (Farouk & Weliczko, 2003; Grujiic´, Petrovic, Pikula, & Amidzˇic, 1993). 4.2. Effect of frozen storage length and post-mortem ageing on colour and lipid oxidation Important changes have been observed in colour parameters and lipid oxidation levels with post-mortem ageing and length of frozen storage. Lipid oxidation and colour deterioration are reasons for meat quality deterioration, and contribute to the development of unacceptable organoleptic characteristics, which are more important in preserved frozen meat where microbiological spoilage is not typically a concern (Frankel, 1998). Contrary to Cifuni, Napolitano, Riviezi, Braghieri, and Girolami’s (2004) report, ageing time did not affect TBARs values, which can be explained since the ageing process took place under vacuum and the normal oxidation process was minimized. Notwithstanding this, for all frozen storage times and ageing periods, MDA content was below the threshold value for rancidity of 2 mg MDA/kg (Watts, 1962). In our study, no differences were observed in hue values as regards post-mortem ageing (p > 0.05). This observation could be due to vacuum packing which increases colour stability maintaining met-

myoglobin levels lower than those found in samples only wrapped in polyethylene (Kenawi, 1994; Lanari, Belvilacqua, & Zarizky, 1989). However, TBARs values increased from 30 days of frozen storage, but statistical differences with respect to fresh meat only appeared after 90 days under frozen storage. Oxidative processes are associated with discolouration of meat, as lipid oxidation results in the formation of pro-oxidation capable of reacting with oxymyoglobin, which leads to metmyoglobin formation (Farouk & Swan, 1998a; Frankel, 1998). Colour deterioration is related to higher contents of metmyoglobin and changes in colorimetric parameters, especially in hue, with higher values corresponding to more discolouration (Clydesdale, 1998; Liu et al., 1996). Other studies (Farouk & Swan, 1998a; Farouk & Weliczko, 2003) also observed that the thawed beef became brown (hue angle increased) with storage time. These authors attributed the increase in hue values, to reduced activity of metmyoglobin reducing enzymes or increased lipid oxidation with storage time. Our results agree with several studies (Abdallah, Marchello, & Ahmad, 1999; Farouk & Swan, 1998b; Farouk et al., 2003) which have observed other colour parameter changes in thawed meat, as lower Chroma, L* and a* values. These authors stated that frozen–thawed beef samples experienced less ‘‘blooming” and more rapid discolouration than fresh cuts during storage. It is important to note that in our study significant colour changes were only observed after 90 days of storage, which is in agreement with most studies mentioned above, where more significant colour changes were obtained as the frozen storage time increased. Nevertheless, the values of colorimetric parameters obtained were, in all cases, within the range of desirable meat colour values, so frozen storage under proper conditions preserves meat without causing discolouration. Moreover, studies such as Fernández et al. (2007) stated that freezing can be used together with other technologies for preserving meat, for example high-hydrostatic pressure, in order to reduce the severe discolouration associated with this new technology. 4.3. Effect of frozen storage length and post-mortem ageing on water holding capacity Post-mortem ageing affected press and freezing losses, but not cooking losses. The differences in the response of parameters used to estimate water holding capacity might be due to differences in the origins of the liquid lost in each process. During cooking, the lost liquid comes from constitutive water and from the fat that melts during heating, so the difference in ageing periods could be attenuated (King, Dikeman, Wheeler, Kastner, & Koohmaraie, 2003; Mandell, Gullett, Buchanam, & Campbell, 1997). Losses due to thawing and pressing, however, come mainly from constitutive water and, because of the effect of ageing on muscle structure and, hence, its capacity to retain water, the differences observed between ageing times in water loss are more pronounced. Our results are in agreement with the general belief that claims a progressive decrease in water holding capacity of beef during frozen storage (Ngapo et al., 1999; Farouk & Weliczko, 2003; Damen & Steenbekkers, 2007). According to research developed by Sacks, Casey, Boshof, and Vanzyl (1993) in mutton muscle stored under freezing conditions, drip loss after 2.5 months of frozen storage was at least double the percentage measured in refrigerated meat. In addition, a recent study (Lagersted et al., 2008) reported that water loss was significantly higher in meat kept frozen at 20 °C for two months than in chilled meat. These authors observed that the differences in water loss between chilled and thawed meats decreased as the previous ageing period increased, values for frozen meat aged for 2 days were 25% higher than those for chilled meat, without differences in meat aged for 14 days. Similarly, Farouk et al. (2003) reported that the water holding capacity tended to decrease gradually with the storage time up to 9 months

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in the form of thaw and cooking losses, indicating an increase in protein denaturation and the attendant loss of ability to hold water. According to studies reported, the reason for the differences in water holding capacity in chilled and frozen meats could be related to the disruption of the muscle cell structure upon freezing. It is important to note that the values of thawing losses are lower than those reported by many other studies (Farouk & Weliczko, 2003; Zhang et al., 2005). Nevertheless, it is necessary to take into account that, in the present study, samples were stored under vacuum packaging, and according to several studies (Bustabad, 1999; Campañone, Roche, Salvadori, & Mascheroni, 2006), the effect of packaging on weight losses is marked. In addition, previous studies have characterized meat from Morucha and its crosses as high water holding capacity beef (Albertí et al., 2005; Vieira et al., 2006). In agreement with our results, Bertram et al. (2007), did not find a difference in water holding capacity between pork samples frozen at 20 °C or 80 °C. However, other studies (Farouk et al. (2003), have reported more drip in slowly frozen meat than in fast frozen meat, which was associated with a greater structural damage caused by larger intracellular ice crystals produced during slow freezing. 4.4. Effect of frozen storage length and post-mortem ageing on texture parameters Taking into account the interaction between post-mortem ageing and length of frozen storage, it seems that freezing did not affect shear force values of aged beef whereas it provided a decrease in this parameter in partially aged beef. There is no agreement in the literature with respect to the effect of freezing on meat texture. Whereas some researches (Farouk et al., 2003; Lagersted et al., 2008; Shanks et al., 2002) have found that freezing causes tenderization in beef, others (Pearson & Miller, 1950) have shown a progressive decrease in beef tenderness during frozen storage. Although in the present study a positive effect of frozen storage on texture was found, significant differences in 20% compression values were only observed after 90 days of frozen storage. These results are consistent with the findings of previous studies (Jackobson and Bengston,1973; Lagersted et al., 2008; Reid, 1999; Shanks et al., 2002; Smith, Carpenter, & King, 1969), which concluded that the effect of frozen storage on beef texture is dependent on frozen storage length. Shanks et al. (2002) observed that, after 2 months of frozen storage at 16 °C, the Warner–Braztler values of frozen steaks aged for 6 or 7 days were approximately equal to the mean values of fresh steaks aged for 14 days. As had been reported by Crouse and Koohmaraie (1990), Reid (1999), and Shanks et al. (2002) the increase in tenderness could be due to the breakdown of muscle fibres caused by enzyme activity and by ice crystal formation. The physical effect of ice crystals on muscle fibre structure is reported in studies on the microstructure of fresh and frozen meats (Grujiic´ et al., 1993; Mousavi et al., 2007). This physical effect supported our texture results, taking into account that, whereas a significant effect of frozen storage was observed in compression at 20% and in the Warner–Braztler test, no significant effect was detected in compression at 80%, which has been associated with the resistance of connective tissue (Campo et al., 2000; Lepetit & Culioli, 1994). In relation to enzyme activity, it seems that small intracellular ice crystals, formed by very fast freezing rates, increase the rate of ageing probably due to the release of enzymes (Dransfield, 1986). 4.5. Effect of frozen storage length and post-mortem ageing on sensory analysis Rancidity was not detected in any of the samples, probably due to the low values obtained for MDA. In this sense, Campo et al.

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(2005) pointed out that TBARs value of 2 could be considered the limiting point from where rancid flavour overpowers beef flavour. A recent prospective study (Damen & Steenbekkers, 2007) revealed that most respondents could not differentiate fresh meat from frozen meat when the process had taken place under proper conditions. Likewise for instrumental texture, an interaction between postmortem ageing and frozen storage in tenderness scores was observed. As has been discussed above, these results are in line with the findings of some studies (Bustabad, 1999; Farouk et al., 2003) comparing non-chilled meats and previously chilled frozen meats, the toughest was non-chilled frozen beef. By contrast, divergent results in tenderness and instrumental shear force were observed by Lagersted et al. (2008), because higher tenderness scores were obtained in chilled meat than in frozen meat but higher peak force values were observed in chilled meat. These authors justified their results because better scores in other sensory parameters such as juiciness and meat taste could have led to a perception of higher tenderness in chilled meat in comparison with frozen meat. Nevertheless, other studies reported that freezing cuts of meat over a wide range of conditions has little effect on the eating quality (Hildrum et al., 1999; Wheeler et al., 1990). In spite of the differences found in drip losses, no effect of ageing or frozen storage was observed on juiciness. This finding was explained by the fact that the parameter evaluated was maintained juiciness, which is dependent on the intramuscular fat content (Destefanis, Barge, & Brugiapaglia, 1996; Vieira et al., 2006). With respect to palatability, a positive effect of the length of post-mortem ageing was obtained, which is consistent with the general belief that the ageing process increases perceived palatability, mainly due to its effect on tenderness (Dransfield, 1998; Monson, Sañudo, & Sierra, 2004; Shanks et al., 2002). 5. Conclusion The results suggest that, in beef from Morucha  Charolais breed, frozen storage up to 3 months under vacuum resulted in changes in colour parameters and lipid oxidation, although the quality was satisfactory in all cases. The most important effect occurred in texture properties of cooked beef, where the effect of the length of frozen storage was dependent on post-mortem ageing. Whereas in 10-day aged meat any positive effect of frozen storage was found, in beef aged for shorter times, better texture values were detected in beef that had been stored frozen for 75 or 90 days. Acknowledgements This study was supported by the Agronomic Technological Institute of Castilla and Leon (ITACyL). The authors would like to thank the laboratory staff of Estación Tecnológica de la Carne (Guijuelo–Salamanca) for their technical assistance and the panellists for their participation in the sensory analysis.

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