Effect of packaging conditions on shelf-life of ostrich steaks

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MEAT SCIENCE Meat Science 78 (2008) 143–152 www.elsevier.com/locate/meatsci

Effect of packaging conditions on shelf-life of ostrich steaks J. Ferna´ndez-Lo´pez *, E. Sayas-Barbera´, T. Mun˜oz, E. Sendra, C. Navarro, J.A. Pe´rez-Alvarez Departamento de Tecnologı´a Agroalimentaria, Escuela Polite´cnica Superior de Orihuela, Universidad Miguel Herna´ndez, Ctra. a Beniel Km 3, 2 03312 Orihuela, Alicante, Spain Received 30 November 2006; received in revised form 5 September 2007; accepted 5 September 2007

Abstract This study was conducted to establish the shelf-life of ostrich steaks stored in four different packaging types: (i) air exposure, (ii) vacuum, and two different modified atmospheres packages (iii) MAP: 80% CO2 + 20% N2, and (iv) MAP + CO: 30% CO2 + 69.8% argon + 0.2% CO. Shelf-life evaluation was based on colour, lipid and hemopigments oxidation, microbial counts and sensory assessment of odour and colour. Samples stored under air exposure showed the highest lipid and hemopigments oxidation rate. Based on aerobic bacteria counts, the shelf life of ostrich steaks stored under aerobic conditions would be 8 d at most, whereas under vacuum, MAP or MAP + CO it would be 12 d. The presence of CO extends the shelf life of ostrich steaks by stabilisation of red colour measured by instrumental and sensory techniques, and maintenance of fresh meat odour by slowing down off-odour perception.  2007 Elsevier Ltd. All rights reserved. Keywords: Ostrich; Meat; Shelf-life; Modified atmosphere packaging; Spoilage

1. Introduction Meat from the ostrich (Struthio camelus) is perceived and marketed as a healthy alternative to other red meats due to favourable nutritional properties (low cholesterol, and intramuscular fat contents and high percentage of polyunsaturated fatty acids) (Fisher, Hoffman, & Mellet, 2000). In Spain, ostrich meat production reached in 2004 1000 t (ACADE., 2005) and shows an important annual growth rate, principally due to its healthy properties, which encourages ostrich meat consumption (25–30% increased in 2004 compared to 2003). To allow competitive marketing of ostrich meat, both for internal consumption and external purposes, it is necessary to know its spoilage characteristics under different conditions in order to extend its shelf-life. Ostrich meat processors sell fresh and frozen meat, as well as processed meat products, to a variety of markets *

Corresponding author. Tel.: +34 966749734; fax: +34 966749677. E-mail address: [email protected] (J. Ferna´ndez-Lo´pez).

0309-1740/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2007.09.003

generally following recent retail packaging practises (Alonso-Calleja, Martı´nez-Ferna´ndez, Prieto, & Capita, 2004). Modern meat packaging techniques are intended to maintain microbial and sensory quality of the product. Changes in the packaging atmosphere (aerobic, vacuum or modified atmosphere) are used in the food industry to extend products shelf-life. Modified atmosphere packaging using a high carbon dioxide (CO2) environment is an effective means of prolonging microbial shelf-life of meat during extended storage (Silliker & Wolfe, 1980; Sørheim, Nissen, & Nesbakken, 1999). Nevertheless, several researchers have stressed that the use of high levels of CO2, with a consequent low O2 concentration, can cause meat discolouration (Silliker, Woodruff, Lugg, Wolfe, & Brown, 1977). Discolouration of the characteristic bright red meat colour is related to the conversion of oxymyoglobin to metmyoglobin (Ledward, 1984). This phenomenon may be counteracted by incorporation of CO to the atmosphere (Lun˜o, Beltra´n, & Roncale´s, 1998; Silliker & Wolfe, 1980). CO combines with myoglo-

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bin to form carboxymyoglobin (MbCO), which is more stable to oxidation than OMb and gives an attractive cherry-red colour to meat. As a consequence of the colour stabilisation, CO has been suggested as a complementary gas for meat packaging (Parry, 1993). The only undesirable effect of using CO on meats is related to human safety (Marquardt & Scha¨fer, 1994). Regulatory authorities in the European Union do not allow the use of CO in meat packaging. However, The Norwegian meat industry is using a gas mixture containing 0.3–0.4% CO to prolong shelf-life for displaying and selling fresh retail meat in all parts of the country (Norwegian Food Control Authority., 2001). The Scientific Committee on Food (European Commission) was asked to evaluate the safety of CO as a packaging gas for meat, and it concluded that there is no health concern associated with the use of 0.3– 0.5% CO in a gas mixture as a modified atmosphere packaging gas for fresh meat, provided that the temperature during storage and transport does not exceed 4 C (SCF., 2001). The aim of our work was to establish the shelf-life of ostrich steaks stored under different packaging conditions at 2 ± 1 C. The evaluation of the shelf-life was based on colour, lipids and hemopigments oxidation, microbial counts and sensory assessment of odour and colour.

CO2 + 20% N2 (MAP). The fourth batch was also packed in modified atmosphere, but using a different gas mixture: 30% CO2 + 69.8% argon + 0.2% CO (MAP + CO). Both gas mixtures were supplied by Abello´ Linde (Barcelona, Spain). All packs were stored at 2 ± 1 C, in a HOTCOLD-GL refrigerated cabinet (J.P. SELECTA S.A., Barcelona, Spain) simulating retail conditions at supermarket. This cabinet was illuminated by a standard supermarket fluorescent lamp (OSRAM, Germany). The samples were exposed to lighting continuously at 1000 lux at the surface. Light intensity was measured using a luxometer Lutron LX-102 (Taiwan). The positions of the samples in the cabinet were rotated every 24 h to minimize light intensity differences and possible temperature variations at the surface of meat. Twelve samples (3 for each lot) were removed from the cabinet at 0, 4, 8, 12, and 18 d for subsequent analysis. 2.3. Physico-chemical analysis The CIELAB 1976 colour space was studied following the procedure of Cassens et al. (1995). The following colour coordinates were determined: lightness (L*), a* coordinate (redness/greenness, +/) and b* coordinate (yellowness/ blueness, +/). From these coordinates, hue (h*) and chroma (C*) were calculated as follows:

2. Materials and methods Hue ¼ tan1 b =a Chroma ¼ ða2 þ b2 Þ

1=2

2.1. Raw materials Iliofibularis muscles were removed from 8 ostrich carcasses after removal of external fat and epimysial connective tissue. The diet history and production practices of the ostrich meat were unknown. Samples were transported to the Food Technology Pilot Plant (Miguel Herna´ndez University) under hygienic conditions and processed immediately upon arrival. 2.2. Storage conditions Ostrich meat was immediately sliced (eight steaks about 2.5 cm thick were cut from each muscle). Each pack (a total of 60 packs) was prepared by placing two steaks (randomly distributed) in a spanded polystyrene tray (318 · 235 · 38 mm), which was overwrapped with a high barrier film of water vapor permeability 1.1 g/m2/24 h at 23 C/50% RH, nitrogen permeability 2.7 cm3/m2/24 h at 23 C/50% RH, carbon dioxide permeability 23 cm3/m2/24 h at 23 C/50% RH and oxygen permeability <5 cm3/m2/24 h at 23 C/50% RH (W.K. Thomas Spain S.L., Rubı´, Barcelona, Spain). These packs were separated in 4 batches of 15 packs. Each batch was brought to different packaging atmospheres using an EGARVAC packaging machine (Barcelona, Spain). One batch was packaged directly by sealing the film upon the tray (AIR). A second batch was vacuum packed (VACUUM). The third batch was packed in modified atmosphere using a gas mixture of 80%

Colour determinations were made at 10 ± 2 C by means of a Minolta CM-2600 (Minolta Camera Co., Osaka, Japan) spectrophotometer with illuminant D65, 10 observer, SCI mode, 11 mm aperture of the instrument for illumination and 8 mm for measurement. American Meat Science Association guidelines for colour measurements were followed (Hunt et al., 1991) and spectrally pure glass (CR-A51, Minolta Co., Osaka, Japan) was put between the samples and the equipment. pH was measured by blending a 15 g sample with 150 mL deionized water for 2 min. The pH of the resultant suspension was measured with a Crison pH meter (Model 507, Crison, Barcelona, Spain) equipped with a Crison combination electrode (Cat. No. 52, Crison, Barcelona, Spain). 2.4. Lipid oxidation Thiobarbituric acid reactive substances (TBARS) assay was performed as described by Rosmini et al. (1996). The mixture was heated for 10 min in a boiling water bath (95–100 C) to develop a pink color, cooled with tap water, centrifuged at 5500 rpm for 25 min in a centrifuge (Alresa HZ50, Orto Alresa, Aljavit, Madrid), and the absorbance of the supernatant was measured spectrophotometrically at 532 nm using a UV spectrophotometer (Unicam Limited, Cambridge, UK). The results were expressed as TBARS (mg malonaldehyde/kg sample).

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2.5. Metmyoglobin (MMb) Five grams of minced meat were used to determine MMb concentration in each sample. Myoglobin was extracted with cold 0.04 M phosphate buffer, pH 6.8, with a sample to buffer ratio of 1:10. Samples were homogenized for 15 s with a Polytron homogenizer (Brinkman Instruments, NY, USA) at 10,800 rpm. The homogenates were then centrifuged for 30 min (50,000g) (Alresa HZ50, Orto Alresa, Aljavit, Madrid) at 5 C. The absorbance of the filtered supernatant was read at 525, 572 and 730 nm. Percentage of MMb was determining using the formula of Krzywicki (1979): 

MMbð%Þ ¼ 1:395–ððA572 –A730 Þ=ðA525 –A730 ÞÞ 100 Samples were kept on ice during the whole assay.

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1.5% peptone water in a Stomacher 400 (Colworth, London, UK) for 1.5 min. Aliquotes were serially diluted in peptone water and plated out following standard methodologies (ICSMF (International Commission on Microbiological Specifications), 1983). Total viable counts were determined on Plate Count Agar incubated at 37 C for 48 h. Enterobacteriaceae were determined using Violet Red Bile Glucose Agar (VRBGA) incubated at 37 C for 24 h. Lactic acid bacteria (LAB) counts were determined on MRS Agar (pH 5.6), with the plates incubated under anaerobic conditions (gas generating kit anaerobic system, Oxoid Unipath Ltd. Basingtoke, Hampshire, UK) at 30 C for 48 h. Psychrotrophic microbiota were determined on Plate Count Agar, and the plates were incubated at 7 C for 10 days. Culture media were from Oxoid (Oxoid Unipath Ltd. Basingtoke, Hampshire, UK). Results were expressed as log10 CFU/g.

2.6. Total iron 2.9. Sensory assessment of odour and colour Total iron concentration was determined in wet-ashed samples using the ferrozine assay (Stookey, 1970). Ostrich meat (0.2–0.3 g) was placed in a test tube and digested with concentrated nitric acid and 30% hydrogen peroxide on a hot plate until a white ash was formed. The ash was dissolved in 0.2 mL of 1.0 N HCl and diluted with 0.8 mL deionized water. Ascorbic acid (1 mL, 1%) was added, and the tube contents were mixed with a vortex mixer for test tubes (Kisker, Steinfer, Germany). After 20 min, 1 mL 10% ammonium acetate buffer and 1 mL of 1 mM ferrozine color reagent were added, and the mixture was shaken. The mixture was allowed to stand at room temperature for 45 min, and then the absorbance was determined from a standard curve made with iron standard solution (Sigma diagnostics, St. Louis, MO 63178 USA). 2.7. Heme iron Heme iron was determined using the method of Hornsey (1956). Meat (2 g) was transferred into a 50 mL polypropylene tube, and 9 mL of acid acetone (90% acetone + 8% deionized water + 2% HCl) was added. The meat was macerated with a glass rod and allowed to stand 1h in a dark cabinet at room temperature. The extract was filtered through Whatman filter paper #42, and the absorbance was read at 640 nm against the acid acetone blank. Total pigments, as acid hematin, were calculated using the formula: Total pigmentðppmÞ ¼ A640  680 and heme iron was calculated as follows: Heme ironðppmÞ ¼ total pigmentðppmÞ  8:82=100 2.8. Microbiological analysis A 10 g aliquot of each meat sample was aseptically obtained. It was then homogeneized with 90 mL of sterile

Sensory evaluation of meat samples of all package types was conducted at 0, 4, 8, 12 and 18 d of refrigerated storage. The sensory panel was composed of 6 experienced and trained panellists from the staff and students of the Food Technology Department at Miguel Herna´ndez University. Panelists had been previously trained to detect off-odour and colour differences related to meat deterioration, according to the method of Cross, Moen, and Stanfield, (1978). The attributes ‘‘off-odour’’ and ‘‘ red colour’’ were rated using five-point descriptive scales as described by Djenane, Sa´nchez-Escalante, Beltra´n, and Roncale´s (2001). Each panellist removed the meat samples from the package by opening it with a knife and placing the sample on a white plastic plate. The samples were then evaluated for offodours. Scores for ‘‘off-odour’’ referred to the intensity of off-odours associated with meat deterioration: 1 = none, 2 = slight, 3 = small, 4 = moderate and 5 = extreme. After allowing the colour to develop for about 5 min, the panellists evaluated the meat samples for red colour. The colour scale used to evaluate meat red colour was a five point scale that had been previously created by the panellists: 5 = extremely intense, 4 = moderately intense, 3 = acceptable, 2 = moderately weak and 1 = extremely weak. Sensory evaluation sessions were held under fluorescent lighting (2000 lx; Philips 40W Cool White). 2.10. Statistical Analysis Analysis of variance was conducted for each variable measured to investigate the effect of storage time and packaging type, and the interaction of both. This was a repeated measures design using ‘‘between-subject’’ factor. The effect of storage day was measured ‘‘within-subjects’’. Tukey’s test for multiple comparisons was preformed using the general linear model of SPSS 14.0 for Windows (SPSS, Chicago, Ill., USA) software package. The level of statistical significance was P < 0.05.

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3. Results and discussion 3.1. pH pH values show differences (P < 0.05) due to storage time and storage conditions (Fig. 1). The initial pH value of ostrich meat was 6.04 ± 0.10. A final pH above 6.0 was also found for ostrich carcasses at 2–6 h after bleeding (Sales & Horbanczuk, 1998). Berge, Lepetit, Renerre, and Touraille (1997) reported that depletion of glycogen reserves following ante-mortem stress in ratites might be the major reason for relative high pH values compared to other meats. pH values decreased during storage time for all storage conditions (Fig. 1). Initial pH values dropped slowly in air packed samples the first 8 days, but at a faster rate on further storage when lactic acid bacteria reached their maximal counts (Fig. 6). The highest decrease rates were found in vacuum and modified atmosphere packed samples, which is in accordance with their highest counts of lactic acid bacteria. Meat pH can be affected by many factors; however, growth of lactic acid bacteria resulting in lactic acid production is the major factor in pH decrease in packaged meats (Gill, 1996). At day 18 of storage, pH differences (P < 0.05) were found between meat air packed and the others, but not between vacuum, MAP and MAP + CO packed ones.

steaks packaged in different packaging conditions are shown in Fig. 2. For all samples, surface L* values increased during storage period. Several authors have reported lightness increases in different meat and meat products during refrigerated storage (Kusmider, Sebranek, Lonergan, & Honeyman, 2002; Ferna´ndez-Lo´pez et al., 2006). Vacuum, air and MAP packaged samples showed similar (P > 0.05) lightness values during the storage period. Ostrich steaks packaged in MAP + CO were lighter (P < 0.05) than the other packed samples. Changes in surface CIE a* values throughout the storage period of ostrich steaks packaged in different packaging conditions are shown in Fig. 2. Redness values for air, vacuum and MAP packed samples decreased (P < 0.05) during storage time. This decrease was higher (P < 0.05) for vacuum and MAP packed samples than for air-packed ones. In a high oxygen atmosphere, oxymyoglobin is rapidly formed, which provides the typical cherry-red visual colour

55 50 45

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Time (days) 20 18 16 14 12

a*

Lightness, redness and hue showed differences (P < 0.05) during storage time and between storage conditions. Yellowness and chroma showed no differences (P > 0.05) for the two factors studied. Initial mean surface L* value for ostrich meat (38.4 ± 1.65) was similar to values reported for other authors (Navarro et al., 2000; Marco, 2004). Changes in CIE L* values throughout the storage period of ostrich

10 8 6 4 2 0

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Fig. 1. pH evolution in ostrich steaks packaged under different conditions of: air, vacuum and modified atmospheres of 80% CO2 + 20% N2 (MAP) or 30% CO2 + 69.8% argon + 0.2% CO (MAP + CO) during storage time at 2 C.

vacuum

air

MAP

MAP+CO

Fig. 2. Lightness (L*), redness (a*) and hue (H*) evolution in ostrich steaks packaged under different conditions of: air, vacuum and modified atmospheres of 80% CO2 + 20% N2 (MAP) or 30% CO2 + 69.8% argon + 0.2% CO (MAP + CO) during storage time at 2 C.

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3.3. Pigment and lipid oxidation Pigment and lipid oxidation are major deteriorative reactions in meat and meat products during storage. They are responsible for a significant loss in quality characteristics such as colour, flavour, texture and nutritive value (Akamittath, Brekke, & Schanus, 1990). The relationship between oxidation of lipids and hemopigments has been well documented in fresh meat during storage (Mercier, Gatellier, & Renerre, 1995). However, the cause-effect relationship between the two processes is not completely clarified and it is not known when or whether lipid oxidation causes pigment oxidation or vice versa.

3.3.1. Lipid oxidation TBARS showed differences (P < 0.05) during storage time and between storage conditions (Fig. 3). Ostrich steaks exposed to air showed the highest (P < 0.05) TBARS values. Steaks in vacuum, MAP or MAP+CO packages showed no differences (P > 0.05) in TBARS values. These results agree with the statements of Ordonez and Ledward (1977) who stated that the concentration of oxygen in the package atmosphere is the determining factor for the rate of lipid oxidation. Excluding or limiting the oxygen content in the vacuum, MAP and MAP + CO packaging atmospheres limited oxidation and thus resulted in lower TBARS values for these meat samples. The increase in TBARS increase was fastest in air packed meat. In vacuum, MAP or MAP + CO packed steaks, differences in TBARS values were not significant until 8 days of storage were reached; while under air exposure, differences were significant (P < 0.05) after only 4 days of storage. Afterwards, TBARS values of air exposed samples dramatically increased. At the end of storage (day 18), the lowest TBARS values (P < 0.05) were obtained in MAP and MAP + CO packed steaks. Reduction in the amount of oxygen in vacuum, MAP and MAP + CO packed steaks and the barrier characteristics of the packaging film accounted for the lower TBARS values. Lo´pezLorenzo, Herna´ndez, Sanz-Pe´rez, and Ordon˜ez (1980) reported that CO2 decreased lipid oxidation rate in pig meat. This effect was attributed to meat pH reduction due to the absorption of CO2 (Jakobsen & Bertelsen, 2002). Some authors attributed an antioxidant activity to CO, but it was only demonstrated at concentration higher than 0.25% (Lun˜o et al., 2000). 3.3.2. Metmyoglobin (MMb) Renerre, Anton, and Gatellier (1992) reported that the susceptibility of myoglobin to autoxidation is the main factor in explaining colour stability in meat and meat products. In meat systems, the identities of the substances

1.6

TBARS (mg MA/kg sample)

of ostrich meat (Seydim, Acton, Hall, & Dawson, 2006). The loss of redness due to oxidation of myoglobin in packaged meat was already expected as a consequence of meat’s high pH (>6.0). At high pH values, mitochondrial enzyme systems (cytochrome, succinate and pyruvate-malate oxidase) do not shut down and have the ability to consume available oxygen (Lawrie, 1998). Bendall and Taylor (1972) reported that the oxygen consumption rate of high pH muscle is higher than that of normal pH muscle. Bembers and Satterlee (1975) also noted that the rate of the conversion of myoglobin to MMb was 1.5–2.0 time faster in high pH systems than that of muscle having more normal pH. The a* values of the MAP + CO packages were considerable higher (P < 0.05) than the other packaging environments, and did not change (P > 0.05) throughout the entire storage period. So, samples packaged in MAP + CO provided a significant stabilisation of red meat colour. Sørheim, Nissen, and Nesbakken (1997) and Lun˜o, Roncale´s, Djenane, and Beltra´n (2000) obtained similar results for beef loin steaks packaged in modified atmospheres with CO. This stabilization of the red colour of meat has been attributed to the formation of carboxymyoglobin due to the reaction of CO + myoglobin. Carboxymyoglobin is more stable to oxidation than oxymyoglobin and gives an attractive cherry-red colour to meat (El-Badawi, Cain, Samuels, & Anglemeier, 1964; Lun˜o et al., 1998). Because of the high stability of carboxymyoglobin, only relatively low levels of CO are needed in order to maintain red colour of meat (Lun˜o et al., 2000). Initial mean b* (8.4 ± 1.06) and C* values (15.8 ± 1.10) in ostrich steaks are in agreement with those reported by other authors (Marco, 2004; Navarro et al., 2000). Changes in hue values during the storage of ostrich steaks, shown in Fig 2, confirmed that the influence of atmosphere type on surface colour follow the same behaviour that were observed for redness a* values. The best performing (P < 0.05) atmosphere for the stabilization of meat colour (based on hue and redness values) was the MAP + CO atmosphere and the worst were the vacuum and MAP atmospheres (with no differences (P > 0.05) between them).

147

1.2

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0.4

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4

8

12

18

Time (days) air

vacuum

MAP

MAP+CO

Fig. 3. TBARS evolution in ostrich steaks packaged under different conditions of: air, vacuum and modified atmospheres of 80% CO2 + 20% N2 (MAP) or 30% CO2 + 69.8% argon + 0.2% CO (MAP + CO) during storage time at 2 C.

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capable of oxidizing OMb to MMb are not clear. Several authors concluded that O 2 can initiate lipid peroxidation, leading to the formation of prooxidant substances capable of reacting with OMb and resulting in MMb formation (Anton et al., 1993). They postulated that OMb could be oxidized not only by lipid-oxy radicals but by other prooxidant radicals generated by O 2. MMb content showed differences (P < 0.05) during storage time and between storage conditions (Fig. 4). Differences (P < 0.05) in MMb content were found between air, vacuum and modified atmosphere packed samples (MAP and MAP + CO) after 4 days, but not (P > 0.05) between MAP and MAP + CO packed ones. The highest (P < 0.05) MMb content was observed for air packed samples, and the lowest (P < 0.05) for the ones packed in modified atmospheres. High oxygen levels can promote not only lipid oxidation but also MMb accumulation (O’Grady, Monahan, Burke, & Allen, 2000). MMb formation was time-dependent when meat samples were stored for 18 days at 2 C. Similar behaviour has been reported for other meat and meat products stored under refrigerated conditions (Bekhit, Geesink, Ilian, Morton, & Bickerstaffe, 2003; Ferna´ndez-Lo´pez et al., 2006). This increase in MMb formation during storage time was more pronounced at the end of storage (12–18 days). In samples packed under vacuum or modified atmosphere conditions, MMb formation was inhibited (P < 0.05). This effect was greater (P < 0.05) in modified atmosphere packed samples than in vacuum packed ones. As can be observed in Fig. 3, meat samples stored under air exposure showed the highest oxidation rate, which corresponds with the highest MMb formation in these samples, likely due to a higher amount of free radicals. Greene, Hsin, and Zipser (1971) reported that 40% MMb caused meat rejection by consumers, so according to these data, samples that may cause rejection with values higher that 40% MMb were the air packed samples stored more than 12 days.

3.2.3. Heme and non heme iron Iron content of ostrich meat (3.2 ± 0.03 mg/100g) is similar to beef meat and approximately three times higher than that of pork and chicken meat (American Ostrich Association., 1998). Therefore, ostrich meat, as other red meats, is a rich source of iron which is an essential mineral. Moreover, the iron in meat is 90% heme iron, which is several times more absorbable than non heme iron present in other foods. Although the mechanism for heme iron release in meat has not been determined, oxidation of the porphyrin ring and denaturation of myoglobin (Kristensen & Purslow, 2001) are probably involved. Some authors have reported that, during refrigerated storage of meat, there is a release of iron from the heme group, with a consequent increase in non heme iron, which speeds up lipid oxidation (Lee, Hendricks, & Cornforth, 1998). Heme iron content showed differences (P < 0.05) due to storage time and storage conditions (Fig. 5). Differences (P < 0.05) in heme iron content were found between air, vacuum and modified atmospheres packed samples, but not (P > 0.05) between MAP and MAP + CO packed ones. The lowest (P < 0.05) heme iron content was observed in air packed samples, and the highest (P < 0.05) in modified atmospheres packed samples (MAP and MAP + CO). This increase in non heme iron content in air packed samples is related to the highest TBA values found in these samples. Heme iron content in ostrich meat decreased (P < 0.05) with increasing storage time but at a different rate, depending on packing conditions. This decrease of heme iron content was lower (P < 0.05) in both vacuum and modified atmosphere packed samples (MAP and MAP + CO). Between MAP and MAP + CO packed meat, the decrease in heme iron content was not significant (P > 0.05) until the end of storage (day 18); in vacuum packed samples, this decrease was already significant (P < 0.05) after 12 days of storage, and in air packed ones after only 4 days.

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Fig. 4. Metmyglobin (%) evolution in ostrich steaks packaged under different conditions of: air, vacuum and modified atmospheres of 80% CO2 + 20% N2 (MAP) or 30% CO2 + 69.8% argon + 0.2% CO (MAP + CO) during storage time at 2 C.

air

vacuum

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Fig. 5. Heme iron content (mg/100 g) evolution in ostrich steaks packaged under different conditions of: air, vacuum and modified atmospheres of 80% CO2 + 20% N2 (MAP) or 30% CO2 + 69.8% argon + 0.2% CO (MAP + CO) during storage time at 2 C.

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Aerobic bacteria (log CFU/g)

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Fig. 6. Bacterial group evolution in ostrich steaks packaged under different conditions of: air, vacuum and modified atmospheres of 80% CO2 + 20% N2 (MAP) or 30% CO2 + 69.8% argon + 0.2% CO (MAP + CO) during storage time at 2 C.

3.3. Microbial quality Microbial counts at day 0 of storage were (log CFU/g): Enterobacteriaceae 2.0 ± 0.2, Lactic acid bacteria 2.9 ± 0.3, Aerobic plate counts 3.7 ± 0.5 and Psychrotrophic counts 3.0 ± 0.4. These microbial counts are similar to previously reported counts on ostrich carcasses (Harris et al., 1994) and higher than those reported for other red meats (Blixt and Borch (2002)). The higher microbial load found in ostrich meat in relation to other red meats has been attributed to the characteristic high pH of this type of meat which, creates and ideal environment for rapid microbial spoilage in some packaging conditions (Alonso-Calleja et al., 2004; Sales & Mellet, 1996; Seydim et al., 2006). Aerobic, lactic acid and psychrotrophic bacteria counts showed differences (P < 0.05) during storage time and

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between packaging conditions (Fig. 6), while enterobacteriaceae showed differences (P < 0.05) only during storage time. The evolution of counts of enterobacteria was similar (P > 0.05) for all samples stored under aerobic conditions, vacuum or modified atmosphere (MAP and MAP + CO). Enterobacteriaceae counts increased (P < 0.05) during storage period, reaching at the end of the storage period (18 day) an average value of 7.0 ± 0.2 log CFU/g. The growth of Enterobacteriaceae throughout storage on all batches could be attributed to the high initial pH of the meat as microorganisms normally compete better in meat of high pH (>6.0) even in vacuum packaging (Bern, Hechelmann, & Leistner, 1976), and to their microaerophilic condition. Aerobic plate counts showed differences (P < 0.05) between air packed samples and the others, but not (P > 0.05) between vacuum, MAP and MAP + CO packed meat. Air packed samples showed higher (P < 0.05) aerobic plate counts than vacuum, MAP or MAP + CO packed ones. It is assumed that vacuum reduces the number of aerobic bacteria during meat storage due to the reduction of oxygen availability. This also applies to modified atmospheres packed without oxygen. In this study, a mixture of CO2 and N2 (MAP) or CO2, argon and CO (MAP + CO) was used to remove the oxygen from package headspace, but it must be also taken into account the ability of CO2 to inhibit the growth of a wide range of microorganisms (Farber, 1991). At the same time, O2 restraint would have favoured lactic acid bacteria (Fig. 6) as will be discussed later. The evolution of aerobic bacteria was different depending on packing conditions. This evolution was similar (P > 0.05) in vacuum, MAP and MAP+CO packed samples. In these packing conditions, there were a decrease (P < 0.05) in aerobic counts during the first 4 days and afterwards, counts increased (P < 0.05) although the counts were always kept at lower levels (P < 0.05) than those of air packed samples during the entire storage period. As soon as spoilage can be detected by odour, taste or appearance, most foods have more than 6 log CFU/g of bacterial load. It is accepted that counts of 7 log CFU/g is the approximated point from which the meat would be unacceptable (Dainty & Mackey, 1992). Therefore, the shelf-life of ostrich meat stored under aerobic conditions would be 8 days, while under vacuum, or modified atmospheres (MAP or MAP + CO) it would be 12 days. Higher shelf life has been reported for other red meat packed under different conditions: values ranging from 14 to 21 days of shelf life have been reported by Blixt and Borch (2002) for beef and pork meat packed under vacuum conditions and by Djenane et al. (2001) for beef meat packed in MA. Lactic acid bacteria counts were different (P < 0.05) between air packed samples and the others, but not (P > 0.05) among vacuum, MAP and MAP + CO packed

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attributes showed differences for packed atmosphere and storage time. For ‘‘red colour’’ evaluation, MAP + CO packed meat showed the best scores (P < 0.05) throughout all the storage time (18 days). Air packed samples showed similar scores to MAP + CO packed ones but only until day 4 of storage; afterwards, colour scores decreased, although the colour perception at the end of storage were better than vacuum and MAP packed samples. No differences (P > 0.05) for colour perception were found between vacuum and MAP steaks throughout storage. The literature indicates that, in packed meat, the presence of oxygen is necessary to have a better perception of the characteristic red colour of fresh meat due to oxymyoglobin pigment (Lun˜o et al., 1998; Renerre et al., 1992), and also that CO is able to produce and stabilise red colour (Lun˜o et al. 1998; Sørheim et al., 1997). These results are in accordance with a* values obtained with instrumental analysis (redness, Fig. 2) that MAP + CO packed samples showed the highest a* values during storage. It is important to observe that although for these samples (MAP + CO) redness values were not modified during storage, a decrease in colour evaluation was detected by panellist up to day 12. It could be related to the detection of superficial lime and discoloration observed in these samples at day 18, principally due to high microbial counts (Fig. 6). ‘‘Off-odour’’ evaluation showed differences (P < 0.05) between air, MAP + CO and the others, but not (P > 0.05) between MAP or vacuum packed ones. The detected offodours were mainly putrefactive and sour. Air packed samples showed the highest scores (P < 0.05) for off-odour, which is in accordance with the highest TBARS values and microbial growth in these samples. Vacuum and MAP packed samples showed a similar behaviour of off-odour perception during storage. In both types of packaging, there was no modification for off-odour perception during the first 4 days of storage, and the highest alteration was obtained between the days 12 and 18, reaching at this time scores higher than 4. However, for air packed samples, this level

samples. Air packed samples showed lower (P < 0.05) counts than vacuum, MAP or MAP + CO packed ones, because lactic acid bacteria are favoured for their anaerobic condition. Lactic acid bacteria counts increased during storage time for all samples, but the evolution of counts in air packed samples was 1 log cycle lower than in the others. These results are in agreement with those reported by other authors about meat stored under anaerobic conditions where lactic acid bacteria represented the dominant microbiota (Blixt & Borch, 2002). Counts of psychrotrophs showed differences (P < 0.05) among packaging systems. The highest (P < 0.05) counts were found in air packed samples, and the lowest (P < 0.05) in modified atmospheres packed ones (MAP and MAP + CO). Vacuum meat presented intermediate counts of psychrotrophs. For all samples, counts of psychrotrophs increased during storage time, although for vacuum and modified atmosphere packed samples (MAP and MAP + CO) there was a difference (P < 0.05) only up to 4 days of storage time. Similar results were found for other authors in meat stored under vacuum conditions, which reported storage times higher than 4 days to detect significant increases in psychrotrophic counts (Lee, Simard, Laleye, & Holley, 1985; Otremba, Dikerman, & Boyle, 1999). Higher rates of microbial growth were reported by Capita, Dı´az-Rodrı´guez, Prieto, and Alonso-Calleja (2006) in ostrich steaks stored under air and vacuum conditions; a behaviour that was attributed to the high initial microbial loads and pH found in the raw meat. It is important to observe that for all type of bacteria analyzed, the presence of 0.2% CO in the atmosphere had no effect (P > 0.05) on microbial growth, in agreement with Lun˜o et al. (1998). 3.4. Sensory assessment of odour and colour Results of sensory analysis of meat samples for colour perception and off-odour, are summarised in Table 1. Both

Table 1 Influence of packing conditions on sensory panel scores (means ± SD) of ostrich steaks during refrigerated storage (2 C) Attribute

Packed atmosphere b

Days of storage 0a

4

8

12

18

Red colour

Air Vacuum MAP MAP + CO

5.0ax 5.0ax 5.0ax 5.0ax

5.0 ± 0.0ax 3.1 ± 0.4by 3.2 ± 0.6by 5.0 ± 0.0ax

3.4 ± 0.4by 2.2 ± 0.3cz 2.0 ± 0.6cz 5.0 ± 0.0ax

3.5 ± 0.2by 1.1 ± 0.3cv 1.3 ± 0.2cv 4.1 ± 0.2ay

2.1 ± 0.5bz 1.0 ± 0.6cv 1.1 ± 0.4cv 3.0 ± 0.0az

Off-odourc

Air Vacuum MAP MAP + CO

1.0av 1.0av 1.0av 1.0av

2.1 ± 0.4az 1.0 ± 0.0bv 1.0 ± 0.0bv 1.0 ± 0.0bv

3.2 ± 0.4ay 2.5 ± 0.3bz 2.5 ± 0.4bz 1.5 ± 0.3cz

4.6 ± 0.3ax 3.8 ± 0.4by 3.9 ± 0.3by 2.5 ± 0.5cy

4.9 ± 0.1ax 4.7 ± 0.3ax 4.8 ± 0.2ax 4.0 ± 0.3bx

a–c Means within a row with different letters are significantly different (P < 0.05) x–z For each attribute, means within a column with different letters are significantly different (P < 0.05). a Sensory analysis at day 0 was run on steaks before packaging. b For red colour: 5 = extremely intense, 4 = moderately intense, 3 = acceptable, 2 = moderately weak, 1 = extremely weak. c For off-odour: 1 = none, 2 = slight, 3 = small, 4 = moderate, 5 = extreme.

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of off-odour was already perceived at day 8 of storage. It is important to observe the particular behaviour of off-odour perception in MAP + CO packed samples. Although these samples showed similar values (P > 0.05) of TBARS and microbial growth during storage than vacuum and MAP packed samples, the scores for off-odour were lower. Some authors reported that 1% CO improved odour shelf life of meat, an effect that was related to the antioxidant effect of high concentrations of CO (Clark, Lentz, & Roth, 1976; Lun˜o et al., 1998). Probably, the concentration of CO used in this study (0.2%) had a low antioxidant effect that, although it was not detected by TBARS analysis, could be involved in extending meat odour shelf life. 4. Conclusions The results of this study indicate that the shelf life of fresh ostrich meat stored at 2 C can be extended by packaging the product under anaerobic conditions (vacuum, MAP or MAP + CO packed). This increase in shelf life is due to the reduction of not only microbial spoilage but also lipid and hemopigment oxidation. Moreover, the presence of CO extends the shelf life of ostrich steaks by stabilisation of red colour measured by instrumental and sensory techniques, and maintenance of fresh meat odour by slowing down off-odour perception. Acknowledgments This research was supported by BANCAJA and Miguel Herna´ndez University through the research project IPLP01. The authors wish to express gratitude to ‘‘Avestruces El Rinco´n’’ (Valencia, Spain) for providing meat samples. References ACADE. (2005). Informe anual de la Asociacio´n de Criadores de Avestruces de Espan˜a. Madrid, Spain. Akamittath, J. G., Brekke, C. J., & Schanus, E. G. (1990). Lipid peroxidation and color stability in restructured meat systems during frozen storage. Journal of Food Science, 55, 1507–1513. Alonso-Calleja, C., Martı´nez-Ferna´ndez, B., Prieto, M., & Capita, R. (2004). Microbiological quality of vacuum-packed retail ostrich meat in Spain. Food Microbiology, 21, 241–246. American Ostrich Association. 1998. http://www.ostriches.org. ` tude des relations Anton, M., Salgues, C., & Renerre, M. (1993). E oxidatives entre les lipides membranaires et la myoglobine in vitro. Science Alimentaire, 13, 261–274. Bekhit, A. E. D., Geesink, G. H., Ilian, M. A., Morton, J. D., & Bickerstaffe, R. (2003). The effects of natural antioxidants on oxidative processes and metmyoglobin reducing activity in beef patties. Food Chemistry, 81, 175–187. Bembers, M., & Satterlee, L. D. (1975). Physico-chemical characterization of normal and PSE porcine muscle myoglobins. Journal of Food Science, 40, 40–43. Bendall, J. R., & Taylor, A. A. (1972). Consumption of oxygen by the muscle of beef animals and related species. Journal of the Science Food and Agriculture, 23, 707–719. Berge, P., Lepetit, J., Renerre, M., & Touraille, C. (1997). Meat quality traits in the emu (Dromaius novaehollandiae) as affected by muscle type and animal age. Meat Science, 45, 209–221.

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