Oxidative stability of chicken meat at different oxygen concentrations in the packaging

The aim of this study was to preserve the quality and prevent the colour, lipid and protein oxida - tion in chicken breast fillet (pectoralis major muscle) during storage. For this purpose, modified atmos - pheres (MA) with four different gas mixtures (vol. %) of oxygen (O 2 ), carbon dioxide (CO 2 ) and nitrogen (N 2 ) were used: oxygen free (0: 20: 80), low (20: 20: 60), medium (40: 20: 40), and high oxygen MA (70: 20: 10). After 9 days of storage in a refrigerator at 2 ± 1 °C, pH was measured, colour was evaluated instrumen - tally and sensorially, the basic chemical composition, thiobarbituric acid reactive substances (TBARs), and protein carbonyl content of raw chicken breast were determined. After thermal treatment (82 °C, 1 hour, sous-vide) of the breast fillet, the sensory characteristics of odour and aroma were evaluated, and the texture (Warner-Bratzler shear force) was measured instrumentally. The composition of the gas mixture affected all oxidation dependent parameters: (i) colour: L * and b * values were the highest under all MA with oxygen, and the a * value under MA without, medium and high-oxygen, (ii) lipids: the TBARs increased significantly under high oxygen MA after 7 days of storage (below the sensory thresholds value for rancidity), (iii) proteins: protein carbonyl content was the highest under medium and high oxygen MA, and (iv) sensory properties: colour oxidation and intensity of odd aromas under all MA with oxygen, as well as odour and aroma of stale in oxygen-free MA were increased. For chicken breast the most suita - ble is packaging under modified atmosphere without O 2 , because the oxidation rate of colour, lipids and proteins during storage was the lowest.


Introduction
Chicken meat is one of the most popular meats due to its low price and favourable nutrient composition (Patsias et al., 2006). The consumption of chicken meat is increasing compared to beef and pork mainly because of its low price, easy and quick preparation, and desirable nutritional value, as it contains many highly usable proteins, low fat, and many polyunsaturated fatty acids (Brenes and Roura, 2010). Despite the above advantages, chicken meat has a relatively short shelf life, even when stored in a cold environment. Therefore, scientists and the meat industry are looking for new ways to extend the shelf life of chicken meat (Patsias et al., 2008). Due to the increasing demand for chicken meat, it is crucial to choose the optimal packaging method to ensure adequate safety and quality of the meat during storage and transportation to the final consumer (Zhang et al., 2015).
Although the packaging requirements for fresh chicken meat are similar to those for red meat, their packaging is particularly challenging due to physiological and biological factors. Raw poultry meat is perishable due to its relatively high pH (5.7-6.7), which allows rapid growth of microorganisms both at room temperature and during refrigeration. In addition, poultry meat is rich in protein and fat with a high content of unsaturated fatty acids and is therefore susceptible to oxidative reactions.
The high content of polyunsaturated fatty acids is the main reason why chicken meat is more susceptible to oxidation, which in turn influences the shorter shelf life due to the deterioration of sensory quality (Cortinas et al., 2004;Xiao et al., 2011). Oxidation of meat leads to negative changes in colour, odour, aroma, and texture, as well as the appearance of potentially toxic substances, so oxidative spoilage must be stopped or prevented. One of the most successful methods to prevent the oxidation of colour, lipids and proteins and the deterioration of sensory quality of meat is proper packaging.
Modified atmosphere (MA) packaging (MAP) is a packaging method that is increasingly used today for packaging a variety of foods, especially raw meat and meat products. The air atmosphere is replaced by a mixture of gases, usually consisting of oxygen, carbon dioxide and nitrogen. The concentration of each gas in the gas mixture depends on the food to be packaged and the desired effect. It is important to choose the right atmosphere for each food to extend the shelf life while maintaining safety and sensory quality. Raw meat is usually packaged in a very low-oxygen (possibly with additional oxygen scavengers) or very high-oxygen atmosphere.
The aim of our study was to maintain quality during 9 days of storage and to prevent the oxidation of colour, lipids and proteins in chicken breast fillets by packing them in a modified atmosphere. For this purpose, different gas mixtures, nitrogen, carbon dioxide and different concentrations of oxygen were tested.

Material and experiment design
Chicken breast muscles (musculus pectoralis major) were packaged in four different atmospheres at a local meat processing plant. The approximate ratio of gases (vol. %, oxygen (O 2 ): carbon dioxide (CO 2 ): nitrogen (N 2 )) during packaging was as follows: modified atmosphere (MA) without oxygen (0: 20: 80), with low oxygen MA (20: 20: 60), with medium oxygen MA (40: 20: 40), and with high oxygen MA (70: 20: 10). For each atmosphere, 9 packaging units (each unit consisted of two breasts) were prepared, as the analyses were repeated on the 2 nd , 7 th and 9 th day after slaughter, taking three units each time (4 atmospheres × 9 packaging units = 36 samples).
On the 2 nd day after slaughter, the meat samples were delivered to the Biotechnical Faculty, where they were stored in a refrigerator at 2 ± 1 °C. First, the composition of the gas mixture in the atmosphere of the packaging units was measured (3 units from each atmosphere). One chicken breast from each of the twelve packaging units was used for the analysis of physicochemical parameters. The pH and instrumental colour values were then measured. Breast fillets were homogenised and used for immediate determination of nutrient composition (water, protein and fat content). Further chemical analyses (thiobarbituric acid reactive substances (TBARs) and protein carbonyl content) were performed after one week of storage of the frozen homogenised samples at 18 ± 1 °C. Chemical analyses were performed in parallel, measuring nutrient composition in one parallel and colour and texture in four parallels. The colour of the second chicken breast from each of the twelve packaging units was sensory evaluated, and each piece was vacuum-packed in a high-barrier sealable lidding film for food trays made of polypropylene and polyethylene (PP/PE) (thickness 50 µm) (Lin Top PP HB A 50; Linpac Plastic Pointivy, France): O 2 transmission, < 5 ×10 −6 m 3 (m 3 × 24 h × bar) −1 ; carbon dioxide transmission, < 25 ×10 −6 m 3 (m 3 × 24 h × bar) −1 ; water vapour transmission, < 0.003 kg (m 2 × 24 h × bar) −1 . This was followed by a one-hour heat treatment in a combi oven (Rational FRIMA (SCC61)) at a moist heat of 82 °C. The technique used is referred to as sous vide cooking or vacuum cooking. The heat-treated samples were subjected to sensory analysis focusing on texture, odour and aroma attributes, and texture (shear force) was measured instrumentally after cooling to room temperature (20 ± 1 °C).
The whole procedure was repeated on the 7 th and 9 th day after slaughter.

Methods
Instrumental methods: A mobile gas analyser for the testing of modified atmospheres in food packaging, calibrated OXYBABY® V instrument (WITT Gasetechnik GmbH & Co. KG, Austria), was used to determine the gas compositions in the packages under each of the packaging atmosphere conditions as % O 2 and % CO 2 . An absolute accuracy of 0.1 % was achieved at O 2 concentrations < 10 vol. %, with < 1 % relative accuracy at O 2 concentrations of 10-100 vol. %, at 20 °C . Water, protein and fat content in the samples were determined using the Food ScanTM Analyser with self-calibration (FOSS, Denmark), pH values were measured directly using a combined glass-gel spear electrode (Type 03, Testo Pty Ltd, Australia), colour L* (lightness), a* (±, red to green) and b* (±, yellow to blue) values on the surface of a 2.54 cm raw slice were measured by CR-400 colorimeter (Konica Minolta Optics, Inc., Osaka, Japan), and instrumental analysis of texture was performed using the TA-XT Plus texture analyser (Stable Micro Systems Ltd., Godalming, Surrey, UK) as described in Zahija et al. (2020).
Chemical analyses: Approximately 100 g of a representative sample was homogenised for 20 s with a Grindomix homogeniser (GM 200; Retch, Germany) at 5000-6000 rpm (ISO 3100-1, 1991). The extent of lipid oxidation of the chicken samples was monitored by measuring the thiobarbituric acid reactive substances (TBARs) as described by Penko et al. (2015). A slightly modified (Rotar, 2019) spectrophotometric method by Soglia et al. (2016) was used to determine the protein carbonyl content. The absorbance was measured with a spectrophotometer (Agilent Technologies, Cary 8454 UV-Vis) at two wavelengths, 280 nm and 370 nm. The content of protein carbonyls was expressed in nmol/mg protein.
Sensory evaluation: A panel of five qualified and experienced panellists in the field of meat products was used to assess the sensory qualities. The sensory evaluation of chicken breast samples was performed according to international standards (ISO 8589:2007, ISO 8586:2012). The panel evaluated the samples separately in three session (days) consisting of 12 samples. The analytical-descriptive test (Golob et al., 2005) was performed by scoring the sensory attributes on a structured scale from 0 to 4 points, where 0 means that the attribute is not expressed, 1 defines a slightly expressed attribute, 2 medium (strongly) expressed, 3 strongly expressed and 4 very strongly expressed attribute of the sample. The panel could also use half values (i.e., 0.5, 1.5, 2.5, and 3.5). The sensory profile of the chicken breast samples was evaluated using 7 descriptors, which were divided into three blocks. The first block related to the visual attribute on the surface of a raw breast fillets as oxidation rate of colour; at higher values, the grey-brown oxidised colour appeared. The second block related to the olfactory attributes, such as rancid odour, stale odour (appearance of stale components, cardboard odour, wet dog hair odour, etc.) and odd odours (presence of foreign, undesirable components). The third block was related to the aroma attributes, such as the aroma of rancid and stale/old (warm over flavour) as well as odd aromas (appearance of undefined acidic, bitter perception).
Data analysis: The data were analysed for normal distributions using the UNIVARIATE procedure (SAS/STAT). The differences according to the atmospheric composition (oxygen MA: without, low, medium and high), time of storage (on day 2, 7 and 9 (d)), as well as to their interaction (without_2 d, low_2 d, medium_2 d, high_2 d, without_7 d, low_7 d, medium_7 d, high_7 d, without_ d, low_9 d, medium_9 d, high_9 d) were analysed through a general linear model procedure and least squares mean test (SAS/STAT), with a 0.05 level of significance. The effect of production repetition (1-3) was not significant and was excluded from the model.

Basic chemical composition
On average, 100 g wet weight of chicken breast contained 24.28 ± 0.59 g protein, 76.94 ± 0.45 g moisture and 1.73 ± 0.36 g fat (not shown in tables). The data agree well with the reference values from the Slovenian nutritional tables (Golob et al., 2006) for 100 g of boneless and skinless chicken breast: 22.8 g of protein, 74.8 g of moisture and 1.5 g of fat. Chmiel et al. (2019) confirmed that the duration and conditions of storage, as well as the type of packaging, did not affect the basic chemical composition of chicken breast.

Gas composition in the packages
After 9 days of storage, the concentrations of O 2 , CO 2 , and N 2 in the packaging units changed significantly (P ≤ 0.001) in all experimental groups, depending on the composition of the gases at filling ( Table 1). Changes in the composition of the atmosphere during storage may be due to the absorption of gases into the tissues (meat), the production of gases during muscle respiration, and some gases could also be produced during bacterial growth in the meat (Keokamnerd et al., 2008;Penko, 2015). In MA without oxygen, CO 2 content decreased and N 2 content increased significantly after the 9-day storage period, while O 2 content remained unchanged. In low oxygen MA, O 2 content decreased by more than half and N 2 content increased by more Table 1 Composition of gas mixture in packaging units during 9-day storage period at 2 ± 1 °C of raw chicken breast muscle (n = 36) than 20 %, while CO 2 content remained unchanged during storage. In MA with medium oxygen content, a decrease in O 2 content (about 9 %) and an increase in N 2 concentration (about 10 %) were also observed. In high oxygen MA the gas ratios did not change significantly during the storage period. Gill (1988) noted that CO 2 is highly soluble in water and oils, so it is easily absorbed into muscle and fat during storage (Penko, 2015). However, Demirhan and Candoğan (2017) found that CO 2 concentration also decreases during storage due to the transfer of CO 2 from inside the packaging unit to the external environment and vice versa (Tománková et

Instrumentally measured texture
The composition of the gas mixture in the packaging unit had no significant effect (P = 0.397) on the Warner-Bratzler shear force (Table 3). However, shear force significantly decreased (P ≤ 0.05) during 9 days of storage under three of the four MA: without, low and high oxygen MA (27 %, 40 %, and 40 %, respectively). Jongberg et al. (2013) found, based on sensory evaluation of texture, that breast stored for 9 days under high oxygen MA (80 % O 2 and 20 % CO 2 ) was significantly less tender than breast stored without oxygen. The author's results did not agree with our results at MA of 70 % O 2 .

Instrumentally measured colour
The effects of 9-day storage period in four different atmospheres on the instrumentally measured surface colour of raw chicken breast were observed ( Table 3). The interaction of storage time and gas mixture composition had a significant effect on all measured colour values. For samples packed under MA without oxygen, after 9 days of storage, the b* value decreased and the a* value increased, the samples were less yellow and redder, while the L* value did not change significantly. Under low oxygen MA, chicken breast L* value increased and b* value decreased during storage, samples became brighter and less yellow. The colour of the samples under medium and high oxygen MA did not change significantly after storage. Jongberg et al. (2013) found that breast meat stored under high oxygen MA (80 % O 2 , 20 % CO 2 ) showed yellow discolouration, whereas this was not the case for thigh and breast meat under MA without oxygen (80 % N 2 , 20 % CO 2 ) and for vacuum packed samples. Penko et al. (2015) found that chicken burg- Table 3. Effect of the atmosphere composition in the packaging unit during 9-day storage period at a temperature of 2 ± 1 °C on instrumentally measured texture and colour, as well as oxidation parameters of chicken breast muscle value of parameter (LSM ± SD) according the atmosphere:

Thiobarbituric acid reactive substances
The effects of the atmosphere composition in the packaging unit and 9-day storage period on thiobarbituric acid reactive substances (TBARs) of raw chicken breast muscle were significant ( Table  3). The highest TBARs were determined for breast samples stored under high and without oxygen MA after 7 and 9 days of storage, respectively, the lowest for breast samples from all modified atmosphere groups after 2 days post mortem, and for samples stored under medium and low oxygen MA after 9 days of storage. Significant increase in TBARs can be observed only under high oxygen MA after 7 days of storage. Significantly less (21 %) secondary products of lipid oxidation (0.22-0.21 mg malondialdehyde (MDA)/kg) were formed in the samples under low and medium MA after 9 days of storage than under without and high-oxygen MA (0.28 mg MDA/kg). An increase in TBARs means a higher degree of oxidation, but not always noticeable changes in sensory properties. An appropriate TBARs value is up to 0.70 mg MDA/kg, and a TBARs value above 1 mg MDA/kg predicts sensory perceived rancidity (Nam in Ahn, 2003;Penko et al., 2015). In this experiment, TBARs values did not exceed 0.70 mg MDA/kg. Demirhan and Candoğan (2017) found that TBARs values of chicken thighs increased during storage under the following atmospheres: air > MA 70 % CO 2 and 30 % N 2 > MA 70 % CO 2 and 30 % N 2 + oxygen scavenger), but did not exceed the threshold value for rancidity. Oxidation of thigh meat lipids occurred to a lesser extent, probably due to the low oxygen concentration in the packaging units

Protein carbonyls
The effects of the atmosphere composition in the packaging unit, 9 days of storage and their interaction on the protein carbonyl content in raw chicken breast were significant (Table 3). On the day 2 after slaughter, significantly higher protein carbonyl content was determined in the samples stored under highest oxygen MA than in the samples stored under medium, low or without oxygen. Carbonyl content in chicken breast under modified atmosphere increased with increasing storage time, except for MA without oxygen, where it remained unchanged for 9 days. Breast meat stored under medium oxygen MA for 7 days and under low and high oxygen MA for 9 days showed a significant increase (72 %, 65, and 27 %, respectively) in protein carbonyl content. Therefore, the highest protein carbonyl content was observed in breast samples under high oxygen MA after 9 days of storage and under medium oxygen MA after 7 and 9 days of storage, respectively.
Protein and lipid oxidation increased after 9 days of storage for both breast and thigh meat packed under high oxygen MA (80 % O 2 , 20 % CO 2 ), but this increase was greater in thigh than in breast meat, indicating lower oxidative stability of chicken thighs. Breast and thigh meat packed under MA without oxygen or vacuum were almost completely protected from oxidation during 9 days of storage (Jongberg et al., 2013). Higher concentrations of O 2 in the packaging atmosphere also resulted in greater oxidation of proteins during storage of beef steaks (Zakrys et al., 2008). This is consistent with the results of this study, as maximum protein carbonyl levels were determined under medium and high oxygen MA. Increased protein carbonyl levels in air-packed chicken thighs, under high oxygen MA (70 % CO 2 and 30 % N 2 ) and under MA with oxygen scavengers were also found by Demirhan and Candoğan (2017). It is obvious that MAP and the application of oxygen scavenger could successfully delay oxidation of chicken thigh meat proteins considering the low carbonyl content of the samples during storage (less than 2 nmol/mg protein). Table 4 shows the professional panel data for sensory analysis of the thermally treated chicken breast samples with respect to the composition of the atmosphere in the packaging unit after storage at a temperature of 2 ± 1 °C. The data show that the composition of the gas mixture affected the sensory properties: oxidation of colour and odd aromas were most expressed in meat stored under oxygen MA (low, medium, and high), stale odours and aromas were most expressed at MA without oxygen. However, during the 9-day storage period, some sensory properties of the meat changed. Rate of colour oxidation increased (most noticeable under low and medium oxygen MA, least noticeable under without oxygen MA), odour and aroma of stale became more expressed under without oxygen and under high oxygen MA.

Sensory properties
Before storage, the odd odours on the breast meat were more expressed under an oxygen-free than under an oxygen-containing MA, but by the 9 day of storage they had decreased significantly. Rancidity during the 9-day storage period was not noted by the testers in any of the samples. More than rancidity and stale aroma, the testers noticed distinct odd aromas, after 7 days of storage under low and high oxygen MA. After the 9 day of storage, odd aromas were similar in all atmospheres (the differences are not significant). The testers associated odd odours and odd aromas detected after 7 days of storage under low oxygen MA with the descriptor of the odour and aroma of the soup.
Based on sensory evaluation, oxygen was found to have no positive effect on the colour of poultry meat (Rossaint et al., 2015). This is likely due to the fact that poultry meat has a lower myoglobin content, and thus the ability to form oxymyoglobin than beef or pork (Millar et al., 1994). In addition to packaging conditions, the colour of poultry meat is influenced by several factors such as sex, age, water content, pre-slaughter conditions, and processing (Faustman and Cassens, 1990).
In their study, Penko et al. (2015) compared the instrumentally measured colour of raw chicken patties with a sensory evaluation of colour oxidation. It was confirmed that the patties packed under high oxygen MA were brighter and more expressed in colour, while patties packed under low oxygen MA were described as greyish. The rancid odour and aroma were also more expressed in the patties packaged under high oxygen MA, which was to be expected since the highest TBARs levels were also detected in these patties. In contrast, Jongberg et al. (2013) could not confirm any correlation between TBARs and sensory analysis of rancid odour. Higher TBARs values were found for thighs packed under high oxygen MA (80 % O 2 , 20 % CO 2 ), while a higher expression of rancid odour was found for packed breast. The breasts have a less expressed natural odour than the thighs, so the rancid odour was more expressed in the breast, while the rancid odour remained masked in the thighs (Jongberg et al., 2013).

Conclusion
Producers of meat and meat preparations are often faced with a task that seems simple at first glance, but often turns out to be not so simple. Poultry can become discoloured under a low oxygen modified atmosphere, and under a high oxygen modified atmosphere it becomes odour-tainted, rancid, or stale Table 4. Sensory attributes of thermally treated chicken breast samples regarding the composition of the atmosphere in the packaging unit during 9 days storage period at a temperature of 2 ± 1 °C evaluated by descriptive analysis with professional panel. 0.6 ± 0.9 cb 1.5 ± 0.6 a 0.9 ± 0.6 b 1.4 ± 0.7 a P S ≤ 0.001 9 0.6 ± 0.5 cb 1.0 ± 0.6 b 0.5 ± 0.5 cbd 0.5 ± 0.5 cbd P A×S = 0.01 and rancid in aroma after a period of storage under refrigerated conditions. Therefore, producers must find a compromising atmosphere composition in the packaging unit that maintains the acceptable colour of the meat while minimising the extent of oxidation processes.
Based on the results of our experiment, we can say that a modified atmosphere with high oxygen content (70 % O 2 , 20 % CO 2 , 10 % N 2 ) is the least suitable for packaging chicken breast, because due to the high oxygen concentration, the oxidation of lipids and proteins was the greatest. As for colour acceptance, chicken breast contains very little myoglobin, so the bright red or pink colour produced by myoglobin at high oxygen concentrations is not such an important parameter that would significantly influence consumer purchase. For these reasons, it is better to use a modified atmosphere with low or without oxygen to pack chicken, especially breast. In addition, it was found that storing chicken meat under low-oxygen modified atmosphere (20 % O 2 , 20 % CO 2 , 60 % N 2 ), which is the first choice in the meat industry, is not suitable because the rate of colour oxidation is the highest. A modified atmosphere without oxygen would probably be the best choice, since protein and colour oxidation were minimal in the breast stored in this way. Although the determined TBARs values after 9 days of storage under modified atmosphere without oxygen were as high as under high oxygen MA (0.28 mg MDA/kg) and still below the sensory threshold for rancidity (1 mg MDA/kg), so lipid oxidation would be minimised and sensory properties would not be affected.
Acknowledgements: The authors gratefully acknowledge the company Pivka Perutninarstvo, d. d. and the Slovenian Research Agency (P4-0234, Integrated food technology and nutrition) for the financial support. *This article is part of a MSc. thesis named Oxidative stability of chicken meat at different oxygen concentrations in the packaging unit, issued by Eva Golob.